Triggering the all-or-nothing switch into mitosis.

The past decade of cell cycle investigations has identified many roads not taken. The kinase that drives mitosis can be modulated by cyclins, by activating phosphorylation, by inhibitory phosphorylation and by binding of inhibitors, but one of these regulatory options controls the transition from G2 phase to mitosis in most circumstances. A switch-like mechanism integrates signals of cellular status and commits the cell to mitosis by abruptly removing inhibitory phosphate from preformed cyclin:Cdk1 complexes. The pathways that flip this switch alter the balance of modifying reactions to favor dephosphorylation, thereby generating a flood of mitotic kinase.

Cell cycle roles for two 14-3-3 proteins during Drosophila development.

Drosophila 14-3-3 epsilon and 14-3-3 zeta proteins have been shown to function in RAS/MAP kinase pathways that influence the differentiation of the adult eye and the embryo. Because 14-3-3 proteins have a conserved involvement in cell cycle checkpoints in other systems, we asked (1) whether Drosophila 14-3-3 proteins also function in cell cycle regulation, and (2) whether cell proliferation during Drosophila development has different requirements for the two 14-3-3 proteins. We find that antibody staining for 14-3-3 family members is cytoplasmic in interphase and perichromosomal in mitosis. Using mutants of cyclins, Cdk1 and Cdc25(string) to manipulate Cdk1 activity, we found that the localization of 14-3-3 proteins is coupled to Cdk1 activity and cell cycle stage. Relocalization of 14-3-3 proteins with cell cycle progression suggested cell-cycle-specific roles. This notion is confirmed by the phenotypes of 14-3-3 epsilon and 14-3-3 zeta mutants: 14-3-3 epsilon is required to time mitosis in undisturbed post-blastoderm cell cycles and to delay mitosis following irradiation; 14-3-3 zeta is required for normal chromosome separation during syncytial mitoses. We suggest a model in which 14-3-3 proteins act in the undisturbed cell cycle to set a threshold for entry into mitosis by suppressing Cdk1 activity, to block mitosis following radiation damage and to facilitate proper exit from mitosis.

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

BACKGROUND: 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. We tested a recent proposal that the destruction of the different cyclins guides progress through mitosis. RESULTS: Real-time imaging revealed that arrest phenotypes differ because each stable cyclin affects specific mitotic events differently. Stable cyclin A prolonged or blocked chromosome disjunction, leading to metaphase arrest. Stable cyclin B allowed the transition to anaphase, but anaphase A chromosome movements were slowed, anaphase B spindle elongation did not occur, and the monooriented disjoined chromosomes began to oscillate between the spindle poles. Stable cyclin B3 prevented normal spindle maturation and blocked major mitotic exit events such as chromosome decondensation but nonetheless allowed chromosome disjunction, anaphase B, and formation of a cytokinetic furrow, which split the spindle. CONCLUSIONS: We conclude 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. We suggest that the schedule of degradation of cyclin A, cyclin B, and then cyclin B3 contributes to the temporal coordination of mitotic events.

Hypoxia and nitric oxide induce a rapid, reversible cell cycle arrest of the Drosophila syncytial divisions.

Cells can respond to reductions in oxygen (hypoxia) by metabolic adaptations, quiescence or cell death. The nuclear division cycles of syncytial stage Drosophila melanogaster embryos reversibly arrest upon hypoxia. We examined this rapid arrest in real time using a fusion of green fluorescent protein and histone 2A. In addition to an interphase arrest, mitosis was specifically blocked in metaphase, much like a checkpoint arrest. Nitric oxide, recently proposed as a hypoxia signal in Drosophila, induced a reversible arrest of the nuclear divisions comparable with that induced by hypoxia. Syncytial stage embryos die during prolonged hypoxia, whereas post-gastrulation embryos (cellularized) survive. We examined ATP levels and morphology of syncytial and cellularized embryos arrested by hypoxia, nitric oxide, or cyanide. Upon oxygen deprivation, the ATP levels declined only slightly in cellularized embryos and more substantially in syncytial embryos. Reversal of hypoxia restored ATP levels and relieved the cell cycle and developmental arrests. However, morphological abnormalities suggested that syncytial embryos suffered irreversible disruption of developmental programs. Our results suggest that nitric oxide plays a role in the response of the syncytial embryo to hypoxia but that it is not the sole mediator of these responses.

