Cyclin B

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 cell cycles 8-13, 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 cyclin accumulation influences the timing of 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 B is present at high levels prior to fertilization whereas Cyclin A is undetectable. Following cellularization, Cyclin B, like Cyclin A accumulates during G2. Cyclin B is maintained at its maximum from prophase until metaphase. While prophase cells often show a more uniform distribution of Cyclin B over the whole cell, degradation of the protein does not occur until the metaphase-anaphase transition, well after the disappearance of Cyclin A, which peaks in prophase and then declines (Whitfield, 1990).

Cyclin A accumulates and is degraded in the same manner 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).

Both Cyclin B levels and DNA-replication checkpoint control the early embryonic mitoses in Drosophila

The earliest embryonic mitoses in Drosophila, as in other animals except mammals, are viewed as synchronous and of equal duration. However, total cell-cycle length steadily increases after cycle 7, solely owing to the extension of interphase. Between cycle 7 and cycle 10, this extension is DNA-replication checkpoint independent, but correlates with the onset of Cyclin B oscillation. In addition, nuclei in the middle of embryos have longer metaphase and shorter anaphase than nuclei at the two polar regions. Interestingly, sister chromatids move faster in anaphase in the middle rather than the posterior region. These regional differences correlate with local differences in Cyclin B concentration. After cycle 10, interphase and total cycle duration of nuclei in the middle of the embryo are longer than at the poles. Because interphase also extends in checkpoint mutant (grapes) embryos after cycle 10, although less dramatic than wild-type embryos, interphase extension after cycle 10 is probably controlled by both Cyclin B limitation and the DNA-replication checkpoint (Ji, 2004).

When analyzing fixed wild-type embryos, embryos with nuclei at different cell-cycle phases were observed, indicating metasynchronous mitoses before cycle 10. Thus, it was asked whether nuclei in different regions extend interphase coordinately. To exclude fixation artifacts and to measure cell-cycle phases, live preblastoderm embryos were examined with two-photon laser-scanning microscopy (TPLSM). The cell-cycle phases of nuclei in different regions of single embryos at cycle 7 were determined. At this stage, nuclei are spread along the anteroposterior axis. Prophase-metaphase of single medial nuclei were observed to be 40 seconds longer than those of more posterior nuclei although interphase showed little regional difference. Metasynchrony was observed in nine embryos analyzed at cycle 7. The average prophase-metaphase time of medial nuclei was 20 seconds longer compared with nuclei in the posterior region. This temporal difference was compensated by a 20 second shorter anaphase-telophase in the middle region so that nuclei entered the next interphase at similar times. Therefore, total cell-cycle duration of medial and posterior nuclei is not different (Ji, 2004).

Evidence points to the conclusion that prior to cycle 10, regional differences in timing of cell-cycle progression are due to changes in metaphase and anaphase duration, but not interphase duration, as it is after cycle 10. Thus, the metasynchronous mitoses before and after cycle 10 are probably generated by different mechanisms. Findings that local differences of CycB levels correlate with metasynchronous mitoses in the preblastoderm cycles raise the question: how are higher CycB levels in the middle of the embryo generated during each prophase and anaphase of cycles 4-8? During these cycles, a gradual decline of CycB in the interior of the embryo was observed. Between late interphase and early anaphase of cycle 4 to cycle 8, cytoplasm in the interior, containing less CycB, flows toward the polar regions. This cytoplasmic movement may cause slightly lower CycB levels at the polar regions during metaphase and anaphase. Meanwhile, cytoplasm at the cortical region with more CycB flows in an opposite direction towards the anterior-medial region and then moves slightly inward. It is therefore proposed that the inward cytoplasmic flow might cause the temporal and local increase of CycB observed in the middle region of the embryo from prophase to anaphase (Ji, 2004).

A model to explain the axial expansion process (between cycle 4 and cycle 8) is based on solation-contraction of the microfilament network within the embryo. Briefly, according to this model, local solation (disassembly) of the contractile microfilament network in the center of embryo will cause the microfilament network attached to the cortex to contract away from the solated site in the center of the embryo. Nuclei and cytoplasm in the center of the embryo move towards the two polar regions because the tension generated by the contraction of the microfilament network is greatest along the anteroposterior axis. This poleward cytoplasmic movement in the interior would force cortical material to flow from poles towards the middle region, where the cytoplasm then moves inwards. This then generates two circular cytoplasmic movements during prophase and metaphase. However, the nature of the 'solating agent' remains unknown in this model. Cdk1-CycB affects both microtubule and microfilament dynamics and CycB is higher in the middle region from prophase to anaphase, suggesting that Cdk1-CycB is a likely candidate for the solating agent that initiates axial expansion. Therefore, a positive feedback loop is seen between CycB distribution and cytoplasmic flow. This feedback loop is disrupted by CycB degradation at anaphase, when a slight backward cytoplasmic flow is observed (Ji, 2004).

According to this scenario, global cytoplasmic movement and local oscillation of mitotic cyclin concentration are the two key factors generating metasynchronous mitoses during preblastoderm cycles. Interestingly, axial expansion only occurs between cycle 4 and 8, the same period during which regional differences are observe in metaphase and anaphase duration. This global cytoplasmic movement is not observed after cycle 8, correlating with the observation of little regional difference in metaphase and anaphase duration after cycle 8. Direct observation of CycB movement in embryos with CycB-GFP fusion proteins would be ideal. However, CycB-GFP signal could not be detected prior to cycle 10 (Ji, 2004).

Interphase extension after cycle 10 has been explained in two ways. Decreasing CycB has been observed to correlate with longer interphase after cycle 10, and it was thus proposed that interphase extension after cycle 10 was due to CycB limitation. However, based on the observation that fast cycles continue after cycle 10 in grp mutant embryos it has been proposed that in wild-type embryos depletion of factors involved in DNA replication causes longer interphase after cycle 10 and the interphase extensions are regulated by the DNA-replication checkpoint pathway (Ji, 2004 and references therein).

Several observations of the current study might resolve this controversy. Interphase extension was observed to occur in grp mutant embryos before cycle 10, thus it is proposed that interphase extension before cycle 10 is solely due to CycB limitation. This is further supported by the following observations: (1) interphase extension occurs at an earlier cycle when maternal CycB is reduced and later when CycB is increased; (2) looking within a specific cycle, interphase is longer when CycB is lower and shorter when CycB is higher; (3) global CycB levels start to oscillate at the beginning of cycle 6 or 7 in wild-type embryos, exactly the same time when interphase duration starts to increase (Ji, 2004).

It is also proposed that after cycle 10, interphase extension is under control of both CycB limitation and the DNA-replication checkpoint. It was reported that grp¹ is a null allele. Interphase continuously extends in grp¹ embryos after cycle 10, although this extension is not as extensive as in wild-type embryos. This observation supports the idea that limitation of CycB is responsible for this increase (Ji, 2004).

With these two proposals in mind, the interphase extension was re-examined. Before cycle 10, interphase extension occurs coordinately in all nuclei and the nuclei doubling time shows no regional difference. After cycle 10, nuclei divide slower in the middle because interphase in this region is longer -- this correlates with an increase of nuclear density in this region of the embryo after cycle 10. In many organisms, a higher nucleocytoplasmic ratio correlates with a slower cell cycle. A venerable hypothesis is that higher nuclear density could result in an earlier depletion of factors necessary for DNA replication, such as deoxynucleotide triphosphates. This may result in slower DNA replication, thereby specifically prolonging interphase and ultimately total cell-cycle length (Ji, 2004).

Currently, there are two major mechanisms proposed to regulate sister chromatid separation in anaphase. (1) Disassembly of microtubules in kinetochore regions shortens kinetochore microtubules and generates the force that pulls the sister chromatids apart once cohesin is cleaved by separase. (2) The disassembly of spindle microtubules at the centrosomal region induces the poleward microtubule movement, which then generates the force that separates the sister chromatids. In syncytial blastoderm Drosophila embryos (cycles 10 to 13), it has been documented that the poleward microtubule movement is the key component that separates the sister chromatids in anaphase A, whereas the disassembly of microtubules at the kinetochore is a minor factor (Ji, 2004).

How does Cdk1-CycB affect velocity of sister chromatid separation? CycB levels negatively affect microtubule stability: higher Cdk1-CycB levels lead to less stable microtubules and, correspondingly, lower Cdk1-CycB levels lead to more stable microtubules. It also takes a longer time to form a stable metaphase configuration when CycB levels are elevated. Furthermore, when more CycB is present the microtubules of the metaphase spindle are weaker than when less CycB is available. It is speculated that the disassembly of spindle microtubule is faster or more efficient at the centrosomal regions because there are fewer microtubules at the centrosomal regions in embryo with higher Cdk1-CycB activity, contributing to the faster poleward microtubule movement. Alternatively, Cdk1-CycB might affect the sister chromatid movement via its target proteins in the centrosomal regions and/or the midzone where the interpolar microtubules overlap. For example, Cdk1-CycB phosphorylates p93dis1 (Drosophila homolog: mini spindles), which is enriched in the centrosomal regions and kinesin Eg5 (Drosophila homolog KLP61F), which accumulates on the midzone after phosphorylation. Mutation of p93dis1 in fission yeast results in failure in sister chromatid separation, while the phosphorylated bipolar kinesin KLP61F is thought to be involved in sister chromatid separation by regulating sliding of the interpolar microtubules in anaphase (Ji, 2004).

The observation that Cdk1-CycB affects the velocity of sister chromatid movement during anaphase supports the idea that microtubule dynamics contribute to the mechanical force for sister chromatid separation. This novel observation provides an entry point to further investigate the molecular mechanism that leads to disassembly of spindle microtubules by analyzing, for example, the possible functions of the target proteins of Cdk1-CycB in the centrosomal region (Ji, 2004).

The TPLSM provides new opportunities for precisely analyzing the timing of biological events. Its major advantages over conventional confocal imaging are that the two-photon excitation generates less photo damage to thick living objects, and is particularly successful at imaging fluorescent signals deep in the specimen. Indeed, it was found that with traditional confocal microscopy, GFP signals prior to cycle 10 are difficult to detect. In addition, cell-cycle arrest and chromosome bridges were frequently observed in the region of focus, indicating phototoxic effects (Ji, 2004).

Using TPLSM, it was found that in the earliest embryonic stages, an abundance of many maternal proteins obscure the observation of the product at the target. However, after the first few cycles, depending on the protein, the maternal storage declines and the GFP signal from the fusion protein becomes target specific. The time point of detection depends on the localization, the amount and function of the protein. For example, histone-GFP on chromosome is recognized after cycle 4, while tubulin-GFP can only be recognized on microtubules after cycle 8 (Ji, 2004).

The ability to detect fluorescent label within the preblastoderm embryo and follow changes in this signal over a relatively long period of time without affecting viability provides opportunity to study this early developmental stage that has been previously inaccessible. For example, there are an abundance of maternal effect mutations that develop apparently normal up to cycle 5 or 6, after which development arrests, a phenomenon referred to as 'epigenetic crisis'. This phenomenon is observed in many vertebrates and invertebrates, indicating that mid-cleavage is a critical developmental stage. Although some of the Drosophila mutations have been molecularly identified, a phenotypic analysis using the TPLSM to assess the function of these genes will increase the understanding of this developmentally critical period (Ji, 2004).

