org Interactive Fly, Drosophila



Transcriptional Regulation

DNA replication in G2 does not normally occur, due to the checkpoint control. To elucidate its mechanism, the functions of the Drosophila genes escargot and cdc2 were studied. When escargot function is eliminated, diploid imaginal cells that are arrested in G2 lose Cyclin A, a regulatory subunit of G2/M cdk, and entered an endocycle. escargot genetically interacts with cdc2, which encodes a catalytic subunit of G2/M cdk. The mutant phenotypes of cdc2 itself are similar to those of escargot: many diploid cells in imaginal discs, salivary glands and the central nervous system enter an endocycle and sometimes form polytene chromosomes. Since mitotically quiescent abdominal histoblasts (the cells that give rise to adult cuticle) still require cdc2 to remain diploid, the inhibitory activity of G2/M cdk on DNA replication appears to be separable from its activity as the mitosis promoting factor. These results suggest that in G2, escargot is required to maintain a high level of G2/M cdk, which actively inhibits the entry into S phase (Hayashi, 1996).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

A double-assurance mechanism controls cell cycle exit upon terminal differentiation in Drosophila

Terminal differentiation is often coupled with permanent exit from the cell cycle, yet it is unclear how cell proliferation is blocked in differentiated tissues. The process of cell cycle exit was examined in Drosophila wings and eyes; cell cycle exit can be prevented or even reversed in terminally differentiating cells by the simultaneous activation of E2F1 and either Cyclin E/Cdk2 or Cyclin D/Cdk4. Enforcing both E2F and Cyclin/Cdk activities is required to bypass exit because feedback between E2F and Cyclin E/Cdk2 is inhibited after cells differentiate, ensuring that cell cycle exit is robust. In some differentiating cell types (e.g., neurons), known inhibitors including the retinoblastoma homolog Rbf and the p27 homolog Dacapo contribute to parallel repression of E2F and Cyclin E/Cdk2. In other cell types, however (e.g., wing epithelial cells), unknown mechanisms inhibit E2F and Cyclin/Cdk activity in parallel to enforce permanent cell cycle exit upon terminal differentiation (Buttitta, 2007).

Current models for cell cycle exit invoke repression of Cyclin/Cdk activity by CKIs or repression of E2F-mediated transcription by RBs as the proximal mechanisms by which cell cycle progression is arrested. Since these models include the potential for positive feedback between E2F and CycE/Cdk2, they predict that the induction of either E2F or a G1 Cyclin/Cdk complex should be sufficient to maintain the activity of the other and thereby sustain the proliferative state. However, in differentiating Drosophila tissues, both E2F and G1 Cyclin/Cdk activities had to be simultaneously upregulated to bypass or reverse cell cycle exit. An explanation for this resides in two observations. First, the ability of Cyclin/Cdk activity to promote E2F-dependent transcription is lost or reduced in the wing and eye after terminal differentiation. Second, increased E2F cannot sustain functional levels of CycE/Cdk2 activity after terminal differentiation, despite an increase in cycE and cdk2 mRNA to levels higher than those observed in proliferative-stage wings. Thus, crosstalk between E2F and Cyclin/Cdk activity appears to be limited, in both directions, as a consequence of differentiation (Buttitta, 2007).

How are these two regulatory interactions altered? One possibility is that Rbf2- or E2F2-dependent repression prevents ectopic Cyclin/Cdk activity from promoting E2F-dependent transcription after prolonged exit. While mRNA expression data and the existing genetic data on E2F2 and Rbf2 do not support this possibility, the roles of Rbf2 or E2F2 have not been tested in the presence of continued Cyclin/Cdk activity. Therefore, transcriptional repression of E2F targets by Rbf2 or E2F2 remains an important issue to address in future experiments (Buttitta, 2007).

More enigmatic is the inability of the ectopic CycE/Cdk2 provided by overexpressed E2F to promote cell cycle progression. One plausible explanation for this is that novel inhibitors of CycE are expressed with the onset of differentiation, and that these raise the threshold of Cyclin/Cdk activity required to promote cell cycle progression. Such inhibitors might make the critical substrates of CycE/Cdk2, which reside on chromatin in DNA-replication and -transcription initiation complexes, less accessible or otherwise recalcitrant to activation. The notion of an increased Cdk threshold is consistent with the observation that the >10-fold increase in CycE/Cdk2 provided by direct overexpression of the kinase bypassed cell cycle exit in conjunction with E2F, while the ~4-fold increase provided indirectly by ectopic E2F is insufficient to drive the cell cycle. Although a >10-fold increase in Cdk activity as applied in these experiments is far above the normal physiological range, such dramatic deregulation of cell cycle genes may be physiologically relevant to cancers, in which gene expression can be greatly amplified (Buttitta, 2007).

Recent studies of cycle exit in larval Drosophila eyes have concluded that Rbf1 and Dap are required to inhibit E2F and CycE/Cdk2 in differentiating photoreceptors. Other studies document the roles of Ago/Fbw7 and components of the Hippo/Warts-signaling pathway in downregulating CycE for cell cycle exit in nonneural cells in the eye. Although the data are consistent with these studies in the eye, Ago and the Hippo/Warts pathway are dispensable for cell cycle exit in the wing. Furthermore, deletion of Rbf1 did not prevent cell cycle exit in the epithelial wing, even when high levels of CycE/Cdk2 were provided. Conversely, deletion of Dap was not sufficient to keep wing cells cycling, even when excessive E2F activity was provided. These observations suggest that unknown inhibitors of E2F and Cyclin/Cdk activity mediate cell cycle exit in specific contexts, such as the wing (Buttitta, 2007).

In attempts to identify upstream factors regulating cell cycle exit, a variety of growth and patterning signals were manipulated in the pupal wing and eye, and their effects on cell cycle exit were examined. Surprisingly, signals that act as potent inducers of proliferation in wings and eyes at earlier stages did not prevent or even delay cell cycle exit upon terminal differentiation. Thus, an important focus for future studies will be the nature of the signals upstream of E2F and CycE that mediate cell cycle exit. These could be novel signals, or combinations of known signals delivered in unappreciated ways (Buttitta, 2007).

How general is double assurance? Studies of cell cycle exit in mammals do not offer a consistent answer to this question. S phase re-entry can be achieved in differentiated cells by activating E2F, CycE/Cdk2, or CycD/Cdk4 alone, but this does not lead to cell division or continued proliferation. Several studies with mammalian cells in vivo have shown that neither increased E2F nor Cyclin/Cdk activity alone is sufficient to fully reverse differentiation-associated quiescence, consistent with the double-assurance model propose in this study. Also consistent with this model is the ability of proteins from DNA tumor viruses, such as adenovirus E1A, SV40 LargeT, and HPV E6 and E7, to fully reverse differentiation-associated cell cycle exit in many cell types. These viral onco-proteins stimulate cell cycle progression by targeting multiple cell cycle factors, which ultimately increase both E2F and G1 Cyclin/Cdk activities simultaneously. For example, LargeT and E1A inhibit both RBs and CKIs, such as p21Cip1 and p27Kip1 (Buttitta, 2007).

There are some instances, however, in which differentiation-associated cell cycle exit has been bypassed, not just delayed, by the deletion of CKIs or RBs. In one such case, p19Ink4d and p27Kip1 were knocked out in the mouse brain, and ectopic mitoses were documented in neuronal cells weeks after they normally become quiescent. Similar results have been obtained with hair and support cells in the mouse inner ear, where deletion of p19Ink4d, p27Kip1, or pRB can bypass developmentally programmed cell cycle exit. In light of these findings, it is interesting to speculate that certain differentiated tissues may retain some ability to repair or regenerate by maintaining the capacity for positive feedback between E2F and CycE/Cdk2 activity. Inner-ear hair cells may be such an example, since in many vertebrates they are capable of regeneration, although this ability has been lost in mammals. Although the mammalian brain has a very limited capacity for regeneration, the cell cycle can be reactivated in the brains of other vertebrates, such as fish, in response to injury. Thus, the retention of crosstalk between E2F and Cyclin/Cdk activities in the evolutionary descendents of regeneration-competent cells might explain some of the tissue-specific sensitivities to loss of CKIs or RBs observed in mammals (Buttitta, 2007).

Regulation of cell proliferation and wing development by Drosophila SIN3 and String

The transcriptional corepressor SIN3 is an essential gene in metazoans. In cell culture experiments, loss of SIN3 leads to defects in cell proliferation. Whether and how SIN3 may regulate the cell cycle during development has not been explored. To gain insight into this relationship, conditional knock down of Drosophila SIN3 was generated and effects on growth and development were analyzed in the wing imaginal disc. It was found that loss of SIN3 affects normal cell growth and leads to down regulation of expression of the cell cycle regulator gene String (Stg). A SIN3 knock down phenotype can be suppressed by overexpression either of Stg or of Cdk1, the target of Stg phosphatase. These data link SIN3 and Stg in a genetic pathway that affects cell cycle progression in a developing tissue (Swaminathan, 2010).

Histone acetylation levels are regulated by the opposing activities of histone lysine (K) acetyltransferases (KATs) and histone deacetylases (HDACs). The SIN3 complex is one of two major class I containing HDAC complexes present in cells. The corepressor SIN3 and the HDAC RPD3 (HDAC1 and 2 in mammals) are two important components of the multi-subunit complex (Silverstein, 2005). Mutations in either SIN3 or RPD3 result in lethality in both Drosophila and mouse model systems (Cowley, 2005; Dannenberg, 2005; David, 2008; Neufeld, 1998b; Pennetta, 1998). Accordingly, establishment and/or maintenance of histone acetylation levels are critical for metazoan development and viability (Swaminathan, 2010).

SIN3 has been shown to be important for cell proliferation. In Drosophila tissue culture cells, reduction of SIN3 protein expression by RNA interference (RNAi) resulted in a G2 phase delay in cell cycle progression (Pile, 2002). A comparison of gene expression profiles from wild type and RNAi-induced SIN3 knock down cells revealed differences in expression of genes encoding proteins that control multiple cellular processes, including cell cycle progression, transcription, mitochondrial activity and signal transduction (Pile, 2003). Expression of two genes critical for the G2/M transition of the cell cycle, String (Stg) and cyclin B (CycB), was reduced in the SIN3 knock down tissue culture cells. Stg is the Drosophila homolog of Schizosaccharomyces pombe Cdc25, a conserved protein phosphatase that dephosphorylates and activates the cyclin dependent kinase, Cdk1 (also known as DmCdc2), which is critical for entry into M phase. CycB interacts with Cdk1 and promotes the G2/M transition (Swaminathan, 2010).

In mouse, knock out of either SIN3 gene, mSin3a or mSin3b, by gene disruption revealed links to cell cycle regulation. Analysis of SIN3-deficient mouse embryonic fibroblasts (MEFs) indicated that mSin3A is important for cell proliferation (Cowley, 2005; Dannenberg, 2005). The mSin3A-deficient MEFs exhibited reduced proliferative capacity relative to their wild type counterparts. Analysis of the DNA content of the MEFs indicated a reduction in the number of cells in S phase with an increase in the number of cells in the G2/M phase of the cell cycle. Although mSin3b is highly similar to mSin3a, the proteins are non-redundant since loss of either gene by targeted gene disruption resulted in embryonic lethality (David, 2008). Furthermore, mSin3B-deficient, but not mSin3a-deficient, MEFs proliferated similarly to the wild type cells under standard culture conditions (David, 2008). Upon serum starvation, however, wild type cells ceased to proliferate while the mSin3B-deficient cells continued to cycle, indicating that mSin3B is necessary for cell cycle exit at the start of differentiation (Swaminathan, 2010).

Null mutations in Drosophila Sin3A result in embryonic lethality with only a few animals surviving to the first larval instar stage (Neufeld, 1998b; Pennetta, 1998). Using an RNAi conditional mutant, it has been determined that SIN3 is also necessary for post-embryonic development (Sharma, 2008). To study the role of SIN3 during the process of cellular proliferation and differentiation, an RNAi conditional mutant was used to eliminate SIN3 in wing imaginal disc cells. SIN3 knock down cells were analyzed during larval and adult stages of development. Loss of SIN3 resulted in fewer cells in the wing blade and a curled wing phenotype in the adult. The curly wing phenotype was partially suppressed by overexpression of the cell cycle regulator Stg and its target Cdk1. These data suggest that SIN3 and G2 to M regulators work in a similar pathway to affect cell cycle progression (Swaminathan, 2010).

Loss of SIN3 from wing imaginal disc cells resulted in a number of observable phenotypes, including smaller imaginal discs and smaller, curly adult wings. The SIN3 knock down curly wing phenotype could be modified by reduction in the level of PCAF, a KAT enzyme that carries out the opposing reaction to histone deacetylation. The curly wing phenotype was also partially suppressed by overexpression of the cell cycle regulatory factors Stg and Cdk1 (Swaminathan, 2010).

SIN3 and proteins associated with the SIN3 complex have been linked to cell cycle regulation in multiple model systems. Loss of Drosophila SIN3 or RPD3 in tissue culture cells resulted in loss of cell proliferation (Pile, 2003). SIN3 has also been implicated in cell survival or proliferation during eye development; generation of homozygous null SIN3 clones resulted in scars across the eye (Neufeld, 1998b). In mouse model systems, genetic knock out of mSin3a from embryonic fibroblasts resulted in loss of cell proliferation (Cowley, 2005; Dannenberg, 2005). Knock out of mSin3b from mouse embryonic fibroblasts resulted in loss of ability of the cells to exit the cell cycle at the start of differentiation (David, 2008). Recent work has indicated that mSin3 is recruited to cell cycle regulated E2F4 target genes in terminally differentiated myoblasts to keep these genes in a repressed state (van Oevelen, 2008). In this study it was observed that reduction of SIN3 in wing imaginal disc cells results in fewer mitotic cells in the wing disc and fewer cells in the adult wing. These results suggest that SIN3 is required for cell proliferation and/or cell survival in the context of a developing organism, as well as in tissue culture cells (Swaminathan, 2010).

Loss of SIN3 in both tissue culture cells and wing imaginal disc tissue results in a decrease of stg mRNA expression. Overexpression of Stg in the background of SIN3 knock down is able to partially suppress the small wing and curly wing phenotypes. Stg is a key regulator of the cell cycle, specifically of the G2 to M transition. Loss of Stg in clones in wing imaginal discs resulted in loss of cell proliferation while overproduction of dE2F resulted in increased Stg expression and accelerated cell proliferation, thus implicating dE2F as a transcriptional activator of stg (Neufeld, 1998a). stg has also been shown to be regulated at the level of transcription by the action of the activator Pointed and the repressor Tramtrack 69 (ttk69). Additional activators including eyes absent and Sine oculis were found to bind to the stg regulatory region in eye imaginal disc cells. Taken together, these results suggest that stg expression is likely regulated by the combinatorial action of multiple activators and repressors, the binding of which may vary with cell cycle stage and tissue (Swaminathan, 2010).

Because SIN3 is a transcriptional corepressor and loss of SIN3 leads to reduced stg expression rather than activation of stg, it is hypothesized that the effect of SIN3 on stg gene expression is indirect. One possible model to explain this effect is that loss of SIN3 leads to an increase in expression of a repressor of stg. If this model is accurate, then loss of this repressor may be able to suppress the SIN3 knock down curly wing phenotype. A second possible model is that loss of SIN3 leads to increased acetylation of a transcription factor necessary for appropriate stg expression. Numerous transcription factors, including p53, have been found to be acetylated. Acetylation of these factors can affect protein stability, localization, interactions with other proteins and DNA binding activity. Experiments to test the possible models linking SIN3 and Stg are currently underway (Swaminathan, 2010).

Genetic interactions were also observed between SIN3 and Cdk1, the substrate of Stg and another important G2/M regulatory factor. Overexpression of Cdk1 suppressed the SIN3 knock down curly wing phenotype. A reduction of Cdk1 levels using the cdc2c03495 allele resulted in enhanced abnormal adult wing morphology as compared to the SIN3 mutants alone. Cdk1 must be dephosphorylated by Stg in order for cells to pass from the G2 to M phase of the cell cycle. Increasing the amount of the substrate for Stg may permit formation of enough active CycB-Cdk1 complexes to drive cell proliferation in the SIN3 knock down cells. A similar suppression of a cell proliferation defect has been previously reported. In Aspergillus nidulans, introduction of an extra copy of cyclin B into a cdc25 (Stg homolog) mutant partially rescued the cell cycle defect of the cdc25 mutant cells (Swaminathan, 2010).

