InteractiveFly: GeneBrief

Cyclin B3: Biological Overview | References

In mitosis and meiosis, chromosome segregation is triggered by the Anaphase-Promoting Complex/Cyclosome (APC/C), a multi-subunit ubiquitin ligase that targets proteins for degradation, leading to the separation of chromatids. APC/C activation requires phosphorylation of its APC3 and APC1 subunits, which allows the APC/C to bind its co-activator Cdc20. The identity of the kinase(s) responsible for APC/C activation in vivo is unclear. Cyclin B3 (CycB3) is an activator of the Cyclin-Dependent Kinase 1 (Cdk1) that is required for meiotic anaphase in flies, worms and vertebrates. It has been hypothesized that CycB3-Cdk1 may be responsible for APC/C activation in meiosis but this remains to be determined. Using Drosophila, this study found that mutations in CycB3 genetically enhance mutations in tws, which encodes the B55 regulatory subunit of Protein Phosphatase 2A (PP2A) known to promote mitotic exit. Females heterozygous for CycB3 and tws loss-of-function alleles lay embryos that arrest in mitotic metaphase in a maternal effect, indicating that CycB3 promotes anaphase in mitosis in addition to meiosis. This metaphase arrest is not due to the Spindle Assembly Checkpoint (SAC) because mutation of mad2 that inactivates the SAC does not rescue the development of embryos from CycB3-/+, tws-/+ females. Moreover, CycB3 was found to promote APC/C activity and anaphase in cells in culture. CycB3 physically associates with the APC/C, is required for phosphorylation of APC3, and promotes APC/C association with its Cdc20 co-activators Fizzy and Cortex. These results strongly suggest that CycB3-Cdk1 directly activates the APC/C to promote anaphase in both meiosis and mitosis (Garrido, 2020).

Mitosis and meiosis (collectively referred to as M-phase) are distinct modes of nuclear division resulting in diploid or haploid products, respectively. In animals, both require the breakdown of the nuclear envelope, the condensation of chromosomes and their correct attachment on a microtubule-based spindle, where chromosomes are under tension and chromatids are held together by cohesins. Progression through these initial phases requires multiple phosphorylation events of various protein substrates by mitotic kinases including Cyclin-Dependent Kinases (CDKs) activated by their mitotic cyclin partners. M-phase completion from this point (mitotic exit) requires the degradation of mitotic cyclins, and the dephosphorylation of several mitotic phosphoproteins by phosphatases including Protein Phosphatase 2A (PP2A). Mitotic exit begins with the segregation of chromosomes in anaphase. In mitosis, sister chromatids segregate. In meiosis I, replicated homologous chromosomes segregate, and in the subsequent meiosis II, sister chromatids segregate. Nuclear divisions are completed with the reassembly of a nuclear envelope concomitant with the decondensation of chromosomes. How mitosis and meiosis are alike and differ in the molecular mechanisms of their exit programs is not completely understood (Garrido, 2020).

Chromosome segregation is triggered by the Anaphase-Promoting Complex/Cyclosome (APC/C), a multi-subunit E3 ubiquitin ligase. By catalysing the addition of ubiquitin chains on the separase inhibitor securin, the APC/C targets it for degradation by the proteasome. As a result, separase cleaves cohesins, allowing separated chromosomes to migrate towards opposing poles of the spindle. Activation of the APC/C in mitosis requires its recruitment of its co-factor Cdc20. This recruitment can be prevented by the Spindle-Assembly Checkpoint (SAC), a complex mechanism that allows the sequestration of Cdc20 until all chromosomes are correctly attached on the spindle. Cdc20 binding to the APC/C is also inhibited by its phosphorylation at CDK sites. Phosphatase activity is then required to dephosphorylate Cdc20 and allow its binding of the APC/C for its activation of anaphase. In addition, phosphorylation of the APC/C itself is required to allow Cdc20 binding. Phosphorylation of APC3/Cdc27 and APC1 is key to this process. Phosphorylation of APC3 at CDK sites promotes the subsequent phosphorylation of APC1, inducing a conformational change in APC1 that opens the Cdc20 binding site. However, the precise identity of the kinase(s) involved in this process in vivo is unknown (Garrido, 2020).

At least 3 types of cyclins contribute to M-phase in animals: Cyclins A, B and B3. The Cyclin A type (A1 and A2 in mammals) can activate Cdk1 or Cdk2 and is required for mitotic entry, at least in part by allowing the phosphorylation of Cdc20 to prevent its binding and activation of the APC/C. This allows mitotic cyclins to accumulate without being ubiquitinated prematurely by the APC/C and degraded. The Cyclin B type (B1 and B2 in mammals) also promotes mitotic entry and is required for mitotic progression by allowing the phosphorylation of several substrates by Cdk1. Mammalian Cyclin B3, which can associate with both Cdk1 and Cdk2, is required for meiosis but its contribution to mitosis is less clear in view of its low expression in somatic cells. Drosophila possesses a single gene for each M-phase cyclin: CycA (Cyclin A), CycB (Cyclin B) and CycB3 (Cyclin B3) that collaborate to ensure mitotic progression by activating Cdk1. Genetic and RNAi results suggest that they act sequentially, CycA being required before prometaphase, CycB before metaphase and CycB3 at the metaphase-anaphase transition. CycA is the only essential cyclin, as it is required for mitotic entry. CycB and CycB3 mutants are viable, but mutations of CycB and CycB3 are synthetic-lethal, suggesting redundant roles in mitosis. However, mutation of CycB renders females sterile due to defects in ovary development, and mutant males are also sterile (Garrido, 2020).

Drosophila CycB3 associates with Cdk1 and is required for female meiosis (Jacobs, 1998). In Drosophila, eggs normally stay arrested in metaphase I of meiosis until egg laying triggers entry into anaphase I and the subsequent meiosis II. However, CycB3 mutant eggs predominantly stay arrested in meiosis I (Bourouh, 2016). In addition, silencing CycB3 expression in early embryos delays anaphase onset during the syncytial mitotic divisions (Yuan, 2015). Cyclin B3 is also required for anaphase in female meiosis of vertebrates and worms. In mice, RNAi Knock-down of Cyclin B3 in oocytes inhibits the metaphase-anaphase transition in meiosis I. Recently, two groups independently knocked out the Cyclin B3-coding Ccnb3 gene in mice and found that they were viable but female-sterile due to a highly penetrant arrest in meiotic metaphase I. In C. elegans, the closest Cyclin B3 homolog, CYB-3 is required for anaphase in meiosis and mitosis (Garrido, 2020).

How Cyclin B3 promotes anaphase in any system is unknown. One possibility is that it is required for Cdk1 to phosphorylate the APC/C on at least one of its activating subunits, APC3 or APC1. This has not been investigated. Another possibility is that inactivation of Cyclin B3 leads to an early mitotic defect that activates the SAC. This appears to be the case in C. elegans, because inactivation of the SAC rescues normal anaphase onset in the absence of CYB-3. However, in Drosophila, inactivation of the SAC by the mutation of mad2 did not eliminate the delay in anaphase onset observed when CycB3 is silenced in syncytial embryos. Similarly, in mouse oocytes, silencing Mad2 does not rescue the meiotic metaphase arrest upon Cyclin B3 depletion. In other studies, SAC markers on kinetochores did not persist in metaphase-arrested Ccnb3 KO oocytes, and SAC inactivation by chemical inhibition of Mps1 did not restore anaphase. Finally, it is also possible that Cyclin B3 is required upstream of another event required for APC/C activation, for example the activation of a phosphatase required for Cdc20 dephosphorylation and subsequent recruitment to the APC/C (Garrido, 2020).

