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Targets of Activity

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).

Protein Interactions

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).

Other APC/C components

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).

Phenotypic characterization of Drosophila ida mutants: defining the role of APC5 in cell cycle progression

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).

Effete-mediated degradation of Cyclin A is essential for the maintenance of germline stem cells in Drosophila

Increasing evidence supports the idea that the regulation of stem cells requires both extrinsic and intrinsic mechanisms. However, much less is known about how intrinsic signals regulate the fate of stem cells. Studies on germline stem cells (GSCs) in the Drosophila ovary have provided novel insights into the regulatory mechanisms of stem cell maintenance. This study demonstrates that a ubiquitin-dependent pathway mediated by the Drosophila eff gene, which encodes the E2 ubiquitin-conjugating enzyme. Effete (Eff), plays an essential role in GSC maintenance. Eff both physically and genetically interacts with dAPC2, a key component of the anaphase-promoting complex (APC), which acts as a multisubunit E3 ligase and plays an essential role in targeting mitotic regulators for degradation during exit from mitosis. This interaction indicates that Eff regulates the APC/C-mediated proteolysis pathway in GSCs. Moreover, expression of a stable form of Cyclin A, but not full-length Cyclin A, results in GSC loss. Finally it was shown that, in common with APC2, Eff is required for the ubiquitylation of Cyclin A, and overexpression of full-length Cyclin A accelerates the loss of GSCs in the eff mutant background. Collectively, these data support the idea that Effete/APC-mediated degradation of Cyclin A is essential for the maintenance of germline stem cells in Drosophila. Given that the regulation of mitotic Cyclins is evolutionarily conserved between flies and mammals, this study also implies that a similar mechanism may be conserved in mammals (Chen, 2009).

Germline stem cells (GSCs) of the Drosophila ovary provide an excellent model system for studying the molecular mechanisms of stem cell regulation in vivo. In adult Drosophila females, two to three GSCs are easily recognized by their molecular markers (either a spherical spectrosome, or an extending fusome when GSCs are dividing) and their location at the apical region of the germarium in close contact with surrounding somatic cells, the terminal filament and cap cells, which together generate a specific micro-environment, or niche, for GSC regulation. The GSC divisions take place along the anteroposterior axis of the ovary to produce an anterior GSC, which remains attached to the niche cells, and a posterior cystoblast (Cb). The Cb divides precisely four times by incomplete cytokinesis to generate 16 interconnected cells that form the germline cyst of the follicle and sustain oogenesis (Chen, 2009).

Genetic analyses have revealed that the stem cell state of GSCs is maintained by both extrinsic and intrinsic mechanisms that repress their differentiation. BMP ligands (Dpp, Gbb) from the niche cells maintain GSCs by suppressing Cb differentiation in the anteriormost cells. This is achieved by silencing the transcription of the bam gene, which encodes a GSC/Cb differentiation-promoting factor. In the GSCs, BMP signaling activates cytoplasmic Mad and Medea, the Drosophila Smads, and promotes their nuclear translocation. In the nucleus, the Smads complex physically interacts with both the bam silencer element and nuclear lamin-associated protein (Ote), resulting in bam transcriptional silencing. Thus, BMP/Dpp-dependent bam transcriptional control serves as the primary pathway for the regulation of GSC fate. Independent of the niche-based regulation of the bam silencing mechanism, the fate of GSCs is also intrinsically controlled by other GSC maintenance factors that repress their differentiation. It has been demonstrated that Pum/Nos-mediated and microRNA-mediated translational repression pathways are not required for bam silencing, suggesting that these pathways act either downstream of or parallel to bam action. Although it is proposed that the Pum/Nos-mediated and microRNA-mediated pathways repress the translation of key differentiation factors to prevent GSC differentiation, the targets of these translational pathways in GSCs are not identified. Thus, the intrinsic mechanisms that repress GSC differentiation are still poorly understood. In addition, as loss of function of the components in these translational repression pathways is not sufficient to completely cause bam mutant germ-cell differentiation, it is speculated that the repression of GSC differentiation may also be controlled by other unknown intrinsic mechanisms (Chen, 2009).