Overexpression of Stg does not fully suppress the SIN3 knock down phenotype, possibly because not all cells in larval wing imaginal discs are sensitive to ectopic Stg expression. Consistent with a cell type specific response to Stg, it was found that Stg overexpression in tissue culture cells is unable to suppress the strong RNAi-induced SIN3-deficient cell proliferation defect. It is also possible that other factors interact with SIN3 to affect wing morphology. Experiments are being conducted to identify other novel factors in the SIN3 regulatory network that may contribute to the role of SIN3 in development (Swaminathan, 2010).

The SIN3 complex is one of the two major class I HDAC complexes conserved from Drosophila to human. The current results have uncovered a genetic link between transcription repression by SIN3 and G2/M cell cycle progression by Stg and Cdk1. Further investigation of this interaction is expected to shed light on the role that histone acetylation plays in the regulation of cell proliferation and differentiation (Swaminathan, 2010).

Protein Interactions

In Drosophila, the maternally expressed mei-41 and grapes 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. Mutations have been identified in Drosophila wee. Like mei-41 and grp, wee is zygotically dispensable but is required maternally for completing the embryonic nuclear cycles. The arrest phenotype of wee mutants, as well as genetic interactions between wee, grp, and mei-41 mutations, suggest that wee is functioning in the same regulatory pathway as these genes. These findings imply that inhibitory phosphorylation of Drosophila Cdk1 (alternatively termed Cdc2) by Wee is required for proper regulation of the early syncytial cycles of embryogenesis (Price, 2000).

The sequence conservation of Drosophila Wee strongly suggests that this protein acts as a Cdc2 inhibitory kinase, and this premise was examined directly in vitro. Assays were developed for Cdc2 phosphorylation, using haemmaglutinin (HA) epitope-tagged Drosophila Cdc2 produced by reticulocyte lysate translation, and GST-tagged Drosophila Cyclin B expressed in E. coli. Nuclear and cytoplasmic extracts were prepared from 0-12 hr Drosophila embryos, and tested for enzymatic modification of HA-Cdc2/Cyclin B complexes. Nuclear extracts contain an activity that tyrosine phosphorylated HA-DmCdc2, as detected by probing Western blots with an antibody to phosphotryosine. This phosphorylation is strongly stimulated by Drosophila Cyclin B. Phosphorylation of Drosophila Cdc2 alters its electrophoretic mobility. S35 labelling of the in vitro translated DmCdc2 allows detection of total Cdc2, and assessment of the efficiency with which input Cdc2 is modified. The shift in the mobility of the protein indicates that the majority of the protein is modified. While a number of new forms are seen, only two bands containing phospho-tyrosine were detected by the antibody (Campbell, 1995).

Like the nuclear extract, in vitro translated Drosophila Wee stimulates tryosine phosphorylation of Cdc2, but the latter reaction is more complete. Only the lower mobility phospho-tyrosine containing band is produced, and strong phosphorylation is seen in the absence of cyclin B. The more rapidly migrating phosphotryosine containing band is seen if cytoplasmic extract is included during the reaction, suggesting that it is produced by secondary modification by a separate activity. Indeed, even though the cytoplasmic extract on its own does not cause tryosine phosphorylation, it does modify Cdc2 to produce a more rapidly migrating form. Based on previous characterization this more rapidly migrating form is phosphorylated on Thr161. The Thr161 modifying activity appears to present in both cytoplasmic and nuclear extracts, but not in reticulocyte-translated protein extracts. Mutation of Tyr15 of Drosophila Cdc2 to Phe15 (Y15 to F15) blocks tryosine phosphorylation, suggesting that this is the residue that is modified by Dwee1. Furthermore, the effect of this mutation on the migration of the higher mobility form of Cdc2 suggests that efficient phosphorylation of both Tyr15 and Thr161 occurs when both in vitro translated Wee and cytoplasmic extract are present (Campbell, 1995).

It is concluded that Wee encodes a tyosine kinase that adds inhibitory phosphates to Cdc2 and that this activity is nuclear in embryos. Thus, the catalytic activity of Drosophila Wee is consistent with that expected for a Cdc2 inhibitory kinase and with its ability to compensate for loss of wee1 and mik1 functions in fission yeast (Campbell, 1995).

In Cdk7 mutant fly embryos, the level of Thr-161 phosphorylation and activity of the Cyclin B-bound Cdc2 was shown to be reduced, and both activities are restored by incubation with purified Cdk7/Cyclin H. This indicates that the major difference between Cdc2 isolated from wild-type and Cdk7 mutant embryos is the extent of Thr-161 phosphorylation. Therefore, Cdk7 is essential for in vivo CAK activity. Although Cdc2/Cyclin B complexes form normally in Cdk7ts mutant embryos, Cdc2 and Cyclin A fail to form a stable complex in the Cdk7 mutant. This is likely attributable to the fact that this event requires the phosphorylation of Cdc2 on Thr-161, as even in the wild type only the phosphorylated form is associated with Cyclin A. These in vivo results correlate well with the finding that human Cdc2 needs to be phosphorylated by CAK to form a stable complex with Cyclin A in vitro, whereas stable Cdc2/Cyclin B and Cdk2/Cyclin E complexes can form in the absence of Thr-161 (or 160) phosphorylation. The Cdc2/Cyclin A complex seems to be more sensitive to a reduction in CAK activity than the Cdc2/Cyclin B complex, as the loss of Cyclin A binding occurs more rapidly than the reduction of Thr-161 phosphorylation of Cyclin B-associated Cdc2 (Larochelle, 1998).

If Drosophila Cdk7 is required specifically for mitosis, it would be expected that the ovarian phenotype resulting from lack of cdk7 would be similar to the one resulting from lack of Cdc2. Therefore, the Cdc2 phenotype was analyzed using the temperature-sensitive transgene Dmcdc2A171T (Sigrist, 1995). Females carrying two copies of this temperature-sensitive allele in the Dmcdc2B47 background show a rapid depletion of follicle cells when transferred to the restrictive temperature after eclosion. This depletion of follicle cells is identical to the one observed in Cdk7ts ovaries. Also, as noted for the Cdk7ts mutant ovaries, mitotic proliferation of the germ line stops but the capacity of the germ-line cells to replicate their DNA is not affected by the loss of Cdc2 activity. Polyploidization of the germ cells usually occurs only when the mitotic division program is terminated and the 16-cell cyst is formed. In both cdc2 and cdk7 mutants the polyploidization of the germ line occurs prematurely. Because Cdc2 mediates this block of endoreplication in mitotic tissues, these results also suggest that the premature endoreplication observed in cdk7 mutant ovaries may be attributable to lack of Cdc2 activity (Larochelle, 1998).

The conserved regulators of cell cycle progression--Cyclins, Cdc2 kinase, and String phosphatase (Cdc25)--accommodate multiple modes of regulation during Drosophila embryogenesis. During cell cycles 2-7, Cdc2/Cyclin complexes are continuously present and show little fluctuation in abundance, phosphomodification, or activity. This suggests that cycling of the mitotic apparatus does not require cytoplasmic oscillations of known regulatory activities. During cycles 8-13, a progressive increase in the degradation of Cyclins at mitosis leads to increasing oscillations of Cdc2 kinase activity. Mutants deficient in cyclin mRNAs suffer cell cycle delays during this period, suggesting that the accumulation of cyclins controls 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).

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, germ cell precursors (called pole cells) proliferate early in embryogenesis and then enter a prolonged quiescence. Pole cell formation begins when nuclei at the posterior end of the embryo pinch off to form pole buds during cycle 9. Pole buds divide twice while somatic nuclei undergo nuclear cycles 9 and 10. The pole buds then cellularize at the end of cycle 10. The pole cells divide asynchronously, zero to two times as suggested by cell counts, while somatic nuclei divide three more times (somatic cycles 11-13). Thus, polar nuclear divisions are asynchronous and lag behind somatic nuclear divisions during syncytial cycles 9 and 10. The polar division program deviates from the somatic division program at a time when pole nuclei and somatic nuclei still share a common cytoplasm: this is earlier than had previously been thought to occur. 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. Cyclin A accumulates in arrested pole cells, consistent with a G2 arrest. Quiescent pole cells can be driven into mitosis by induction of either an activator of Cdc2 (Cdc25 [string] phosphatase) or a mutant form of Cdc2 that cannot be inhibited by phosphorylation. In contrast, induction of wild-type Cdc2 with a mitotic cyclin does not induce mitosis in pole cells. It is proposed 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. Although the data suggest that expression of String is likely to contribute to the reentry of pole cells into the cell cycle, how this reentry occurs during normal development is not understood. It is not clear exactly when quiescence is terminated in pole cells; although pole cell division is known to occur in late embryogenesis. These studies represent the first molecular characterization of proliferation in embryonic germ cells of Drosophila (Su, 1998).

In Drosophila embryos, Cyclin E is the normal inducer of S phase in G1 cells. Stable G1 quiescence requires the downregulation both of cyclin E and of other factors that can bypass the normal regulation of cell cycle progression. High-level expression of Cyclin A triggers the G1/S transition in wild-type embryos and in mutant embryos lacking Cyclin E. Three types of control downregulate this Cyclin A activity: (1) cyclin destruction limits the accumulation of Cyclin A protein in G1;(2) inhibitory phosphorylation of cdc2, the kinase partner of Cyclin A, reduces the S-phase promoting activity of Cyclin A in G1, and (3) Roughex, a protein with unknown biochemical function, limits Cyclin A function in G1. Overexpression of rux blocks S phase induction by coexpressed Cyclin A and promotes the degradation of Cyclin A. Rux also prevents a stable Cyclin A mutant from inducing S phase, indicating that inhibition does not require cyclin destruction, and instead drives the nuclear localization of Cyclin A. It is concluded that Cyclin A can drive the G1/S transition, but this function is suppressed by three types of control: Cyclin A destruction, inhibitory phosphorylation of cdc2, and inhibition by rux. The partly redundant contributions of these three inhibitory mechanisms safeguard the stability of G1 quiescence until the induction of Cyclin E. The action of rux during G1 resembles the action of inhibitors of mitotic kinases present during G1 in yeast, although no obvious sequence similarity exists (Sprenger, 1997).

Elaborate mechanisms have evolved for regulating Cdc2 activity so that mitosis occurs in a timely manner, when preparations for its execution are complete. In Schizosaccharomyces pombe, Wee1 and a related Mik1 kinase are Cdc2-inhibitory kinases that are required for preventing premature activation of the mitotic program. To identify Cdc2-inhibitory kinases in Drosophila, a screen was performed for cDNA clones that rescue S. pombe wee1- mik1- mutants from lethal mitotic catastrophe. One of the genes identified in this screen, Drosophila wee1 (Dwee1), encodes a new Wee1 homolog. Dwee1 kinase is closely related to human and Xenopus Wee1 homologs, and can inhibit Cdc2 activity by phosphorylating a critical tyrosine residue. Dwee1 mRNA is maternally provided to embryos, and is zygotically expressed during the postblastoderm divisions of embryogenesis. Expression remains high in the proliferating cells of the central nervous system well after cells in the rest of the embryo have ceased dividing. The loss of zygotically expressed Dwee1 does not lead to mitotic catastrophe during postblastoderm cycles 14 to 16. This result may indicate that maternally provided Dwee1 is sufficient for regulating Cdc2 during embryogenesis, or it may reflect the presence of a redundant Cdc2 inhibitory kinase, as in fission yeast (Campbell, 1995).

The cdc2+ gene product (p34cdc2) is a protein kinase that regulates entry into mitosis in all eukaryotic cells. The role that p34cdc2 plays in the cell cycle has been extensively investigated in a number of organisms, including the fission yeast S. pombe. To study the degree of functional conservation among evolutionarily distant p34cdc2 proteins, an S. pombe strain has been constructed in which the yeast cdc2+ gene has been replaced by its Drosophila homolog CDC2Dm (the CDC2Dm strain). This CDC2Dm S. pombe strain is viable, capable of mating and producing four viable meiotic products, indicating that the fly p34CDC2Dm recognizes all the essential S. pombe cdc2+ substrates, and that cdc2 is recognized by cyclin partners and other elements required for its activity. The p34CDC2Dm protein yields a lethal phenotype in combination with the mutant B-type cyclin p56cdc13-117, suggesting that this S. pombe cyclin might interact less efficiently with the Drosophila protein than with its native p34cdc2 counterpart. This CDC2Dm strain also responds to nutritional starvation and to incomplete DNA synthesis, indicating that proteins involved in these signal transduction pathways interact properly with p34CDC2Dm (and/or that p34cdc2-independent pathways are used). The CDC2Dm gene produces a "wee" phenotype: it is largely insensitive to the action of the S. pombe wee1+ mitotic inhibitor, suggesting that the Drosophila wee1+ homolog might not be functionally conserved. This CDC2Dm strain is hypersensitive to UV irradiation, to the same degree as wee1-deficient mutants. A strain that co-expresses the Drosophila and yeast cdc2+ genes shows a dominant wee phenotype, but displays a wild-type sensitivity to UV irradiation, suggesting that p34cdc2 triggers mitosis and influences the UV sensitivity by independent mechanisms (Bejarano, 1995).

Cell cycle checkpoints maintain the fidelity of the somatic cell cycle by ensuring that one step in the cell cycle is not initiated until a previous step has been completed. The extent to which cell cycle checkpoints play a role in the initial rapid embryonic divisions of higher eukaryotes is unclear. The initial syncytial divisions of Drosophila embryogenesis provide an excellent opportunity to address this issue as they are amenable to both genetic and cellular analysis. In order to study the relevance of cell cycle checkpoints in early Drosophila embryogenesis, the maternal-effect grapes (grp) mutation was characterized. This mutation may affect feedback control during early syncytial divisions. The Drosophila grp gene encodes a predicted serine/threonine kinase and has significant homology to chk1/rad27, a gene required for a DNA damage checkpoint in Schizosaccharomyces pombe. In S. pombe, p107/wee1 is phosphorylated by p56chk1 in vivo, and this results in maintenance of cdc2 Y15 phosphorylation and hence G2 delay. Relative to normal embryos, embryos derived from grp-mutant mothers exhibit elevated levels of DNA damage. During nuclear cycles 12 and 13, alignment of the chromosomes on the metaphase plate is disrupted in grp-derived embryos; the embryos undergo a progression of cytological events that are indistinguishable from those observed in normal syncytial embryos exposed to X-irradiation. The mutant embryos also fail to progress through a regulatory transition in Cdc2 activity, which normally occurs during interphase of nuclear cycle 14. It is proposed that the primary defect in grp-derived embryos is a failure to replicate or repair DNA completely before mitotic entry during the late syncytial divisions. This suggests that wild-type grp functions in a developmentally regulated DNA replication/damage checkpoint operating during the late syncytial divisions. These results are discussed with respect to the proposed function of the chk1/rad27 gene (Fogarty, 1997).

Kussel (1995) describes the dynamic intracellular localization of Drosophila Pendulin and its role in the control of cell proliferation. Pendulin is a new member of a superfamily of proteins that contains Armadillo (Arm) repeats and displays extensive sequence similarity with the Srp1 protein from yeast, with RAG-1 interacting proteins from humans, and with the importin protein from Xenopus. Almost the entire polypeptide chain of Pendulin is composed of degenerate tandem repeats of approximately 42 amino acids each. A short NH2-terminal domain contains adjacent consensus sequences for nuclear localization and cdc2 kinase phosphorylation. The subcellular distribution of Pendulin is dependent on cell cycle phase. During interphase, Pendulin protein is found exclusively in the cytoplasm of embryonic cells. At the transition between G2 and M-phase, Pendulin rapidly translocates into the nuclei, where it is distributed throughout the nucleoplasm and the areas around the chromosomes. In the larval CNS, Pendulin is predominantly expressed in the dividing neuroblasts, where it undergoes the same cell cycle-dependent redistribution as in embryos. Pendulin is encoded by the oho31 locus and is expressed both maternally and zygotically. Recessive lethal mutations in the oho31 gene result in a massive decrease or loss of zygeotic Pendulin expression. Hematopoietic cells of mutant larvae overproliferate and form melanotic tumors, suggesting that Pendulin normally acts as a blood cell tumor suppressor. In contrast, growth and proliferation in imaginal tissues are reduced and irregular, resulting in abnormal development of imaginal discs and the CNS of the larvae. This phenotype shows that Pendulin is required for normal growth regulation. Based on the structure of the protein, it is proposed that Pendulin may serve as an adaptor molecule to form complexes with other proteins. The sequence similarity with importin indicates that Pendulin may play a role in the nuclear import of karyophilic proteins and some of these may be required for the normal transmission and function of proliferative signals in the cells (Kussel, 1995).

