retina aberrant in pattern/fizzy-related


REGULATION

Notch-dependent Fizzy-related expression is required for the mitotic-to-endocycle transition in follicle cells

During Drosophila oogenesis, Notch function regulates the transition from mitotic cell cycle to endocycle in follicle cells at stage 6. Loss of either Notch function or its ligand Delta (Dl) disrupts the normal transition; this disruption causes mitotic cycling to continue and leads to an overproliferation phenotype. In this context, the only known cell cycle component that responds to the Notch pathway is String/Cdc25 (Stg), a G2/M cell cycle regulator. Prolonged expression of string is not sufficient to keep cells efficiently in mitotic cell cycle past stage 6, suggesting that Notch also regulates other cell cycle components in the transition. By using an expression screen, such a component was found: Fizzy-related/Hec1/Cdh1 (Fzr), a WD40 repeat protein. Fzr regulates the anaphase-promoting complex/cyclosome (APC/C) and is expressed at the mitotic-to-endocycle transition in a Notch-dependent manner. Mutant clones of Fzr have revealed that Fzr is dispensable for mitosis but essential for endocycles. Unlike in Notch clones, in Fzr mutant cells mitotic markers are absent past stage 6. Only a combined reduction of Fzr and ectopic Stg expression prolongs mitotic cycles in follicle cells, suggesting that these two cell cycle regulators, Fzr and Stg, are important mediators of the Notch pathway in the mitotic-to-endocycle transition (Schaeffer, 2004).

In Drosophila, nurse and follicle cells in the adult ovary endocycle in a regulated manner. It has been suggested that endocycling requires the loss of M-phase cyclin-dependent kinase (Cdk) activity and oscillations in the activity of S-phase Cdk. In Drosophila follicle cells, the function of the Notch pathway in the mitotic-to-endocycle transition has been well established. Lack of Notch activity in Drosophila follicle cells leads to prolonged mitosis at the expense of endocycles, suggesting that Notch functions in this context as a tumor suppressor. Because very few signaling pathways that stop the mitotic cell cycle have been identified, it is important to understand the relationship between the Notch pathway and known cell cycle regulators in more detail (Schaeffer, 2004).

String encodes the Drosophila homolog of the yeast cell cycle regulator Cdc25, a phosphatase whose role is to activate the Cdk-cyclin complex at the G2/M transition by dephosphorylating the inhibitory sites. As a consequence, cells are propelled into mitosis. Notch signaling downregulates string at stage 6 of oogenesis to allow the cells to transit into the endocycle. The 4.9 kb and 6.4 kb elements found in the 50 kb-long string promoter drive string expression in follicle cells from germarium to stage 3 and from stage 4 to stage 6, respectively. The Notch-Delta cascade achieves the tight downregulation of the 6.4 kb element at stage 6, when the mitotic-to-endocycle transition takes place. A string rescue construct that contains 15.3 kb of the string promoter restores only the early string expression pattern between germarium and stage 1-2 egg chambers (because of the 4.9 kb element) but does not contain the control element active between stages 3 and 6 (the 6.4 kb element). Although stg clones produce cells arrested in G2, the mutant nuclei were larger than in the wild-type cells when stg clones were produced in the background of the 15.3 kb rescue construct. Furthermore, the mutant clones are half the size of sister clones, suggesting that the mutant cells stop division and possibly enter endocycle too early. If downregulating String leads the follicle cells to enter an endocycle rather than to completely arrest, then the sole role of Notch, which downregulates string expression at the switch, is to act on string to promote endocycling. If this is the case, string expression is the only limiting factor in the mitotic-to-endocycle transition. Also, because ectopic expression of stg in Drosophila embryos and discs is capable of driving cells blocked in G2 into mitosis, continuous string expression should keep most cells in the mitotic phase (Schaeffer, 2004).

stg (either with a heat-shock-inducible promoter or with one or two copies of the UAS-stg transgene) was overexpressed via the flip-out Gal4 system to analyze whether String is sufficient to prolong division of follicle cells past stage 6. Overexpression of string with a transgene driven by a heat-shock promoter did not show any ectopic Cyclin B or Phospho-Histone 3 (PH3) expression. With one copy of the UAS string transgene, prolonged mitotic divisions were rarely observed in follicle cells past stage 6, except in the posterior region, where 10% of the clones that overexpressed string showed ectopic Cyclin B or PH3 expression. When two copies of the UAS-stg construct were present, leading to higher string expression levels, a higher incidence of Cyclin B and PH3 expression was seen in posterior clones. However, only in a few cases did the ectopic expression of string in lateral and anterior follicle cells prolong their mitotic state. In addition, in most cells, overexpression of string did not affect endocycling (Schaeffer, 2004).

Because the String protein is not enough to create extra cell divisions, except in the highly sensitized posterior area, it was proposed that the mitotic-to-endocycle transition is regulated by a combination of String and other Notch-controlled components yet to be uncovered. Lack of String generally arrests cells in G2, when high levels of mitotic cyclins can be found. Cyclin A and Cdc2 have been implicated in inhibiting the assembly of prereplication complexes in G2. Furthermore, when Cdc2 or Cyclin A activity is eliminated, mutant cells enter endocycles in Drosophila because the assembly of prereplication complexes is then allowed. The hypothesis here is that the Notch signaling pathway allows cells to bypass this inhibition by activating a specific gene/genes that would allow cells to continue to cycle without undergoing the mitotic phase. Expression of such a gene would be activated after/during the mitotic-to-endocycle transition and possibly act on mitotic cyclin regulation and/or the mitotic cyclin-associated kinase, Cdc2. In order to find these genes, an expression screen was performed for genes differentially expressed before and after the transition (Schaeffer, 2004).

400 lethal X chromosome P element enhancer trap lines were screened for changes in expression levels at stage 7 by using the β-gal reporter gene. Three interesting functional groups were obtained from this screen: adhesion molecules, transcriptional control proteins, and cell cycle regulators. Premature expression of fzr caused formation of enlarged nuclei, a potential indication of precocious endocycles. Therefore the cell cycle regulator Fzr was analyzed in more detail in the mitotic-to-endocycle transition (Schaeffer, 2004).

The lines fzrG0326 and fzrG0418 have the P{lacW} element inserted in the first intron and at the 5'-end of the Fizzy-related gene, respectively, and are hypomorphic alleles of fzr. These constructs drive expression of the reporter gene after the transition, from stage 6-7 onward. This expression is tightly correlated with the end of mitotic cycles; no fzr expression is observed in follicle cells that show PH3 staining. A similar expression pattern of Fzr was observed with the Fzr specific antibody, and the fzr mRNA pattern in follicle cells reflects this pattern as well (Schaeffer, 2004).

Fzr, also known as Retina aberrant in pattern, is a conserved WD domain protein that is required during G1 for proteolysis of mitotic regulators such as Aurora-A kinase and Cyclins A, B, and B3 in an APC/C-dependent manner. Loss of Fzr in Drosophila causes cells to progress through an extra division cycle in the epidermis and inhibits endoreduplication in the salivary gland cells, whereas fzr overexpression inhibits mitosis and transforms mitotic cycles into endoreduplication cycles. This finding suggests that, in at least some cell types, the Fzr protein is essential for the mitotic-to-endocycle transition. Because fzr expression is upregulated in follicle cells when the Notch cascade is activated, whether the fzr expression was responsive to Notch activity was tested by using the fzrG0326 (fzr-LacZ) enhancer trap line to analyze fzr expression levels in follicle cells that surround the Dl germline clones. A clear reduction of fzr expression was observed in all Dl germline clones past stage 6, demonstrating that fzr expression is dependent on Notch activity in the mitotic-to-endocycle transition (Schaeffer, 2004).

Mitotic-cyclin protein levels are downregulated at the mitotic-to-endocycle transition. Cyclin A protein levels are reduced at the end of mitotic cycles in the follicle cells. Similarly, Cyclin B is downregulated at the protein level at the mitotic-to-endocycle transition. In situ hybridization studies indicated that neither gene was regulated at the transcriptional level during or after the transition but showed mRNA expression in the follicle cells throughout oogenesis until stage 10. Thus, both the Cyclin A and the Cyclin B protein levels are regulated posttrancriptionally at the mitotic-to-endocycle transition. This regulation is critical for the mitotic-to-endocycle transition because continuous expression of cyclin A in posterior follicle cells results in small nuclei and a reduced DNA level, indicative of a defect in the transition to endocycles. This supports previous reports showing that overexpression of cyclin A inhibits the progression of endoreplication cycles in Drosophila salivary glands (Schaeffer, 2004).

Because the downregulation of Cyclin A and B expression coincides with the upregulation of Fzr and because Fzr is required for proteolysis of Cyclin A and Cyclin B in embryonic epidermal cells, clones for a fzr null allele (fzrie28) were generated to test whether Fzr might be responsible for the mitotic-to-endocycle transition by downregulating the mitotic cyclin levels. Initially, the ovaries were immunostained with antibodies against Cyclin B. The clonal cells lacking Fzr function showed a limited but consistent increase of Cyclin B level after stage 6, when Cyclin B is normally absent. In a similar manner, fzrie28 mutant cells strongly upregulated Cyclin A after the mitotic-to-endocycle transition. It is therefore concluded that, as seen in other systems, Fzr function in follicle cells is to degrade mitotic cyclins (Schaeffer, 2004).

Upregulation of the mitotic Cyclin A during endocycles has been shown to inhibit endoreplication. The ovaries bearing fzrie28 mutant cells were stained with DAPI to observe nuclei size and shape. In addition to showing a failure of Cyclin A and B removal, the fzrie28 mutant cells showed phenotypes that indicated endocycle inhibition. The small nuclei size and reduced DNA level seen in the fzrie28 mutant cells are reminiscent of the Notch phenotype and thereby show that Fzr is required for the mitotic-to-endocycle transition. Unlike the Notch clones in which Cyclin B and PH3 expression were detected after stage 6, the fzrie28 mutant cells do not shown signs of overproliferation. No PH3 staining is ever observed in mutant clones after the transition, suggesting that the fzrie28 mutant cells do not continue to divide past stage 6. The 6.4 kb Stg-LacZ transgene, abruptly downregulated by Notch at the mitotic-to-endocycle transition, did not show any prolonged expression in fzrie28 mutant cells after stage 6, indicating that cells are not in a mitotic phase. The number of cells in mutant and sister clones (two copies of GFP) were counted. The same number of cells was observed in mutant clones as in the associated twin spot; the ratio varied from 0.64 to 3, with a mean of 1.01). All these clues led to the idea that despite mitotic cyclins' upregulation in fzr-/- mutant cells, those cells do not divide past stage 6 (Schaeffer, 2004).

Because the number of cells in the mutant follicle cell clones and the associated twin spots are the same and because the expression of the 6.4 kb stg-LacZ transgene is normal prior to the switch to endocycling, it is concluded that, even though Fzr is required for endocycles, it is dispensable for the mitotic stages in the Drosophila ovary. Similarly, it has been shown that completion of mitosis does not require Fzr in embryos (Schaeffer, 2004).

