Cyclin A

DEVELOPMENTAL BIOLOGY

Embryonic

Maternally contributed cyclin A and B proteins are initially distributed uniformly throughout the syncytial Drosophila embryo. As dividing nuclei migrate to the cortex of the embryo, the A and B cyclins become concentrated in surface layers extending to depths of approximately 30-40 microns and 5-10 microns, respectively. The initiation of nuclear envelope breakdown, spindle formation, and the initial congression of the centromeric regions of the chromosomes onto the metaphase plate all take place within the surface layer occupied by cyclin B on the apical side of the blastoderm nuclei. Cyclin B is seen mainly, but not exclusively, in the vicinity of microtubules throughout the mitotic cycle. It is most conspicuous around the centrosomes. Cyclin A is present at its highest concentrations throughout the cytoplasm during the interphase periods of the blastoderm cycles, although weak punctate staining can also be detected in the nucleus. It associates with the condensing chromosomes during prophase, segregates into daughter nuclei in association with chromosomes during anaphase, to redistribute into the cytoplasm after telophase. In contrast to the cycles following cellularization, neither cyclin is completely degraded upon the metaphase-anaphase transition (Maldonado-Codina, 1992).

During mitotic cycles 8-13, prior to cellularization, a progressive increase in the degradation of cyclins at mitosis leads to increasing oscillations of cdc2 kinase activity. Quantitative measurements indicate that less than 10% of Cyclin A and B are degraded at mitosis 5, and that about 30%, 60%, and 80% are degraded at mitoses 8, 11 and 13 respectively. It appears that cyclin synthesis is limiting for mitoses 10-13. Mutants deficient in cyclin mRNAs suffer cell cycle delays during this period, suggesting that the accumulation of cyclins times these cycles. During interphase 14, programmed degradation of maternal String protein leads to inhibitory phosphorylation of cdc2 and cell cycle arrest. Subsequently, mitoses 14-16 are triggered by pulses of zygotic string transcription (Edgar, 1994).

Cyclin A mRNA levels are roughly correlated with the mitotic activity in the embryo. Maternal transcripts are abundant in early embryos, but levels are lower at the beginning of cell cycle 14. During cycles 15 and 16 there is reaccumulation by zygotic transcription, while only very low levels are present in older embryos. After 13 cleavage divisions Cyclin A is completely degraded during subsequent cell divisions (Lehner, 1989).

During interphase 14 Cyclin A accumulates in the cytoplasm. During prophase the most intense Cyclin A staining is localized in the region of the condensing chromatin. Metaphase cells are either brightly stained, intermediately stained or unstained, indicating that Cyclin A is completely degraded within metaphase. No Cyclin A is detected in anaphase, telophase and early in the following interphase (Lehner, 1989).

The divisions during cellularization are not synchronous, and Cyclin A staining reflects the spatio-temporal program of embryonic cell divisions. For example, the first cells to enter the 14th mitosis are in a cluster in the head region, and these are the first labelled with anti-Cyclin A. When these cells have passed metaphase, Cyclin A is no longer detectable (Lehner, 1989).

Cyclin A accumulates and is degraded in a similar fashion, in both colchicine treated cells and untreated control cells. Whereas Cyclin B accumulation seems to be independent of spindle function, its degradation is inhibited by colchicine treatment (Whitfield, 1990).

Degradation of the mitotic cyclins is a hallmark of the exit from mitosis. Induction of stable versions of each of the three mitotic cyclins of Drosophila, cyclins A, B, and B3, arrests mitosis with different phenotypes. A recent proposal that the destruction of the different cyclins guides progress through mitosis has been tested. Real-time imaging has revealed that arrest phenotypes differ because each stable cyclin affects specific mitotic events differently. Stable cyclin A (CYC-A^S) prolongs or blocks chromosome disjunction, leading to metaphase arrest. Stable cyclin B allows the transition to anaphase, but anaphase A chromosome movements are slowed, anaphase B spindle elongation does not occur, and the monooriented disjoined chromosomes begin to oscillate between the spindle poles. Stable cyclin B3 prevents normal spindle maturation and blocks major mitotic exit events such as chromosome decondensation but nonetheless allows chromosome disjunction, anaphase B, and formation of a cytokinetic furrow, which splits the spindle. It is concluded that degradation of distinct mitotic cyclins is required to transit specific steps of mitosis: Cyclin A degradation facilitates chromosome disjunction; Cyclin B destruction is required for anaphase B and cytokinesis and for directional stability of univalent chromosome movements, and Cyclin B3 degradation is required for proper spindle reorganization and restoration of the interphase nucleus. It is suggested that the schedule of degradation of cyclin A, cyclin B, and then cyclin B3 contributes to the temporal coordination of mitotic events (Parry, 2001).

