InteractiveFly: GeneBrief

cortex: Biological Overview | References

Meiosis is a highly specialized cell division that requires significant reorganization of the canonical cell-cycle machinery and the use of meiosis-specific cell-cycle regulators. The Anaphase-promoting complex (APC, a machine for degrading proteins; see APC subunits Cdc27 and morula; for review see Acquaviva, 2006) and a conserved APC adaptor/activator, Cdc20 (also known as Fizzy), are required for anaphase progression in mitotic cells. The APC has also been implicated in meiosis, although it is not yet understood how it mediates these non-canonical divisions. Cortex (Cort) is a diverged Fzy homologue that is expressed in the female germline of Drosophila, where it functions with the Cdk1-interacting protein Cks30A to drive anaphase in meiosis II. This study shows that Cort functions together with the canonical mitotic APC adaptor Fzy to target the three mitotic cyclins (A, B and B3) for destruction in the egg and drive anaphase progression in both meiotic divisions. In addition to controlling cyclin destruction globally in the egg, Cort and Fzy appear to both be required for the local destruction of cyclin B on spindles. Cyclin B associates with spindle microtubules throughout meiosis I and meiosis II, and dissociates from the meiotic spindle in anaphase II. Fzy and Cort are required for this loss of cyclin B from the meiotic spindle. These results lead to a model in which the germline-specific APC^Cort cooperates with the more general APC^Fzy, both locally on the meiotic spindle and globally in the egg cytoplasm, to target cyclins for destruction and drive progression through the two meiotic divisions (Swan, 2007).

The cell divisions of female meiosis and the ensuing mitotic cycles of early embryogenesis represent two examples of non-canonical cell cycles. Meiosis differs from the typical mitotic cycle in several respects. Most notably, two divisions occur in sequence without an intervening S-phase, resulting in the production of four haploid gametes. Additionally, the first meiotic division involves the segregation of homologous chromosomes and occurs without sister chromatid segregation, whereas the second meiotic division involves the segregation of sister chromatids, as occurs in mitosis. The regulation of meiosis requires a significant reorganization of the canonical cell-cycle machinery and the use of a number of meiosis-specific cell-cycle regulators. One example is in the regulation of anaphase - the coordinated series of events that results in the segregation of chromosomes to produce two daughter nuclei. In mitotically dividing cells, anaphase progression crucially depends on the inactivation of the mitotic kinase Cdk1 (also known as Cdc2) and on the subsequent release of sister chromatid cohesion through the destruction of cohesin complexes. These events are controlled by an E3 ubiquitin ligase -- the anaphase-promoting complex (APC) -- in association with an adaptor protein, Fzy, and this complex targets mitotic cyclins and securin (potential Drosophila homolog; Pimples) for destruction (reviewed in Peters, 2002). The role of the APC in meiosis appears to be more complex than in mitotic cells. For example, the APC only partially inhibits Cdk1 activity between meiotic divisions and sister chromatid cohesion persists at centromeres through anaphase I. It is not yet clear how the activity of the APC is modified in these specialized cell divisions (Swan, 2007).

In most eukaryotes, the meiotic cell cycle is followed by another atypical cell cycle -- the cleavage divisions of early embryogenesis. In Drosophila, these cleavage cycles occur as a series of synchronized, rapid nuclear divisions and are referred to as syncytial divisions. The female meiotic cell cycle is not only closely linked to the syncytial mitotic cell cycle in time, but it also occurs within a shared cytoplasm -- that of the egg. Therefore, these two distinct cell cycles share a common pool of cell-cycle regulators, and may share common strategies for spatially and temporally regulating cell-cycle progression within a syncytium (Swan, 2007).

One way in which the syncytial cell cycle is modified appears to be in the limited destruction of mitotic cyclins in each cell cycle, apparently by restricting their destruction to the area of the mitotic nuclei. Although there is evidence that cyclin destruction is spatially regulated in somatic cells, this strategy appears to be of particular importance in the syncytial embryo of Drosophila as a means to conserve mitotic cyclins for the duration of the rapid syncytial divisions. Several lines of evidence suggest that at least one cyclin, cyclin B, undergoes limited local destruction on mitotic spindles in the syncytial embryo. It is not yet known what mediates this local cyclin B destruction, and it is also not known whether this is unique to the syncytial mitotic cell cycle or if it occurs in the preceding meiotic divisions (Swan, 2007).

Drosophila represents an excellent model system for understanding how the canonical cell-cycle machinery is developmentally modified, and how novel cell-cycle regulators are used to control meiosis and syncytial divisions. cortex (cort) encodes a Cdc20/Cdh1 (Cdh1 is also known as Fzr and Rap)-related protein, that appears to be required specifically in female meiosis (Chu, 2001; Lieberfarb, 1996; Page, 1996) and functions with a germline-specific Cks gene, Cks30A, to mediate the destruction of cyclin A (Swan, 2005a; Swan, 2005b). This study shows that the canonical APC adaptor Fzy functions together with Cort to target mitotic cyclins for destruction, and to drive anaphase in both meiosis I and meiosis II. Female meiosis, like the subsequent syncytial mitotic cell cycles, appears to involve the local destruction of cyclin B, and both Cort and Fzy were found to be required for this process (Swan, 2007).

