BIOLOGICAL OVERVIEW

Cyclin B, along with its dimerization partner cdc2, plays a significant role in cell division in Drosophila, but this fact is easy to overlook for several reasons. First, mutational studies can be misleading, with respect to the vital role played by Cyclin B during cell division. A biological fail safe mechanism appears to exist between Cyclin B and Cyclin A: a single mutational deficiency in either of these two cyclins involved in the G2-M transition can be compensated for by the unmutated cyclin. One might reasonably suppose that because Cyclin B is thus replaceable, it is less than essential to the process. The compensation is not 100% effective however; mitotic spindles are abnormal and progression through mitosis is delayed in cyclin B deficient embryos (Knoblich, 1993).

Two other factors mitigate against finding a substantial role for Cyclin B in Drosophila development: first, maternal cyclins are involved in development through mitosis 14, at which time they are degraded. Second, and occuring after this time, the dynamics of String protein limit the rate of mitosis. Despite these limiting factors, Cyclins A and B are produced in G2 both before and after cell cycle 14.

The strongest evidence that Cyclin B is crucial to mitosis is provided by the measurement of cyclin levels throughout the process. During cycles 8-13 a progressive increase in the degradation of cyclins at mitosis leads to increasing oscillations of cdc2 kinase activity. Quantitative measurements indicate that less than 10% of Cyclin A and B are degraded at mitosis 5, and that about 30%, 60%, and 80% are degraded at mitoses 8, 11 and 13 respectively. It appears that cyclin synthesis is limiting for mitoses 10-13 (Edgar, 1994).

During interphase 14, programmed degradation of maternal String protein leads to inhibitory phosphorylation of cdc2 and cell cycle arrest. Subsequently, mitoses 14-16 are triggered by pulses of zygotic string transcription. The use of an N-terminally truncated Cyclin B in Drosophila provides evidence for the role of Cyclin B in timing mitosis. Cyclin A is degraded during metaphase and Cyclin B degradation occurs at approximately the metaphase-anaphase transition (Whitfield, 1990). The N-terminally truncated Cyclin B fails to degrade due to absence of a 'destruction box' in the truncated N-terminal region. The truncated Cyclin B results in mitotic delay at late anaphase (Rimmington, 1994). The gene fizzy, homologous to cdc20 in S. cerevisiae, is required for metaphase-anaphase transition in Drosophila, and is required for normal cyclin A and B degradation. It is suggested that fizzy functions to promote the ubiquitin-dependent proteolytic events that occur during mitosis (Dawson, 1995).

Looking to yeast and to vertebrates for clues as to the role of Cyclin B in mitosis, one finds a bewildering array of information. There appears to be a connection between G1-S cyclins and G2-M cyclins. Cdk2 kinase, the partner for G1-S cyclins, is a positive regulator of cdc2-Cyclin B complexes in Xenopus. When cdk2 kinase activity is inhibited by the cdk-specific inhibitor, a block to mitosis occurs, and inactive cdc2-cyclin B accumulates (Guadagno, 1996). Eukaryotic cells have evolved regulatory mechanisms to ensure the strict alternation of DNA replication and mitosis. Cdc2/cyclin B has a role in preventing re-replication of the genome before mitosis. A Xenopus homolog of S. pombe, cdc21, exhibits cell-cycle dependent chromatin binding and phosphorylation in association with S-phase control. Cdc21 remains bound to chromatin during the initiation of DNA replication and is displaced with the progression of the replication fork. Cytoplasmic cdc21 remains underphosphorylated but at the beginning of mitosis the entire pool of cdc21 is hyperphosphorylated, possibly by the cdc2/cyclin B kinase. These properties identify Xenopus cdc21 as a possible component of the DNA licensing factor (Coué, 1996). For more information about licensing factor see the DNA replication site.

Several targets of cdc2-Cyclin B kinase have been identified. The dimer cdc2-Cyclin B targets Wee1 kinase. Wee1 then inhibits cell division by phosphorylating cdc2. In each cell cycle, the mouse Wee1 kinase is phosphorylated at M-phase resulting in inactivation of Wee kinase. The N-terminal domain or entire molecule is extensively phosphorylated by the cdc2-Cyclin B dimer (Honda, 1995). Drosophila Cyclin B has a consensus cAMP-dependent protein kinase site. Evidence from Xenopus suggests that cyclin degradation and exit from mitosis requires Cyclin B/cdc2-dependent activation of the cAMP-PKA pathway. The concentration of cAMP and the activity of PKA decrease at the onset of mitosis and increase at the transition between mitosis and interphase (Grieco, 1996). Proteins of the mitotic apparatus are direct targets of cdc2-Cyclin B dimer.

A human homolog of Xenopus Eg5, (a kinesin-related motor protein) is implicated in the assembly and dynamics of the mitotic spindle. An evolutionarily conserved cdc2 phosphorylation site in HsEg5 is phosphorylated specifically during mitosis in HeLa cells and in vitro by p34cdc2/Cyclin B. Phosphorylation controls the association of this motor protein with the spindle apparatus (Blangy, 1995).

Cdc2/Cyclin B targets the ubiquitin proteins involved in cyclin destruction. Two specific components are required for the ubiquitination of mitotic cyclins: E2-C, a cyclin-selective ubiquitin carrier protein that is constitutively active during the cell cycle, and E3-C, a cyclin-selective ubiquitin ligase (termed the cyclosome) that purifies as part of an approximately 1500-kDa complex and is active only near the end of mitosis. The cyclosome has been separated from its ultimate upstream activator, cdc2, that activates the cyclosome complex by means of phosphorlyation (Lahav-Baratz, 1995).