Although fzrie28 mutant cells show upregulation of the mitotic cyclins, these cells do not divide. In all dividing cells, the G2/M transition depends on String to activate the kinase activity of the mitotic cyclin-Cdc2 complexes. Because the 6.4 kb stg-LacZ transgene is downregulated by the Notch pathway after the transition, the fzrie28 mutant cells might require Stg to prolong mitosis. To test this, string was overexpressed by using a heat shock promoter in the fzrie28 follicle cell clones. The flies were heat shocked twice, once to promote the formation of fzrie28 mutant clones and again, 12 hr prior to dissection, in order to induce stg expression. In a wild-type fzr background, this low level of string expression alone is insufficient for prolonging mitosis past the mitotic-to-endocycle transition (no upregulation of Cyclin B and PH3 markers). However, this prolonged stg expression is enough to push the fzrie28 mutant cells into mitosis, as shown by the PH3 staining and mitotic figures in egg chambers at stage 9. More strikingly, PH3-positive cells as well as mitotic figures were seen in nonclonal areas heterozygous for fzrie28, which prompted a test to see whether reducing the level of Fzr to one copy while overexpressing stg by heat shock is sufficient to produce the PH3-positive cells. In order to demonstrate a direct stg effect, the flies were examined 2 hr after heat shock. The fzrG0326 enhancer trap line (fzr LacZ) was used to reduce the level of Fzr and to mark the stages precisely. It was found that 39% of ovarioles of the experimental group fzrG0326;;Hs Stg displayed PH3-positive cells and mitotic figures at stage 7-8, whereas 0%-2% did so in the control groups. In order to further determine whether the ratios of cells in a mitotic stage in mutant and control situations were similar, the number of PH3-positive cells observed in a single focal plan was quantified before and at stage 7-8, in the experimental group fzrG0326;;Hs Stg as well as in three control groups. On average, 12%-15% of the cells showed PH3 staining at mitotic stages before the transition (before stage 7), but none did so after the transition (stage 7-8). In contrast, ovaries with reduced fzr and prolonged stg expression showed PH3 staining after stage 7, whereas the control groups did not. In comparison, 8.5% of cells in the egg chamber did exhibit PH3 staining. It is possible that the percentage of mitotic cells observed in mutant egg chambers past the transition was somewhat lower than the percentage of mitotic cells observed in wild-type egg chambers before the transition because of the low level of string expression given by the heat shock construct or subtle effects of yet-unraveled components in the transition. However, these data strongly suggest that reducing the Fzr level in combination with prolonged stg expression can prolong the mitotic stage in follicle cells (Schaeffer, 2004).

In Drosophila, loss of Fzr causes progression through an extra division cycle in the epidermis and inhibition of endoreplication in the salivary glands, in addition to the upregulation of mitotic cyclins. In follicle cells loss of Fzr causes an inhibition of endoreplication as well as an upregulation of the mitotic cyclins, particularly Cyclin A, but no prolonged mitosis. This difference might be due to the lack of String in follicle cells. It is possible that in the epidermis, residual String might dephosphorylate and therefore activate the mitotic cyclin/Cdk complexes and allow an extra mitosis to proceed, whereas in follicle cells the absence of String might result in G2-arrest. This is supported by the fact that overexpressing a string transgene under the control of a heat shock promoter rescues cell division in a fzr mutant (Schaeffer, 2004).

Notch mutant cells are mitotic: in those cells, Stg is upregulated, and Fzr is not activated. Those two events (upregulation of String and downregulation of Fzr) are able to keep the cells in mitotic cycle in 39% of stage 7-8 egg chambers. It is therefore possible that Notch controls the mitotic to endocycle transition by repressing String to block mitosis and by activating Fzr to allow endocycle progression (Schaeffer, 2004).

Based on earlier studies, it has been proposed that endocycle is induced by lack of M-phase Cdk activity. However, the regulation and exact manifestation of this task has not been previously uncovered. This study shows that in Drosophila follicle cells the Notch pathway executes the task by first freezing the mitotic cyclin/Cdk complex in an inactive, phosphorylated form and thereafter inducing the degradation of the mitotic cyclins to allow progression to S phase. Further studies will reveal whether Notch action is also required for G1-to-S-phase transition or whether these two alterations, lack of String, and expression of Fzr are sufficient to transform mitotic cells to endocycling cells (Schaeffer, 2004).

The roles of Fzy/Cdc20 and Fzr/Cdh1 in regulating the destruction of cyclin B in space and time

In Drosophila cells cyclin B is normally degraded in two phases: (1) destruction of the spindle-associated cyclin B initiates at centrosomes and spreads to the spindle equator, and (2) any remaining cytoplasmic cyclin B is degraded slightly later in mitosis. The APC/C regulators Fizzy (Fzy)/Cdc20 and Fzy-related (Fzr)/Cdh1 bind to microtubules in vitro and associate with spindles in vivo. Fzy/Cdc20 is concentrated at kinetochores and centrosomes early in mitosis, whereas Fzr/Cdh1 is concentrated at centrosomes throughout the cell cycle. In syncytial embryos, only Fzy/Cdc20 is present, and only the spindle-associated cyclin B is degraded at the end of mitosis. A destruction box-mutated form of cyclin B (cyclin B triple-point mutant [CBTPM]-GFP) that cannot be targeted for destruction by Fzy/Cdc20, is no longer degraded on spindles in syncytial embryos. However, CBTPM-GFP can be targeted for destruction by Fzr/Cdh1. In cellularized embryos, which normally express Fzr/Cdh1, CBTPM-GFP is degraded throughout the cell but with slowed kinetics. These findings suggest that Fzy/Cdc20 is responsible for catalyzing the first phase of cyclin B destruction that occurs on the mitotic spindle, whereas Fzr/Cdh1 is responsible for catalyzing the second phase of cyclin B destruction that occurs throughout the cell. These observations have important implications for the mechanisms of the spindle checkpoint (Raff, 2002).

This study follows the subcellular localization of Fzy/Cdc20 and Fzr/Cdh1 throughout the cell cycle in living Drosophila embryos. GFP-Fzy is concentrated on kinetochores, centrosomes, and spindles early in mitosis, and starts to disappear from these structures once the chromosomes align at the metaphase plate. This localization is similar to that reported for p55cdc20 in fixed human cells, and it fits in well with the proposed role of Fzy/Cdc20 in linking the spindle assembly checkpoint to the APC/C. In higher eukaryotes, the spindle checkpoint system consists of several proteins, including the Mad and Bub proteins as well as CenpE, Mps1, Rod, and ZW10. As cells enter mitosis, most of these proteins accumulate on unattached kinetochores, and are then lost from the kinetochores once the chromosomes align at the metaphase plate. Several of these checkpoint proteins can bind to Fzy/Cdc20, and this appears to inhibit the ability of Fzy/Cdc20 to activate the APC/C. Therefore, an unattached kinetochore is thought to continuously generate inhibitory checkpoint protein/Fzy (Cdc20) complexes, thus ensuring that the APC/C is not activated until all of the chromosomes have aligned properly at the metaphase plate (Raff, 2002).

The checkpoint proteins Mad2, BubR1, CENP-E, Rod, and ZW10 have all been shown to bind to kinetochores and then move along microtubules to the centrosomes in a dynein-dependent manner. During mitosis, the localization of GFP-Fzy to kinetochores is microtubule independent, whereas its localization at centrosomes is microtubule dependent. This is consistent with the possibility that Fzy/Cdc20 may also load onto kinetochores and then move along microtubules to the centrosomes (Raff, 2002).

In contrast to GFP-Fzy, GFP-Fzr is strongly concentrated at centrosomes throughout the cell cycle, apparently in a microtubule-independent fashion. The concentration of Fzr/Cdh1 at centrosomes was unexpected, since it had been previously proposed that Fzr/Cdh1 catalyzes the second phase of cyclin B destruction that occurs in the cytoplasm. However, the Fluorescence Redistribution After Photobleaching (FRAP) analysis suggested that Fzr/Cdh1 is rapidly turned over at centrosomes. Although the significance of this turnover is unclear, it is possible that Fzr (Cdh1)-APC/C complexes activated at centrosomes could diffuse throughout the cell to catalyze the destruction of cyclin B (Raff, 2002).

Fzy/Cdc20 protein is abundant in syncytial embryos, whereas Fzr/Cdh1 protein is virtually undetectable. Moreover, a D-box-mutated form of cyclin B (CBTPM-GFP), which cannot be targeted for destruction by Fzy/Cdc20, is not degraded on spindles in syncytial embryos. CBTPM-GFP can be targeted for destruction by Fzr/Cdh1, and, in cellularized embryos, where Fzr/Cdh1 is normally present, CBTPM-GFP is destroyed throughout the cell but with slowed kinetics. Taken together, these findings indicate that Fzy/Cdc20 alone is responsible for catalyzing the destruction of cyclin B on the spindle in syncytial embryos, whereas Fzr/Cdh1 can catalyze the destruction of cyclin B throughout the cell in cellularized embryos (Raff, 2002).

These results suggest a model of how the destruction of Drosophila cyclin B is regulated in space and time. Early in mitosis, inhibitory checkpoint protein/Fzy (Cdc20) complexes form at unattached kinetochores. It is proposed that these complexes are restricted to the spindle microtubules, and spread from the kinetochore to the centrosome, and then throughout the spindle. As the kinetochores align at the metaphase plate, inhibitory complexes no longer form, and this leads to the activation of Fzy (Cdc20)-APC/C complexes. Exactly where and how this activation occurs is unclear, but it is proposed that only the specific pool of Fzy/Cdc20 that has passed through the kinetochore (and so is restricted to the spindle) is activated to degrade cyclin B. The destruction of cyclin B on the spindle then initiates the second phase of cyclin B destruction by activating Fzr/Cdh1-APC/C complexes. Unlike the Fzy/Cdc20 complexes, activated Fzr/Cdh1 complexes are not restricted to spindle microtubules, and can target cyclin B for destruction throughout the cell (Raff, 2002).

Since the destruction of cyclin B appears to initiate at centrosomes, it is suspected that the Fzy (Cdc20)-APC/C complexes initially become activated to degrade cyclin B at centrosomes. Presumably, the activated complexes then spread along the microtubules toward the spindle equator. This would explain why, in syncytial Drosophila embryos where only Fzy/Cdc20 is present, the attachment between centrosomes and spindles appears to be essential for the destruction of the spindle-associated cyclin B. Why Fzy/Cdc20 might initially be activated at centrosomes is unclear. Perhaps the disassembly of the inhibitory checkpoint protein/Fzy (Cdc20) oligomers that form at the unattached kinetochores requires some activity that is concentrated at centrosomes (Raff, 2002).

It is stressed that this model applies only to the destruction of cyclin B. For example, cyclin A is also targeted for destruction by Fzy (Cdc20)-APC/C complexes, but it is not concentrated on spindles. It seems unlikely that Fzy/Cdc20 also catalyzes the destruction of cyclin A only on the spindle. Therefore, it is speculated that there must be separate pools of Fzy/Cdc20 that are responsible for degrading cyclin A and B. An attractive aspect of this model is that it explains how these different pools are generated. Only the pool of Fzy/Cdc20 that passes through the kinetochore is inhibited from activating the APC/C by the spindle checkpoint system, and only this pool of Fzy/Cdc20 is competent to catalyze the destruction of cyclin B. In this way, the destruction of cyclin B is inhibited by the spindle checkpoint system, whereas the destruction of cyclin A is not (Raff, 2002).

Could this mechanism for regulating cyclin B destruction in Drosophila embryos apply to other systems? If two vertebrate mitotic cells are fused to form a single cell, the presence of an unattached kinetochore on one spindle (spindle A) does not block the exit from mitosis on the other spindle (spindle B) once the chromosomes on spindle B have aligned. Moreover, once spindle B exits mitosis, spindle A exits mitosis soon afterwards, even if some of its kinetochores remain unattached. These observations are consistent with the model. It would be predicted that the Fzy (Cdc20)/checkpoint-protein complexes generated at the unattached kinetochores of spindle A are restricted to microtubules and so cannot inhibit the exit from mitosis on the neighboring spindle B. Moreover, the activation of Fzy/Cdc20 on spindle B would eventually activate Fzr (Cdh1)-APC/C complexes on spindle B. These complexes can then spread throughout the cell, ultimately degrading cyclin B on spindle A and forcing it to exit mitosis. The degradation of clb2 in S. cerevisiae also occurs in two phases that appear to be catalyzed sequentially by Fzy/Cdc20 and Fzr/Cdh1, although the spatial organization of this destruction has not been investigated (Raff, 2002).