The schedule of destruction of three mitotic cyclins can dictate the timing of events during exit from mitosis

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).

CYC-B antibody stains all metaphase cells but fails to stain cells that have clearly progressed to anaphase, arguing that CYC-B is normally degraded rapidly at the metaphase/anaphase transition. Consistent with this, spindle-associated fluorescence from a CYC-B-GFP fusion protein disappears before anaphase, although a weaker cytoplasmic fluorescence persists. Onset of CYC-B degradation at metaphase/anaphase implies that CYC-B^S should have its earliest impact after metaphase. Fitting this prediction, CYC-B^S arrests mitosis with disjoined, but not separated, chromosomes. However, this seems unlikely to be a simple arrest configuration. Since a balance of tension is thought to retain chromosomes at the metaphase plate, disjunction ought to free the sister chromosomes to move to the poles. Indeed, real-time results reveal that early mitotic events are normal and that anaphase movements follow chromosome disjunction. The unusual terminal configuration follows specific disruptions in anaphase (Parry, 2001).

The earliest defect following expression of CYC-B^S is the slowing of anaphase A chromosome movements. Coordinated action of motor proteins at the kinetochore and spindle microtubule depolymerization usually drive these movements. The data do not identify the aspect of movement that is inhibited, but they show that timely degradation of CYC-B is essential for normal chromosome movement (Parry, 2001).

CYC-B persistence also blocks anaphase B pole separation. Spindle elaboration relies on a balance of forces from numerous motor proteins. Recent work suggests that the balance of forces tips toward pole separation upon downregulation of the minus end-directed motor Ncd. Accordingly, the observations could be explained if CYC-B degradation triggers downregulation of Ncd and so induces pole movements. Regardless of the mechanism, the findings show that anaphase B initiation is regulated and that it can be timed by the schedule of CYC-B degradation (Parry, 2001).

Mitosis does not simply arrest at the point of its inhibition by CYC-B^S. After the slowed anaphase movements, the chromosomes pause, then move discoordinately back to the midpoint of the spindle. From there, the chromosomes take frequent excursions toward the poles, often oscillating back and forth between one pole and the other. These oscillations resemble those of the unpaired sex chromosome in male meiosis in grasshoppers. Without a balancing pull toward the opposite pole, chromosomes move toward the pole to which they are attached. But why do they oscillate? According to one interpretation, instability in kinetochore-spindle interactions leads to a cycle of detachment and eventual reattachment to spindle fibers from the opposite pole and subsequent movement to that pole. Physical pulling indicates that chromosomes are not always attached to the spindle but also demonstrate that tension stabilizes attachment. It has been inferred that these features cooperate to promote the attachment of the two kinetochores of normal chromosomes to opposite poles during prometaphase, as this ought to be the only stable outcome. Because of its contribution to the alignment of chromosomes, tension-dependent kinetochore-spindle attachment is thought to be an essential feature of prometaphase (Parry, 2001).

The stability of kinetochore-spindle attachment becomes independent of tension by the time of anaphase. This is necessary because tension is lost when chromosomes become univalent upon disjunction, yet chromosomes must retain their orientation to a single pole for orderly distribution to daughter nuclei. It is not known what controls the change in stability of kinetochore-spindle attachment between prometaphase and anaphase. The finding that persistence of CYC-B results in continued unstable attachment suggests that degradation of CYC-B is required for this transition in stability. In contrast to the unstable kinetochore-spindle interaction at the CYC-B^S arrest, chromosomes are held at the poles for as long as 20 min at the CYC-B3^S arrest. It is proposed that the unstable kinetochore-spindle attachment that prevails in prometaphase can be countered by tension during prometaphase/metaphase or by degradation of CYC-B at the metaphase/anaphase transition. Additionally, it is suggested that the period between the degradation of CYC-B and destruction of CYC-B3 provides a mitotic phase of stable kinetochore-spindle attachment during which chromosomes can be faithfully segregated. To probe the mechanism of this effect, it will be interesting to examine the influence of CYC-B/Cdk1 on putative regulators of chromosome congression such as CENP-E (Parry, 2001).

The CYC-B^S results differ from some related analyses in other systems. Stabilization of the S. cerevisiae Clb2 arrests cells with an elongated spindle, which suggests the completion of anaphase. However, differences in the biology and uncertain analogies among cyclins make comparisons to yeast difficult. Introduction of a stable sea urchin delta-90 cyclin B into Xenopus extract systems and into mammalian tissue culture cells allows chromosome separation to the poles. However, when tested in sea urchin, this echinoderm delta-90 cyclin B induces a phenotype consistent with the one described in Drosophila. Furthermore, a stable version of vertebrate cyclin B2, one of the two closely related B type cyclins in vertebrates, blocks HeLa cells with a metaphase-like arrest with chromosomes 'not properly aligned at the metaphase plate', which is consistent with the arrest characterized in Drosophila. Based on observations that heterologous cyclin and Cdk combinations, particularly the echinoderm cyclin B with Drosophila Cdk, have limited activity, it is suggested that discordant observations result from the use of heterologous cyclins and that the results reported here will extend to other metazoan systems (Parry, 2001 and references therein).

Following the expression of CYC-B3^S, prophase, metaphase, and anaphase A and B all occur with the normal timing and kinetics. Once anaphase is complete, however, CYC-B3^S blocks further nuclear and spindle events: chromosomes remain condensed, the nuclear membrane fails to reform, and the spindle remains intact without maturing to its late mitotic configuration with a prominent midbody. In many arrested cells, cytokinesis proceeds, bifurcating the spindle and separating the two polar clusters of chromosomes. Furrowing is detected as chromosomes reach the poles, and both live and fixed data suggest that furrows are deep, stable, and perhaps complete. Double labeling reveals cells that stain for CYC-B3, but not CYC-B, indicating that CYC-B3 is normally degraded after CYC-B and hence after the metaphase/anaphase transition. In addition, CYC-B3 labeling has disappeared by the time anaphase separation of chromosomes is complete. Mitosis progresses normally to late anaphase in the presence of CYC-B3^S, and only at this point is an arrest of multiple late mitotic events seen (Parry, 2001).

Cytokinetic furrow formation in cells with condensed chromosomes implies that cytokinesis is not coupled to the progress of nuclear or cytoplasmic events by direct dependencies. Instead, the results suggest that the onset of cytokinesis is regulated by the schedule of cyclin destruction. Perhaps the different abilities of CYC-B and CYC-B3 to inhibit furrow formation allow a window of opportunity for timely initiation of furrow formation. This window would be created by degradation of CYC-B before CYC-B3 but might be offset from the time of degradation by a lag time required for the dephosphorylation of relevant targets. Consistent with this idea, furrow formation commences earlier than normal in embryos deficient in CYC-B (Parry, 2001).

Cytokinesis without mitotic progression produces a furrow that appears to cut through the spindle that lacks the compact microtubular midbody structure. Thus, in the context of the CYC-B3^S arrest, furrow formation can occur in the absence of a spindle midbody, a structure that has been proposed to be essential for cytokinesis. Nonetheless, the gene products normally recruited to the midbody may be recruited to the midzone in the absence of morphological specialization and thereby play their proposed roles in the stabilization of the cytokinetic furrow (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).

Cyclin B expression in the optic lobe

Lamina precursor cells (LPCs) of the optic lobe are derived from a set of neighboring neuroblasts in the anterior segment of the outer proliferative center (aOPC). The aOPC and LPCs form an epithelial sheet on the surface of the brain. OPC neuroblasts that produce LPCs are at the anteriormost extent of this epithelium, with the lamina marking its posterior limit. LPCs are produced continuously from OPC neuroblasts and complete two cell cycles before differentiating into neurons. LPCs therefore enter the cell cycle as they are produced from aOPC neuroblasts and exit after the second division to differentiate into lamina neurons. As a result of this "assembly line" organization, LPCs in successive phases of the cell cycle are found in sequence along the proliferative epithelium. LPCs in different phases of the cycle are found at discrete positions relative to anatomical landmarks. Most notable of these is a furrow in the aOPC/LPC epithelium, located at the anterior boundary of the developing lamina. This lamina furrow, like the morphogenetic furrow (MF) in the eye disc, sweeps forward as neurons are added to the differentiating lamina. LPC divisions are synchronized across the furrow, with cells in different phases of the cell cycle at specific positions. The distribution of LPCs can be revealed using two markers, antibodies to Cyclin B, which is expressed at highest levels in late G2-early M phase, and propidium iodide, a fluorescent dye that binds to DNA and permits the visualization of mitotic chromosomes. The two LPC division cycles are evident as two regions of peak cyclin B expression corresponding to two domains of mitotic figures, along the anterior and posterior segments of the lamina furrow respectively. Cells between the two Cyclin B-expressing domains reside in G1, (low levels of cyclin B) and the subsequent S phase. Newly arrived photoreceptor axons specifically run along the base of the G1-phase LPCs and trigger their entry into S phase (Nakato, 1995).

Third instar larvae homozygous for several division abnormally delayed (dally) alleles were evaluated for the organization and cell cycle progression of LPC divisions. All dally mutants examined showed the same constellation of defects, with varying degrees of abnormalities either as homozygotes, or in combination with dally P2. In every brain examined the G2- and M-phase cells of the second LPC division were absent. In wild-type larvae, G2- and M-phase cells of the second division are located near the surface of the brain, at the posterior limit of the proliferative epithelium. Cyclin B-expressing cells and mitotic figures are not found in this region of dally P2 homozygous larval brains. The complete absence of the second LPC division in dally homozygotes, as evidenced by the loss of the second cyclin B-expressing domain, is particularly clear from lateral views of brain lobes. Abnormalities in the first LPC division cycle are also found. Normally, G2- and M-phase cells of the first division are found exclusively along the anterior segment of the lamina furrow. In dally P2 homozygous larvae, mitotic figures are frequently found in the posterior part of the furrow. The domain of Cyclin B immunoreactivity marking the G2 phase of the first division extends up to these abnormally positioned mitotic cells. The extended Cyclin B domain, and the misplacement of the M phase cells of the first division, suggests that this division cycle is delayed somewhere along the G2-M transition in dally P2 mutants (Nakato, 1995).

Distribution during oogenesis and localization to pole cells

Drosophila oocytes develop within cysts containing 16 cells that are interconnected by cytoplasmic bridges. Although the cysts are syncytial, the 16 cells differentiate to form a single oocyte and 15 nurse cells; several mRNAs that are synthesized in the nurse cells accumulate specifically in the oocyte. An examination of cytoskeletal organization during oocyte differentiation provides insight into the mechanisms that generate the cytoplasmic asymmetry within these cysts. Shortly after formation of the 16 cell cysts, a prominent microtubule organizing center (MTOC) is established within the syncytial cytoplasm. At the time the oocyte is determined, a single microtubule cytoskeleton connects the oocyte with the remaining 15 cells of each cyst. Recessive mutations at the Bicaudal-D (Bic-D) and egalitarian (egl) loci block oocyte differentiation and disrupt the formation and maintenance of this polarized microtubule cytoskeleton. Microtubule assembly-inhibitors phenocopy these mutations, and prevent oocyte-specific accumulation of Oskar, Cyclin B and 65FmRNAs. Formation of the polarized microtubule cytoskeleton is now thought to be required for oocyte differentiation; this structure appears to mediate the asymmetric accumulation of mRNAs within the syncytial cysts (Theurkauf, 1993).

