BIOLOGICAL OVERVIEW

Drosophila Cdc2 (cdc stands for cell division control) is the catalytic subunit (the cyclin dependent kinase or cdk) of the cyclin/cdk heterodimer responsible for the execution of the mitotic (M) phase of the cell cycle. While cyclin (the regulatory subunit in all cyclin/cdk heterodimers) alternates between synthesis and degradation during the cell cycle, steady levels of the cdk are maintained; cdk activity is regulated through phosphorylation. The cyclin dependent kinase Cdc2, also known as Cdk1, is maintained in an inactive hyperphosphorylated state during G1, S and G2 stages. The inhibition of Cdc2 activation during these stages involves phosphorylation at conserved residues threonine 14 and tyrosine 15, which overlap the ATP binding site of Cdc2.

The dephosphorylation of Cdc2 is highly regulated by what is termed checkpoint control. Checkpoint control ensures that M phase entry depends on the successful completion of S phase; entry into M phase is allowed only if DNA is fully replicated and is not damaged. Cdc2 is suddenly dephosphorylated by the phosphatase known as String at the boundary between G2 and M, thus driving the cell into mitosis. At this point Cdc2 fulfills a second biological role; the promotion of M phase. Cdc2 targets many cellular proteins involved in mitosis, activating them by catalytically adding phosphate residues to threonine and serine residues. In this second biological role, the Cdc2/Cyclin heterodimer is termed the mitosis promoting factor, or MPF. For more information about the G2 checkpoint, see Cyclin B.

Although the activity of Cdc2 is central to checkpoint control, inputs from developmentally regulated transcription factors, such as Escargot, are required. Escargot is involved in maintaining diploidy in imaginal cells, ensuring that these cells retain the ability to proliferate and differentiation upon metamorphosis. This requirement exemplifies the complex regulation that occurs at the G2/M boundary (Hayashi, 1996).

The mutant phenotypes of cdc2 are similar to those of escargot: many diploid cells in imaginal discs, salivary glands and the central nervous system enter an endocycle, characterized by DNA replication without a subsequent mitotic phase. Such endocycling cells are often polytene, possessing thick chromosomes with DNA replicated many times over. When escargot function is eliminated, diploid imaginal cells that were arrested in G2 lose Cyclin A, a regulatory subunit of G2/M cdk, and entered endocycle. escargot genetically interacts with cdc2, suggesting an intimate biological interaction. Since mitotically quiescent abdominal histoblasts still require cdc2 to remain diploid, the inhibitory activity of Cdc2 on DNA replication appears to be separable from its activity as the mitosis promoting factor. These results suggest that in G2, escargot is required to maintain a high level of G2/M cdk, which actively inhibits the entry into S phase. Expression of Cyclin A is lost in escargot mutants, suggesting that Cdc2 activity (dependent on its regulatory subunit Cyclin A), indirectly depends on escargot (Hayashi, 1996). >The notion of a correlation between cell fate and time of cell division remains a cornerstone of theories that explain the origin of patterns during development. For example, in the pupal wing disc, at pupariation, cells arrest in G2 before progressing through two programmed cell divisions, separated by an intervening S-phase. Cells in vein regions divide in a reproducible pattern before the cells in the adjacent intervein regions. In the early developing embryo, cells with a common developmental fate can be recognized as mitotic domains after cellularization because they divide almost synchronously at defined developmental stages. In the growing amphibian limb, treatment with colchicine inhibits cell divisions, reduces the size of the limb and also reduces the number of digets formed (references in Weigmenn, 1997).

ring larval development, Drosophila imaginal discs increase in size about 1000-fold; cells are instructed to acquire distinct fates as a function of their position. The secreted signaling molecules Wingless and Decapentaplegic have been implicated as sources of positional information that globally control growth and patterning. Evidence has also been presented that local cell interactions play an important role in controlling cell proliferation in imaginal discs. A test was made of the role of cell division in pupal wing patterning in Drosophila by inactivating the mitotic kinase Cdc2 in developing imaginal discs. Clones of temperature sensitive cdc2 mutant cells were generated by mitotic recombination and the resulting patterns of tissue development were observed (Weigmenn, 1997).

C growth was seen to continue after inactivation of Cdc2, with little effect on overall patterning. The final size and shape of the pupal wing is not affected by inhibiting pupal cell division. Blocking cell division does not affect the spatial and temporal patterning of DNA replication in pupal wings. DNA replication is seen to continue at its normal pace in the absence of cell division, and larger than normal cells are generated, these cells attaining the architectural arrangement that smaller, normal cells achieve in the wild-type situation. Thus cell growth continues in the absence of cell division during larval stages and inhibition of cell division in mid-larval stages fails to block the patterning process. It is concluded that the mechanisms that regulate the size of the disc do not function by regulating cell division, but appear to act primarily by regulating size in terms of physical distance or tissue volume. In other words, overall size and shape of discs with mutant clones is unaffected and therefore disc size and structure is independent of cell number and pattern of cell division (Weigmann, 1997).

Ther are clear limitations to the universality of the above observations. If larvae are shifted to restrictive temperatures at early stages of development, wings fail to grow to normal size. In this case, where the cell number and overall dimensions of the disc are greatly reduced, pattern elements of gene expression do not resolve properly. This is consistent with the results of experiments with growing amphibian limbs (Weigmann, 1997).

There is already some precedence for these findings in the literature of Drosophila development. Simpson and Morata (1981) produced small clones of genetically marked, but otherwise wild-type cells in discs carrying large clones of Minute+ cells (which had been induced earlier). Mutant Minute cells divide more slowly than wild-type Mutant+ cells. Clones of wild-type cells located near the edge of the Minute+ clone grow larger than genetically equivalent clones far from the clone border. These observations suggest that cells within the Mutant+ territory but near the Minute mutant territory are stimulated to proliferate more strongly than cells surrounded by wild-type cells. It is concluded that the ability of cells to respond appropriately to long range patterning cues is relatively independent of cell number and that cell size can continue to increase if cell division is blocked, thus compensating for the presence of fewer cells (Weigmann, 1997). It might be that within a certain range, normal patterning mechanisms and compensatory mechanisms take presence over a deficiency in cell number to attain a genetically preprogrammed pattern.

Studies in unicellular systems have established that DNA damage by irradiation invokes a checkpoint that acts to stall cell division. During metazoan development, the modulation of cell division by checkpoints must occur in the context of gastrulation, differential gene expression and changes in cell cycle regulation. To understand the effects of checkpoint activation in a developmental context, a study was performed of the effect of X-rays on post-blastoderm Drosophila embryos. In Drosophila, DNA damage delays anaphase chromosome separation during cleavage cycles that lack a G2 phase. In post-blastoderm cycles that include a G2 phase, irradiation delays the entry into mitosis. Gastrulation and the developmental program of string (Cdc25) gene expression, which normally regulates the timing of mitosis, occurs normally after irradiation. The radiation-induced delay of mitosis accompanies the exclusion of mitotic cyclins from the nucleus. Furthermore, a mutant form of the mitotic kinase Cdk1 that cannot be inhibited by phosphorylation drives a mitotic cyclin into the nucleus and overcomes the delay of mitosis induced by irradiation. It is concluded that developmental changes in the cell cycle, for example, the introduction of a G2 phase, dictate the response to checkpoint activation, for example, delaying mitosis instead of or in addition to delaying anaphase. This unprecedented finding suggests that different mechanisms are used at different points during metazoan development to stall cell division in response to checkpoint activation. The delay of mitosis in post-blastoderm embryos is due primarily to inhibitory phosphorylation of Cdk1, whereas nuclear exclusion of a cyclin-Cdk1 complex might play a secondary role. Delaying cell division has little effect on gastrulation and developmentally regulated string gene expression, supporting the view that development generally dictates cell proliferation and not vice versa (Su, 2000).

To examine the effect of DNA damage on the progression of the cell cycle during Drosophila embryogenesis, embryos 0-4.5 hours of age were exposed to 570 rads of X-rays. At this dose, 40%-60% of cellular embryos die and fail to hatch into larvae. This dose therefore corresponds to the half-maximal lethal dose (LD50). When syncytial embryos are exposed to X-rays: nuclei enter mitosis normally but chromosome segregation is delayed. The delay is transient such that nuclei enter the next interphase without completely separating sister chromosomes, resulting in polyploid nuclei (Su, 2000).