However, the model cannot explain how cyclin B is degraded in early Xenopus embryo extracts. Like early Drosophila embryos, these extracts contain Fzy/Cdc20, but lack Fzr/Cdh1. Nonetheless, cyclin B is completely degraded at the end of mitosis in these extracts, even if no nuclei or spindles are present. Thus, Fzy/Cdc20 can catalyze the destruction of cyclin B that is not spindle associated in Xenopus extracts. The reason for this apparent difference is unclear. However, it is noted that early Xenopus extracts do not have a functional spindle checkpoint. The mechanisms that link the destruction of cyclin B to the spindle checkpoint may also be required to restrict Fzy/Cdc20 complexes to the mitotic spindle (Raff, 2002).

The finding that Fzy/Cdc20 and Fzr/Cdh1 are concentrated at centrosomes highlights the potential importance of this organelle in regulating the exit from mitosis. It is speculated that the concentration of these proteins at centrosomes might serve two purposes. (1) It might enhance the fidelity of their sequential activation. The inactivation of cyclin B/cdc2 triggered by Fzy/Cdc20 seems to start at centrosomes, and cyclin B levels might only have to fall below a certain threshold level at the centrosome (rather than throughout the whole cell) to trigger the activation of the centrosomal Fzr/Cdh1. (2) In budding yeast there is a second, Bub2-dependent checkpoint that monitors the positioning of the spindle between the mother and daughter cell. Bub2 is concentrated at the spindle pole body where it is thought to suppress the activation of the mitotic exit network, and so block the activation of Fzr/Cdh1 and the exit from mitosis. It is not clear if mammalian cells also have a spindle orientation checkpoint, but if they do, the concentration of Fzr/Cdh1 at centrosomes may be important for the function of this checkpoint (Raff, 2002).

CycA-dependent kinase activity is required to inhibit one inhibitor of mitosis, the Fzr protein

Cyclin A (CycA), the only essential mitotic cyclin in Drosophila, is cytoplasmic during interphase and accumulates in the nucleus during prophase. Interphase localization is mediated by Leptomycin B (LMB)-sensitive nuclear export. This is a feature shared with human CyclinB1, and it is assumed that nuclear accumulation is necessary for mitotic entry. Whether the unique mitotic function of CycA requires nuclear accumulation has been tested. Subcellular localization signals were fused to CycA and their mitotic capability was tested. Surprisingly, nuclear accumulation was not required, and even a membrane-tethered form of CycA is able to induce mitosis. It was noted that Cyclin B (CycB) protein disappears prematurely in CycA mutants, reminiscent of rca1 mutants. Rca1 is an inhibitor of Fizzy-related-APC/C activity, and in rca1 mutants, mitotic cyclins are degraded in G2 of the 16th embryonic cell cycle. Overexpression of Rca1 can restore mitosis in CycA mutants, indicating that the mitotic failure of CycA mutants is caused by premature activation of the APC/C. The essential mitotic function of CycA is therefore not the activation of numerous mitotic substrates by Cdk1-dependent phosphorylation. Rather, CycA-dependent kinase activity is required to inhibit one inhibitor of mitosis, the Fzr protein (Dienemann, 2004).

Drosophila CycA displays a striking change in its subcellular localization at the onset of mitosis. This is true for a HA-tagged version of CycA (HA-CycA), whose localization and destruction is indistinguishable from the endogenous CycA. HA-CycA is cytoplasmic in interphase and accumulates in the nucleus at prophase. This nuclear accumulation of HA-CycA correlates with chromosome condensation in prophase cells. Therefore, nuclear CycA-Cdk1 activity might trigger mitotic events in the nucleus, and this might be the essential function of CycA for mitosis (Dienemann, 2004).

Human Cyclin B1 (CycB1) displays a similar subcellular distribution throughout the cell cycle caused by a dynamic shuttling between nucleus and cytoplasm. During interphase, export from the nucleus prevails and results in cytoplasmic localization of CycB1. Preventing nuclear export by using LMB, which is a well-established inhibitor of nuclear export, results in nuclear accumulation even in interphase. To test if CycA localization is mediated by nuclear export, Drosophila embryos were treated with LMB and nuclear accumulation of CycA was observed in interphase cells. Thus localization of CycA is mediated by a controlled balance between nuclear import and export, similar to what has been shown for CycB1 (Dienemann, 2004).

To test the functional significance of the subcellular dynamics of CycA, its localization was changed by forcing constitutive nuclear accumulation or preventing prophase accumulation. This was realized by the fusion of heterologous localization signals onto the N terminus of CycA. Such N-terminal fusions did not impair the ability of these constructs to activate Cdk1. The expression of the constructs was accomplished by the UAS-Gal4 system. Expression levels were comparable and below that of endogenous CycA. Thus, any effects caused by these constructs are not due to overexpression artifacts (Dienemann, 2004).

To achieve a constitutive nuclear localization, CycA was fused to the nuclear localization sequence (NLS) of the SV 40 large T antigen. This results in constitutive nuclear CycA even during interphase. Yet, no premature mitotic events were observed. Obviously, nuclear accumulation of CycA is not sufficient to induce mitosis since inhibitory phosphorylations are present on Cdk1 and expression of the phosphatase CDC25String is limiting for Cdk1 activation (Dienemann, 2004).

To enhance nuclear export of CycA even during prophase, the nuclear export signal (NES) of human PKI was fused to CycA. This construct delayed nuclear prophase accumulation. In comparison with the endogenous CycA, HA-NES-CycA was nuclear only in an advanced state of prophase probably after nuclear envelope breakdown. This shows that nuclear export is not globally shut down during prophase, allowing the continuing export of HA-NES-CycA at early prophase stages. Thus, nuclear export of wild-type CycA must be regulated to allow nuclear accumulation during prophase, which can be counteracted by the fusion of an exogenous export signal that is apparently not subject to this regulation (Dienemann, 2004).

In order to completely exclude CycA from the nucleus, CycA was tethered to the membrane. The Torso receptor tyrosine kinase was used and the cytoplasmic region with the HA-CycA coding region was replaced. Initial localization studies were carried out by a transient expression assay in which mRNA encoding Tor-HA-CycA was injected into Drosophila embryos. The distribution of Tor-HA-CycA in such embryos displays the expected plasma membrane localization pattern. Importantly, even mitotic cells show membrane localized HA staining of the Tor-HA-CycA construct. Similarly, in older embryos in which expression was induced by the UAS-Gal4 system, Tor-HA-CycA was never found to accumulate in the nucleus. However, this construct is not restricted to the outer rim of the plasma membrane. Extracts from embryos expressing Tor-HA-CycA were fractionated into membrane and cytoplasmic fractions and Tor-HA-CycA was found specifically enriched in the membrane fraction. This indicates that Tor-HA-CycA is confined to the membrane compartment within the cell, and the observed staining in older embryos likely reflects the presence of this construct in the endomembrane system. These localization data show that the used heterologous localization signals are functional in Drosophila, redirecting the CycA constructs to the desired locations (Dienemann, 2004).

It was then asked if the differently localized CycA constructs were able to fulfill the mitotic function of CycA. Epidermal cells lacking CycA fail to execute the 16th mitosis. As a consequence, mutant embryos have fewer but bigger cells compared to wild-type. Expression of CycA from a transgene can overcome the mitotic defect best seen by a comparison of CycA mutant cells with those that express CycA. The prdGal4 driver line was used to achieve expression in every other segment. Abdominal segment A1, in which prd is active, was compared with segment A2. Expression of HA-CycA results in increasing cell numbers and reduction in cell size, indicating that HA-CycA can restore mitosis in CycA mutant embryos (Dienemann, 2004).

Whether HA-NLS-CycA and HA-NES-CycA are able to overcome the CycA mutant phenotype was tested in epidermal cells. Both constructs induced cell divisions in the CycA mutant background. HA-NES-CycA expression in segment A1 resulted in cell numbers comparable to wild-type, and HA-NLS-CycA expression even slightly increased cell numbers. Thus, neither cytoplasmic nor nuclear localization during early prophase stages is required for mitosis 16 in epidermal cells. Whether CycA localization is important for any of the other mitoses that occur during development was tested. Unfortunately, the endogenous CycA promoter is large and not well characterized. Therefore, being aware that unpatterned expression of CycA might disturb development, the ubiquitous daGal4 driver line was used. When expression was at moderate levels, CycA mutant flies were recovered that express HA-CycA ubiquitously. As expected from an unpatterned CycA expression, flies were recovered at low frequency and showed various abnormalities, like rough eyes, bristle defects, and reduced viability. But this experimental setup allowed a test of whether HA-NES-CycA or HA-NLS-CycA are able to support all mitoses during embryonic and larval life. In both cases, CycA mutant flies were recoved at similar frequencies, indicating that they can mediate proliferation throughout development. This shows that the normal subcellular dynamic of CycA is not essential for proliferation (Dienemann, 2004).

The expression of Tor-HA-CycA during embryogenesis in a CycA mutant did result only in a limited increase in cell number. In contrast, a high number of cells positive for the mitotic marker PH3 was observed, indicating that this construct was able to induce mitosis. This, and additional evidence leads to the conclusion that a membrane-anchored form of CycA is able to induce mitosis (Dienemann, 2004).

This raises the following question: how can nuclear mitotic events be triggered when CycA-dependent Cdk1 activity is prevented from entering the nucleus? The three mitotic cyclins in Drosophila, CycA, CycB and CycB3, have partially redundant functions. However, in CycA mutants, the presence of CycB and CycB3 is clearly not sufficient to induce mitosis. To further corroborate this, additional, HA-tagged CycB (HA-CycB) or a nuclear-localized CycB (NLS-CycB) were expressed in a CycA mutant. Both activate Cdk1 in vitro, but neither induced proliferation in a CycA mutant background. CycB protein distribution was analyzed in CycA mutant embryos; it was noticed that CycB was degraded prematurely in cells that would normally go into mitosis shortly -- i.e., in the G2 stage of cell cycle 16. This can be seen best in CycA mutant embryos in which every other segment is 'rescued' by HA-CycA, or even Tor-HA-CycA. This phenotype is reminiscent of the rca1 mutant phenotype. In rca1 mutants, mitotic cyclins are degraded prematurely in G2 during the 16th embryonic cell cycle. Rca1 is an inhibitor of Fizzy-related (Fzr)-dependent APC/C activity (Grosskortenhaus, 2002). As cells prepare for the first G1 phase during embryogenesis, Fzr, which is required for the establishment of G1, is upregulated. Several partially redundant mechanisms prevent Fzr-APC/C activity in G2. Besides Rca1, CycA-Cdk1 contributes to Fzr inactivation. The disappearance of CycB in CycA mutants suggests that Fzr becomes activated prematurely. To test if this is the case, Rca1 was overexpressed in CycA mutants to prevent Fzr activation. Indeed, Rca1 overexpression is sufficient to prevent premature CycB degradation and cell divisions could occur. Rca1 overexpression could not completely restore cell numbers; indicating that CycA inhibition of Fzr is of greater importance in this situation. This function of CycA can apparently not be fulfilled by the endogenous CycB or even after overexpression of CycB. Human Cyclin A can interact with Fzr through a so-called RXL motif in Fzr and a hydrophic patch in Cyclin A. Such a motif is also present in Drosophila Fzr, possibly causing its CycA-Cdk1-dependent phosphorylation. Apparently, this function of CycA is not necessary in the nucleus, in agreement with findings that Fzr is predominantly localized to the cytoplasm. When CycA is tethered to the membrane, inhibition of Fzr might be sufficient to allow entry into mitosis. Presumably, the Fzr protein itself is shuttling between the cytoplasm and the nucleus, thereby allowing inactivation wherever CycA is localized (Dienemann, 2004).