This study has investigated how CycB3 promotes anaphase in Drosophila. Several lines of evidence are reported indicating that CycB3 directly activates the APC/C in both meiosis and mitosis (Garrido, 2020).

Altogether, the results strongly suggest that CycB3-Cdk1 directly activates the APC/C by phosphorylation, promoting its function at the metaphase-anaphase transition in meiosis and in both maternally driven early embryonic mitoses and somatic cell divisions. This regulation is likely mediated by the phosphorylation in the activation loop of APC3 by CycB3-Cdk1 that ultimately promotes the recruitment of the Cdc20-type co-activators Fizzy and Cortex. Previous work has shown that APC3 phosphorylation and APC/C activation by cyclin-CDK complexes require their CKS subunit (see Cks30A). CKS subunits can act as processivity factors that bind phosphorylated sites to promote additional phosphorylation by the CDK. Thus, phosphorylation of APC3 would prime the binding of a cyclin-CDK-CKS complex to promote the additional phosphorylation of APC1, allowing for Cdc20 binding. It has been shown that mutation of phosphorylation sites into Asp or Glu residues cannot substitute for the presence of phosphate in the CKS binding site. Therefore, it was not possible to generate a mutation in APC3 that would have mimicked phosphorylation at S316 to enhance cyclin-CDK-CKS binding. Such a mutation in APC3, if it were possible, would have potentially rescued APC/C activity in the absence of CycB3 according to this model. However, it is likely that this analysis did not detect all phosphorylation sites in the APC/C. Thus, the possibility cannot be exclustion that other phosphorylation events, mediated by CycB3-Cdk1 or another kinase, may be required for complete APC/C activation. For example, other phosphorylation events have been proposed to regulate APC/C localization. It is even formally possible that CycB3-Cdk1 is required to activate another proline-directed kinase that phosphorylates APC3 at S316. The interdependence between CycB3 and Tws that this study uncovered may reflect a role of PP2A-Tws in the recruitment of Cdc20 co-activators to the APC/C. Cdc20 must be dephosphorylated at CDK sites before binding the APC/C, and in human cells both PP2A-B55 and PP2A-B56 promote this event (Garrido, 2020).

CycB3 is strongly required for APC/C activation in meiosis and in the early syncytial mitoses, and to a lesser extent in other mitotic divisions, despite the presence of two additional mitotic cyclins, CycA and CycB, capable of activating Cdk1. There are many possible reasons for this requirement. Overexpression of stabilized forms of CycA or CycB can block or slow down anaphase, suggesting that they may interfere with APC/C function in this transition. However, under normal expression levels, CycA or CycB or both may contribute to activate the APC/C like CycB3. CycB3 mutant flies develop until adulthood, which implies that the APC/C can be activated to induce anaphase in at least a vast proportion of mitotic cells, and this activation could be mediated by CycA and/or CycB. CycA is essential for viability and CycB mutants show strong female germline development defects, complicating the examination of potential roles for these cyclins at the metaphase-anaphase transition. Thus, in principle, the requirements for CycB3 in female meiosis, in embryos and in mitotic cells in culture could merely reflect the need for a minimal threshold of total mitotic cyclins. This possibility is considered unlikely because CycB3 is expressed at much lower levels than CycB in early embryos. Moreover, while maternal heterozygosity for mutations in CycB3 and tws causes a metaphase arrest in embryos, heterozygosity for mutations in CycB and tws does not cause embryonic defects. In fact, genetic results suggest that the function of CycB is antagonized by PP2A-Tws in embryos, while CycB3 and PP2A-Tws collaborate for APC/C activation in embryos. Thus, although it is possible that CycA and CycB can participate in APC/C activation, CycB3 probably has some unique feature that makes it particularly capable of promoting APC/C activation (Garrido, 2020).

By what mechanism could CycB3 be particularly suited for APC/C activation? Cyclins can play specific roles by contributing to CDK substrate recognition or by directing CDK activity in space and time. This study did not investigate the precise nature of the molecular recognition of the APC/C by CycB3. It may be that CycB3 possesses a specific binding site for the APC/C that is lacking in CycA and CycB. Another possibility is that differences in localization between cyclins dictate their requirements. In particular, while CycA and CycB are cytoplasmic in interphase, CycB3 is nuclear. It is surmised that the nuclear localization of CycB3 may help concentrate CycB3 in the spindle area upon germinal vesicle breakdown, when the very large oocyte enters meiosis. In future studies, it will be interesting to compare the ability of different mitotic cyclins to activate the APC/C and to determine the molecular basis of potential differences (Garrido, 2020).

In any case, the results show that CycB3 activates the APC/C and that this regulation is essential in Drosophila. Cyclin B3 has been shown to be required for anaphase in female meiosis of insects (Drosophila), worms (C. elegans) and vertebrates (mice). It is tempting to conclude that the activation of the APC/C is a function of Cyclin B3 conserved in all these species. However, in C. elegans embryos, the metaphase arrest upon CYB-3 (Cyclin B3) inactivation requires SAC activity. The underlying mechanism and whether it also occurs in other systems remain to be determined. However, CYB-3 plays roles in C. elegans that have not been detected for Cyclin B3 in flies or vertebrates, including a major role in mitotic entry, where CYB-3 mediates the inhibitory phosphorylation of Cdc20. In this regard, C. elegans CYB-3 may be more orthologous to Cyclin A. Yet, given that Cyclin B3 is required for anaphase in a SAC-independent manner in flies and mice, it seems reasonable to suggest that the direct activation of the APC/C by Cyclin B3 is conserved in vertebrates (Garrido, 2020).

Cyclin B3 deficiency impairs germline stem cell maintenance and its overexpression delays cystoblast differentiation in Drosophila ovary

It is well known that cyclinB3 (cycB3) plays a key role in the control of cell cycle progression. However, whether cycB3 is involved in stem cell fate determination remains unknown. The Drosophila ovary provides an exclusive model for studying the intrinsic and extrinsic factors that modulate the fate of germline stem cells (GSCs). Here, using this model, Drosophila cycB3 was shown to plays a new role in controlling the fate of germline stem cells (GSC). Results from cycB3 genetic analyses demonstrate that cycB3 is intrinsically required for GSC maintenance. Results from green fluorescent protein (GFP)-transgene reporter assays show that cycB3 is not involved in Dad-mediated regulation of Bmp signaling, or required for dpp-induced bam transcriptional silencing. Double mutants of bam and cycB3 phenocopied bam single mutants, suggesting that cycB3 functions in a bam-dependent manner in GSCs. Deficiency of cycB3 fails to cause apoptosis in GSCs or influence cystoblast (CB) differentiation into oocytes. Furthermore, overexpression of cycB3 dramatically increases the CB number in Drosophila ovaries, suggesting that an excess of cycB3 function delays CB differentiation. Given that the cycB3 gene is evolutionarily conserved, from insects to humans, cycB3 may also be involved in controlling the fate of GSCs in humans (Chen, 2018).

The cycB3 gene is evolutionarily-conserved among higher eukaryotic organisms examined, from insects to mammalians. The cycB3 protein is present as Cyclin A and B (two other B-type Cyclins) in mitotically-proliferating cells, and is involved in the regulation of mitosis, where it cooperates with Cyclin A and B. It is reported that Cyclin A and B are involved in the regulation of ovarian GSC maintenance in Drosophila. Earlier observation showed that cycB3² homozygous mutant females partially exhibit thinned ovaries. Given reports on Cyclin activity in stem cells, the thinned ovaries prompted a further exploration of the potential involvement of cycB3 in the maintenance of germline stem cells, in the Drosophila ovary. The phenotypic assays indicate that a cycB3 deficiency leads to GSC loss with ageing. The rescue assays and genetic mosaic analyses convincingly suggest that CycB3 functions as an intrinsic factor for controlling the fate of GSC (Chen, 2018).