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

wee has an essential maternal function during the nuclear division cycles of embryogenesis and also implicates zygotic wee function in a cell cycle checkpoint that responds to inhibition of DNA replication. The demonstration that wee has a role during the early syncytial nuclear cycles calls into question a previous assumption that inhibitory phosphorylation does not control these cycles. Analyses of the state of phosphorylation during the early cycles had failed to detect inhibitory phosphorylation of Cdk1 prior to cycle 13. Furthermore, because reduction in the gene dose of cyclin A and cyclin B slows the late nuclear cycles, it has been suggested that progress of these cycles is regulated by accumulation of cyclins to a threshold level. The finding that wee is required for completing the nuclear division cycles suggests that inhibitory phosphorylation plays a role in their regulation after all. The failure to detect inhibitory phosphorylation during these cycles can be explained if only a small pool of Cdk1 is subject to this modification. Wee1-type kinases are predominantly nuclear in Drosophila and other organisms and nuclear Wee1 activity is sufficient to block entry into mitosis even in the presence of high cytoplasmic Cdk1 activity. Hence, it is suggested that inhibitory phosphorylation of a small nuclear pool of Cdk1 contributes importantly to the control of the syncytial cycles. The proposal that inhibitory phosphorylation regulates syncytial cycles is an implicit component of a recently proposed model for the mechanism by which mei-41 and grp regulate the progressive lengthening of these cycles. In response to incompletely replicated DNA, the recognized activities of these conserved checkpoint kinases arrest the cell cycle by preventing the removal of inhibitory phosphates from Cdk1. While this model appears to be at odds with the lack of detectable inhibitory phosphorylation of Cdk1 during the syncytial cycles, the findings that Drosophila wee is required for the early nuclear division cycles supports this proposal. Indeed, the apparent parallels in the phenotypes of mei-41, grp, and wee maternal mutants suggest that these genes operate by a similar mechanism. Because the results implicate this pathway without defining precisely how it is induced, it remains possible that the same pathway could be used in a unique regulatory circuit. In either case, the lesson seems to be that the remarkable conservation of the eukaryotic cell cycle regulatory machinery is coupled with an equally remarkable flexibility in how that machinery can be deployed, depending on the particular developmental constraints of each organism. In early Drosophila embryos, a regulatory pathway that usually serves a surveillance function plays an essential cell cycle role (Price, 2000).

Ectopic expression of the Drosophila cdk1 inhibitory kinases, Wee1 and Myt1, interferes with the second mitotic wave and disrupts pattern formation during eye development

Wee1 kinases catalyze inhibitory phosphorylation of the mitotic regulator Cdk1, preventing mitosis during S phase and delaying it in response to DNA damage or developmental signals during G2. Unlike yeast, metazoans have two distinct Wee1-like kinases, a nuclear protein (Wee1) and a cytoplasmic protein (Myt1). The genes encoding Drosophila Wee1 and Myt1 have been isolated and genetic approaches are being used to dissect their functions during normal development. Overexpression of Dwee1 or Dmyt1 during eye development generates a rough adult eye phenotype. The phenotype can be modified by altering the gene dosage of known regulators of the G2/M transition, suggesting that these transgenic strains can be used in modifier screens to identify potential regulators of Wee1 and Myt1. To confirm this idea, a collection of deletions for loci that can modify the eye overexpression phenotypes was tested and several loci were identified as dominant modifiers. Mutations affecting the Delta/Notch signaling pathway strongly enhance a GMR-Dmyt1 eye phenotype but do not affect a GMR-Dwee1 eye phenotype, suggesting that Myt1 is potentially a downstream target for Notch activity during eye development. Interactions with p53 were observed, suggesting that Wee1 and Myt1 activity can block apoptosis (Price, 2002).

The G1/S and G2/M cell cycle transitions are temporally and spatially controlled during metazoan development, allowing growth and cell division to be coordinated with patterning and differentiation. Studies of G2/M checkpoint controls in metazoans have emphasized regulatory mechanisms affecting the Cdc25-like phosphatases, which activate the mitotic regulator Cdk1 by removing inhibitory phosphorylation. Regulatory mechanisms affecting the activity and protein stability of the Cdk1 inhibitory kinases are still poorly understood, but are probably just as important. There are ample precedents for these mechanisms from studies of Wee1 and Mik1 kinases in S. pombe and SWE1 in S. cerevisiae (Price, 2002).

During the third larval instar, the Drosophila eye disc undergoes progressive transformation from a relatively amorphous epithelial sac into the complex arrangement of ommatidial facets that comprises the adult compound eye. This transformation is marked by passage of a constriction called the morphogenetic furrow (MF) across the eye disc. Cells within the MF normally arrest in G1 and failure to synchronize cells at this stage disrupts ommatidial patterning. Following the MF, a population of cells called the second mitotic wave (SMW) undergoes a final cell cycle. If cells are blocked in G1 by overexpression of a p21 CKI homolog, insufficient cells are left to form all of the cell types required for normal ommatidia, resulting in a rough adult eye phenotype. In this report, GMR-driven misexpression of Dmyt1 immediately after the MF both delays the SMW divisions and reduces the numbers of mitotic cells, also resulting in a rough eye phenotype (Price, 2002).

Dwee1 and Dmyt1 overexpression eye phenotypes are sensitive to modification by mutations in known cell cycle regulatory genes, illustrating the feasibility of screening for mutations of genes that are potential regulators of either Wee1 or Myt1. Mutations in genes that promote mitosis, such as cdc2 and cdc25string, should dominantly enhance these overexpression phenotypes and this expectation was confirmed for both of these genes with Dmyt1. Although a GMR-Dwee1 eye phenotype is also enhanced by mutations in cdc2, it is not enhanced by mutations in cdc25string, providing evidence that Wee1 and Myt1 kinases have distinct Cdk1 regulatory effects in this developmental context. This result could be explained by a requirement for higher levels of cdc25string activity to overcome GMR-Dmyt1 inhibition of Cdk1 relative to GMR-Dwee1, perhaps because it is inherently more difficult to dephosphorylate Cdk1 inhibited on both T14 and Y15 by Myt1 activity, compared with Cdk1 inhibited on Y15 alone by Wee1 (Price, 2002).

The rux gene encodes a novel Cdk1 inhibitor that controls the onset of S phase during embryogenesis, eye development, and spermatogenesis. rux also plays a novel role in mitosis, by an unknown mechanism. Rux and Wee1 both negatively regulate Cdk1 activity; thus the observation that coexpression of these genes generates more extreme rough eye phenotypes than seen with either alone is consistent with known functions for these genes. Surprisingly, flies lacking both zygotic Dwee1 and rux functions show nearly complete synthetic lethality, with rare escapers exhibiting extensive adult bristle phenotypes. This interaction suggests that rux and Dwee1 may also cooperate in some other, as yet undefined regulatory mechanism. The extensive bristle phenotypes seen in rux;Dwee1 double mutant escapers could indicate disruption of cell cycle timing or abrogation of genome integrity checkpoints, similar to the phenotypes seen in mus304 mutants exposed to ionizing radiation, which are associated with increased genome instability. Another piece of evidence suggesting a role for Wee1 kinases in regulating genome stability is the interaction observed with Drosophila p53. In humans, the p53 tumor suppressor promotes apoptosis in cells that have suffered DNA damage. Overexpression of Drosophila p53 in the eye promotes extensive cell death by apoptosis, resulting in extremely defective eyes. There is significant suppression of the p53 overexpression eye phenotype by coexpression of either GMR-Dwee1 or GMR-Dmyt1, suggesting that these Cdk1 inhibitory kinases can negatively regulate p53-induced apoptosis. Since Cdk1 activity has been implicated in promoting apoptosis, this effect would be consistent with known functions of Wee1 and Myt1 in Cdk1 inhibition. Other reports relevant to this issue are somewhat contradictory, however. In human cell culture, Wee1 can inhibit granzyme B-induced apoptosis; furthermore, Wee1 appears to be downregulated through a p53-dependent mechanism, suggesting that p53 regulation of Wee1 might normally occur during this process. In contrast, Wee1 activity can actually promote apoptosis in a Xenopus oocyte extract system. Further studies are clearly needed to establish the physiological significance of any purported roles for Wee1 or Myt1 in regulating apoptosis, p53-dependent or otherwise (Price, 2002).

A screen for modulators of wee1 overexpression has been conducted in S. pombe, by isolating suppressors of wee1-induced lethality. These studies identified mutations in the gene encoding the Hsp90 chaperone as potent suppressors, suggesting a role for Hsp90 in promoting the assembly and/or disassembly of functional Wee1 protein complexes. In contrast, no hsp83 mutant alleles (encoding Drosophila Hsp90) were found to act as suppressors of a combined GMR-Dmyt1/GMR-Dwee1 transgene eye phenotype. Several other genetic loci have been identified as specific enhancers of eye phenotypes generated by GMR-Dwee1 or GMR-Dmyt1 alone, indicating that phenotypic effects mediated by Wee1 and Myt1 are responsive to lowered expression of different genes. These observations may reflect differences in threshold requirements for the relevant gene products in promoting mitosis (as suggested by the interactions with cdc25string) or they may signify differences in the regulation of Wee1 and Myt1 kinases that it will now be possible to dissect by identifying and characterizing the relevant modifier loci. Direct genetic screens to address this issue are being undertaken for mutations in genes that modify GMR-Dwee1 and GMR-Myt1 eye phenotypes. One of the loci identified as a specific enhancer of the GMR-Dmyt1 eye phenotype is Delta. This interaction could reflect defects in Dl-dependent neuronal specification that are enhanced by GMR-Dmyt1 activity, or it may indicate a novel role for Delta/Notch signaling in regulating Myt1 activity (Price, 2002).

In S. pombe, the DNA damage and DNA replication checkpoint pathways that regulate Cdk1 by inhibitory phosphorylation act by controlling the activity and stability of Wee1 and Mik1 kinases, as well as Cdc25 phosphatases. Although metazoan homologs of components of these checkpoint pathways show significant sequence conservation with their yeast homologs, the actual functions and interactions of individual components are not necessarily conserved. For example, Xenopus homologs of the checkpoint kinases Chk1 and Cds1, which respond to DNA damage and block DNA replication, respectively, in S. pombe, respond in the exact opposite manner to these stresses in Xenopus egg extracts. This example serves as a warning that simple predictions of metazoan gene function based on extrapolation from known functions of yeast genes can be misleading. Metazoan development requires that novel regulatory mechanisms exist to link specific developmental processes with the basic cell cycle machinery. Drosophila represents an ideal model for analyzing these developmental controls of the cell cycle, since the effects of specific mutations on complex processes like morphogenesis and differentiation can be established. The recent characterization of the tribbles gene in Drosophila illustrates this point. Trbl activity delays mitosis in invaginating G2 cells (mitotic domain 10) in a cycle 14 embryo. Although cdc25string transcription initiates in domain 10 before it is transcribed in other cells, these cells remain G2 arrested until they are completely internalized, well after cells in nine other mitotic domains have subsequently expressed cdc25string and entered mitosis. Trbl activity downregulates Cdc25string protein stability, providing an explanation for these observations. A similar purpose could be served by Trbl simultaneously upregulating Dwee1 or Dmyt1 activity. Intriguingly, Trbl contains motifs reminiscent of Nim1-type kinases, which negatively regulate Wee1 and Swe1 kinase activity and stability in S. pombe and S. cerevisiae. Despite these sequence similarities, the Trbl protein apparently lacks a functional catalytic domain, raising the possibility that Trbl could act in a 'dominant negative' manner to activate Wee1 (or Myt1) by interfering with the activities of Nim1-like inhibitors. Genetic interactions described in this study are consistent with this possibility (Price, 2002).

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

Drosophila 14-3-3γ and 14-3-3ζ 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, it was 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. Antibody staining for 14-3-3 family members is cytoplasmic in interphase and perichromosomal in mitosis. Using mutants of cyclins, Cdk1 and Cdc25string to manipulate Cdk1 activity, it was 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 14-3-3γ and 14-3-3ζ mutants: 14-3-3γ is required to time mitosis in undisturbed post-blastoderm cell cycles and to delay mitosis following irradiation; 14-3-3ζ is required for normal chromosome separation during syncytial mitoses. A model is suggested 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 (Su, 2001).

In a previous study of 14-3-3γ localization in the embryo, this protein was reported to become nuclear-localized in infolding cells (Tien, 1999). However, a close examination of the published data revealed that the localization was in pre-mitotic cells (the publication featured mitotic domain 14 that borders the ventral furrow). In fact, a close correspondence of cells that show nuclear-localized 14-3-3γ in this publication (Tien, 1999) and cells that compose the mitotic domains is what led to further examination of the role of 14-3-3 proteins in the cell cycle. Using the same antibody and the same conditions, similar staining patterns were demonstrated (Tien, 1999). A different interpretation of these data is being offered. No correlation of the localized staining with the movement of cells or folding of the epithelium was found. Instead, the findings that 14-3-3 proteins localize to the perichromosomal region during mitosis and that this localization is coupled to Cdk1 activity demonstrate that localization is coupled to cell cycle progression and suggest that 14-3-3 proteins have a cell cycle role (Su, 2001).

One striking set of data presented in this study concern the localization of 14-3-3 proteins to the neighborhood of chromosomes in mitosis. Although the perinuclear localization of Drosophila 14-3-3 proteins is unprecedented, the interphase location and activity are consistent with reports from other systems. S. pombe Rad24 remains exclusively cytoplasmic throughout the cell cycle and this localization appears to be important for blocking mitosis upon checkpoint activation. Similarly, it has been proposed that cytoplasmic human 14-3-3sigma inhibits mitosis by retaining Cdk1/cyclin B in the cytoplasm (Chan, 1999). Like their homologs in other systems, Drosophila 14-3-3 proteins are cytoplasmic in interphase, and analysis of mutations suggests that Drosophila 14-3-3γ also inhibits entry into mitosis in response to activation of DNA damage checkpoint in embryos. This is in agreement with its proposed role in other species and consistent with a recent report (Brodsky, 2000) of a role for 14-3-3γ in preventing mitosis after DNA damage in Drosophila larvae (Su, 2001).

In addition, observations indicate a role for 14-3-3γ in the normal timing of embryonic mitoses. The precise schedule of mitotic times of cells in various positions in the Drosophila embryo made possible detection of deviations from normal timing that are as small as a few minutes. Defects can occur in the normally rigid stereotypical order with which different regions of the embryo progress into mitosis. For example, recent reports described the premature mitosis of mesodermal cells, normally domain 10, in a mutant tribbles. When embryos deficient in 14-3-3γ were examined, a different type of timing defect was found. The normal order of the mitotic domains was retained, but the entire schedule of mitosis was advanced relative to germ-band extension, a major morphological marker of developmental progression. Because there was no detectable slowing of germ-band extension in 14-3-3γ mutant embryos, it is infered that mitosis is advanced in embryos that lack 14-3-3γ. Thus, 14-3-3γ might set physiologically relevant thresholds for entry into mitosis in Drosophila, and this activity might be amplified in response to irradiation. S. pombe mutants in a 14-3-3 homolog show smaller cell size at division; because cellular growth in this organism occurs mainly in G2, it has been proposed that G2 is shorter in these 14-3-3 mutants (Ford, 1994), although precise measurements of this period have not been reported. Thus, it remains to be seen whether 14-3-3 proteins have a similar ability to set the threshold for normal mitosis in other species where only its checkpoint function has been detected (Su, 2001).

14-3-3ζ mutants show defective mitoses in the syncytium, indicating a requirement for this protein in syncytial divisions. Embryos that lack checkpoint functions such as Grapes (Chk1 homolog) and Mei-41 (an ATR homolog) also show mitotic defects, and it has been proposed that these defects are secondary to entry into mitosis with unreplicated DNA. However, loss of 14-3-3ζ functions affects early cycles. By contrast, the dramatic phenotypes of checkpoint defects occur at later syncytial stages (around cycle 12) when checkpoints are thought to become essential to postpone mitosis as S phase takes longer to complete. Thus, the early phenotype of 14-3-3ζ mutant embryos suggests that 14-3-3ζ has roles beyond its likely function in the checkpoint. Perhaps, like 14-3-3γ, 14-3-3ζ might contribute to the normal timing of mitosis even when checkpoints are not operating. Alternatively, incomplete separation of chromosomes in 14-3-3ζ mutants could indicate a more direct involvement of 14-3-3ζ in mitotic progression, an idea that is supported by the localization of the proteins around the mitotic chromosomes and their dispersal after chromosome separation. A direct test of these models will require specific inactivation of 14-3-3ζ in mitosis (as opposed to interphase) (Su, 2001).

Drosophila 14-3-3γ and 14-3-3ζ have documented roles in RAS signaling. Recent data implicate a MAP kinase pathway in cell cycle control in Xenopus, raising the possibility that Drosophila 14-3-3 proteins function through a MAPK pathway to affect their cell cycle roles. This is thought to be unlikely because treatment of Drosophila embryos with pharmacological inhibitors of MAPK pathway did not phenocopy either 14-3-3γ or 14-3-3ζ mutations (Su, 2001).