Ubiquitin-mediated protein degradation plays a variety of roles in the regulation of many developmental processes. The enzymatic reaction of protein ubiquitylation is a coordinated three-step process involving three classes of enzymes known as E1 (Uba1 -- FlyBase), E2 (UbcD4 -- FlyBase) and E3. Firstly, E1 (Ubiquitin activating enzyme 1) catalyzes the formation of a thiolester bond linkage between the active-site cysteine residue on E1 and the C terminus of ubiquitin. Secondly, the activated ubiquitin (E1-Ub) is then transferred to E2 (Ubiquitin conjugating enzyme 4) via formation of an E2-Ub thiolester. Thirdly, E3 (ubiquitin ligase) promotes the transfer of the ubiquitin from E2-Ub to a lysine residue of the target protein through an isopeptide bond. Repeated cycles of this reaction can result in polyubiquitylation of the target protein, which is finally targeted for degradation by the 26S proteasome. The Drosophila effete (eff) gene encodes a class I ubiquitin-conjugating enzyme that was first shown to be required for proper telomere behavior (Cenci, 1997). Early studies also showed that eff is required for proper cyst formation in ovary (Lilly, 2000). However, whether eff is involved in the regulation of GSC fate remains unknown (Chen, 2009). Mitotic cyclins, in concert with their Cyclin-dependent kinase (CDK) partners, play important roles in the promotion of mitosis, such as entry into mitosis and the alignment of chromosomes at metaphase. The degradation of mitotic cyclins by the anaphase-promoting complex/cyclosome (APC/C), a large multisubunit E3 ligase, leads to inactivation of CDKs during the metaphase-anaphase transition and is essential for the completion of mitosis in all eukaryotes. In Drosophila, it has been shown that induction of stable versions of the three mitotic cyclins, Cyclin A, B and B3, arrests mitosis with different phenotypes, suggesting that degradation of distinct mitotic cyclins is required for specific mitotic events. It has been shown that the asymmetric division of GSCs requires the activity of Cyclin B; however, it remains unknown whether the regulation of mitotic cyclins also contributes to fate determination in GSCs. This study demonstrates that the eff gene, which encodes an E2 ubiquitin-conjugating enzyme (Eff), is intrinsic to and essential for Drosophila GSC maintenance. Moreover, Eff both genetically and physically interacts with dAPC2, a key component of APC/C complexes. dCDC20/Fzy, a key regulator of APC/C complexes, is shown to be involved in GSC regulation. Finally, constant expression of a stable form of Cyclin A results in the loss of GSCs. Notably, in common with APC2, Eff is required for the ubiquitylation of Cyclin A, and overexpression of full-length Cyclin A accelerates the loss of GSCs in the eff mutant background. Thus, these data support the idea that Effete/APC-mediated degradation of Cyclin A is essential for the maintenance of GSCs in Drosophila (Chen, 2009).

To identify new factors that regulate the self-renewal or differentiation of GSCs in the Drosophila ovary, female sterile lines, or weak fertile lines with P-element insertion, available from Bloomington Stock Center, were screened. The typical characteristic of GSC maintenance defects is a reduction in germ-cell number. This eventually results in an empty germarium lacking germ cells, and a decline in the production of egg chambers. Based on these criteria, a line with a P-element insertion in the third chromosome, P{PZ}eff8 was identified, that exhibits severe defects in germline development, including the loss of germ cells. To systematically study the behavior of GSCs and early germ cells in the eff8 mutant, anti-Vasa and anti-Hts antibodies were used to visualize germ cells and fusomes, respectively. In the tip of wild-type germarium, two or three GSCs were readily recognized by anti-Vasa antibody, and fusomes were morphologically spherical and anchored between the GSCs and cap cells. In addition, a normal germline lineage with sequentially differentiated cells marked by branched fusomes was also observed. However, in the 7-day-old ovaries from eff8 homozygous females, about 30% of mutant ovarioles contained either empty or abnormal germaria. Furthermore, the germ-cell defect phenotype became much more severe with age. These findings suggest that the loss of eff may affect the maintenance of GSCs. To determine whether the GSC maintenance defect associated with eff8 was indeed due to the loss of eff function rather than other genetic backgrounds, the phenotypes resulting from removal of eff were analyzed in several allelic combinations, eff8/effs1782, eff8/effmer1 and eff8/effmer4. The number of GSCs in the available eff allelic combinations were quantified at days 1, 7 and 14 after eclosion. Compared to wild type, the average number of GSCs in all eff mutants either rapidly or progressively declined during the testing period, indicating that the loss of eff resulted in the loss of GSCs. To further confirm this observation, a transgene, P{effP-eff}, was generated in which an eff cDNA was placed under the control of a 5.8 kb eff promoter. The GSC loss phenotype in different eff allelic backgrounds was fully rescued by the transgene line, P{effP-eff}. Taken together, these findings indicate that the eff gene plays an essential role in the maintenance of GSCs (Chen, 2009).