In most cell types, in both Drosophila and in other metazoans, the APC^Fzy drives anaphase progression by targeting mitotic cyclins and other mitotic proteins for destruction. This study shows that the female germline is an exception in that the APC^Fzy is not sufficient. A germline-specific APC adaptor, Cort, cooperates with Fzy to mediate cyclin destruction in meiosis (Swan, 2007).

The cort gene encodes a diverged member of the Fzy/Cdh1 family (Chu, 2001). Fzy/Cdh1 homologues interact with the APC and with specific sequences (D-box, KEN box or A-box) found on cyclins and on other APC targets. As such, Fzy/Cdh1 proteins act as specificity factors to target proteins for ubiquitination and eventual destruction. Cort protein, like all Fzy/Cdh1-family proteins, contains seven WD domains in the C-terminal-half of the protein, implicated in substrate recognition (Pfleger, 2001). Cort has an N-terminal C-box (amino acids 482, 483) and a C-terminal IR tail (amino acids 54-60), both implicated in binding to the APC. In addition to containing these conserved functional domains, Cort displays a conserved ability to mediate cyclin destruction. cort mutations result in the overaccumulation of cyclin A, cyclin B and cyclin B3 in the egg (Swan, 2005a), whereas the ectopic expression of Cort in the wing disc leads to a reduction in the levels of these mitotic cyclins. Taken together, these results indicate that Cortex encodes a functional member of the Fzy/Cdh1 family (Swan, 2007).

Although the Drosophila genome has four genes that encode Fzy/Cdh1 proteins, only two of these proteins, Fzy and Cort, are expressed at detectable levels in the female germline (Raff, 2002; Jacobs et al., 2002; Chu, 2001). The role of these two APC adaptors has been studied both individually and in double mutants, and it was found that they function together to promote anaphase in both the first and second meiotic divisions of female meiosis. In most cell types in Drosophila and other eukaryotes, a single APC complex, APC^Fzy, is responsible for cyclin destruction and anaphase progression. It is therefore surprising that, in the female germline of Drosophila, two APC adaptors are necessary for meiotic progression. In the case of meiosis I, Cort and Fzy appear to play largely redundant roles, since only removing both genes results in a significant block in meiosis I. The two APC complexes may also be functionally redundant with respect to global cyclin levels. Mutations in either fzy or cort result in an increase in the levels of cyclin A, cyclin B and cyclin B3, whereas mutation in both genes results in even-further increases in cyclin levels (Swan, 2007).

Although Cort and Fzy have overlapping roles in promoting anaphase I, both are essential for meiosis II. This could simply reflect a greater quantitative requirement for APC activity in meiosis II. Alternatively, the two APC complexes could have distinct roles in the second meiotic division. Consistent with this latter possibility, mutations in either cort or fzy both result in arrest at different stages of meiosis II: cort mutants arrest with the sister chromatids associated, and therefore in metaphase, whereas fzy mutants almost invariably arrest with separated sister chromatids, and are therefore in anaphase. cort and fzy also result in different patterns of cyclin B stabilization on the arrested spindles, suggesting roles in metaphase and anaphase, respectively. Therefore, Cort may function to initiate sister chromatid separation at the onset of anaphase II and Fzy may primarily function later, in anaphase II. Alternatively, the later arrest observed in fzy could simply reflect the fact that the fzy alleles that have been used are not nulls, and it is possible that a complete loss of Fzy activity would also result in a metaphase arrest, as seen in cort. However, comparing the meiosis II phenotypes of fzy with Cks30A-null mutants suggests that the later arrest in fzy is not simply due to residual activity. Cks30A-null mutants have a weaker meiotic arrest than fzy; they complete meiosis at high frequency (Swan, 2005a), but they display a higher frequency of metaphase arrest or delay. The fact that fzy does not similarly cause a delay in metaphase of meiosis II suggests that it is only required at anaphase. Therefore, it is possible that Fzy is crucial at anaphase, whereas Cort is necessary for the metaphase to anaphase transition (Swan, 2007).

The different temporal requirements for Cort and Fzy prior to and after sister chromatid separation, respectively, could be related to their apparent differences in substrate specificity. Western analysis reveals that Cort is more important for the destruction of cyclin A and cyclin B3, whereas Fzy appears to play a greater role in cyclin B destruction in the egg. In mitotic cells, cyclin destruction occurs sequentially. Cyclin A is destroyed first, in prometaphase, and this is a prerequisite for sister chromatid separation. Cyclin B destruction occurs at anaphase onset and is necessary for later anaphase events, subsequent to sister chromatid separation. Therefore, it is possible that Cort promotes the early stages of meiotic anaphase by targeting cyclin A for destruction, whereas Fzy is more crucial later, through its targeting of cyclin B for destruction (Swan, 2007).