The cdc2/Cyclin B heterodimer was first characterized as maturation promoting factor, or MPF. Unfertilized frog eggs manifest cytoplasmic activity that can induce immature oocytes to undergo meiotic maturation. MPF activity drives the events of early mitosis such as nuclear breakdown, chromosome condensation and spindle formation by phosphorylating cellular substrates. While cdc2 (the catalytic subunit of the MPF heterodimer) is required to drive the events of early mitosis, it must be inactivated to allow the events of late mitosis to proceed. The metaphase-anaphase transition is a checkpoint during mitosis. At this juncture the cell is able to monitor the integrity of its spindle before proceeding to inactivate MPF and initiate chromosome separation (Murray, 1992 and references). What is the link between phosphorlyation and this checkpoint? A common mitotic error is the attachment of a chromosome to only one spindle pole rather than to both poles. Even a single such chromosomal error delays anaphase in cells. Recently it has been shown that when the tension associated with proper attachment is absent the kinetochore becomes phosphorylated and anaphase is delayed. It has been proposed that the kinetochore protein dephosphorylation caused by tension is the all-clear signal to the checkpoint. The involvement of Cyclin B/cdc2 in the events surrounding this mitotic checkpoint have yet to be documented, but the fact that Cyclin B/cdc2 is degraded at the metaphase-anaphase transition, suggests that MPF is the direct target of this checkpoint (Nicklas, 1995 and Dawson, 1995).

Translational control of maternal Cyclin B mRNA by Nanos in the Drosophila germline

In the Drosophila embryo, Nanos and Pumilio collaborate to repress the translation of hunchback mRNA in the somatic cytoplasm. Both proteins are also required for repression of maternal Cyclin B mRNA in the germline; it has not been clear whether they act directly on Cyclin B mRNA, and if so, whether regulation in the presumptive somatic and germline cytoplasm proceeds by similar or fundamentally different mechanisms. This report shows that Pumilio and Nanos bind to an element in the 3' UTR to repress Cyclin B mRNA. Regulation of Cyclin B and hunchback differ in two significant respects. (1) Pumilio is dispensable for repression of Cyclin B (but not hunchback) if Nanos is tethered via an exogenous RNA-binding domain. Nanos probably acts, at least in part, by recruiting the CCR4-Pop2-NOT deadenylase complex, interacting directly with the NOT4 subunit. (2) Although Nanos is the sole spatially limiting factor for regulation of hunchback, regulation of Cyclin B requires another Oskar-dependent factor in addition to Nanos. Ectopic repression of Cyclin B in the presumptive somatic cytoplasm causes lethal nuclear division defects. It is suggested that a requirement for two spatially restricted factors is a mechanism for ensuring that Cyclin B regulation is strictly limited to the germline (Kadyrova, 2007).

Thus Nos and Pum directly regulate maternal CycB mRNA, binding to an NRE in its 3' UTR. Differences in the spacing and arrangement of protein-binding sites within the hb and CycB NREs appear to account for the regulation of hb but not CycB by Brat. For regulation of CycB, the main function of Pum is to recruit Nos, a role that can be bypassed by tethering Nos via an exogenous RNA-binding domain. CycB-bound Nos is then likely to act, at least in part, by recruiting a deadenylase complex, interacting with its NOT4 subunit. Regulation of CycB is limited to the PGCs to avoid the deleterious consequences of repression in the presumptive somatic cytoplasm. The requirement for both Nos plus at least one additional germline-restricted factor may be part of a mechanism to ensure that CycB regulation is strictly limited to the PGCs (Kadyrova, 2007).

The co-crystal structure of human Pum bound to a fragment of the hb NRE shows that a single Pum RBD directly contacts eight nucleotides of the RNA. However, Puf proteins bind with essentially wild-type affinity to many mutant sites, suggesting that all eight nucleotides are not rigidly specified. How, then, do Puf proteins recognize specific mRNA targets in vivo (Kadyrova, 2007)?

Part of the answer appears to be that, within functional NREs, more than eight nucleotides are recognized, at least by Drosophila Pum. Mutations that simultaneously disrupt Pum binding in vitro and regulation in vivo are spread over 20 nts of the hb NRE and 18 nts of the CycB NRE. These extended Pum mutational 'footprints' are too large to be accounted for by binding of a single RBD; it is suggested that two or more Pum RBDs bind each NRE, an idea supported by the detection of two RNA-protein complexes in gel mobility shift experiments using both the CycB and hb NREs. This model disagrees with earlier experiments that suggested only a single Pum RBD binds to the hb NRE. Further biochemical and structural studies will be required to resolve the issue (Kadyrova, 2007).

The distribution of Pum- and Nos-binding sites within the CycB and hb NREs is different. In the former, the Nos binding site lies 5' to the Pum-binding site(s), whereas in the latter, the Nos-binding site is flanked by nucleotides recognized by Pum. It is assumed that the different arrangement of Nos- and Pum-binding sites is responsible for the assembly of Pum-NRE-Nos complexes with different topographies, such that Brat is recruited to hb but not to CycB. Further definition of each RNP structure will ultimately be required to understand the combinatorial assembly of different repressor complexes on each NRE (Kadyrova, 2007).