Attempts were made to understand the molecular mechanism underlying the action of Eff in GSCs by searching for Eff-interacting partners. Given that Eff functions as an E2 ubiquitin-conjugating enzyme, an E2/E3-based small-scale candidate screen was carried out by performing yeast two-hybrid experiments in which Eff was used as the bait to screen Eff-interacting E3. Notably, it was found that, among the candidates, dAPC2, encoded by the Drosophila morula (mr) gene, can strongly interact with Eff protein. To confirm this yeast two-hybrid interaction, whether Eff interacts with dAPC2 in Drosophila S2 cells was investigated by performing immunoprecipitation experiments. It was shown that Eff and dAPC2 can co-immunoprecipitate each other in transfected S2 cells, suggesting that Eff and dAPC2 are physically associated. To test whether dAPC2 physically associates with endogenous Eff in germ cells, a transgene, P{nosP-myc:dAPC2}, was generated. Results from co-immunoprecipitation showed that endogenous Eff physically associated with Myc:dAPC2, supporting further the argument that Eff interacts with dAPC2 in germ cells. In mitosis, the anaphase-promoting complex/cyclosome APC/C), a multisubunit complex that functions as an E3 ligase, plays important roles in ubiquitylating mitotic regulators such as mitotic cyclins and thus targets them for degradation by 26S proteasome. During this process, APC2, a cullin domain-containing protein, has been shown to function as a key mediator of APC/C complex activity. To test whether eff genetically interacts with mr (dAPC2) in the regulation of GSCs, the number of GSCs in both eff single-mutant and mr; eff double-mutant backgrounds were quantified at different time points. A weak allelic combination of mr (mr1/mr2) exhibited no apparent defect in GSC maintenance. However, mr; eff double-mutant ovaries showed more rapid GSC loss than the eff mutant alone, suggesting that dAPC2/mr enhances the phenotype of GSC loss in eff. Together, these results demonstrate that Eff interacts both physically and genetically with dAPC2 (Chen, 2009).

The ubiquitin-mediated proteolysis mechanism, which is evolutionarily conserved for the regulation of protein turnover, has been shown to play important roles in numerous biological processes, such as the cell cycle, pattern formation and tissue homeostasis. Drosophila Eff, which was initially identified as a class I E2 ubiquitin-conjugating enzyme encoded by the eff gene, has been shown to be involved in several cellular and developmental processes, including chromosome segregation, chromatin remodeling and protection against cell death. This study found that loss of eff function results in the depletion of GSCs, revealing a new role for eff in the regulation of GSC fate. Using germline clonal analysis and rescue experiments, it was further defined that the eff gene is an intrinsic, rather than extrinsic, factor for the maintenance of GSCs. Previous studies have shown that Eff is involved in the rpr-induced apoptosis pathway through a physical interaction with DIAP1 that stimulates DIAP1 auto-ubiquitylation. Therefore, it is possible that loss of GSCs in eff mutants may be due to reduced viability of GSCs. The results clearly show that eff-/- GSCs undergo differentiation rather than apoptosis, thus supporting the idea that the role of eff is to repress the premature differentiation of GSCs (Chen, 2009).