Previous studies have discovered that the Dpp/Bam pathway is the essential signaling pathway for maintaining GSCs in the Drosophila ovary. The bam gene is a key switch in regulating the fate of GSC. Combining these results, a model is proposed to explain how CycB3 is involved in regulation of GSC/CB fate determination. In GSCs, the data show that CycB3 is not involved in Dpp-mediated bam transcriptional silencing. The cycB3 deficiency triggers GSC pre-differentiation and eventually causes its loss phenotype. In CBs, the bam gene exhibits a high expression level, due to loss of the inhibition by Dpp signaling, and the Bam protein can promote CB differentiation. Genetic interaction analyses strongly shows that cycB3 function is positioned upstream of Bam action in CBs. The excess cycB3 come from cycB3 overexpression, which specifically suppresses CB differentiation, probably through repressing the activity of Bam. However, what are the factors that functionally position upstream of cycB3 in CBs of Drosophila ovary? This still remains elusive (Chen, 2018).

It is reported that cycB3 promotes metaphase–anaphase transition in Drosophila embryos. The current data show that overexpression of cycB3 fails to increase the number of GSCs, suggesting that the excess CycB3 may fail to influence transition into the GSC system, whereas the excess CycB3 is sufficient to delay CB differentiation. The underlying molecular mechanism might be due to the fact that the increased CycB3 activity is sufficient to enhance CB proliferation, by promoting metaphase-anaphase transition (Chen, 2018).

Distinct and Overlapping Requirements for Cyclins A, B, and B3 in Drosophila Female Meiosis

Meiosis, like mitosis depends on the activity of the mitotic Cyclin dependent kinase, Cdk1 and its cyclin partners. This study examined the specific requirements for the three mitotic cyclins, Cyclin A, Cyclin B and Cyclin B3> in meiosis of Drosophila melanogaster. All three cyclins were found to contribute redundantly to nuclear envelope breakdown, though Cyclin A appears to make the most important individual contribution. Cyclin A is also required for bi-orientation of homologues in meiosis I. Cyclin B3, as previously reported, is required for anaphase progression in meiosis I and in meiosis II. Cyclin B3 also plays a redundant role, with Cyclin A, in preventing DNA replication during meiosis. Cyclin B is required for maintenance of the metaphase I arrest in mature oocytes, for spindle organization and for timely progression through the 2nd meiotic division. It is also essential for polar body formation at the completion of meiosis. With the exception of its redundant role in meiotic maturation, Cyclin B appears to function independently of Cyclins A and B3 through most of meiosis. The study concludes that the 3 mitotic Cyclin-Cdk complexes have distinct and overlapping functions in Drosophila female meiosis (Bourouh, 2016).

Cyclin B3 is a mitotic cyclin that promotes the metaphase-anaphase transition

The timing mechanism for mitotic progression is still poorly understood. The ">spindle assembly checkpoint (SAC), whose reversal upon chromosome alignment is thought to time anaphase, is functional during the rapid mitotic cycles of the Drosophila embryo; but its genetic inactivation had no consequence on the timing of the early mitoses. Mitotic cyclins-Cyclin A, Cyclin B, and Cyclin B3-influence mitotic progression and are degraded in a stereotyped sequence. RNAi knockdown of Cyclins A and B resulted in a Cyclin B3-only mitosis in which anaphase initiated prior to chromosome alignment. Furthermore, in such a Cyclin B3-only mitosis, colchicine-induced SAC activation failed to block Cyclin B3 destruction, chromosome decondensation, or nuclear membrane re-assembly. Injection of Cyclin B proteins restored the ability of SAC to prevent Cyclin B3 destruction. Thus, SAC function depends on particular cyclin types. Changing Cyclin B3 levels showed that it accelerated progress to anaphase, even in the absence of SAC function. The impact of Cyclin B3 on anaphase initiation appeared to decline with developmental progress. These results show that different cyclin types affect anaphase timing differently in the early embryonic divisions. The early-destroyed cyclins-Cyclins A and B-restrain anaphase-promoting complex/cyclosome (APC/C) function, whereas the late-destroyed cyclin, Cyclin B3, stimulates function. It is proposed that the destruction schedule of cyclin types guides mitotic exit by affecting both Cdk1 and APC/C, whose activities change as each cyclin type is lost (Yuan, 2015).

Work in tissue culture cells suggested a 'wait-until-ready' model for the control of anaphase onset, wherein unattached chromosomes activate the spindle assembly checkpoint (SAC) to prevent anaphase-promoting complex/cyclosome (APC/C) activation until all the chromosomes are attached (i.e., ready for anaphase). A shortcoming of this mechanism appears in a multinucleate cell where the first spindle to satisfy the SAC activates anaphase in the entire cell. Since this activation by the lead nucleus short-circuits regulation in all slower nuclei, this mode of timing control appears inappropriate for the syncytial Drosophila embryo (Yuan, 2015).

The mitotic cyclins are degraded in an orderly sequence, with Cyclin A disappearing in metaphase, Cyclin B near the time of onset of anaphase, and Cyclin B3 during anaphase. Moreover, destruction of each cyclin is required for progress to the next stage of mitosis. Hence, the schedule of destruction ought to pace the progress of mitotic exit. To test the basis of timing control of anaphase initiation during the syncytial mitotic cycles of early Drosophila embryos, SAC was inactivated or levels of the different mitotic cyclins were manipulted, and the duration of metaphase was analyzed (Yuan, 2015).

The function of the SAC is dispensable for Drosophila, as mad2 null and bubR1ΔKEN mad2 double mutants were viable and fertile. Although loss of the SAC slightly accelerated progress to anaphase in larval neuroblasts, no difference was detected in metaphase length in early embryos. Thus, the SAC did not contribute to the timing of metaphase-anaphase transition at this developmental stage. As reported previously, RNAi knockdown of the three mitotic cyclins in embryos blocks the cell cycle rapidly and effectively (McCleland, 2008), and knockdown of individual or pairs of cyclins gives distinct phenotypes (McCleland, 2009; Yuan, 2012). Knocking down the early-degraded cyclins, Cyclin A and Cyclin B, accelerated progress to anaphase and led to mitoses without metaphase. Embryos entered anaphase prematurely and chromosomes were randomly segregated. Reciprocally, injection of mRNA encoding these early-degraded cyclins delayed chromosome segregation. Surprisingly, Cyclin B3 knockdown moderately extended metaphase, and injection of Cyclin B3 mRNA slightly advanced anaphase. It is concluded that the early-degraded cyclins delay anaphase, whereas Cyclin B3 advances it (Yuan, 2015).

How do the early cyclins inhibit anaphase entry? The APC/C system has a poorly understood capacity to degrade different substrates in an orderly progression, and Cyclin A and Cyclin B are among the most preferred substrates. These cyclins enjoy several cyclin-type-specific interactions that promote their early recruitment to the APC/C. These include a binding interaction between cyclin-dependent kinase regulatory subunit 1 (Cks1) and phosphorylated APC/C and complex interactions with a category of APC/C inhibitory proteins, Rca1/Emi1/Emi2. The strong interactions may commit APC/C to these preferred substrates to enforce ordered destruction. As long as the APC/C is preoccupied with destruction of these early cyclins, its action on other substrates will be inhibited, deferring their destruction. If the early cyclins suppress APC/C-mediated destruction of later substrates, perhaps they also contribute to checkpoint suppression of APC/C activity (Yuan, 2015).