In addition to the NRE, Pum also binds with high affinity to at least two other sites in the CycB 3' UTR; however, binding to these sites does not mediate translational repression in the PGCs, perhaps because neither supports recruitment of Nos. These sites may simply bind Pum fortuitously, or they may mediate Nos-independent regulation at other stages of development. Pum has been suggested to destabilize bcd mRNA at the anterior of the embryo in a Nos-independent manner. Another Nos-independent function of Pum is the repression of CycB translation throughout the prospective somatic cytoplasm during the early syncitial nuclear cleavages. These processes might be mediated by elements in Fragments A and F of the 3' UTR, that bind Pum but not Nos (Kadyrova, 2007).

A general framework has been provided for understanding how Puf proteins act to control either the translation or stability of target mRNAs (Goldstrohm, 2006). The yeast Puf protein MPT5 interacts directly with Pop2, one of the catalytically active subunits of a large deadenylase complex. Subsequent deadenylation could either silence the mRNA or cause its degradation, depending on other signals in the transcript or the composition of the deadenylase complex (or both). The Puf-Pop2 interaction is conserved across species (including Drosophila), supporting the idea that the mechanism uncovered for MPT5 might generally be applicable to Puf proteins (Kadyrova, 2007).

In this context, it is surprising that Pum is dispensable if Nanos is tethered to CycB via MS2 CP. It is suggested that yeast Puf proteins both recognize target mRNAs and recruit the deadenylase, but that in the Drosophila germline these functions are partitioned, with Pum primarily responsible for target mRNA recognition and Nos primarily responsible for effector recruitment. This model has the attraction of attributing an important role to Nos, which is essential for Puf-mediated regulation in Drosophila, and probably other metazoans as well. What, then, might be the role of the conserved interaction between Pum and Pop2? One possibility is that it acts cooperatively with Nos to recruit the deadenylase; unlike CycB, other mRNA targets (e.g. hb) might require recruitment by both Nos and Pum to ensure efficient deadenylation. Another possibility is that it plays an essential role for mRNAs regulated by Pum but not Nos (Kadyrova, 2007).

Oscillations in CycB activity underlie normal cell cycle progression. During the early embryonic syncitial nuclear cleavages, degradation in the vicinity of the nuclei is thought to deplete CycB locally. Recent work has shown that Pum can inappropriately repress de novo translation of CycB mRNA during the initial nuclear cleavages if not antagonized by the PNG kinase, resulting in mitotic failure. This early Pum-dependent repression is thought to be Nos-independent, as it occurs efficiently in the anterior, where Nos activity is undetectable (Kadyrova, 2007).

The results show that if CycB is inappropriately subjected to Pum+Nos-dependent repression via the hb NRE, CycB is locally depleted, resulting in mitotic failure during nuclear division cycles 10-13. Since it is thought to be the case during the early cycles (1-7), de novo synthesis of CycB apparently is required to counteract the local degradation that probably occurs during M phase of each cycle. The CycB NRE must therefore be precisely tuned to repress translation only in the PGCs and not in the presumptive somatic cytoplasm (Kadyrova, 2007).

Osk is the limiting factor for assembly of pole plasm in the embryo; the results suggest that it stimulates the accumulation or activity of at least one factor in addition to Nos that is required for repression of CycB in the PGCs. The existence of a co-factor is inferred from the finding that ectopic Nos can repress CycB in the somatic cytoplasm only in the presence of ectopic Osk. Regulation of CycB may depend on more than one germline-restricted factor to ensure that potentially deleterious repression does not occur in the somatic cytoplasm (Kadyrova, 2007).

A germline Nos co-factor might act in a variety of ways. It could bind to the CycB NRE adjacent to Pum and Nos, substituting functionally for Brat, which is recruited to the Pum-hb NRE-Nos complex. The 50 nt CycB NRE is inactivated by a truncation at both ends that leaves the Pum- and Nos-binding sites intact, consistent with the idea that another factor binds to the element. Another possibility is that the co-factor is a germline-specific component of the adenylation/deadenylation machinery, as is the case for the GLD-2 cytoplasmic poly(A)-polymerase in C. elegans. Distinguishing among these ideas awaits identification of the cofactor (Kadyrova, 2007).

Onset of the DNA replication checkpoint in the early Drosophila embryo

The Drosophila embryo is a promising model for isolating gene products that coordinate S phase and mitosis. Increasing maternal Cyclin B dosage to up to six copies (six cycB) increases Cdk1-Cyclin B (CycB) levels and activity in the embryo, delays nuclear migration at cycle 10, and produces abnormal nuclei at cycle 14. The level of CycB in the embryo inversely correlates with the ability to lengthen interphase as the embryo transits from preblastoderm to blastoderm stages and defines the onset of a checkpoint that regulates mitosis when DNA replication is blocked with aphidicolin. A screen for modifiers of the six cycB phenotypes identified 10 new suppressor deficiencies. In addition, heterozygote dRPA2 (a DNA replication gene) mutants suppressed only the abnormal nuclear phenotype at cycle 14. Reduction of dRPA2 also restored interphase duration and checkpoint efficacy to control levels. It is proposed that lowered dRPA2 levels activate Grp/Chk1 to counteract excess Cdk1-CycB activity and restore interphase duration and the ability to block mitosis in response to aphidicolin. These results suggest an antagonistic interaction between DNA replication checkpoint activation and Cdk1-CycB activity during the transition from preblastoderm to blastoderm cycles (Crest, 2007).