In the ubiquitin pathway, E2 conjugating enzymes have much lower specificity compared with E3 ligases. Certain E2s are known to function together with distinct type E3 ligases for substrate ubiquitylation and degradation. Eff is involved in protein degradation mediated by various RING finger-containing E3 ligases [e.g., Sina, Neur and DIAP (Iap2 -- FlyBase)], and regulates several signaling transduction pathways (Kuo, 2006; Ryoo, 2002; Tang, 1997). Since Eff plays a role downstream of, or parallel to, bam function, it is important to know what biochemical functions Eff performs in the regulation of GSCs. This work provided biochemical evidence showing that Eff not only physically interacts with the dAPC2 protein, but is also crucial for the ubiquitylation and degradation of Cyclin A. Moreover, genetic analyses revealed that eff interacts with both dAPC2 and cyclin A with respect to the regulation of GSCs. In addition, this study shows that dCDC20/Fzy, a key regulator of APC/C complexes, is involved in GSC regulation. Together, these data strongly support a model in which Eff facilitates the E3 ligase function of APC/CDC20 to ensure the self-renewal of GSCs (Chen, 2009).

Early studies in Xenopus and clam extracts demonstrated that both UBC4, a homolog of Eff, and UBCx/E2-C equally supported APC-mediated ubiquitylation reactions in vitro (. However, an in vivo study showed that these two classes of E2 are not functionally equivalent but exhibit distinct functions in mitotic cyclin degradation, suggesting that different E2 family members probably execute distinct functions (Seino, 2003). The Drosophila ortholog of UBCx/E2-C, Vihar E2, has been reported to be involved in Cyclin B degradation during the metaphase-anaphase transition. This work presents both genetic and biochemical evidence that Eff, the Drosophila homolog of UBC4, is essential for Cyclin A degradation in GSCs. Because the mitosis-related ubiquitin-conjugating enzyme, Vihar E2, is involved in APC/C-mediated ubiquitylation that potentially regulates Cyclin A degradation, it would be interesting to determine whether and/or how different E2 family members (e.g. Eff and Vihar E2) coordinately support specific APC-mediated mitotic cyclin destruction with respect to GSC regulation (Chen, 2009).

It has been shown that APC/C activity is required for the asymmetric localization of Miranda and its cargo proteins during neuroblast division. In Drosophila ovary, previous studies have demonstrated that Cyclin B plays important roles in GSC division and is essential role for GSC maintenance. However, it still remains unexplored whether the tight regulation of cyclins is also required for the fate determination of GSCs. The regulatory roles of mitotic cyclins at the cellular level during mitosis have been explored in detail. It has been reported that the sequential degradation of Cyclin A, Cyclin B and Cyclin B3 completes mitotic exit, which is mediated by APC/CDC20 in early M phase and by APC/Cdh1 during late M phase (Zachariae, 1999). Interestingly, the expression of stable forms of each cyclin leads to distinct mitosis defects, suggesting that the degradation of distinct mitotic cyclins is responsible for specific steps of mitosis. However, the biological basis for the control of the cyclin destruction remains elusive. Given that the APC-mediated pathway plays important roles in the proper cell mitosis, as loss of function of components in the pathway results in upregulation of mitotic cyclins that cause mitosis delay/or arrest, the question becomes whether the maintenance of GSCs requires the proper cell mitosis mediated by the regulation of mitotic cyclins. This study has shown that the forced expression of a stable form of Cyclin A leads to defects in GSC maintenance, suggesting that blocking mitotic progression may force germline stem cells to precociously differentiate, essentially altering their fate. Although the forced expression of a stable form of Cyclin B or Cyclin B3 does not give rise to any apparent defect in GSCs, one explanation is that stabilized Cyclin A may block cell-cycle progression more severely than the other stabilized Cyclins and prolonged M phase might be unfavorable for stem cell maintenance (Chen, 2009).

Taken together, these findings support a mechanism underlying the fate determination of stem cells that is linked to the control of the proper cell mitosis. Since the control of degradation of mitotic cyclins is evolutionarily conserved between flies and mammals, it would be interesting to also determine whether the control of proper cell mitosis is important for the maintenance of stem cells from other organisms, including mammals (Chen, 2009).

lemmingA encodes the Apc11 subunit of the APC/C in Drosophila melanogaster that forms a ternary complex with the E2-C type ubiquitin conjugating enzyme, Vihar and Morula/Apc2

