The initiation of DNA synthesis is thought to occur at sites bound by a heteromeric origin recognition complex (ORC). Previously, it has been shown that the level of the large subunit of Drosophila, ORC1, is modulated during cell cycle progression and that changes in ORC1 concentration alter origin utilization during development. The mechanisms underlying cell cycle-dependent degradation of ORC1 have been investigated. Signals in the non-conserved N-terminal domain of ORC1 mediate its degradation upon exit from mitosis and in G1 phase by the anaphase-promoting complex (APC) in vivo. Degradation appears to be the result of direct action of the APC, as the N-terminal domain is ubiquitylated by purified APC in vitro. This regulated proteolysis is potent, sufficient to generate a normal temporal distribution of protein even when transcription of ORC1 is driven by strong constitutive promoters. These observations suggest that in Drosophila, ORC1 regulates origin utilization much as does Cdc6 in budding yeast (Araki, 2003).
The behavior of a cyclin B-green fluorescent protein (GFP) fusion protein has been studied in living Drosophila embryos in order to study how the localization and destruction of cyclin B is regulated in space and time. The fusion protein accumulates at centrosomes in interphase, in the nucleus in prophase, on the mitotic spindle in prometaphase and on the microtubules that overlap in the middle of the spindle in metaphase. In cellularized embryos, toward the end of metaphase, the spindle-associated cyclin B-GFP disappears from the spindle in a wave that starts at the spindle poles and spreads to the spindle equator; when the cyclin B-GFP on the spindle is almost undetectable, the chromosomes enter anaphase, and any remaining cytoplasmic cyclin B-GFP then disappears over the next few minutes. The endogenous cyclin B protein appears to behave in a similar manner. These findings suggest that the inactivation of cyclin B is regulated spatially in Drosophila cells. The anaphase-promoting complex/cyclosome (APC/C) specifically interacts with microtubules in embryo extracts, but it is not confined to the spindle in mitosis, suggesting that the spatially regulated disappearance of cyclin B may reflect the spatially regulated activation of the APC/C (Huang, 1999).
To study the distribution of the APC/C in Drosophila embryos, a cDNA that encodes the Drosophila homolog of the APC/C component cdc16 (Dmcdc16) was cloned. Antibodies against the Dmcdc16 protein were prepared, as well as against the Drosophila homolog of the APC/C component cdc27 (Dmcdc27). Antibodies against two regions of the Dmcdc27 protein, and a single region of the Dmcdc16 protein were examined. In Western blots of embryo extracts, both of the antibodies raised against the Dmcdc27 protein recognize a prominent band of ~100 kDa, which is close to the predicted size (102 kDa) of the Dmcdc27 protein, as well as a number of other bands. Only the 100 kDa protein is co-precipitated with the anti-Dmcdc16 antibodies. The anti-Dmcdc16 antibodies recognize a prominent band of ~80 kDa, which is close to the predicted size (82 kDa) of the Dmcdc16 protein, as well as a band of ~58 kDa. Only the ~80 kDa protein is co-precipitated with anti-Dmcdc27 antibodies. In sucrose gradient sedimentation experiments, fractions of the Dmcdc16 and Dmcdc27 proteins appeared to co-migrate as a large complex (>19S), although a large fraction of the Dmcdc16 protein behaves as a much smaller protein. These results suggest that Dmcdc16 and Dmcdc27 are both components of the same higher molecular weight complex, presumably the Drosophila APC/C. These proteins interact biochemically with microtubules in early embryo extracts. The majority of the Dmcdc27, and a substantial fraction of Dmcdc16, interact with microtubules. The majority of cyclin B also interacts with microtubules in this type of experiment. The interaction of these proteins with microtubules appeared to be specific, as none of them associated with actin filaments polymerized in similar extracts. The anti-Dmcdc16 and anti-Dmcdc27 antibodies were then used to stain syncytial Drosophila embryos, however, they only weakly label centrosomes and microtubules in methanol-fixed embryos. A weak punctate staining is also visible in the chromosomal regions during mitosis, but the bulk of the Dmcdc16 and Dmcdc27 appears to be distributed in a punctate fashion throughout the cytoplasm at all stages of the cell cycle. Thus, the APC/C appears to be located at multiple sites within the embryo (Huang, 1999).