Regardless of the mechanism of action of 14-3-3ζ, it is notable that it has essential cell cycle roles in the absence of perturbations that normally provoke checkpoint responses. This reinforces other findings in Drosophila and in mammals that suggest that functions normally considered to be checkpoint functions have essential roles in regulating the cell cycle early in development (Su, 2001).

Based on the cytoplasmic localization of 14-3-3γ and cyclin/Cdk1 during interphase, it is proposed that 14-3-3γ acts to keep Cdk1 in check during interphase. As Cdk1 becomes active (owing to the accumulation of its activator Stg or after recovery from DNA damage) and cells enter mitosis, accumulating cyclin/Cdk1 activity promotes and maintains, probably indirectly, 14-3-3 protein localization near chromosomes. Upon the transition to anaphase, the localized 14-3-3 proteins can contribute to chromosome separation. The decline in Cdk1 activity allows 14-3-3 proteins to return to their interphase distribution. Thus, during interphase, 14-3-3γ can act to keep Cdk1 inactive in the cytoplasm but, once Cdk1 is active, it can act in turn to localize 14-3-3 proteins in preparation for their action during the exit from mitosis. No physical interaction has been detected between 14-3-3 proteins and Drosophila homologs of cell cycle regulators known to interact with 14-3-3 proteins in other systems (Cdc25string and cyclin B). Thus, understanding the mechanism of 14-3-3 action might require the identification of novel target molecules (Su, 2001).

The results do not rule out the possibility that 14-3-3ζ also functions to regulate the entry into mitosis in cellular embryos. This possibility cannot be addressed because 14-3-3ζ mutants arrest before G2/M control is first seen in embryogenesis, and the fraction of embryos that do progress to cellular stages are too defective with respect to cell cycle progression and gastrulation. In addition, the fact that these embryos progressed to cellular stages might reflect an incomplete loss of maternal 14-3-3ζ, thus precluding meaningful experiments. What is certain, however, is that 14-3-3γ cannot substitute for 14-3-3ζ during the nuclear divisions of syncytial stages, and that 14-3-3ζ cannot substitute 14-3-3γ for regulating the entry into mitosis during cellular stages (Su, 2001).

In summary, three lines of data indicate that Drosophila 14-3-3 proteins function in normal cell cycle progression, in addition to checkpoint regulation. These are: (1) cell cycle stage specific localization, which is dictated by Cdk1; (2) advancement of mitotic entry in 14-3-3γ mutants; and (3) defective mitoses in 14-3-3ζ mutants. This is the first clear evidence for the requirement for 14-3-3 proteins in normal mitosis in a eukaryote. Furthermore, the fact that mutations in two 14-3-3 proteins lead to different outcomes and at different stages in embryogenesis indicates that these proteins are not functionally redundant. Instead, the results provide strong evidence that, during metazoan development, cell division and its regulation might have different requirements for two members of the 14-3-3 family (Su, 2001).

Identification of Drosophila Myt1 kinase and its role in Golgi during mitosis

Entry into mitosis is regulated by inhibitory phosphorylation of cdc2/cyclin B, and these phosphorylations can be mediated by the Wee kinase family. This study presents the identification of Drosophila Myt1 kinase and examines the relationship of Myt1 and Wee activities in the context of Cdc2 phosphorylation. Myt1 kinase was found by BLAST-searching the complete Drosophila genome using the amino acid sequence of human Myt1 kinase. A single predicted polypeptide was identified that shared a 48% identity within the kinase domain with human and Xenopus Myt1. Consistent with its putative role as negative regulator of mitotic entry, overexpression of this protein in Drosophila S2 cells results in a reduced rate of cellular proliferation while the loss of expression via RNA interference (RNAi) results in an increased rate of proliferation. In addition, loss of Myt1 alone or in combination with Drosophila Wee1 (Wee1) results in a reduction of cells in G2/M phase and an increase in G1 phase cells. Finally, loss of Myt1 alone results in a significant reduction of phosphorylation of cdc2 on the threonine-14 (Thr-14) residue as expected. Surprisingly however, a reduction in the phosphorylation of Cdc2 on the tyrosine-15 (Tyr-15) residue is observed only when expression of both Myt1 and Wee expression is reduced via RNAi and not loss of expression of Wee alone. Most strikingly, in the absence of Myt1, Golgi fragmentation during mitosis is incomplete. These findings suggest that Myt1 and Wee have distinct roles in the regulation of Cdc2 phosphorylation and the regulation of mitotic events (Cornwell, 2002).

This study shows that loss of dMyt1 expression results in reduced phosphorylation of the Thr-14 residue of cdc2, an increased rate of cell proliferation, and a reduction of cells in G2/M with an increased number of cells in G1. The most biologically interesting observation was that loss of dMyt1 resulted in the incomplete fragmentation of the Golgi in mitotic cells. Taken together, these results indicate that dMyt1 kinase is involved in cdc2 regulation and necessary for proper Golgi fragmentation in mitosis (Cornwell, 2002).

RNAi in Drosophila S2 cells was used as a means to provide insight into the functional role of dMyt1 and dWee1 in the cell cycle beyond previously published biochemical studies. Consistent with Myt1 biochemical studies (i.e., negative regulation of cdc2/cyclin B, it was found that loss of dMyt1 expression but not of other genes (dWee1, Polo, or Dif) affects the phosphorylation state of cdc2 at the Thr-14 residue. The loss of dWee1 alone is insufficient to result in a reduced phosphorylation of the cdc2 Tyr-15 residue. These results are interesting in the context of previous biochemical analyses demonstrating that Myt1, although capable of phosphorylating both Thr-14 and Tyr-15, has a preference for phosphorylation of cdc2/Thr-14 and Wee1 is restricted to phosphorylation of cdc2/Tyr-15 only. However, the data suggest that in S2 cells the role of dMyt1 in Tyr-15 phosphorylation is greater than anticipated since loss of dWee1 did not significantly reduce Tyr-15 phosphorylation. Perhaps, this dual specific activity of dMyt1 is necessary to ensure that cdc2 localized to cytoplasmic compartments is fully inhibited until the onset of mitosis. Additional evidence that dMyt1 is a Myt1 kinase ortholog came from recombinant expression of tagged dMyt1 and localization to the Golgi (Cornwell, 2002).

Additional consequences were observed with the loss of dMyt1 expression, such as, alterations in the cell cycle. It was found that S2 cells RNAi-treated for dMyt1 contained fewer cells in G2/M by FACS and an increased number of mitotic cells via immunostaining. This suggests (as would be predicted) that the loss of dMyt1 shortens the G2 phase of the cell cycle by allowing premature activation of cdc2/cyclin B (Cornwell, 2002).

Because dMyt1 kinase is expressed in discrete cytoplasmic subcompartments that appear to overlap with the Golgi and active cdc2 has been shown to phosphorylate several Golgi-specific proteins at the onset of mitosis, it was of interest to exploring the impact on the Golgi following the loss of dMyt1 expression. At the onset of mitosis, the immunofluorescent vesicular (punctate-stained) Golgi is redistributed to a dispersed uniform staining pattern. Most interestingly, in S2 cells RNAi-treated for dMyt1, the Golgi staining pattern fails to redistribute and this phenotype persists throughout mitosis. This suggests that the Golgi is not devesiculating in the normal manner that occurs during mitosis. Several possible explanations to this observation are proposed. Although dMyt1 is a negative regulator of cdc2, it may also be required to sequester cdc2 to the Golgi. Previous studies have shown that Myt1 and cdc2/cyclin B can physically associate via domains other than the kinase domain. Myt1 may act as a sink to localize cdc2 to the Golgi or to certain Golgi compartments so that at mitosis cdc2/cyclin B is present and once activated can phosphorylate Golgi proteins before being transported to the nucleus. Alternatively, prematurely active cdc2 (due to the absence of dMyt1) drives events that block subsequent steps of the Golgi devesiculation process. Finally, these two models may overlap. The question of cdc2 transport and localization in the absence of dMyt1 are obvious and will be the focus of future studies. However, these data do not exclude the possibility that Myt1 has a novel function unrelated to cdc2 regulation that is required for Golgi devesiculation (Cornwell, 2002).

Human homologue of the Drosophila melanogaster lats tumour suppressor modulates CDC2 activity

The relationship between Warts and Drosophila Cdc2 was examined in Drosophila. Although Drosophila Cdc2 remains at a constant level during the cell cycle, Cyclins A and B are degraded when Cdc2/cyclin complexes are inactivated. Thus, the levels of Cyclins A and B are sensitive indicators of Cdc2/cyclin activities. By staining eye imaginal discs containing clones of warts mutant cells with either anti-Cyclin A or B antibodies, it was found that inactivation of warts leads to abnormal accumulation of Cyclin A, but not Cyclin B. This provides further evidence that inactivation of warts deregulates Cdc2/Cyclin A activity, and suggests that the warts mutant phenotype could be suppressed by reducing Cdc2/Cyclin A activity. Indeed, both lethality and overproliferation phenotypes of various warts mutants can be suppressed by mutations in cdc2. For example, removing one copy of cdc2 is sufficient to rescue the pupal lethality and tissue overproliferation phenotypes of wts mutants. cyclin A behaves similar to cdc2 in its interaction with wts, whereas reduction of the dosage of cyclin B has no effect on the wts mutant phenotype. Furthermore, mutations in cell-cycle regulator genes such as dEF2, cyclin E and the Drosophila CDK2 homolog cdc2 do not interact with wts mutants. These observations show that the genetic interaction between wts, cdc2 and cyclin A is specific, and support the conclusion that Warts negatively modulates the activity of the CDC2/CyclinA complex (Tao, 1999).

Cyclin D-Cdk4 and cyclin E-Cdk2 regulate the Jak/STAT signal transduction pathway in Drosophila

The JAK/STAT signal transduction pathway regulates many developmental processes in Drosophila. However, the functional mechanism of this pathway is poorly understood. The Drosophila cyclin-dependent kinase 4 (Cdk4) exhibits embryonic mutant phenotypes identical to those in the Hopscotch/JAK kinase and stat92E/STAT mutations. Specific genetic interactions between Cdk4 and hop mutations suggest that Cdk4 functions downstream of the HOP tyrosine kinase. Cyclin D-Cdk4 (as well as Cyclin E-Cdk2) binds and regulates STAT92E protein stability. STAT92E regulates gene expression for various biological processes, including the endocycle S phase. These data suggest that Cyclin D-Cdk4 and Cyclin E-Cdk2 play more versatile roles in Drosophila development (Chen, 2003).

In a large screen for autosomal P element-induced zygotic lethal mutations associated with specific maternal effect lethal phenotypes, a mutation, l(2)sh0671, located at 53C, was identified that showed a maternal effect segmentation phenotype. The phenotype is similar to the effect of loss of hop and stat92E gene activity during oogenesis. The P element, l(2)sh0671, was inserted into the second intron of the Cdk4 gene before the ATG translation initiation code (Chen, 2003).

In mammals and Drosophila, Cdk4 forms a protein complex that regulates the cell cycle progression. The Cyclin D and Cdk4 complex (CycD-Cdk4) phosphorylates and releases RB from RB/E2F; free E2F then activates gene expression, including Cyclin E (CycE). Cyclin E and Cdk2 form a complex (CycE-Cdk2) and regulate the cell cycle at the G1-S transition point. To further examine relations between the HOP/STAT92E signal transduction pathway and cell cycle regulation, the genetic interaction of hop with CycE was tested. Like HS-Cdk4, HS-CycE rescues hopC111 embryo segmentation defects but has no effect on stat92E mutant embryos (Chen, 2003).

The viability and formation of melanotic tumors at 29°C were compared in females heterozygous for hopTum-l and CycE with females heterozygous only for hopTum-l. An improved survival rate was observed by removing a single copy of CycE in hopTum-l heterozygous females. As in the case of Cdk4, the formation of melanotic tumors is less affected by removing a single copy of CycE in hopTum-l heterozygous females. These results suggest that CycD-Cdk4 and CycE-Cdk2 complexes are members of the HOP/STAT92E signal transduction pathway and function downstream of the HOP tyrosine kinase and either upstream of or parallel to the STAT92E transcription factor (Chen, 2003).

Thus Cdk4 functions in the HOP/STAT92E pathway and regulates embryonic segmentation, tracheal formation, eye development, and melanotic tumor formation. Specific genetic interactions between Cdk4 and hop or stat92E mutations suggest that Cdk4 functions upstream of STAT and parallel to or downstream of the HOP tyrosine kinase. Furthermore, CycD-Cdk4 and CycE-Cdk2 bind and regulate STAT92E protein stability. These data demonstrate that, besides their role in regulating the cell cycle, CycD-Cdk4 and CycE-Cdk2 have a role in regulating cell fate determination and proliferation via STAT signaling (Chen, 2003).

Genome-wide RNAi analysis of JAK/STAT signaling components in Drosophila: RNAi knockdown of cdc2 results in a decrease in STAT92E tyrosine phosphorylation

The cytokine-activated Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway plays an important role in the control of a wide variety of biological processes. When misregulated, JAK/STAT signaling is associated with various human diseases, such as immune disorders and tumorigenesis. To gain insights into the mechanisms by which JAK/STAT signaling participates in these diverse biological responses, a genome-wide RNA interference (RNAi) screen was carried out in cultured Drosophila cells. One hundred and twenty-one genes were identified whose double-stranded RNA (dsRNA)-mediated knockdowns affected STAT92E activity. Of the 29 positive regulators, 13 are required for the tyrosine phosphorylation of STAT92E. Furthermore, it was found that the Drosophila homologs of RanBP3 and RanBP10 are negative regulators of JAK/STAT signaling through their control of nucleocytoplasmic transport of STAT92E. In addition, a key negative regulator of Drosophila JAK/STAT signaling was identified, protein tyrosine phosphatase PTP61F; it is a transcriptional target of JAK/STAT signaling, thus revealing a novel negative feedback loop. This study has uncovered many uncharacterized genes required for different steps of the JAK/STAT signaling pathway (Baeg, 2005).

Interestingly, this assay revealed that RNAi knockdown of the cyclin-dependent kinase 2 gene (cdc2) resulted in a decrease in STAT92E tyrosine phosphorylation, suggesting that cdc2 modulates JAK/STAT signaling by affecting tyrosine phosphorylation of STAT92E. Consistent with this observation, Warts/Lats, which has been shown both biochemically and genetically to interact with cdc2 and to negatively regulate its kinase activity, was identified in the screen as a potential negative regulator of JAK/STAT signaling. These results suggest that STAT92E plays an important role in Warts/Lats-mediated inhibition of cell proliferation (Baeg, 2005).

Drosophila Cks30A interacts with Cdk1 to target Cyclin A for destruction in the female germline

Cks is a small highly conserved protein that plays an important role in cell cycle control in different eukaryotes. Cks proteins have been implicated in entry into and exit from mitosis, by promoting Cyclin-dependent kinase (Cdk) activity on mitotic substrates. In yeast, Cks can promote exit from mitosis by transcriptional regulation of cell cycle regulators. Cks proteins have also been found to promote S-phase via an interaction with the SCFSkp2 Ubiquitination complex. The Drosophila Cks gene, Cks30A (corresponding to the gene remnants), is required for progression through female meiosis and the mitotic divisions of the early embryo through an interaction with Cdk1 (Cdc2). Cks30A mutants are compromised for Cyclin A destruction, resulting in an arrest or delay at the metaphase/anaphase transition, both in female meiosis and in the early syncytial embryo. Cks30A appears to regulate Cyclin A levels through the activity of a female germline-specific anaphase-promoting complex, CDC20-Cortex. A second closely related Cks gene, Cks85A, plays a distinct, non-overlapping role in Drosophila, and the two genes cannot functionally replace each other (Swan, 2005a).

The maternal effect lethal gene, remnants, corresponds to one of the two Drosophila Cks genes (Cks30A). Analysis of two hypomorphic alleles and a null allele made by homologous recombination confirmed that Cks30A is not essential for cell cycle regulation in most tissue types. Rather, Cks30A functions in specialized cell cycles: the abdominal histoblast divisions, female meiosis and the syncytial divisions of the early embryo (Swan, 2005a).

Cks30A mutants displayed a strikingly simple mitotic phenotype: most embryos from mutant females arrested in metaphase of the first mitotic division. Similarly, Cks30A mutants display a pronounced delay or arrest in metaphase of female meiosis II. Therefore, there is a common requirement for Cks30A in metaphase to anaphase progression in female meiosis II and in early embryonic mitosis. The second meiotic division is similar to a mitotic division in that it involves the segregation of sister chromatids, and therefore Cks30A may be part of a conserved machinery that is required for both of these processes (Swan, 2005a).