The meiotic cell cycle differs in many respects from the standard mitotic cycle. Whereas APC-mediated destruction of mitotic regulators appears to be required for anaphase progression in most or all mitotic cells, the role of the APC and cyclin destruction in meiosis is not as well-understood. This analysis of the two APC adaptors Cort and Fzy has permitted an evaluation of the role of the APC complex in female meiosis in Drosophila. The APC is required for anaphase progression in both meiotic divisions. Correlating with its requirement for the completion of meiosis, the APC is required for the destruction of mitotic cyclins. At least one of these cyclins, cyclin B, is a crucial substrate in meiosis, because the expression of a stabilized form of cyclin B disrupts this process. Therefore, APC activity and cyclin destruction are required for anaphase progression in both meiotic divisions, in addition to in mitosis. APC activity has been implicated in both meiotic divisions in C. elegans and in the mouse, and in the second, but not the first, meiotic division in Xenopus. In yeast, two APC complexes, the mitotic APC^Fzy and a meiosis-specific complex (APC^Ama1 in S. cerevisiae and APC^Mfr1 in S. pombe) function together to mediate protein destruction in meiosis. It now appears that Drosophila also uses two APC complexes in female meiosis, and this may turn out to be a common strategy in other eukaryotes (Swan, 2007).

Cks30A belongs to a highly conserved family of proteins that bind to and stimulate the activity of the mitotic kinase Cdk1. In Xenopus, the Cks30A homologue Xep9 stimulates the Cdk-dependent phosphorylation of APC subunits, and thereby promotes the activation of the APC^Fzy complex (Patra, 1998). The current results suggest that Cks30A may have a similar role in stimulating both the APC^Fzy and APC^Cort in female meiosis in Drosophila. (1) Cks30A, like cort and fzy, is required for the completion of meiosis II and, like fzy, it is required for the completion of the first mitotic division of embryogenesis (Lieberfarb, 1996; Page, 1996; Swan, 2005). (2) Cks30A, as are Cort and Fzy, is necessary for global cyclin destruction in the Drosophila egg and for local cyclin B destruction on the meiotic spindle. Global levels of cyclin A and cyclin B3 are elevated to a greater extent in Cks30A mutants than in single mutants for cort or fzy, consistent with the idea of Cks30A activating both Cort and Fzy. (3) Cks30A is necessary for the activity of ectopically expressed Cort in the adult wing. Cks30A may also play a role in activating APC^Fzy in mitotic cells. the temperature-sensitive fzy⁶ allele is lethal at all temperatures in a Cks30A-null background, suggesting that the Cks30A-dependent activation of APC^Fzy becomes essential when Fzy activity is compromised (Swan, 2007).

Although Cks30A appears to promote the activity of the APC^Cort and the APC^Fzy, these complexes seems to retain some activity in the absence of Cks30A. Whereas cort and fzy cause an arrest in meiosis II, Cks30A-null mutants are typically delayed only in meiosis II (Swan, 2005a). Also, although cyclin A and cyclin B3 levels are elevated more in Cks30A eggs than in either fzy or cort, their levels are still not as high as in fzy; cort double mutants, indicating that Fzy and Cort can destroy cyclin A and cyclin B3 to some degree in the absence of Cks30A. Cyclin B destruction is even less dependent on Cks30A, because cyclin B levels are affected less in Cks30A mutants than in either cort or fzy single mutants. Therefore, Cks30A may be more crucial for the activity of APC^Cort and APC^Fzy complexes on cyclin A and cyclin B3, and less crucial for their activity on cyclin B. The relatively weaker meiotic arrest in Cks30A mutants compared to fzy; cort double mutants may also indicate that the APC has other meiotic targets that can be destroyed in the absence of Cks30A (Swan, 2007).

Cyclin B undergoes local oscillations in its association with mitotic spindles in syncytial embryos, appearing transiently along the full length of the mitotic spindle in early metaphase and gradually disappearing from the spindle starting at the centrosomes and ending at the kinetochores. The timing of this loss of cyclin B from the spindle, at the onset of anaphase, corresponds with the timing of cyclin B destruction in other cell types, suggesting the possibility that cyclin B is locally destroyed on the spindle in anaphase. This study shows that cyclin B is subject to similar local oscillations in the female meiotic cycles, and that cyclin B destruction is necessary for the completion of female meiosis. Importantly, the local loss of cyclin B from the spindle in meiosis is dependent on the APC adaptors Cort and Fzy, and that the local loss of cyclin B from the spindle in mitosis depends on Fzy. These results strongly suggest that the local loss of cyclin B from the spindle in anaphase of meiosis II and anaphase of mitosis is actually due to its local destruction (Swan, 2007).