How might the activation of the APC/C to degrade cyclin B be regulated spatially? Recent experiments suggest that the temporal control of APC/C activation toward specific substrates depends on its association with members of the Fizzy (Fzy)/CDC20 and Fizzy-related (Fzr)/CDH1 family of proteins. In Drosophila, for example, fzy is required for the destruction of Cyclin A, Cyclin B and Cyclin B3 at around metaphase/anaphase, whereas Fzr is required for the destruction of these proteins in late mitosis/G1. Perhaps the spatial regulation of cyclin B destruction is also regulated by the association of the APC/C with members of this family of proteins: APC/C-Fzy complexes might accumulate on the spindle and ubiquitinate the spindle-associated cyclin B toward the end of metaphase, for example, while APC/C-Fzr complexes might remain in the cytoplasm and ubiquitinate the cytoplasmic cyclin B later in mitosis. In syncytial embryos, Fzr might not be present or might be inactivate, explaining why only the spindle-associated cyclin B is degraded. Although Fzy and Fzr have not been seen associated with specific organelles in Drosophila embryos, a fraction of the p55CDC protein, a Fzy/CDC20 family member, appears to be located on centrosomes and spindles in mammalian cells (Huang, 1999 and references).
If this interpretation that the disappearance of cyclin B-GFP directly reflects its degradation is correct, then the results suggest that a substantial fraction of cyclin B is being degraded while the cell is still in metaphase. This contradicts the prevailing idea that cyclin B is degraded abruptly at the metaphase-anaphase transition. However, recent experiments studying the disappearance of cyclin B-GFP fusion proteins in mammalian cells (P. Clute and J .Pines, personal communication to Huang, 1999) and in S.pombe (M.Yanagida, personal communication to Huang, 1999) have also concluded that a substantial fraction of the cyclin B-GFP disappears from cells while they are in metaphase, and this disappearance also appears to be regulated spatially in these systems. While the spatially regulated activation of the APC/C is an attractive way to explain the spatially regulated disappearance of cyclin B, there are other possible explanations. The degradation of cyclin B, for example, could be initiated in the cytoplasm, but the release of cyclin B from the spindle might initially maintain a relatively constant level of the protein in the cytoplasm. This explanation, however, would not easily account for the partial degradation of cyclin B that is observed in syncytial embryos. Another possibility is that the APC/C is activated globally in the cell to target cyclin B for destruction, but it is concentrated on the spindle in metaphase and then moves into the cytoplasm during the latter stages of mitosis. This explanation is not consistent with observations as to the localization of Dmcdc16 and Dmcdc27 in fixed embryos, but this localization may not reflect accurately the distribution of these proteins in living cells. It is also possible that the targeting of cyclin B for degradation and the degradation itself may be separable events: the protein may be polyubiquitinated by the APC/C on the spindle, for example, but it may have to leave the spindle to be degraded by the 26S proteosome. More experiments will be required to analyse how the spatially regulated disappearance of cyclin B is related to its polyubiquitination and ultimate degradation. Moreover, it has been shown recently that cyclin B is not essential for mitosis in cellularized embryos if cyclin B3 is present (Jacobs, 1998). Cyclin B3 appears to be a nuclear protein; it will be interesting to investigate how cyclin B3 compensates for the loss of cyclin B (Huang, 1999).
The gene makos (mks) encodes the Drosophila counterpart of the Cdc27 subunit of the anaphase promoting complex (APC/C). Neuroblasts from third-larval-instar mks mutants arrest mitosis in a metaphase-like state but show some separation of sister chromatids. In contrast to metaphase-checkpoint-arrested cells, such mutant neuroblasts contain elevated levels not only of cyclin B but also of cyclin A. Mutations in mks enhance the reduced ability of hypomorphic polo mutant alleles to recruit and/or maintain the centrosomal antigens gamma-tubulin and CP190 at the spindle poles. Absence of the MPM2 epitope from the spindle poles in such double mutants suggests Polo kinase is not fully activated at this location. Thus, it appears that spindle pole functions of Polo kinase require the degradation of early mitotic targets of the APC/C, such as cyclin A, or other specific proteins. The metaphase-like arrest of mks mutants cannot be overcome by mutations in the spindle integrity checkpoint gene bub1, confirming this surveillance pathway has to operate through the APC/C. However, mutations in the twins/aar gene, which encodes the 55kDa regulatory subunit of PP2A, do suppress the mks metaphase arrest and so permit an alternative means of initiating anaphase. Thus the APC/C might normally be required to inactivate wild-type twins/aar gene product (Deak, 2003).