Ubiquitin-dependent protein degradation is a critical step in key cell cycle events, such as metaphase-anaphase transition and mitotic exit. The anaphase promoting complex/cyclosome (APC/C) plays a pivotal role in these transitions by recognizing and marking regulatory proteins for proteasomal degradation. Its overall structure and function has been elucidated mostly in yeasts and mammalian cell lines. The APC/C is, however, a multisubunit assembly with at least 13 subunits and their function and interaction within the complex is still relatively uncharacterized, particularly in metazoan systems. This study used lemming(lmg) mutants to study the APC/C subunit, Apc11, and its interaction partners in Drosophila. The lmg gene was initially identified through a pharate adult lethal P element insertion mutation expressing developmental abnormalities and widespread apoptosis in larval imaginal discs and pupal abdominal histoblasts. Larval neuroblasts were observed to arrest mitosis in a metaphase-like state with highly condensed, scattered chromosomes and frequent polyploidy. These neuroblasts contain high levels of both cyclin A and cyclin B. The lmg gene was cloned by virtue of the lmg03424 P element insertion which is located in the 5' untranslated region. The lemming locus is transcribed to give a 2.0 kb mRNA that contains two ORFs, lmgA and lmgB. The lmgA ORF codes for a putative protein with more than 80% sequence homology to the APC11 subunit of the human APC/C. The 85 amino acid protein also contains a RING-finger motif characteristic of known APC11 subunits. The lmgA ORF alone was sufficient to rescue the lethal and mitotic phenotypes of the lmg138 null allele and to complement the temperature sensitive lethal phenotype of the APC11-myc9 budding yeast mutant. The LmgA protein interacts with Mr/Apc2, and they together form a binding site for Vihar, the E2-C type ubiquitin conjugating enzyme. Despite being conserved among Drosophila species, the LmgB protein is not required for viability or fertility. This work provides insight into the subunit structure of the Drosophila APC/C with implications for its function. Based on the presented data, it is suggested that the Lmg/Apc11 subunit recruits the E2-C type ubiquitin conjugating enzyme, Vihar, to the APC/C together with Mr/Apc2 by forming a ternary complex (Nagy, 2012).

The APC/C belongs to the cullin-RING family of multisubunit ubiquitin ligases. Previous studies of the budding yeast and human APC/C indicated that the cullin-related Apc2 and the RING-finger-containing Apc11 subunits together form the minimal ubiquitin ligase modul. This study shows that, in Drosophila, the Apc11 subunit is encoded by the dicistronic lemming locus. The upstream ORF, lmgA, encodes a putative protein containing a RING-finger motif characteristic of known APC11 subunits and shows more than 80% sequence similarity with the APC11 subunit of the human APC/C. Since the Apc11 subunit is proposed to play a role in the catalytic center of the APC/C, mutations in lmg are expected to lead to loss of APC function, and therefore to aberrant cell cycle progression. The lmg mitotic phenotype presented in this paper is consistent with the Lmg protein being a subunit of the APC. The mitotic defects observed in lmg larval neuroblasts, including metaphase-like arrest, chromosome overcondensation and polyploidy, in addition to widespread apoptosis of mitotically-active cells, are very similar to those reported for loss of other subunits of the Drosophila APC/C. A role of LmgA in the APC/C is further supported by the elevated levels of cyclin A and B observed in lmg neuroblasts. Another line of supporting evidence comes from the synergistic genetic interaction between lmgA and mr/Apc2 and lmgA and vihar and from the physical interactions among these proteins, since it is known that, in yeasts and vertebrates, these proteins form the catalytic module of the APC/C. These data, together with its ability to complement the mutant phenotype of yeast Apc11-deficient cells support the designation of lmgA as a true Apc11 orthologue (Nagy, 2012).

The APC/C requires special E2 enzymes for activity and has been demonstrated to function with Ubc4/5 and E2-C type E2 enzymes in vitro . Whereas in yeast and human cells the E2 enzymes bind to either Apc2 or Apc11, the current data suggest that in Drosophila, both of these subunits are required for effective E2 binding. This could represent an architectural variation in the catalytic subcomplex of different APC/C ligases (Nagy, 2012).