The pattern of accumulation and loss of cyclin B from the spindle in meiosis differs in some respects compared to syncytial mitotic cycles. (1) In metaphase of mitosis, cyclin B initially accumulates throughout the spindle microtubules, whereas, in metaphase of the meiotic divisions, cyclin B first appears exclusively at the spindle mid-zone. This difference may reflect the fact that the meiotic spindle does not contain centrosomes and cyclin B may, therefore, not load onto spindles from centrosomes and progress along the spindles to the kinetochores, as has been proposed for mitosis. (2) The timing of cyclin B destruction appears to be different between the meiotic and mitotic cycles. Most strikingly, there is no loss of cyclin B from the spindle in anaphase of meiosis I, implying that local cyclin B destruction is not necessary for the completion of the first meiotic division. In addition, the loss of cyclin B from the spindle following meiosis II occurs only late in anaphase rather than at the onset of anaphase, as occurs in the syncytial mitotic cycles. It is not yet known how cyclin B destruction is prevented in anaphase I and early in anaphase of meiosis II. One possibility is that the spindle-assembly checkpoint is locally active during these stages. This checkpoint is required for the proper completion of female meiosis in Drosophila, and it will be interesting to see if this requirement reflects a role in inhibiting either APC^Fzy or APC^Cort activity (Swan, 2007).

The specific accumulation of cyclin B at the spindle mid-zone in meiosis may reflect the unique properties of the meiotic spindle. The mid-zone microtubules or central spindle microtubules are a subset of spindle microtubules that do not end in kinetochores, but instead overlap at the mid-zone with microtubules from the other pole. In dividing cells, the central spindle is crucial for cytokinesis, but, in female meiosis, it appears to have a role in spindle assembly. Along with cyclin B, the chromosomal passenger proteins Aurora B and Incenp are recruited to the spindle mid-zone. It will be of great interest to determine what these proteins do at the mid-zone and how cyclin B destruction at this site may be important for anaphase in meiosis. It will also be important to determine how the APC^Cort targets cyclin B at the spindle mid-zone. It has not been possible to detect any specific localization of GFP or HA-tagged Cortex in meiosis or in the syncytial embryo, but it is possible that its activity is spatially regulated (Swan, 2007).

In conclusion, these results support a model in which two APC complexes, APC^Fzy and APC^Cort, cooperate to mediate the destruction of meiotic cyclins and allow progression through female meiosis (Swan, 2007).

Developmental role and regulation of cortex, a meiosis-specific anaphase-promoting complex/cyclosome activator

During oogenesis in metazoans, the meiotic divisions must be coordinated with development of the oocyte to ensure successful fertilization and subsequent embryogenesis. The ways in which the mitotic machinery is specialized for meiosis are not fully understood. cortex, which encodes a putative female meiosis-specific anaphase-promoting complex/cyclosome (APC/C) activator, is required for proper meiosis in Drosophila. Cort physically associates with core subunits of the APC/C in ovaries. APC/C(Cort) targets Cyclin A for degradation prior to the metaphase I arrest, while Cyclins B and B3 are not targeted until after egg activation. The regulation of Cort was investigated and it was found that Cort protein is specifically expressed during the meiotic divisions in the oocyte. Polyadenylation of cort mRNA is correlated with appearance of Cort protein at oocyte maturation, while deadenylation of cort mRNA occurs in the early embryo. Cort protein is targeted for degradation by the APC/C following egg activation, and this degradation is dependent on an intact D-box in the C terminus of CORT. These studies reveal the mechanism for developmental regulation of an APC/C activator and suggest it is one strategy for control of the female meiotic cell cycle in a multicellular organism (Pesin, 2007; full text of article).

This study demonstrates a physical interaction between Cort and the APC/C strengthens and confirms previous suggestions that cort encodes a functional meiosis-specific APC/C activator. A strong metaphase II arrest phenotype in cort mutant eggs and distant homology to the Cdc20/FZY protein family initially suggested that Cort might function as an APC/C activator (Chu, 2001; Page, 1996). cort has been shown to negatively regulate levels of mitotic cyclin proteins, which is consistent with a role for Cort in activating the APC/C (Swan, 2007). However, biochemical evidence linking Cort to the APC/C in vivo is crucial for this argument. This study has shown that Cort physically associates with core subunits of the APC/C in ovaries, strongly supporting Cort's role as an APC/C activator (Pesin, 2007).

Coordination of the meiotic divisions with oogenesis and the transition from meiosis to restart of the mitotic cell cycle in embryogenesis present unique regulatory challenges for the organism. This study of cortex in Drosophila suggests that developmental control of levels of a meiosis-specific APC/C activator is one way in which meiosis is developmentally regulated. This strategy exploits ongoing regulatory mechanisms occurring during meiosis and embryogenesis: cytoplasmic polyadenylation during oocyte maturation, deadenylation after egg activation, and APC/C-dependent degradation in the early embryo (Pesin, 2007).

Cytoplasmic polyadenylation upon oocyte maturation has been shown to translationally activate maternal transcripts of genes that are required for meiotic entry, transition between meiosis I and meiosis II, and metaphase II arrest in vertebrates. cort mRNA is polyadenylated at oocyte maturation; polyadenlyation adds cort to the group of transcripts that are translationally unmasked for entry into the meiotic divisions. What is the signal for polyadenylation of cort? Masked transcripts contain a cis-acting cytoplasmic polyadenylation element (CPE) to which CPE binding protein (CPEB) is bound. Phosphorylation of CPEB upon oocyte maturation triggers elongation of the poly(A) tail and activation of translation. A CPE has not been identified in the 3' UTR of cort, although CPE sequences are quite variable. In addition, no dependence of cort polyadenylation on orb, the CPEB homolog in Drosophila has been identified. Because the orb alleles that were used are hypomorphic, the possibility that polyadenylation of cort is orb/CPEB-dependent cannot be ruled out (Pesin, 2007).