An alternative explanation for the mitotic arrest in Cks30A mutants is that it is a secondary effect of a prior failure in meiosis or pronuclear fusion. However, FISH experiments indicate that this mitotic arrest occurs even in embryos that successfully undergo pronuclear fusion. Also, mutations in alpha-Tubulin67C, that block pronuclear fusion, do not lead to a mitotic arrest in the embryo, indicating that these events are not coupled (Swan, 2005a).

In addition to a crucial role in exit from mitosis, Cks30A is important in at least one aspect of entry into mitosis: spindle formation. Cks30AKO mutants are severely delayed in assembly of the first mitotic spindle. Cks30A was also required for proper assembly of the female meiotic spindle and the specialized spindle-like microtubule aster of the polar bodies. Therefore, Cks30A appears to be required at two points in the mitotic (or meiotic) cell cycle: in prometaphase for spindle assembly and at the metaphase-to-anaphase transition (Swan, 2005a).

These dual roles for Cks30A in meiosis appear to be at least partially conserved in other metazoans. Xenopus Cks2 (Xe-p9), like Cks30A, is required for the metaphase-to-anaphase transition in meiosis II. Xenopus Cks2 is also required in vitro for entry into mitosis, and this may be related to the in vivo requirement for Cks30A in spindle assembly in meiosis and mitosis. Cks genes in other eukaryotes also appear to have related but distinct functions in meiosis. Mouse cks2 is essential for anaphase progression in meiosis I of both male and female meiosis, while in C. elegans cks-1 is not required for entry into anaphase, but is necessary for proper chromosome segregation in meiosis I, possibly reflecting a role in meiotic spindle assembly. Therefore, Cks genes appear to share a common requirement in entry into and exit from meiosis in different eukaryotes (Swan, 2005a).

The two roles for Cks30A appear to reflect a conserved role in promoting Cdk1 activity. Cdk1 is the central mitotic Cdk, and its kinase activity on specific mitotic proteins is required for entry into mitosis, including spindle assembly, and in maintaining the metaphase state. Cdk1 activity is also required for exit from mitosis through activation of the APC, which in turn promotes anaphase progression by targeting mitotic cyclins for destruction. In Drosophila Cks30A and Cdk1 interact in vivo, and mutations in Cks30A that disrupt Cdk1 binding are compromised for activity in vivo. Cks30A also interacts genetically with Cdk1 in another cell type, the abdominal histoblasts. Therefore, genetic and physical evidence supports the conclusion that the observed interaction with Cdk1 is required for Cks30A function (Swan, 2005a).

Cks30A is required for the destruction of Cyclin A in the ovary and in the syncytial embryo and two observations argue that it is this failure to degrade Cyclin A that results in the observed delay or arrest in metaphase of meiosis II and in mitosis: (1) a similar metaphase arrest is seen in cellularized embryos expressing non-degradable Cyclin A, while syncytial embryos with a slight excess of Cyclin A (approximately 1-3 x wild type) due to mutations in grapes display a metaphase delay; (2) the Cks30A mutant phenotype can be partially rescued by lowering Cyclin A levels. The CDC20 homolog, cortex, is also required for exit from meiosis II, and cortex is also required for Cyclin A destruction in the ovary. These results argue that Cks30A and the APCCortex function in the same pathway leading to Cyclin A destruction, although the possibility that Cks30A and Cortex act in independent pathways to promote Cyclin A destruction cannot be ruled out (Swan, 2005a).

In vitro studies of Cks2 in Xenopus have led to a model in which Cks bound to Cdk1 recruits phosphorylated Cdk1 substrates to the kinase, allowing these substrates to be more efficiently recognized and thereby further phosphorylated by Cdk1. The CDC27 and CDC16 components of the APC are key targets of Cks-Cdk1 phosphorylation in Xenopus. While it is not yet clear how APC phosphorylation leads to its activation, there is evidence that one of the effects of phosphorylation is to stimulate CDC20 binding to the APC. Therefore it is possible that in Drosophila Cks30A-Cdk1 phosphorylates the APC, and this phosphorylation specifically stimulates the association of Cortex with the APC. Alternatively, Cks30A-Cdk1 may directly phosphorylate and activate Cortex (Swan, 2005a).

In mammalian cells and in the cellularized embryo, the completion of mitosis depends on the sequential destruction of the three mitotic cyclins by the APCFzy. Cyclin A is destroyed first in prometaphase, dependent on APCFzy activity. Although the APCFzy is active, the spindle checkpoint is thought to inhibit its activity on Cyclin B. Upon spindle assembly, the checkpoint is relieved and APCFzy can mediate Cyclin B destruction (Swan, 2005a).

It now appears that some but not all aspects of anaphase progression are conserved in the second meiotic division and in the nuclear divisions of the syncytial embryo. Although overall Cyclin B levels do not oscillate during the early syncytial divisions (cycles 1 to 8), Cyclin B appears to undergo local degradation on the mitotic spindle at anaphase, and the injection of stabilized Cyclin B into syncytial embryos results in an early anaphase arrest. By contrast to the early anaphase arrest upon Cyclin B stabilization, the injection of an APC-inhibiting peptide into early embryos results in a metaphase arrest. The results suggest that this metaphase arrest is due to the failure of APCCortex-mediated Cyclin A destruction. Like Cyclin B levels, Cyclin A levels do not oscillate detectably in cycles 1 to 7, although unlike Cyclin B, this appears to be due to a balance between constant destruction and new protein synthesis. Despite this difference, it remains possible that local oscillations in Cyclin A and Cyclin B could drive these syncytial cell cycles (Swan, 2005a).

While the importance of cyclin destruction may be conserved in the early embryo, the means by which the cyclins are destroyed appears to be different. In cellularized embryos, the APCFzy is responsible for the sequential destruction of all three mitotic cyclins. In the syncytial embryo, Fzy is not required for Cyclin A destruction. Cortex, a diverged, female germline-specific CDC20, targets Cyclin A for destruction, but has no detectable effect on Cyclin B or B3 levels in the syncytial embryo. It remains possible that Cortex is responsible for the destruction of local pools of maternal Cyclin B (and possibly B3). Alternatively, the known maternal requirement for fzy may reflect a role in the local destruction of these cyclins. This would suggest a model in which the germline utilizes two CDC20 homologs, Cortex and Fzy, to mediate the sequential destruction of Cyclins A, B and possibly B3 in the syncytial embryo. Further work will be needed to test this model. It is also not clear if Cyclin A is the only target of the APCCortex and if the APCCortex is the only target of Cks30A-Cdk1. In addition to metaphase arrest, Cks30A mutants have spindle assembly delays or defects, a phenotype that has not been observed in other cell types to result from a failure to degrade Cyclin A. Interestingly, cortex mutants also have abnormal meiosis II spindles and fail to assemble a mitotic spindle around the male pronucleus, suggesting the possibility that the sole function of Cks30A in the female germline is to activate Cortex. Like Cks30A, C. elegans cks-1 and mouse cks2 appear to be predominantly required for meiosis, and this may also reflect specific roles in activating meiosis-specific APC complexes. The histoblast requirement for Cks30A, in contrast, is unlikely to represent a role in Cortex activation (or subsequent Cyclin A destruction), since cortex mutants, either alone or in combination with Cks30A, have no effect on abdominal development (Swan, 2005a).

A specific requirement for Cks30A in activation of the maternal-specific APCCortex would explain why Cks30A is essential for anaphase progression in female meiosis and the syncytial embryo but not in most cell types. An alternative possibility, that Cks30A is functionally redundant with the other Drosophila Cks, Cks85A, cannot be ruled out. Cks85A mutants alone or in combination with Cks30A, do not have obvious defects in exit from mitosis. Furthermore, while closely related to Cks30A, Cks85A cannot replace Cks30A when expressed in the female germline, and Cks30A cannot replace Cks85A when expressed zygotically. Therefore, it is concluded that the two Drosophila Cks genes have distinct and non-overlapping functions. Recently, Cks85A was found to interact with a Drosophila Skp2 homolog in a genome-wide yeast two-hybrid screen. Two residues on Cks1 have recently been found to be crucial for Skp2 binding in vitro, and these residues are conserved or similar in Drosophila Cks85A. If indeed Cks85A represents the Drosophila Cks1 ortholog, it is perhaps not surprising that Cks30A cannot functionally replace Cks85A, since it has been found that Cks2 orthologs cannot bind Skp2 in vitro. However, it is unexpected that Cks85A cannot substitute for Cks30A in vivo. To date all Cks proteins tested can stimulate the Cdk-dependent phosphorylation of mitotic proteins in vitro, and the mouse Cks1 can functionally replace Cks2 in vivo. The failure to rescue Cks30A mutant phenotypes cannot be due to an inability of Cks85A to interact with Cdks, since Cks85A binds Cdks with even greater affinity than does Cks30A. It is possible that, analogous to the Cks1/Skp2 interaction, Cks30A has an as-yet-to-be-identified partner that is necessary for its mitotic activities. Cks85A would, therefore, be unable to carry out the mitotic activities because of an inability to bind this putative Cks30A partner (Swan, 2005a).

In conclusion, Drosophila Cks30A is crucial for Cdk1 activity in spindle assembly and anaphase progression in female meiosis and early embryonic mitosis, and at least part of this activity appears to be to regulate Cyclin A levels. Cks30A functions non-redundantly with another closely related Drosophila cks, Cks85A (Swan, 2005a).

Female meiosis and the rapid mitotic cycle of early embryos are two non-canonical cell cycles that occur sequentially in the same cell, the egg, and utilize the same pool of cell cycle proteins. Using a genetic approach to identify genes that are specifically required for these cell cycles in Drosophila, it was found that a Drosophila Cks gene, Cks30A is required for spindle assembly and anaphase progression in both female meiosis and in the syncytial embryo. Cks30A interacts with Cdk1 to target cyclin A for destruction in the female germline, possibly through the activation of a novel germline specific CDC20 protein, Cortex. These results indicate that anaphase progression in female meiosis and the early embryo are under unique control in Drosophila (Swan, 2005b).

A pre-anaphase role for a Cks/Suc1 in acentrosomal spindle formation of Drosophila female meiosis

Conventional centrosomes are absent from a female meiotic spindle in many animals. Instead, chromosomes drive spindle assembly, but the molecular mechanism of this acentrosomal spindle formation is not well understood. Female sterile mutations were screened for defects in acentrosomal spindle formation in Drosophila female meiosis. One of them, remnants (rem), disrupts bipolar spindle morphology and chromosome alignment in non-activated oocytes. rem encodes a conserved subunit of Cdc2 (Cks30A). Since Drosophila oocytes arrest in metaphase I, the defect represents a new Cks function before metaphase-anaphase transition. In addition, the essential pole components, Msps and D-TACC, were often mislocalized to the equator, which may explain part of the spindle defect. In contrast, the second cks gene cks85A has an important role in mitosis. In conclusion, this study describes a new pre-anaphase role for a Cks in acentrosomal meiotic spindle formation (Pearson, 2005).

This study shows a new pre-anaphase function of a Cks protein in acentrosomal spindle formation during Drosophila female meiosis. Through a cytological screen, spindle defects were found in remnants among female sterile mutants and remnants was found to encode one of two Cks proteins (Cks30A) in Drosophila. Cytological analysis showed that Cks30A is required for correct formation of the acentrosomal spindle and chromosome alignment in female meiosis I. Observation on mislocalization of the essential pole components, Msps and D-TACC, in the mutant provides a molecular insight into a role of Cks30A in spindle morphogenesis (Pearson, 2005).

Cks/Suc1 protein is the third subunit of the Cdc2-cyclin B complex that is conserved across eukaryotes. Although it has been known to be essential for the cell cycle, the function seems to be less straightforward than that of the other subunits of the Cdc2 complex. One reason is that Cks also interacts with other Cdks and has Cdk-independent functions. Even if Cks is limited to roles in mitosis/meiosis, Cks proteins are implicated in entry into mitosis/meiosis, metaphase-anaphase transition and also exit from mitosis/meiosis. Furthermore, the roles of Cks are further complicated by the fact that animal genomes encode two Cks homologues (Pearson, 2005).

Studies in Caenorhabditis elegans and mice have shown that one of two cks genes is required for female fertility. Similarly, the results indicate that one of two Drosophila cks homologues, cks30A, is expressed maternally and is required for female meiosis. Further analysis indicates that Cks30A is required for proper bipolar spindle formation and chromosome alignment in mature oocytes arrested in metaphase I. In C. elegans, depletion of one of the Cks proteins by RNAi results in a failure to complete meiosis I. Similarly, in mice, oocytes from a Cks2 knockout cannot progress past metaphase I and a small percentage of oocytes show chromosome congression failure. In both cases, the defects have been interpreted mainly as post-metaphase defects. Since Drosophila non-activated oocytes are arrested in metaphase I until ovulation, pre-anaphase function of Cks30A can be distinguised from possible post-metaphase function. This study clearly shows that Drosophila Cks30A has a function in establishing metaphase I, in addition to later functions (Pearson, 2005).

At the moment, it is not known how the cks30A mutation disrupts spindle formation and chromosome alignment in female meiosis. It has been thought that a loss of Cks function affects the Cdc2 activity towards certain substrates. The essential pole components, Msps and D-TACC, mislocalize to the spindle equator in the mutant. Previously, it was hypothesized that Msps is transported by the Ncd motor and anchored to the poles by D-TACC. D-TACC localizes to the poles independently from Ncd, but may also be transported from the spindle equator along microtubules by other motors. Cks30A-dependent Cdc2 activity may be required for activating the transport system at the onset of spindle formation in female meiosis. Consistently, it was found that cyclin B is concentrated around the equator of the metaphase I spindle. Msps is the XMAP215 homologue and belongs to a family of conserved microtubule-associated proteins. It is a major microtubule regulator, both in mitosis/meiosis and interphase. The mislocalization of this microtubule-regulating activity could lead to the disruption of spindle organization in the mutant (Pearson, 2005).

Wee1 interacts with members of the γTURC and is required for proper mitotic-spindle morphogenesis and positioning: Some of the abnormalities in dwee1 mutant embryos cannot be explained by premature entry into mitosis or bulk elevation of Cdk1 activity

Wee1 kinases delay entry into mitosis by phosphorylating and inactivating cyclin-dependent kinase 1 (Cdk1). Loss of this activity in many systems, including Drosophila, leads to premature mitotic entry. Drosophila Wee1 (dwee1) mutant embryos show mitotic-spindle defects that include ectopic foci of microtubule organization, formation of multipolar spindles from adjacent centrosome pairs, and promiscuous interactions between neighboring spindles. Furthermore, centrosomes are displaced from the embryo cortex in mutants. These defects are not observed to the same extent in embryos in which nuclei also enter mitosis prematurely as a result of a lack of checkpoint control or in embryos with elevated Cdk1 activity. dWee1 physically interacts with members of the γ-tubulin ring complex (γTuRC), and γ-tubulin is phosphorylated in a dwee1-dependent manner in embryo extracts. Some of the abnormalities in dwee1 mutant embryos cannot be explained by premature entry into mitosis or bulk elevation of Cdk1 activity. Instead, dWee1 is also required for phosphorylation of gamma-tubulin, centrosome positioning, and mitotic-spindle integrity. A model is proposed to account for these requirements (Stempff, 2005).

Cdk1 activity is elevated during cortical syncytial cycles in dwee1 mutant embryos, and elevation of Cdk1 activity has been shown previously to affect spindle morphogenesis in precortical syncytial cycles. Therefore, the possibility that elevated Cdk1 activity is the cause of dwee1-specific spindle defects was addressed. To this end, fixed embryos from a fly stock with six copies of cyclin B, six cycB, were analyzed. Increasing cyclin B levels in embryos is known to increase Cdk1 activity. Consistently, it was found that (six cycB) embryos harbor higher CycB-Cdk1 activity than do wild-type embryos. More Cdk1 coprecipitates with cyclin B from six cycB embryos than from wild-type or dwee1 mutant embryos, suggesting that this increase in activity is due to the presence of more-active complexes, an idea that is consistent with previous observations that Cdk1 levels are not limiting in embryos. six cycB embryos display defects such as asynchronous divisions, but dwee1-specific spindle defects were not detected. It is concluded that a bulk elevation of Cdk1 activity cannot account for dwee1-specific spindle defects (Stempff, 2005).

CycB levels are reproducibly lower in dwee1 embryos than in wild-type embryos. The reason for this finding is not known, but nonetheless the possibility that reduction of CycB levels is the cause of dwee1-specific phenotypes was ruled out. This was done by analyzing embryos from mothers that are hemizygous for cycB and therefore have lower CycB levels. No evidence was found of spindle interactions or centrosome-positioning changes that resemble those of dwee1 mutant embryos (Stempff, 2005).