The dicistronic nature of the lmg locus is a notable but puzzling fact. Whereas the upstream lmgA ORF encodes the Apc11 subunit of the Drosophila APC/C, the existence and function of the predicted downstream lmgB ORF product remains unknown. No sequence or functional relationship could be found between lmgA and lmgB, though such relationships are characteristic of many dicistronic genes. Genomes of other species from Drosophilidae (especially in the melanogaster group) contain both these ORFs and the intercistronic sequence in a similar arrangement. Moreover, the high evolutionary conservation of LmgA and LmgB and significant conservation of both ICS and 3'-UTR suggest functional relevance. However, it was found that the putative LmgB is dispensable for the organism and lacks known protein motifs. In addition to this, no apparent LmgB interaction partners could be found in yeast two hybrid screen and LmgB could not be efficiently translated from the dicistronic mRNA in S2 cells. lmgA contains three in-frame AUG codons in addition to its initiating AUG codon. It has been shown for two Drosophila dicistronic transcripts, of the stoned and snapin loci, that such in-frame AUG codons effectively attenuate the translation of the second ORF. However, the rationale for the dicistronic arrangement of the lmgA and lmgB cistrons and the function of the lmgB ORF remains obscure (Nagy, 2012).

The mechanism by which loss of APC/C function leads to apoptosis is unknown but it may be significant that lmg mutant cells entered apoptosis directly, and rapidly, from arrested cells, without a return to the interphase state. There is accumulating evidence that mitosis and apoptosis share components. It has been suggested that apoptosis is a default pathway and proteins such as survivin are required to counteract this pathway during mitosis. Cells treated with drugs which alter microtubule dynamics, such as paclitaxel (Taxol) also undergo mitotic arrest and enter apoptosis rapidly, and directly, from mitosis. Since these drugs are thought to trigger the spindle assembly checkpoint which in turn acts by inhibiting the APC/C , it is possible that loss of APC/C activity is responsible for triggering apoptosis. Inactivation of the APC/C by cleavage of the CDC27 component by caspases has also been shown to occur during apoptosis triggered by Fas ligand in Jurkat cells, contributing to an increase in Cdk activity. There have been several reports of increased Cdk activity during apoptosis, suggesting that these enzymes form part of the apoptotic pathway. Increased mitotic cyclin levels, and Cdk activity, may therefore play a role in apoptosis triggered by loss of APC/C function. Apoptosis, however, does not normally occur when cyclin levels are high at metaphase. This may be because of protective factors such as survivin. A loss of protective activity during anaphase may allow cells to respond to abnormally high levels of Cdk activity and undergo apoptosis. Alternatively, if the APC/C itself plays a protective role, simultaneous loss of this protection and elevated Cdk levels would result in apoptosis (Nagy, 2012).

The polyploid cells observed in larval brain squashes may be cells that have escaped apoptosis, exited mitosis without cytokinesis, and then duplicated their chromosomes before re-entering mitosis again. If so, some cells can clearly repeat the process several times, as cells were observed that were highly polyploid. Furthermore, no G2-arrested interphase larval abdominal histoblasts were observed undergoing apoptosis. This suggests that there is a phase, during mitosis, when cells are particularly sensitive to loss of lmg function and respond by undergoing apoptosis. This might be expected if loss of APC/C function is playing a relatively direct role in triggering apoptosis (Nagy, 2012).

The data presented in this paper demonstrate that the upstream member of a dicistronic gene, lmgA codes for the Apc11 subunit of the APC/C in a multicellular metazoan species, Drosophila melanogaster. Its genetic and physical interactions with Mr/Apc2 and the E2-C type ubiquitin-conjugating enzyme, Vihar, suggest that their ternary complex represents the same catalytic module of the APC/C that was identified in yeast and mammalian cells by functional means (Nagy, 2012).

Transcription-independent function of Polycomb group protein PSC in cell cycle control

Polycomb group (PcG) proteins control development and cell proliferation through chromatin-mediated transcriptional repression. A transcription-independent function is described for PcG protein Posterior sex combs (PSC) in regulating the destruction of cyclin B (CYC-B). A substantial portion of PSC was found outside canonical PcG complexes, instead associated with CYC-B and the anaphase-promoting complex (APC). Cell-based experiments and reconstituted reactions have established that PSC and Lemming (LMG, also called APC11) associate and ubiquitylate CYC-B cooperatively, marking it for proteosomal degradation. Thus, PSC appears to mediate both developmental gene silencing and posttranslational control of mitosis. Direct regulation of cell cycle progression might be a crucial part of the PcG system's function in development and cancer (Mohd-Sarip, 2012).