Egg activation triggers maternal transcript destabilization in several organisms, some of which occurs through ccr4-dependent deadenylation, and this is likely to be important for localization of maternal transcripts in the embryo and proper zygotic development (Tadros, 2005). This study shows that cort mRNA is deadenylated in the early embryo in a ccr4-dependent manner, but this deadenylation is not required for lowering Cort protein levels. However, it might not be possible to detect a difference in protein levels because of the rapid APC/C-dependent degradation of Cort protein that occurs after release of the metaphase I arrest. Deadenylation could serve as a backup mechanism to ensure that Cort protein levels remain low in the early embryo by destabilizing cort mRNA (Pesin, 2007).

The APC/C drives degradation of Cyclin B and other substrates during the rapid syncytial mitotic divisions of early embryogenesis in Drosophila. This study found that Cort is targeted for APC/C-dependent degradation by the completion of meiosis in the early embryo. The targeting of an APC/C activator for degradation by another form of APC/C is not unprecedented; APC/C^Cdh1 targets Cdc20 for degradation in G1 (Pesin, 2007).

These data support the conclusion that Cort is targeted by APC/C^FZY: (1) FZY is thought to be the only activator present in early embryos; (2) this study shows that cort and fzy interact genetically in a way that is consistent with cort being a negative downstream target of fzy in embryos; (3) in embryo injection experiments, exogenous MYC-Cort is degraded in a D-box-dependent manner in injected embryos. Because the only APC/C activator in early embryos is FZY, degradation of MYC-Cort is likely to occur through APC/C^FZY in this assay (Pesin, 2007).

It is also possible the APC/C^Cort regulates itself in a negative feedback loop by targeting Cort for degradation when levels of Cort reach a certain threshold at the end of meiosis. To address this possibility, the degradation of Cort was examined in a homozygous cort^QW55 background in which there is no functional Cort protein. Cort^QW55 mutant protein is not degraded at the transition from mature stage 14 oocytes to unfertilized eggs, unlike in a heterozygous control background. These results suggest that Cort could be targeted by itself, but it remains a possibility that the lesion in the cort^QW55 allele prevents an interaction between Cort^QW55 mutant protein and the APC/C machinery. The lesion does not disrupt the D-box, but it could affect proper folding and structure of the protein. In summary, it is concluded that Cort is targeted for degradation by the APC/C. It is most likely that FZY is the participating APC/C activator, but Cort may also contribute to targeting itself for degradation (Pesin, 2007).

Recent work has shown that both cort and fzy are required for the meiotic divisions in Drosophila female meiosis. Mutant analysis suggests that cort and fzy act redundantly to control the metaphase I to anaphase I transition, whereas they seem to act with different temporal and spatial specificity in targeting Cyclin B for destruction along the meiosis II spindles. cort cannot functionally substitute for fzy in the early embryo, suggesting that they target nonredundant sets of substrates. However, the possibility that MYC-Cort was not present in sufficient levels in early embryos for rescue because of low expression levels or protein instability cannot be ruled out. Although MYC-Cort is expressed at high levels in stage 14 oocytes, it appears to be subject to degradation after the completion of meiosis, like the endogenous Cort protein (Pesin, 2007).

Furthermore, homozygous cort mutants alone exhibit a strong metaphase II arrest, indicating that the wild-type levels of fzy in this background are not able to act in place of cort to control passage through metaphase II. Finally, FZY is expressed at a uniform level during oogenesis and embryogenesis, which is in contrast to the results in this study showing that Cort expression is specifically upregulated during the meiotic divisions. On the basis of all of these observations, it is thought likely that in addition to the mitotic cyclins, APC/C^Cort targets a unique set of substrates in meiosis that are not recognized by APC/C^FZY. The identification of these meiotic substrates will be crucial for understanding how the meiotic divisions are controlled in the oocyte (Pesin, 2007).

The study of meiotic control of the APC/C is especially intriguing in Drosophila, because in addition to cort, a female meiosis-specific activator, the genome contains fizzy-related 2 (fzr2), another member of the Cdc20/FZY family. fzr2 is expressed exclusively in testes and may act as a male meiosis-specific activator. Further study of both cort and fzr2 will be important for understanding differential developmental regulation of the APC/C during meiosis in females versus males (Pesin, 2007).

In mitosis, cyclins are targeted sequentially for destruction by the APC/C. Degradation of Cyclin A begins just after nuclear envelope breakdown in prometaphase, while degradation of Cyclin B does not occur until the metaphase to anaphase. Sequential degradation of Cyclin A, Cyclin B, and, finally, Cyclin B3 in Drosophila triggers a series of distinct events leading to exit from mitosis. A similar situation exists in Drosophila female meiosis, in which degradation of Cyclin A by APC/C^Cort initiates upon nuclear envelope breakdown, but degradation of Cyclin B and Cyclin B3 does not occur until after the metaphase I to anaphase I transition (Pesin, 2007).