The interactions between neighboring centrosomes and spindles in dwee1 mutants are limited to cortical syncytial divisions. These are a subset of syncytial divisions that follow the migration of nuclei to the embryo cortex. During cortical divisions, nuclei and centrosomes closely abut the cortex, and their position is maintained by microtubule-filament- and actin-filament-dependent mechanisms. Specifically, astral microtubules nucleated by the centrosomes are proposed to interact with cortical actin to mediate the attachment of centrosomes, along with their associated nuclei, to the cortex. Physical separation of mitotic spindles in a syncytium is thought to occur by reorganization of F-actin caps into pseudocleavage furrows that surround each dividing nucleus. Pseudocleavage furrows form during prophase and metaphase, retract in anaphase, and are mostly absent in late anaphase and telophase (Stempff, 2005).

Pseudocleavage furrows in dwee1 mutants form with normal timing and reach similar depths as in wild-type embryos. Nuclei in dwee1 mutants, however, are positioned beyond the deepest part of the furrows in metaphase. Quantification of centrosome-cortex distance in wild-type embryos and dwee1 mutants in cycle 12 illustrates this phenotype. This cycle was chosen because it occurs after completion of cortical nuclear migration but before the onset of the centrosome-inactivation checkpoint, as evidenced by anastral spindles. Displacement of centrosomes from the cortex is observed in both interphase and mitosis of cycle 12 in dwee1 mutants and is only partially rescued by the chk2 mutation. It is suggested that centrosome and nuclear displacement in dwee1 mutants has two underlying components: one is a consequence of the Chk2-mediated checkpoint, and the other is a more direct result of loss of dwee1. That is, dwee1 is required to promote centrosome-cortex interaction, which is thought to be dependent on centrosomal microtubules and cortical actin. The displacement of centrosomes from the cortex in dwee1 mutants could distance mitotic spindles from the protection of pseudocleavage furrows in prophase and metaphase, thereby allowing interactions between adjacent spindles (Stempff, 2005).

six cycB embryos show normal localization of nuclei and centrosomes, suggesting that bulk elevation of Cdk1 activity in dwee1 mutants cannot account for the cortical detachment of centrosomes. Additionally, spindle interactions in dwee1 mutants also initiate when pseudocleavage furrows are normally absent (anaphase and telophase). Therefore, a furrow-independent mechanism may operate to keep spindles apart in anaphase and telophase in a dwee1-dependent fashion (Stempff, 2005).

To further address the requirement for dWee1 in centrosome and spindle function, dWee1-containing protein complexes were purified and dWee1-interacting proteins were identified by mass spectrometry. The heat-inducible HA-dWee1 transgene used as a source of dWee1 has been shown to partially rescue dwee1 mutant embryos when expressed in the mothers, indicating that the product is functional. HA-dWee1 was induced in embryos, purified on an anti-HA antibody column, and eluted with a HA-dipeptide. Analysis of eluates by SDS-PAGE and mass spectrometry identified peptides that matched dWee1 and Drosophila γ-tubulin ring proteins (Dgrips) 163, 128, 91, 84, and 71. The identities of Dgrip84, Dgrip91, and HA-dWee1 were confirmed by Western blotting. Note that γ-tubulin was not detected in the HA-dWee1 eluates because a strong background band in the 50 kDa range (the MW of γ-tubulin), likely the IgG heavy chain, prevented analysis of that region of the gel (Stempff, 2005).

Because the above experiments were performed with overexpressed, tagged dWee1, it was necessary to ensure that endogenous dWee1 interacts with γTuRC. Dgrip91 and γ-tubulin was readily detected in immunoprecipitates by using an antibody against dWee1. It was not possible, however, to detect dWee1 in immunoprecipitates by using an antibody against γ-tubulin or Dgrip91. This may be because it is possible, at best, to precipitate approximately 5% of total protein present with each antibody. The presence of γ-tubulin, a structural component of the cytoskeleton, at a higher concentration than dWee1, a regulatory kinase, is a likely scenario, and it could explain why no dWee1 is seen in immunoprecipitates of γ-tubulin (Stempff, 2005).

To determine whether the kinase dWee1 influences the phosphorylation status of proteins it binds to, γ-tubulin (which is known to be phosphorylated in budding yeast) was examined. Two-dimensional (2D) gel electrophoresis followed by Western blotting revealed that Drosophila γ-tubulin separates as a series of five spots in the first dimension. Two of the more acidic isoforms are phosphatase sensitive, suggesting that Drosophila γ-tubulin is a phosphoprotein. The phosphatase-sensitive acidic forms are absent or severely diminished in extracts from dwee1 mutant embryos (Stempff, 2005).

Given that interphase is truncated in dwee1 mutants, the possibility was addressed that loss of γ-tubulin phosphorylation is a consequence of changes in cell cycle profile. However, extracts from grp mutant embryos that exhibit truncated interphases retain the phosphatase-sensitive γ-tubulin isoforms. It is concluded that interphase shortening does not lead to loss of γ-tubulin phosphorylation and that dwee1 is required for γ-tubulin phosphorylation in vivo. Despite testing a range of conditions, it has not been possible to phosphorylate γ-tubulin with recombinant dWee1 in vitro, although GST-dWee1 readily autophosphorylates and phosphorylates Cdk1 in these assays. Either dWee1 regulates γ-tubulin phosphorylation indirectly or γ-tubulin phosphorylation by dWee1 requires a cofactor (Stempff, 2005).

The known role of Wee1 homologs in cell cycle regulation is accomplished through a single substrate, Cdk1. Elevation of bulk Cdk1 activity in an otherwise wild-type background, however, does not produce dwee1-specific phenotypes. Therefore, if dWee1 influences spindle organization or positioning via Cdk1, it would have to regulate Cdk1 locally, at the embryo cortex for example. The attachment of centrosomes to the cortex is mediated by microtubules. In the absence of dWee1, Cdk1 activity would be higher locally, i.e., between the centrosome and the cortex, and could inhibit microtubule growth in this region, leading to the displacement of centrosomes. This idea is consistent with observations that increased Cdk1 activity destabilizes microtubules during nuclear migration in Drosophila embryos. In six cycB embryos, dWee1 could still inhibit Cdk1 locally to allow normal nuclear and spindle positioning. This possibility can be addressed with a Cdk1 mutant that cannot be phosphorylated by dwee1 and should mimic the loss of dwee1. Attempts were made to introduce into syncytial embryos (which are prezygotic transcription) such a mutant in which Y14 and T15 have been altered, Cdk1AF, by expressing it in females. Unfortunately, females fail to lay eggs after induction of Cdk1AF, suggesting disruption of oogenesis and precluding further analysis (Stempff, 2005).

An alternate approach to introduction of Cdk1AF is to induce dWee1-antagonizing phosphatases, Cdc25string and Cdc25twine, in embryos. Indeed, such experiments have been described before. Increasing the maternal Cdc25 gene dose by up to 4-fold in various combinations of the two Drosophila Cdc25 homologs produces increased mRNA and protein in embryos and leads to an extra syncytial nuclear division before cellularization. This division and preceding syncytial divisions, however, are normal in fixed and live embryos. No mitotic abnormalities, which are readily apparent throughout dwee1 mutant embryos, were seen in embryos with elevated Cdc25. This is consistent with the observation that bulk elevation of Cdk1 activity does not produce dwee1-specific phenotypes. Instead, localized regulation of Cdk1 by dWee1, which would still be present in embryos harboring extra Cdc25, could explain the apparently normal divisions in these embryos (Stempff, 2005).

Another explanation for spindle phenotypes in dwee1 mutants is suggested by the finding that dWee1 shows physical interaction with components of the γTuRC and that dwee1 influences the phosphorylation status of γ-tubulin in vivo. In this model, dWee1 promotes the phosphorylation of γ-tubulin, either directly or indirectly. In dwee1 mutants, loss of γ-tubulin phosphorylation could compromise microtubule-dependent attachments between centrosomes and the cortex. A test of this model will require identification and mutation of dwee1-dependent phosphoacceptor residues in γ-tubulin. Interestingly, the budding-yeast γ-tubulin homolog, Tub4p, is phosphorylated on a tyrosine residue during G1, but the responsible kinase has yet to be identified. A phosphomimetic mutation of Tub4p affects the number and organization of microtubules and causes transient nuclear-positioning abnormalities. Thus, it is possible that in both yeast and fly, the phosphorylation status of γ-tubulin plays a role in centrosome and nuclear positioning via interactions with the cortex (Stempff, 2005).

At present, it is not possible to distinguish between the two above explanations for dwee1 phenotypes; the explanations are not mutually exclusive. However, neither is predicted by previous models that describe how Wee1 homologs act to regulate entry into mitosis. Human Wee1, for example, resides in the nucleus during interphase and is proposed to prevent nuclear accumulation of Cdk1 activity and nuclear-envelope breakdown, an initiating event in mitosis. Such models can explain Wee1's role in regulating when mitosis occurs, but the current results indicate that Wee1 can also regulate where (relative to the cortex) mitosis occurs (Stempff, 2005).

The first 11 nuclear divisions proceed normally in dwee1 mutants. Therefore, dwee1-dependent regulation of spindle organization or positioning is not essential for mitosis per se. In cycle 12, nuclei are at the cortex and at twice the density (i.e., closer together) compared to those in cycle 11. It is reasoned that manifestation of dwee1-specific spindle interactions in later cortical cycles is a consequence of increasing nuclear density with each cycle that brings neighboring spindles closer together. In such a situation, protection offered by actin furrows may be essential to keep spindles apart. Detachment of centrosomes from the cortex would distance the spindles from furrows, allowing neighbors to interact (Stempff, 2005).

It is not known whether dwee1 also plays a role in spindle morphogenesis and centrosome positioning in cell cycles beyond cortical syncytial cycles. It is known, however, that dwee1 is needed to ensure fidelity of cell division in larvae; larval neuroblasts in dwee1 mutants show elevated mitotic index and ploidy. It would be interesting to determine the basis for this requirement and whether dwee1 has a role in the positioning of the spindle in cell divisions where a specific cortical attachment of the spindle is required, such as in the asymmetric cell divisions of neuroblast lineages (Stempff, 2005).

This study presents several lines of data that collectively suggest a requirement for dWee1 in centrosome function and spindle morphogenesis. Importantly, these roles translate into a requirement for dWee1 in not only temporal but also spatial regulation of mitosis. Two mechanistic models, which are not mutually exclusive, have been offered to account for these results: localized regulation of Cdk1 by dWee1 and phosphoregulation of γTuRC. Further analysis will be needed to test these models, but it is clear that the requirement for dWee1 cannot be explained by simple regulation of bulk Cdk1 activity. In this regard, Wee1 homologs may be likened to other kinases, such as Plk and Aurora B, that have multiple roles in mitosis through multiple substrates. Localized activity of master regulatory kinases such as these is likely to coordinate many distinct cell-division events (such as spindle movements, chromosome segregation, and cytokinesis) to allow faithful segregation of genetic information into daughter cells (Stempff, 2005).

Myt1 is a Cdk1 inhibitory kinase that regulates multiple aspects of cell cycle behavior during gametogenesis

Two myt1 mutant alleles (originally designated as myt11 and myt12) were isolated in a genetic screen for hemizygous mutants with phenotypic defects that could be rescued by a P{myt1+} transgene. These alleles exhibit markedly different viability as hemizygotes; however, these differences were removed by out-crossing, indicating they were due to secondary lesions. Viable hemizygous myt1 mutants [myt/Df(3L)64D-F] exhibit bristle defects affecting the dorsal thorax, head and eye, and are male sterile. Although myt1 females are fertile, variable maternal effect lethal embryonic phenotypes were observed in their progeny. Genomic sequencing of the myt1 alleles identified identical mutations in each: a single nucleotide deletion at position 514 (amino acid 173). The fact that EMS mutagenesis usually causes CG-->TA transitions, combined with the unlikelihood that this mutation would have occurred twice independently, suggests that a spontaneous mutation occurred in the previously isogenized stock used for the screen. The myt1 mutation is predicted to cause a frame-shift alteration in the sequence of the protein, followed by a premature stop codon at nucleotide 689 (amino acid 232). This would truncate the protein within the kinase domain and also delete other conserved sequence motifs near the C terminus of the protein, suggesting that the mutants are likely functionally null. Moreover, myt1/Df(3L)64D-F hemizygotes display identical phenotypes as transheterozygous combinations of the original alleles, fulfilling classical genetic criteria that these myt1 alleles are functionally amorphic (Jin, 2005).

The male sterility of myt1 mutants led to a search for specific cell cycle defects during spermatogenesis, Male germline development begins with stem cell divisions that generate gonialblasts, which then undergo four synchronous mitotic divisions to produce cysts of 16 primary spermatocytes. These primary spermatocytes remain in G2 phase for ~90 hours before undergoing meiotic divisions to produce cysts containing 64 syncytial spermatids that differentiate into mature sperm. To analyze how loss of Myt1 function affects these cell divisions, an antibody was used that recognizes a phosphorylated form of histone H3 (PH3) as a marker for mitotic or meiotic cells. In control testes, small numbers of mitotic cells were usually seen near the tip of the testis. More distally along a control testis, one often observes a single PH3-positive meiotic cyst. A striking increase was observed in the numbers of PH3-positive cells in myt1 mutants. In addition to clearly demarcated germline cysts, isolated PH3-positive cells were observed along the length of myt1 mutant testes, as well as PH3-positive cells at the distal end of the testes that were never seen in controls. These cell proliferation defects were suppressed and male fertility was restored when a P{myt1+} transgene was introduced into the myt1 mutant background, confirming that these mutant phenotypes were due to a loss of Myt1 activity. The adult bristle phenotype observed in myt1 mutants was also rescued by this transgene (Jin, 2005).

To determine if there are other proliferation defects observable in the myt1 mutants, BrdU incorporation to assay for DNA replication was used in short-term (30 minute) cultures of dissected testes. BrdU incorporation was seen only in the cells near the tip of the testes in the controls, implying that pre-meiotic S phase is essentially complete by the time the primary spermatocyte cysts move away from the tip. A marked increase in BrdU-incorporating cells was observed near the apical tip of the testes in the myt1 mutants, relative to controls. These observations could be explained if cells in the myt1 mutants cycle faster than normal, or if they continue to cycle instead of undergoing developmental cell cycle arrest. Delays during S phase and mitosis could also contribute to these effects (Jin, 2005).

Male-sterile mutants with overproliferation defects can result from germline stem cells (GSC) or spermatogonia failing to differentiate properly, so that they continue to proliferate instead of entering meiosis. To determine if overproliferation in myt1 mutants is attributable to similar defects, established cell fate markers were used to examine germline stem cells, spermatogonia and spermatocytes. myt1 mutants have normal numbers of germline stem cells, assessed by immunostaining for the germline-specific marker Vasa and Fas3, which marks the somatic hub that GSCs associate with. There was, however, a significant increase in secondary spermatogonial cells marked by antibodies against BamC in the myt1 mutants, relative to controls. This could occur if the secondary spermatogonia undergo one or more extra rounds of cell division, in which case PH3-positive spermatogonial cysts with 16 or more cells would be expected (Jin, 2005).

To test this possibility, a BamC and PH3 colocalization experiment was performed. In controls, cysts were never observed containing more than eight cells that were both BamC and PH3 positive, consistent with spermatogonia only undergoing four mitotic divisions. By contrast, ~30% of the myt1 mutant testes examined contained at least one 16-cell cyst that was both BamC and PH3-positive, implying that these spermatogonia were undergoing an extra round of cell division. To further test this idea, germ cell cysts were examined by phase contrast microscopy, and the numbers of cells in each cyst were quantified. As expected, ~10% of the mutant cysts contain twice the expected numbers of primary spermatocytes or spermatids, a phenotype that is never seen in controls. In both 64-cell and 128-cell myt1 mutant spermatid cysts, there was consistent evidence of variable, aberrant-looking nuclei and nebenkern. These observations suggest that loss of Myt1 activity affects segregation of chromosomes and mitochondria during meiosis, in addition to the mitotic defects described earlier (Jin, 2005).