In vertebrates as well as in Drosophila, overexpression of CycA results in ectopic S phases. In addition, nuclear CycB1 was shown to be able to induce S phase in vertebrates. Tests were performed to see if the subcellular localization of CycA is important for S phase induction by expressing the different CycA constructs during eye imaginal disc development. The different CycA constructs were expressed in postmitotic cells by using the sevGal4 driver. Expression of HA-CycA as well as all other CycA constructs used in this study but none of the CycB constructs, including the NLS-CycB construct, did result in ectopic S phases. At present, it is not known how membrane anchored CycA can induce S phase, which is a clear nuclear event. Possibly, Fzr is inactivated in G1 by the Tor-HA-CycA that is not degraded efficiently during mitosis and persists in the G1 state. After Fzr inactivation, the half-life of endogenous CycA during G1 would be increased and could trigger the observed S phases (Dienemann, 2004).

In conclusion, these data show that the dynamic changes in the subcellular localization of CycA are not essential for its mitotic function. It is suggested that the unique function of CycA for mitosis does not lie in the activation of specific mitotic substrates by Cdk1-dependent phosphorylation. Rather, CycA dependent kinase activity is required to inhibit one inhibitor of mitosis, namely the Fzr protein. In the absence of CycA premature APC/C activation results in the degradation of substrates that are required for mitotic entry, like CycB. Since overexpression of Cyclin B is not sufficient to restore mitosis, other substrates that are necessary for mitotic entry might by degraded by Fzr-dependent APC/C activity as well -- one candidate being Cdc25, whose levels are regulated by the APC/C during the cell cycle. The Drosophila system allowed a test the functional requirements for CycA in a mutant background. Such an analysis is difficult in vertebrate cells since CycB1 mutant mice die very early in utero and functional studies are complicated by the fact that sites that are required for nuclear entry are also required for CycB1 activation. While nuclear accumulation of CycA at prophase might not be essential, whether it is important for the normal kinetics of mitotic progression and whether its cytoplasmic location during interphase is important in checkpoint controls as it was shown for CycB1 in vertebrates is currently being investigated (Dienemann, 2004).

Terminal mitoses require negative regulation of Fzr/Cdh1 by Cyclin A, preventing premature degradation of mitotic cyclins and String/Cdc25

Cyclin A expression is only required for particular cell divisions during Drosophila embryogenesis. In the epidermis, Cyclin A is strictly required for progression through mitosis 16 in cells that become post-mitotic after this division. By contrast, Cyclin A is not absolutely required in epidermal cells that are developmentally programmed for continuation of cell cycle progression after mitosis 16. These analyses suggest the following explanation for the special Cyclin A requirement during terminal division cycles. Cyclin E is known to be downregulated during terminal division cycles to allow a timely cell cycle exit after the final mitosis. Cyclin E is therefore no longer available before terminal mitoses to prevent premature Fizzy-related/Cdh1 activation. As a consequence, Cyclin A, which can also function as a negative regulator of Fizzy-related/Cdh1, becomes essential to provide this inhibition before terminal mitoses. In the absence of Cyclin A, premature Fizzy-related/Cdh1 activity results in the premature degradation of the Cdk1 activators Cyclin B and Cyclin B3, and apparently of String/Cdc25 phosphatase as well. Without these activators, entry into terminal mitoses is not possible. However, entry into terminal mitoses can be restored by the simultaneous expression of versions of Cyclin B and Cyclin B3 without destruction boxes, along with a Cdk1 mutant that escapes inhibitory phosphorylation on T14 and Y15. Moreover, terminal mitoses are also restored in Cyclin A mutants by either the elimination of Fizzy-related/Cdh1 function or Cyclin E overexpression (Reber, 2006).

Mitotic cyclins accumulate during the S and G2 phases of the cell cycle. Their C-terminal cyclin boxes mediate binding to cyclin-dependent kinase 1 (Cdk1). Their rapid degradation during late M and G1 phase depends on the D- and KEN-boxes in their N-terminal domains. These degradation signals are recognized by Fizzy/Cdc20 (Fzy) and Fizzy-related/Cdh1 (Fzr), which recruit the mitotic cyclins to the anaphase-promoting complex/cyclosome (APC/C) during M and G1, respectively. The ubiquitin ligase activity of the APC/C allows cyclin poly-ubiquitination and consequential proteolysis (Reber, 2006).

Metazoan species express three different types of mitotic cyclins: A, B and B3. The specific functions of these different cyclins are not understood in detail. The presence of single genes coding for either Cyclin A (CycA), Cyclin B (CycB) or Cyclin B3 (CycB3) has facilitated a genetic dissection of their functional specificity in Drosophila melanogaster. In this organism, development to the adult stage requires the zygotic function of CycA, but not of CycB or CycB3. Initial analysis of the embryonic cell proliferation program in CycA mutants revealed that epidermal cells fail to progress through the sixteenth round of mitosis. Cyclin A is also required for mitosis 16 in the epidermis of dup/Cdt1 mutant embryos, in which mitosis 16 is no longer dependent upon completion of the preceding S phase. The failure of mitosis 16 in CycA mutants therefore does not simply result from the activation of a DNA replication or damage checkpoint -- a possibility suggested by evidence obtained in vertebrate cells in which Cyclin A binds not only to Cdk1 but also to Cdk2, and provides crucial functions during S phase (Reber, 2006 and references therein).

The accumulation of Cyclin B and Cyclin B3 during cycle 16, which also occurs in CycA mutants, complicates the explanation of why mitosis 16 in the epidermis requires Cyclin A. In Xenopus egg extracts, Cyclin B can trigger entry into mitosis in the absence of Cyclin A. Conversely, mitosis is clearly inhibited in cultured human cells after the microinjection of antibodies against cyclin A. Cyclin A-Cdk1 complexes are thought to have special properties, important for starting up a positive-feedback loop that confers a switch-like behavior on the Cdk1 activation process. In this feedback loop, Cdk1 activity results in phosphorylation and suppression of the inhibitory Wee1 kinase, as well as in phosphorylation and activation of the String/Cdc25 phosphatase, which removes the inhibitory phosphate modifications from Cdk1. However, the analyses described in this study indicate that the Cyclin A requirement in Drosophila is not linked to this positive-feedback loop. Rather, it is linked to the fact that the sixteenth round of mitosis during embryogenesis is the last cell division for the great majority of the epidermal cells (Reber, 2006).

After mitosis 16, most epidermal cells enter a G1 phase and become mitotically quiescent. By contrast, all the previous embryonic divisions (mitoses 1-15) are followed by an immediate onset of S phase. The G1 phase after mitosis 16 is therefore the first G1 phase during development. Entry into this G1 phase is dependent upon a complete, developmentally controlled inactivation of Cyclin E-Cdk2 and Cyclin A-Cdk1, because both complexes can trigger entry into S phase. Cyclin E-Cdk2 inactivation results from transcriptional CycE downregulation and concomitant upregulation of dacapo, which encodes the single Drosophila CIP/KIP-type inhibitor specific for Cyclin E-Cdk2. Cyclin A-Cdk1 inactivation is dependent on Fzr, which is also transcriptionally upregulated. Moreover, Fzr is activated as a consequence of Cyclin E-Cdk2 inactivation. Importantly, this cell cycle exit program is initiated already during G2 of the final division cycle (Reber, 2006).

Although cycle 16 is the final division cycle for most epidermal cells, some defined regions do not activate the cell cycle exit program during cycle 16. Instead, they maintain CycE expression, enter S phase immediately after mitosis 16 and complete an additional division cycle 17. In these regions, mitosis 16 is not fully inhibited in CycA mutants. Cyclin A is therefore especially important for terminal mitoses preceding G1 and cell cycle exit. This study shows that the downregulation of Cyclin E-Cdk2 before terminal divisions, in preparation for the imminent cell cycle exit, converts Cyclin A from a redundant into an indispensable, negative regulator of Fizzy-related/Cdh1, preventing premature degradation of the mitotic inducers String/Cdc25 and the mitotic cyclins. The significance of the basic cell cycle regulator Cyclin A therefore depends on the developmental context (Reber, 2006).

The phenotypical characterization of mutations in the Drosophila genes encoding the A-, B- and B3-type cyclins have indicated that Cyclin A is the most crucial of these co-expressed mitotic cyclins. Although zygotic CycB or CycB3 function is not essential for cell proliferation and development to the adult stage, null mutations in CycA result in embryonic lethality. This study has clarified the molecular basis of the distinct importance of Cyclin A. The results indicate that the crucial role of Cyclin A is linked to its ability to inhibit Fzr-APC/C-mediated degradation. Moreover, this Cyclin A-dependent negative regulation of the Fzr-APC/C-degradation pathway is of particular importance for progression through the very last mitotic division preceding cell cycle exit and the proliferative quiescence of epidermal cells during embryogenesis. This particular Cyclin A requirement during terminal divisions is caused by a cell cycle exit program that is initiated already before the terminal mitosis. The cell cycle exit program includes downregulation of Cyclin E-Cdk2, which has a comparable ability to inhibit the Fzr-APC/C-degradation pathway to Cyclin A. The downregulation of Cyclin E-Cdk2 by the cell cycle exit program turns Cyclin A into an indispensable inhibitor of the premature degradation of mitotic cyclins and String/Cdc25 via Fzr-APC/C before the terminal mitosis. Accordingly, the terminal mitosis in the epidermis of CycA mutants can be restored by overexpression of Cyclin E, by genetic elimination of Fzr, or by simultaneous expression of the String/Cdc25-independent Cdk1AF mutant and B-type cyclin versions that are no longer Fzr-APC/C substrates (Reber, 2006).

The fact that Cyclin A is also a substrate of Fzr-APC/C-mediated degradation complicates the interpretation of the results. Two findings, however, strongly suggest that Cyclin A functions not just downstream of Fzr, but also upstream as a negative regulator. The observed premature loss of B-type cyclins in CycA mutants is readily explained by a negative effect of Cyclin A on Fzr-APC/C activity and is difficult to explain if Cyclin A was only a Fzr-APC/C substrate. Moreover, the suppression of the UAS-fzr overexpression phenotype by co-expression of UAS-CycA, which is described here, includes the re-accumulation of B-type cyclins and not just the restoration of terminal mitosis 16 (Reber, 2006).

Work in mammalian cells has clearly established that Cyclin A functions as a negative regulator of Fzr/Cdh1. Human Cyclin A can bind directly to Cdh1. Moreover, Cyclin A-dependent Cdk activity phosphorylates Cdh1, resulting in the dissociation of Cdh1 from APC/C. Conversely, mutations in Cdk consensus phosphorylation sites of human CDH1 were reported to abolish inhibition by Cyclin A. The current findings point to alternative modes of Fzr-APC/C-inhibition by Cyclin A. Fzrpsm variant no longer contains canonical Cdk consensus phosphorylation sites (S/T P) and yet its activity is still suppressed by CycA overexpression. Fzr inhibition by CyclinA-dependent phosphorylation of non-consensus sites remains a possibility in Drosophila. However, it is pointed out that, apart from a potential control by Cdk phosphorylation, Fzr is inhibited by the Emi1-like Drosophila protein Rca1. Rca1 overexpression has been shown to prevent premature Cyclin B degradation and restore mitosis 16 in the epidermis of CycA mutant embryos. Based on these observations, the failure of mitosis 16 in CycA mutants was proposed to reflect premature Fzr activation, a suggestion fully confirmed by the current work. It is conceivable, therefore, that the Cyclin A-mediated suppression of Fzrpsm activity involves Rca1 or other unknown targets. The fact that not only Cyclin A, but also Cyclin E, effectively suppresses Drosophila Fzr and Fzrpsm provides further support of additional regulatory complexity. In vertebrate systems, only Cyclin A and not Cyclin E was shown to bind and inhibit Cdh1 (Reber, 2006).