In Drosophila cells, the destruction of cyclin B is spatially regulated. In cellularized embryos, cyclin B is initially degraded on the mitotic spindle and is then degraded in the cytoplasm. In syncytial embryos, only the spindle-associated cyclin B is degraded at the end of mitosis. The anaphase promoting complex/cyclosome (APC/C) targets cyclin B for destruction, but its subcellular localization remains controversial. GFP fusions of two core APC/C subunits, Cdc16 and Cdc27, were constructed. These fusion proteins were incorporated into the endogenous APC/C and were largely localized in the cytoplasm during interphase in living syncytial embryos. Both fusion proteins rapidly accumulate in the nucleus prior to nuclear envelope breakdown but only weakly associate with mitotic spindles throughout mitosis. Thus, the global activation of a spatially restricted APC/C cannot explain the spatially regulated destruction of cyclin B. Instead, different subpopulations of the APC/C must be activated at different times to degrade cyclin B. Surprisingly, it was noticed that GFP-Cdc27 associates with mitotic chromosomes, whereas GFP-Cdc16 does not. Moreover, reducing the levels of Cdc16 or Cdc27 by >90% in tissue culture cells leads to a transient mitotic arrest that is both biochemically and morphologically distinct. Taken together, these results raise the intriguing possibility that there could be multiple forms of the APC/C that are differentially localized and perform distinct functions (Huang, 2002).
The destruction of cyclin B in Drosophila cells is spatially regulated and occurs in two phases. The destruction of cyclin B initiates at the centrosomes and then spreads to the spindle equator. Once the cyclin B on the spindle has been degraded, the remaining cytoplasmic cyclin B is then degraded. These phases appear to be separable, since in syncytial embryos only the spindle-associated cyclin B is degraded at the end of mitosis. The localization of GFP-Cdc16 and GFP-Cdc27 in living syncytial embryos suggests that only a small fraction of the APC/C is associated with mitotic spindles. Thus, the APC/C cannot be globally activated to degrade cyclin B at the end of mitosis. Instead, subpopulations of the APC/C must be activated at different times and at different places in order to explain the spatially regulated destruction of cyclin B. It is speculated that the APC/C is present in excess relative to two of its key regulators, Fzy/Cdc20 and Fzr/Cdh1. The targeting of Fzy and Fzr to different locations in the cell may explain how the destruction of cyclin B is regulated in space and time (Raff, 2002).
Surprisingly, GFP-Cdc27 associates with mitotic chromatin whereas GFP-Cdc16 does not, suggesting that these two core APC/C components are not always associated with one another in Drosophila embryos. This prompted a test to see whether these proteins might perform distinct functions. Depleting either protein by >90% from cells in culture produces a mitotic arrest that is both morphologically and biochemically distinct. These data raise the intriguing possibility that the APC/C may exist as several related complexes that could perform different functions (Raff, 2002).
A crucial question in interpreting these data is whether the localization of GFP-Cdc16 and GFP-Cdc27 accurately reflects the localization of the endogenous proteins. This is likely for several reasons. (1) Both fusion proteins are expressed at levels roughly comparable to the endogenous proteins, no fraction of either protein is found that does not behave as though it is part of a large complex that largely co-migrates with the endogenous Cdc16 and Cdc27 on a gel filtration column. Anti-GFP antibodies can precipitate the endogenous Cdc16 and Cdc27 from extracts expressing either fusion protein, demonstrating that these complexes also contain endogenous APC/C components. (2) The GFP-Cdc27 fusion protein can rescue a cdc27 mutation, suggesting that it is functional. (3) Although the distribution of GFP-Cdc16 and GFP-Cdc27 are not identical, they are very similar, and to date, it appears that no other GFP fusion proteins have been described that have this localization pattern. It seems unlikely that both fusion proteins would be artifactually mislocalized in such a similar way (Raff, 2002).