Next, whether additional defects in the cell cycle behavior of somatic cells might contribute to the myt1 over-proliferation phenotype was examined. Somatic stem cells located at the apical tip of the testes divide to generate cyst cells whose fate is intimately coupled with male germline development. Two cyst cells associate with each gonialblast and remain associated with the descendant cyst for the remainder of spermatogenesis. Normally, these somatic cyst cells do not undergo further cell division, suggesting that their differentiation is coupled with exit from the cell cycle. Antibodies against Eya were used to mark the cyst cells. There was a marked increase in the number of cyst cell nuclei in the myt1 mutants, relative to controls. Because the cyst cells are quiescent, they are PH3-negative in the controls. In myt1 mutants, however, cyst cell nuclei can be double-labeled with antibodies to PH3 and Eya. When Aly was used as a marker for spermatocytes, Aly-positive cysts with more than two Eya-positive cyst nuclei were never observed in controls, but were often seen in the mutants, implying that these extra nuclei remain associated with their germline cysts during meiosis. In addition to the ectopic division of cyst cells, it was also observed that terminal epithelial cells located at the distal end of the testes ectopically label with PH3 antibodies, unlike controls. These observations further distinguish myt1 mutants from previously described male-sterile over-proliferation mutants and implicate Myt1 in a molecular mechanism that promotes cell cycle exit during terminal differentiation. These defects may affect the ability of cyst and terminal cells to provide essential cell signaling or other support functions to their associated germline cells. Such effects could conceivably compromise sperm maturation or translocation and contribute to the observed sterility of male myt1 mutants. Mature sperm translocate into the seminal vesicle after spermatid differentiation and can be visualized by DNA staining. In myt1 mutants, the seminal vesicle appeared to be empty (Jin, 2005).

Previous studies in Drosophila have demonstrated that regulation of entry into mitosis and meiosis is controlled by inhibitory phosphorylation of Cdk1. Given that Myt1 is a Cdk1 inhibitory kinase, it was expected that phenotypic defects of myt1 mutants would be due to a defect in Cdk1 regulation. To test this idea, a heat shock-inducible, non-inhibitable allele of Cdk1 (hs-Cdk1AF) was expressed in testes, to see if it would phenocopy any of the defects observed in myt1 mutants. Expression of this transgene has previously been used to bypass a developmentally regulated G2 arrest in embryonic germline cells. As predicted, heat-shock induced expression of Cdk1AF caused germline over-proliferation defects similar to those seen in myt1 mutants. These included increased numbers of PH3-positive cells, relative to controls. It was confirmed, by BamC antibody co-localization, that some of these PH3-positive cells in the Cdk1AF-expressing testes were secondary spermatogonia undergoing an extra round of mitosis. Induction of hs-Cdk1AF also phenocopied the defects seen in germline-associated somatic cyst cells labeled with Eya. These results demonstrate that Myt1 inhibitory phosphorylation of Cdk1 is required for regulating multiple aspects of cell cycle behavior during spermatogenesis (Jin, 2005).

Female myt1 mutants are fertile; however, a high incidence of early lethality in maternally affected mutant embryos suggests that Myt1 might also function during oogenesis. Oogenesis initiates with stem cell divisions that produce cystoblasts that then undergo four synchronous mitotic divisions, to generate 16 cell cysts. A single cell in each cyst differentiates into an oocyte and progresses into prophase of meiosis I, where it remains arrested until ovulation. The 15 remaining cells in the cyst differentiate as nurse cells. Two or three germline stem cells (GSCs) are located at the tip of the germarium, each containing a ball-shaped fusome-related structure called a spectrosome. Fusomes are germline-specific membranous organelles that interconnect the cyst cells and are thought to coordinate their mitotic cell divisions. Using the position of GSCs and antibodies against Hts to label spectrosomes, it was determined that the number of GSCs is comparable in myt1 mutants and controls. The numbers of dividing GSCs, cystoblasts and cystocytes were counted, and a mitotic index was calculated for each cell type, using antibodies against Cnn and Hts to mark spindle poles and spectrosomes (or fusomes, in cystocytes), respectively, as well as the DNA-labeling dye Hoechst 33258 to mark condensed mitotic chromosomes. Female GSCs typically undergo one cell cycle per day, consequently mitotic GSCs are rarely observed and the mitotic index is very low. Only two mitotic GSCs were found in 130 germaria from control ovarioles. By contrast, 15 mitotic GSCs were found among 50 myt1 mutant germaria, a 20-fold increase in the mitotic index. The mutant cystoblasts and their cystocyte descendants also had a significantly higher mitotic index than normal, so it was not uncommon to find a metaphase stem cell and a metaphase cystoblast or cystocyte in a single myt1 mutant germarium. In the controls, at most a single dividing cyst was seen in each germarium. These results show that loss of myt1 activity causes germline overproliferation in females, as well as males. No egg chambers were observed with greater than 16 cells in myt1 mutants, however, indicating that ectopic germline cell divisions do not account for this defect, in females (Jin, 2005).

Oocyte and nurse cell differentiation appeared normal in the myt1 mutants, as assessed by antibody staining of Orb and Gurken in oocytes, and by nuclear morphology of the nurse cells. When myt1 mutant germaria were examined with antibodies against the Vasa germline marker, a significant increase was noted in the number of cysts (one- to two-fold), relative to controls. Although myt1 mutant cystoblasts and cystocytes are similar in size to controls, the GSCs appear slightly smaller. These data suggest that myt1 mutant GSCs are cycling more rapidly and therefore produce more germline cysts. As the differences are not as extreme as the GSC mitotic index measurements would predict, these results also imply that compensatory delays probably occur during these mitotic cell cycles that can account for this discrepancy (Jin, 2005).

To investigate whether there are effects on homologous chromosome segregation during female meiosis in myt1 mutant females, standard genetic tests were undertaken to identify non-disjunction (NDJ) events. These tests show that loss of Myt1 causes elevated non-disjunction, implicating Myt1 as a regulator of female meiosis. The NDJ frequency for myt1 mutants is much higher than controls for the X chromosome and the 4th chromosome. Further experiments indicated that exceptional progeny derive from NDJ during meiosis I in myt1 females. These data demonstrate that loss of Myt1 activity compromises female meiosis, specifically meiosis I (Jin, 2005).

Female germline cells are associated with somatic follicle cells derived from stem cell precursors. When each 16-cell germline cyst buds off from the germarium as an egg chamber, a layer of undifferentiated follicle cells surrounds it. These follicle cells then differentiate into functionally distinct subclasses. The stalk cells (located between each egg chamber) and the polar cells (located at each end of the chamber), cease dividing immediately after the egg chamber forms, whereas the remaining follicle cells proliferate asynchronously until stage 6 of oogenesis. Accordingly, early egg chambers have only small numbers of PH3-positive follicle cells. In myt1 mutant egg chambers, there was a marked increase in PH3-positive follicle cells before stage 6, as well as ectopic PH3-positive follicle cells after stage 6. Curiously, these ectopic PH3-positive follicle cells primarily appeared at the anterior and posterior ends of each egg chamber (Jin, 2005).

Each of the four major types of follicle cells can be distinguished by their cell shape and by expression of distinct molecular markers. Stalk cells have a unique disc-like shape and inter-egg chamber location; polar cells are located at the end of each egg chamber and express Fas3 before stage 9; border cells maintain Fas3 expression and migrate towards the posterior after stage 9, and stretched cells extend over the 15 nurse cells and express Eya. By these criteria, the different follicle cell types all appeared to be represented in myt1 mutants; however, unlike the controls, some of these cells were PH3 positive, suggesting that they were undergoing ectopic cell divisions. Consistent with this interpretation, there were more Eya-expressing cells in the mutants than in controls by stage 9, indicating that some of these cells are able to complete cell division. The typical 'stretched' morphology characteristic of this cell type was disrupted, presumably as a result of cytoskeleton reorganization accompanying mitosis. Also ectopic PH3-positive main body follicle cells were observed in mutant egg chambers after stage 9, long after these cells normally cease dividing. Thus, loss of Myt1 function causes germline-associated somatic cells to undergo ectopic cell division, in both males and females (Jin, 2005).

Mitotic activation of the kinase Aurora-A requires its binding partner Bora, which is activated by Cdc2

The protein kinase Aurora-A is required for centrosome maturation, spindle assembly, and asymmetric protein localization during mitosis. Borealis (Bora, so named for aurora borealis to indicate its similarity with aurora-A) is a conserved protein that is required for the activation of Aurora-A at the onset of mitosis. In the Drosophila peripheral nervous system, bora mutants show defects during asymmetric cell division identical to those observed in aurora-A. Furthermore, overexpression of bora can rescue defects caused by mutations in aurora-A. Bora is conserved in vertebrates, and both Drosophila and human Bora can bind to Aurora-A and activate the kinase in vitro. In interphase cells, Bora is a nuclear protein, but upon entry into mitosis, Bora is excluded from the nucleus and translocates into the cytoplasm in a Cdc2-dependent manner. A model is presented here in which activation of Cdc2 initiates the release of Bora into the cytoplasm where it can bind and activate Aurora-A (Hutterer, 2006).

To test whether the genetic interaction reflects a physical interaction between Bora and Aurora-A, binding assays were performed in Drosophila tissue culture cells. Drosophila S2 cells were transfected with Aurora-A and Bora-GFP, and protein lysates were subjected to immunoprecipitation by anti-GFP. Since Aurora-A is specifically detected in the immunoprecipitate, it is concluded that Bora can bind to Aurora-A in vivo. To test whether this is due to a direct interaction, in vitro binding experiments were performed. In vitro translated Aurora-A binds to a GST-Bora fusion-protein but not to GST alone. While the nonconserved C terminus of Bora is dispensible for Aurora-A binding, the interaction is abrogated by deleting the conserved region (BoraΔ2) or a region N-terminal to the conserved part (BoraΔ1). Interestingly, the interaction is also observed between in vitro translated human Aurora-A and MBP-HsBora. Human Aurora-A can even bind to Drosophila MBP-Bora in vitro. The interaction with Aurora-A seems to be essential for Bora function since the N-terminal 404 amino acids of Bora (almost identical to BoraΔ3) can rescue the bora and aurA37 mutant phenotypes, while the C terminus (amino acids 404–539) does not. Thus, Bora and its homologs act as binding partners of Aurora-A (Hutterer, 2006).

Several Aurora-A regulators—like TPX2 also act as substrates for the kinase. To test whether Bora can be phosphorylated by Aurora-A, in vitro kinase assays were performed. Drosophila Aurora-A expressed and purified from E. coli can phosphorylate bacterially expressed myelin basic protein tagged Bora (MBP-Bora) but not MBP alone. Interestingly, the kinase activity of Aurora-A toward Bora is as potent as toward myelin basic protein, which is often used as a model substrate. Similarly, human Aurora-A can phosphorylate the human Bora homolog. To test which region of Bora is phosphorylated, Bora deletions were used in the kinase assay. Deletion of 125 amino acids from the N terminus of Bora (BoraΔ2) eliminates phosphorylation by Aurora-A, while deletion of the C terminus from amino acid 209 onward (BoraΔ5) does not affect it. Interestingly, Bora is still phosphorylated when the N-terminal 67 amino acids are deleted (BoraΔ1), suggesting that direct binding to Aurora-A is not necessary for Bora to act as a substrate. These experiments suggest that the N terminus of Bora is phosphorylated by Aurora-A (Hutterer, 2006).

To test whether Bora can influence the kinase activity of Aurora-A, recombinant human Bora was used in an in vitro kinase assay with myelin basic protein as a substrate. Addition of Bora increases Aurora-A activity in a dose-dependent manner, and a 2.5-fold maximum increase in kinase activity was observed. Aurora-A is regulated by phosphorylation in the activation loop of the kinase. Since Aurora-A can autophosphorylate, any kinase preparation may be partially active, and this might explain the modest degree of activation by recombinant Bora. Consistent with this, when Aurora-A is inactivated by pretreatment with protein phosphatase 1 (PP1), addition of Bora induces an over 7-fold increase in kinase activity. Analogous experiments with the Drosophila homologs reveal that Drosophila Bora similarly activates the Drosophila kinase, showing that it acts as a kinase activator as well. Taken together, these results demonstrate that Bora is an activator of Aurora-A (Hutterer, 2006).

Mutation of the autophosphorylation site of Aurora-A to alanine renders the kinase inactive, and an interesting question is whether the stimulation of Aurora-A by Bora bypasses the need for autophosphorylation. It was found that addition of Bora does not restore activity to the mutant kinase, suggesting that activation by Bora requires autophosphorylation of Aurora-A (Hutterer, 2006).

To determine the subcellular localization of Bora in SOP cells, live imaging was performed of a Bora-GFP fusion protein, which can rescue both bora and aurA37 mutant phenotypes. Histone-RFP is used to label chromosomes and indicates the cell-cycle stage. Constructs were specifically expressed by neuralized-Gal4 in SOP cells and dividing cells were imaged in whole living pupae. In interphase, Bora is a nuclear protein. When chromosomes condense, however, Bora is released from the nucleus. It is completely excluded from the nucleus by late prophase and is uniformly distributed in the cytoplasm after nuclear envelope breakdown. In telophase, Bora enters both daughter cells where it relocates into the nucleus. Bora does not have an obvious nuclear localization signal. However, it was found that the first 125 amino acids of the protein are sufficient for nuclear retention, suggesting that they contain the sequence that mediates nuclear import. Live imaging of GFP-Aurora-A together with Histone-RFP allows correlation of the localization of Aurora-A with Bora. In interphase, the two proteins are in distinct compartments. Nuclear release of Bora coincides with centrosome separation and strong recruitment of Aurora-A to the maturing centrosomes. Since both centrosome separation and maturation defects are observed in aurora-A mutants, these results suggest that release of Bora coincides with Aurora-A activation (Hutterer, 2006).

While Aurora-A is required for a subset of mitotic events, Cdc2 is essential for all steps of mitosis. How Cdc2 activates Aurora-A is unclear. To test whether Cdc2 regulates the release of Bora into the cytoplasm, Bora localization was examined in string mutants. String is the Drosophila homolog of the Cdc25 phosphatase, and in string mutants, Cdc2 is not activated. Antibody staining of Drosophila embryos reveals that endogenous Bora shows the same dynamic localization during the cell cycle as the functional GFP fusion protein. In string mutant embryos, however, Bora was never observed in the cytoplasm, indicating that Cdc2 activation is required for the release of Bora from the nucleus. To test whether Cdc2 might directly phosphorylate Bora, in vitro kinase assays were performed. Both Bora and HsBora are phosphorylated by recombinant Cdk1. Although the in vivo relevance of Cdk1 phosphorylation remains to be tested, these experiments show that Bora is released into the cytoplasm at the onset of mitosis in a Cdc2-dependent manner (Hutterer, 2006).

Drosophila myt1 is the major cdk1 inhibitory kinase for wing imaginal disc development

Mitosis is triggered by activation of Cdk1, a cyclin-dependent kinase. Conserved checkpoint mechanisms normally inhibit Cdk1 by inhibitory phosphorylation during interphase, ensuring that DNA replication and repair is completed before cells begin mitosis. In metazoans, this regulatory mechanism is also used to coordinate cell division with critical developmental processes, such as cell invagination. Two types of Cdk1 inhibitory kinases have been found in metazoans. They differ in subcellular localization and Cdk1 target-site specificity: one (Wee1) being nuclear and the other (Myt1), membrane-associated and cytoplasmic. Drosophila has one representative of each: dMyt1 and dWee1. Although dWee1 and dMyt1 are not essential for zygotic viability, loss of both resulted in synthetic lethality, indicating that they are partially functionally redundant. Bristle defects in myt1 mutant adult flies prompted a phenotypic analysis that revealed cell-cycle defects, ectopic apoptosis, and abnormal responses to ionizing radiation in the myt1 mutant imaginal wing discs that give rise to these mechanosensory organs. Cdk1 inhibitory phosphorylation was also aberrant in these myt1 mutant imaginal wing discs, indicating that dMyt1 serves Cdk1 regulatory functions that are important both for normal cell-cycle progression and for coordinating mitosis with critical developmental processes (Jin, 2008).

Multicellular organisms regulate Cdk1 by inhibitory phosphorylation to prevent mitosis when DNA is being replicated or repaired and to ensure that mitosis does not interfere with critical developmental processes that require remodeling of the cytoskeleton. Previous studies of Drosophila Wee1 and Myt1 revealed that these conserved Cdk1 inhibitory kinases were required during early embryogenesis and gametogenesis, respectively. This study has characterized imaginal and adult developmental defects caused by loss of dMyt1 activity (and to a much lesser extent, dWee1), that confirm the importance of Cdk1 inhibitory phosphorylation for coordinating cell-cycle events with critical developmental processes (Jin, 2008).

In Drosophila and other organisms, G2/M delays can be induced by overexpression of Myt1 kinases, suggesting a specific role for Myt1 in regulating this stage of the cell cycle. Further evidence of a role for Myt1 in G2/M regulation comes from studies of oocyte maturation in frogs, starfish, and nematodes. Not all data indicate that Myt1 is required for G2 phase arrest, however, and there is no evidence that dMyt1 regulates oocyte maturation in Drosophila. Nor is there evidence that dMyt1 activity is responsible for the timing of the G2/M meiotic transition that follows a prolonged 4-day-long G2 phase arrest, in Drosophila primary spermatocytes. Moreover, a recent study showed that functional depletion of human Myt1 by siRNA did not affect the proportion of cells in G2 phase, but instead affected membrane dynamics during mitotic exit (Nakajima, 2008). More needs to be learned about Myt1 mediated regulatory mechanisms before these apparent discrepancies in Myt1 functions are resolved (Jin, 2008).

