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).
To test whether Cdc16 and Cdc27 could perform distinct functions, the levels of each protein was reduced in Drosophila tissue culture cells using RNAi. Although this procedure depletes both proteins by >90%, the affect of depleting Cdc27 was 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 (Huang, 2002).
It is possible, however, that the different phenotypes induced by depleting Cdc16 and Cdc27 could be explained if depleting Cdc27 simply inactivated the APC/C more efficiently than depleting Cdc16. This would be surprising, as previous studies 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 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 affects on total APC/C activity. In addition, two lines of evidence suggest that in these 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 stabilises 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 (Huang, 2002).
The interpretation of this RNAi data is complicated, however, because the behavior is being studied of a population of cells that appears to only transiently arrest in mitosis as the cells 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 only a maximum of ~25% of RNAi treated cells arrested in mitosis was seen. 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 (Bentley, 2002). Therefore the interpretation of these experiments should be taken cautiously. 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 (Huang, 2002).
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 (Huang, 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 migrates at a slightly smaller size on gel filtration columns than Cdc27 (and the same is 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 (Huang, 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. These 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 (Huang, 2002).
Rca1 is an essential inhibitor of the anaphase-promoting complex/cyclosome (APC) in Drosophila. APC activity is restricted to mitotic stages and G1 by its activators Cdc20-Fizzy [Cdc20(Fzy)] and Cdh1-Fizzy-related [Cdh1(Fzr)], respectively. In rca1 mutants, cyclins are degraded prematurely in G2 by APC-Cdh1(Fzr)-dependent proteolysis, and cells fail to execute mitosis. Overexpression of Cdh1(Fzr) mimics the rca1 phenotype, and coexpression of Rca1 blocks this Cdh1(Fzr) function. Rca1 and Cdh1(Fzr) are in a complex that also includes the APC component Cdc27. Previous studies have shown that phosphorylation of Cdh1 prevents its interaction with the APC. These data reveal a different mode of APC regulation by Rca1 at the G2 stage, when low Cdk activity is unable to inhibit Cdh1(Fzr) interaction. Rca1 might be able to inhibit preformed APC-Cdh1Fzr complexes. Additionally it could prevent a fruitful association of Cdh1Fzr with the APC. Regardless of the exact biochemical composition of the Rca1-containing complex, all of the data support the conclusion that Rca1 is a specific inhibitor of Cdh1Fzr-dependent APC activity. This function of Rca1 is necessary during the G2 stage of the cell cycle to prevent a premature activation of the APC-Cdh1Fzr complex (Grosskortenhaus, 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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