Cyclin A

Promoter

Polycomb group (PcG) and trithorax group (trxG) proteins are well known for their role in the maintenance of silent and active expression states of homeotic genes. However, PcG proteins may also be required for the control of cellular proliferation in vertebrates. In Drosophila, PcG factors act by associating with specific DNA regions termed PcG response elements (PREs). This study investigated whether Drosophila cell cycle genes are directly regulated by PcG proteins through PREs. A PRE was isolated that regulates Cyclin A expression. This sequence is bound by the Polycomb (PC) and Polyhomeotic (PH) proteins of the PcG, and also by GAGA factor (GAF), a trxG protein that is usually found associated with PREs. This sequence causes PcG- and trxG-dependent variegation of the mini-white reporter gene in transgenic flies. The combination of FISH with PC immunostaining in embryonic cells shows that the endogenous CycA gene colocalizes with PC at foci of high PC concentration named PcG bodies. Finally, loss of function of the Pc gene and overexpression of Pc and ph trigger up-regulation and down-regulation, respectively, of CycA expression in embryos. These results demonstrate that CycA is directly regulated by PcG proteins, linking them to cell cycle control in vivo (Martinez, 2006).

Given the well-described nature of homeotic gene silencing by PcG proteins (i.e., stable maintenance of repression throughout development), PcG genes would not appear at first glance to be obvious candidates for factors controlling the dynamic expression of cell cycle genes. Indeed, actively proliferating cells must reexpress their rate-limiting division components with each cell cycle. It was observed, however, that RNAi-mediated depletion of PC in cycling S2 cells modifies their cell cycle profile, although it does not affect the overall rate of cell proliferation. The Drosophila CycA gene was identified as a direct target in vivo for PC, PH, and GAF in cycling S2 and embryonic cells. In ChIP experiments, the PcG-binding element was precisely mapped in the CycA gene to a region spanning from the promoter to the first intron. This CycA region shares some but not all properties with homeotic PREs. First, the sequence is sufficient to silence the mini-white reporter gene in vivo, producing a characteristic eye variegation phenotype. Second, as expected for a PRE, mini-white silencing is genetically dependent on the activities of the PcG and trxG genes. Third, it was demonstrated that the endogenous CycA gene is repressed in a Pc-dependent manner during embryonic development: In homozygous Pc mutants CycA expression is derepressed in late (stages 11/12/13) embryos. Finally, stable repression of CycA in normal embryos can be visualized as a colocalization between the CycA locus and PcG bodies that gradually increases, reaching a maximum at the time when cells totally stop dividing and begin to differentiate. Together, these results are consistent with PcG proteins playing a functional role in the stable repression of the CycA gene in vitro and in vivo (Martinez, 2006).

In addition, Pc and ph overexpression in rapidly proliferating cells during early embryonic development causes a systematic decrease in the expression of the CycA gene. This suggests that PcG proteins may play a dual molecular role in the regulation of CycA, acting as stable silencing factors in mitotically quiescent cells and as modulators of promoter output in proliferating cells (Martinez, 2006).

In these experiments, PcG members bound the CycA PRE in actively dividing S2 cells. This binding is most likely functionally relevant, since depletion of PC in S2 cells reproducibly modified the cell cycle division profile, correlating with increased CycA levels. An accumulation of cells in the G2/M phase of the cell cycle was found in PC-depleted cells in comparison to control cells. This accumulation is reminiscent of the phenotype observed in the Drosophila dally mutant, in which the cell division pattern is altered in the nervous system and G2/M progression is disrupted in specific sets of dividing cells in the larval brain and eye disc. In this mutant, lamina precursor cells retain high levels of CycA for a prolonged period of time. Although these experiments do not allow a precise definition of exactly which step within the G2/M transition is abnormal, it is proposed that elevated levels of CycA, or an abnormally long persistence of CycA, might cause a delay in exit from mitosis. Accumulation of CycA has been previously shown to accelerate the G1/S transition. Consistent with this finding, in these experiments the population of S2 cells in G1 and S phases was largely decreased after PC depletion (Martinez, 2006).

The implication of PcG members in cell cycle control during active proliferation is surprising. Interestingly, Beuchle (2001) removed individual PcG proteins from clones of proliferating cells in imaginal discs and showed that Psc-Su(z)2 and ph⁰ mutant clones are large and round, reminiscent of clones of mutations that cause disc tumors. While the exact nature of the defect was unknown, it could be rescued by resupplying Psc and Su(z)2 several hours after the induction of the clone. This suggests that the effects produced by altering PcG-mediated regulation of cell proliferation/growth might be reversible (Martinez, 2006).