The difference in timing of Cyclin A and Cyclin B degradation in mitosis is due to regulation of the APC/C by the spindle assembly checkpoint. The spindle assembly checkpoint inhibits APC/C^Cdc20 from initiating anaphase until all chromosomes are bioriented on the spindle, in part through direct binding of Cdc20 to Mad2 and BubR1. Spindle assembly checkpoint proteins specifically inhibit APC/C-dependent ubiquitination of Cyclin B but not of Cyclin A. APC/C^Cort may be regulated in a similar manner during meiosis I. Indeed, the spindle assembly checkpoint is likely to function during meiosis I in Drosophila; the conserved spindle checkpoint kinase Mps1 is required for delaying entry into anaphase I to allow for proper segregation of achiasmate homologs and maintenance of chiasmate homolog connections in Drosophila oocytes. Furthermore, a functional Mad2-dependent checkpoint exists during meiosis I in mouse oocytes, and spindle checkpoint components have been shown to regulate the APC/C during meiosis I in C. elegans (Pesin, 2007).

To determine whether APC/C^Cort is regulated by the spindle checkpoint, it was asked if BubR1 or Mad2 physically associate with Cort in stage 14-enriched ovaries. No association with BubR1 or Mad2 was detected. Although this negative result does not rule out the possibility of regulation of APC/C^Cort by the spindle checkpoint, it suggests that APC/C^Cort may be subject to other types of regulation that inhibit it from targeting Cyclin B and Cyclin B3 for degradation until after the metaphase I arrest (Pesin, 2007).

In conclusion, through the investigation of cortex, a meiosis-specific APC/C activator, one way was found in which the meiotic cell cycle may be developmentally controlled during oogenesis. cort is developmentally regulated by existing post-transcriptional and post-translational mechanisms, resulting in expression of Cort protein being restricted to the meiotic divisions. Further study of APC/C^Cort will continue to elucidate the ways in which developmental control of the APC/C contributes to proper female meiosis in a metazoan (Pesin, 2007).

Phospho-regulation pathways during egg activation in Drosophila melanogaster

Egg activation is the series of events that transition a mature oocyte to an egg capable of supporting embryogenesis. Increasing evidence points toward phosphorylation as a critical regulator of these events. This study used Drosophila melanogaster to investigate the relationship between known egg activation genes and phosphorylation changes that occur upon egg activation. Using the phosphorylation states of four proteins-Giant Nuclei, Young Arrest, Spindly, and Vap-33-1-as molecular markers, this study showed that the egg activation genes sarah, CanB2, and cortex are required for the phospho-regulation of multiple proteins. This study showed that an additional egg activation gene, prage, regulates the phosphorylation state of a subset of these proteins. Finally, this study shows that Sarah and calcineurin are required for the Anaphase Promoting Complex/Cyclosome (APC/C)-dependent degradation of Cortex following egg activation. From these data, a model is presented in which Sarah, through the activation of calcineurin, positively regulates the APC/C at the time of egg activation, which leads to a change in phosphorylation state of numerous downstream proteins (Krauchunas, 2013).

A Meiosis-Specific Form of the APC/C Promotes the Oocyte-to-Embryo Transition by Decreasing Levels of the Polo Kinase Inhibitor Matrimony

Oocytes are stockpiled with proteins and mRNA that are required to drive the initial mitotic divisions of embryogenesis. But are there proteins specific to meiosis whose levels must be decreased to begin embryogenesis properly? The Drosophila protein Cortex (Cort) is a female, meiosis-specific activator of the Anaphase Promoting Complex/Cyclosome (APC/C), an E3 ubiquitin ligase. Immunoprecipitation of Cortex followed by mass spectrometry was performed, and the Polo kinase inhibitor Matrimony (Mtrm) was identified as a potential interactor with Cort. In vitro binding assays showed Mtrm and Cort can bind directly. Mtrm protein levels are reduced dramatically during the oocyte-to-embryo transition, and this downregulation does not take place in cort mutant eggs, consistent with Mtrm being a substrate of APC^Cort. Mtrm was shown to be subject to APC^Cort-mediated proteasomal degradation, and a putative APC/C recognition motif was identified in Mtrm that when mutated partially stabilized the protein in the embryo. Furthermore, overexpression of Mtrm in the early embryo caused aberrant nuclear divisions and developmental defects, and these were enhanced by decreasing levels of active Polo. These data indicate APC(Cort) ubiquitylates Mtrm at the oocyte-to-embryo transition, thus preventing excessive inhibition of Polo kinase activity due to Mtrm's presence (Whitfield, 2013).

Despite its pivotal role in development, regulation of the oocyte-to-embryo transition is poorly understood. Given the maternal stockpiles in the oocyte, mechanistic differences between meiosis and mitosis, and meiosis-specific forms of the APC/C, it is crucial to determine which proteins need to be degraded to switch correctly from meiosis to mitosis. The meiosis-specific activator Cort is essential for the transition from oocyte to embryo despite Fzy/Cdc20's presence. Cortex's existence raised the possibility that degradation of particular meiosis-specific proteins may be necessary for the onset of embryogenesis. This study shows this to be the case: the Cort form of the APC/C is required for Mtrm's destruction at the oocyte-to-embryo transition. Furthermore, reduced levels of Mtrm heading into embryogenesis are necessary for proper development, indicative of requirements for differential levels of the protein in meiosis and mitosis (Whitfield, 2013).

