Gene name - fizzy
Cytological map position-35F1-35F1
Function - signaling
Symbol - fzy
FlyBase ID: FBgn0001086
Genetic map position - 2L
Classification - WD40 domain protein
Cellular location - cytoplasmic
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 APCCort cooperates with the more general APCFzy, 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 APCFzy 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 APCFzy 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, APCFzy, 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 APCFzy and a meiosis-specific complex (APCAma1 in S. cerevisiae and APCMfr1 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 APCFzy complex (Patra, 1998). The current results suggest that Cks30A may have a similar role in stimulating both the APCFzy and APCCort 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 APCFzy 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 APCFzy becomes essential when Fzy activity is compromised (Swan, 2007).
Although Cks30A appears to promote the activity of the APCCort and the APCFzy, 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 APCCort and APCFzy 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 APCFzy or APCCort 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 APCCort 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, APCFzy and APCCort, cooperate to mediate the destruction of meiotic cyclins and allow progression through female meiosis (Swan, 2007).
Computer searches of protein sequence databases using the BLASTP program revealed homology between fzy and many other proteins all of which contained WD-40 repeat domains. The best match is between Fzy and p55CDC, a mammalian gene that appears to be a cell cycle component, overall Fzy and p55CDC are 50% identical and 59% similar. The second best match is between Fzy and the Saccharomyces cerevisiae cell cycle gene product Cdc20, overall they are 32% identical and 46% similar. Although the homology between Fzy, p55CDC and Cdc20 is most striking within the WD-40 repeat region of these proteins, they also show blocks of conservation outside this region. Moreover, the extent of homology between the different WD-40 repeats of Fzy, p55CDC and Cdc20 correlated with their position within these proteins: i.e., the first repeat of Fzy is most homologous to the first repeat of p55CDC and Cdc20, the second with the second and so on. These comparisons suggest that Fzy, p55CDC and CDC20 may represent a family of orthologous genes, within the WD-40 repeat superfamily, which are involved in cell cycle regulation (Dawson, 1995).
To test if fzy is functionally as well as structurally homologous to CDC20, the fzy coding sequence from cDNA fzym5 was placed in a yeast expression vector and transformed into the temperature-sensitive cdc20-1 strain. However, it was not possible to rescue the temperature sensitive lethality of the cdc20-1 mutant with this fzy construct even though it is known to be expressed, and produces full-length fzy protein. Moreover, wild-type CDC20 expressed from the same vector under the same conditions will rescue (Dawson, 1995).
date revised: 20 May 2007
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