Previous work showed that Cdk1 inhibitory phosphorylation is required for proper development of thoracic mechanosensory organs. This study has now identified dMyt1 as the primary Cdk1 inhibitory kinase for this developmental program. Several molecular mechanisms could explain the role of dMyt1 in mechanosensory bristle development. One obvious possibility is that myt1 mutant sensory organ precursor (SOP) cells and their descendants might divide prematurely due to a defect in G2/M regulation, resulting in aberrant segregation of cell fate determinants. If there was a relatively narrow window for coordinating specific developmental events with the G2/M transition, disrupting this regulatory mechanism could account for the observed loss and duplication of bristles and socket cells in myt1 mutants. Live analysis of mechanosensory organ development could test this possibility (Jin, 2008).

Alternatively, myt1 mutant phenotypes could reflect defects in Myt1-mediated regulatory mechanisms that are important for the control of intracellular membrane dynamics during mitosis, particularly the Golgi apparatus and endoplasmic reticulum. The Drosophila Golgi apparatus undergoes significant morphological changes that have been linked to specific developmental states and so the observed myt1 mutant developmental defects might reflect problems in the structure or function of this organelle. Further support for this idea comes from a recent study showing that asymmetrical segregation of mouse Numb (a conserved cell fate determinant) requires the Golgi apparatus, leading to the suggestion that Golgi fragmentation and reconstitution could represent a mechanism for coupling cell-fate specification and cell-cycle progression (Jin, 2008).

Another possible explanation for myt1 mutant defects concerns the large quantities of actin that are synthesized and packaged to form the large mechanosensory bristle shafts. This process involves extensive reorganization of the endoplasmic reticulum and Golgi apparatus to accommodate increased membrane trafficking. Defects in the structure or function of the Golgi apparatus and ER caused by loss of dMyt1 activity could therefore account for defects or diminution in these bristles. Resolving which of these potential mechanisms best explain the role of dMyt1 during mechanosensory organ development will be a major challenge of future research (Jin, 2008).

Intriguing cell-cycle defects (higher mitotic index, aberrant chromatin condensation, and ectopic apoptosis), as well as defects in responses to ionizing radiation in proliferating cells, were observed in myt1 mutant imaginal wing discs. These observations suggest an important role for dMyt1 in conserved cell-cycle checkpoint responses that target Cdk1 by inhibitory phosphorylation. It was not anticipated that dMyt1 would serve such functions, since Wee1 kinases are generally assumed to be responsible for checkpoint responses that protect the nucleus from premature Cdk1 activity. It was not clear that myt1 mutants were deficient in conventional premitotic checkpoint responses, however. Indeed, the partial decline in myt1 mutant PH3-labeled cells observed immediately after exposure to ionizing radiation could reflect activation of an otherwise dispensable Wee1-regulated premitotic checkpoint mechanism. The remaining PH3-positive cells that persisted long after irradiation in myt1 mutant discs could be arrested in mitosis by an alternative regulatory mechanism that was responsive to DNA damage. Further studies will be needed to clarify the respective roles of dMyt1 and dWee1 in cellular responses to DNA damage (Jin, 2008).

This study also observed profound defects in Cdk1 inhibitory phosphorylation in myt1 mutant imaginal discs. Phosphorylation of the T14 residue of Cdk1 was eliminated, demonstrating that dMyt1 is solely responsible for this regulatory modification, like Myt1 homologs described in other organisms. It was also observed that phosphorylation of the Y15 residue of Cdk1 was markedly reduced in myt1 mutant extracts, demonstrating for the first time that dMyt1 functions as a dual specificity Cdk1 inhibitory kinase, in vivo. Why dWee1 activity is insufficient for maintaining normal levels of phosphorylation of the Y15 residue is not clear, since Cdk1 complexes are thought to shuttle between the nucleus and cytoplasm. One possible explanation is that the doubly phosphorylated Cdk1 isoform may be more refractory to dephosphorylation by Cdc25 phosphatases, and hence more stably inhibited, than Cdk1 phosphorylated on a single residue. Another possibility is that the kinase-independent Myt1 mechanism proposed to tether phospho-inhibited Cdk1 complexes in the cytoplasm until cells are ready for mitosis might also protect them from dephosphorylation. Loss of either of these regulatory mechanisms could therefore underlie the cell-cycle defects observed in myt1 mutants. Testing these hypotheses promises to yield interesting new insights into cell-cycle regulation and the diverse developmental roles of dMyt1 and similar regulatory kinases in other organisms (Jin, 2008).

Dual phosphorylation of Cdk1 coordinates cell proliferation with key developmental processes in Drosophila

Eukaryotic organisms use conserved checkpoint mechanisms that regulate Cdk1 by inhibitory phosphorylation to prevent mitosis from interfering with DNA replication or repair. In metazoans, this checkpoint mechanism is also used for coordinating mitosis with dynamic developmental processes. Inhibitory phosphorylation of Cdk1 is catalyzed by Wee1 kinases that phosphorylate tyrosine 15 (Y15) and dual-specificity Myt1 kinases found only in metazoans that phosphorylate Y15 and the adjacent threonine (T14) residue. Despite partially redundant roles in Cdk1 inhibitory phosphorylation, Wee1 and Myt1 serve specialized developmental functions that are not well understood. Wild type and phospho-acceptor mutant Cdk1 proteins were expresses in order to investigate how biochemical differences in Cdk1 inhibitory phosphorylation influence Drosophila imaginal development. Phosphorylation of Cdk1 on Y15 appeared to be crucial for developmental and DNA damage-induced G2 phase checkpoint arrest, consistent with other evidence that Myt1 is the major Y15-directed Cdk1 inhibitory kinase at this stage of development. Expression of non-inhibitable Cdk1 also caused chromosome defects in larval neuroblasts that were not observed with Cdk1(Y15F) mutant proteins that were phosphorylated on T14, implicating Myt1 in a novel mechanism promoting genome stability. Collectively, these results suggest that dual inhibitory phosphorylation of Cdk1 by Myt1 serves at least two functions during development. Phosphorylation of Y15 is essential for the pre-mitotic checkpoint mechanism, whereas T14 phosphorylation facilitates accumulation of dually inhibited Cdk1-Cyclin B complexes that can be rapidly activated once checkpoint-arrested G2 phase cells are ready for mitosis (Ayeni, 2013).

Integrated stability and activity control of the Drosophila Rbf1 retinoblastoma protein

Retinoblastoma (RB) family transcriptional corepressors regulate diverse cellular events including cell cycle, senescence, and differentiation. The activity and stability of these proteins are mediated by post-translational modifications, however there is no general understanding of how distinct modifications coordinately impact both of these properties. Previous work has shown that protein turnover and activity are tightly linked through an evolutionarily conserved C-terminal instability element (IE) in the Drosophila RB-related protein Rbf1; surprisingly, mutant proteins with enhanced stability were less, not more active. To better understand how activity and turnover are controlled in this model RB protein, the impact of Cyclin-Cdk kinase regulation on Rbf1 was assessed. An evolutionarily conserved N-terminal threonine residue is required for Cyclin-Cdk response, and showed a dominant impact on turnover and activity, however specific residues in the C terminal IE differentially impacted Rbf1 activity and turnover, indicating an additional level of regulation. Strikingly, specific IE mutations that impaired turnover but not activity induced dramatic developmental phenotypes in the Drosophila eye. Mutation of the highly conserved K774 residue induced hypermorphic phenotypes that mimicked the loss of phosphorylation control; mutation of the corresponding codon of the human RBL2 gene has been reported in lung tumors. These data supports a model in which closely intermingled residues within the conserved IE govern protein turnover, presumably through interactions with E3 ligases, and protein activity, via contacts with E2F transcription partners. Such functional relationships are likely to similarly impact mammalian RB family proteins, with important implications for development and disease (Zhang, 2014).

TARANIS functions with Cyclin A and Cdk1 in a novel arousal center to control sleep in Drosophila

Sleep is an essential and conserved behavior whose regulation at the molecular and anatomical level remains to be elucidated. This study identifies Taranis (Tara), a Drosophila homolog of the Trip-Br (SERTAD) family of transcriptional coregulators, as a molecule that is required for normal sleep patterns. Through a forward-genetic screen, tara was isolated as a novel sleep gene associated with a marked reduction in sleep amount. Targeted knockdown of tara suggests that it functions in cholinergic neurons to promote sleep. tara encodes a conserved cell-cycle protein that contains a Cyclin A (CycA)-binding homology domain. Tara regulates CycA protein levels and genetically and physically interacts with CycA to promote sleep. Furthermore, decreased levels of Cyclin-dependent kinase 1 (Cdk1), a kinase partner of CycA, rescue the short-sleeping phenotype of tara and CycA mutants, while increased Cdk1 activity mimics the tara and CycA phenotypes, suggesting that Cdk1 mediates the role of Tare and CycA in sleep regulation. Finally, a novel wake-promoting role was described for a cluster of ∼14 CycA-expressing neurons in the pars lateralis (PL), previously proposed to be analogous to the mammalian hypothalamus. The study proposes that Taranis controls sleep amount by regulating CycA protein levels and inhibiting Cdk1 activity in a novel arousal center (Afonso, 2015).

Most animals sleep, and evidence for the essential nature of this behavior is accumulating. However, how sleep is controlled at a molecular and neural level is far from understood. The fruit fly, Drosophila, has emerged as a powerful model system for understanding complex behaviors such as sleep. Mutations in several Drosophila genes have been identified that cause significant alterations in sleep. Some of these genes were selected as candidates because they were implicated in mammalian sleep. However, others (such as Shaker and CREB) whose role in sleep was first discovered in Drosophila have later been shown to be involved in mammalian sleep, validating the use of Drosophila as a model system for sleep research. Since the strength of the Drosophila model system is the relative efficiency of large-scale screens, unbiased forward-genetic screens have been conducted to identify novel genes involved in sleep regulation. Previous genetic screens for short-sleeping fly mutants have identified genes that affect neuronal excitability, protein degradation, and cell-cycle progression. However, major gaps remain in understanding of the molecular and anatomical basis of sleep regulation by these and other genes (Afonso, 2015).

Identifying the underlying neural circuits would facilitate the investigation of sleep regulation. The relative simplicity of the Drosophila brain provides an opportunity to dissect these sleep circuits at a level of resolution that would be difficult to achieve in the more complex mammalian brain. Several brain regions, including the mushroom bodies, pars intercerebralis, dorsal fan-shaped body, clock neurons, and subsets of octopaminergic and dopaminergic neurons, have been shown to regulate sleep. However, the recent discovery that Cyclin A (CycA) has a sleep-promoting role and is expressed in a small number of neurons distinct from brain regions suggests the existence of additional neural clusters involved in sleep regulation (Afonso, 2015).

From an unbiased forward-genetic screen, this study discovered taranis (tara), a mutant that exhibits markedly reduced sleep amount. tara encodes a Drosophila homolog of the Trip-Br (SERTAD) family of mammalian transcriptional coregulators that are known primarily for their role in cell-cycle progression. TARA and Trip-Br proteins contain a conserved domain found in several CycA-binding proteins. This research shows that tara regulates CycA levels and genetically interacts with CycA and its kinase partner Cyclin-dependent kinase 1 (Cdk1) to regulate sleep. Furthermore, a cluster of CycA-expressing neurons in the dorsal brain was shown to lie in the pars lateralis (PL), a neurosecretory cluster previously proposed to be analogous to the mammalian hypothalamus, a major sleep center. Knockdown of tara and increased Cdk1 activity in CycA-expressing PL neurons, as well as activation of these cells, reduces sleep. Collectively, these data suggest that TARA promotes sleep through its interaction with CycA and Cdk1 in a novel arousal center (Afonso, 2015).

From an unbiased forward genetic screen, this study has identified a novel sleep regulatory gene, tara. The data demonstrate that TARA interacts with CycA to regulate its levels and promote sleep. Cdk1 was also identified as a wake-promoting molecule that interacts antagonistically with TARA. Given the fact that TARA regulates CycA levels, the interaction between TARA and Cdk1 may be mediated by CycA. The finding that Cdk1 and CycA also exhibit an antagonistic interaction supports this view. The previous discovery that CycE sequesters its binding partner Cdk5 to repress its kinase activity in the adult mouse brain points to a potential mechanism, namely that TARA regulates CycA levels, which in turn sequesters and inhibits Cdk1 activity. TARA and its mammalian homologs (the Trip-Br family of proteins) are known for their role in cell-cycle progression. However, recent data have shown that Trip-Br2 is involved in lipid and oxidative metabolism in adult mice, demonstrating a role beyond cell-cycle control. Other cell-cycle proteins have also been implicated in processes unrelated to the cell cycle. For example, CycE functions in the adult mouse brain to regulate learning and memory. Based on the finding that CycA and its regulator Rca1 control sleep, it was hypothesized that a network of cell-cycle genes was appropriated for sleep regulation. The current data showing that two additional cell-cycle proteins, TARA and Cdk1, control sleep and wakefulness provide support for that hypothesis. Moreover, the fact that TARA and CycA, factors identified in two independent unbiased genetic screens, interact with each other highlights the importance of a network of cell-cycle genes in sleep regulation (Afonso, 2015).

There are two main regulatory mechanisms for sleep: the circadian mechanism that controls the timing of sleep and the homeostatic mechanism that controls the sleep amount. This study has shown that TARA has a profound effect on total sleep time. TARA also affects rhythmic locomotor behavior. Since TARA is expressed in clock cells, whereas CycA is not, it is possible that TARA plays a non-CycA dependent role in clock cells to control rhythm strength. The finding that tara mutants exhibit severely reduced sleep in constant light suggests that the effect of TARA on sleep amount is not linked to its effect on rhythmicity. Instead, TARA may have a role in the sleep homeostatic machinery, which will be examined in an ongoing investigation (Afonso, 2015).

To fully elucidate how sleep is regulated, it is important to identify the underlying neural circuits. This study has shown that activation of the CycA-expressing neurons in the PL suppresses sleep while blocking their activity increases sleep, which establishes them as a novel wake-promoting center. Importantly, knockdown of tara and increased Cdk1 activity specifically in the PL neurons leads to decreased sleep. A simple hypothesis, consistent with the finding that both activation of PL neurons and increased Cdk1 activity in these neurons suppress sleep is that Cdk1 affects neuronal excitability and synaptic transmission. Interestingly, large-scale screens for short-sleeping mutants in fruit flies and zebrafish have identified several channel proteins such as SHAKER, REDEYE, and ETHER-A-GO-GO and channel modulators such as SLEEPLESS and WIDE AWAKE. Thus, it is plausible that Cdk1 regulates sleep by phosphorylating substrates that modulate the function of synaptic ion channels or proteins involved in synaptic vesicle fusion, as has previously been demonstrated for Cdk5 at mammalian synapses (Afonso, 2015).

Whereas the data mapped some of TARA’s role in sleep regulation to a small neuronal cluster, the fact that pan-neuronal tara knockdown results in a stronger effect on sleep than specific knockdown in PL neurons suggests that TARA may act in multiple neuronal clusters. PL-specific restoration of TARA expression did not rescue the tara sleep phenotype (data not shown), further implying that the PL cluster may not be the sole anatomical locus for TARA function. Given that CycA is expressed in a few additional clusters, TARA may act in all CycA-expressing neurons including those not covered by PL-Gal4. TARA may also act in non-CycA-expressing neurons. The data demonstrate that tara knockdown using Cha-Gal4 produces as strong an effect on sleep as pan-neuronal knockdown. This finding suggests that TARA acts in cholinergic neurons, although the possibility cannot be ruled out that the Cha-Gal4 expression pattern includes some non-cholinergic cells. Taken together, these data suggest that TARA acts in PL neurons as well as unidentified clusters of cholinergic neurons to regulate sleep (Afonso, 2015).

Based on genetic interaction studies, tara has been classified as a member of the trithorax group genes, which typically act as transcriptional coactivators. However, TARA and Trip-Br1 have been shown to up- or downregulate the activity of E2F1 transcription factor depending on the cellular context, raising the possibility that they also function as transcriptional corepressors. Interestingly, TARA physically interacts with CycA and affects CycA protein levels but not its mRNA expression. These findings suggest a novel non-transcriptional role for TARA, although an indirect transcriptional mechanism cannot be ruled out. The hypothesis that TARA plays a non-transcriptional role in regulating CycA levels and Cdk1 activity at the synapse may provide an exciting new avenue for future research (Afonso, 2015).

cdc2: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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