Polycomb group (PcG) proteins are transcriptional repressors that maintain cell-fate decisions and control cell proliferation. They function as part of distinct multiprotein complexes that modulate chromatin structure. The RING domain protein Posterior sex combs (PSC) is a subunit of Polycomb repressive complex 1 (PRC1) and dRING–associated factors (dRAF), which mediate monoubiquitylation of histone H2A. A substantial portion of PSC is part of neither PRC1 nor dRAF, suggesting that PSC might have additional functions. The effects of depleting either Polycomb (PC), Polyhomeotic (PH), PSC, or dRING were compared by treating S2 cells with the appropriate double-stranded RNAs (dsRNAs). PC, PH, PSC, and dRING form the core of PRC1, whereas dRING, PSC, and dKDM2 are the central subunits of dRAF. Knock-down (KD) of PH or PSC decreased cell accumulation, whereas depletion of PC or dRING had no appreciable effects. Fluorescence-activated cell sorter (FACS) analysis indicated that cells lacking PSC primarily accumulated at the G2-M phase of the cell cycle. Loss of other PcG proteins did not give a clear cell cycle arrest. Therefore, PSC might function in cell cycle regulation, independent of PRC1 or dRAF (Mohd-Sarip, 2012).

Consistent with the G2-M arrest caused by loss of PSC, maternal effect mutations of Psc cause mitotic segregation defects in early Drosophila embryos. This is illustrated by the mitotic chromosome bridges, frequently detected in the progeny of Psch27 mutant mothers. Because early embryos have nonconventional checkpoint mechanisms, problems at either S phase or mitosis can lead to segregation defects. In S2 cells, which have a conventional cell cycle, depletion of PSC caused severe mitotic defects. After depletion of PSC, ~68% of mitotic cells displayed an abnormal phenotype, whereas loss of the other PcG proteins did not affect mitosis. The PcG system has been implicated in the regulation of cell cycle genes. Yet, because the integrity of PcG complexes is required for silencing, it was suspected that PSC's role in mitosis extends beyond transcription repression. Indeed, a portion of cellular PSC does not appear to be part of PRC1 nor dRAF (Mohd-Sarip, 2012).

To identify interaction partners, three distinct affinity-purified antibodies were used to isolate PSC from whole-cell extracts of 0- to 12-hour-old Drosophila embryos. Mass spectrometric analysis revealed that, in addition to PRC1 and dRAF subunits, cyclin B (CYC-B), cell division cycle 2 (CDC2, also called cyclin-dependent protein kinase 1), and key subunits of the anaphase-promoting complex (APC) associate with PSC. Although PSC was present in PC, dRING, and PH purifications, CYC-B and the APC were absent. The APC is a multisubunit E3 ubiquitin ligase that is pivotal to cell cycle regulation. CYC-B ubiquitylation by the APC, marking it for destruction by the proteasome, is required for completion of anaphase and cytokinesis. This study confirmed the selective association of PSC with CYC-B and APC by a series of immunoprecipitations (IPs) combined with protein immunoblotting. IPs of PcG proteins showed that only PSC associates with CYC-B; CDC2; and the APC subunits Morula (Mr, also called APC2), CDC23 (APC8), and Lemming (Lmg, also called APC11). Reverse IPs further established the unique association of PSC with CYC-B and the APC. Lmg is a small 85–amino acid protein, comprising mainly a RING domain, that is essential for the ubiquitin ligase activity of the APC. Many RING domain proteins are E3 ubiquitin ligases and frequently function as homo- or heterodimers. For example, PSC and its mammalian homolog BMI1 bind dRING or RING1B, respectively, and stimulate histone H2A ubiquitylation. This study found that the RING domain of PSC was necessary and sufficient to bind LMG, whereas its C-terminal region bound CYC-B. Thus, PSC appears to associate with Lmg and CYC-B directly (Mohd-Sarip, 2012).