In cellularized embryos, changes in cell cycle indicators that are consistent with a delay in the entry into mitosis are observed. In untreated embryos at these stages, cells divide in stereotypical clusters termed 'mitotic domains'. Both the location of a mitotic domain within the embryo and the time at which it goes through mitosis are invariant from embryo to embryo. The timing of morphogenetic movements that comprise gastrulation is likewise invariant from embryo to embryo. Thus, the wild-type pattern of mitotic cells at any specific time during this period, as indicated by the extent of gastrulation, is easily recognizable. In irradiated samples, embryos were found in which expected mitotic domains were not in mitosis, as judged by the absence of condensed chromosomes and mitotic figures. Antibody staining for a mitotic-specific phospho-epitope on histone H3 (PH3), and staining with wheatgerm agglutinin (WGA) to detect nuclear envelope breakdown, has confirmed the absence of mitoses in these embryos. It is inferred that irradiation delays the entry into mitosis in cellularized embryos, whereas under identical conditions, chromosome segregation is delayed in syncytial embryos. Treatment of cellularized embryos with a DNA-damaging agent, methyl methane sulfonate, results in a similar delay of mitosis. Therefore, the observed effect of irradiation on mitosis is probably due to the DNA-damaging activity of X-rays (Su, 2000).

It is an unprecedented finding that irradiation leads to two different cell cycle responses in a single organism: either the delay of anaphase chromosome segregation or the delay of mitosis. Mitotic chromosome segregation and the initiation of mitosis are regulated by different mechanisms. The former requires the proteolysis of proteins, such as PDS1 in budding yeast and cyclin A in Drosophila, whereas the latter requires the activation of mitotic cyclin-Cdk complexes. It is suggested that checkpoint activation by the same dose of radiation under identical conditions must have used different downstream mechanisms in order to delay chromosome segregation in the syncytium and mitosis in the cellularized embryos. Although mechanisms that operate in the syncytium remain elusive, the mechanisms used by cellularized embryos were addressed in this study (Su, 2000).

Despite the finding that irradiation does not interfere with String expression, it might have antagonized String activity. Cdc25Stg activates Cdk1 by removing the inhibitory phosphates on Thr14 and Tyr15. A Cdk1 mutant in which these residues have been mutated (Cdk1AF) bypasses the requirement for String. If the mechanism by which radiation delays mitosis solely involves inhibitory phosphorylation of Cdk1, Cdk1AF should bypass the radiation-induced delay. To test this hypothesis, Cdk1 or Cdk1AF, in conjunction with a mitotic cyclin, was expressed from a heat-inducible (hs) promoter during interphase 14. It was then asked whether irradiation could delay the onset of mitosis 14 in embryos expressing these transgenes. It was found that many cells of heat-shocked embryos that carried hs-Cdk1AF and hs-mitotic cyclin transgenes fail to delay mitosis after irradiation. This effect was seen with mitotic cyclins A, B or Bs -- a truncated version of cyclin B that is resistant to proteolysis. In contrast, embryos carrying hs-Cdk1, in combination with the same cyclins, behave like wild-type embryos and delay mitosis. It is concluded that Cdk1AF, and not Cdk1, can overcome the radiation-induced delay in mitosis. It is inferred that inhibitory phosphorylation on Cdk1 is required to delay mitosis in response to DNA damage, in agreement with previous results from fission yeast and vertebrates (Su, 2000).

Interestingly, the ability of Cdk1AF and cyclins to overcome the delay of mitosis in Drosophila was seen only in certain cells of the embryos, and these cells represent mitotic domains, for example, domain 4. Cells of mitotic domains are distinguished from their neighbors by their accumulation of String protein. Although further experiments are required to demonstrate the importance of String, the perfect coincidence of clusters of irradiated cells that entered mitosis in the presence of Cdk1AF as well as accumulated String, has led to the following suggestion: although cyclin-Cdk1AF activity is not present in sufficient quantities to promote mitosis by itself under these experimental conditions, this activity can induce endogenous String to activate endogenous Cdk1 and induce mitosis. A similar feedback mechanism has been proposed for human Cdk1 and Cdc25. It follows then that endogenous String and Cdk1 might be inhibited by irradiation, but that this inhibition can be overcome by a small amount of Cdk1AF activity (Su, 2000).

The same amount of Cdk1AF activity overcomes another consequence of irradiation, namely, the nuclear exclusion of a mitotic cyclin. Nuclear cyclin/Cdk1 activity is a prerequisite to mitosis and the exclusion of cyclin B1 from the nucleus appears to contribute to the delay in mitosis after irradiation in human cells. In cellular-stage Drosophila embryos, cyclins A and B remain enriched in the cytoplasm in interphase. Cyclin A accumulates in the nucleus of cells that initiate mitosis, as does cyclin B. In irradiated embryos, both cyclins A and B are excluded from nuclei although their levels remain unchanged. In cells that express Cdk1AF (with a mitotic cyclin) that enter mitosis even after irradiation, nuclear accumulation of cyclin A is evident. Thus, a low level of Cdk1 activity, provided by Cdk1AF in these experiments, leads to both the nuclear accumulation of a cyclin and the entry into mitosis (Su, 2000).

Given these two observations -- that Cdk1AF drives the nuclear accumulation of cyclin A and that nuclear accumulation of mitotic cyclins coincides with the entry into mitosis in unperturbed cell cycles -- it has been proposed that Cdk1 activity normally drives the nuclear accumulation of cyclin-Cdk1 complexes. In support of this idea, Cyclin A remains excluded from nuclei in string mutants. In accordance with this, Cdk1AF, in conjunction with endogenous String, overcomes the radiation-induced delay of mitosis because Cdk1AF can start the feedback loop that activates endogenous Cdk1 by endogenous String and Cdk1 activity can drive the nuclear accumulation of cyclin-Cdk1. These ideas help explain previous observations in human cells. In the latter, although the exclusion of cyclin B1 from nuclei appears to be of some importance to regulating mitotic entry, Cdk1AF can overcome the checkpoint-induced delay of mitosis, regardless of whether cyclin B1 or NLS-cyclin B1, which is constitutively localized to the nucleus, is co-expressed. Thus, Cdk1AF in human cells, as in Drosophila, might also drive the nuclear accumulation of cyclin-Cdk complexes and the entry into mitosis by initiating a positive feedback loop for the activation of endogenous Cdk1. Whether a similar feedback loop of Cdk1, String and cyclin-localization operates to control mitosis in other tissues, such as larval imaginal discs, remains to be seen (Su, 2000).

Drosophila myt1 is the major cdk1 inhibitory kinase for wing imaginal disc development

Mitosis is triggered by activation of Cdk1, a cyclin-dependent kinase. Conserved checkpoint mechanisms normally inhibit Cdk1 by inhibitory phosphorylation during interphase, ensuring that DNA replication and repair is completed before cells begin mitosis. In metazoans, this regulatory mechanism is also used to coordinate cell division with critical developmental processes, such as cell invagination. Two types of Cdk1 inhibitory kinases have been found in metazoans. They differ in subcellular localization and Cdk1 target-site specificity: one (Wee1) being nuclear and the other (Myt1), membrane-associated and cytoplasmic. Drosophila has one representative of each: dMyt1 and dWee1. Although dWee1 and dMyt1 are not essential for zygotic viability, loss of both resulted in synthetic lethality, indicating that they are partially functionally redundant. Bristle defects in myt1 mutant adult flies prompted a phenotypic analysis that revealed cell-cycle defects, ectopic apoptosis, and abnormal responses to ionizing radiation in the myt1 mutant imaginal wing discs that give rise to these mechanosensory organs. Cdk1 inhibitory phosphorylation was also aberrant in these myt1 mutant imaginal wing discs, indicating that dMyt1 serves Cdk1 regulatory functions that are important both for normal cell-cycle progression and for coordinating mitosis with critical developmental processes (Jin, 2008).

Multicellular organisms regulate Cdk1 by inhibitory phosphorylation to prevent mitosis when DNA is being replicated or repaired and to ensure that mitosis does not interfere with critical developmental processes that require remodeling of the cytoskeleton. Previous studies of Drosophila Wee1 and Myt1 revealed that these conserved Cdk1 inhibitory kinases were required during early embryogenesis and gametogenesis, respectively. This study has characterized imaginal and adult developmental defects caused by loss of dMyt1 activity (and to a much lesser extent, dWee1), that confirm the importance of Cdk1 inhibitory phosphorylation for coordinating cell-cycle events with critical developmental processes (Jin, 2008).