In the current experiments, Pc and ph overexpression in cycling embryonic cells were found to silence endogenous CycA expression. This result demonstrates that the effect of PcG proteins on the endogenous CycA PRE is dose-dependent in cycling cells, and suggests that CycA maintains an intrinsic capacity to be silenced despite being normally transcribed. In normal proliferating cells, induced transcription through the CycA locus, which would necessarily transverse the PRE, might be sufficient to counteract the PRE silencing activity of the CycA PRE. Indeed, it has recently been shown that intergenic transcription through a PRE counteracts silencing (Martinez, 2006).

The results suggest that the CycA PRE might present dual functional properties depending on whether cells are cycling or are arrested in the cell cycle. The CycA PRE might behave as a transcriptional attenuator element in cycling cells and as a stable silencer in a subset of mitotically quiescent cells. Recent data suggest the existence of functionally distinct PcG protein complexes that differ in composition as a function of developmental stage and cellular proliferation status. It would thus be of great interest to biochemically characterize the composition of PcG complexes present at different phases of the cell cycle or during different developmental stages in Drosophila (Martinez, 2006).

Although PcG proteins can repress CycA in mitotically arrested embryonic cells, this does not account for all aspects of stable CycA repression. For example, terminally differentiated cells of the salivary glands from third instar larvae do not express CycA, but neither the endogenous gene nor the isolated PRE are able to attract PcG proteins in this tissue. This situation is similar to the hh gene, which is a known target of PcG proteins. Another chromatin-silencing activity must therefore be responsible for this silencing. One possible candidate is the recently described dREAM complex (Korenjak, 2004), which contains the Drosophila E2F and RBF (pRb homolog) factors and binds to silent E2F-binding-site-containing genes during development, including in salivary glands. Whether or not this is the case, silencing of the CycA gene seems to be regulated in a complex manner that might change during different phases of the cell cycle and might depend on the developmental stage and the tissue under analysis (Martinez, 2006).

In addition to CycA being regulated by PcG members, the converse might also be possible; i.e., PcG-binding and/or silencing activity might be regulated in a cell cycle-dependent manner. In a preliminary genetic analysis involving trans-heterozygous allelic combinations, it was found that the homeotic phenotypes of extra sex combs in the T2 and T3 thoracic legs in males and the pigmentation of the A4 tergite (Mcp phenotype) associated with mutations in the Pc and ph genes are enhanced when combined with a CycA mutation. This may suggest the existence of a feedback regulatory loop between PcG genes and CycA (Martinez, 2006).

From studies in vertebrates, it is clear that PcG proteins repress p16ink4a and p19arf, although a strict demonstration of direct repression is still missing. It is not known whether plutonium, the putative Drosophila homolog of p16ink4a, is silenced by PcG proteins. However, a 'ChIP-on-chip' analysis was carried out of the binding profiles of PC, PH, and GAF proteins in a region covering 10% of the Drosophila melanogaster genome. This analysis led to the identification, among others, of several potential PcG target genes that play a role in the control of proliferation and growth. These include the escargot (esg), elbowB (elB), and no ocelli (noc) genes, in addition to a p53-like factor encoded by bifid. Interestingly, esg and elB, as well as the known PcG target gene hh, have been coidentified as potential tumor suppressors in a protein overexpression screen. Finally, recent evidence suggests that hh regulates both proliferation and differentiation in the developing Drosophila retina (Martinez, 2006).

Together with the role of PcG proteins in the regulation of CycA, this evidence suggests that PcG proteins may be globally involved in the coupling of cell proliferation with growth or differentiation during development in Drosophila and perhaps also in vertebrates. This intriguing possibility warrants future investigation (Martinez, 2006).

Drosophila MCRS2 associates with RNA polymerase II complexes to regulate transcription

Drosophila MCRS2 (dMCRS2; MCRS2/MSP58 and its splice variant MCRS1/p78 in humans) belongs to a family of forkhead-associated (FHA) domain proteins. Whereas human MCRS2 proteins have been associated with a variety of cellular processes, including RNA polymerase I transcription and cell cycle progression, dMCRS2 has been largely uncharacterized. Recent data show that MCRS2 is purified as part of a complex containing the histone acetyltransferase MOF (males absent on first) in both humans and flies. MOF mediates H4K16 acetylation and regulates the expression of a large number of genes, suggesting that MCRS2 could also have a function in transcription regulation. This study shows that dMCRS2 copurifies with RNA polymerase II (RNAP II) complexes and localizes to the 5' ends of genes. Moreover, dMCRS2 is required for optimal recruitment of RNAP II to the promoter regions of cyclin genes. In agreement with this, dMCRS2 is required for normal levels of cyclin gene expression. A model is proposed whereby dMCRS2 promotes gene transcription by facilitating the recruitment of RNAP II preinitiation complexes (PICs) to the promoter regions of target genes (Anderson, 2010).