The current findings demonstrate that the Cyclin A requirement in epidermal cells is maximal for progression through the last mitosis of Drosophila embryogenesis, which precedes cell cycle exit and proliferative quiescence. A prominent Cyclin A requirement for terminal mitoses appears to exist in neuroblast lineages during development of the embryonic CNS, although definitive proof will require further work. On the basis of this analysis in epidermal cells, a high Cyclin A requirement for entry into mitosis is expected whenever Fzr levels are high and Cyclin E levels low. During the comparatively slow postembryonic cell cycles of imaginal cells, the periodicity of Cyclin E expression is presumably far more pronounced than during the rapid embryonic cycles in which the persistent presence of maternally contributed Cyclin E eliminates G1 phases. In imaginal cell cycles, which have a G1 phase, Cyclin E expression might therefore be low before each mitosis, and not just before terminal divisions. In combination with Fzr expression, every imaginal mitosis might therefore be strongly dependent upon Cyclin A. By contrast, in the absence of Fzr, progression through mitosis appears to be almost completely independent of Cyclin A, as is evidenced by the observation that the epidermal cells in fzr CycA double mutant embryos not only progress successfully through mitosis 16, but also complete an extra division cycle 17. Nevertheless, 10% of the late mitosis 17 figures in these double mutants displayed lagging chromosomes, indicating that cell cycle progression is not entirely normal in the absence of Cyclin A (Reber, 2006).

The cell cycle exit program, which is activated during the final division cycle in the embryonic epidermis, includes the strong transcriptional upregulation of the CIP/KIP-type Cyclin E-Cdk2 inhibitor Dacapo, apart from the downregulation of Cyclin E and the upregulation of Fzr. Accordingly, genetic elimination of dacapo function should also restore progression through terminal mitosis 16 in CycA mutants. However, mitosis 16 was not observed in the epidermis of dacapo CycA double mutants. The contribution of Dacapo to Cyclin E-Cdk2 inhibition appears to be insignificant before mitosis 16. After the stage of mitosis 16, however, the epidermal cells in these double mutants entered an endoreduplication cycle, a behavior that is also displayed by some cells in the prospective anterior spiracle region of CycA single mutants. This region does not downregulate Cyclin E during cycle 16 in the wild type, it does not upregulate Dacapo, and it progresses through an additional cycle 17 instead of becoming postmitotic after mitosis 16, in contrast to the great majority of the other epidermal cells. The premature activation of Fzr in CycA mutants, therefore, appears to result in DNA replication origin re-licensing, perhaps as a result of B-type cyclin and geminin degradation. Cyclin E-Cdk2 activity might subsequently trigger endoreduplication in cells in which it is not effectively eliminated by both Cyclin E downregulation and Dacapo upregulation. Importantly, not all cells in the anterior spiracle region of CycA mutants endoreduplicate, some of the cells still manage to divide. This variability could reflect minor differences in the onset and strength of the zygotic Cyclin E expression. The outcome of insufficient Cyclin A levels appears to be highly dependent on the levels of Cyclin E and Fzr, which, in turn, are subject to developmental regulation, in particular during cell cycle exit. The significance of basic cell cycle regulators in vivo is therefore different in various tissues and developmental stages, and most likely in various cultured mammalian cell types as well (Reber, 2006).

APC/CFzr/Cdh1 promotes cell cycle progression during the Drosophila endocycle

The endocycle is a commonly observed variant cell cycle in which cells undergo repeated rounds of DNA replication with no intervening mitosis. How the cell cycle machinery is modified to transform a mitotic cycle into endocycle has long been a matter of interest. In both plants and animals, the transition from the mitotic cycle to the endocycle requires Fzr/Cdh1, a positive regulator of the Anaphase-Promoting Complex/Cyclosome (APC/C). However, because many of its targets are transcriptionally downregulated upon entry into the endocycle, it remains unclear whether the APC/C functions beyond the mitotic/endocycle boundary. This study reports that APC/CFzr/Cdh1 activity is required to promote the G/S oscillation of the Drosophila endocycle. Compromising APC/C activity, after cells have entered the endocycle, inhibits DNA replication and results in the accumulation of multiple APC/C targets, including the mitotic cyclins and Geminin. Notably, the data suggest that the activity of APC/CFzr/Cdh1 during the endocycle is not continuous but is cyclic, as demonstrated by the APC/C-dependent oscillation of the pre-replication complex component Orc1. Taken together, these data suggest a model in which the cyclic activity of APC/CFzr/Cdh1 during the Drosophila endocycle is driven by the periodic inhibition of Fzr/Cdh1 by Cyclin E/Cdk2. It is proposed that, as is observed in mitotic cycles, during endocycles, APC/CFzr/Cdh1 functions to reduce the levels of the mitotic cyclins and Geminin in order to facilitate the relicensing of DNA replication origins and cell cycle progression (Narbonne-Reveau, 2008).

The endocycle provides a useful model for determining the minimum cell cycle inputs required to achieve a G/S oscillation and the once-per-cell-cycle replication of the genome. This study demonstrates that APC/C activity is required for endocycle progression. During the endocycle, mitotic activities are repressed. This is accomplished, at least in part, by preventing the accumulation of the mitotic activators Cyclin A, Cyclin B and Cdc25, which function to activate the mitotic kinase Cdk1. During the mitotic cycle, the mitotic cyclins are periodically targeted for regulated proteolysis by the E3-Ubiquintin ligase the APC/C. Yet the transcriptional downregulation of several APC/C targets at the mitotic/endocycle boundary, including the mitotic cyclins and String/Cdc25, suggested that the proteolytic activity of the APC/C might not be necessary during endocycles. However, this study found that compromising APC/C activity, after cells have entered the endocycle, results in the accumulation of Geminin and the mitotic cyclins, and in a block of DNA replication. Thus, the transcriptional downregulation of APC/C targets observed at the mitotic/endocycle transition is either downstream of APC/C activity and/or not sufficient to maintain low levels of these proteins. Taken together, these data suggest a model in which APC/C promotes the G/S oscillation of the endocycle by preventing the unscheduled accumulation of Geminin and the mitotic cyclins (Narbonne-Reveau, 2008).

During endocycles, APC/C activity prevents the inappropriate accumulation of Geminin, an inhibitor of the DNA replication-licensing factor Cdt1/Dup. When directly expressed in endocycling cells, Geminin efficiently inhibits DNA replication. These results strongly suggest that an essential function of the APC/C during the endocycle is to prevent the unregulated accumulation of Geminin. A similar role has been proposed for the APC/C during endoreplicative cycles of mouse trophoblasts (Gonzalez, 2006). However, the current data indicate that Geminin is not the only essential target of the APC/C during endocycles. A candidate for a second important target of the APC/C during endocycles is Cyclin A. Previous studies have shown that the overexpression of Cyclin A in the salivary gland, between the first and second endocycle, results in variable inhibitory effects on endoreplication. Although the majority of salivary gland cells that overexpress Cyclin A appear to be unaffected, a small percentage show a marked decrease in ploidy values. The reason for this variability is not clear. However, if the inhibitory influence of Cyclin A is mediated through binding and activation of Cdk1, this effect may be greatly amplified in the presence of high levels of String/Cdc25, which removes an inhibitory phosphate from Cdk1. Recent studies indicate that String/Cdc25, which contains both a consensus Ken box and D-box, is a target of the APC/C (Barbara Thomas, personal communication to Narbonne-Reveau, 2008). Therefore, an essential function of the APC/C during endocycles may involve restricting the activity of the mitotic kinase Cdk1, by preventing the accumulation of both Cyclin A and String/Cdc25. Finally, it is noted that the APC/C may have additional essential targets during the endocycle, which have yet to be identified (Narbonne-Reveau, 2008).

The periodic accumulation of the Orc1 protein during endocycles strongly suggests that the activity of the APC/CFzy/Cdh1 may not be continuous but cyclical. Previous work indicates that in Drosophila Cyclin E/Cdk2 inhibits the activity of APC/CFzy/Cdh1. These data are consistent with the observation that phosphorylation of Fzr/Cdh1 by Cdks inhibits the ability of Fzr/Cdh1 to bind and activate the APC/C in yeast, Xenopus and mammals. During the endocycle, the levels of Cyclin E oscillate. Taken together, these observations suggest a model in which APC/CFzy/Cdh1 is regulated by the periodicity of Cyclin E/Cdk2 activity, with high levels of Cyclin E resulting in the inhibition of APC/CFzy/Cdh1 activity and low levels of Cyclin E permitting full APC/CFzy/Cdh1 activity. The current data support this hypothesis. First, it was found that the periodicity of Orc1 levels during the endocycle requires a functional O-box, consistent with the cyclic destruction of Orc1 by APC/CFzy/Cdh1. Second, the levels of Orc1 are sensitive to Cyclin E. Specifically, overexpressing Cyclin E after cells have entered the endocycle results in the accumulation of APC/CFzy/Cdh1 targets, including Orc1, Cyclin A, Cyclin B and Geminin. Thus, the regulatory relationship observed between Cyclin E/Cdk2 and Fzr/Cdh1 that has been reported during mitotic cycles is conserved during endocycles. Finally, in endocycling cells the accumulation of Orc1 occurs during periods of high Cyclin E/Cdk2 activity, when APC/CFzy/Cdh1 dependent proteolysis would be predicted to be low. These data support the idea that the oscillations of Cyclin E/Cdk2 activity drive the periodicity of APC/CFzy/Cdh1 activity during the endocycle (Narbonne-Reveau, 2008).

Although a requirement for the oscillation of APC/CFzy/Cdh1 activity during the Drosophila endocycle has not been formally demonstrated, it is interesting to speculate on how the cyclic, rather than the continuous, activity of the APC/C might serve to facilitate endocycle progression. The data indicate that a period of high APC/CFzy/Cdh1 activity is required during the G phase of the endocycle in order to degrade the mitotic cyclins and Geminin, which can function to inhibit the formation of pre-RCs. However, a period of low APC/C activity may also be important. The continuous activation of APC/CCdh1 significantly slows DNA replication in mouse tissue culture cells. This inhibition may reflect the inability of a cell to accumulate adequate levels of proteins required for DNA replication, such as the APC/CCdh1 target and pre-replication complex component CDC6, in the presence of a constitutively active APC/CCdh1. In Drosophila, continuous APC/CFzy/Cdh1 activity might prevent the accumulation of two pre-RC components, CDC6 and Orc1. Intriguingly, APC/C activity also appears to oscillate during mammalian endocycles. In endocycling mouse trophoblasts, the levels of Cyclin A oscillate, consistent with the regulated destruction of the Cyclin A protein by the APC/C. Additionally, the inhibition of APC/C activity in endocycling trophoblasts results in the accumulation of the APC/C targets Cyclin A and Geminin. Taken together, these observations support a model in which the oscillation of APC/CFzy/Cdh1 activity, which is driven by the regulatory influences of Cdks, promotes efficient cell cycle progression during the endocycle (Narbonne-Reveau, 2008).