In Drosophila and other organisms, G2/M delays can be induced by overexpression of Myt1 kinases, suggesting a specific role for Myt1 in regulating this stage of the cell cycle. Further evidence of a role for Myt1 in G2/M regulation comes from studies of oocyte maturation in frogs, starfish, and nematodes. Not all data indicate that Myt1 is required for G2 phase arrest, however, and there is no evidence that dMyt1 regulates oocyte maturation in Drosophila. Nor is there evidence that dMyt1 activity is responsible for the timing of the G2/M meiotic transition that follows a prolonged 4-day-long G2 phase arrest, in Drosophila primary spermatocytes. Moreover, a recent study showed that functional depletion of human Myt1 by siRNA did not affect the proportion of cells in G2 phase, but instead affected membrane dynamics during mitotic exit (Nakajima, 2008). More needs to be learned about Myt1 mediated regulatory mechanisms before these apparent discrepancies in Myt1 functions are resolved (Jin, 2008).

Previous work showed that Cdk1 inhibitory phosphorylation is required for proper development of thoracic mechanosensory organs. This study has now identified dMyt1 as the primary Cdk1 inhibitory kinase for this developmental program. Several molecular mechanisms could explain the role of dMyt1 in mechanosensory bristle development. One obvious possibility is that myt1 mutant sensory organ precursor (SOP) cells and their descendants might divide prematurely due to a defect in G2/M regulation, resulting in aberrant segregation of cell fate determinants. If there was a relatively narrow window for coordinating specific developmental events with the G2/M transition, disrupting this regulatory mechanism could account for the observed loss and duplication of bristles and socket cells in myt1 mutants. Live analysis of mechanosensory organ development could test this possibility (Jin, 2008).

Alternatively, myt1 mutant phenotypes could reflect defects in Myt1-mediated regulatory mechanisms that are important for the control of intracellular membrane dynamics during mitosis, particularly the Golgi apparatus and endoplasmic reticulum. The Drosophila Golgi apparatus undergoes significant morphological changes that have been linked to specific developmental states and so the observed myt1 mutant developmental defects might reflect problems in the structure or function of this organelle. Further support for this idea comes from a recent study showing that asymmetrical segregation of mouse Numb (a conserved cell fate determinant) requires the Golgi apparatus, leading to the suggestion that Golgi fragmentation and reconstitution could represent a mechanism for coupling cell-fate specification and cell-cycle progression (Jin, 2008).

Another possible explanation for myt1 mutant defects concerns the large quantities of actin that are synthesized and packaged to form the large mechanosensory bristle shafts. This process involves extensive reorganization of the endoplasmic reticulum and Golgi apparatus to accommodate increased membrane trafficking. Defects in the structure or function of the Golgi apparatus and ER caused by loss of dMyt1 activity could therefore account for defects or diminution in these bristles. Resolving which of these potential mechanisms best explain the role of dMyt1 during mechanosensory organ development will be a major challenge of future research (Jin, 2008).

Intriguing cell-cycle defects (higher mitotic index, aberrant chromatin condensation, and ectopic apoptosis), as well as defects in responses to ionizing radiation in proliferating cells, were observed in myt1 mutant imaginal wing discs. These observations suggest an important role for dMyt1 in conserved cell-cycle checkpoint responses that target Cdk1 by inhibitory phosphorylation. It was not anticipated that dMyt1 would serve such functions, since Wee1 kinases are generally assumed to be responsible for checkpoint responses that protect the nucleus from premature Cdk1 activity. It was not clear that myt1 mutants were deficient in conventional premitotic checkpoint responses, however. Indeed, the partial decline in myt1 mutant PH3-labeled cells observed immediately after exposure to ionizing radiation could reflect activation of an otherwise dispensable Wee1-regulated premitotic checkpoint mechanism. The remaining PH3-positive cells that persisted long after irradiation in myt1 mutant discs could be arrested in mitosis by an alternative regulatory mechanism that was responsive to DNA damage. Further studies will be needed to clarify the respective roles of dMyt1 and dWee1 in cellular responses to DNA damage (Jin, 2008).

This study also observed profound defects in Cdk1 inhibitory phosphorylation in myt1 mutant imaginal discs. Phosphorylation of the T14 residue of Cdk1 was eliminated, demonstrating that dMyt1 is solely responsible for this regulatory modification, like Myt1 homologs described in other organisms. It was also observed that phosphorylation of the Y15 residue of Cdk1 was markedly reduced in myt1 mutant extracts, demonstrating for the first time that dMyt1 functions as a dual specificity Cdk1 inhibitory kinase, in vivo. Why dWee1 activity is insufficient for maintaining normal levels of phosphorylation of the Y15 residue is not clear, since Cdk1 complexes are thought to shuttle between the nucleus and cytoplasm. One possible explanation is that the doubly phosphorylated Cdk1 isoform may be more refractory to dephosphorylation by Cdc25 phosphatases, and hence more stably inhibited, than Cdk1 phosphorylated on a single residue. Another possibility is that the kinase-independent Myt1 mechanism proposed to tether phospho-inhibited Cdk1 complexes in the cytoplasm until cells are ready for mitosis might also protect them from dephosphorylation. Loss of either of these regulatory mechanisms could therefore underlie the cell-cycle defects observed in myt1 mutants. Testing these hypotheses promises to yield interesting new insights into cell-cycle regulation and the diverse developmental roles of dMyt1 and similar regulatory kinases in other organisms (Jin, 2008).

Control of PNG kinase, a key regulator of mRNA translation, is coupled to meiosis completion at egg activation

The oocyte-to-embryo transition involves extensive changes in mRNA translation, regulated in Drosophila by the PNG kinase complex whose activity is shown in this study to be under precise developmental control. Despite presence of the catalytic PNG subunit and the PLU and GNU activating subunits in the mature oocyte, GNU is phosphorylated at Cyclin B/CDK1 sites and unable to bind PNG and PLU. In vitro phosphorylation of GNU by CyclinB/CDK1 blocks activation of PNG. Meiotic completion promotes GNU dephosphorylation and PNG kinase activation to regulate translation. The critical regulatory effect of phosphorylation is shown by replacement in the oocyte with a phosphorylation-resistant form of GNU, which promotes PNG-GNU complex formation, elevation of Cyclin B, and meiotic defects consistent with premature PNG activation. After PNG activation GNU is destabilized, thus inactivating PNG. This short-lived burst in kinase activity links development with maternal mRNA translation and ensures irreversibility of the oocyte-to-embryo transition (Hara, 2017).

The massive changes in mRNA translation accompanying egg activation occur in a matter of minutes and must be linked to completion of meiosis in the oocyte. This study found that in Drosophila the solution to this developmental challenge is the regulation of PNG kinase activity. The results show that GNU is phosphorylated at CycB/CDK1 sites in mature oocytes, and in vitro CycB/CDK1 can directly phosphorylate GNU and thereby inhibit its ability to activate PNG kinase via inhibition of formation of the complex. In mature oocytes that are arrested at metaphase I, GNU is phosphorylated at CDK1 consensus sites and prevented from interaction with the PNG-PLU sub-complex. Following egg activation, as CycB protein and H1 kinase activity decline, GNU is dephosphorylated. This corresponds to the completion of meiosis and activation of the PNG kinase complex. Consistent with dephosphorylation of GNU being the crucial event for activation of PNG, substitution of a phosphorylation-resistant form of GNU into the oocyte results in premature elevation of CycB protein, implying PNG activation. Thus it is proposed that control of PNG kinase activity via GNU phosphorylation by CycB/CDK1 links meiotic completion and translational control of maternal mRNA to coordinate their timing precisely during egg activation. Active PNG leads to decreased GNU protein levels. This makes a negative feedback to shut down PNG kinase activity, thereby ensuring PNG kinase activity is constrained to the short developmental window of the oocyte-to-embryo transition (Hara, 2017).