Drosophila wee1 has an essential role in the nuclear divisions of early embryogenesis.

In Drosophila, the maternally expressed mei-41 and grp genes are required for successful execution of the nuclear division cycles of early embryogenesis. In fission yeast, genes encoding similar kinases (rad3 and chk1, respectively) are components of a cell cycle checkpoint that delays mitosis by inhibitory phosphorylation of Cdk1. We have identified mutations in a gene encoding a Cdk1 inhibitory kinase, Drosophila wee1 (Dwee1). Like mei-41 and grp, Dwee1 is zygotically dispensable but is required maternally for completing the embryonic nuclear cycles. The arrest phenotype of Dwee1 mutants, as well as genetic interactions between Dwee1, grp, and mei-41 mutations, suggest that Dwee1 is functioning in the same regulatory pathway as these genes. These findings imply that inhibitory phosphorylation of Cdk1 by Dwee1 is required for proper regulation of the early syncytial cycles of embryogenesis.

Comparative genomics of the eukaryotes.

A comparative analysis of the genomes of Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae-and the proteins they are predicted to encode-was undertaken in the context of cellular, developmental, and evolutionary processes. The nonredundant protein sets of flies and worms are similar in size and are only twice that of yeast, but different gene families are expanded in each genome, and the multidomain proteins and signaling pathways of the fly and worm are far more complex than those of yeast. The fly has orthologs to 177 of the 289 human disease genes examined and provides the foundation for rapid analysis of some of the basic processes involved in human disease.

Rux is a cyclin-dependent kinase inhibitor (CKI) specific for mitotic cyclin-Cdk complexes.

BACKGROUND: Roughex (Rux) is a cell-cycle regulator that contributes to the establishment and maintenance of the G1 state in the fruit fly Drosophila. Genetic data show that Rux inhibits the S-phase function of the cyclin A (CycA)-cyclin-dependent kinase 1 (Cdk1) complex; in addition, it can prevent the mitotic functions of CycA and CycB when overexpressed. Rux has no homology to known Cdk inhibitors (CKIs), and the molecular mechanism of Rux function is not known. RESULTS: Rux interacted with CycA and CycB in coprecipitation experiments. Expression of Rux caused nuclear translocation of CycA and CycB, and inhibited Cdk1 but not Cdk2 kinase activity. Cdk1 inhibition by Rux did not rely on inhibitory phosphorylation, disruption of cyclin-Cdk complex formation or changes in subcellular localization. Rux inhibited Cdk1 kinase in two ways: Rux prevented the activating phosphorylation on Cdk1 and also inhibited activated Cdk1 complexes. Surprisingly, Rux had a stimulating effect on CycA-Cdk1 activity when present in low concentrations. CONCLUSIONS: Rux fulfils all the criteria for a CKI. This is the first description in a multicellular organism of a CKI that specifically inhibits mitotic cyclin-Cdk complexes. This function of Rux is required for the G1 state and male meiosis and could also be involved in mitotic regulation, while the stimulating effect of Rux might assist in any S-phase function of CycA-Cdk1.

Mitotic regulators govern progress through steps in the centrosome duplication cycle.

Centrosome duplication is marked by discrete changes in centriole structure that occur in lockstep with cell cycle transitions. We show that mitotic regulators govern steps in centriole replication in Drosophila embryos. Cdc25(string), the expression of which initiates mitosis, is required for completion of daughter centriole assembly. Cdc20(fizzy), which is required for the metaphase-anaphase transition, is required for timely disengagement of mother and daughter centrioles. Stabilization of mitotic cyclins, which prevents exit from mitosis, blocks assembly of new daughter centrioles. Common regulation of the nuclear and centrosome cycles by mitotic regulators may ensure precise duplication of the centrosome.