It is possible, however, that the localization of both fusion proteins is largely correct, but the differences observed in the localization of the two fusion proteins are artefactual. Perhaps, for example, a fraction of GFP-Cdc27 is not incorporated into the APC/C and can bind non-specifically to mitotic chromatin. This is unlikely for two reasons: (1) no pool of monomeric GFP-Cdc27 can be detected on gel filtration columns; (2) a C-terminal fusion of GFP with Cdc27 (Cdc27-GFP) has been expressed. This fusion protein is non-functional: it is not incorporated into the endogenous APC/C; it does not rescue a cdc27 mutation, and it does not bind to mitotic chromatin but is instead localized throughout the cytoplasm. Thus, even if there were a small pool of monomeric GFP-Cdc27, it seems unlikely that it would bind to mitotic chromatin. Alternatively, perhaps GFP-Cdc16 is incorporated into the APC/C, but the GFP moiety specifically prevents the complex interacting with chromatin. Although this would be surprising, since the presence of even multiple copies of the GFP-Cdc16 transgene does not appear to have any deleterious affects on flies, this possibility cannot presently be ruled out. A previous study has shown that mammalian Cdc27 biochemically co-purifies with mitotic chromatin whereas Cdc16 does not. Thus, in both Drosophila and mammalian cells there is evidence that Cdc27 associates with mitotic chromatin whereas Cdc16 does not (Raff, 2002).
To test whether Cdc16 and Cdc27 could perform distinct functions, the levels of each protein were reduced in Drosophila tissue culture cells using RNAi. Although this procedure depletes both proteins by >90%, the affect of depleting Cdc27 is always much more deleterious to cells than depleting Cdc16. Moreover, cyclin A is normally undetectable on metaphase chromosomes, and this was true in Cdc16RNAi cells but not in Cdc27RNAi cells. This suggests that a chromosome-associated fraction of cyclin A can be degraded when Cdc16 is depleted but not when Cdc27 is depleted, correlating with the observation that Cdc27 associates with mitotic chromatin whereas Cdc16 does not. Intriguingly, a slower migrating form of cyclin A was also reproducibly detectable in Western blots of Cdc27RNAi cells but not Cdc16RNAi cells. Perhaps this slower migrating form of cyclin A represents a chromatin-bound form of cyclin A that is not degraded properly when Cdc27 is depleted (Raff, 2002).
It is possible, however, that the different phenotypes induced by depleting Cdc16 and Cdc27 could be explained if depleting Cdc27 simply inactivates the APC/C more efficiently than depleting Cdc16. This would be surprising, since previous studies in yeast have suggested that both proteins are 'core' components of the APC/C that are present in roughly stoichiometric amounts. And, perturbing the function of either yeast protein by mutation or antibody injection causes the same phenotype -- a strong metaphase arrest. Thus, one would not predict that depleting either protein by >90% would produce such different effects on total APC/C activity. In addition, two lines of evidence suggest that in the Drosophila experiments depleting Cdc27 is not simply inducing a stronger version of the same phenotype induced by depleting Cdc16. (1) Depleting either protein weakly stabilizes cyclin B and strongly stabilizes Fzy/Cdc20 to about the same extent, suggesting that at least some aspects of APC/C function are equally inhibited by the depletion of either protein. (2) Cells in which both proteins are simultaneously depleted by >90% appear to have an intermediate chromosome/spindle morphology phenotype, arguing that the Cdc27RNAi phenotype is not simply a more extreme version of the Cdc16RNAi phenotype (Raff, 2002).
