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Endocycles are variant cell cycles comprised of DNA synthesis (S)- and gap (G)-phases but lacking mitosis. Such cycles facilitate post-mitotic growth in many invertebrate and plant cells, and are so ubiquitous that they may account for up to half the world's biomass. DNA replication in endocycling Drosophila cells is triggered by cyclin E/cyclin dependent kinase 2 (CYCE/CDK2), but this kinase must be inactivated during each G-phase to allow the assembly of pre-Replication Complexes (preRCs) for the next S-phase. How CYCE/CDK2 is periodically silenced to allow re-replication has not been established. Here, using genetic tests in parallel with computational modelling, we show that the endocycles of Drosophila are driven by a molecular oscillator in which the E2F1 transcription factor promotes CycE expression and S-phase initiation, S-phase then activates the CRL4(CDT2) ubiquitin ligase, and this in turn mediates the destruction of E2F1 (ref. 7). We propose that it is the transient loss of E2F1 during S phases that creates the window of low Cdk activity required for preRC formation. In support of this model overexpressed E2F1 accelerated endocycling, whereas a stabilized variant of E2F1 blocked endocycling by deregulating target genes, including CycE, as well as Cdk1 and mitotic cyclins. Moreover, we find that altering cell growth by changing nutrition or target of rapamycin (TOR) signalling impacts E2F1 translation, thereby making endocycle progression growth-dependent. Many of the regulatory interactions essential to this novel cell cycle oscillator are conserved in animals and plants, indicating that elements of this mechanism act in most growth-dependent cell cycles.

The kinetochore is assembled during mitotic and meiotic divisions within the centromeric region of chromosomes. It is composed of more than eighty different proteins. Spc105 (also designated as Spc7, KNL-1 or Blinkin in different eukaryotes) is a comparatively large kinetochore protein, which can bind to the Mis12/MIND and Ndc80 complexes and to the spindle assembly checkpoint components Bub1 and BubR1. Our genetic characterization of Drosophila Spc105 shows that a truncated version lacking the rapidly evolving, repetitive central third still provides all essential functions. Moreover, in comparison with Cenp-C that has previously been observed to extend from the inner to the outer kinetochore region, full-length Spc105 is positioned further out and is not similarly extended along the spindle axis. Thus, our results indicate that Spc105 forms neither an extended link connecting inner Cenp-A chromatin with outer kinetochore regions nor a scaffold constraining kinetochore subcomplexes and spindle assembly checkpoint components together into a geometrically rigid supercomplex. Spc105 seems to provide a platform within the outer kinetochore allowing independent assembly of various kinetochore components.

The destruction box (D-box) consensus sequence has been defined as a motif mediating polyubiquitylation and proteolysis of B-type cyclins during mitosis. We show here that the regions with similarity to D-boxes are not required for mitotic degradation of Drosophila Cyclin A. Instead of a simple D-box, a complex N-terminal degradation signal is present in this cyclin. Mutations that impair or abolish mitotic Cyclin A destruction delay progression through metaphase, but only when overexpressed. Moreover, these mutations prevent epidermal cells from entering the first G1 phase of embryogenesis and lead to a complete extra division cycle instead of a timely cell proliferation arrest. Residual Cyclin A activity after mitosis, therefore, has S phase-promoting activity. In principle, an S phase defect could also explain why epidermal cells fail to enter mitosis 16 in mutants lacking zygotic Cyclin A function. However, we demonstrate that this failure of mitosis is not caused simply by DNA replication or damage checkpoints. Entry into mitosis requires a function of Cyclin A that does not depend on the presence of the N-terminal region.