A requirement for reduction in levels of Mtrm is illustrated by the deleterious effects of overexpression of the protein in the embryo. A crucial role for Mtrm degradation in the transition from oocyte to embryo is supported by the observation that reduction in levels of Mtrm protein can suppress the developmental block caused by low activity of Cort. In the grau mutants, levels of Cort are reduced, and the mutant oocytes arrest in meiosis. By mutating a single copy of the mtrm gene, this arrest was overcome, the eggs progressed, and several nuclear divisions occurred (Whitfield, 2013).

Mtrm provides key insights into how protein degradation can be regulated at the oocyte-to-embryo transition. Mtrm is not completely removed from the embryo, illustrating that its protein levels are important and degradation does not have to be an all-or-none process. In this case, APC^Cort acts as a rheostat, allowing for high levels of Mtrm in meiosis and low levels in mitosis. Consistent with this, it is interesting that stabilized forms of Mtrm present at lower levels than the overexpressed wild-type form did not exhibit an embryonic phenotype. mCherry-Mtrm also is present at levels lower than endogenous Mtrm in stage 14 oocytes, and therefore may never reach high enough levels to be able to cause the developmental defects seen with the overexpressed form of Mtrm. This offers evidence for a specific threshold of Mtrm that can be tolerated in the early embryo (Whitfield, 2013).

Polo kinase is a critical regulator of both mitosis and meiosis, and is conserved from yeast to humans. polo (and its orthologs) help regulate mitotic/meiotic entry, chromosome segregation, centrosome dynamics, and cytokinesis. With such diverse roles during mitosis and meiosis, Polo function must be carefully regulated. Up-regulation of human Polo-like kinase (Plk1) is prevalent in many human cancers, and identifying potent inhibitors of Plk1 is the focus of much research. In Drosophila, without inhibition by Mtrm during prophase of meiosis I, Polo prematurely triggers nuclear envelope breakdown (through activation of the Cdc25 phosphatase) and eventually leads to chromosome nondisjunction. Mutation of polo has direct consequences on female meiotic progression as well. During Drosophila embryogenesis, expression of Scant, a hyperactive form of the Polo antagonist Greatwall kinase, leads to dissociated centrosomes from prophase nuclei. Embryos homozygous for polo¹ show a wide array of defects, including irregular DNA masses with disorganized spindles, reminiscent of the mtrm overexpression phenotype. These data illustrate the importance of Polo kinase in both mitosis and meiosis, and that improper regulation of its activity can have disastrous consequences on cell division (Whitfield, 2013).

Current evidence suggests that Mtrm regulates Polo activity during both meiosis and mitosis. The current results shed light on how the oocyte/embryo might use the same protein to regulate Polo during such drastically different cell divisions. The data indicate meiosis requires high levels of Mtrm protein/Polo inhibition, while low levels of Mtrm are needed for early embryogenesis. This is likely a mechanism to allow for fine tuning of Polo activity during the rapid divisions of the syncytial embryo (Whitfield, 2013).

The results of this study provide an interesting biological counterpoint to a recent study on the S. cerevisiae meiosis-specific APC/C activator Ama1. Previously, Ama1 had been known to act later in meiosis, regulating spore formation and Cdc20 degradation at meiosis II, It has been shown that APC^Ama1 also acts earlier in meiosis to clear out mitotic regulators (including Polo/Cdc5) during the extended meiotic prophase I. Consequently, cells lacking Ama1 exit prematurely from prophase I. It is interesting that two meiosis-specific APC/C activators have now been tied to regulation of Polo kinase. Ama1 has a direct, inhibitory effect early in meiosis, whereas Cort seemingly activates Polo indirectly through degradation of Mtrm late in meiosis (Whitfield, 2013).

Mtrm is not likely to be the only specific substrate of Cort, and it will be exciting to search for more APC^Cort substrates in the future. It will also be interesting to examine whether Cort targets continue to follow a graded versus all-or-none pattern of degradation during the oocyte-to-embryo transition. Further study of meiosis-specific APC/C activators will give valuable insight into the distinctions between meiotic and mitotic regulation and the control of the onset of embryogenesis (Whitfield, 2013).