The interpretation of this RNAi data is complicated, however, since the behavior is being analyzed of a population of cells that appear to only transiently arrest in mitosis as they run out of Cdc16 or Cdc27. How these cells eventually exit mitosis is unknown, but it is noted that Drosophila tissue culture cells are notoriously difficult to arrest in mitosis, even with microtubule destabilizing agents. This 'mitotic slippage' mechanism probably explains why a maximum of ~25% of RNAi treated cells are observed arrested in mitosis. A similar failure to completely arrest cells in mitosis has been made in Drosophila larval neuroblasts mutant in the ida/APC5 subunit of the APC/C. Therefore the interpretation of these experiments must remain cautious. Nevertheless, these data are at least consistent with the possibility that Cdc16 and Cdc27 could exist in multiple complexes that perform at least partially non-overlapping functions (Raff, 2002).
Are there multiple APC/C complexes? The APC/C has been purified from several systems, and in all cases it has been found to contain homologs of Cdc16 and Cdc27. In human cells, APC/C complexes are homogeneous enough that a structure has been derived from cryo-electron microscopy and angular reconstitution studies. Moreover, previous studies in several systems have shown that perturbing APC/C activity always produces a similar phenotype -- a strong mitotic arrest. How can these findings be reconciled with the suggestion that the APC/C could exist in several complexes (Raff, 2002)?
The finding that anti-GFP antibodies can immunoprecipitate Cdc16 from extracts expressing GFP-Cdc16 and can immunoprecipitate Cdc27 from extracts expressing GFP-Cdc27 may give a clue to this apparent paradox. This finding suggests that the APC/C either contains multiple copies of both proteins or that multiple APC/Cs can bind to each other during purification. If the APC/C contains multiple copies of Cdc16 and Cdc27 then different forms of the APC/C could vary in their ratio of Cdc27 to Cdc16. Perhaps a form with a high ratio of Cdc27 to Cdc16 might interact with mitotic chromatin, whereas a form with a low Cdc27 to Cdc16 ratio might not. In this study, Cdc16 reproducibly migrated at a slightly smaller size on gel filtration columns than Cdc27 (and the same was true of GFP-Cdc16 compared with GFP-Cdc27), supporting the idea that the two proteins may not always exist in identical complexes. Such subtly different complexes, however, might be difficult to detect in purified APC/C preparations. Similarly, if multiple APC/Cs can bind to each other during purification, this might obscure the existence of several related complexes in purified preparations. Interestingly, Cdc16, Cdc27 and another APC/C component, Cdc23, all contain TPR repeats and can bind to themselves and to each other. This could explain how the APC/C can contain multiple copies of Cdc16 and Cdc27 or how different APC/C complexes might bind to each other during purification (Raff, 2002).
In summary, it has widely been assumed that the APC/C exists as a single complex, although there is little direct evidence to support this assumption. The data raise the possibility that the APC/C may exist as several related complexes that perform at least partially non-overlapping functions. The observations suggest that there must be subpopulations of the APC/C that are independently activated to degrade cyclin B at different times and at different places. A requirement to regulate overall APC/C activity in a temporally and spatially co-ordinated fashion could explain why the APC/C is so structurally complex (Raff, 2002).
The Imaginal discs arrested (ida) gene that is required for proliferation of imaginal disc cells during Drosophila development has been cloned and characterized. Ida is homologous to APC5, a subunit of the anaphase-promoting complex (APC/cyclosome). ida mRNA is detected in most cell types throughout development, but it accumulates to its highest levels during early embryogenesis. A maternal component of Ida is required for the production of eggs and viable embryos. Homozygous ida mutants display mitotic defects: they die during prepupal development, lack all mature imaginal disc structures, and have abnormally small optic lobes. Cytological observations show that ida mutant brains have a high mitotic index and many imaginal cells contain an aneuploid number of aberrant overcondensed chromosomes. However, cells are not stalled in metaphase, as evidenced by the observation that mitotic stages in which chromosomes are oriented at the equatorial plate are never observed. Interestingly, some APC/C-target substrates such as cyclin B are not degraded in ida mutants, whereas others controlling sister-chromatid separation appear to be turned over. Taken together, these results suggest a model in which IDA/APC5 controls regulatory subfunctions of the anaphase-promoting complex (Bentley, 2002).