To investigate cyclin influence on SAC function, embryos with were treated with colchicine after RNAi knockdown of pairs of cyclins. Control embryos treated with colchicine stably arrested with condensed chromosomes and had no detectable nuclear membrane. Embryos with only Cyclin B also exhibited persistent chromosome condensation. The chromosomes in embryos with only Cyclin A started to decondense after a moderate arrest, consistent with the previously reported continued degradation of Cyclin A at a checkpoint arrest. The chromosomes of embryos with only Cyclin B3 began to decondense after a short arrest, and nuclear membrane staining appeared. This study concluded that spindle disruption does not stably arrest a Cyclin B3-only mitosis despite the expectation that SAC should stabilize this late-degrading cyclin (Yuan, 2015)

The failure of SAC in the Cyclin B3-only mitosis might reflect an inability to activate the spindle checkpoint. However, Mad2 recruitment to the prometaphase kinetochores, a hallmark of SAC activation, still occurs in the Cyclin B3-only mitosis. This focused the attention of this study on the function of the SAC (Yuan, 2015).

Since a stable form of Cyclin B3 arrests cells in late mitosis with condensed chromosomes, escape from a mitotic arrest ought to be associated with Cyclin B3 destruction. To characterize cyclin degradation, mRNAs encoding mitotic cyclins were made with an EGFP tag fused to their C termini. Cyclin A-GFP and Cyclin B-GFP were enriched on centrosomes and kinetochores in mitosis. Interestingly, Cyclin B3-GFP was enriched on nuclear envelope/ER-like membranous structures that bracket the spindle in early Drosophila mitoses. Unlike Cyclin A, Cyclin B3 was stabilized in colchicine-injected wild-type embryos. However, when Cyclin A and Cyclin B were knocked down, Cyclin B3 was degraded in the presence of colchicine. Injection of recombinant Cyclin B proteins into these embryos blocked Cyclin B3 degradation. Degradation of Cyclin B3 during a Cyclin B3-only mitosis was mediated by APC/C, as inhibition of APC/C activity by injecting UbcH10C114S prevented its destruction. It is concluded that SAC needs the early cyclins in order to stabilize the late-degrading Cyclin B3 (Yuan, 2015).

The prevailing model for SAC is not sufficient to explain why the SAC requires early-degrading cyclins. Substrate is recruited to APC/C via the interaction between the destruction box of substrate and the WD40 domains of Cdc20. The SAC, once activated, is thought to function by blocking substrate binding to Cdc20. SAC inhibition of Cdc20 should inhibit Cyclin B3 destruction without reliance on an early cyclin. Apparently, the inhibition of APC/C by the SAC is more complex. It is suggested that Cyclin B continues to engage the APC/C during an SAC arrest, perhaps via Cdc20-independent interactions, and that SAC suppresses the ability of APC/C to degrade Cyclin B, effectively freezing ordered degradation and holding the APC/C in abeyance (Yuan, 2015).

Cyclin B3 knockdown extended metaphase. Perhaps, this is the result of activation of SAC upon Cyclin B3 knockdown. The Cyclin B3 RNAi experiment was repeated in the mad2 null embryos. Cyclin B3 knockdown still caused a metaphase delay in the SAC-deficient embryos (Yuan, 2015).

Although independent of SAC, it was thought that Cyclin B3 might alter early mitotic events with secondary effects on the APC/C and transition to anaphase. The mad2 mutant offered a context in which it was possible disrupt the spindle and normal events of mitotic progress with colchicine and still assess mitotic exit. To test whether Cyclin B3 influences mitotic progression in these embryos, Cyclin B3 was knocked down and the duration of the 'mitotic phase' was followed based on the degree of DNA condensation. Cyclin B3 knockdown, but not knockdown of the other cyclins, extended this mitotic phase. It is concluded that Cyclin B3 normally promotes the mitotic exit program and that this action is independent of both SAC and the spindle. If Cyclin B3 directly regulates APC/C function, it might influence its cellular dynamics. Cdc27 is an APC/C subunit whose phosphorylation by Cdk1-cyclin appears to activate APC/C function and direct it to anaphase chromosomes. The anaphase localization of Cdc27 was studied in both wild-type and single cyclin RNAi-treated embryos. Cdc27 was recruited to anaphase chromosomes in wild-type, and Cyclin A or Cyclin B knockdown embryos; however, Cyclin B3 knockdown greatly reduced the anaphase chromosomal localization of Cdc27 (Yuan, 2015).

Cyclins generally inhibit mitotic exit. At least their destruction underlies downregulation of the mitotic Cdk, and their stabilization blocks mitotic exit. This makes acceleration of anaphase onset by Cyclin B3 appear incongruous. However, cyclins are thought to promote their own demise by activating the APC/C, and the best APC/C activator among the mitotic cyclins will promote mitotic exit. Suppression of APC/C by early-degraded cyclins and activation by a late-degraded cyclin would first stabilize the metaphase state, but initial cyclin destruction would ramp up APC/C activity in a decisive transition. As a late-degrading cyclin that does not block anaphase, Cyclin B3 is particularly well suited as an activator of the APC/C (Yuan, 2015).

It is not known how Cyclin B3 stimulates anaphase. However, cyclin:Cdk kinase activity phosphorylates certain APC/C subunits, such as Cdc27, as well as phosphorylating and inactivating an inhibitor of the APC/C, Emi2. It is suggest that Cyclin B3:Cdk1 has unique substrate specificity and spatial-temporal distribution that make it an effective activator of APC/C. Regardless of the detailed mechanism, this study shows that cyclin types influence APC/C function, and hence regulate their own destruction schedule (Yuan, 2015).

Flies without zygotic Cyclin B3 can develop into healthy adults, but the females are infertile. Cyclin B3 null females had normal ovaries and laid fertilized eggs, but these exhibited early cell-cycle defects. It is hypothesized that the female sterility, or more properly maternal-effect lethality, in the Cyclin B3 null flies resulted from a deficiency in exit from mitosis or meiosis (Yuan, 2015).

To test this, eggs were imaged from homozygous Cyclin B3 mutant mothers and controls. Eggs from Cyclin B3 null mothers were reported to be defective in exiting from meiosis. However, this study observed some eggs with centrosomes and polar bodies, indicators of fertilization and completion of meiosis, respectively. A Cyclin B3-deficient egg with a seemingly normal polar body and a metaphase spindle with four centrosomes adjacent to it is illustrated. These centrosomes were detached from the spindle, and the metaphase spindle was acentrosomal. In the older eggs, numerous microtubule-organizing centers were formed and DNA became highly fragmented. The data suggested that some of the mutant eggs finished meiosis but then arrested in metaphase of the subsequent mitotic division. These observations are consistent with a role of Cyclin B3 in stimulation of anaphase as is seen in later-staged embryos. It is suggested that Cyclin B3 is so important in meiosis and in the earliest mitoses that its absence disrupts restoration of interphase at these stages (Yuan, 2015).