These findings highlight the linchpin role the GNU subunit plays in the developmental control of PNG kinase activity. This regulation is exerted both at the levels of GNU protein and via its phosphorylation state. Although the presence of all three PNG kinase complex proteins is limited to late oogenesis through the early embryo, GNU is present over a narrower time window. GNU protein is undetectable in stage 10 oocytes and is rapidly accumulated during oocyte maturation. A previous genome-wide study showed that GNU protein accumulation during oocytes maturation relies on translational activation of its mRNA. Although the regulatory mechanisms for gnu translational activation remain to be defined, it is clearly dependent on CDK1, but not MOS, activity. Thus CDK1 promotes the appearance of GNU, permitting all PNG subunits to be present in the mature oocyte and poised for activation, while it simultaneously prevents activation by phosphorylation of GNU (Hara, 2017).

Several previous experimental observations now have clear significance in support of the role of CDK1 phosphorylation of GNU in inhibiting PNG complex formation and kinase activation. Ectopic GNU protein expression in early stage oocytes, which are in prophase I, causes png-dependent premature CycB protein expression and actin disorganization. Importantly, these defects result from expression of GNU at a developmental stage when although PNG and PLU are present at low levels, CycB/CDK1 is not active, and thus would not be able to block formation of the PNG complex. Analysis of the phosphorylation state of GNU in mutants for the calcipressin Sarah (sra) or the meiosis-specific APC/C activator Cortex (cort) showed that GNU remains hyperphosphorylated in eggs laid from both types of mutant mothers. Interestingly, both of these mutants fail to complete meiosis, with sra mutant eggs arrested in anaphase I, and cort mutants arrested in metaphase II. The failure of GNU to be dephosphorylated in both of these mutants agrees with the demonstration that GNU becomes hypophosphorylated after the completion of meiosis (Hara, 2017).

Although PNG and PLU levels in embryos are gradually decreased 2-4 hr after laying GNU seems to disappear after the completion of meiosis but before the initiation of embryogenesis. This is evidenced by GNU protein levels being decreased in unfertilized in vivo activated eggs, which complete meiosis II but do not enter into the embryonic cycles. This rapid GNU disappearance requires PNG kinase activity, revealing the existence of a negative feedback to shut off PNG activity shortly after its activation. It remains to be determined how GNU protein levels decline. PNG phosphorylates GNU on sites other than the CDK1 sites, and this phosphorylation may target GNU for degradation mediated by an E3 ubiquitin ligase of the SCF or the APC/C families. It is noted that GNU levels fail to decline in cort mutants, which lack one form of the APC/C. This may be an indirect effect of failure of dephosphorylation of the CDK1 sites in GNU and thus absence of PNG activation, but it is also possible that GNU is targeted directly to the APC/Ccort after completion of meiosis (Hara, 2017).

A key question raised by the demonstration of the regulatory role of GNU phosphorylation is whether developmental control of phosphatase activity is required. One possibility is that the phosphatase responsible for dephosphorylation of the CDK1 sites in GNU is constitutively active. CycB/CDK1 activity is high in the metaphase I-arrested mature oocyte, but egg activation triggers meiotic resumption and CDK1 inactivation. Reduction of CycB/CDK1 activity in the presence of active phosphatase may be sufficient for hypophosphorylated GNU. Alternatively, egg activation could lead to activation of a phosphatase. The identity of the GNU phosphatase for the CDK1 sites awaits elucidation. Although it has been shown that PP1 is capable of dephosphorylating GNU in mature oocytes, PP2A also is a possible GNU phosphatase during the process, particularly given it is the major phosphatase that removes phosphates on CDK1 substrates in mitotic exit (Hara, 2017).

The partial activity of the GNU 9A protein form provides insights into the mechanism by which GNU activates the PNG kinase. This mutant protein binds to the PNG-PLU sub-complex even in high CDK activity, however, it does not activate PNG kinase to the full extent that wild-type GNU does. This implies that there is an additional step to PNG kinase activation after GNU and the PNG-PLU sub-complex interaction. It is likely that the kinase activation following complex formation involves the N-terminus of GNU. A point mutation in the GNU N-terminal region (changing Pro 17 to Leu) retains ability to bind the PNG-PLU sub-complex but not does not activate PNG kinase activity in vitro (Hara, 2017).

The PNG kinase is significant for the understanding of how a kinase can rapidly control translation of hundreds of mRNAs. In addition to these insights into mRNA translation, identifying and defining the role of regulators involved in triggering the profound changes accompanying the oocyte-to-embryo transition is crucial for understanding of the onset of development, with implications for human fertility. This study has shown two forms of regulation of PNG kinase activity: one being regulation of protein expression of PNG kinase complex components and another being regulation of its activity. Strikingly, a cell cycle regulator, CDK1, controls both. This implies that CDK1 precisely regulates PNG kinase activity, a translational regulator, thus coordinating cell cycle progression and the translational landscape change during the oocyte-to-embryo transition. There are interesting parallels between the current findings and those in C. elegans. In C. elegans another kinase, the MBK-2 member of the conserved DYRK family of dual specificity tyrosine kinases, like PNG is crucial for the oocyte-to-embryo transition. MBK-2 activation is linked to the meiotic cell cycle by being downstream of the APC/C. MBK-2 controls proteolysis of oocyte proteins through the SCF E3 ubiquitin ligase, and it also affects RNA granule dynamics and thus likely impacts translation. Although PNG and MBK-2 are distinct kinases, these distantly related invertebrates utilize parallel approaches of coupling to cell cycle regulators to limit kinase activity to the oocyte-to-embryo transition. In both organisms, this links meiotic progression to gene expression changes after egg activation. This conservation suggests that this strategy may be employed for the onset of mammalian development (Hara, 2017).

Spatiotemporal control of mitotic exit during anaphase by an aurora B-Cdk1 crosstalk

According to the prevailing 'clock' model, chromosome decondensation and nuclear envelope reformation when cells exit mitosis are byproducts of Cdk1 inactivation at the metaphase-anaphase transition, controlled by the spindle assembly checkpoint. However, mitotic exit was recently shown to be a function of chromosome separation during anaphase, assisted by a midzone Aurora B phosphorylation gradient - the 'ruler' model. This study found that Cdk1 remains active during anaphase due to ongoing APC/C(Cdc20)- and APC/C(Cdh1)-mediated degradation of B-type Cyclins in Drosophila and human cells. Failure to degrade B-type Cyclins during anaphase prevented mitotic exit in a Cdk1-dependent manner. Cyclin B1-Cdk1 localized at the spindle midzone in an Aurora B-dependent manner, with incompletely separated chromosomes showing the highest Cdk1 activity. Slowing down anaphase chromosome motion delayed Cyclin B1 degradation and mitotic exit in an Aurora B-dependent manner. Thus, a crosstalk between molecular 'rulers' and 'clocks' licenses mitotic exit only after proper chromosome separation (Afonso, 2019).

Taken together, this work reveals that degradation of B-type Cyclins specifically during anaphase is rate-limiting for mitotic exit among animals that diverged more than 900 million years ago. Most importantly, Cdk1 activity during anaphase was shown to be a function of chromosome separation and is spatially regulated by Aurora B localization and activity at the spindle midzone. In concert with previous work, the findings unveil an unexpected crosstalk between molecular 'rulers' (Aurora B) and 'clocks' (B-type Cyclins-Cdk1) that ensures that cells only exit mitosis after proper chromosome separation during anaphase, consistent with the previously proposed chromosome separation checkpoint hypothesis. An Aurora B-dependent spatial control mechanism regulating normal NER in human cells has been recently confirmed. However, nuclear envelope defects associated with incomplete chromosome separation during anaphase (namely, anaphase lagging chromosomes due to mitotic errors) were proposed as an inevitable pathological condition. The present work provides yet additional evidence for a molecular network operating during anaphase that promotes chromosome segregation fidelity by controlling mitotic exit in space and time. According to this model, APC/CCdc20 mediates the initial degradation of Cyclin B1 during metaphase under SAC control. The consequent decrease in Cdk1 activity as cells enter anaphase targets Aurora B to the spindle midzone (via Subito/Mklp2/kinesin-6); Aurora B at the spindle midzone (counteracted by PP1/PP2A phosphatases on chromatin establishes a phosphorylation gradient that locally delays APC/CCdc20- and APC/CCdh1-mediated degradation of residual Cyclin B1 (and possibly B3) at the spindle midzone, at least in Drosophila cells. Localization experiments in human cells suggest that Cdk1 itself might be enriched at the spindle midzone. Consequently, as chromosomes separate and move away from the spindle midzone, Cdk1 activity decreases, allowing the PP1/PP2A-mediated dephosphorylation of Cdk1 and Aurora B substrates (e.g., Lamin B and Condensin I) necessary for mitotic exit. This model is consistent with the recent demonstration that Cdk1 inactivation promotes the recruitment of PP1 phosphatase to chromosomes to locally oppose Aurora B phosphorylation and recent findings in budding yeast demonstrating equivalent phosphorylation and dephosphorylation events during mitotic exit. It is also consistent with a premature Greatwall inactivation and PP2A:B55 reactivation that would be predicted after acute Cdk1 inactivation during anaphase. Most important, this model provides an explanation for the coordinated action of two unrelated protein kinases that likely regulate multiple substrates required for mitotic exit (Afonso, 2019).