The observation that CYC-A^S arrests cells in metaphase suggests that, normally, CYC-A must be degraded in order to disjoin sister chromosomes. Consistent with this interpretation, recent reports have implicated cyclin degradation in the regulation of the metaphase/anaphase transition. Irradiation of Drosophila embryos induces a prolonged metaphase, during which damage appears to be repaired. CYC-A has been implicated in this arrest by the finding that mutant embryos with reduced CYC-A levels fail to arrest at metaphase. Similarly, the ability of DNA damage to induce an arrest prior to DNA segregation in S. cerevisiae requires the presence of either Clb5 or Clb6. While homology is not sufficient to define a homolog of metazoan cyclin A among S. cerevisiae cyclins, common functional attributes suggest that Clb5/6 and cyclin A are analogs. It is proposed that A-like cyclins in diverse species act to inhibit the metaphase/anaphase transition and that progress to later steps requires degradation of these cyclins (Parry, 2001).

The degree to which CYC-A^S prolongs mitosis depends on the experimental conditions. The differences appear to be attributable to a limited ability of CYC-A^S to compete with preexisting endogenous CYC-A. Delayed metaphase/anaphase transitions are gradual, with disjunction drawn out over a few minutes, and sometimes ineffective. Ordinarily, the disjunction of chromosomes is abrupt and synchronous, occurring within several seconds in Drosophila. The abruptness of the transition suggests that a switch-like regulatory circuit must govern its execution. The finding that CYC-A^S can prolong disjunction suggests that CYC-A inhibits events that are downstream of the switch or part of the switch that usually imparts all-or-nothing behavior (Parry, 2001).

The abrupt activation of chromosome disjunction is likely to involve multiple regulatory inputs. Indeed, identification of Drosophila analogs of the securins, separins, and cohesins suggests that Drosophila uses a conserved pathway to regulate chromosome disjunction. Consistent with this, stabilization of the securin, Pimples (Pim; Stratmann, 1996) shows that degradation of this protein promotes disjunction of sister chromosomes. How might cyclin degradation interface with securin degradation to activate sister chromosome disjunction? CYC-A appears to block chromosome disjunction independently of securin, because persistence of CYC-A^S does not block PIM degradation. While there is not enough data in either S. cerevisiae or Drosophila to define the regulatory relationship, a model is suggested as a simple proposal to account for data from both systems. This proposal suggests that separins are inhibited incompletely by securin or CYC-A/Cdk1 alone, but effectively by the combination (Parry, 2001).

The timing of CYC-A destruction suggests that it precedes PIM destruction in a pathway leading to chromosome disjunction. CYC-A immunofluorescence shows both stained and unstained metaphases, and the proportion of unstained metaphases (about 30%) suggests that CYC-A is degraded about 15-35 s prior to chromosome disjunction. During this interval, it is expected that a target of CYC-A/Cdk1 kinase activity is dephosphorylated, separins are activated, and cohesins are cleaved. It seems likely that numerous regulatory interactions might operate to make cohesin cleavage fast and the metaphase/anaphase transition abrupt. The degradation of PIM, which takes place around the time of the metaphase/anaphase transition, might be one of the changes promoting rapid activation of the separins (Parry, 2001).

Unlike the arrests induced by the other stable cyclins, the metaphase arrest induced by CYC-A^S appears to be a normal mitotic configuration. This is interpreted to mean that CYC-A can arrest progression of all mitotic events in a concerted fashion. The ability to impose a concerted arrest is consistent with a role in checkpoint arrest of the cycle; however, reversal of such a block would have to be abrupt to avoid the defects associated with prolongation of chromosome disjunction (Parry, 2001).

The three stable cyclins differ in their abilities to block specific mitotic events, each blocking event occurring at the time of degradation of the endogenous cognate cyclin. Since the action of stable cyclins, in those cases in which it has been tested, is enhanced by coexpression of the kinase partner Cdk1, it is believed that these blocks are mediated by the corresponding cyclin/Cdk1 complexes. It is proposed that the specificity results because the different cyclin/Cdk1 complexes differ in their ability to target particular cellular substrates, as suggested by numerous biochemical investigations. During the exit from mitosis, differences in cyclin/Cdk1 specificity, in conjunction with the ordered degradation of the different cyclins, may be an important way of coupling the mechanics of cell division to the regulatory machinery. By degrading each cyclin in turn, the inhibitions established at each transition point are relieved and the cellular processes of chromosome disjunction, anaphase movements, and cytokinesis can proceed with the proper kinetics (Parry, 2001).