Next, Cyclin B3 function was examined in larval neuroblasts, a more differentiated cell type that gives rise to neurons. Surprisingly, the metaphase duration in the neuroblasts of Cyclin B3 null larvae was indistinguishable from that in the control heterozygous siblings. Thus it is concluded that the impact of Cyclin B3 on anaphase onset is developmentally regulated. Meiosis and early embryonic mitoses need Cyclin B3, but later neuroblast divisions do not. Interestingly, the constitution of the APC/C holoenzyme is also under developmental regulation. During female meiosis and early embryonic mitoses, a distinct Cdc20-like protein, CORT, is expressed. It would be of great interest to explore the interaction between Cyclin B3 and this novel APC/C activator (Yuan, 2015).

The lack of an effect of Cyclin B3 in later cell cycles is not due to lack of Cyclin B3. It is expressed widely in Drosophila and was shown to regulate onset of cytokinesis in later cycles. This suggests that Cyclin B3 is not always important for timing and that timing duty shifts from a program of cyclin destruction at early stages to a SAC-dependent wait-until-ready mechanism. This raises the issue of how these two mechanisms are controlled to guarantee accurate mitotic progress. While it might seem dangerous to have different mechanisms timing mitosis come and go, it should be recalled that the checkpoint mechanism is beautifully appropriate as a backup mechanism. It is suggested that whenever spindle establishment slows to the point that misaligned chromosomes are still present after the cyclin-based timer has run its course, the SAC takes over. By removing the danger of misregulation, the SAC may have freed evolution and development to alter timing inputs governing mitotic progress (Yuan, 2015).

Different cyclin types collaborate to reverse the S-phase checkpoint and permit prompt mitosis

Precise timing coordinates cell proliferation with embryonic morphogenesis. As Drosophila embryos approach cell cycle 14 and the midblastula transition, rapid embryonic cell cycles slow because S phase lengthens, which delays mitosis via the S-phase checkpoint. This study probed the contributions of each of the three mitotic cyclins to this timing of interphase duration. Each pairwise RNA interference knockdown of two cyclins lengthened interphase 13 by introducing a G2 phase of a distinct duration. In contrast, pairwise cyclin knockdowns failed to introduce a G2 in embryos that lacked an S-phase checkpoint. Thus, the single remaining cyclin is sufficient to induce early mitotic entry, but reversal of the S-phase checkpoint is compromised by pairwise cyclin knockdown. Manipulating cyclin levels revealed that the diversity of cyclin types rather than cyclin level influenced checkpoint reversal. It is concluded that different cyclin types have distinct abilities to reverse the checkpoint but that they collaborate to do so rapidly (Yuan, 2012).

Pairwise knockdown of cyclins in embryos lacking Chk1/Grapes (embryos from grp mutant mothers) modestly extends interphase in the grp embryo. This knockdown also substantially suppresses the mitosis 13 defects in grp embryos. The mitotic catastrophe in grp embryos has long been thought to be caused by entry into mitosis with incompletely replicated DNA. Indeed, analysis of PCNA localization supports a proposal that the small extension of interphase allows completion of S phase and, hence, suppression of the catastrophe. This suppression of the grp phenotype, which extends to partial restoration of gastrulation, is reminiscent of suppression of hypomorphic mei-41 mutations when the maternal dose of CycA and/or CycB was reduced. However, the most important feature of this analysis is that the extension of interphase by pairwise cyclin knockdown in grp embryos is so slight that interphase remains shorter than a wild-type interphase 13. Thus, each individual cyclin type can drive rapid advance to mitosis in the absence of functional Chk1. Furthermore, the G2 that was introduced by cyclin knockdown was absent in grp embryos, and cyclin type-specific differences in interphase length were minor. These results demonstrate that mitotic entry is timed primarily by the Grapes-dependent checkpoint in cyclin knockdown embryos and, moreover, that the G2 induced in these embryos results from action of the checkpoint (Yuan, 2012).

How might S phase govern the time of mitosis when mitosis begins well after completion of S phase? To test whether the S-phase checkpoint delayed accumulation of the remaining cyclin, single knockdown embryos were immunoblotted to follow accumulation in wild-type and grp embryos. Cyclin accumulated during S phase in both wild-type and grp mutant embryo. Thus, Grapes function does not delay the production of cyclin. Instead, the difference between grp⁺ and grp⁻ embryos suggests that persistent activity of the checkpoint prevents the checkpoint-competent embryos from going into mitosis after pairwise cyclin knockdown. Because wild-type embryos enter mitosis immediately after S phase, the checkpoint is rapidly reversed when there is a full complement of cyclin types, but its reversal is delayed upon pairwise cyclin knockdown. Apparently, the different cyclin types ordinarily collaborate to rapidly reverse the checkpoint (Yuan, 2012).

This study shows that a persistent action or consequence of the DNA replication checkpoint underlies a G2 phase that is introduced by pairwise knockdown of cyclins. One might propose that cyclin knockdown doesn't really cause persistence of the checkpoint activity but simply makes a slowly decaying checkpoint function longer by compromising the cyclin-Cdk1 activity that must be suppressed by the checkpoint. Such an interpretation is disfavored because it is quantitative, and the data argue that neither reduction nor increase in the remaining cyclin affects the duration of interphase. Instead, it is argued that cyclin-Cdk1 contributes to shutting off the checkpoint, and it is proposed that efficient shutoff of the checkpoint requires multiple cyclin types. One way to explain this is based on the distinct subcellular localizations of mitotic cyclins. Once activated, the checkpoint can operate in multiple cellular compartments, such as the nucleus and the cytoplasm. Although signals coordinate entry into mitosis in the cytoplasm and nucleus, persistent nuclear checkpoint activity can prevent mitotic entry despite cytoplasmic Cdk activity. Individual cyclins would not be able to act on their own to reverse the checkpoint in all compartments if each is excluded from one compartment. For example, cyclin B is efficiently excluded from the nucleus in cycle 13 embryos and presumably would not contribute to checkpoint reversal in this compartment, whereas cyclin B3 is nuclear. In embryos with only CycB, the checkpoint should be reversed first in the cytoplasm; however, progress to mitosis should depend on slower reversal in the nucleus, which might be based on communication between compartments. Consistent with this proposal, injection of CycB protein has been shown to preferentially drive cytoplasmic, but not nuclear, mitotic events. The full complement of cyclins with distinct localizations, however, appears to reverse the checkpoint promptly and coordinately in all the compartments (Yuan, 2012).

Influence of cyclin type and dose on mitotic entry and progression in the early Drosophila embryo

Cyclins are key cell cycle regulators, yet few analyses test their role in timing the events that they regulate. RNA interference and real-time visualization in embryos were used to define the events regulated by each of the three mitotic cyclins of Drosophila melanogaster, CycA, CycB, and CycB3. Each individual and pairwise knockdown results in distinct mitotic phenotypes. For example, mitosis without metaphase occurs upon knockdown of CycA and CycB. To separate the role of cyclin levels from the influences of cyclin type, two cyclins were knocked down and the gene dose of the one remaining cyclin was reduced. This reduction did not prolong interphase but instead interrupted mitotic progression. Mitotic prophase chromosomes formed, centrosomes divided, and nuclei exited mitosis without executing later events. This prompt but curtailed mitosis shows that accumulation of cyclin function does not directly time mitotic entry in these early embryonic cycles and that cyclin function can be sufficient for some mitotic events although inadequate for others (McCleland, 2009).