Transcribed genes are localized according to chromosomal position within polarized Drosophila embryonic nuclei.

When some genes are silenced, their positions within the nucleus can change dramatically [1] [2]. It is unclear, however, whether genes move to new positions when they are activated [3]. The chromosomes within the polarized nuclei of the fruit fly Drosophila have a well-characterized apical-basal orientation (the Rabl configuration [4]). Using a high-resolution in situ hybridization method [5], we found that each of 15 transcribed genes was localized as predicted by their chromosomal position and by the known polarized organization of the chromosomes. We also found that, within their specific apical-basal plane, most nascent transcript foci could occupy any radial position. There was no correlation between the apical-basal position of the transcribed locus and the final cytoplasmic site of localization of the RNA along the apical-basal axis of the cell. There was also no relationship between the distance of loci from the nuclear periphery and the amount of nascent mRNA decorating the gene. Our results are consistent with the view that effective transcription can occur without major re-localization of the genes themselves.

Cytoskeleton: centrosom-in absentia.

Recent results challenge long-held assumptions that centrosomes are essential organizers of mitotic spindles, but suggest that they couple spindle behavior with developmental and cellular events, perhaps by nucleating astral microtubules which mediate interactions with other cytoskeletal components.

Drosophila grapes/CHK1 mutants are defective in cyclin proteolysis and coordination of mitotic events.

The Drosophila grapes (grp) gene, which encodes a homolog of the Schizosaccharomyces pombe Chk1 kinase, provides a cell-cycle checkpoint that delays mitosis in response to inhibition of DNA replication [1]. Grp is also required in the undisturbed early embryonic cycles: in its absence, mitotic abnormalities appear in cycle 12 and chromosomes fail to fully separate in subsequent cycles [2] [3]. In other systems, Chk1 kinase phosphorylates and suppresses the activity of Cdc25 phosphatase: the resulting failure to remove inhibitory phosphate from cyclin-dependent kinase 1 (Cdk1) prevents entry into mitosis [4] [5]. Because in Drosophila embryos Cdk1 lacks inhibitory phosphate during cycles 11-13 [6], it is not clear that known actions of Grp/Chk1 suffice in these cycles. We found that the loss of grp compromised cyclin A proteolysis and delayed mitotic disjunction of sister chromosomes. These defects occurred before previously reported grp phenotypes. We conclude that Grp activates cyclin A degradation, and functions to time the disjunction of chromosomes in the early embryo. As cyclin A destruction is required for sister chromosome separation [7], a failure in Grp-promoted cyclin destruction can also explain the mitotic phenotype. The mitotic failure described previously for cycle 12 grp embryos might be a more severe form of the phenotypes that we describe in earlier embryos and we suggest that the underlying defect is reduced degradation of cyclin A.

Nitric oxide contributes to behavioral, cellular, and developmental responses to low oxygen in Drosophila.

A nitric oxide (NO)/cyclic GMP (cGMP) signaling pathway is thought to play an important role in mammalian vasodilation during hypoxia. We show that Drosophila utilizes components of this pathway to respond to hypoxia. Hypoxic exposure rapidly induced exploratory behavior in larvae and arrested the cell cycle. These behavioral and cellular responses were diminished by an inhibitor of NO synthase and by a polymorphism affecting a form of cGMP-dependent protein kinase. Conversely, these responses were induced by ectopic expression of NO synthase. Perturbing components of the NO/cGMP pathway altered both tracheal development and survival during prolonged hypoxia. These results indicate that NO and protein kinase G contribute to Drosophila's ability to respond to oxygen deprivation.

Size control: cell proliferation does not equal growth.

Division subdivides mass without increasing it. So one should not expect that an increase in cell division would make an organism bigger. Both classic and recent experiments confirm this simple rationale: altering proliferation produces normally sized body structures with either especially small or exceptionally large cells.

The cell cycle program in germ cells of the Drosophila embryo.