The data raises important questions. Why do levels of some APC/CFzy/Cdh1 targets, such as Cyclin A, Cyclin B and Geminin, remain below the level of detection while the levels of Orc1 protein oscillate? What might account for these different modes of regulation? Currently, there is no definitive explanation. However, at least three possibilities, which are not mutually exclusive, are envisaged, that may contribute to this differential behavior. First, it was found that relative to the Cyclin A and geminin, the levels of Orc1 transcript are only minimally downregulated upon entry into the endocycle. Transcriptional downregulation, or changes in transcript stability, may help contribute to the low levels of Geminin and Cyclin A proteins observed during the endocycle. Second, the translational efficiency of a subset of transcripts may be reduced upon entry into the endocycle. Finally, it is possible that the Orc1 protein is not as efficiently targeted by the APC/CFzy/Cdh1 as the mitotic cyclins or Geminin. Indeed the cis-acting sequences that target these proteins for destruction show considerable variability. Orc1 is targeted for APC/CFzy/Cdh1 destruction via a novel motif called the O-box (Araki, 2005). By contrast, Cyclin B and Geminin are targeted by a similar but unique sequence called the destruction-box (D-box), while Drosophila Cyclin A is targeted for destruction by a large complex N-terminal degradation sequence. There is precedence for post-translational regulation of APC/CFzy/Cdh1 targets, resulting in differential expression. In mammalian cells the pre-RC component CDC6, which is structurally related to Orc1, is protected from APC/CFzr/Cdh1 degradation by phosphorylation by Cyclin E/Cdk2. One or all of these potential mechanisms may contribute to the differential expression of various APC/CFzr/Cdh1 targets during the endocycle (Narbonne-Reveau, 2008).

Recent evidence from mice indicates that the depletion of the APC/C inhibitor Emi1/Rca1, results in both a strong decrease in E2F target mRNAs, such as geminin and Cyclin A, as well as APC/C activation. This study suggested that the regulation of APC/C activity, by the inhibitor Emi1/Rca1, drives a positive feedback circuit that controls both protein stability and mRNA expression. Thus, the observed decrease in the levels of at least some APC/C targets that occurs upon depletion of Emi1/Rca1, including Geminin and Cyclin A, are controlled at the levels of transcription and protein stability. Developmentally programmed endocycles may provide a natural example where cell cycle progression occurs in the context of increased APC/CFzr/Cdh1 activity. Thus, a similar positive-feedback circuit may be operating during Drosophila endocycles to downregulate the transcription of E2F target genes. Determining the precise regulatory relationships between the upregulation of APC/CFzr/Cdh1 activity and the transcriptional downregulation of genes such as Cyclin A and geminin, during the Drosophila endocycle represents an exciting area for future research (Narbonne-Reveau, 2008).

The requirement for APC/C activity to promote endocycle progression may help answer several longstanding questions concerning the regulation of the Drosophila endocycle. For example, why does the continuous expression of Cyclin E inhibit cell cycle progression during the endocycle but not the mitotic cycle? Several models have been proposed to explain this difference. First, the breakdown of the nuclear envelope that occurs during the mitotic cycle, but not the endocycle, may allow for a transient decrease in local Cyclin E/Cdk2 activity, thus allowing for the relicensing of DNA replication origins. Alternatively, there may be differences in the machinery required to produce a functional pre-RC in mitotic versus endocycling cells. The current results suggest an alternative model for why endocycles are unusually sensitive to continuous Cyclin E expression. This model is based on the demonstration that endocycle progression requires APC/C activity. Both Fzy/Cdc20 and Fzr/Cdh1 function as activators of the APC/C. However, the regulation of these APC/C activators is very distinct. During the mitotic cycle, the binding of Fzy/Cdc20 to the APC/C is dependent on the phosphorylation of several APC/C subunits by the mitotic kinase Cdk1. By contrast, a Cdk-dependent inhibitory phosphorylation on Fzr/Cdh1 relegates APC/CFzr/Cdh1 activity to late M phase and G1. Because of its requirement for Cdk1 activity, APC/CFzy/Cdc20 is unlikely to be active during most endocycles. Indeed, Drosophila endocycles proceed normally in fzy mutants. Thus, the only available activator of the APC/C during the endocycle is Fzr/Cdh1. As previously discussed, Fzr/Cdh1 is inhibited by Cyclin E/Cdk2 activity. Therefore, it is proposed that during the endocycle, continuous Cyclin E/Cdk2 activity results in the permanent inhibition of the only available activator of the APC/C, Fzr/Cdh1. This leads to the accumulation of Geminin, Cyclin A and other potential targets, which act to block cell cycle progression. Thus, the ability of continuous Cyclin E to inhibit DNA replication during the endocycle may reflect differences in the available activators of the APC/C present in mitotic versus endocycling cells (Narbonne-Reveau, 2008).

Genome-wide analysis reveals a cell cycle-dependent mechanism controlling centromere propagation

Centromeres are the structural and functional foundation for kinetochore formation, spindle attachment, and chromosome segregation. In this study, factors required for centromere propagation were isolated using genome-wide RNA interference screening for defects in centromere protein A (CENP-A; centromere identifier [CID]) localization in Drosophila. The proteins CAL1 and CENP-C were identified as essential factors for CID assembly at the centromere. CID, CAL1, and CENP-C coimmunoprecipitate and are mutually dependent for centromere localization and function. The mitotic cyclin A (CYCA) and the anaphase-promoting complex (APC) inhibitor RCA1/Emi1 were identified as regulators of centromere propagation. CYCA was shown to be centromere localized, and CYCA and RCA1/Emi1 were shown to couple centromere assembly to the cell cycle through regulation of the fizzy-related/CDH1 subunit of the APC. These findings identify essential components of the epigenetic machinery that ensures proper specification and propagation of the centromere and suggest a mechanism for coordinating centromere inheritance with cell division (Erhardt, 2008).

This is the first example of a genome-wide RNAi screen for mislocalization of an endogenous chromosomal protein and provides the distinct advantage that the primary screen output is a direct readout of the phenotype of interest. This approach identified novel and known factors that control the assembly of centromeric chromatin and link centromere assembly and propagation to the cell cycle (Erhardt, 2008).

Although centromere assembly has been described as a hierarchical process directed by CENP-A, the data show that CID, CENP-C, and CAL1 are interdependent for centromere propagation, which is consistent with experiments in vertebrate cells showing interdependence between the CENP-H-CENP-I complex and CENP-A. However, studies in C. elegans and vertebrates have not detected a role for CENP-C in CENP-A chromatin assembly, suggesting that CENP-C plays a more prominent role in regulating centromere propagation in flies. Collectively, these results suggest that CENPs that depend on CENP-A for their localization may 'feed back' to control CENP-A assembly. Histone variants are assembled into chromatin both by histone chaperones (e.g., the histone H3.3-specific chaperone HIRA [histone regulatory A] that provides specificity to the CHD1 chromatin-remodeling ATPase) and by histone variant-specific ATPases (e.g., Swr1 that can use the general chaperone Nap1 or the specific chaperone Chz1 to assemble H2A.Z). CENP-C or CAL1 might facilitate centromere-specific CID localization by providing centromere specificity to a chromatin-remodeling ATPase in a manner analogous to HIRA or might direct the localization of chromatin assembly factors to the centromere. It will be interesting to determine what factors associate with CAL1 and CENP-C as a route to elucidating the mechanisms of centromere assembly and propagation (Erhardt, 2008).

The loading of CENP-A in human somatic cells and in Drosophila embryos occurs after anaphase initiation when APCFZR/CDH1 activity is high. Ubiquitin-mediated proteolysis facilitates formation of a single centromere by degrading noncentromeric CENP-A, and subunits of the APC are localized to kinetochores. The results demonstrate that normal regulation of APCFZR/CDH1 activity is required for centromere propagation, providing a link between centromere assembly and cell cycle regulation (Erhardt, 2008).

Two alternative models are proposed for the role of APCFZR/CDH1 in centromere function. The first model is that CYCA is the relevant substrate of APCFZR/CDH1 and that the kinase activity of the CYCA-Cdk1 complex is required for the localization of CID, CENP-C, and CAL1 to the centromere. CYCA is normally degraded as cells proceed through mitosis, suggesting that CYCA-Cdk1 would likely act during G2 or early M to phosphorylate a substrate involved in centromere assembly. The CID and CENP-C localization defect caused by CYCA depletion was rescued by the simultaneous depletion of FZR/CDH1 even though the levels of CYCA protein remained low in the double depletion. The rescue of the CID and CENP-C localization defect in cells with low CYCA protein suggests that maintaining high levels of CYCA-Cdk1 activity is not required for centromere propagation, but it cannot be ruled out that the residual CYCA protein in these cells is sufficient to rescue the centromeric phenotype when APC activity is compromised by FZR/CDH1 depletion (Erhardt, 2008).

The second model that is consistent with these observations is that one or more APCFZR/CDH1 substrates (“X”) regulate the interdependent localization of CID, CENP-C, and CAL1 to the centromere. RCA1 and CYCA inhibit the APC in G2 to allow mitotic cyclin accumulation. An APCFZR/CDH1 substrate could repress centromere assembly until anaphase/G1, when proteolysis would remove the repression in a manner analogous to replication licensing. If an APCFZR/CDH1 substrate acted solely as a negative regulator of centromere assembly, FZR/CDH1 depletion should prevent CID assembly at centromeres, and premature APCFZR/CDH1 activation by CYCA or RCA1 depletion might cause an increase of CID at centromeres as a result of premature assembly. It was observed that neither CDH1 nor CDC20 depletion alone impacted CID, CAL1, or CENP-C assembly at centromeres or the overall levels of these proteins but that premature APC activation resulted in failed centromere assembly (Erhardt, 2008).

A simple interpretation of the results is that CYCA-Cdk1 or another APCFZR/CDH1 substrate acts during G2/metaphase before APCFZR/CDH1 activation to make centromeres competent for assembly during anaphase and/or G1. Premature removal of the APCFZR/CDH1 substrate would cause failure to relicense the centromeres for assembly in the next G1 phase. When compared with the process of replication licensing, in which the positive regulator CDC6 and the negative regulators geminin and CYCA are all substrates of APCFZR/CDH1, the model of a single APCFZR/CDH1 substrate that controls centromere licensing or propagation may be oversimplified. This study observed that defective centromere localization of CID and CENP-C after CYCA or RCA1 depletion was not rescued by CDC20 depletion, but a role for APCFZY/CDC20 in centromere propagation cannot be ruled out because premature APCFZR/CDH1 activation could mask a subsequent role for FZY/CDC20, which is activated at the metaphase/anaphase transition (Erhardt, 2008).

It is not yet known whether the localization of CYCA at centromeres is important for the regulation of centromere assembly. In Drosophila, it has been demonstrated that the subcellular localization of CYCA is not important for proper progression through the cell cycle; however, these experiments did not directly address whether mislocalization of CYCA prevented the association of CYCA with centromeres. It will be interesting to determine whether CID, CENP-C, and CAL1 localization require centromere-localized CYCA-Cdk1 activity or whether any of these proteins are a direct target of CYCA-Cdk1 (Erhardt, 2008).

The results suggest that CID or CAL1 levels are indirectly controlled by APC activity. Interestingly, the human M18BP1 has recently been proposed to act as a 'licensing factor' for centromere assembly. Although no clear homologues of M18BP1/KNL2 have been identified in Drosophila, both CAL1 in flies and M18BP1/KNL2 in other species are interdependent with CENP-A for centromere localization. Strikingly, levels of CAL1 and M18BP1/KNL2 are reduced on metaphase centromeres and increase coincident with CENP-A loading in late anaphase/telophase. Further analysis is required to determine whether CAL1 and M18BP1/KNL2 function analogously in centromere assembly. It will be important to determine whether fly homologues of other Mis18 complex components are associated with CAL1 and important for centromere assembly. Identifying the APC substrates involved in centromere assembly will be necessary to distinguish between these models and to determine how these proteins epigenetically regulate centromere assembly and couple this essential process to the cell cycle (Erhardt, 2008).