It has been proposed (model 1) that the ability of the DNA replication checkpoint to regulate the entry into M-phase is not active before cycle 11 in wild-type (two cycB) embryos. This model is based on observations that the earliest defects in interphase duration can be detected only after cycle 10 in grp (dChk1) or mei-41 (dATR) mutant embryos. In this view, before cycle 10 in two cycB embryos, nuclei have normal morphology because the DNA replication machinery is abundant. The exponentially increasing numbers of nuclei, however, titrate components of this machinery to a critical level after cycle 10, thereby inducing the checkpoint activation, interphase extension, and delay of M-phase in a Grp- and Mei-41-dependent manner (Crest, 2007).

An alternative (model 2) is proposed: Checkpoint function is active all the time, but before cycle 10 checkpoint activity is too low and is overridden by the high level of maternal Cdk1-CycB. The difference between the two models is that in model 1, checkpoint is activated by a critical amount of the replication machinery. In model 2, at a critical concentration of Cdk1-CycB, this kinase (Cdk1) can no longer override checkpoint activity. Several observations are not compatible with model 1, but are with model 2. First, here this study shows that checkpoint activity does not depend on a specific number of nuclei, number of rounds of divisions, or time after fertilization, but on the amount of CycB-Cdk1. It occurs earlier in one cycB embryos with fewer nuclei and later in six cycB embryos with more nuclei (Crest, 2007).

Second, model 1 does not explain interphase extension before cycle 10 and the slight increase of interphase even in grp mutant embryos after cycle 10 (Crest, 2007).

Third, Grp protein is required for the degradation of cyclin A in the presence of cycloheximide as early as cycle 4. Although the effect of Grp on CycA degradation may be different from its effect on replication checkpoint activation, this observation suggests that Grp is present and functional before cycle 10. How then do nuclei enter M-phase at the normal time when aph. is applied before cycle 10, i.e., show no DNA replication checkpoint? It is reasoned that successful execution of the checkpoint requires the inhibitory effect of Grp to overcome the M-phase-promoting effect of Cdk1-CycB. Early embryos might have too few nuclei, thus limited numbers of replication forks, to trigger sufficient Grp/Chk1 activity necessary to overcome the relatively high levels of Cdk1-CycB. Despite this situation, nuclear morphology is normal because replication machinery is not limited before cycle 10, S-phase is rapidly completed, and nuclei can successfully undergo a normal mitosis (Crest, 2007).

Fourth, during normal S-phase of either yeast or mammalian cells, a low level of replication checkpoint activity is observed. This low level of checkpoint activity can be detected as phosphorylation on Chk1 in S-phase cells without any replication stress or DNA damage. Physiological regulation of Chk1 is under the control of similar factors of the DNA replication checkpoint machinery, and thus it was proposed that DNA replication per se generates lesions that activated the checkpoint pathway. Alternatively, but not mutually exclusively, this constitutively low level of replication checkpoint activation may be due to transient signaling from the replication forks, which does not lead to cell cycle arrest, but serves as a mechanism to coordinate the firing of replication origins, thereby moderating the rate of S-phase (Crest, 2007).

Model 2 not only accounts for observations with the aph. experiments, but also accounts for observation of the CycB effects on the replication checkpoint effect. In one cycB embryos the Grp/Chk1 effect is observed earlier, presumably because levels of Cdk1-CycB become limiting earlier, whereas in six cycB embryos, these occur later. Using PH3 staining on anaphase chromosomes as a measurement for Chk1-dependent Cyclin A degradation indicates that Chk1 is not functioning in six cycB embryos before cycle 11, possibly because it is overridden by the abundance of Cdk1-CycB in these embryos. It is proposed that in six cycB embryos at cycle 11 or later, Cdk1-CycB activity is still too high and forces the nuclei into mitosis at a time when the DNA replication machinery is limited, resulting in precocious M-phase and abnormal nuclei (Crest, 2007).

This study addresses how dRPA2 might suppress the six cycB phenotype at cycle 14. dRPA2 is a subunit of a highly conserved heterotrimeric complex of proteins that make up the RPA complex. All three subunits contain DNA-binding domains, which stabilize ssDNA as it is unwound at the replication fork. This stabilized DNA allows for Cdc45 and DNA polymerase-α to initiate DNA replication. Additional roles for RPA have been implicated in DNA repair and recombination (Crest, 2007).

In six cycB embryos, during the blastoderm cycles, elevated levels of Cdk1-CycB override Chk1 and the nuclei divide before DNA replication is completed, leading to abnormal nuclei. It is speculated that reducing dRPA2 is likely to slow DNA replication because RPA can facilitate DNA replication by unwinding dsDNA and by modulating the activities of several enzymes, such as DNA helicases, DNA polymerases, and primases. This would result in less RPA coated, primed DNA and ssDNA. Such a DNA structure may potentiate the TopBP1-mediated ATR-ATRIP kinase activation, leading to stronger Chk1 activation. Thus in dRPA2/six cycB embryos, a stronger Chk1 activation would have a stronger inhibitory effect on Cdk1-CycB activity that cancels out the effect caused by extra Cdk1-CycB. This interpretation for the suppressive effect of RPA2 on the six cycB phenotype suggests an antagonistic relationship between the DNA replication checkpoint activation and Cdk1-CycB activity in regulating the transition from the preblastoderm cycles to the blastoderm cycles (Crest, 2007).