The concentration of Cyclin B transcripts at the posterior pole of the Drosophila oocyte occurs at a late stage of oogenesis, depending upon the sequence in the 3' untranslated part of the RNA. These transcripts are incorporated into the pole cells of the developing embryo and persist through a subsequent period of embryogenesis in which these cells are not dividing. RNA injected into the posterior cytoplasm of syncytial embryos accumulates in the pole cells if it contains sequences present in the 3' untranslated region of maternal Cyclin B transcripts. The injected RNA is not translated until a point prior to the resumption of mitosis by these cells, that is, not until the cells have become incorporated into the gonads. Zygotic transcription directed from the Cyclin B promoter does not begin in the pole cells until the first instar larva has hatched. Deletion of a small sequence element from the 3' untranslated region of an epitope tagged Cyclin B RNA does not affect its posterior accumulation but results in its premature translation (Dalby, 1993).

Drosophila germ granules are structured and contain homotypic mRNA clusters

Germ granules, specialized ribonucleoprotein particles, are a hallmark of all germ cells. In Drosophila, an estimated 200 mRNAs are enriched in the germ plasm, and some of these have important, often conserved roles in germ cell formation, specification, survival and migration. How mRNAs are spatially distributed within a germ granule and whether their position defines functional properties is unclear. This study, using single-molecule FISH and structured illumination microscopy, a super-resolution approach, shows that mRNAs are spatially organized within the granule whereas core germ plasm proteins are distributed evenly throughout the granule. Multiple copies of single mRNAs organize into 'homotypic clusters' that occupy defined positions within the center or periphery of the granule. This organization, which is maintained during embryogenesis and independent of the translational or degradation activity of mRNAs, reveals new regulatory mechanisms for germ plasm mRNAs that may be applicable to other mRNA granules (Trcek, 2015).

This study combined single-molecule FISH with structured illumination microscopy (SIM), a super-resolution technique, to gain a high-resolution view of the mRNA-bound germ granule. This combinatorial approach allowed the determination that germ granule-localized mRNAs occupy distinct positions within the granule and relative to each other, while germ granule proteins are homogeneously distributed within the granular space. Multiple localized mRNAs group to form homotypic cluster, which gives the germ granule its structure. This structure does not change through early embryonic development and does not correlate with the translational onset of localized mRNAs or with the ability of germ granules to protect bound mRNAs from decay (Trcek, 2015).

This analysis of the organizational structure of germ plasm focused on core germ granule protein components, Vas, Osk, Tud and Aub, and on cycB, nos, pgc, gcl and osk mRNA. cycB, nos, pgc, gcl and osk serve as prototypes for mRNA localization to the germ granules because their localization to the germ plasm, their regulation in the germ plasm and biological significance for germ cell biology are understood best. While mRNA localization studies suggest that up to 200 mRNAs may be localized to the posterior pole of the early embryo, it is assumed that regulatory mechanisms revealed by the study of cycB, nos, pgc and gcl are shared among other germ plasm-localized mRNAs. The study of germ plasm-localized mRNA regulation revealed that only localized mRNAs translate, while their unlocalized counterparts are translationally silent, that localized mRNAs are protected from mRNA decay, and that the 3′ UTRs of localized mRNAs are necessary and often sufficient to localize mRNAs to the posterior and render them translationally competent. The experiments demonstrate that cycB, nos, pgc and gcl mRNAs concentrate in homotypic clusters, assume specific positions within the germ granules, and can organize into separate granules. The results make it unlikely that cycB, nos, pgc and gcl clusters contain more than one type of mRNA. If clustering between heterotypic mRNAs was a common organizational strategy, the pairwise analysis with cycB, nos, pgc and gcl would have not yielded the distinct volumes observed. Thus, despite the fact that only a limited number of localized RNAs were sampled, it is anticipated that germ granule organization observed for cycB, nos, pgc and gcl is also shared by other germ granule-localized mRNAs, which are similarly regulated (Trcek, 2015).

Given that the core germ plasm proteins Osk, Vasa, Aub and Tud recruit other germ granule components and are themselves homogeneously distributed within the granule, it is unlikely that the germ granule structure is dictated by proteins alone. Homotypic clustering could also be driven by intramolecular RNA–RNA interactions, similar to those found in the localized bicoid mRNA at the anterior pole and in the co-packaged osk mRNA during transport to the oocyte posterior. The dramatic increase in mRNA concentration in the granule compared with rest of the embryo may raise the likelihood for two mRNAs to interact or even induce RNA–RNA interactions by altering mRNA conformation thus driving homotypic clustering (Trcek, 2015).

In yeast, the movement of mRNAs in and out of stress granules and processing bodies determines their translatability and stability and in Drosophila oocytes the position of bicoid and gurken mRNA within the sponge body correlates with their translational activity. This study found, however, that the mRNA position within the germ granule is independent of translational or degradation activity of localized mRNAs. Some translational and decay regulators found in germ granules are also found in sponge bodies, stress granules and processing bodies. Thus the data imply that in germ granules these proteins may regulate transcripts differently to allow for the dynamic regulation of different mRNAs. Alternatively, sorting of mRNAs into distinct granules could specify their activity. For example, pgc co-localizes with core germ-granule components as well as with osk mRNA. Thus, the pool of pgc associated with osk could be functionally different from the one that associates with Vasa, Osk, cycB, nos and gcl. Indeed, in older embryos just before pgc becomes translated, pgc moves away from osk, but not from VasaGFP, cycB, nos and gcl. Speculatively, this could be the mechanism that determines the onset of pgc translation (Trcek, 2015).

mRNA clustering could also enhance biochemical reactions locally either by enabling protein complex formation, by quick re-binding of a regulator to a neighbouring mRNA or by increasing the concentration of a regulator of the cluster RNAs. For example, it has been proposed that the repression of cycB translation by Nanos protein (Nos) depends on a high local concentration of Nos in the germ plasm. Multiple nos mRNAs within the cluster could increase the local concentration of Nos thus counteracting the loss of the unbound Nos due to diffusion into the embryo. Once bound to cycB, Nos could also be quickly re-bound by the neighbouring cycB mRNAs thus maintaining high Nos concentration and ensuring efficient cycB repression. In this way each mRNA cluster in the granule would resemble a biochemical territory, consistent with the recent observations showing that germ granules in Caenorhabditis elegans, which behave like liquid droplets, are also not homogeneous. It is proposed that an mRNA-protein granule organization similar to the one described in this study for Drosophila germ granules could be a conserved feature of larger ribonucleoprotein granules (Trcek, 2015).

Effects of Mutation or Deletion

In eukaryotes, mitotic cyclins localize differently in the cell and regulate different aspects of the cell cycle. The relationship between subcellular localization of Cyclins A and B and their functions in syncytial preblastoderm Drosophila embryos has been investigated. During early embryonic cycles, Cyclin A is always concentrated in the nucleus and present at a low level in the cytoplasm. Cyclin B is predominantly cytoplasmic, and localized within nuclei only during late prophase. Also, Cyclin B colocalizes with metaphase but not anaphase spindle microtubules. Maternal gene doses of Cyclins A and B were changed to test their functions in preblastoderm embryos. Increasing doses of Cyclin B increases Cyclin B-Cdk1 activity, which correlates with shorter microtubules and slower microtubule-dependent nuclear movements. This provides in vivo evidence that Cyclin B-Cdk1 regulates microtubule dynamics. In addition, the overall duration of the early nuclear cycles is affected by Cyclin A but not Cyclin B levels. Taken together, these observations support the hypothesis that Cyclin B regulates cytoskeletal changes while Cyclin A regulates the nuclear cycles. Varying the relative levels of Cyclins A and B uncouples the cytoskeletal and nuclear events, so it is speculated that a balance of cyclins is necessary for proper coordination during these embryonic cycles (Stiffler, 1999).

Axial expansion occurs during cycles 4-6 when nuclei move along the anterior/posterior axis of the embryo. In cycles 7-10, nuclei migrate to the embryonic cortex. The microfilament breakdown during axial expansion was investigated. It has been proposed that microtubules regulate the stability of the microfilaments and direct their breakdown, thus controlling the movement of the nuclei. The network of astral microtubules pushes nuclei to the cortex of the embryo. These movements occur periodically and correlate with dynamic changes of the network. Microtubules undergo dramatic phase-specific morphological changes during cycles 4-7. Interphase astral microtubules radiate from centrosomes and form a network that generates the positioning of evenly spaced nuclei. This network breaks down in prophase and microtubules reorganize into mitotic spindles in metaphase. In anaphase the microtubules bring chromatids to the poles and the mitotic spindle breaks down; a midbody remains between the separating chromatids. Finally, microtubules reform asters as division is completed in telophase, and the midbody persists through interphase (Stiffler, 1999).

Cyclin B has been shown to regulate microtubule dynamics in Xenopus extracts, and cyclin B localizes to microtubules in blastoderm stage Drosophila embryos. Microtubular dynamics affects nuclear migration in the preblastoderm embryos. Whether or not cyclin B levels affect axial expansion and cortical migration during preblastoderm cycles was tested. To do this, the volume of interphase asters was calculated and microtubule configurations were observed in embryos possessing different doses of Cyclin B (1-, 2- and 4-cyclin B embryos). Astral microtubule volume is inversely proportional to Cyclin B dose. Confocal images reveal a similar correlation between microtubule length and cyclin B dose during interphase and metaphase. In interphase, microtubules are longer in 1- and 2-cyclin B embryos, when compared to those of 4-cyclin B embryos. During prophase in 1-cyclin B embryos, asters do not break down as completely as they do in 4-cyclin B embryos. During metaphase, mitotic spindles appear larger in 1-cyclin B embryos and smaller in 4-cyclin B embryos, as compared to 2-cyclin B embryos. In addition, metaphase asters are longer and clearly visible in 1-cyclin B compared to those in 2-cyclin B, but they are not detectable in 4-cyclin B embryos. During anaphase and telophase, larger microtubule asters are observed in 1-cyclin B embryos compared to those in 2- and 4-cyclin B embryos. To amplify the effects of cyclin B on microtubule morphology, 0- and 10-cyclin B embryos were examined. In 0-cyclin B embryos, microtubules are often elongated and organized into thick bundles throughout the embryo. Astral microtubules are often observed without nuclei; centrosomes appear to divide and migrate without associated nuclei. In 10-cyclin B embryos, microtubules are severely reduced in all phases of the cycle and nuclei remain close together. Microtubule morphology in 0- and 10-cyclin B embryos is more severely altered than that of 1- and 4-cyclin B embryos. Thus cyclin B levels correlate with microtubule abundance in different cycle phases (Stiffler, 1999).

Because cyclin A localized in nuclei, a test was performed to see whether reducing cyclin A affects cell-cycle duration. Decreased Cyclin A correlates with a significant increase in cell-cycle time during cycles 4-6, which is independent of cyclin B levels.There is a slight increase in the length of time from egg deposition to cycle 10 in 1-cyclin A embryos. In contrast, increased cyclin B does not have a linear effect on cycle time. These observations show that overall cycle length is sensitive to cyclin A levels. To analyze cell-cycle progression, the mitotic index for embryos was calculated at varying doses of cyclins A and B. During these early cycles, divisions are synchronous. Therefore, the mitotic index is the fraction of embryos in a specific cell-cycle phase, and represents the relative duration that embryos spend in that phase. During cycles 2 to 8, 4-cyclin B embryos have a longer metaphase and shorter interphase, compared to 1- or 2-cyclin B embryos, but other phases are not different. This shows that metaphase is longer in 4-cyclin B embryos while interphase is shorter; therefore the overall cell-cycle duration is the same. 1-cyclin B embryos spend less time in metaphase and more in interphase; thus the overall cell-cycle duration is again unaffected. However, less cyclin A affects the overall cycle duration, but the mitotic index is not different from controls. It is concluded that cyclin B dose affects metaphase and interphase duration while cyclin A regulates the overall cycle duration (Stiffler, 1999).