When mitosis is bypassed, as in some cancer cells or in natural endocycles, sister chromosomes remain paired and produce four-stranded diplochromosomes or polytene chromosomes. Cyclin/Cdk1 inactivation blocks entry into mitosis and can reset G2 cells to G1, allowing another round of replication. Reciprocally, persistent expression of Cyclin A/Cdk1 or Cyclin E/Cdk2 blocks Drosophila endocycles. Inactivation of Cyclin A/Cdk1 by mutation or overexpression of the Cyclin/Cdk1 inhibitor, Roughex (Rux), converts the 16th embryonic mitotic cycle to an endocycle; however, Rux expression fails to convert earlier cell cycles unless Cyclin E is also downregulated. Following induction of a Rux transgene in Cyclin E mutant embryos during G2 of cell cycle 14 (G214), Cyclins A, B, and B3 disappear and cells reenter S phase. This rereplication produced diplochromosomes that segregated abnormally at a subsequent mitosis. Thus, like the yeast CKIs Rum1 and Sic1, Drosophila Rux can reset G2 cells to G1. The observed cyclin destruction suggests that cell cycle resetting by Rux is associated with activation of the anaphase-promoting complex (APC), while the presence of diplochromosomes implies that this activation of APC outside of mitosis is not sufficient to trigger sister disjunction (Vidwans, 2002).

The abrupt disappearance of mitotic cyclins is generally taken as an indication of activation of the mitotic ubiquitin ligase APC and subsequent degradation. Since Rux-mediated resetting from G2 to G1 was accompanied by cyclin disappearance, it is speculated that APC activation occurs during resetting but that it is insufficient to promote sister disjunction in this context. Consistent with this, studies in Saccharomyces cerevisiae have suggested that mitotic phosphorylation acts in conjunction with APC-targeted degradation to promote chromatid disjunction. The widespread occurrence of polytene and diplochromosomes suggests that disjunction can be bypassed in diverse systems. The variety of cell cycle defects and polyploid phenotypes in cancer cell lines has suggested that production of diplochromosomes involves the complete bypass of mitosis. Perhaps the dependence of disjunction on mitotic events is general (Vidwans, 2002).

Larval

In Drosophila, the sensory mother cells of macrochaetes are chosen from among the mitotically quiescent clusters of cells in wing imaginal discs, where other cells are proliferating. The pattern of cyclin A, one of the G2 cyclins, reveals that mitotically quiescent clusters of cells are arrested in G2. When precocious mitoses are induced during sensory mother cell determination by the ectopic expression of string, a known G2/M transition regulator, the formation of sensory mother cells is disturbed, resulting in the loss of macrochaetes in the adult notum. This suggests that G2 arrest of the cell cycle ensures the proper determination of sensory mother cells, and that G2 arrest in mitotically quiescent clusters of cells is controlled by the down-regulation of string transcription (Kimura, 1997).

Control of cell proliferation in the Drosophila eye by Notch signaling

Cell proliferation in animals must be precisely controlled, but the signaling mechanisms that regulate the cell cycle are not well characterized. A regulated terminal mitosis, called the second mitotic wave (SMW), occurs during Drosophila eye development, providing a model for the genetic analysis of proliferation control. This study reports a cell cycle checkpoint at the G1-S transition that initiates the SMW; Notch signaling is required for cells to overcome this checkpoint. Notch triggers the onset of proliferation by multiple pathways, including the activation of dE2F1, a member of the E2F transcription factor family. Delta to Notch signaling derepresses the inhibition of dE2F1 by RBF, and Delta expression depends on the secreted proteins Hedgehog and Dpp. Notch is also required for the expression of Cyclin A in the SMW (Baonza, 2005).

This work identifies a new cell cycle checkpoint in the second mitotic wave and describes how intercellular signaling overcomes this checkpoint. Delta signaling to Notch triggers a progression from G1 arrest in the morphogenetic furrow into the S phase of the terminal mitosis. Two effectors of this Notch requirement have been identified, dE2F1 transcriptional activity and cyclin A expression. Although the data imply that at least one other target also exists, this is unidentified. The data preclude this additional factor from being Cyclin E. Previous work has identified a later SMW checkpoint, at the G2-M transition. Together, Notch and the EGFR therefore coordinately provide spatial and temporal control of the cell cycle in the SMW (Baonza, 2005).