The molecular chaperone Hsp90 is required for cell cycle exit in Drosophila

The coordination of cell proliferation and differentiation is crucial for proper development. In particular, robust mechanisms exist to ensure that cells permanently exit the cell cycle upon terminal differentiation, and these include restraining the activities of both the E2F/DP transcription factor and Cyclin/Cdk kinases. A genetic screen in Drosophila was designed to identify genes required for cell cycle exit. This screen utilized a reporter that is highly E2F-responsive and results in a darker red eye color when crossed into genetic backgrounds that delay cell cycle exit. Mutation of Hsp83, the Drosophila homolog of mammalian Hsp90, results in increased E2F-dependent transcription and ectopic cell proliferation in pupal tissues at a time when neighboring wild-type cells are postmitotic. Further, these Hsp83 mutant cells have increased Cyclin/Cdk activity and accumulate proteins normally targeted for proteolysis by the anaphase-promoting complex/cyclosome (APC/C), suggesting that APC/C function is inhibited. Indeed, reducing the gene dosage of an inhibitor of Cdh1/Fzr, an activating subunit of the APC/C that is required for timely cell cycle exit, can genetically suppress the Hsp83 cell cycle exit phenotype. Based on these data, it is proposed that Cdh1/Fzr is a client protein of Hsp83. The results reveal that Hsp83 plays a heretofore unappreciated role in promoting APC/C function during cell cycle exit and suggest a mechanism by which Hsp90 inhibition could promote genomic instability and carcinogenesis (Bandura, 2013).

Analysis of Hsp83 mutant clones indicates that the cell cycle exit delay is primarily due to increased Cyclin/Cdk activity. In addition, the levels of several APC/C target proteins, including Cyclin A, are increased in cells lacking Hsp83. Removing one copy of an inhibitor of the APC/CCdh1 suppresses the Hsp83 cell cycle exit phenotype. These data are consistent with a model in which Cdh1/Fzr is a client protein of Hsp83, and Hsp83 restrains Cyclin/Cdk activity after cell cycle exit by ensuring maximal activity of the APC/CCdh1. In this way, Hsp83 could provide yet another layer of control over the core cell cycle machinery to ensure that cells stop dividing on schedule during development (Bandura, 2013).

Several mechanisms have been implicated in controlling permanent cell cycle exit upon terminal differentiation. E2F and Rb family members influence cell proliferation by transcriptional regulation, Cyclin/Cdks and CKIs regulate cell proliferation via control of cell cycle protein phosphorylation, and the APC/C targets pro-proliferative proteins for proteasome-dependent degradation. However, it has been clear that additional factors must exist that modulate the activity of known cell cycle regulators to ensure timely cell cycle exit. This study has provide evidence of an additional layer of control imposed on the cell cycle machinery by the molecular chaperone Hsp90 (Bandura, 2013).

Cells mutant for the Drosophila Hsp90 homologue, Hsp83, or expressing an RNAi against Hsp83 experienced a delay in cell cycle exit. Hsp83 mutant cells also experienced ectopic increases in both E2F and Cyclin/Cdk activity. The data indicate that the increased Cyclin/Cdk activity was the primary defect, which secondarily caused an increase in E2F activity only at time points shortly after wild-type cells have exited the cell cycle. It was further found that the Hsp83 mutant cells contained increased levels of Cyclin A, Cyclin B and Geminin proteins, consistent with reduced APC/C function. This finding has led to a hypothesis that Cdh1/Fzr may be a client protein of Hsp83. The cell cycle exit effect resulting from Hsp83 knock-down was suppressed by reducing the dosage of Rca1, which provided support to this hypothesis (Bandura, 2013).

Although the number of cells undergoing mitosis in the mutant and RNAi clones was significantly increased compared to control clones (1-1.24% versus 0%), Hsp83 loss-of-function resulted in a rather small number of cells delaying cell cycle exit. Why was this? One reason is that the cell cycle exit mechanism is quite robust. To provide a comparison, rbf1 mutant clones in pupal tissues exhibit a mitotic index of ~9% only from 24-28 APF and dap mutant cells do not experience any cell cycle exit delay. Although the Hsp83 mutant cells have a mitotic index of only ~1%, ectopic cell divisions continue until approximately 40 APF, timing consistent with one extra cell division. In addition, direct overexpression of Rca1 to inhibit Fzr delays cell cycle exit with similar timing as the Hsp83 mutation, and also results in a modest increase in mitotic index in pupal tissues. It is not surprising that Hsp836-55, a hypomorphic mutation in something that is thought to optimize the function of Cdh1/Fzr, would produce a much more subtle effect (Bandura, 2013).

This study is the first providing evidence that Cdh1/Fzr could be an Hsp90 client. In budding yeast, it has been demonstrated that another chaperone, the CCT chaperonin, is required for the folding of both Cdc20 and Cdh1 and therefore is necessary for all APC/C activity. While the CCT chaperonin functions in the bulk folding of nascent proteins, the Hsp90 family of chaperones generally promotes more subtle structural changes to potentiate the function of its clientele. It is proposed that Hsp90 is specifically required to optimize the function of APC/CCdh1 and not APC/CCdc20. Indeed, the fact that it was possible to observe Hsp90 mutant cells undergoing mitosis indicates that the function of the APC/CCdc20 is intact, as cells lacking functional Cdc20/Fzy arrest in mitosis (Bandura, 2013).

Several studies have demonstrated that mutation or inhibition of fzr can delay cell cycle exit, but it is not clear which target protein(s) of the APC/CCdh1 are responsible for this delay. One likely candidate for the crucial target protein that causes the delay in cell cycle exit is Cyclin A. In Drosophila, Cyclin A normally functions during mitosis, but when ectopically expressed it can also induce entry into S phase. Cyclin A overexpression can drive the G1/S transition even in the absence of Cyclin E, suggesting that Cyclin A/Cdk complexes can directly phosphorylate Cyclin E/Cdk2 targets important for S phase. Further, in mammals it has been demonstrated that Cdk1 is the only essential Cdk and that it is sufficient to drive the entire cell cycle in the absence of interphase Cdks. Indeed, direct overexpression of Cyclin A can cause one complete ectopic cell cycle in differentiating pupal eyes and wings. However, the extra cell cycle induced in the Drosophila embryonic epidermis by mutation of fzr cannot be rescued by also mutating cyclin A, suggesting there may be other crucial target proteins of the APC/CCdh1 in addition to Cyclin A that must be restrained in order to initiate cell cycle exit. APC/C targets that could potentially play a role in cell cycle exit include Cyclins B and B3, Cdc25/Stg phosphatase and the DNA replication factor Orc1. Further studies are needed to address whether restraining these target proteins comprises an important part of the cell cycle exit requirement for APC/CCdh1 (Bandura, 2013).

The current model is that Hsp90 facilitates the function of the Cdh1/Fzr. What could be the purpose for this regulation of the APC/CCdh1? Unlike other ubiquitin ligases that recognize their targets only when they have been post-translationally modified, for example by phosphorylation or hydroxylation, the APC/C recognizes different unmodified substrates at distinct points in the cell cycle. Although it is not entirely understood how this change in substrate specificity occurs, it is clear that it is partially dependent on which co-activator, Cdc20/Fzy or Cdh1/Fzr, is associated with the complex. While Cdc20/Fzy is expressed periodically and this may partially control activation of the APC/CCdc20, Cdh1/Fzr is not periodically transcribed. Cdh1/Fzr is known to be negatively regulated by both Cyclin/Cdk phosphorylation and binding of inhibitors. In addition, activation of Cdh1/Fzr by interaction with Hsp90 in a cell cycle-dependent manner could potentially also be responsible for the timely activation of the APC/CCdh1 (Bandura, 2013).

How could a protein such as Hsp90 that is expressed ubiquitously and at high levels regulate another protein so that it only acts at a specific place and time? Hsp90 function is modulated both by association with co-chaperones and via post-translational modifications, including phosphorylation, acetylation and S-nitrosylation, all of which can direct Hsp90 to particular client proteins. A recent study has even indicated that a portion of cellular Hsp90 is phosphorylated by Wee1, causing it to selectively associate with only some of its clients in a cell cycle-dependent manner. Although it has not been demonstrated that this specific phosphorylation event plays a role during exit from the cell cycle, these data combined with data indicating that Hsp90 is subject to many post-translational modification raise the possibility that a particular combination of modifications on Hsp90 could create a G1-specific Hsp90 activity that optimizes the function of clients important for cell cycle exit in G1 (Bandura, 2013).

Hsp90 is needed to protect a number of mutated and overexpressed oncoproteins, such as ErbB2/HER2, v-Src, Akt and Bcr-Abl, from misfolding and degradation. Therefore, inhibition of Hsp90 is an efficient way to silence multiple oncogenic signaling pathways simultaneously. As a result, several Hsp90 inhibitors are currently being developed and tested as anti-cancer therapeutics. However, the pleiotropic effects of Hsp90 inhibitors may complicate their clinical efficacy. For example, Hsp90 inhibition results in the release and activation of the heat shock transcription factor 1 (HSF1), and HSF1 has a clear role in supporting the proliferation and survival of transformed cells. In addition, in Drosophila, is had been demonstrated that mutation or inhibition of Hsp90 relieves the suppression of transposon activity, resulting in de novo mutations and revealing that Hsp90 inhibition can be mutagenic. The current findings, which suggest that APC/CCdh1 function is reduced in the absence of Hsp90, identify an additional mechanism by which Hsp90 inhibition could promote genomic instability and carcinogenesis. Mouse and human cells lacking Cdh1/Fzr exhibit multiple markers of genomic instability, including chromosome breaks, anaphase bridges and aneuploidy. Further, Cdh1/Fzr heterozygous mice displayed an increased propensity to develop epithelial tumors, suggesting a tumor suppression function for Cdh1/Fzr. Overall, these data highlight a need to target Hsp90 inhibitors not only to tumor cells, but specifically to Hsp90-oncoprotein client interactions within tumor cells. Current efforts by researchers to design Hsp90 inhibitors that disrupt specific Hsp90-co-chaperone interactions, or to develop Hsp90 isoform- and cell location-specific inhibitors are underway. This approach of designing more precisely targeted Hsp90 inhibitors will be important to reduce undesirable pro-proliferative and mutagenic effects of Hsp90 inhibition (Bandura, 2013).

Protein Interactions

Regulator of cyclin A1 (Rca1) specifically inhibits Cdh1Fzr-dependent anaphase-promoting complex/cyclosome activity and prevents cyclin degredation in G2. In the absence of rca1 function, mitotic cyclins are prematurely degraded, and cells fail to enter mitosis. This phenotype is reminiscent of the phenotype produced by overexpression of Fizzy-related, here termed Cdh1Fzr. Double-mutant analysis demonstrates that premature cyclin destruction in rca1 mutants is mediated by Cdh1Fzr. Furthermore, Rca1 can block the effects of Cdh1Fzr overexpression, supporting the notion that Rca1 inhibits Cdh1Fzr-dependent APC activity. Coimmunoprecipitation experiments reveal that Rca1 and Cdh1Fzr are in a complex that also contains the APC component Cdc27. Collectively, these data show that Rca1 is a negative regulator of Cdh1Fzr-dependent APC activity. It is suggested that a similar function is required in all cells in which kinase activity is low during G2 to prevent a premature activation of the APC by Cdh1 (Grosskortenhaus, 2002).

Mitotic cyclins remain unstable during G1, mediated by APC-Cdh1Fzr-dependent degradation (Sigrist, 1997). The first G1 phase during embryogenesis is not established in fzr mutants, and cells perform an additional S phase, presumably triggered by the S phase activity of the CycA/Cdk1 complex (Sigrist, 1997; Sprenger, 1997). Cdh1Fzr mRNA expression cannot be detected during the cellular blastoderm stages, but low levels of Cdh1Fzr are presumably present. High levels of Cdh1Fzr are expressed during stage 11 of embryogenesis, when most cells are in the 16th cell cycle, shortly before they enter the first G1 phase. In contrast to Cdc20Fzy, APC activation by Cdh1Fzr can be induced ectopically by its overexpression (Sigrist, 1997). During embryogenesis, this results in degradation of mitotic cyclins in G2 of cell cycle 16 and a failure to execute mitosis 16 (Grosskortenhaus, 2002 and references therein).