To complement these biochemical experiments with a genetic-interaction assay, the GAL4-UAS system was used in Drosophila.The glass multimer reporter (GMR) was used to drive ectopic CYC-B expression (GMR>CYC-B) in the developing eye. Ectopic CYC-B caused a mild rough-eye phenotype, characterized by disorganized ommatidia and loss of bristles. Concomitant expression of dsRNA directed against Psc mRNA [GMR>CYC-B; GMR>PSCRNAi (RNAi, RNA interference)] enhanced the GMR>CYC-B phenotype, consistent with the notion that PSC is a negative regulator of CYC-B. In contrast, expression of dsRNA directed against Pc had no effect on the CYC-B overexpression phenotype. Alone, neither reduction of PSC levels nor PC depletion had an appreciable effect on eye development. Thus, PSC interacts both genetically and biochemically with CYC-B (Mohd-Sarip, 2012).

To test whether PSC regulates abundance of CYC-B in vivo, the Patched (Ptc) driver was used to direct the expression of dsRNA directed against Psc mRNA in a central band across the wing imaginal disc of third instar larvae. Immunostaining of CYC-B (red) and PSC (green) revealed a strong increase in CYC-B, precisely in the area of the disc where PSC was depleted. CYC-B abundance was also reported to increase in cellular clones that lack both Psc and Su(z)2, but not in Pc or dRing mutant clones. The effect of PSC on CYC-B was transcription-independent because expression of cyc-B mRNA was not affected by PSC depletion. Likewise, loss of PSC or LMG in S2 cells caused accumulation of CYC-B, which was even greater when both factors were depleted. However, the abundance of cyc-B mRNA in S2 cells was not affected by depletion of PSC or LMG. Thus, CYC-B accumulation appears to be caused by a transcription-independent mechanism, possibly involving PSC-directed ubiquitylation (Mohd-Sarip, 2012).

To investigate the role of PSC in CYC-B ubiquitylation, CYC-B was immunopurified from cells that were depleted of PSC or LMG and treated with proteasome inhibitors. Immunoblotting revealed that the loss of either PSC or LMG caused decreased levels of polyubiquitylated CYC-B (Ub-CYC-B). Almost no Ub-CYC-B was detectable in cells lacking both PSC and LMG. To test whether failed CYC-B destruction could explain the mitotic defects after the loss of PSC, CYC-B was overexpressed in S2 cells. After ectopic expression of CYC-B, ~70% of mitotic cells displayed a variety of defects. Concomitant overexpression of either PSC or LMG almost completely reversed the CYC-B misexpression phenotype. In contrast, extra PC had no effect. Collectively, these results suggest that PSC-mediated CYC-B ubiquitylation is crucial for normal mitosis (Mohd-Sarip, 2012).

Purified PSC, LMG, and CYC-B were used in a reconstituted ubiquitylation system, which was dependent on E1 and E2 enzymes, to test the ability of PSC to act as a ubiquitin E3 ligase for CYC-B. Approximately equimolar amounts of either PSC or LMG could direct CYC-B ubiquitylation. But together, PSC and LMG generated higher levels of Ub–CYC-B. Determination of the CYC-B ubiquitylation rate revealed a more-than-additive effect of combining PSC and LMG, indicating that they function cooperatively. A substitution mutation replacing a signature cysteine residue of the RING consensus with an alanine [PSC-C287A (C287A: Cys287→Ala287)] abrogated PSC's ability to ubiquitylate CYC-B. PSC-C287A blocked ubiquitylation of CYC-B by LMG, suggesting that it acts as a dominant negative. Indeed, the C287A mutation did not affect PSC binding to LMG or CYC-B. In contrast to ectopic PSC, expression of PSC-C287A caused severe mitotic defects in S2 cells. This mitotic phenotype was relieved by concomitant overexpression of LMG, suggesting that extra LMG squelches the dominant-negative PSC. Whereas ectopic expression of either PSC or LMG in S2 cells did not affect mitosis, overexpression of both PSC and LMG caused mitotic defects. These results suggest that PSC and LMG cooperate in the ubiquitylation of CYC-B, marking it for destruction by the proteasome (Mohd-Sarip, 2012).

Regulated protein destruction is fundamental to cell cycle progression. The work reported in this study shows that, in addition to transcriptional repression, PSC cooperates with LMG in the APC to direct CYC-B degradation. During mitosis, PSC (and its mammalian homologs) and key PRC1 subunits PH and PC dissociate from the chromatin, making a transcriptional function at that time unlikely. Like PSC, other chromatin regulators may also target proteins that are neither involved in chromatin dynamics nor transcription (Mohd-Sarip, 2012).

morula: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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