The earliest divisions in the embryo (with the exception of mammals) are maternally regulated until the zygotic genome takes over. On the basis of many observations with different animals such as Xenopus and Drosophila, a simple concept developed: Early cell cycles are invariant, synchronous, and lack both gap phases until the transition to zygotic control occurs. The time point at which this change happens has been called the midblastula transition (MBT). It is assigned to the specific cycle when zygotic transcription is activated. Furthermore, G2 is induced in Drosophila and G1 and G2 in Xenopus into the abbreviated cell cycles and cell division is patterned (Crest, 2007).

This concept is attractive; however, as with many simple concepts, the more that is learned the harder it is to accept the concept at face value. For example, synchronous divisions have never been observed in Caenorhabditis elegans. Even in Drosophila, the simplification that early cell cycles are synchronous and equal in length is incorrect since interphase durations steadily increase after cycle 7 and metasynchronous mitoses are observed as early as cycle 4. These changes occur long before cycle 14, the time that has been designated by many as the MBT. The data presented in this study clearly demonstrate a change in the maternal program as the embryo develops: A DNA replication checkpoint is first detectable after cycle 10, but becomes increasingly robust in the subsequent cycles, indicating that the ability to regulate M-phase by checkpoints is not completely'off' or 'on'. In addition, it was found that the DNA replication checkpoint is detectable earlier in one cycB and later in six cycB embryos, clearly indicating that changes do not have to occur at a specific stage. A gradual attainment of full checkpoint function is also supported by the fact that aph. injections before cycle 11 can stall/delay the nuclear cycle, but not the centrosomal cycle. These results are not compatible with the idea of an invariant maternal program for pre-MBT cycles (Crest, 2007).

Another simplification is that changes that define the MBT are events that envelope the entire embryo. In sea urchins the deceleration of the cell cycle in the micromeres occurs long before these changes have been observed in the macromeres. Thus an MBT has not been proposed for this animal. But differences are also observed among the blastoderm cycles in Drosophila, where divisions in the middle of the embryo are slower than at the poles, correlating with their nuclear densities (Crest, 2007).

The initiation of the zygotic program also does not occur suddenly from off to on. fushi tarazu (ftz) transcripts are first observed at cycle 9 in one or the other embryo and in one or the other nucleus. Transcription gradually increases over the next three cycles (cycles 9-12). This gradual increase is a consequence of the dose-dependent repressor Tramtrack (TTK), where with one gene dose of maternal ttk, initial transcription of ftz occurs one cycle earlier, and conversely extra copies of ttk result in initial transcription of ftz one cycle later. These data are interpreted as a decline of TTK during cycles 8-10 to a threshold level where TTK repression is insufficient, enabling low-level transcription of ftz (Crest, 2007).

Despite the many observations that do not fit the MBT concept, textbooks, reviews, research articles, and grant proposals still hold on to it, hindering progress in the understanding of how maternally controlled development declines and terminates and zygotic programming gradually takes over (Crest, 2007).

Cell type-specific translational repression of Cyclin B during meiosis in males

The unique cell cycle dynamics of meiosis are controlled by layers of regulation imposed on core mitotic cell cycle machinery components by the program of germ cell development. Although the mechanisms that regulate Cdk1/Cyclin Bactivity in meiosis in oocytes have been well studied, little is known about the trans-acting factors responsible for developmental control of these factors in male gametogenesis. During meiotic prophase in Drosophila males, transcript for the core cell cycle protein Cyclin B1 (CycB) is expressed in spermatocytes, but the protein does not accumulate in spermatocytes until just before the meiotic divisions. This study shows that two interacting proteins, Rbp4 and Fest, expressed at the onset of spermatocyte differentiation under control of the developmental program of male gametogenesis, function to direct cell type- and stage-specific repression of translation of the core G2/M cell cycle component cycB during the specialized cell cycle of male meiosis. Binding of Fest to Rbp4 requires a 31-amino acid region within Rbp4. Rbp4 and Fest are required for translational repression of cycB in immature spermatocytes, with Rbp4 binding sequences in a cell type-specific shortened form of the cycB 3' UTR. Finally, it was shown that Fest is required for proper execution of meiosis I (Baker, 2015).

This study shows that the developmental program of male gametogenesis imposes several levels of cell type- and stage-specific post-transcriptional control on expression of the key G2/M cell cycle regulatory component CycB during meiotic prophase and identifies two key developmentally regulated trans-acting factors involved. First, the cycB RNA expressed in spermatocytes has a short 3' UTR, only 130 nt long and missing previously identified translational regulatory sequences used in other cell types. Second, the RNA-binding protein Rbp4, expressed starting early in meiotic prophase soon after completion of pre-meiotic DNA synthesis, binds the short 3' UTR and blocks translation of cycB in immature spermatocytes. Third, the Rbp4-interacting protein Fest, also upregulated early in the spermatocyte period, is also required for blocking CycB expression in immature spermatocytes (Baker, 2015).