An N-terminally truncated form of Cyclin B was introduced into the Drosophila germ-line downstream of the yeast upstream activator that responds to GAL4. When such lines of flies are crossed to lines in which GAL4 is expressed in imaginal discs and larval brain, the majority of the resulting progeny die at the late pupal stage of development. Very rarely (< 0.1% of progeny) adults emerge that have a mutant phenotype typical of flies with mutations in genes required for the cell cycle: they have rough eyes, deformed wings, abnormal bristles, and die within hours of emergence. The brains of third instar larval progeny show an abnormally high proportion of mitotic cells containing overcondensed chromatids that have undergone anaphase separation, together with cells that cannot be assigned to a particular mitotic stage. Immunostaining indicates that these anaphase cells contain moderate levels of Cyclin B, suggesting that persistent p34cdc2 kinase activity can prevent progression from anaphase into telophase (Rimmington, 1994).

Cyclin A performs a mitotic function: it acts synergistically with Cyclin B during the G2-M transition. In double mutant embryos that express neither Cyclin A nor Cyclin B zygotically, cell cycle progression is blocked just before the exhaustion of the maternally contributed Cyclin A and B stores. BrdU-labeling experiments indicate that cell cycle progression is blocked in G2 before entry into the fifteenth round of mitosis. Expression of either Cyclin A or B from heat-inducible transgenes is sufficient to overcome this cell cycle block. This block is also not observed in single mutant embryos deficient for either Cyclin A or B. In Cyclin B deficient embryos, cell cycle progression continues after the apparent exhaustion of the maternal contribution, suggesting that Cyclin B might not be essential for mitosis. However, mitotic spindles are clearly abnormal and progression through mitosis is delayed in these Cyclin B deficient embryos (Knoblich, 1993).

The Drosophila body pattern is laid down by maternal and zygotic factors acting during the early phase of embryonic development. During this period, nascent zygotic transcripts longer than about 6 kilobases are aborted between the rapid mitotic cycles. Resurrector1 (Res1) and Godzilla1 (God1), two newly identified dominant zygotic suppressor mutations, and a heterozygous maternal deficiency of the Cyclin B locus, complement the partial loss of function of the segmentation gene knirps (kni) by extending the length of mitotic cycles at blastoderm. The mitotic delay caused by Res1 and God1 zygotically and by the deficiency of the Cyclin B locus maternally allows the expression of a much longer transcript of a kni cognate gene, one normally aborted between the short mitotic cycles. A consequence of this is the surival of kni mutant progeny (Ruden 1995).

A potential functional overlap between two cyclin dependent kinases (Dmcdc2 and Dmcdc2c) and two cyclins (Cyclin A and Cyclin B) shows no functional overlap between the Dmcdc2 and the Dmcdc2c kinases. The phenotype resulting from mutations in Dmcdc2 is not affected by altering the level of Dmcdc2c. Cyclin A and Cyclin B have largely overlapping functions. Cell proliferation is observed in the absence of either Cyclin A or Cyclin B, but not if both cyclins are absent. Cyclin A also has essential functions that cannot be taken over by Cyclin B, but these functions appear to be required at defined developmental stages in specific tissues only (Lehner, 1992).

Cyclin B3 has been conserved during higher eukaryote evolution as evidenced by its identification in chicken, nematodes, and insects. Except for the destruction box, similarities are restricted to the cyclin box where the B3 types share between 39% and 46% identity. Cyclin B3 is present in addition to Cyclins A and B in mitotically proliferating cells and is not detectable in endoreduplicating tissues of Drosophila embryos. Cyclin B3 is coimmunoprecipitated with Cdk1(Cdc2) but not with Cdk2(Cdc2c). It is degraded abruptly during mitosis like Cyclins A and B. In contrast to these latter cyclins, which accumulate predominantly in the cytoplasm during interphase, Cyclin B3 is a nuclear protein. Genetic analyses indicate functional redundancies. Double and triple mutant analyses demonstrate that Cyclins A, B, and B3 cooperate to regulate mitosis, but surprisingly single mutants reveal that neither Cyclin B3 nor Cyclin B is required for mitosis. However, both are required for female fertility and Cyclin B also for male fertility (Jacobs, 1998).

Surprisingly, Cyclin B and Cyclin B3 are not essential for viability, demonstrating that cell divisions can proceed without either Cyclin B or Cyclin B3. No mitotic abnormalities are detected in Cyclin B3 mutant embryos. In the absence of Cyclin B, however, cell divisions are slower than in wild type. Mitotic figures are enriched and mitotic spindles are unusual in Cyclin B mutant embryos. Spindle fibers are not as strong and straight as in wild type. Cyclin B, therefore, appears to be required for an efficient reorganization of interphase microtubules into a mitotic spindle. Consistent with this notion, Cyclin B/Cdk1 is known to increase microtubule dynamics in vitro. Mutant embryos lacking both Cyclin B and Cyclin B3 zygotically show much more severe mitotic defects than single mutants. Entry into mitosis is delayed. Mitotic spindle formation is severely disturbed as suggested by the drastic enrichment of prophase-like figures with condensed chromosomes and immature spindles. The spindles that are eventually formed appear to be largely incapable of segregating sister chromatids to the poles. The rare anaphase figures do not reveal the characteristic chromosome orientation indicative of the strong mechanical pull evident in wild-type anaphase, where chromosome arms trail behind leading centromeric regions on their way to the spindle poles. In addition to cooperation of Cyclins B and B3, synergy of Cyclins A and B3 can be demonstrated. Although chromatin condensation is particularly slow, spindle formation appears to be less affected in Cyclin A;Cyclin B3 double mutants compared to Cyclin B:Cyclin B3 double mutants (Jacobs, 1998).

On the basis the double mutant analyses it has been concluded that all of the mitotic cyclins cooperate to bring about progression through mitosis. These results are consistent with the idea that Cyclin A is most potent in triggering mitosis, whereas Cyclin B has an intermediate and Cyclin B3 the lowest potency. The results also confirm the suggestion that Cyclin B is particularly important for the organization of mitotic spindles. In addition, they suggest that Cyclin A, which translocates into the nucleus very early during prophase, and Cyclin B3, which accumulates in the nucleus already during interphase, are particularly important for chromosome condensation (Jacobs, 1998).

It has been proposed that different mitotic processes that occur after the metaphase-anaphase transition might be temporally arranged by the ordered disappearance of Cyclins A, B, and B3, such disappearances resulting from proteolytic degradation. This proposal is based not only on the ordered disappearance of the different mitotic cyclins, but also on the specific mitotic defects resulting after the expression of mitotic cyclins with amino-terminal deletions, which also remove the destruction box. These mutant nondestructible Cyclins A, B, and B3 are found to delay mitotic progression before sister chromatid separation, sister chromatid segregation to the poles, and telophase including cytokinesis, respectively. On the basis of this proposal, all these processes are expected to start simultaneously in Cyclin B;Cyclin B3 double mutants after the disappearance of Cyclin A during mitosis. In fact, chromosome segregation to the poles, chromosome decondensation, and cytokinesis are all attempted simultaneously in these double mutant embryos. However, the interpretation of these phenotypes is complicated by the fact that mitotic progression until metaphase is already severely affected, and not just exit from mitosis. This functional overlap between the cyclins is less pronounced in the germ line and in particular during oogenesis where both Cyclin B and Cyclin B3 are essential. Therefore, a future detailed characterization of the role of different mitotic cyclins in oogenesis should be especially informative with regard to cyclin type-specific functions and regulatory differences between mitosis and meiosis (Jacobs, 1998).

cdc2 links the Drosophila cell cycle and asymmetric division machineries; Maintaining the asymmetric localization of the apical components during mitosis requires Cdc2/B-type cyclin complexes

Asymmetric cell divisions can be mediated by the preferential segregation of cell-fate determinants into one of two sibling daughters. In Drosophila neural progenitors, Inscuteable, Partner of Inscuteable and Bazooka localize as an apical cortical complex at interphase, which directs the apical-basal orientation of the mitotic spindle as well as the basal/cortical localization of the cell-fate determinants Numb during mitosis. Although localization of these proteins shows dependence on the cell cycle, the involvement of cell-cycle components in asymmetric divisions has not been demonstrated. Neural progenitor asymmetric divisions require the cell-cycle regulator cdc2. By attenuating Drosophila cdc2 function without blocking mitosis, normally asymmetric progenitor divisions become defective, failing to correctly localize asymmetric components during mitosis and/or to resolve distinct sibling fates. cdc2 is not necessary for initiating apical complex formation during interphase; however, maintaining the asymmetric localization of the apical components during mitosis requires Cdc2/B-type cyclin complexes. These findings link cdc2 with asymmetric divisions, and explain why the asymmetric localization of molecules like Inscuteable show cell-cycle dependence (Tio, 2001).

There are three known mitotic cyclins in Drosophila -- cyclin A, B and B3; these need to be destroyed for mitotic exit to occur. Is a subset of these cyclins preferentially required for maintaining the apical components? The temporal profile of Insc localization was followed with respect to the time of cyclin A (metaphase), cyclin B (early anaphase) and cyclin B3 (late anaphase) destruction. The results from anti-Insc/anti-cyclin-A/DNA-stain triple-labelling experiments show that Insc remains apically localized at metaphase/anaphase after destruction of cyclin A. Supporting the idea that cyclin A is dispensable, no evidence of defective Insc localization was detected in cyclin A single mutants and cyclin A;cyclin B3 or cyclin A;cyclin B double mutants. In wild-type NBs, Insc delocalization occurs after chromosome separation, coinciding with the time when cyclin B3 becomes undetectable. Single mutants in cyclin B and cyclin B3 do not show defects in Insc localization; however, in cyclin B;cyclin B3 double mutants, mislocalization of Insc can be seen in most mitotic NBs (72% for prophase; 71% for metaphase). These results indicate that whereas cyclin A appears dispensable, the B-type cyclins are required to maintain asymmetric localization of Insc during mitosis (Tio, 2001).

PAN GU: a protein kinase that inhibits S phase and promotes mitosis in early Drosophila development: PNG controls the cell cycle by permiting the accumulation of adequate levels of Cyclin B protein

Following completion of meiosis, DNA replication must be repressed until fertilization. In Drosophila, this replication block requires the products of the pan gu (png), plutonium (plu) and giant nuclei (gnu) genes. (For information on the Pan Gu legend, see Pan Gu Creates the World). These genes also ensure that S phase oscillates with mitosis in the early division cycles of the embryo. png encodes a Ser/Thr protein kinase expressed only in ovaries and early embryos; the predicted extent of kinase activity in png mutants inversely correlates with the severity of the mutant phenotypes. The Plu and Png proteins form a complex that has Png-dependent kinase activity, and this activity is necessary for normal levels of mitotic cyclins. Cyclin B is a key Png target. Mutations in cyclin B dominantly enhance png, whereas png is suppressed by cyclin B overexpression. Suppression occurs via restoration of Cyclin B protein levels that are decreased in png mutants. plu and gnu phenotypes are also suppressed by cyclin B overexpression. These studies demonstrate that a crucial function of PNG in controlling the cell cycle is to permit the accumulation of adequate levels of Cyclin B protein. These results reveal a novel protein kinase complex that controls S phase at the onset of development apparently by stabilizing mitotic cyclins (Fenger, 2000 and Lee, 2001).