RNAi of mitotic cyclins in Drosophila uncouples the nuclear and centrosome cycle

Successful cell duplication requires orderly progression through a succession of dramatic cell-cycle events. Disruption of this precise coupling can compromise genomic integrity. The coordination of cell-cycle events is thought to arise from control by a single master regulator, cyclin:Cdk, whose activity oscillates. However, very little is known of how individual cell-cycle events are coupled to this oscillator and how the timing of each event is controlled. This study developed an approach with RNA interference (RNAi) and real-time imaging to study cyclin contributions to the rapid syncytial divisions of Drosophila embryos. Simultaneous knockdown of all three mitotic cyclins blocked nuclei from entering mitosis. Despite nuclear arrest, centrosomes and associated myosin cages continued to divide until the midblastula transition. Centrosome division was synchronous throughout the embryo and the period of the uncoupled duplication cycle increased over successive divisions. In contrast to its normal actions, injection of a competitive inhibitor of the anaphase-promoting complex/cyclosome (APC/C) after knockdown of the mitotic cyclins did not interfere with the centrosome-duplication cycles. Finally, this study examined how cyclin knockdown affects the onset of cellularization at the midblastula transition and found that nuclear cell-cycle arrest did not advance or delay onset of cellularization. This study has shown that knockdown of mitotic cyclins allows centrosomes to duplicate in a cycle that is uncoupled from other cell-cycle events. It is suggested that high mitotic cyclin normally ensures that the centrosome cycle remains entrained to the nuclear cycle (McCleland, 2008).

Drosophila Cyclin B3 is required for female fertility and is dispensable for mitosis like Cyclin B

Cyclin B3 has been conserved during higher eukaryote evolution as evidenced by its identification in chicken, nematodes, and insects. This study demonstrate that Cyclin B3 is present in addition to Cyclins A and B in mitotically proliferating cells and not detectable in endoreduplicating tissues of Drosophila embryos. Cyclin B3 is coimmunoprecipitated with Cdk1(Cdc2) but not with Cdk2(Cdc2c). It is degraded abruptly during mitosis like Cyclins A and B. In contrast to these latter cyclins, which accumulate predominantly in the cytoplasm during interphase, Cyclin B3 is a nuclear protein. Genetic analyses indicate functional redundancies. Double and triple mutant analyses demonstrate that Cyclins A, B, and B3 cooperate to regulate mitosis, but surprisingly single mutants reveal that neither Cyclin B3 nor Cyclin B is required for mitosis. However, both are required for female fertility and Cyclin B also for male fertility (Jacobs, 1998).

The G2-to-M Transition Is Ensured by a Dual Mechanism that Protects Cyclin B from Degradation by Cdc20-Activated APC/C

In the eukaryotic cell cycle, a threshold level of cyclin B accumulation triggers the G2-to-M transition, and subsequent cyclin B destruction triggers mitotic exit. The anaphase-promoting complex/cyclosome (APC/C) is the E3 ubiquitin ligase that, together with its co-activator Cdc20, targets cyclin B for destruction during mitotic exit. This study shows that two pathways act in concert to protect cyclin B from Cdc20-activated APC/C in G2, in order to enable cyclin B accumulation and the G2-to-M transition. The first pathway involves the Mad1-Mad2 spindle checkpoint complex, acting in a distinct manner from checkpoint signaling after mitotic entry but employing a common molecular mechanism-the promotion of Mad2-Cdc20 complex formation. The second pathway involves cyclin-dependent kinase phosphorylation of Cdc20, which is known to reduce Cdc20's affinity for the APC/C. Cooperation of these two mechanisms, which target distinct APC/C binding interfaces of Cdc20, enables cyclin B accumulation and the G2-to-M transition (Lara-Gonzalez, 2019).

The rise and decay in Cdk1-cyclin B activity drive eukaryotic cells into and out of M phase in the cell cycle. While Cdk1 protein levels are relatively constant during the cell cycle, cyclin B periodically accumulates until it reaches a critical threshold that, through a series of feedback mechanisms, leads to high Cdk1-cyclin B kinase activity. High Cdk1-cyclin B kinase activity drives striking cellular changes associated with mitosis—genome compaction, nuclear envelope breakdown, centrosome separation, bipolar spindle formation, and assembly of kinetochores that couple chromosomes to spindle microtubules. Once all chromosomes have attached to spindle microtubules, cyclin B degradation is initiated to drive mitotic exit. The anaphase-promoting complex/cyclosome (APC/C) is the multi-subunit E3 ubiquitin ligase that, when bound to its co-activator Cdc20, targets cyclin B as well as securin, an inhibitor of sister chromatid separation, for proteolytic degradation (Lara-Gonzalez, 2019).

To achieve the periodic accumulation and decline of cyclin B levels that demarcates M phase in the cell cycle, APC/CCdc20 must be restrained to enable sufficient cyclin B accumulation to trigger the G2-to-M transition. Once mitosis is initiated, APC/CCdc20 must also be restrained until all chromosomes are connected to the spindle, in order to prevent errors in chromosome segregation. Recent work has greatly advanced understanding of how unattached chromosomes inhibit APC/CCdc20 during mitosis via the spindle checkpoint pathway that is activated at kinetochores. The kinetochore-based spindle checkpoint produces a diffusible inhibitor of the APC/CCdc20, known as the mitotic checkpoint complex. A central component of the mitotic checkpoint complex is the protein Mad2, which exists in two conformations: open and closed. The closed form of Mad2 is bound to related peptide motifs from its cellular partners, Mad1 and Cdc20. When the spindle checkpoint is active, Mad1-Mad2 complexes concentrate at unattached kinetochores where they recruit soluble open Mad2 and catalyze its conversion to form a closed complex with Cdc20. Kinetochore-generated Mad2-Cdc20 complex then associates with Mad3 (known as BubR1 in vertebrates)—Bub3 to generate the mitotic checkpoint complex. The mitotic checkpoint complex is a potent inhibitor of already-active APC/CCdc20 that acts by binding to the APC/CCdc20 holocomplex and interfering with its ability to recruit E2 ubiquitin-conjugating enzymes and to recognize substrates. Early in mitosis, prior to assembly of mature kinetochores, Mad1-Mad2 complex localized at nuclear pores is proposed to generate the mitotic checkpoint complex via a similar reaction (Lara-Gonzalez, 2019).

In contrast to APC/CCdc20 regulation by kinetochores in mitosis, how APC/CCdc20 is restrained during G2 to allow cyclin B accumulation and the G2-to-M transition is not understood. The APC/C inhibitor Emi1 was initially proposed to block APC/CCdc20 activity in G2. However, subsequent work showed that Emi1 targets APC/C bound by its other co-activator, Cdh1, and its primary function is to prevent re-replication. Emi2, related to Emi1, was shown to inhibit APC/CCdc20 during oocyte meiotic arrest; however, knockout analysis has shown that Emi2 is dispensable for somatic divisions in mammals. In addition, Emi proteins are not as widely conserved as cyclin B and the APC/C. Other mechanisms suggested to restrain APC/CCdc20 include the N-terminal region of BubR1(Mad3), phosphorylation of Cdc20 by cyclin-dependent kinases, and requirement for phosphorylation of core APC/C subunits by the mitotic kinases Cdk1 and Plk1 to promote Cdc20 binding. The last idea has strong support from biochemical, structural, and Xenopus egg extract studies—egg extracts rapidly cycle between S and M phases without an intervening G2 phase—but its importance in wider cellular contexts has not been assessed. Thus, in contrast to the mechanistic understanding of how active APC/CCdc20 is regulated by the spindle checkpoint after mitotic entry, there is less clarity on how APC/CCdc20 is restrained to allow sufficient cyclin B accumulation for the G2-to-M transition (Lara-Gonzalez, 2019).