Rbp4 and Fest RNA and protein are expressed in very early spermatocytes prior to onset of transcription of cycB, which depends on action of the tMAC complex (White-Cooper, 1998; Beall 2007). As a result, when the cycB RNA is expressed, it arrives in a cytoplasm already primed for its proper cell type- and stage-specific translational repression. Expression of Cyclin B3 (Clb3) protein in budding yeast has been shown to be restricted to meiosis II via sequences in the CLB3 3' UTR that block translation during meiosis I. Although translational repression of CLB3 in meiosis I was important to prevent premature separation of sister chromatids, an event appropriate for meiosis II rather than meiosis I, trans-acting factors responsible for the stage-specific translational repression have yet to be identified. The current data reveal that translational repression of a cyclin (in this case CycB) is also a key feature of meiotic prophase during spermatogenesis in a metazoan animal. Surprisingly, expression of CycB in immature spermatocytes -- either in rbp4 or fest mutants or by a mutated CycB-eYFP reporter -- was insufficient to drive those cells immediately into meiotic division. This might be because action of the Cdc25 cell cycle phosphatase encoded by twine, which is also translationally repressed in immature spermatocytes and becomes translationally activated by the RNA-binding protein Boule only in mature spermatocytes, is also required to generate active Cdk1/CycB. (Baker, 2015)

One general model for Fest function invokes the possibility that Rbp4 recruits Fest to the cycB 3' UTR, where Fest is able to interfere with cycB translation. However, no compelling evidence was found of specific binding of Fest to the cycB 3' UTR in biotin pull-down experiments from testis extracts from flies expressing eYFP-Fest either with or without functional Rbp4, which suggests that either Fest is not recruited to the cycB 3' UTR, or that the biotin pull-down assay has limitations in detecting indirect RNA-protein interactions. As a result, it is important to consider other mechanisms for Fest function, including the possibility that binding of Fest to Rbp4 is needed only briefly to enact a post-translational modification of or conformational change within Rbp4 to promote its ability to recruit partners and/or repress translation. It is also technically possible that Fest and Rbp4 act in parallel pathways to regulate CycB. Finally, as the fest germ cell phenotype is dramatically stronger than that of rbp4, it is likely that Fest regulates other proteins in addition to Rbp4 (Baker, 2015)

It is not yet known how information about spermatocyte maturation is communicated to Rbp4 or Fest to allow translation of cycB in mature spermatocytes. One or more proteins could respond to input regarding cell size, given that spermatocytes grow 25-fold in volume during meiotic G2. Alternatively, given that translation of cycB in mature spermatocytes requires function of the testis TAF proteins, signals indicating the completion of the spermatocyte transcription program (not merely its onset) could trigger the reprieve from translational repression. Another possibility might be a meiotic arrest checkpoint mechanism triggered by transcriptional activity from unpaired chromatin, as seen in mammalian spermatocytes. Whatever the stimulus, it is clear that through stage-specific expression of the translational regulators Rbp4 and Fest in very early spermatocytes, the developmental program of male germ cell differentiation exerts additional layers of control over the core cell cycle machinery (Baker, 2015)

Asymmetric assembly of centromeres epigenetically regulates stem cell fate

Centromeres are epigenetically defined by CENP-A-containing chromatin and are essential for cell division. Previous studies suggest asymmetric inheritance of centromeric proteins upon stem cell division; however, the mechanism and implications of selective chromosome segregation remain unexplored. This study shows that Drosophila female germline stem cells (GSCs) and neuroblasts assemble centromeres after replication and before segregation. Specifically, CENP-A deposition is promoted by CYCLIN A, while excessive CENP-A deposition is prevented by CYCLIN B, through the HASPIN kinase. Furthermore, chromosomes inherited by GSCs incorporate more CENP-A, making stronger kinetochores that capture more spindle microtubules and bias segregation. Importantly, symmetric incorporation of CENP-A on sister chromatids via HASPIN knockdown or overexpression of CENP-A, either alone or together with its assembly factor CAL1, drives stem cell self-renewal. Finally, continued CENP-A assembly in differentiated cells is nonessential for egg development. This work shows that centromere assembly epigenetically drives GSC maintenance and occurs before oocyte meiosis (Dattoli, 2020).

Stem cells are fundamental for the generation of all tissues during embryogenesis and replace lost or damaged cells throughout the life of an organism. At division, stem cells generate two cells with distinct fates: (1) a cell that is an exact copy of its precursor, maintaining the 'stemness,' and (2) a daughter cell that will subsequently differentiate. Epigenetic mechanisms, heritable chemical modifications of the DNA/nucleosome that do not alter the primary genomic nucleotide sequence, regulate the process of self-renewal and differentiation of stem cells. In Drosophila male germline stem cells (GSCs), before division, phosphorylation at threonine 3 of histone H3 (H3T3P) preferentially associates with chromosomes that are inherited by the future stem cell (Xie, 2015). Furthermore, centromeric proteins seem to be asymmetrically distributed between stem and daughter cells in the Drosophila intestine and germline. These findings support the 'silent sister hypothesis', according to which epigenetic variations differentially mark sister chromatids driving selective chromosome segregation during stem cell mitosis. Centromeres, the primary constriction of chromosomes, are crucial for cell division, providing the chromatin surface where the kinetochore assembles. In turn, the kinetochore ensures the correct attachment of spindle microtubules and faithful chromosome partition into the two daughter cells upon division. Centromeric chromatin contains different kinds of DNA repeats (satellite and centromeric retrotransposons) wrapped around nucleosomes containing the histone H3 variant centromere protein A (CENP-A). Centromeres are not specified by a particular DNA sequence. Rather, they are specified epigenetically by CENP-A. Centromere assembly, classically measured as CENP-A deposition to generate centromeric nucleosomes, occurs at the end of mitosis (between telophase and G1) in humans. Additional cell cycle timings for centromere assembly have been reported in flies. Interestingly, Drosophila spermatocytes and starfish oocytes are the only cells known to date to assemble centromeres before chromosome segregation, during prophase of meiosis I. These examples show that centromere assembly dynamics can differ among metazoans and also among different cell types in the same organism (Dattoli, 2020).