During the first seven cell cycles in Drosophila, the mitotic cyclins A and B, and CDC2 activity are present at high levels with no detectable fluctuation in overall levels during the cell cycle. Localized degradation of Cyclin B has been detected, though, and it is thought that localization of cyclins A and B may be important in coordinating the nuclear cycles with the microtubules. Furthermore, inhibition of the cyclin degradation machinery with a destruction box peptide or injection of non-destructible Cyclin B causes mitotic arrest in the early embryo. All these results suggest that cyclins play a crucial role in coordinating S phase and mitosis in the early embryo. How the cyclins are controlled is unclear (Fenger, 2000).

The plu gene has been shown to encode a 19 kDa ankyrin-repeat protein. png encodes a Ser/Thr protein kinase that exists in vivo in a complex with Plu. This protein kinase complex is present specifically in the egg and early embryo, and thus it appears to control S phase uniquely in response to fertilization at the onset of development. Analysis of cyclins A and B in png mutants shows that Png affects the levels and post-translational modification of mitotic cyclins, as well as the extent of histone H3 phosphorylation and CDC2 kinase activity. Therefore Png controls chromosome condensation and mitotic progression in the early embryo (Fenger, 2000).

The phenotypes of plu, png and gnu mutants suggest that these genes normally function to inhibit initiation of DNA replication during early embryogenesis. Activating mitosis is one means by which this could be accomplished. If mitotic functions were not activated in the mutant embryos then repeated rounds of DNA replication could occur. This phenomenon would be similar to the repeated rounds of DNA replication that result from loss-of-function of cdc2 and cyclin B in S. pombe. Mitotic cyclin/Cdk activity is needed to inactivate replication origins, and chromosome condensation in mitosis may also serve to block DNA replication. To test if png, plu and gnu affect regulation of mitosis, the protein levels and forms of CDC2 and the mitotic cyclins A and B were examined in mutant embryos. Interestingly, the levels of mitotic cyclins are decreased in mutant embryonic extracts, and the effect is allele specific with respect to png. Three forms of Cyclin A were detected on immunoblots, and the fastest migrating form was predominantly decreased in the mutants: barely detectable levels of this fast migrating form were present in weak png mutant embryos, and none was detected in strong png, plu or gnu mutants. The slower two forms of Cyclin A were less affected, although they also showed allele-specific reduction: the greatest decrease was seen in png¹⁰⁵⁸, png¹⁷², png¹⁹²⁰ , plu and gnu, and the highest levels of Cyclin A were seen in the three weak png alleles. This experiment was repeated twice, and the same decreases in Cyclin A levels were observed. It was also found that levels of Cyclin A were reduced in unfertilized eggs from png¹⁰⁵⁸ mutant mothers (Fenger, 2000).

To determine if the missing fast-migrating Cyclin A form was phosphorylated, and thus a potential Png substrate, wild-type embryonic extracts were treated with lambda-phosphatase. Compared with the 'no phosphatase' control and to wild-type extracts homogenized directly in urea sample buffer, phosphatase-treated extracts showed a loss of the slowest migrating form and an accumulation of the fastest migrating form, indicating that the fast form was actually unphosphorylated. If Cyclin A were a substrate of Png, the phosphorylated form of Cyclin A would be the most decreased of the forms. Since the unphosphorylated form appeared to be preferentially lost in png mutants, it is unlikely that Cyclin A is a Png substrate (Fenger, 2000).

An allele-dependent decrease in Cyclin B levels of mutant embryos was also detected. Again, all of the alleles of png, gnu and the plu null mutation showed decreased cyclin levels compared with wild-type embryos, and of the mutants, the highest levels were seen in the three weak png alleles. The plu null and gnu mutants had the lowest levels, and of the png alleles, the lowest levels were seen in png¹⁷², png¹⁹²⁰, png¹⁵⁸ and png¹⁰⁵⁸. It is interesting that png¹⁷² and png¹⁹²⁰ (which were both predicted to have the lowest kinase activity based on the nature of the mutations) also have the lowest levels of cyclins A and B. Cyclin B levels also were decreased in unfertilized eggs from png¹⁰⁵⁸ mutant mothers. Consistent with the decrease in levels of mitotic cyclins, it was found that CDC2 kinase activity was also decreased (Fenger, 2000).

Examination of the levels and forms of CDC2 showed no difference between wild-type and mutant embryos. It has been observed that Histone H3 phosphorylation correlates with CDC2 kinase activity in early Drosophila embryos, so anti-phospho-H3 antibodies were used to examine levels of H3 phosphorylation in mutant embryos. Phospho-H3 levels were decreased in all the mutants, and again the degree of decrease correlated with allele strength. The lowest levels of phospho-H3 were seen in plu, gnu, png¹⁷², png¹⁹²⁰ and png¹⁰⁵⁸. The highest levels were observed in the three weak png alleles and png¹⁵⁸. MPM2 antibodies also recognize phospho-epitopes present in mitotic cells and dependent on CDC2 activity. A decrease in at least one MPM2 epitope was observed in png¹⁰⁵⁸ and plu⁶ embryonic extracts compared with wild. To assay CDC2 kinase levels directly, CDC2 was immunoprecipitated and levels of histone H1 kinase activity were tested in the pellets. The proteins in the pellet were immunoblotted and the levels of phosphorylated histone H1 were compared with the amount of CDC2 immunoprecipitated, as measured by probing the membrane with antibodies against CDC2. In the mutants the levels of CDC2 kinase activity were decreased about twofold. In conclusion, these results show that mitotic cyclins and CDC2 activity are decreased in png, plu and gnu mutants, and the decrease is allele specific and consistent with predicted levels of Png kinase activity (Fenger, 2000).

The primary defect in png, plu and gnu mutants is likely caused by a decrease in the levels of mitotic cyclins A and B and associated CDK activity. The over-replication phenotype might result because mitotic cyclins are required to block re-replication in the unfertilized egg and early embryo, because the embryo requires chromosome condensation to block re-replication, or a combination of both. Work in Schizosaccharomyces pombe has shown that Cyclin B can function to inhibit DNA replication, since strains deleted for cdc13, which encodes Cyclin B, undergo repeated rounds of S phase without mitoses. Moreover, it has been shown that CDC2 and Cyclin A inhibit endoreplication in diploid cells during Drosophila larval development. The replication of centrosomes that are unattached to nuclei, seen in these mutants, is observed also in embryos lacking Cyclin B, so this mutant cytoskeletal defect may be due to decreased Cyclin B levels (Fenger, 2000).

It is unlikely that Png interacts directly with cyclins because Png and Plu do not co-immunoprecipitate with Cyclin A or Cyclin B, and the unphosphorylated form of Cyclin A appears to be predominantly lost in png mutants. The differential decrease of the different forms of Cyclin A suggests that it is unstable in the mutants, particularly the faster migrating form, and that the decrease is not due to decreased cyclin translation. One possibility is that Png controls cyclin stability by acting through the protein degradation machinery of the APC/cyclosome. It is known that yeast Cdh1, an activator of the APC and subsequent Clb degradation, is inactivated by phosphorylation, so Fizzy-related (Fzr), the Drosophila Cdh1 homolog, could be a substrate of Png. The identification of the Plu-Png protein kinase complex opens the way to identify its regulators and substrate targets in controlling the S-M cell cycles. In a survey of the genome to identify genes that genetically interact to suppress or enhance png mutations, several intervals and loci have been identified. Recovery of Png interactors will permit the unraveling of the mechanism by which Png stabilizes mitotic cyclins and inhibits S phase at this time in development (Fenger, 2000).

A genetic screen for suppressors and enhancers of the Drosophila PAN GU cell cycle kinase identifies Cyclin B as a target

Two deficiencies that remove the 59AB region on chromosome 2R strongly enhanced the pan gu mutant phenotype. This region contains the cyclin B gene, a strong candidate for the interacting locus given the observation that Cyclin B protein levels are decreased in png mutants in proportion to the strength of the allele. Further reduction of Cyclin B protein by mutation of one copy of the gene would be predicted to enhance the png phenotype. To test whether the enhancement was due to a decreased gene dosage of Cyclin B, an allele of cyclin B was obtained that is a small deletion generated by imprecise excision of a P element. This mutation strongly enhances the png phenotype (Lee, 2001).

Conversely, it was of interest to test whether the phenotype could be suppressed by increasing the level of Cyclin B protein in the png mutant embryos, so multiple copies (six to eight) of the wild-type cyclin B gene were crossed into the png mutant background. Strains with extra copies of the cyclin B gene in the png mutant background produce increased amounts of Cyclin B protein. Overexpression of cyclin B strongly suppresses the png mutants. In the weak png³³¹⁸ mutants with extra copies of the cyclin B gene, there is a striking increase in the number of nuclei in the embryos as well as in the number of embryos with multiple nuclei. In addition, the polar bodies contain condensed chromosomes with a normal rosette arrangement. This extent of suppression was not observed with any other deficiency or mutant tested. Moreover, the chromosomes of the zygotic nuclei were condensed, and mitotic figures were visible. The suppression of the zygotic nuclei was not complete, however, because all of the embryos contained polyploid nuclei, and none survived past embryogenesis. Thus, the linkage between S and M phases was ultimately broken, and nuclear division ceased. Overexpression of cyclin B dramatically suppresses the strong png¹⁰⁵⁸ mutants as well, resulting in multiple nuclei in these embryos. However, unlike the weak png³³¹⁸ mutants with extra cyclin B, normal polar bodies were not observed (Lee, 2001).

Cyclin B levels were also increased in png mutants by heat-shock induction and by induction in the germline using GAL4 under the control of the nanos promoter. The cyclin B gene in the latter construct lacks the N-terminal destruction box and thus would promote elevated levels of Cyclin B both from transcriptional induction as well as lack of degradation by the ubiquitin-APC/C pathway. Both the weak and strong png phenotypes are suppressed by overexpression of cyclin B by either of these two methods. However, the degree of suppression of the zygotic nuclear phenotype of png is less than that for lines carrying extra copies of the wild-type cyclin B gene, and restoration of normal polar body morphology was not observed (Lee, 2001).