These results led to a proposal of the following course of events. Notch is activated by the uniform band of Delta in all cells as they emerge from the morphogenetic furrow. Cells that are uncommitted thereby enter S phase, whereas cells that are part of the precluster are blocked from responding and remain in G1. It has been shown that the G1 arrest of precluster cells is dependent on EGFR activation, although the details of the mechanism remain unclear. One of the consequences of EGFR activation in precluster cells is the upregulation of Delta expression. Cells between the preclusters would therefore end up initially receiving low-level uniform Delta, later reinforced by the upregulated Delta in the adjacent preclusters. Together, these provide a robust and modulated activation of Notch in cells that will enter the SMW (Baonza, 2005).

It is emphasized that this work uncovers a normal developmental function for Notch signaling only in the control of a specific terminal mitotic cycle. The fact that clones of Notch and Delta mutant cells can be generated implies that they are not required for the earlier, unpatterned proliferation ahead of the morphogenetic furrow. Similarly, the ability to make clones in other imaginal discs indicates that there is no requirement for Delta/Notch signaling in most cell proliferation in Drosophila. Rather, this signal requirement, and the subsequent EGFR-dependent entry into mitosis, is superimposed upon normal controls in this regulated terminal mitosis. Moreover, the ability of Notch signaling to initiate S phase is restricted to a short period. Notch has other functions later in eye development, and it has been shown that later ectopic signaling does not lead to additional proliferation. Nevertheless, Delta-expressing clones in other tissues also hyperproliferate, suggesting that ectopic Notch activity has a wider ability to trigger inappropriate proliferation (Baonza, 2005).

The EGFR and Notch signal systems play distinct roles in regulating the SMW. After completing its preliminary role in maintaining cells in G1 arrest, EGFR signaling ensures that cells only undergo mitosis if they are adjacent to developing clusters, thereby matching the number of cells born with the number that will be required to complete ommatidial differentiation. In contrast, Notch initiates the whole process by regulating whether cells emerging from the morphogenetic furrow enter the SMW or remain arrested in G1 and start to differentiate (Baonza, 2005).

The secreted protein Hedgehog has a primary role in the forward movement of the morphogenetic furrow. Hedgehog also has an important function in initiating and coordinating the onset of the SMW, specifically the initiation of S phase. Hh and Dpp together lead to the expression of Delta in the furrow. Furthermore, Hh is essential for the expression of cyclin D and cyclin E in the morphogenetic furrow, whereas Cyclin E is the main cyclin that regulates S phase onset. These data imply that Hedgehog signaling activates several independent branches of the pathway that lead to the onset of S phase in the SMW. Incidentally, the observation that Cyclin E accumulates in Notch mutant clones, which lack dE2F1 activity, indicates that, at least in this context, Cyclin E is not sufficient to inhibit RBF and thereby activate dE2F1 activity (Baonza, 2005).

Cyclin A is best characterized as a mitotic cyclin, and its destruction is a key step in the completion of mitosis. An additional function in the onset of S phase in Drosophila remains enigmatic. Mammalian Cyclin A and its associated kinase Cdk2 can drive G1 cell extracts into S phase, and anti-Cyclin A antibodies can block S phase in injected cells. But, in Drosophila, S phase can proceed normally in the absence of Cyclin A and Cyclin A does not bind Cdk2. Nevertheless, when overexpressed, Cyclin A can overcome the lack of Cyclin E and allow cells to enter S phase. Furthermore, overexpression of the Cyclin A inhibitor Roughex blocks entry into S phase in embryos, and roughex mutants show precocious S phase entry in the SMW. Ectopic BrdU incorporation is observed in the eye disc when Cyclin A is misexpressed. The data indicating that Cyclin A is one of the targets of Notch signaling further support the idea that Cyclin A is part of the machinery that controls the onset of S phase in the SMW (Baonza, 2005).