Previous landmark work has carefully monitored the kinetics of Cyclin B1 degradation in living human HeLa and rat kangaroo Ptk1 cells during mitosis, and concluded that Cyclin B1 was degraded by the end of metaphase, becoming essentially undetectable as cells entered anaphase. However, it was noticed that, consistent with the current findings, a small pool of Cyclin B1 continued to be degraded during anaphase in Ptk1 cells. Subsequent work investigating cellular response to anti-mitotic drugs has also shown that human DLD-1 cells undergoing normal mitosis entered anaphase with as much as 32% of Cyclin B1 compared to metaphase levels, suggesting that human cells enter anaphase with significant Cdk1 activity. Indeed, quantitative analysis with a FRET biosensor in human HeLa cells also revealed residual Cdk1 activity during anaphase. However, the significance of persistent Cdk1 activity for the control of anaphase duration and mitotic exit was not investigated in these original studies. Previous works also clearly demonstrated that forcing Cdk1 activity during anaphase through expression of non-degradable Cyclin B1 (and Cyclin B3 in Drosophila) prevents chromosome decondensation and NER. However, while these works suggested the existence of different Cyclin B1 thresholds that regulate distinct mitotic transitions, expression of non-degradable Cyclin B1 could be interpreted as an artificial gain of function that preserves Cdk1 activity during anaphase. For example, it was shown that expression of non-degradable Cyclin B1 during anaphase 'reactivates' the SAC, inhibiting APC/CCdc20. The current work demonstrates in five different experimental systems, from flies to humans, including primary tissues, that Cdk1 activity persists during anaphase and is rate-limiting for the control of mitotic exit. Failure to degrade B-type Cyclins during anaphase blocked cells in an anaphase-like state with separated sister chromatids that remained condensed for several hours, whereas complete Cdk1 inactivation in anaphase triggered chromosome decondensation and NER. Importantly, if a positive feedback loop imposed by phosphatases was sufficient to drive mitotic exit simply by reverting the effect of Cdk1 phosphorylation prior to anaphase, cells would exit mitosis regardless of the remaining pool of B-type Cyclins that sustains Cdk1 activity during anaphase. The main conceptual implication of these findings is that, contrary to what was previously assumed, mitotic exit is determined during anaphase and not at the metaphase-anaphase transition under SAC control (Afonso, 2019).

This model also implies that persistent Cyclin B1-Cdk1 in anaphase is spatially regulated by a midzone Aurora B gradient. Indeed, a residual pool of Cyclin B1-Cdk1 was identified enriched at the spindle midzone and midbody, this localization was dependent on Aurora B activity and localization at the spindle midzone. Interestingly, human Cyclin B2 (which contains a recognizable KEN box), as well as Cdk1, were identified at the midbody and Cdk1 inactivation during late mitosis was required for the timely completion of cytokinesis in human cells. Thus, it is possible that in human cells, Cdk1 activity during anaphase is regulated not only by Cyclin B1, but also by Cyclin B2. Importantly, this model predicted the existence of a midzone-centered Cdk1 activity gradient during anaphase, which was confirmed experimentally by targeting a FRET reporter of Cdk1 activity to chromosomes (Afonso, 2019).

Finally, the experiments indicate that Aurora B activity regulates Cyclin B1 homeostasis and consequently anaphase duration in the presence of incompletely separated chromosomes. One possibility is that direct Cyclin B1 phosphorylation by Aurora B spatially regulates Cyclin B1 degradation during anaphase, mediated by both APC/CCdc20 and APC/CCdh1. Another non-mutually exclusive possibility is that Aurora B indirectly controls Cyclin B1 during anaphase by regulating APC/CCdc20 and/or APC/CCdh1 activity, as recently shown for Cdk1. Future work will be necessary to test these hypotheses (Afonso, 2019).

In conclusion, this study has uncovered an unexpected level of regulation at the end of mitosis in metazoans and reconciled what were thought to be antagonistic models of mitotic exit relying either on molecular 'clocks' or on 'rulers'. These findings have profound implications to fundamental understanding of how tissue homeostasis is regulated, perturbation of which is a hallmark of human cancers (Afonso, 2019).

Cyclin B3 activates the Anaphase-Promoting Complex/Cyclosome in meiosis and mitosis

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).

Cullin-5 mutants reveal collective sensing of the nucleocytoplasmic ratio in Drosophila embryogenesis

In most metazoans, early embryonic development is characterized by rapid division cycles that pause before gastrulation at the midblastula transition (MBT). These cleavage divisions are accompanied by cytoskeletal rearrangements that ensure proper nuclear positioning. However, the molecular mechanisms controlling nuclear positioning are not fully elucidated. In Drosophila, early embryogenesis unfolds in a multinucleated syncytium. Nuclei rapidly move across the anterior-posterior (AP) axis at cell cycles 4-6 in a process driven by actomyosin contractility and cytoplasmic flows. In shackleton (shkl) mutants, this axial spreading is impaired. This study shows that shkl mutants carry mutations in the cullin-5 (cul-5) gene. Live imaging experiments show that Cul-5 is downstream of the cell cycle but is required for cortical actomyosin contractility. The nuclear spreading phenotype of cul-5 mutants can be rescued by reducing Src activity, suggesting that a major target of cul-5 is Src kinase. cul-5 mutants display gradients of nuclear density across the AP axis that were exploited to study cell-cycle control as a function of the N/C ratio. The N/C ratio is sensed collectively in neighborhoods of about 100 μm, and such collective sensing is required for a precise MBT, in which all the nuclei in the embryo pause their division cycle. Moreover, it was found that the response to the N/C ratio is slightly graded along the AP axis. These two features can be linked to Cdk1 dynamics. Collectively, this study revealed a new pathway controlling nuclear positioning and provides a dissection of how nuclear cycles respond to the N/C ratio (Hayden, 2022).

The tight control of the cell cycle and nuclear (cell) positioning and number is a ubiquitous feature of metazoan development and is crucial to the proper development of early embryos. This work has taken advantage of shkl mutants that have defects in nuclear spreading to identify a novel pathway involved in the control of cortical contractility and gain insights into how nuclei respond to changes in the N/C ratio. Through DNA sequencing and complementation tests, this study has identified shkl mutants as mutations of the ubiquitin ligase Cul-5. In the early embryo, Cul-5 does not regulate the cell-cycle oscillator but is required for Rho and myosin activities. Cul-5 restricts the levels of active Src kinase, which is a known regulator of the actomyosin cytoskeleton. Indeed, it was found that the cullin-5 phenotype could be largely rescued through a genetic reduction in Src activity and recapitulated through Src overexpression, indicating that a main function of Cul-5 is to downregulate Src activity. These results implicate the Cul-5/Src axis as a crucial pathway involved in the control of cortical contractility in early Drosophila embryos (Hayden, 2022).

In the early embryo, nuclei regulate their own positioning through PP1 activity that spreads from the nuclei to the cortex. This localized PP1 activity drives activation of Rho and myosin II accumulation in turn. The current results argue that Cul-5 and Src act in a pathway downstream or parallel to the cell cycle to regulate Rho activity. The molecular mechanisms by which Cul-5 and Src control Rho remain to be elucidated, as is the possible connection between the cell-cycle oscillator and Cul-5/Src activities. Since Src has been shown to regulate Rho GTPases in several contexts, these mechanisms are natural candidates for the regulation of cortical actomyosin regulation via the Cul-5/Src pathway (Hayden, 2022).