Mammalian cyclin D-Cdk4 complexes have been characterized as growth factor-responsive cell cycle regulators. Their levels rise upon growth factor stimulation, and they can phosphorylate and thus neutralize Retinoblastoma (Rb) family proteins to promote an E2F-dependent transcriptional program and S-phase entry. Here we characterize the in vivo function of Drosophila Cyclin D (CycD). We find that Drosophila CycD-Cdk4 does not act as a direct G(1)/S-phase regulator, but instead promotes cellular growth (accumulation of mass). The cellular response to CycD-Cdk4-driven growth varied according to cell type. In undifferentiated proliferating wing imaginal cells, CycD-Cdk4 caused accelerated cell division (hyperplasia) without affecting cell cycle phasing or cell size. In endoreplicating salivary gland cells, CycD-Cdk4 caused excessive DNA replication and cell enlargement (hypertrophy). In differentiating eyes, CycD-Cdk4 caused cell enlargement (hypertrophy) in post-mitotic cells. Interaction tests with a Drosophila Rb homolog, RBF, indicate that CycD-Cdk4 can counteract the cell cycle suppressive effects of RBF, but that its growth promoting activity is mediated at least in part via other targets.

Complexes of D-type cyclins and cdk4 or 6 are thought to govern progression through the G(1) phase of the cell cycle. In Drosophila, single genes for Cyclin D and Cdk4 have been identified, simplifying genetic analysis. Here, we show that Drosophila Cdk4 interacts with Cyclin D and the Rb homolog RBF as expected, but is not absolutely essential. Flies homozygous for null mutations develop to the adult stage and are fertile, although only to a very limited degree. Overexpression of inactive mutant Cdk4, which is able to bind Cyclin D, does not enhance the Cdk4 mutant phenotype, confirming the absence of additional Cyclin D-dependent cdks. Our results indicate, therefore, that progression into and through the cell cycle can occur in the absence of Cdk4. However, the growth of cells and of the organism is reduced in Cdk4 mutants, indicating a role of D-type cyclin-dependent protein kinases in the modulation of growth rates.

While entry into mitosis is triggered by activation of cdc2 kinase, exit from mitosis requires inactivation of this kinase. Inactivation results from proteolytic degradation of the regulatory cyclin subunits during mitosis. At least three different cyclin types, cyclins A, B and B3, associate with cdc2 kinase in higher eukaryotes and are sequentially degraded in mitosis. We show here that mutations in the Drosophila gene fizzy (fzy) block the mitotic degradation of these cyclins. Moreover, expression of mutant cyclins (delta cyclins) lacking the destruction box motif required for mitotic degradation affects mitotic progression at distinct stages. Deltacyclin A results in a delay in metaphase, deltacyclin B in an early anaphase arrest and deltacyclin B3 in a late anaphase arrest, suggesting that mitotic progression beyond metaphase is ordered by the sequential degradation of these different cyclins. Coexpression of deltacyclins A, B and B3 allows a delayed separation of sister chromosomes, but interferes wit chromosome segregation to the poles. Mutations in fzy block both sister chromosome separation and segregation, indicating that fzy plays a crucial role in the metaphase/anaphase transition.

A variety of different cyclin proteins have been identified in higher eukaryotes. In the case of cyclin B, functional analyses have clearly demonstrated an important role in the control of entry into mitosis. The function of cyclin A is more complex. It appears to function in the control of both S- and M-phase. The results of our genetic analyses in Drosophila demonstrate that cyclin A has a mitotic function and that 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.

Using probes obtained by PCR amplification, we have cloned Drosophila cDNAs encoding structural homologs of the p34cdc2 cell cycle kinase. Southern blot experiments and in situ hybridization to polytene chromosomes demonstrated that the isolated cDNAs, were derived from two distinct genes, Dm cdc2 (31E) and Dm cdc2c (92F). Northern blot and in situ hybridization experiments revealed that these two genes are coexpressed during embryogenesis and that expression is correlated with cell proliferation. However, despite the similarity in structure and expression, the two gene products differed in functional assays in yeasts. Expression of Dm cdc2 in Schizosaccharomyces pombe and Saccharomyces cerevisiae rescued cell cycle arrest caused by mutations in cdc2+ and CDC28, the genes encoding the p34cdc2 kinase homologs of these yeasts. In contrast, the Dm cdc2c gene product did not restore cell cycle progression. Thus, in addition to the identification of a functional homolog in Drosophila, our results indicate the presence of a closely related cognate of the p34cdc2 cell cycle kinase.