The germ cells of metazoans follow a program of proliferation that is distinct from proliferation programs of somatic cells. Despite their developmental importance, the cell proliferation program in the metazoan primordial germ cells is not well characterized and the regulatory controls are not understood. In Drosophila melanogaster, germ cell precursors (called pole cells) proliferate early in embryogenesis and then enter a prolonged quiescence. We found that polar nuclear divisions are asynchronous and lag behind somatic nuclear divisions during syncytial cycles 9 and 10. Thus, the polar division program deviates from the somatic division program when pole nuclei and somatic nuclei still share a common cytoplasm, earlier than previously thought. The lag in polar nuclear divisions is independent of grapes, which is required for lengthening somatic cell cycles 10-13. Mapping of the last S phase in pole cells and measurement of their DNA content indicate that pole cells become quiescent in G2 phase of the cell cycle. We were able to drive quiescent pole cells into mitosis by induction of either an activator of Cdc2 (Cdc25string phosphatase) or a mutant form of Cdc2 that cannot be inhibited by phosphorylation. In contrast, induction of wild-type Cdc2 with a mitotic cyclin did not induce mitosis in pole cells. We propose that inhibition of Cdc2 by phosphorylation contributes to G2 arrest in pole cells during embryogenesis. Furthermore, pole cells enter G1 following induced mitoses, indicating that entry into both mitosis and S phase is blocked in quiescent pole cells. These studies represent the first molecular characterization of proliferation in embryonic germ cells of Drosophila.

Exit from mitosis in Drosophila syncytial embryos requires proteolysis and cyclin degradation, and is associated with localized dephosphorylation.

The cyclin proteolysis that accompanies the exit from mitosis in diverse systems appears to be essential for restoration of interphase. The early syncytial divisions of Drosophila embryos, however, occur without detectable oscillations in the total cyclin level or Cdk1 activity. Nonetheless, we found that injection of an established inhibitor of cyclin proteolysis, a cyclin B amino-terminal peptide, prevents exit from mitosis in syncytial embryos. Similarly, injection of a version of Drosophila cyclin B that is refractory to proteolysis results in mitotic arrest. We infer that proteolysis of cyclins is required for exit from syncytial mitoses. This inference can be reconciled with the failure to observe oscillations in total cyclin levels if only a small pool of cyclins is destroyed in each cycle. We find that antibody detection of histone H3 phosphorylation (PH3) acts as a reporter for Cdk1 activity. A gradient of PH3 along anaphase chromosomes suggests local Cdk1 inactivation near the spindle poles in syncytial embryos. This pattern of Cdk1 inactivation would be consistent with local cyclin destruction at centrosomes or kinetochores. The local loss of PH3 during anaphase is specific to the syncytial divisions and is not observed after cellularization. We suggest that exit from mitosis in syncytial cycles is modified to allow nuclear autonomy within a common cytoplasm.

Fluctuations in cyclin E levels are required for multiple rounds of endocycle S phase in Drosophila.

The precise cell-cycle alternation of S phase and mitosis is controlled by alternating competence of nuclei to respond to S-phase-inducing factors [1]. Nuclei acquire competence to replicate at the low point in cyclin-dependent kinase (Cdk) activities that follows mitotic destruction of cyclins. The elevation of Cdk activity late in G1 is thought to drive cells into S phase and to block replicated DNA from re-acquiring replication competence [2]. Whereas mitosis is normally required to eliminate the cyclins prior to another cycle of replication, experimental elimination of Cdk activity in G2 can restore competence to replicate [3-6]. Here, we examine the roles of Cdks in the endocycies of Drosophila [7]. In these cycles, rounds of discrete S phases without intervening mitoses result in polyteny. Cyclins A and B are lost in cells as they enter endocycles [8,9], and pulses of Cyclin E expression drive endocycle S phases [10-12]. To address whether oscillations of Cyclin E expression are required for endocycles, we expressed Cyclin E continuously in Drosophila salivary glands. Growth of the cells was severely inhibited, and a period of DNA replication was induced but further replication was inhibited. This replication inhibition could be overcome by the kinase inhibitor 6-dimethylaminopurine (6-DMAP), but not by expression of subunits of the transcription factor E2F. These results indicate that endocycle S phases require oscillations in Cdk activity, but, in contrast to oscillations in mitotic cells, these occur independently of mitosis.