Control of the MBT by the N/C ratio is important in several species, including Drosophila and Xenopus but likely excluding zebrafish. This density of DNA (as well as nuclear size) can directly or indirectly impact multiple aspects of the MBT, namely zygotic gene expression and cell-cycle control. Previous experiments with embryos irradiated to generate different nuclear densities across the AP axis argued that nuclear cycles and zygotic activation of a large set of genes respond to the local N/C ratio. This study has exploited the changes in nuclear positioning in shkl embryos to generate a continuous range of nuclear densities. This property has lead insights into how the decision of nuclei to pause their cell cycles at the MBT is affected by the N/C ratio. The threshold for nuclear division was found to be about 70% of the density at nuclear cycle 14, which confirms previous results. This value-about halfway between the density at cycles 13 and 14-likely contributes to the robustness of the MBT. However, it is not sufficient for the robustness of the MBT. To ensure reliable lengthening of cycle 14 in all nuclei, the sensing of the N/C ratio must be averaged over hundreds of nuclei. Consistently, the results suggest that nuclei sense the local N/C ratio in neighborhoods of ~100 &mi;m. This length essentially coincides with the correlation length of the Cdk1 activity field, which is established via reaction-diffusion mechanisms. Additionally, it was found that a model based on uniform sensing of the N/C ratio fails to predict the behavior of a large fraction of nuclei. However, a model assuming a slightly higher N/C ratio threshold in the posterior is highly predictable and mainly misses the behavior of nuclei at the interface between the region of extra division and that of normal division. Thus, it is proposed that the N/C ratio is the major regulator of the cell cycle at the MBT and that no mechanism other than a slight spatial modulation of the N/C threshold is needed to account for nuclear behaviors. This spatial modulation likely reflects the fact that the rate of Cdk1 activation is also slightly graded across the AP axis. The Cdk1 activation gradient is dependent on the DNA replication checkpoint, which argues that the gradient might be controlled by an asymmetric distribution of factors controlling DNA replication and/or Chk1 activity. Alternatively, the DNA replication checkpoint and Cdk1 activity might be influenced by factors controlling AP patterning and expressed in gradients across the embryos. In the future, it will be interesting to understand the mechanisms and possible functional significance of this gradient (Hayden, 2022).

The precise coordination of biochemical and mechanical signals is a ubiquitous feature of embryonic development. In early Drosophila embryogenesis, it is necessary for the uniform positioning of nuclei and timing of the MBT. This work has identified a new pathway wherein Cul-5 regulates cortical contractility by restricting Src activity. The results investigating embryos with patchy divisions indicate that nuclei sense the N/C ratio in neighborhoods of ~100 μm and pause the cell cycle when the local density exceeds a threshold around 70% of the normal density at the MBT. Moreover, the threshold required to arrest the cell cycle is slightly graded across the AP axis and is coupled to the spatiotemporal dynamics of Cdk1. Quantitatively measuring biochemical and physical dynamics during specific morphogenic events will undoubtedly continue to reveal new insights into the mechanisms and regulations of these pathways (Hayden, 2022).

Cyclin B Export to the Cytoplasm via the Nup62 Subcomplex and Subsequent Rapid Nuclear Import Are Required for the Initiation of Drosophila Male Meiosis

The cyclin-dependent kinase 1 (Cdk1)-cyclin B (CycB) complex plays critical roles in cell-cycle regulation. Before Drosophila male meiosis, CycB is exported from the nucleus to the cytoplasm via the nuclear porin 62kD (Nup62) subcomplex of the nuclear pore complex. When this export is inhibited, Cdk1 is not activated, and meiosis does not initiate. This study investigated the mechanism that controls the cellular localization and activation of Cdk1. Cdk1-CycB continuously shuttled into and out of the nucleus before meiosis. Overexpression of CycB, but not that of CycB with nuclear localization signal sequences, rescued reduced cytoplasmic CycB and inhibition of meiosis in Nup62-silenced cells. Full-scale Cdk1 activation occurred in the nucleus shortly after its rapid nuclear entry. Cdk1-dependent centrosome separation did not occur in Nup62-silenced cells, whereas Cdk1 interacted with Cdk-activating kinase and Twine/Cdc25C in the nuclei of Nup62-silenced cells, suggesting the involvement of another suppression mechanism. Silencing of roughex rescued Cdk1 inhibition and initiated meiosis. Nuclear export of Cdk1 ensured its escape from inhibition by a cyclin-dependent kinase inhibitor. The complex re-entered the nucleus via importin β at the onset of meiosis. A model is proposed regarding the dynamics and activation mechanism of Cdk1-CycB to initiate male meiosis (Yamazoe, 2023).

A conserved molecular mechanism that controls the initiation of cell division in eukaryotes involves the activation of cyclin-dependent kinase 1 (Cdk1), which serves as a master regulator of the M phase of mitosis and meiosis. In eukaryotes, the following three conditions are indispensable for activating this protein kinase: complex formation with its regulatory subunit, cyclin B (CycB); phosphorylation of Thr161 of Cdk1; and removal of phosphate groups from Thr14 and Tyr15, both of which are involved in the negative regulation of the kinase phosphorylated by Wee1/Myt1. Cdk1 is activated at the onset of the M phase via dephosphorylation of Thr14 and Tyr15 by cell division cycle 25 (Cdc25) orthologues. Thr161 of Cdk1 also needs to be phosphorylated by Cdk- activating kinase (CAK). In addition to Cdk1 modification, another type of inhibitors known as Cdk inhibitors (CKIs), such as p21, play an important role in controlling the cell cycle. CKIs were originally identified as negative factors that bind to suppress Cdk activity at the G1/S phase and also affect CycB-Cdk1 during the G2/M transition . These inhibitors need to be released from Cdk1 before the onset of the M phase. From later stages of the G2 phase towards the beginning of the M phase, Cdk1 activity depends on an increase in the expression of CycB. In vitro assays using animal oocyte extracts have revealed that Cdks are activated progressively. A small population of the CycB-Cdk1 complex is first activated by a trigger Consequently, the balance between Cdc25 and Wee1/Myt1 activities is shifted so that Cdc25 activity becomes predominant (Yamazoe, 2023).

CycB-Cdk1 further accelerates dephosphorylation of the kinase via positive feedback loops, leading to maximal activation. In contrast, a double-negative feedback loop implemented by the inactivation of a counteracting phosphatase by Cdk1 can also contribute to Cdk1's own activation. In addition, the subcellular localization of Cdk1 and its regulatory factors and the timing of their migration to other compartments are considered critical points for mitotic entry in mammalian cells. In the G2 phase, CYCB1 is enriched in the cytoplasm but continuously shuttles into and out of the nucleus until shortly before the onset of mitosis. Mitosis is triggered by the activation of Cdk1-CycB and its translocation from the cytoplasm to the nucleus. The spatial and feedback regulation ensures a rapid and irreversible transition from interphase to mitosis (Yamazoe, 2023).

Much progress has already been made in elucidating special regulatory activities that control Cdk1 activation during the G2/M transition in mitosis. However, several issues remain to be uncovered regarding the mechanism of meiotic initiation. Meiosis is expected to be highly susceptible to spatial and temporal control of the cell cycle in cooperation with the developmental program. For example, in mouse oocytes, spatial regulation of anaphase-promoting complex (APC)/CCdh1-induced CycB degradation maintains G2 arrest of oocytes for several years. The stepwise activation of Cdk1 may, rather, play a more important role in meiosis than in the mitotic cell cycle. In Drosophila, the developmental program and cell-cycle progression in meiosis have been better studied. As the meiotic cycle in Drosophila generally constitutes a prolonged G2-like growth period, the timing of meiosis initiation is expected to be strictly regulated. However, the mechanism by which Cdk1-dependent phosphorylation is timed to occur shortly before the nuclear envelope breaks down remains to be explored. With reference to the regulatory mechanisms of mitotic initiation, a similar regulatory mechanism can be expected to initiate male meiosis in Drosophila. In contrast, several specific regulations separate from the core regulatory system take place during meiosis. For example, a Cdc25 orthologue encoded by twine plays a meiosis-specific role in activating Cdk1 before the onset of meiosis during oogenesis and spermatogenesis, whereas string is required at the initiation of mitotic events during embryogenesis and those during the development of germline stem cells and their progenitor cells. cycB mRNA is expressed at low levels in the spermatogonia during mitotic proliferation. Then, it is downregulated after the completion of mitotic divisions and re-expressed at high levels in spermatocytes during the growth phase before meiosis. In contrast, CycB protein levels in spermatocytes remain low until spermatocytes enter the G2/M transition after the appearance of mRNA. CycB translation is repressed until before the onset of male meiosis by two proteins that bind to cycB mRNA in spermatocytes. CycB accumulates in the cytoplasm prior to the initiation of chromatin condensation, remains at a high level during prophase, and then enters the nucleus at the onset of meiosis (Yamazoe, 2023).