A key player in centromere assembly in vertebrates is HJURP (holliday junction recognition protein), which localizes at centromeres during the cell cycle window of CENP-A deposition. Furthermore, centromere assembly is regulated by the cell cycle machinery. In flies, deposition of CID (the homologue of CENP-A) requires activation of the anaphase promoting complex/cyclosome (APC/C) and degradation of CYCLIN A (CYCA). In humans, centromere assembly is antagonized by Cdk1 activity, while the kinase Plk1 promotes assembly. Additionally, the CYCLIN B (CYCB)/Cdk1 complex inhibits the binding of CENP-A to HJURP, preventing CENP-A loading at centromeres. To date, little is known about centromere assembly dynamics and functions in stem cell asymmetric divisions. Drosophila melanogaster ovaries provide an excellent model to study stem cells in their native niche. In this tissue, germline stem cells (GSCs) are easily accessible and can be manipulated genetically. Moreover, centromere assembly mechanisms in GSCs and their differentiated cells, cystoblasts (CBs), could be used to epigenetically discriminate between these two cell types. In Drosophila, CID binds to CAL1 (fly functional homologue of HJURP) in a prenucleosomal complex, and its localization to centromeres requires CAL1 and CENP-C (Dattoli, 2020).

This study investigated the dynamics of CENP-A deposition in Drosophila GSCs. GSC centromeres are assembled after replication, but before chromosome segregation, with neural stem cells following the same trend. Centromere assembly in GSCs is tightly linked to the G2/M transition. Indeed, CYCA localizes at centromeres, and its knockdown is responsible for a marked reduction of centromeric CID and CENP-C, but not CAL1. Surprisingly, excessive CID deposition is prevented by CYCB, through the kinase HASPIN. Superresolution microscopy analysis of GSCs at prometaphase and metaphase shows that CID incorporation on sister chromatids occurs asymmetrically, and chromosomes that will be inherited by the stem cell are loaded with more CID. Moreover, GSC chromosomes make stronger kinetochores, which anchor more spindle fibers. This asymmetric distribution of CID between GSC and CB is maintained also at later stages of the cell cycle, while it is not observed in differentiated cells outside of the niche. This study also found that the depletion of CAL1 at centromeres blocks GSC proliferation and differentiation. Notably, overexpression of both CID and CAL1, as well as HASPIN knockdown, promotes stem cell self-renewal and disrupts the asymmetric inheritance of CID. Conversely, overexpression of CAL1 causes GSC-like tumors. Finally, CAL1 and CID knockdown at later stages of egg development have no obvious effect on cell division, suggesting that these cells inherit CID from GSCs. Taken together, these findings establish centromere assembly as a new epigenetic pathway that regulates stem cell fate (Dattoli, 2020).

In this study a detailed characterization of centromere dynamics was performed throughout the cell cycle in Drosophila female GSCs. This analysis reveals that GSCs initiate CID incorporation after replication and that its deposition continues until at least prophase. Drosophila neural stem cells follow the same trend. Notably, this timing is different from existing studies in other metazoans. It was also found that CYCA, CYCB, and HASPIN are critically involved in CID (and CENP-C) loading at centromeres. According to the model, CYCA promotes centromere assembly, while CYCB prevents excessive deposition of CID, through the HASPIN kinase. Moreover, chromosomes that will be inherited by GSCs are labeled with a higher amount of CID and capture more spindle microtubules. Importantly, this study shows that overexpression of CAL1 and CID together, as well as HASPIN knockdown, promotes stem cell self-renewal, disrupting the asymmetric inheritance of CID. Depletion of CAL1 in stem cells blocks cell division, while CAL1 overexpression causes GSC-like tumors, highlighting its crucial role in cell proliferation. Three main points of discussion are raised: (1) the biological significance of centromere assembly in G2-M phase; (2) CAL1 is a cell proliferation marker; and (3) CID incorporation into centromeric chromatin occurs before meiosis (Dattoli, 2020).

According to the data, CID deposition occupies a wide window of time from after replication and early G2 phase to prophase. The assembly of GSC centromeres during the G2/M transition could be due to the contraction of the G1 phase, a characteristic of stem cells. Yet, in fly embryonic divisions, G1 phase is missing, and instead CID loading occurs at anaphase. Therefore, G2/M assembly might be a unique property of stem cells. This timing is also similar to the one found for Drosophila spermatocytes, which assemble centromeres in prophase of meiosis I. These spermatocytes undergo an arrest in prophase I for days, indicating a gradual loading of CID over a long period of time. Intriguingly, a similar phenomenon has been recently observed in G0-arrested human tissue culture cells and starfish oocytes. Given that GSCs are mostly in G2 phase, Drosophila stem cells might show similar properties to quiescent cells. According to the most recent models, there is a dual mechanism for CENP-A deposition: (a) a rapid pulse during G1 in mitotically dividing cells; and (b) a slow but constant CENP-A deposition in nondividing cells to actively maintain centromeres. Indeed, while previous studies in Drosophila NBs show a rapid pulse of CENP-A incorporation at telophase/G1, the majority of the loading could occur between G2 and prophase. The new results also support this model (Dattoli, 2020).