Chromosome association of minichromosome maintenance proteins in Drosophila endoreplication cycles.

Minichromosome maintenance (MCM) proteins are essential eukaryotic DNA replication factors. The binding of MCMs to chromatin oscillates in conjunction with progress through the mitotic cell cycle. This oscillation is thought to play an important role in coupling DNA replication to mitosis and limiting chromosome duplication to once per cell cycle. The coupling of DNA replication to mitosis is absent in Drosophila endoreplication cycles (endocycles), during which discrete rounds of chromosome duplication occur without intervening mitoses. We examined the behavior of MCM proteins in endoreplicating larval salivary glands, to determine whether oscillation of MCM-chromosome localization occurs in conjunction with passage through an endocycle S phase. We found that MCMs in polytene nuclei exist in two states: associated with or dissociated from chromosomes. We demonstrate that cyclin E can drive chromosome association of DmMCM2 and that DNA synthesis erases this association. We conclude that mitosis is not required for oscillations in chromosome binding of MCMs and propose that cycles of MCM-chromosome association normally occur in endocycles. These results are discussed in a model in which the cycle of MCM-chromosome associations is uncoupled from mitosis because of the distinctive program of cyclin expression in endocycles.

Mutations of the Drosophila dDP, dE2F, and cyclin E genes reveal distinct roles for the E2F-DP transcription factor and cyclin E during the G1-S transition.

Activation of heterodimeric E2F-DP transcription factors can drive the G1-S transition. Mutation of the Drosophila melanogaster dE2F gene eliminates transcriptional activation of several replication factors at the G1-S transition and compromises DNA replication. Here we describe a mutation in the Drosophila dDP gene. As expected for a defect in the dE2F partner, this mutation blocks G1-S transcription of DmRNR2 and cyclin E as previously described for mutations of dE2F. Mutation of dDP also causes an incomplete block of DNA replication. When S phase is compromised by reducing the activity of dE2F-dDP by either a dE2F or dDP mutation, the first phenotype detected is a reduction in the intensity of BrdU incorporation and a prolongation of the labeling. Notably, in many cells, there was no detected delay in entry into this compromised S phase. In contrast, when cyclin E function was reduced by a hypomorphic allele combination, BrdU incorporation was robust but the timing of S-phase entry was delayed. We suggest that dE2F-dDP contributes to the expression of two classes of gene products: replication factors, whose abundance has a graded effect on replication, and cyclin E, which triggers an all-or-nothing transition from G1 to S phase.

Chromosome association of minichromosome maintenance proteins in Drosophila mitotic cycles.

Minichromosome maintenance (MCM) proteins are essential DNA replication factors conserved among eukaryotes. MCMs cycle between chromatin bound and dissociated states during each cell cycle. Their absence on chromatin is thought to contribute to the inability of a G2 nucleus to replicate DNA. Passage through mitosis restores the ability of MCMs to bind chromatin and the ability to replicate DNA. In Drosophila early embryonic cell cycles, which lack a G1 phase, MCMs reassociate with condensed chromosomes toward the end of mitosis. To explore the coupling between mitosis and MCM-chromatin interaction, we tested whether this reassociation requires mitotic degradation of cyclins. Arrest of mitosis by induced expression of nondegradable forms of cyclins A and/or B showed that reassociation of MCMs to chromatin requires cyclin A destruction but not cyclin B destruction. In contrast to the earlier mitoses, mitosis 16 (M16) is followed by G1, and MCMs do not reassociate with chromatin at the end of M16. dacapo mutant embryos lack an inhibitor of cyclin E, do not enter G1 quiescence after M16, and show mitotic reassociation of MCM proteins. We propose that cyclin E, inhibited by Dacapo in M16, promotes chromosome binding of MCMs. We suggest that cyclins have both positive and negative roles in controlling MCM-chromatin association.