Notch signaling in mammals, as in flies, is pleiotropic and context dependent. This is highlighted in human cancer, where Notch is oncogenic in a number of cases, particularly in hematopoietic neoplasms, but in other contexts has tumor suppressor functions. Moreover, although the current work highlights a proliferative function, it has been shown that Notch inhibits proliferation in the wing disc. Notwithstanding this caveat, it is striking that Notch activity can be hyperproliferative in humans and in Drosophila, and little is known about this proliferative response. It has recently been shown in the developing Drosophila central nervous system that Notch activity can maintain cells in a proliferative state by antagonizing the p21/p27 homolog Dacapo, thereby maintaining Cyclin E expression. Similarly, the dacapo gene is downregulated in response to Notch in the mitotic-to-endocycle transition in Drosophila follicle cells. This work describes a different mechanism: Notch signaling overcomes a G1-S checkpoint via the activation of universally conserved cell cycle components, RBF1, dE2F1, and possibly Cyclin A. Although tempting to speculate that these data may provide some insight into oncogenic mechanisms, it will be important to ascertain whether the particular relationships between Notch and the core machinery that triggers S phase is indeed conserved. In fact, the data imply that Notch probably also influences the mitotic cycle at other points. If the only role of Notch were to advance cells into S phase, they would simply arrest at the next checkpoint, G2-M. The fact that Notch activity leads to overgrowth therefore implies that Notch can also, directly or indirectly, drive cells through the subsequent G2-M checkpoint (Baonza, 2005).

Oogenesis

Regulated changes in the cell cycle underlie many aspects of growth and differentiation. Prior to meiosis, germ cell cycles in many organisms become accelerated, synchronized, and modified to lack cytokinesis. These changes cause cysts of interconnected germ cells to form that typically contain 2n cells. In Drosophila, developing germ cells during this period contain a distinctive organelle, the fusome, that is required for normal cyst formation. The cell cycle regulator Cyclin A transiently associates with the fusome during the cystocyte cell cycles, suggesting that fusome-associated Cyclin A drives the interconnected cells within each cyst synchronously into mitosis. In the presence of a normal fusome, overexpression of Cyclin A forces cysts through an extra round of cell division to produce cysts with 32 germline cells. Female sterile mutations in UbcD1, encoding an E2 ubiquitin-conjugating enzyme, have a similar effect. These observations suggest that programmed changes in the expression and cytoplasmic localization of key cell cycle regulatory proteins control germline cyst production (Lilly, 2000).

The fusome occupies a key location within growing cysts for mediating synchrony. By the onset of mitosis at each round of cyst division, the fusome has completed its cycle of growth and new segments have fused into a single continuous structure. The branches of the G2 fusome pass uninterrupted through the ring canals that interconnect the cystocytes, providing a single internal interface between all cyst cells. Synchronous cell cycles occur only while the fusome is present. Cell-cell junctions alone are insufficient to guarantee synchrony, since ring canals remain present in older cysts when the cells cycle asynchronously. Also, genetic data support a role for the fusome in cystocyte synchrony. Mutations that disrupt the fusome disrupt synchrony, as indicated by the production of cysts that no longer contain 2n cells (Lilly, 2000).

These studies suggest that the fusome affects cystoctye synchrony through an interaction with CycA. CycA associates with the fusome only in synchronously cycling cystocytes, and not in 16-cell cysts. CycA-fusome association begins in G2 when CycA levels on the fusome rise above those observed in the rest of the cyst. By early prophase CycA levels on the fusome are much higher than in the surrounding cytoplasm. As mitosis progresses CycA is seen at high levels throughout the cyst before being destroyed at approximately the metaphase-to-anaphase transition. The accumulation of CycA on the fusome prior to the activation of CycA/Cdk1 at the G2/M transition suggests that CycA association with the fusome synchronizes the entry of all the interconnected cystocytes within a cyst into mitosis (Lilly, 2000).

How might CycA effect mitotic synchrony through its association with the fusome? The fusome most likely acts in some way to spatially equalize the activation of CycA/ Cdk1 activity in all cystocytes during late G2. In yeast, Cyc/Cdk activation is controlled by changes in the levels of stimulatory and inhibitory phosphorylation at specific target sites. The phosphatases and kinases carrying out these changes are themselves subject to control by cdk-mediated phosphorylation to generate a strong positive feedback loop. It is proposed that enzymes responsible for CycA/Cdk1 activation are present in the fusome and are subject to similar positive feedback control. Activation of CycA/Cdk1 activity in any portion of the fusome might spread to adjacent molecules, causing a wave of activation to spread rapidly throughout the fusome. The ability of the fusome to propagate an active state of CycA/Cdk1 would synchronize the mitotic entry of all cystocytes that retained an intact fusome (Lilly, 2000).

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