The initiation of mRNA transcription involves the assembly of a transcription preinitiation complex (PIC), which as a minimum includes RNA polymerase II (RNAP II), Mediator, and six general transcription factors (TFIIA, -B, -D, -E, -F, and -H) at the core promoter DNA region. PIC assembly is initiated by the binding of the TATA box binding protein (TBP) subunit of TFIID to the promoter, which is stabilized in the presence of TFIIA and Mediator. Subsequently, TFIIB binds to and stabilizes the TFIIA-TFIIB-Mediator-DNA complex and functions as an adaptor that recruits the preformed RNAP II-TFIIF complex to the promoter. TFIIE and TFIIH then join to form the complete PIC (Anderson, 2010).

Once the PIC has been assembled on the promoter, transcription initiation occurs in several steps, which involve extensive phosphorylation of the C-terminal domain (CTD) of RNAP II. Early on in the transition from preinitiation to elongation, phosphorylation of Ser5s in the CTD heptapeptide repeats takes place, and this depends on the activity of the TFIIH-associated kinase cyclin-dependent kinase 7 (Cdk7; mammals)/Kin28 (yeast). Subsequently, Ser2s are phosphorylated by the elongation phase kinase Cdk9 (mammals)/CTDK-1 (yeast) to generate elongation-proficient RNAP II complexes. Another Cdk, Cdk8, can negatively regulate RNAP II transcription, partially via its inhibitory effect on Cdk7 activity. More recently, it has been suggested that Cdk11^p110 regulates RNAP II transcription in humans. Thus, Cdk11^p110 binds to hypo- and hyper-phosphorylated RNAP II, and antibody-mediated repression of Cdk11^p110 activity results in inhibition of RNAP II transcription (Anderson, 2010 and references therein).

In addition to the phosphorylation events that control RNAP II activity, modification of the chromatin structure represents an important mechanism for regulating gene expression. When the chromatin is in its repressed state, the DNA is wrapped tightly around the histones, creating a barrier to the assembly of the RNAP II PIC at the promoter region. Activation of gene expression is associated with a number of histone modifications that loosen the chromatin structure, including acetylation, methylation, ubiquitylation, and phosphorylation. Histone H3 and H4 acetylations are particularly frequent toward the 5' ends of actively transcribed genes and presumably facilitate the initial assembly of the PICs at the promoter region. MOF (males absent on first) is a histone H4 lysine 16 (H4K16)-specific histone acetyltransferase (HAT) in both mammals and Drosophila. MOF is part of several complexes, including the Drosophila male-specific lethal (MSL) complex, which is required for X chromosome dosage compensation, the mammalian counterpart of the MSL complex, and the MOF-MSL1v1 complex, which mediates p53 acetylation at K120. In addition, MOF copurifies with a number of other proteins, such as the forkhead-associated (FHA) domain-containing protein MCRS2, NSL1-3 (for nonspecific lethal 1 to 3), and MBD-R2, as part of the NSL complex (Anderson, 2010).

This study focuses on the function of Drosophila MCRS2 (dMCRS2), the Drosophila ortholog of human MCRS2 (also known as MSP58). Whereas human MCRS1 and -2 proteins have been associated with a variety of cellular processes, including RNA polymerase I transcription and cell cycle progression, dMCRS2 is largely uncharacterized. In addition to the recent observation that human and Drosophila MCRS2s form complexes with MOF (Cai, 2010; Prestel, 2010; Mendjan, 2006; Raja, 2010), several other reports suggest that MCRS1 and -2 proteins could function in transcription regulation via interactions with the transcriptional repressor Daxx or the basic region leucine zipper factor Nrf1 (Anderson, 2010).

Drosophila MCRS2 and its human homologue, MCRS2, are 59% identical, with the highest level of homology being in the FHA domain. Whereas dMCRS2 is largely uncharacterized, MCRS1 and -2 have been linked with a variety of cellular processes, RNAP I-dependent transcription, transcriptional repression, and cell cycle control, though these functions remain poorly understood (Anderson, 2010).

This study shows that dMCRS2 is an essential nuclear protein required for cell cycle progression and growth during development. The data show that dMCRS2 physically associates with Cdk11 and RNAP II and colocalizes with RNAP II PICs on polytene chromosomes in vivo. Consistent with this, dMCRS2 is required for optimal binding of RNAP II components to the cyclin promoter regions and for normal levels of cyclin gene expression (Anderson, 2010).