By analyzing proliferation in the C. elegans germline and supporting key findings in human cultured cells, this study define two parallel-acting mechanisms that target Cdc20 to suppress APC/CCdc20 activation and enable the G2-to-M transition. The first mechanism involves the Mad1-Mad2 complex acting in a manner distinct from its well-defined spindle checkpoint function that requires kinetochore localization. The second mechanism involves phosphorylation of Cdc20 that reduces its binding affinity for the APC/C. Inhibition of both mechanisms disrupts the G2-to-M transition to a similar extent as inhibition of core mitotic entry circuit factors, such as Cdk1 and cyclin B. In addition to identifying a dual inhibitory mechanism that restrains APC/CCdc20 to enable the G2-to-M transition, these results reveal a conserved checkpoint signaling-independent function of the Mad1-Mad2 complex (Lara-Gonzalez, 2019).

The results shown in this study highlight two distinct mechanisms that target Cdc20 and act in concert to enable cyclin B accumulation and promote the G2-to-M transition. The first mechanism involves the Mad1-Mad2 complex and the second phosphorylation of Cdc20 by cyclin-dependent kinases. The findings lead to a model in which Cdc20 phosphorylation and Mad2 binding cooperatively reduce affinity of Cdc20 for the APC/C in G2 by targeting two conserved regions, the C-box and the KILR motif that are employed by Cdc20 to bind to distinct parts of the Apc8B subunit of the APC/C. Mad2 binds adjacent to and sterically blocks the KILR motif whereas Cdc20 phosphorylation electrostatically inhibits interaction of the C-box with Apc8B. This coordinated action prevents formation of active APC/CCdc20 complexes, thereby protecting cyclin B during its synthesis and enabling the G2-to-M transition. These findings illuminate a poorly understood feature of the eukaryotic cell cycle and identify a new conserved function for a central component of the spindle checkpoint pathway (Lara-Gonzalez, 2019).

Using C. elegans and human cultured cells, this study showed that Mad1-Mad2 promotes mitotic entry by restricting the ability of APC/CCdc20 to target cyclin B for degradation. The Mad1-Mad2 reaction that promotes mitotic entry has different requirements from the Mad1-Mad2-based spindle checkpoint pathway. Mad1-Mad2 function in the spindle checkpoint requires docking onto kinetochores and/or nuclear pores, where kinase activities catalyze the formation of a Mad2-Cdc20 complex that associates with Mad3(BubR1)-Bub3 to produce the mitotic checkpoint complex, a potent inhibitor of already-active APC/CCdc20. In contrast, this study shows that the role of Mad1-Mad2 in promoting the G2-to-M transition does not require formation of the mitotic checkpoint complex or localization to the kinetochore or nuclear pore and most likely acts by sequestering Cdc20 from forming active holocomplexes with the core APC/C (Lara-Gonzalez, 2019).

The data do not support the idea that an interphase form of the mitotic checkpoint complex is needed to inhibit APC/CCdc20 prior to the G2-to-M transition. This is evident from imaging of in situ-tagged cyclin B1 in human cells, where cyclin B1 levels prior to NEBD were similar between control and BubR1 depletion, even though cyclin B1 was immediately degraded following mitotic entry in the latter. The findings also challenge a prior study where the N terminus of BubR1, but not Mad2, was suggested to control cyclin B accumulation during mitotic entry in mouse embryonic fibroblasts. While the precise reasons for the difference will require future independent efforts, the conclusion is supported by analysis in the highly divergent contexts of C. elegans germ cells and human cultured cells; in addition, in situ-tagged cyclin B was used, in contrast to transfected cyclin B fusions in the prior study, enabling rigorous comparison between different perturbations. While MCC containing BubR1 does exist at low levels prior to mitosis, it is speculated that in interphase, where the affinity of Cdc20 for the APC/C is low, Mad2 is sufficient to sequester Cdc20, whereas the high affinity of Cdc20 for the APC/C in mitosis due to APC/C phosphorylation requires the full MCC to efficiently inhibit active APC/CCdc20 (Lara-Gonzalez, 2019).

In the spindle checkpoint, concentration of Mad1-Mad2 in the vicinity of kinase activities at the kinetochore accelerates the conformational transition of free Mad2 promoted by the Mad1-Mad2 complex. The current results suggest that, prior to mitotic entry, Mad1-Mad2 complexes are acting in the cytoplasm to promote a similar association of free Mad2 with Cdc20. Inhibition of Mps1, the kinase activating Mad1-Mad2 in human cells at the kinetochore, does not affect the G2-to-M transition. Thus, either a different kinase activity is involved or the basal uncatalyzed rate of Mad2’s conformational transition by Mad1-Mad2 is sufficient to promote the G2-to-M transition. It will be important to elucidate in future work if and how the Mad1-Mad2 complex is regulated to promote the G2-to-M transition (Lara-Gonzalez, 2019).

In addition to Mad1-Mad2 catalyzed Mad2-Cdc20 complex formation, Cdc20 phosphorylation by Cdks, which reduces the affinity of its C-box-mediated interaction with the Apc8B subunit of the APC/C, is important to prevent APC/C activation prior to mitotic entry. The data in C. elegans germ cells highlight the existence of a regulatory relay mechanism, in which a Cdk1-cyclin B1 complex phosphorylates Cdc20 to promote accumulation of cyclin B3, which is the major Cdk1 activator for mitotic entry in this tissue. In mammalian cells, where mitotic entry is driven by cyclin B1, cyclin A2-Cdk2, active in late S and G2 phases, has been proposed to be the kinase complex that phosphorylates Cdc20, suggesting the existence of a conceptually similar relay mechanism. The severity of the defect in the G2-to-M transition when both Mad1-Mad2 and Cdc20 phosphorylation are simultaneously inhibited highlights the importance of keeping Cdc20 from activating the APC/C in order to enable entry into mitosis (Lara-Gonzalez, 2019).

Recent work has identified a different mechanism that controls APC/CCdc20 activity that is intrinsic to the APC/C. In this mechanism, a loop region of Apc1 limits binding of the C-box of Cdc20 until Cdk activity-dependent phosphorylation causes loop displacement. This work suggests that, while this mechanism is likely sufficient for coupling APC/CCdc20 activation to Cdk activity in rapid embryonic divisions that alternate between S and M phases, it does not restrain APC/CCdc20 activation to a sufficient extent to enable the G2-to-M transition in C. elegans germ cells and human somatic cells that have a defined G2 phase. It is speculated that an additional level of control is required in cells with a G2 phase because of Cdk-cyclin activity that is present, e.g., cyclin A2-Cdk2 in human cells and cyclin B1-Cdk1 in C. elegans germ cells, that relieves the APC/C-intrinsic autoinhibition mechanism. Thus, to enable cyclin B accumulation in G2, it becomes essential to prevent Cdc20 from binding to the APC/C. In rapid embryonic cycles, Cdk activity (comprised of Cdk1-cyclin B) is only prominent in M phase, thereby making the APC/C-intrinsic mechanism sufficient. Integration of the two mechanisms described int this with genetic perturbation of the Cdk-mediated relief of APC/C autoinhibition will be an important future goal (Lara-Gonzalez, 2019).

This work does not address how APC/C activated by its other co-activator, Cdh1/Hct1/Fizzy-related, is suppressed during mitotic entry. As Mad2 does not bind to Cdh1, it is unlikely to directly control APC/CCdh1 activity. Emi1 and Cdk phosphorylation have both been suggested to inhibit APC/CCdh1. Notably, Emi1 inhibition of APC/CCdh1 is important to prevent re-replication, highlighting that APC/CCdh1 is suppressed during S and G2 phases. However, Emi1 is not conserved in species like C. elegans, suggesting that Cdk phosphorylation is also a potentially conserved mechanism suppressing APC/CCdh1, a possibility that will need to be addressed in future work (Lara-Gonzalez, 2019).