Given the observation that png, plu, and gnu appear to function in a common pathway, the genetic interactions between png and cyclin B, and the decreased Cyclin B protein levels in plu and gnu mutants, it was of interest to test whether or not the plu and gnu phenotypes could be modified by altering the cyclin B gene dosage. Unlike png, all of the existing mutations in plu and the single gnu mutation appear to be null alleles. Because mitosis occurs only very rarely in embryos from plu and gnu mutant females, it is not possible to identify enhancers of this strong phenotype. Deficiencies identified in this screen as suppressors of the png phenotype did not suppress the plu phenotype, perhaps due to a lack of residual Plu function. Surprisingly, increasing the level of Cyclin B protein in plu⁶ and gnu³⁰⁵ mutants by increasing the cyclin B gene dosage (four extra copies) results in suppression of the mutant phenotypes. For plu females with extra cyclin B, 39% of their embryos were multinucleated compared to 1.5% in sibling controls; for gnu females with extra cyclin B, 65% of their embryos were multinucleated compared to 0.8% in sibling controls. Similar results were obtained by inducing nondegradable Cyclin B in the germline of the plu mutant. Unlike the weak png mutation, overexpression of cyclin B does not correct the polar body defects associated with plu and gnu mutations (Lee, 2001).

The suppression of png, plu, and gnu by overexpressing cyclin B is not complete because ultimately nuclear divisions fail, and the nuclei continue to replicate and become polyploid. It is possible that Cyclin B is the sole target of the Png/Plu Complex and Gnu and that the levels of increased Cyclin B protein in the png, plu, and gnu mutants (via increased copies of the cyclin B gene) are not adequate for completion of all the S-M cycles. However, it seems more likely that, although Cyclin B is a key target, other targets of the Png/Plu complex and Gnu are also important. Cyclin A is a particularly good candidate for two reasons. Cyclin A protein levels are decreased in png, plu, and gnu mutants; for png, the decrease is in proportion to the strength of the allele. Decreasing the dosage of maternal cyclin A to one copy causes an increase in cycle time during the early embryonic divisions. In contrast, decreasing the dosage of cyclin B does not affect the timing of nuclear cycles, whereas it does affect microtubule dynamics. These observations led to the conclusion that Cyclin B controls cytoskeletal events during the S-M cycles, but Cyclin A controls the nuclear cycles. If this model is correct, Cyclin A may be a critical target for the influence of Png, Plu, and Gnu on the nuclear cycles (Lee, 2001).

No enhancement of the png phenotype by mutations in cyclin A was observed, and overexpression of cyclin A unexpectedly enhanced the phenotype. This latter result likely reflects the ability of excess Cyclin A to promote DNA replication, but it is not clear why a reduction in Cyclin A does not affect the png phenotype. Further delineation of the role of Cyclin A levels will likely emerge from identification of Png kinase substrates and elucidation of the mechanism by which Png influences Cyclin A and B protein levels (Lee, 2001).

There are several mechanisms by which Png, Plu, and Gnu could affect Cyclin A and B protein levels, including maternal transcription, mRNA stability or processing, translation, and cyclin protein stability. Mutations in the pathway that target mitotic cyclin proteins for destruction were examined. No suppression of the png mutant phenotype was observed by reducing the dosage of the two known activators of cyclin destruction, fzy or fzr. Similarly, mutation of an APC/C subunit or several genes affecting the ubiquitin pathway did not alter the png mutant phenotype. These negative results do not exclude a role for Png in controlling APC/C-mediated protein degradation, since the dosage reductions may not have reduced protein activity below a crucial threshold. Additional experiments will be required to evaluate how PNG affects Cyclin A and B protein levels (Lee, 2001).

Drosophila p27Dacapo expression during embryogenesis is controlled by a complex regulatory region independent of cell cycle progression; Effects of Cyclin mutation on Dacapo expression

In the embryonic epidermis, dacapo expression starts during G2 of the final division cycle and is required for the arrest of cell cycle progression in G1 after the final mitosis. The onset of dacapo transcription is the earliest event known to be required for the epidermal cell proliferation arrest. To advance an understanding of the regulatory mechanisms that terminate cell proliferation at the appropriate stage, the control of dacapo transcription has been analyzed. dacapo transcription is not coupled to cell cycle progression. It is not affected in mutants where proliferation is arrested either too early or too late. Moreover, upregulation of dacapo expression is not an obligatory event of the cell cycle exit process. During early development of the central nervous system, Dacapo cannot be detected during the final division cycle of ganglion mother cells, while it is expressed at later stages. The control of dacapo expression therefore varies in different stages and tissues. The dacapo regulatory region includes many independent cis-regulatory elements. The elements that control epidermal expression integrate developmental cues that time the arrest of cell proliferation (Meyer, 2002).

In the embryonic epidermis, dap transcripts start to accumulate during G2 before the final mitosis 16. Within the epidermis, the pattern of dap transcript accumulation anticipates the pattern of mitosis 16. It is first observed in the region of the tracheal pits and in the prospective posterior spiracle region, then in the dorsal epidermis and finally also in the ventral epidermis. To determine whether dap expression is dependent on progression through previous divisions, string (stg) mutant embryos were examined. In these embryos, cell proliferation is prematurely arrested in G2 before mitosis 14. Nevertheless, the accumulation of dap transcripts is not delayed in stg embryos. In Cyclin A Cyclin B double mutant embryos. cell proliferation is prematurely arrested in G2 before mitosis 15. Nevertheless, accumulation of dap transcripts occurs normally in these embryos. In contrast to mutations in stg and Cyclin A and B, which result in a premature cell cycle arrest, overexpression of Cyclin E triggers an additional division cycle, as also observed in dap mutants. To address whether Cyclin E overexpression inhibits dap transcription, embryos carrying prd-GAL4 and UAS-Cyclin E were examined. In these embryos, Cyclin E is overexpressed in alternating segments of the epidermis. However, accumulation of dap transcripts starts normally throughout the entire epidermis. It is concluded therefore that the extra division cycle that occurs in the UAS-Cyclin E-expressing segments does not result from inhibition of dap expression, and it is assumed that p27DAP protein levels are simply insufficient to bind and inhibit all of the Cyclin E/Cdk2 complexes present in the overexpressing regions (Meyer, 2002).

The idea that developmental regulators govern dap expression is consistent with the finding that the onset of dap transcription is not dependent on completion of the embryonic cell proliferation program. The accumulation of dap transcripts occurs at the correct stage also in the epidermis of stg and Cyclin A Cyclin B double mutants where cells arrest prematurely. Moreover, the onset of dap transcription in the epidermis is not affected by overexpression of positive regulators of cell proliferation like Cyclin E, Cyclin D/Cdk4 and E2F1/DP (Meyer, 2002).

During development of multicellular organisms, cell proliferation evidently has to be coordinated with other processes (pattern formation, morphogenesis and growth). In principle, coordination could be achieved by developmental control of a single essential cell cycle regulator, while all others might simply be governed by feedback coupling to cell cycle progression. Analyses in Drosophila embryos have clearly demonstrated that multiple cell cycle regulators are controlled by developmental signals. Apart from this work on dap, previous studies have revealed very similar findings in the case of string and Cyclin E. The embryonic expression of both genes is controlled largely independent of cell cycle progression by many independent enhancers within extensive cis-regulatory regions (Meyer, 2002).

Genetic interactions between Cdk1-CyclinB and the Separase complex in Drosophila

Cdk1-CycB plays a key role in regulating many aspects of cell-cycle events, such as cytoskeletal dynamics and chromosome behavior during mitosis. To investigate how Cdk1-CycB controls the coordination of these events, a dosage-sensitive genetic screen was performed that was based on the observations that increased maternal CycB (four extra gene copies) leads to higher Cdk1-CycB activity in early Drosophila embryos, delays anaphase onset, and generates a sensitized non-lethal phenotype at the blastoderm stage (defined as six cycB phenotype). Mutations in the gene three rows (thr) enhance, while mutations in pimples (pim, encoding Drosophila Securin) or separase (Sse) suppress, the sensitized phenotype. In Drosophila, both Pim and Thr are known to regulate Sse activity, and activated Sse cleaves a Cohesin subunit to initiate anaphase. Compared with the six cycB embryos, reducing Thr in embryos with more CycB further delays the initiation of anaphase, whereas reducing either Pim or Sse has the opposite effect. Furthermore, nuclei move slower during cortical migration in embryos with higher Cdk1-CycB activity, whereas reducing either Pim or Sse suppresses this phenotype by causing a novel nuclear migration pattern. Therefore, the genetic screen has identified all three components of the complex that regulates sister chromatid separation, and these observations indicate that interactions between Cdk1-CycB and the Pim-Thr-Sse complex are dosage sensitive (Ji, 2005).

Increasing maternal Cdk1-CycB activity leads to defective mitoses, indicating a disruption in the coordination between the nuclear and cytoplasmic cycle (Ji, 2002). Nevertheless, these embryos develop to adults. Also, higher Cdk1-CycB activity causes shorter microtubules, and longer metaphase but shorter anaphase (Ji, 2002). These observations suggest that a slight delay of anaphase initiation may result in slightly disrupted coordination between nuclear and cytoplasmic events, such as chromatid separation and microtubule dynamics. Thus, in the six cycB genetic background, mutations that worsened the defect in coordination were identified as enhancers, whereas mutations that rectified the defects were identified as suppressors (Ji, 2002) (Ji, 2005).

Indeed, further reducing maternal thr by one copy in embryos with higher Cdk1-CycB activity leads to an even greater delay of anaphase onset, resulting in more frequent and severe nuclear defects. It is proposed that a greater delay of anaphase onset is the result of fewer Thr-Sse dimers, thereby causing an increase in the time taken to cleave Cohesin. This idea is based on the observation that the majority of the thr/six cycB embryos had many macro/micro-nuclei, and had disrupted synchrony and chromosomal bridges both before and after cycle 10, which indicates that these defects result from abnormal chromatid separation. This scenario would explain why thr becomes haplo-insufficient in the presence of higher Cdk1-CycB activity (six cycB background, but not in the wild-type (two cycB) background (Ji, 2005).

Sse and Cdk1-CycB activities have opposite effects on the onset of anaphase: higher Sse activity leads to earlier anaphase onset whereas higher Cdk1-CycB delays it. If this is so, reducing Pim, the inhibitor of Sse, would lead to slightly earlier activation of Sse than in six cycB embryos, and thus correct the timing of anaphase initiation (Ji, 2005).

Alternatively, both Pim and CycB need to be degraded to initiate anaphase -- thus reducing pim in a six cycB genetic background might suppress the six cycB phenotype if Pim and CycB compete for destruction by the ubiquitin/proteasome system. Both CycB and Securin contain a similar N-terminal sequence motif, known as the 'destruction box'. The idea that CycB and Securin compete for degradation is supported by the observation that the N-terminal fragments of CycB and Securin compete with the full-length protein for the destruction machinery in yeast. According to this scenario, Pim degradation would be delayed in six cycB embryos because more CycB needs to be degraded. Reducing Pim, as in pim/six cycB embryos would relieve the inhibition of Pim on Sse, thus suppressing the six cycB phenotype (Ji, 2005).

Both scenarios could explain why reducing Pim in embryos with higher Cdk1-CycB normalizes anaphase onset. However, additional assumptions are necessary for the second hypothesis. For example, it is not known whether Pim degradation is affected by its binding with Thr and/or Sse, or by levels of Thr and/or Sse. Interestingly, there are indications that degradation of Securin may be affected by its binding with Separase in human cells (Ji, 2005).

How can the dominant effect of the pim² allele be explained? Since Pim² can still bind to Thr even though it does not bind to Sse, Pim² may inhibit Thr by titrating it into an ineffective Pim²-Thr complex that cannot recruit Sse. Accordingly, Pim² would inactivate both Pim and Thr, thus it might have a phenotype similar to that seen with other pim alleles when they were combined with a thr mutation (Ji, 2005).