Incorporation of CID before chromosome segregation might reflect a different CYCLIN-CDK activity in these cells. For instance, it has been already shown that in Drosophila GSCs CYCLIN E, a canonical G1/S cyclin, exists in its active form (in combination with Cdk2) throughout the cell cycle, indicating that some of the biological process commonly occurring in G1 phase might actually take place in G2 phase. This is in line with the current functional findings, where depletion of CYCA causes a decreased efficiency in CID and CENP-C assembly. This study also found that this loss might be independent from CAL1. Surprisingly, correct CID deposition in GSCs also requires CYCB and HASPIN. Indeed, an inhibitory mechanism for CID deposition through CYCB has already been proposed in mammals (Stankovic, 2017). Interestingly, in Drosophila male GSCs, centromeric CAL1 is reduced between G2 and prometaphase (Ranjan, 2019), further suggesting a role for additional regulators of CID assembly, such as CYCA/B or HASPIN, at this time (Dattoli, 2020).

According to the current results, asymmetric cell division of GSCs is epigenetically regulated by differential amounts of centromeric proteins deposited at sister chromatids, which in turn can influence the attachment of spindle microtubules and can ultimately bias chromosome segregation. It is interesting to speculate on the temporal sequence of these events. Two scenarios can be proposed: (a) the nucleation of microtubules from the GSC centrosome requires bigger kinetochores; or (b) bigger kinetochores require a higher amount of spindle fibers to attach. The current results together with recent studies support the latter scenario. In fact, in Drosophila male GSCs, asymmetric distribution of centromeric proteins is established before microtubule attachment. Furthermore, microtubule disruption leaves asymmetric loading of CID intact, while it disrupts the asymmetric segregation of sister chromatids. The current data confirm this model, symmetric segregation of CID was observed upon HASPIN knockdown. Indeed, in vertebrates HASPIN knockdown causes spindle defects. Specifically, it was observed that a 1.2-fold difference in CID and CENP-C levels between GSC and CB chromosomes can bias segregation. While this difference is small, it fits with the observation that small changes in CENP-A level (on the order of 2-10% per day) impact on centromere functionality in the long run. In Drosophila male GSCs, an asymmetric distribution of CID on sister chromatids >1.4-fold was reported. This higher value might reflect distinct systems in males and females or the quantitation methods used. Importantly, CID asymmetry in males is established in G2/prophase, in line with the time window this study defines for CID assembly. Further support for unexpected CID loading dynamics comes from the finding that GSCs in G2/prophase contain ~30% more CID on average compared with S phase, indicating that CID is not replenished to 100% each cell cycle. Interestingly, the time course of H3T3P appearance during the GSC cell cycle closely follows the timing of CID incorporation, suggesting that the asymmetric deposition of CID might drive the differential phosphorylation of the histone H3 on sister chromatids. Finally, the results are in line with findings that the long-term retention of CENP-A in mouse oocytes has a role in establishing asymmetric centromere inheritance in meiosis (Dattoli, 2020).

These functional studies support a role for CAL1 in cell proliferation, with no apparent role in asymmetric cell division. Indeed, centromeric proteins have been already proposed as biomarkers for cell proliferation. Specifically, functional analysis of centromeric proteins, as well as the HASPIN kinase, allowed discrimination between the classic role of centromeres in cell division and a role in asymmetric cell division. In a favorite scenario, CAL1 is needed to make functional centromeres crucial for cell division, while the asymmetric distribution of CID sister chromatids regulates asymmetric cell division and might depend on other factors, such as HASPIN. However, it cannot be rule out that the effects on cell fate observed with the functional analysis might reflect alternative CAL1 functions outside of the centromere, for example due to changes in chromosome structure or gene expression (Dattoli, 2020).

Centromeres are crucially assembled in GSCs and therefore before meiosis of the oocyte takes place. Thus, it is possible that the 16-cell cysts inherit centromeric proteins synthesized and deposited in the GSCs, and the rate of new CID loading is reduced. This would explain why CAL1 function at centromeres is dispensable at this developmental stage (Dattoli, 2020).

Ultimately, the results provide the first functional evidence that centromeres have a role in the epigenetic pathway that specifies stem cell identity. Furthermore, these data support the silent sister hypothesis (Lansdorp, 2007), according to which centromeres can drive asymmetric division in stem cells (Dattoli, 2020).

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 -123 or more

Bases in 3' UTR - 776

PROTEIN STRUCTURE

Amino Acids - 530

Structural Domains

Within a central region spanning 206 amino acid residues, the Drosophila Cyclins A and B share 35% identity, whereas clam Cyclin A and Drosophila Cyclin A share 53% identity as do clam Cyclin B and Drosophila Cyclin B. In particular, the Drosophila Cyclin B sequence contains a consensus cAMP-dependent protein kinase site, a feature common to all other Cyclin B sequences (Whitfield, 1990).

date revised: 21 June 2024 

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