Drosophila spermatocytes before or during meiosis I offer several advantages with respect to the investigation of cell-cycle regulation at the G2/M phase. Identifying and observing meiotic cells is easy due to the large cell size, which originates the remarkable cell growth. This facilitates the observation of the subcellular localization of specific regulatory proteins. Nevertheless, comparing the spermatocytes at similar developmental stages in different cysts is not easy. The growth phase has been classified into six stages, based on the chromatin morphology and the intracellular structure of pre-meiotic spermatocytes. Recently, the characteristic size and morphology of the nucleolus in the growth phase have allowed precise identification of the developmental stages of spermatocytes (Yamazoe, 2023).

The small pores that penetrate the nuclear membrane are called nuclear pore complexes (NPCs) and play a critical role in regulating the nuclear-cytoplasmic transport of mRNAs and proteins. The NPCs are constructed of more than 30 types of nucleoporins (Nups), and these are highly conserved among eukaryotes. The protein transport between the cytoplasm and the nucleus through the NPCs requires a group of proteins called Karyopherins. These group proteins are classified into importins, which help proteins get into the nucleus by binding to nuclear localization sequences (NLSs), and exportins, which help proteins get out of the nucleus. Members of the importin-β family bind cargo proteins to transport them while mediating interactions with the NPCs. Surprisingly, no spermatocytes undergoing meiosis have been observed in testes featuring the spermatocyte-specific depletion of components of the nuclear porin 62kD (Nup62) subcomplex, which comprises the central channel of the NPC, although meiosis initiates normally in testes featuring the depletion of other NPC subcomplexes. Moreover, previous research has shown that silencing Nup62 using RNA interference (RNAi) results in the accumulation of CycB in the nucleus during the growth phase, corresponding to a prolonged G2 phase before the initiation of meiosis. This inhibits Cdk1 activation, leading to cell-cycle arrest before male meiosis. However, these results are inconsistent with previous results suggesting that the precocious accumulation of CycB in the nucleus by export-defective CycB expression does not influence mitotic entry in mammalian cells. This unexpected finding highlights the importance of CycB subcellular localization in cell-cycle progression before male meiosis and suggests that selective nuclear-cytoplasmic transport of cell-cycle regulators may be critical for determining the timing of meiotic initiation. A constitutively active Cdk1 mutant (Cdk1^{T14A Y15F}) has failed to rescue the meiotic phenotype caused by Nup62 silencing, suggesting that the removal of inhibitory Cdk1 phosphorylation was not involved in the absence of male meiosis. However, the mechanism by which meiotic initiation is hampered upon inhibition of CycB nuclear export remains unclear (Yamazoe, 2023).

This study aimed to clarify the importance of protein transport in determining when meiosis is initiated in Drosophila males. A time-lapse observation of living pre-meiotic spermatocytes was performed to investigate whether CycB continuously shuttles between the nucleus and cytoplasm before meiosis. Furthermore, the subcellular localization of positive regulators and the formation of possible complexes between the regulatory proteins and Cdk1 were investigated. Whether negative regulators were involved in inhibiting the nuclear export of Cdk1 and its activation by RNAi was also investigated. Additionally, whether importin β is required for the re-entry of Cdk1 into the nucleus was also examined. Based on the results of those investigations, a new model is proposed regarding the intracellular dynamics and stepwise activation of Cdk1-CycB to initiate male meiosis in Drosophila (Yamazoe, 2023).

The Cdk1-CycB complex serves as a common master regulator of the cell-cycle progression into the M phase in mitosis and meiosis. The importance of spatial and feedback regulation in the activation of Cdk1 has been well demonstrated in mitosis. In contrast, the regulatory mechanism in meiotic initiation remained unclear. A previous study reported that Cdk1 is not activated, and that meiosis does not initiate when the export of the kinase complex is inhibited from the nucleus. This study aimed to clarify the importance of the subcellular localization of Cdk1-CycB in determining when meiosis is initiated in Drosophila males. A time-lapse observation of living pre-meiotic spermatocytes was performed, and Cdk1-CycB was found to continuously shuttle into and out of the nucleus before meiosis. Overexpression of CycB, but not that of NLS-CycB, rescued reduced cytoplasmic CycB and inhibition of meiosis in Nup62-silenced cells. Furthermore, the subcellular localization was investigated of positive regulators, and Cdk1 was shown to interact with Cdk-activating kinase and Twine/Cdc25C even in the nuclei of Nup62-silenced cells, suggesting that other regulatory factors were involved in the failure of meiotic initiation. Silencing of one of the CKIs, roughex, rescued the Cdk1 inhibition and initiated meiosis. Full-scale Cdk1 activation occurred in the nucleus shortly after its rapid nuclear entry. The complex re-entered the nucleus via importin β at the onset of meiosis (Yamazoe, 2023).

Cdk1 activation is an essential step for initiating mitotic and meiotic divisions. Depletion of the Nup62 subcomplex of the NPC inhibits Cdk1 activation and meiotic initiation. CycB is accumulated in the nuclei of Nup62-silenced cells, whereas it is localized in the cytoplasm of normal cells before meiotic initiation. This study explored why CycB-Cdk1 was localized in the nucleus upon downregulating the expression of the Nup62 subcomplex or exportin orthologue. After the growth phase in normal spermatocytes, CycB-Cdk1 migrated to the nucleus, and meiosis started. Taking these results together with the previous findings that the full-scale activation of Cdk1 was suppressed in Nup62-silenced cells, it was speculated that abnormal subcellular localization may be involved in inhibiting Cdk1 activation in Nup62-silenced cells. CycB should be transported to the nuclei of spermatocytes in advance during early stages of the growth phase. CYCB1 continuously shuttles in and out of the nucleus before the M phase in human cells. Similarly, it was observed that CycB was transported to the nucleus and then immediately exported from the nucleus during the growth phase in normal spermatocytes. Two reasons for cell-cycle arrest following Nup62 silencing before the initiation of meiosis may be considered: (1) the precocious localization of CycB-Cdk1 at the nucleus as a consequence of Nup62 silencing may dominantly inhibit the activation of endogenous Cdk1 before the onset of meiosis; or (2) the reduced amount of Cdk1-CycB in the cytoplasm of Nup62-silenced cells may inhibit meiotic initiation. Ectopic overexpression of CycB, but not NLS-CycB, rescued the inhibition of meiotic initiation in Nup62-silenced cells. These observations support the second possibility, that cytoplasmic CycB is more important than nuclear CycB for Cdk1 activation (Yamazoe, 2023).

The absence of MPM2 epitopes in the nuclei and cytoplasm of the silenced cells indicated that Cdk1 was not activated in either compartment. Consistently, Cdk1-dependent events, such as centrosome separation, were suppressed in the cytoplasm, suggesting that initial Cdk1 activation takes place in the cytoplasm. Therefore, CycA-Cdk1 can be considered a regulator of initial activation. Drosophila CycA functions as a mitotic cyclin, unlike its mammalian orthologue. It is translated earlier than CycB during the growth phase. In spermatocytes expressing constitutively active Cdk1, which is not suppressed by Myt1, and in myt1 hypomorphic mutant cells, premature centriole disengagement occurs. This meiotic phenotype can be suppressed by the depletion of CycA activity. These previous results suggest that CycA-Cdk1 activity can influence centrosome dynamics in male meiosis. This study observed that CycA depletion results in the accumulation of CycB in the nucleus during the G2 phase and the inhibition of full-scale Cdk1 activation. Further studies are necessary to clarify the possible role of CycA-Cdk1 in meiotic initiation (Yamazoe, 2023).