It has been proposed that after the active Thr-Sse heterodimer cleaves the Cohesin subunit, Thr itself is cleaved by Sse, which presumably inactivates Sse at the end of anaphase. Because of this negative feedback, Thr-Sse heterodimer activity is likely to be limited to a short time after anaphase begins. It is not known whether Thr is cleaved by the same Sse molecule that it binds or by another Thr-Sse dimer. A similar negative-feedback mechanism in Separase regulation was found in Xenopus and human cells, where Separase undergoes auto-cleavage. However, the cleaved fragments are still active and remain associated, thus the function of the auto-cleavage in regulating anaphase onset is not resolved (Ji, 2005).

If the hypothesis that levels of Thr and Pim affect the onset of anaphase by modifying Sse activity is correct, Sse is expected to be an enhancer. However, both the amorphic allele sse^13m and the deficiency Df(3L)SseA are suppressors. This presents a challenge. Two scenarios are proposed to explain this unexpected result. If cleavage of Thr by Thr-Sse inactivates Sse, it is speculated that both Thr-Sse heterodimers and Sse monomers have protease activity to cleave Thr bound to Sse. If so, compared with six cycB embryos, reducing Sse in sse/six cycB embryos would reduce the concentration of Thr-Sse, and thus the cleaveage of Thr and the inactivation of Sse would take longer. The delay in Sse inactivation could have similar effects as does increasing Thr-Sse levels (i.e., Separase activity), helping to overcome the inhibitory effect of a higher Cdk1-CycB activity on sister chromatid separation. The explanation of the effect of Separase activity on the onset of anaphase is consistent with observations that depletion of a Cohesin subunit DRad21/Scc1 in Drosophila cultured cells and embryos by RNAi leads to premature chromatid separation and abnormal spindle morphology, suggesting that the onset of anaphase is defined by the cleavage efficiency of Drad21/Scc1 (Ji, 2005).

Alternatively, the suppressive effect of Sse could be caused by Sse possessing functions other than the ability to cleave the Cohesin subunit. This possibility is supported by the following observations in budding yeast. (1) Besides cleaving Securin, Separase can also cleave the kinetochore and the spindle associated protein Slk19 at the onset of anaphase. Cleaved Slk19 localizes to the spindle midzone and is required to maintain spindle stability in anaphase, preventing elongated spindle from breaking down prematurely. (2) Separase may also promote phosphorylation of Net1, the inhibitor of phosphatase Cdc14, thereby causing the release of Cdc14 from the nucleolus, a key step in mitotic exit. It is still an open question whether Separase has additional substrates. Although it is not known whether similar mechanisms also occur in Drosophila, it is possible that the suppressive effect observed by reducing Sse may be caused by affecting the exit of mitosis through other Sse targets (Ji, 2005).

Reducing either Pim or Sse restores the microtubule morphology in interphase, but not in metaphase. In these embryos, nuclei show a faster and novel pattern in cortical migration, but this still leads to a normal nuclear distribution at cycle 10. Although it is not clear whether levels of Separase, Securin or APC/C modulate microtubule stability, it has been observed that Separase, Securin and components of the APC/C complex co-localize with spindle microtubules. For examples, in budding yeast, phosphorylated Pds1 (Securin) binds with Esp1 (Separase) and the complex is targeted to the spindle apparatus. In Drosophila, both Sse and Pim co-localize with spindle microtubules. Furthermore, components of APC/C, such as CDC16 and CDC27, co-immunoprecipitate with microtubules in Drosophila embryos. Finally, Securin co-localizes with mitotic spindles in HeLa cells (Ji, 2005).

Based on these observations, several hypotheses may explain the dosage effects of Pim and Sse on microtubule morphology at different cell-cycle phases. The most compelling one is that if CycB and Pim compete for poly-ubiquitination by APC/C on microtubules, reducing Pim may lead to faster CycB degradation, resulting in the restoration of microtubule morphology in interphase compared with six cycB embryos. By contrast, because there is no degradation of either Pim or CycB in metaphase, the effect of degradation competition between Pim and CycB is absent, thus explaining why astral microtubule morphology is not restored in pim/six cycB embryos. If Sse levels affect Pim degradation, reducing either Pim or Sse could have similar effects on CycB degradation. Thus it is speculated that the interplay between the different kinetics of Cdk1-CycB activity and Separase activity over the cell cycle may contribute to the different effects of Sse/Pim dosage on microtubule stability (Ji, 2005).

To understand why reducing either Pim or Sse leads to faster nuclear movements and a different nuclear migration pattern, the mechanics involved in the process of cortical migration need to be considered. Two major cytoskeletal networks are reorganized during this process: microtubules are stabilized in late telophase and early interphase; this pushes nuclei to the cortex, where the microfilament network is denser than in the interior. Thus, the velocity and pattern of nuclear movement will be defined both by the pushing force generated by microtubules and by the resistance generated by the microfilament matrix (Ji, 2005).

In embryos with more Cdk1-CycB, microtubules become less stable (Ji, 2002). This may generate a weaker force to push nuclei to the cortex, resulting in the slower and less direct nuclear movement that was observed. When either Pim or Sse is further reduced, microtubule morphology is restored in early interphase. This may contribute to the observation of faster nuclear cortical migration than in the six cycB embryos. However, why do nuclei in Sse/four cycB or pim/four cycB embryos move even faster than in two cycB embryos? This observation is puzzling. The simple explanation would be that the microtubule network is more robust in Sse/four cycB or pim/four cycB embryos than in two cycB embryos. Previously suggested was a model in which microtubule and microfilament networks antagonistically interact with each other, and in which Cdk1-CycB activity negatively affects this interaction in early Drosophila embryos (Ji, 2002). Accordingly, a more robust microtubule network would result in a weaker microfilament network, presumably reducing the resistance for nuclear movement because of the less dense microfilament matrix in the extended cortex. The novel pathway of nuclear movement may reflect the disrupted balance between microtubule and microfilament networks because of the over-corrected microtubules in interphase. Consistent with this scenario, dramatic global cytoplasmic movements are also observed in pim¹/four cycB and sse^13m/four cycB embryos during the nuclear cortical migration. Thus, an increased microtubule network and the less dense microfilament matrix might account for accelerated nuclear movements (Ji, 2005).

This genetic screen has identified modifiers of the six cycB phenotype (Ji, 2002). The studies have documented an interplay between Cdk1-CycB, microtubules and microfilaments. This study reports three new modifiers that affect the six cycB phenotype. One of them, thr, is an enhancer. Interestingly, when the enhancer thr is combined with the suppressor quail (which encodes a villin-like protein), it is found that the six cycB phenotype is restored. This indicates that, at least at the genetic level, the amount of Cdk1-CycB modulates many parameters of gene products regulating nuclear behavior and cytoskeletal stability (Ji, 2005).

Progress in developmental genetics requires the functional analyses of genes, which is best addressed by the description of pleiotropic phenotypes. Increasing Cdk1-CycB in combination with decreasing Pim or Sse almost completely corrects the onset of anaphase and normalizes nuclear distribution at cycle 10. What is not expected and could only be observed by combining live analysis with data from fixed embryos is that microtubule configuration is corrected to wild type in interphase but not metaphase, and that a novel nuclear cortical migration pattern appears. Because this phenotype is only observed in combination with excessive Cdk1-CycB, the term 'heterosis combined with epistasis' is suggested to describe the microtubule phenotype. Such a mechanism may have a selective advantage and therefore might occur in other slightly deleterious genetic combinations (Ji, 2005).

RNAi of mitotic cyclins in Drosophila uncouples the nuclear and centrosome cycle

Successful cell duplication requires orderly progression through a succession of dramatic cell-cycle events. Disruption of this precise coupling can compromise genomic integrity. The coordination of cell-cycle events is thought to arise from control by a single master regulator, cyclin:Cdk, whose activity oscillates. However, very little is known of how individual cell-cycle events are coupled to this oscillator and how the timing of each event is controlled. An approach with RNA interference (RNAi) and real-time imaging was developed to study cyclin contributions to the rapid syncytial divisions of Drosophila embryos. Simultaneous knockdown of all three mitotic cyclins, Cyclin A, Cyclin B, and Cyclin B3, blocked nuclei from entering mitosis. Despite nuclear arrest, centrosomes and associated myosin cages continue to divide until the midblastula transition. Centrosome division is synchronous throughout the embryo and the period of the uncoupled duplication cycle increases over successive divisions. In contrast to its normal actions, injection of a competitive inhibitor of the anaphase-promoting complex/cyclosome (APC/C) after knockdown of the mitotic cyclins does not interfere with the centrosome-duplication cycles. Finally, how cyclin knockdown affects the onset of cellularization at the midblastula transition was studied and it was found that nuclear cell-cycle arrest did not advance or delay onset of cellularization. This study shows that knockdown of mitotic cyclins allows centrosomes to duplicate in a cycle that is uncoupled from other cell-cycle events. It is suggested that high mitotic cyclin normally ensures that the centrosome cycle remains entrained to the nuclear cycle (McCleland, 2008).

Interestingly, these data show that reduction in mitotic cyclin blocks mitosis without blocking centrosome duplication. It is suggested that the uncoupled centrosome cycles represent full duplication cycles, because three cycles of centrosome duplication were observed, which requires more than a division of previously duplicated centrosome components. Furthermore, the appearance and movements of centrosomes is similar to that seen in normal cycles. How might cyclin knockdown bypass the normally tight coordination between centrosome and nuclear division (McCleland, 2008)?

After cyclin knockdown, reduced accumulation is seen of a GFP-cyclin reporter and reduced cyclin A is seen on Western blots. The residual cyclin could retain some function, but it is not adequate to facilitate mitosis. Thus, the level of mitotic cyclin required for centrosome multiplication (if any) is less than the level required for mitosis. Accordingly, in a standard cell-cycle paradigm, cyclin accumulation would first satisfy the threshold for centrosome multiplication and only later reach the mitotic threshold, followed by the resetting of the cycle by mitotic cyclin destruction. A simple interpretation of these results is that cyclin accumulation has been arrested between the two thresholds so that mitosis is blocked but centrosome multiplication proceeds. However, if the centrosome cycle is mechanistically coupled to the mitotic cycle, one might expect that blocking mitosis would also block the centrosome cycle, unless mitotic cyclins are also required for the coupling mechanism. Indeed, there are a number of indications that mitotic cyclins influence the centrosome cycle. Moreover, there are also observations that suggest several points of coupling of the centrosome-cycle and cell-cycle progression (McCleland, 2008).

The centrosome-duplication cycle normally occurs in lockstep with progress through the cell cycle. During syncytial mitoses, the centrioles of a centriole pair disjoin at the transition to anaphase, daughter centriole assembly begins in anaphase, and centrosomes move apart during interphase. Subsequent shifts in this coordination occur in parallel with changes in cyclin:Cdk regulation. When a G2 phase appears in cycle 14, completion and maturation of daughter centrioles is held in abeyance until expression of Cdc25^stg. Furthermore, when a G1 appears in cycle 17, initiation of daughter centrioles is deferred because of cyclin E downregulation. Apparently, multiple steps of the centrosome cycle are coupled to the cell cycle, and previous work suggests various ways that cyclin:Cdk might couple the centrosome and mitotic cycles (McCleland, 2008).