In summary, this study establish the existence of two distinct mechanisms acting on Cdc20 to protect cyclin B during its accumulation and thereby enable the G2-to-M transition in the eukaryotic cell cycle. The fact that cells can still enter mitosis following loss of either mechanism and that these mechanisms likely do not operate in rapid embryonic divisions lacking a G2 phase, explains why they were not appreciated in prior efforts. Notably, one of the mechanisms uncovered in this study involves a core spindle checkpoint complex acting outside of the context of chromosome segregation during mitosis. The mechanisms described are likely to broadly control cell division cycles with a defined G2 phase, during which genome integrity is ensured prior to commitment to mitosis (Lara-Gonzalez, 2019).

Cyclin B3 is required for metaphase to anaphase transition in oocyte meiosis I

Meiosis with a single round of DNA replication and two successive rounds of chromosome segregation requires specific cyclins associated with cyclin-dependent kinases (CDKs) to ensure its fidelity. But how cyclins control the distinctive meiosis is still largely unknown. This study explored the role of cyclin B3 in female meiosis by generating Ccnb3 mutant mice via CRISPR/Cas9. Ccnb3 mutant oocytes characteristically arrested at metaphase I (MetI) with normal spindle assembly and lacked enough anaphase-promoting complex/cyclosome (APC/C) activity, which is spindle assembly checkpoint (SAC) independent, to initiate anaphase I (AnaI). Securin siRNA or CDK1 inhibitor supplements rescued the MetI arrest. Furthermore, CCNB3 directly interacts with CDK1 to exert kinase function. Besides, the MetI arrest oocytes had normal development after intracytoplasmic sperm injection (ICSI) or parthenogenetic activation (PA), along with releasing the sister chromatids, which implies that Ccnb3 exclusively functioned in meiosis I, rather than meiosis II. This study sheds light on the specific cell cycle control of cyclins in meiosis (Li, 2019).

Cyclin B3 promotes anaphase I onset in oocyte meiosis

Meiosis poses unique challenges because two rounds of chromosome segregation must be executed without intervening DNA replication. Mammalian cells express numerous temporally regulated cyclins, but how these proteins collaborate to control meiosis remains poorly understood. This study shows that female mice genetically ablated for cyclin B3 are viable-indicating that the protein is dispensable for mitotic divisions-but are sterile. Mutant oocytes appear normal until metaphase I but then display a highly penetrant failure to transition to anaphase I. They arrest with hallmarks of defective anaphase-promoting complex/cyclosome (APC/C) activation, including no separase activity, high CDK1 activity, and high cyclin B1 and securin levels. Partial APC/C activation occurs, however, as exogenously expressed APC/C substrates can be degraded. Cyclin B3 forms active kinase complexes with CDK1, and meiotic progression requires cyclin B3-associated kinase activity. Cyclin B3 homologues from frog, zebrafish, and fruit fly rescue meiotic progression in cyclin B3-deficient mouse oocytes, indicating conservation of the biochemical properties and possibly cellular functions of this germline-critical cyclin (Karasu, 2019).

Cyclin B3 controls anaphase onset independent of spindle assembly checkpoint in meiotic oocytes

Cyclin B3 is a relatively new member of the cyclin family whose functions are little known. This study found that depletion of cyclin B3 inhibited metaphase-anaphase transition as indicated by a well-sustained MI spindle and cyclin B1 expression in meiotic oocytes after extended culture. This effect was independent of spindle assembly checkpoint activity, since both Bub3 and BubR1 signals were not observed at kinetochores in MI-arrested cells. The metaphase I arrest was not rescued by either Mad2 knockdown or cdc20 overexpression, but it was rescued by securin RNAi. It is concluded that cyclin B3 controls the metaphase-anaphase transition by activating APC/C(cdc20) in meiotic oocytes, a process that does not rely on SAC activity (Zhang, 2015).

Search PubMed for articles about Drosophila Cyclin B3

Bourouh, M., Dhaliwal, R., Rana, K., Sinha, S., Guo, Z. and Swan, A. (2016). Distinct and Overlapping Requirements for Cyclins A, B, and B3 in Drosophila Female Meiosis. G3 (Bethesda) 6(11): 3711-3724. PubMed ID: 27652889

Chen, D., Zhou, L., Sun, F., Sun, M. and Tao, X. (2018). Cyclin B3 Deficiency Impairs Germline Stem Cell Maintenance and Its Overexpression Delays Cystoblast Differentiation in Drosophila Ovary. Int J Mol Sci 19(1). PubMed ID: 29351213

Garrido, D., Bourouh, M., Bonneil, E., Thibault, P., Swan, A. and Archambault, V. (2020). Cyclin B3 activates the Anaphase-Promoting Complex/Cyclosome in meiosis and mitosis. PLoS Genet 16(11): e1009184. PubMed ID: 33137813

Jacobs, H. W., Knoblich, J. A. and Lehner, C. F. (1998). Drosophila Cyclin B3 is required for female fertility and is dispensable for mitosis like Cyclin B. Genes Dev 12(23): 3741-3751. PubMed ID: 9851980

Karasu, M. E., Bouftas, N., Keeney, S. and Wassmann, K. (2019). Cyclin B3 promotes anaphase I onset in oocyte meiosis. J Cell Biol 218(4): 1265-1281. PubMed ID: 30723090

Lara-Gonzalez, P., Moyle, M. W., Budrewicz, J., Mendoza-Lopez, J., Oegema, K. and Desai, A. (2019). The G2-to-M Transition Is Ensured by a Dual Mechanism that Protects Cyclin B from Degradation by Cdc20-Activated APC/C. Dev Cell 51(3): 313-325 e310. PubMed ID: 31588029

Li, Y., Wang, L., Zhang, L., He, Z., Feng, G., Sun, H., Wang, J., Li, Z., Liu, C., Han, J., Mao, J., Li, P., Yuan, X., Jiang, L., Zhang, Y., Zhou, Q. and Li, W. (2019). Cyclin B3 is required for metaphase to anaphase transition in oocyte meiosis I. J Cell Biol 218(5): 1553-1563. PubMed ID: 30770433

McCleland, M. L. and O'Farrell, P. H. (2008). RNAi of mitotic cyclins in Drosophila uncouples the nuclear and centrosome cycle. Curr Biol 18(4): 245-254. PubMed ID: 18291653

McCleland, M. L., Farrell, J. A. and O'Farrell, P. H. (2009). Influence of cyclin type and dose on mitotic entry and progression in the early Drosophila embryo. J Cell Biol 184(5): 639-646. PubMed ID: 19273612

Yuan, K., Farrell, J. A. and O'Farrell, P. H. (2012). Different cyclin types collaborate to reverse the S-phase checkpoint and permit prompt mitosis. J Cell Biol 198(6): 973-980. PubMed ID: 22965907

Yuan, K. and O’Farrell, P.H. (2015). Cyclin B3 is a mitotic cyclin that promotes the metaphase-anaphase transition. Curr. Biol. 25: 811–816. PubMed ID: 25754637

Zhang, T., Qi, S. T., Huang, L., Ma, X. S., Ouyang, Y. C., Hou, Y., Shen, W., Schatten, H. and Sun, Q. Y. (2015). Cyclin B3 controls anaphase onset independent of spindle assembly checkpoint in meiotic oocytes. Cell Cycle 14(16): 2648-2654. PubMed ID: 26125114

date revised: 2 June 2022

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