Rca1 is an essential inhibitor of the anaphase-promoting complex/cyclosome in Drosophila. APC activity is restricted to mitotic stages and G1 by its activators Cdc20-Fizzy (Cdc20Fzy) and Cdh1-Fizzy-related (Cdh1Fzr), respectively. In rca1 mutants, cyclins are degraded prematurely in G2 by APC-Cdh1Fzr-dependent proteolysis, and cells fail to execute mitosis. Overexpression of Cdh1Fzr mimics the rca1 phenotype, and coexpression of Rca1 blocks this Cdh1Fzr function. Previous studies have shown that phosphorylation of Cdh1 prevents its interaction with the APC. The data reveal another mode of APC regulation; this one is fulfilled by Rca1 at the G2 stage, when low Cdk activity is unable to inhibit Cdh1Fzr interaction (Grosskortenhaus, 2002).

In rca1 mutants, levels of mitotic cyclins are reduced during interphase of the 16th cell cycle. This premature cyclin disappearance becomes obvious only when mutant and rescued segments in a given embryo are compared and is more difficult to detect when mutant and wt embryos are compared. The lower levels of mitotic cyclins are not caused by changes in cyclin transcription or translation, since mitotic cyclins accumulate normally at the beginning of cell cycle 16. Mitotic cyclins are usually stable in interphase cells of cellularized Drosophila embryos. It is therefore concluded that their disappearance in rca1 mutants is caused by premature degradation. The remaining cyclin levels are apparently not sufficient to allow entry into mitosis. In Drosophila, CycA and CycB are cytoplasmic during interphase and accumulate in the nucleus only during prophase. It has been speculated that the nuclear accumulation of mitotic cyclins is required for certain mitotic events like DNA condensation. Rca1 is a nuclear protein and could be required to prevent degradation of mitotic cyclins, specifically in the nucleus. Another possibility is that Rca1 sequesters parts of the degradation machinery in the nucleus away from the bulk of mitotic cyclins present in the cytoplasm (Grosskortenhaus, 2002).

In rca1 mutant embryos, residual levels of cytoplasmic CycA and CycB are visible. Supplying additional CycA (but not CycB) is sufficient to rescue the mitotic failure of rca1 mutants. This demonstrates that CycA is the crucial mitotic factor missing in rca1 mutant embryos (Grosskortenhaus, 2002).

The requirements for rca1 function are not restricted to embryogenesis. Clonal analysis of rca1 function shows that imaginal cells lacking rca1 also have reduced cyclin levels and fail to proliferate normally. In these cells, large nuclei are found typical for cells undergoing endocycles. An overreplication has been reported in imaginal discs lacking Cdk1 activity. Since mitotic cyclins are degraded in rca1 mutant cells, it is expected that low Cdk1 kinase activity could result in the lack of proliferation and in overreplication of the genome. A detailed analysis of the DNA content and DNA replication pattern will reveal whether rca1 mutant cells similarly undergo endocycles (Grosskortenhaus, 2002).

The APC targets cyclins for degradation, and its activity is normally restricted to mitotic stages and G1. However, in rca1 mutants, premature cyclin destruction during G2 is observed, indicating that Rca1 is required to inhibit the APC and thus ensures high mitotic cyclin levels for mitotic entry (Grosskortenhaus, 2002).

On a molecular level, Rca1 could inhibit APC ubiquitin ligase activity directly, or it might specifically prevent activation of the APC by Cdc20Fzy and Cdh1Fzr. Several lines of evidence suggest that Rca1 is a specific inhibitor of Cdh1Fzr-dependent APC activity and does not affect APC-Cdc20Fzy. First of all, premature degradation of cyclins in rca1 mutants depends on fzr gene function. Embryos mutant for fzr and rca1 do not degrade cyclins prematurely, and mitosis 16 is restored. Thus, fzr is epistatic to rca1. In contrast, rca1 is epistatic to fzy (P. O'Farrell, personal communication to Grosskortenhaus, 2002). In addition, overexpression studies also support the specificity of Rca1. Overexpression of Cdc20Fzy is without consequences for cyclin levels and cell cycle progression (Sigrist, 1997). In contrast, overexpression of Cdh1Fzr results in premature cyclin degradation, as in rca1 mutants. The coexpression of Rca1 negates this phenotype, indicating that Cdh1Fzr and Rca1 oppose each other (Grosskortenhaus, 2002).

During the first 16 cell cycles in Drosophila, Cdc20Fzy is thought to be the major APC-activating protein, since Cdh1Fzr is present at higher levels only at later stages. Uniform overexpression of HA-Rca1 has no influence on cell cycle progression or cyclin degradation during the first 16 divisions, indicating that Rca1 does not inhibit Cdc20Fzy-dependent APC activity (Grosskortenhaus, 2002).

Ubiquitous overexpression of HA-Rca1 also has no influence on the establishment of the G1 state. This is in contrast to fzr mutants that fail to downregulate mitotic cyclins levels after mitosis 16 and that do not establish a G1 phase. However, HA-Rca1 itself is degraded in G1 cells and thus cannot influence Cdh1Fzr function. It is also suggested that Rca1 activity is subjected to cell cycle-specific regulation and it is expected that Rca1 function is downregulated during mitosis to allow Cdh1Fzr activity during later stages of mitosis and at the beginning of G1 (Grosskortenhaus, 2002).

A biochemical interaction between Rca1 and Cdh1Fzr was seen in coimmunoprecipitation experiments. Using embryonic extracts, it was shown that both proteins are present in a complex. The complex precipitated from embryonic extracts also contains the APC component Cdc27. Thus, 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).

Cdh1 is also regulated by phosphorylation. Only unphosphorylated Cdh1 can bind to and activate the APC, and several kinases have been implicated in the phosphorylation of Cdh1, including Cdk1 and Cdk2. Accordingly, no Cdh1-dependent APC activity was found during S phase and early mitotic stages. When degradation of mitotic cyclins abate Cdk1 activity after the metaphase-anaphase transition, unphosphorylated Cdh1 is thought to activate the APC. This activity is then maintained during the G1 state and turned off when cells start to accumulate Cdk2 activity at the G1-S transition. The G2 state is also characterized by low Cdk kinase activity, yet not in all cell cycles. In Drosophila, Cdk1 is inhibited during G2 stages by tyrosine phosphorylation. But during the first 15 divisions, Cdk kinase activity is provided by CycE/Cdk2 activity that is present throughout the cell cycle, including G2. During cell cycle 16, CycE/Cdk2 kinase activity drops, since CycE mRNA is downregulated and the CycE/Cdk2 inhibitor Dacapo is upregulated. Thus, in G2 of cell cycle 16, Cdk1 as well as Cdk2 activity is expected to be low. At this stage, phosphorylation of Cdh1Fzr cannot prevent its association with the APC, and the requirements for Rca1 become evident. Accordingly, Rca1 is dispensable when CycE is overexpressed during cell cycle 16. Thus, phosphorylation and interaction with Rca1 can control Cdh1Fzr activity during G2. However, Rca1- and CycE-dependent phosphorylation of the APC are not completely redundant. In CycA mutant embryos, maternally provided CycA is normally sufficient to allow execution of mitosis 15, despite low CycA levels. In rca1;CycA double mutants, cells arrest before mitosis 15, likely caused by a further reduction in CycA. During this stage, CycE is still present but apparently cannot substitute completely for the lack of rca1 function (Grosskortenhaus, 2002).

It is expected that Cdh1 inhibition by an Rca1-like function is also required in other organisms. In human cells, Cdh1 phosphorylation is also low when most cells are in late S phase and G2, but only low APC activity is detected. Thus, human cells must also have a mechanism preventing APC-Cdh1 activation when Cdh1 phosphorylation is low (Grosskortenhaus, 2002).

Recently, potential homologs of Rca1 have been identified in Xenopus, mouse, and humans and named Emi1. Emi1 from Xenopus inhibits Cdc20 activity in the Xenopus system. In contrast, Rca1 specifically inhibits APC-Cdh1Fzr, but not APC-Cdc20Fzy activity. Databank searches reveal that the Drosophila genome does not contain an additional protein that resembles Emi1 or Rca1. It is thus believed that Rca1 has specificity different from that of Emi1. This difference might reflect the manner in which cell cycle progression is controlled in these organisms. In Drosophila, CycA- and CycB-containing Cdk1 complexes are targets for inhibitory phosphorylation. In contrast, the CycA/Cdk1 complex in Xenopus is not subject to this modification, and one can expect Cdk1 activity even in G2. This could result in the phosphorylation of the APC and activation by Cdc20, even before the spindle checkpoint is activated. Thus, Emi1 might have been adapted to these specific requirements in Xenopus to suppress Cdc20-dependent APC function before mitosis. Rca1, in contrast, is required to prevent APC-Cdh1Fzr activity when Cdk1 kinase activity is low in G2, an environment that is permissive for APC-Cdh1 complex formation. Significantly, recent work on the human form of Emi1 has revealed that this protein inhibits the APC-Cdh1 complex (P. Jackson, personal communication to Grosskortenhaus, 2002).

In S. pombe, which is also characterized by low Cdk kinase activity in G2, premature APC-Ste9 (the Cdh1 homolog) activity is probably prevented by very low Ste9 protein levels during G2. Thus, different ways of preventing premature APC activation at the G2 stage have been selected in various organisms and by different cell types within an organism. These data reveal the importance of APC downregulation by the Rca1 protein that is specific for the APC-Cdh1Fzr activity. In addition to the Emi1/Rca1 class of proteins, inhibition of the APC-Cdh1 complex is also mediated by a protein related to Mad2 protein. The biological role of this inhibition has not been elucidated so far (Grosskortenhaus, 2002).

Thus, a number of different mechanisms regulating the activity of the APC-Cdh1 have been identified recently. It has been shown that APC activity is restricted to mitotic stages and G1. The data on Rca1 demonstrate a novel control of APC-Cdh1 activity that is necessary to prevent unwanted APC activity at the G2 stage. It is expected that this mechanism of controlling APC-Cdh1 activity is also involved in the downregulation of APC-Cdh1 activity at the G1-S transition (Grosskortenhaus, 2002).

Notch-dependent downregulation of the homeodomain gene cut is required for the mitotic cycle/endocycle switch and cell differentiation in Drosophila follicle cells; Cut promotes Cyclin A expression by negatively regulating Fizzy-related

During Drosophila mid-oogenesis, follicular epithelial cells switch from the mitotic cycle to the specialized endocycle in which the M phase is skipped. The switch, along with cell differentiation in follicle cells, is induced by Notch signaling. The homeodomain gene cut functions as a linker between Notch and genes that are involved in cell-cycle progression. Cut is expressed in proliferating follicle cells but not in cells in the endocycle. Downregulation of Cut expression is controlled by the Notch pathway and is essential for follicle cells to differentiate and to enter the endocycle properly. cut-mutant follicle cells enter the endocycle and differentiate prematurely in a cell-autonomous manner. By contrast, prolonged expression of Cut causes defects in the mitotic cycle/endocycle switch. These cells continue to express an essential mitotic cyclin, Cyclin A, which is normally degraded by the Fizzy-related-APC/C ubiquitin proteosome system during the endocycle. Cut promotes Cyclin A expression by negatively regulating Fizzy-related. These data suggest that Cut functions in regulating both cell differentiation and the cell cycle, and that downregulation of Cut by Notch contributes to the mitotic cycle/endocycle switch and cell differentiation in follicle cells (Sun, 2005).


retina aberrant in pattern/fizzy-related: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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