Quantitative proteomics reveals the dynamics of protein changes during Drosophila oocyte maturation and the oocyte-to-embryo transition

The onset of development is marked by two major, posttranscriptionally controlled, events: oocyte maturation (release of the prophase I primary arrest) and egg activation (release from the secondary meiotic arrest). Using quantitative mass spectrometry, proteome remodeling has been described during Drosophila egg activation. This study describes quantitative mass spectrometry-based analysis of the changes in protein levels during Drosophila oocyte maturation. This study presents the first quantitative survey of proteome changes accompanying oocyte maturation in any organism and provides a powerful resource for identifying both key regulators and biological processes driving this critical developmental window. Muskelin, found to be up-regulated during oocyte maturation, was shown to be required for timely nurse cell nuclei clearing from mature egg chambers. Other proteins up-regulated at maturation are factors needed not only for late oogenesis but also completion of meiosis and early embryogenesis. Interestingly, the down-regulated proteins are predominantly involved in RNA processing, translation, and RNAi. Integrating datasets on the proteome changes at oocyte maturation and egg activation uncovers dynamics in proteome remodeling during the change from oocyte to embryo. Notably, 66 proteins likely act uniquely during late oogenesis, because they are up-regulated at maturation and down-regulated at activation. This study found down-regulation of this class of proteins to be mediated partially by APC/CORT, a meiosis-specific form of the E3 ligase anaphase promoting complex/cyclosome (APC/C) (Kronja, 2014).

Search PubMed for articles about Drosophila Cortex

Chu, T., Henrion, G., Haegeli, V. and Strickland, S. (2001). Cortex, a Drosophila gene required to complete oocyte meiosis, is a member of the Cdc20/fizzy protein family. Genesis 29: 141-152. PubMed ID: 11252055

Jacobs, H., Richter, D., Venkatesh, T. and Lehner, C. (2002). Completion of mitosis requires neither fzr/rap nor fzr2, a male germline-specific Drosophila Cdh1 homolog. Curr. Biol. 12: 1435-1441. PubMed ID: 12194827

Krauchunas, A. R., Sackton, K. L. and Wolfner, M. F. (2013). Phospho-regulation pathways during egg activation in Drosophila melanogaster. Genetics 195(1): 171-180. PubMed ID: 23792954

Kronja, I., Whitfield, Z. J., Yuan, B., Dzeyk, K., Kirkpatrick, J., Krijgsveld, J. and Orr-Weaver, T. L. (2014). Quantitative proteomics reveals the dynamics of protein changes during Drosophila oocyte maturation and the oocyte-to-embryo transition. Proc Natl Acad Sci U S A. PubMed ID: 25349405

Lieberfarb, M. E., Chu, T., Wreden, C., Theurkauf, W., Gergen, J. P. and Strickland, S. (1996). Mutations that perturb poly(A)-dependent maternal mRNA activation block the initiation of development. Development 122: 579-588. PubMed ID: 8625809

Page, A. W. and Orr-Weaver, T. L. (1996). The Drosophila genes grauzone and cortex are necessary for proper female meiosis. J. Cell Sci. 109: 1707-1715. PubMed ID: 8832393

Patra, D. and Dunphy, W. G. (1998). Xe-p9, a Xenopus Suc1/Cks protein, is essential for the Cdc2-dependent phosphorylation of the anaphase- Promoting complex at mitosis. Genes Dev 12: 2549-59. PubMed ID: 9716407

Pfleger, C. M. and Kirschner, M. W. (2000). The KEN box: an APC recognition signal distinct from the D box targeted by Cdh1. Genes Dev 14: 655-665. PubMed ID: 10733526

Peters, J. M. (2002). The anaphase-promoting complex: proteolysis in mitosis and beyond. Mol. Cell 9: 931-943. PubMed ID: 12049731

Pesin, J. A. and Orr-Weaver, T. L. (2007). Developmental role and regulation of cortex, a meiosis-specific anaphase-promoting complex/cyclosome activator. PLoS Genet. 3(11): e202. PubMed ID: 18020708

Raff, J. W., Jeffers, K. and Huang, J.-y. (2002). The roles of Fzy/Cdc20 and Fzr/Cdh1 in regulating the destruction of cyclin B in space and time. J. Cell Biol. 157: 1139-1149. PubMed ID: 12082076

Swan, A., Barcelo, G. and Schupbach, T. (2005a). Drosophila Cks30A interacts with Cdk1 to target Cyclin A for destruction in the female germline. Development 132: 3669-3678. PubMed ID: 16033797

Swan, A. and Schupbach, T. (2005b). Drosophila female meiosis and embryonic syncytial mitosis use specialized Cks and CDC20 proteins for cyclin destruction. Cell Cycle 4: 1332-1334. PubMed ID: 16138012

Swan, A. and Schupbach, T. (2007). The Cdc20 (Fzy)/Cdh1-related protein, Cort, cooperates with Fzy in cyclin destruction and anaphase progression in meiosis I and II in Drosophila. Development 134(5): 891-9. PubMed ID: 17251266

Tadros, W., Lipshitz, H. D. (2005). Setting the stage for development: mRNA translation and stability during oocyte maturation and egg activation in Drosophila. Dev. Dyn. 232: 593-608. PubMed ID: 15704150

Whitfield, Z. J., Chisholm, J., Hawley, R. S. and Orr-Weaver, T. L. (2013). A Meiosis-Specific Form of the APC/C Promotes the Oocyte-to-Embryo Transition by Decreasing Levels of the Polo Kinase Inhibitor Matrimony. PLoS Biol 11: e1001648. PubMed ID: 24019759

date revised: 10 December 2020

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