In Drosophila the APC/C complex is estimated to consist of 11 proteins. However the biochemical function and requirement of so many subunits is unclear. One hypothesis proposes that the large number of subunits reflects the need to identify and target a large number of substrates. The model is supported by the recent characterization of the 3D structure of the human APC/C. The structure has an asymmetric morphology with a large inner cavity surrounded by an outer protein wall. The complexity of the structure suggests that discrete subunits may guide substrates into the inner cavity, where ubiquitination could take place. Thus the removal of a single subunit would disrupt the ubiquitination of only a fraction of substrates. Interestingly, the data suggests that Ida may be involved in the degradation of cyclin B but is not essential for the degradation of Securins. It should be noted that in this model not all subunits need play a role in substrate identification, since some are required for core stability and catalyzing the ubiquitination events. For example, Cdc27 and Cdc16 play critical roles in core stability, and Apc11 is required for the ubiquitination of substrates (Bentley, 2002 and references therein).
Another consideration for the role of APC/C subunits concerns the possibility that they specifically interact with regulators of APC/C activity during the cell cycle. Perhaps the most actively studied regulators of APC/C activity are the components of the spindle checkpoint pathway. Upon detection of DNA damage or unattached kinetochores, the spindle checkpoint pathway will send a 'wait' signal. In response to this signal, Mad2 will bind the APC/C, preventing its activity and halt progression of all mitotic events until the checkpoint has been fulfilled. Positive regulators play an equally important role in driving the cell through coordinated mitotic events. In Drosophila, the WD40-repeat protein, Fizzy (Fzy), binds to and drives APC/C-dependent ubiquitin-ligase activity in vitro. The Fzy homolog in yeast, Cdc20p, positively regulates the destruction of Pds1p, and Fzy is thought to serve a comparable role in Drosophila because Fzy is required for Pimples (Securin) degradation during mitosis. Consistent with these predictions, loss-of-function mutations in fzy prohibit cells from progressing through metaphase, and demonstrate that Fzy is required for metaphase exit and completion of mitosis in Drosophila. Fzy is highly unstable and present only at late S phase and during mitosis, further ensuring that Fzy-dependent APC/C events are temporally regulated. Finally, Fzy degradation is dependent on APC/C subunits, demonstrating that Fzy is also a substrate of the APC/C. An additional WD40-repeat protein, Fizzy-related (Fzy), is also believed to be required for the degradation of B-type cyclins during M and G1 phases, but differs from Fzy in that it is stable throughout the cell cycle (Bentley, 2002 and references therein).
In ida mutants, cyclin B levels are not properly degraded during anaphase. Thus it is possible that the function of Ida, alone or in concert with other subunits, is to direct cyclin B to the APC/C for degradation. However, other defects observed in ida cells are thought to be distinct from those observed in cells expressing a non-degradable cyclin B transgene. Therefore, it is proposed that there are additional regulatory functions for the IDA protein to help explain the lack of metaphase figures, the observed sister-chromatid separation, the high levels of Bub1 staining during anaphase, and the resulting aneuploidy that is observed in ida mutant cells (Bentley, 2002).
In one model, Ida functions as a part of the APC/C that receives a spindle checkpoint 'wait' signal. Thus when IDA is missing, the spindle checkpoint signal is not received, but the cell initiates sister-chromatid separation and anaphase onset prematurely. Presumably, this could occur even in the absence of proper chromosome attachment and alignment at the metaphase plate. Thus, metaphase figures would not be observed in ida mutants, but aberrant anaphases containing lagging chromosomes with high Bub1 staining (equal to signal checkpoint firing) would be detected. The missegregation of the unattached chromatids would also lead to cells containing an aneuploid number of chromosomes (Bentley, 2002).
In an alternative model, IDA plays a role in targeting Fzy for ubiquitin-dependent degradation. In this case, the removal of Ida would result in ectopic levels of Fzy, which would prematurely activate sister-chromatid separation and progression through mitosis. Consistent with this model, mutations in ida suppress the embryonic lethality associated with a fizzy null mutation (Bentley, 2002).
It should be noted that neither model directly address the high mitotic index -- a hallmark of cell cycle stall -- observed in squashes of ida cells. However, it is proposed that as cells become more and more aneuploid, alternative pathways, including the DNA replication checkpoint, may eventually cause a prometaphase stall or arrest (Bentley, 2002).
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