Conceptually, the once-per-cell-cycle duplication of centrosomes is similar to the regulation of DNA replication. DNA replication is coupled to oscillations in cyclin:Cdk activity because cyclin:Cdk inhibits one step of replication but is required to promote another. However, centrosomes have been found to duplicate in experimental conditions apparently lacking oscillations of cyclin:Cdk1. In a Xenopus egg extract arrested with low mitotic cyclin:Cdk1 kinase activity by inhibition of DNA synthesis, centrosomes continued replicating in a cyclin E dependent fashion. Thus, cyclin E:Cdk2 makes a positive contribution to the centrosome cycle, but centrosomes multiplied in its continuous presence, indicating that this cyclin:Cdk does not block duplication. Similarly, upon deletion of S. cerevisiae Clb 1-4 (G2 cyclins), uncoupled duplication of the spindle-pole bodies occurred in the continuous presence of Cln2 (G1 cyclin) or Clb5 (S phase cyclin). Thus, G1 cyclins and/or S phase cyclins promote centrosome duplication without blocking it. Importantly, in normal cycles the G1 cyclins do not provoke multiple rounds of uncoupled centrosome division (McCleland, 2008).

How might such divisions be suppressed? Interestingly, as in the above experiments in Xenopus and yeast, treatments that eliminate or suppress mitotic cyclin:Cdk1 seem to uncouple centrosome replication. Centrioles multiplied without mitosis in Drosophila upon temperature inactivation of a Cdk1^ts, and centrosomes amplified in sea urchin and frog embryos arrested by inhibition of protein synthesis, which presumably blocks cyclin accumulation. Thus, the findings are in accord with previous observations in suggesting that mitotic cyclins are required to enforce coupling of the centrosome cycle to the mitotic cycle (McCleland, 2008).

Suppression of uncoupled centrosome cycles by mitotic cyclin:Cdk1 could be the result of inhibition of one or more steps of the centrosome cycle. Indeed, stabilized versions of the mitotic cyclins or inhibition of the APC/C blocks mitotic exit and blocks daughter-centriole production, showing that mitotic cyclins have either a direct or indirect inhibitory action on centrosome replication (McCleland, 2008).

Because several steps of the centrosome cycle appear coupled to the cell cycle, the cyclin inputs might be complex. For example, the finding that Cdc25^stg promotes daughter-centriole maturation in G2 of cycle 14 suggests that cyclin:Cdk1 activation is required for centrosome maturation. However, this is not easily consistent with the observation that inactivation of Cdk1^ts allows centriole multiplication without a deficit in daughter-centriole growth\. Another possibility is that Cdc25^stg removes an inhibitor of daughter-centriole maturation. Indeed, tyrosine phosphorylated Cdc28 of S. cerevisiae inhibits spindle-pole-body duplication, and Cdc25^Mih1 reverses this inhibition. In summary, present evidence is consistent with direct or indirect inhibition of centrosome duplication by mitotic cyclin (McCleland, 2008).

It is noted that the multiple centrioles produced after inactivation of Drosophila Cdk1^ts did not separate and that the separation of yeast spindle-pole bodies requires active Cdc28. It is suggested that there is also a positive contribution of mitotic cyclin:Cdk1 to centrosome multiplication but that this requirement is either absent in the early syncytial cycles or that it is satisfied by a low level of mitotic cyclin that persists following RNAi. If a residue of Cdk1 promotes the uncoupled centrosome cycle, why does it not also promote mitosis? Perhaps the cyclin level is too low, the residual Cdk1 activity is localized to the centrosome, or the nuclear cycle is prevented by an unappreciated checkpoint (McCleland, 2008).

If cyclin:Cdk1 provides both negative and positive contributions to the centrosome cycle, a simple model could explain coupling of the centrosome cycle to mitosis. G1 cyclins promote centrosome duplication but also trigger mitotic cyclin accumulation. If kinase inactive mitotic cyclin:Cdk1 inhibits a step in centrosome maturation, this would ensure centrosomes do not divide until mitotic entry, whereupon Cdk1 activation would allow completion of centrosome duplication. Furthermore, if active cyclin:Cdk1 kinase and metaphase activities suppressed centrosome separation, separation of the duplicated centrosome would await mitotic exit. Additional studies will be required to define this multistep coupling mechanism (McCleland, 2008).

The switch from maternal to zygotic regulation at the MBT involves a wholesale reorganization of many regulatory circuits. Although there has been great interest in the mechanisms that time and coordinate this transition, little is known about either the timer or the mechanism. Experiments in frog and fly have suggested that the MBT occurs when the exponential multiplication of nuclei increases the nuclear to cytoplasmic ratio to a threshold. But what provides the readout of the increasing nuclear density? In flies, the capacity to promote mitotic cyclin destruction correlates with an increase in the nuclear to cytoplasmic ratio, or, as emphasized by some authors, it also correlates with the increase in centrosomes and mitotic apparatuses. This relationship between cyclin degradation and nuclear concentration might explain the gradual prolongation of the blastoderm cycles and onset of the MBT. This interphase prolongation has been suggested to allow time for transcription of components necessary at the MBT. Accordingly, knockdown of cyclin synthesis should dramatically influence MBT timing, perhaps directly if cyclin:Cdk levels provide a regulatory input or indirectly if cell-cycle length or nuclear density provides an input (McCleland, 2008).

Knockdown of mitotic cyclins blocked mitosis at the injected pole, modestly extended the cell-cycle length in more distal regions, and usually left the cycle unaffected at the most distal pole. When the unaffected end of such chimeric embryos completed cycle 13, ingression of the cellularization membranes occurred in concert in regions of the embryo in cycle 14, cycle 13, and cycle 12. Thus, local knockdown of cyclins, prolongation of the cell cycle, and reduction of local nuclear density was not sufficient to forestall cellularization. Although other MBT parameters have yet to be characterized, the apparently normal gastrulation of embryos arrested in cycle 13 suggests concerted transition of the various MBT events. These findings are not easily consistent with earlier ideas because the experiment alters many parameters thought to contribute to triggering the MBT. It is noted that one parameter that is not changed by cyclin RNAi is the increasing centrosome density, which remains a viable candidate for triggering the MBT (McCleland, 2008).

Different cyclin types collaborate to reverse the S-phase checkpoint and permit prompt mitosis

Precise timing coordinates cell proliferation with embryonic morphogenesis. As Drosophila embryos approach cell cycle 14 and the midblastula transition, rapid embryonic cell cycles slow because S phase lengthens, which delays mitosis via the S-phase checkpoint. This study probed the contributions of each of the three mitotic cyclins to this timing of interphase duration. Each pairwise RNA interference knockdown of two cyclins lengthened interphase 13 by introducing a G2 phase of a distinct duration. In contrast, pairwise cyclin knockdowns failed to introduce a G2 in embryos that lacked an S-phase checkpoint. Thus, the single remaining cyclin is sufficient to induce early mitotic entry, but reversal of the S-phase checkpoint is compromised by pairwise cyclin knockdown. Manipulating cyclin levels revealed that the diversity of cyclin types rather than cyclin level influenced checkpoint reversal. It is concluded that different cyclin types have distinct abilities to reverse the checkpoint but that they collaborate to do so rapidly (Yuan, 2012).

Pairwise knockdown of cyclins in embryos lacking Chk1/Grapes (embryos from grp mutant mothers) modestly extends interphase in the grp embryo. This knockdown also substantially suppresses the mitosis 13 defects in grp embryos. The mitotic catastrophe in grp embryos has long been thought to be caused by entry into mitosis with incompletely replicated DNA. Indeed, analysis of PCNA localization supports a proposal that the small extension of interphase allows completion of S phase and, hence, suppression of the catastrophe. This suppression of the grp phenotype, which extends to partial restoration of gastrulation, is reminiscent of suppression of hypomorphic mei-41 mutations when the maternal dose of CycA and/or CycB was reduced. However, the most important feature of this analysis is that the extension of interphase by pairwise cyclin knockdown in grp embryos is so slight that interphase remains shorter than a wild-type interphase 13. Thus, each individual cyclin type can drive rapid advance to mitosis in the absence of functional Chk1. Furthermore, the G2 that was introduced by cyclin knockdown was absent in grp embryos, and cyclin type-specific differences in interphase length were minor. These results demonstrate that mitotic entry is timed primarily by the Grapes-dependent checkpoint in cyclin knockdown embryos and, moreover, that the G2 induced in these embryos results from action of the checkpoint (Yuan, 2012).

How might S phase govern the time of mitosis when mitosis begins well after completion of S phase? To test whether the S-phase checkpoint delayed accumulation of the remaining cyclin, we immunoblotted single knockdown embryos to follow accumulation in wild-type and grp embryos. Cyclin accumulated during S phase in both wild-type and grp mutant embryo. Thus, Grapes function does not delay the production of cyclin. Instead, the difference between grp⁺ and grp⁻ embryos suggests that persistent activity of the checkpoint prevents the checkpoint-competent embryos from going into mitosis after pairwise cyclin knockdown. Because wild-type embryos enter mitosis immediately after S phase, the checkpoint is rapidly reversed when there is a full complement of cyclin types, but its reversal is delayed upon pairwise cyclin knockdown. Apparently, the different cyclin types ordinarily collaborate to rapidly reverse the checkpoint (Yuan, 2012).

This study shows that a persistent action or consequence of the DNA replication checkpoint underlies a G2 phase that is introduced by pairwise knockdown of cyclins. One might propose that cyclin knockdown doesn't really cause persistence of the checkpoint activity but simply makes a slowly decaying checkpoint function longer by compromising the cyclin-Cdk1 activity that must be suppressed by the checkpoint. Such an interpretation is disfavored because it is quantitative, and the data argue that neither reduction nor increase in the remaining cyclin affects the duration of interphase. Instead, it is argued that cyclin-Cdk1 contributes to shutting off the checkpoint, and it is proposed that efficient shutoff of the checkpoint requires multiple cyclin types. One way to explain this is based on the distinct subcellular localizations of mitotic cyclins. Once activated, the checkpoint can operate in multiple cellular compartments, such as the nucleus and the cytoplasm. Although signals coordinate entry into mitosis in the cytoplasm and nucleus, persistent nuclear checkpoint activity can prevent mitotic entry despite cytoplasmic Cdk activity. Individual cyclins would not be able to act on their own to reverse the checkpoint in all compartments if each is excluded from one compartment. For example, cyclin B is efficiently excluded from the nucleus in cycle 13 embryos and presumably would not contribute to checkpoint reversal in this compartment, whereas cyclin B3 is nuclear. In embryos with only CycB, the checkpoint should be reversed first in the cytoplasm; however, progress to mitosis should depend on slower reversal in the nucleus, which might be based on communication between compartments. Consistent with this proposal, injection of CycB protein has been shown to preferentially drive cytoplasmic, but not nuclear, mitotic events. The full complement of cyclins with distinct localizations, however, appears to reverse the checkpoint promptly and coordinately in all the compartments (Yuan, 2012).

These data demonstrate a cyclin-type effect on reversal of the DNA replication checkpoint, which emphasizes the qualitatively distinct contributions among mitotic cyclins during mitotic entry. This study opens many further questions, such as what causes the checkpoint to inactivate? How do mitotic cyclins promote checkpoint reversal? It is believed that answers to these questions will lead to a fuller understanding of the timing mechanism of the cell division cycle (Yuan, 2012).