Male meiosis does not initiate until Cdk1-CycB is exported from the nucleus to the cytoplasm during the growth phase. The inhibition of meiosis in Nup62-silenced spermatocytes may be not responsible for the aberrant dephosphorylation of Tyr14 and Thr15 residues in Cdk1 by Twine, as previously reported, nor for the aberrant phosphorylation of Thr161 by CAK in the previous study. The data suggesting colocalization and close association of CAK with Cdk1, as well as colocalization and close association of Twine with Cdk1, in the Nup62RNAi spermatocytes are consistent with a model in which CAK activates Cdk1 in the nuclei of Nup62-depleted cells. Even so, they do not directly prove that Cdk1 was phosphorylated and dephosphorylated by the kinase and phosphatase, respectively. Further biochemical experiments are needed to confirm the phosphorylation status of the relevant amino acid residues within Cdk1, although specific antibodies that recognize the phosphorylated peptides in Drosophila are not available. If the speculation above is correct, mechanisms other than protein modification may suppress Cdk1 activation. CKIs directly bind to cyclin-Cdk complexes to suppress their activities and regulate cell-cycle progression. The current genetic analysis suggested that Rux may be involved in inhibiting Cdk1 activation until CycB-Cdk1 is exported from the nucleus to the cytoplasm. Two possible mechanisms by which Rux could suppress Cdk1-cyclins in the nucleus can be considered. First, Rux may inhibit Cdk1-CycA activity in the nucleus. Since CycA is translated and activated earlier than CycB, Cdk1-CycB activity, a major driver of male meiosis, may be suppressed via the inhibition of Cdk1-CycA by Rux until the complex is exported from the nucleus. Alternatively, Rux may directly suppress Cdk1-CycB in spermatocytes before meiosis. Rux can bind to CycB-Cdk1 and suppress its CycB-dependent kinase activity. However, the effects of Rux on mitotic cyclin-Cdk1 complexes open up the possibility that it also contributes to the regulation of mitotic initiation in Drosophila embryos. Whether rux is involved in determining the timing of male meiosis should be investigated (Yamazoe, 2023).

These observations indicate that the nuclear re-entry of CycB is a rapid process. The nuclear transfer machinery may be activated by Cdk1, thereby enabling rapid nucleusto-cytoplasm transport. Mammalian CYCB1 is imported through direct interaction with importin β. Cdk1 phosphorylates importin β, stimulating an interaction between importins α and β to accelerate protein transport. It was noticed that importin β was involved in the rapid nuclear import of CycB, although a typical NLS was not identified in Drosophila CycB. Importin β was not required for slow import in the G2 phase before centrosome separation, as the event was not affected in Fs(2)-silenced cells. Polo-like kinase suppresses the nuclear export of cyclin B1-Cdk1 via phosphorylation of the nuclear export signal of cyclin in animal cells. This kinase is thought to facilitate the rapid accumulation of CycB in the nucleus. However, Polo may not play a critical role in the rapid nuclear import of Cdk1-CycB at the onset of Drosophila male meiosis because the silencing of polo did not affect meiotic initiation and the nuclear export of Cdk1-CycB did not change in Nup62-silenced cells (Yamazoe, 2023).

Evidence was obtained that the subcellular localization of essential cell-cycle regulators plays an important role in Cdk1 activation and meiotic initiation. Cdk1 needs to be activated in the cytoplasm during the G2/M transition; otherwise, meiosis cannot initiate properly. When Cdk1 remains in the nucleus, the level of Cdk1-CycB is reduced in the cytoplasm, becoming insufficient to initiate meiosis. If the positive regulators required for Cdk1 activation are localized to the cytoplasm, Cdk1 must be exported to the cytoplasm for activation. Conversely, if negative regulators are localized to the nucleus, they need to be released from Cdk1 for its activation. CAK and Twine were localized in the nucleus throughout the growth phase. Subcellular localization of these positive factors does not support the first possibility. In contrast, the negative regulators Wee1/Myt1 were also predominantly localized in the nucleus. Rux is localized in the cytoplasm when CycA re-enters the nucleus. Before this developmental stage, the subcellular localization of Rux was not reported. However, it was observed that rux silencing rescued the accumulation of CycB-Cdk1 in the nucleus, thereby suggesting that the Cdk1 complex was suppressed by Rux until it was released from the inhibitor (Yamazoe, 2023).

Cell-cycle regulation differs in some ways between Drosophila male meiosis and mitosis. Before the initiation of mitosis in animal cells, CycB migrates to the nucleus to avoid premature mitosis until DNA damage checkpoints are verified. In contrast, the initiation of meiosis may not be permitted until the clearance of further conditions that the pre-meiotic spermatocytes should fulfill, for example, by ensuring sufficient cell growth (Yamazoe, 2023).

Several proteins and mRNAs are required for meiotic division, and post-meiotic events occur during the growth phase. In a hypomorphic mutant for eIF4G encoding a eukaryotic translation initiation factor, the growth of germline cells was inhibited. Moreover, neither meiosis nor sperm differentiation was observed in mutant testes. Therefore, Cdk1 activation that terminates the growth phase may need to be strictly regulated before meiosis, for example, through additional checkpoints that monitor cell growth (Yamazoe, 2023).

A new model regarding the stepwise activation of Cdk1-cyclins associated with the nuclear-cytoplasmic shuttling of CycB is proposed (see New model for the dynamics of CycB-Cdk1 shuttling in and out of the nucleus during interphase and its rapid nuclear re-entry to initiate male meiosis in Drosophila). During a prolonged G2 phase in spermatocytes, Cdk1-CycB continues to be modified by Wee1/Myt1 and Twine in the cytoplasm and nucleus. The complex has an intrinsic ability to temporally migrate to the nucleus. Simultaneously, it is exported more rapidly back to the cytoplasm through a unique exportin orthologue, Emb, via the Nup62 subcomplex of the NPC. Most kinase complexes are inactivated by Wee1/Myt1, which initially dominates over Twine, and are further suppressed by Rux through the suppression of Cdk1-cyclins (CycA or CycB) in the nucleus. Nevertheless, a small population of Cdk1-cyclins may execute some pre-meiotic events such as centrosome separation. With a sharp increase in CycB expression shortly before the onset of meiosis, a small portion of active Cdk1 initiates the production of a large amount of the active Cdk1 complex through the activation of Twine and inactivation of negative regulators. The kinase complex is rapidly imported into the nucleus via the Fs(2)Ket/importin β-mediated pathway. Through positive and double-negative feedback loops, the resultant CycB-Cdk1 triggers meiotic initiation after the completion of fullscale activation in the nucleus. Further investigations are warranted to validate this model. This study investigated Cdk1 activation using MPM2 antibody, which recognized phosphorylated proteins by several kinases, including Cdk1. Although it can be concluded that Cdk1 was not activated either when no MPM2 epitopes were observed, the detection of the epitopes does not necessarily prove that Cdk1 was activated. If further experiments using another specific probe that can detect the activation of this kinase directly were to become available, a more reliable conclusion could be reached. This is one of the limitations of this study and a challenge for the future. This study demonstrated that colocalization and close association of CAK with Cdk1, as well as colocalization and close association of Twine with Cdk1, could be normally observed even when the nuclear export of CycB-Cdk1 was inhibited. However, these data do not directly prove that Cdk1 was phosphorylated and dephosphorylated by the kinase and phosphatase, respectively. However, the specific antibodies that recognize the phosphorylated peptides in Drosophila Cdk1 are not available (Yamazoe, 2023).

GENE STRUCTURE

Bases in 5' UTR - 45

Bases in 3' UTR - 99

PROTEIN STRUCTURE

Amino Acids - 297

Structural Domains

The two Drosophila Cdc2 proteins, Cdc2 and Cdc2c exhibit a 56% sequence identity. Cdc2 is more closely related to the identified Cdc2 sequences of other species than is Cdc2c. This suggests that the evolutionary separation of Cdc2 and Cdc2c is ancient. The major differences between the Drosophila homologs are confined to regions that are not conserved among known p34cdc2 homologs (Lehner, 1990). Cdc2c functioning during G1 and S phases, partnering Cyclin E, is required for progression through S phase of the mitotic cycle (Knoblich, 1994).

date revised: 21 June 2024 

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