The demonstration of colocalization of dMCRS2 with RNAP II on numerous sites on polytene chromosomes is in agreement with a recent ChIP-seq analysis, which revealed that dMCRS2 is present on the promoters of over 4,000 genes, correlating with 55% of active genes (Raja, 2010). Furthermore, gene expression profiling studies show that dMCRS2 depletion elicits the downregulation of over 5,000 genes. This essential function as a broad-specificity transcriptional regulator is reflected by the extreme growth defect of dMCRS2-depleted cells both in vivo and in cell culture and in the fact that dMCRS2 has been recovered as a hit in RNAi screens for diverse cellular functions such as centrosome maturation and Hedgehog signaling (Anderson, 2010).

In accordance with its pleiotropic function, dMCRS2 can be purified with a number of proteins, from NSL components to members of the RNAP II machinery. Moreover, dMCRS2 colocalizes with RNAP II PICs on polytene chromosomes in vivo, suggesting that it may regulate an early step in the recruitment and/or assembly of RNAP II PICs. This is consistent with the majority of dMCRS2 binding to the promoter regions of autosomal and X-linked genes and the fact that dMCRS2 is required for the loading of RNAP II components to cyclin gene promoters. Thus, dMCRS2 appears to be an important transcriptional regulator, and the data represent the first evidence for a physical connection between dMCRS2 and the core transcriptional machinery. While the results suggest that dMCRS2 associates with RNAP II complexes via protein-protein interactions, future studies will need to establish the exact molecular nature of this connection (Anderson, 2010).

Interestingly, MCRS2 and dMCRS2 copurify with the MOF HAT independently of the dosage compensation MSL complex. Furthermore, it was observed that dMCRS2 coimmunoprecipitates and colocalizes extensively with MOF on polytene chromosomes. MOF, as well as binding to the 3' ends of MSL targets along the male X chromosome, is also found on numerous promoter regions, both on the X chromosome and on autosomes in both sexes. Since MCRS2 also binds to promoters, it is possible that dMCRS2 and MOF could function in concert in transcriptional regulation. However, despite the evidence that MOF regulates a broad range of both X-linked and autosomal genes, no physical connection between the putative dMCRS2-MOF NSL complex and RNAP II complexes has been established so far. This study shows that both dMCRS2 and MOF associate with core RNAP II complexes in cultured cells (Anderson, 2010).

dMCRS2 may promote transcription by different mechanisms. Through its HAT activity, dMCRS2-associated MOF may create a relaxed chromatin state favorable to PIC assembly, either by inducing the physical weakening of DNA/histone or histone/histone interactions or by promoting the recruitment of bromodomain-containing factors. dMCRS2 may also induce PIC formation by recruiting the preformed RNAP II/TFIIF complex and/or promoting transcription elongation through the recruitment of CK2 and the FACT complex, which facilitates transcription elongation by remodeling chromatin. However, whether these different dMCRS2-containing complexes regulate common target genes or whether they represent distinct transcriptional regulators remains to be investigated. In summary, a model is proposed where dMCRS2 binds to multiple sites along the chromosomes and promotes the recruitment of RNAP II PICs to target genes (Anderson, 2010).

Transcriptional Regulation

In escargot mutants, diploid imaginal cells arrested in G2 lose Cyclin A and enter an endocycle, the duplication of DNA without subsequent mitosis, creating a state of polyploidy. Mutants in cdc2, the cyclin dependent kinase, give similar results. Escargot acts to maintain high levels of both Cdc2 and Cyclin A in the active form that inhibits entry into S (the DNA synthetic phase) (Hayashi, 1996).

The Drosophila gene for cyclin A is expressed in dividing cells throughout development. This expression pattern is similar to that of genes related to DNA replication, suggesting involvement of some common control mechanism(s). In the upstream region (-71 to -64 with respect to the transcription initiation site) of the CycA gene, a sequence was found that is identical to the DNA replication-related element (DRE; 5'-TATCGATA), which is important for high level expression of replication-related genes such as those encoding DNA polymerase alpha and proliferating cell nuclear antigen. Deletion or base substitution mutations result in an extensive reduction in Cyclin A expression. Monoclonal antibodies against DRE binding factor (DREF) diminish or supershift the complex of the DREF and the DRE-containing fragment. The results indicate that the Drosophila CycA gene is under the control of a DRE/DREF system, as are DNA replication-related genes (Ohno, 1996).

The function of the neuronal differentiation gene daughterless is required for the proper initiation of neuronal lineage development in all peripheral nervous system (PNS) lineages following the selection of neuronal precursor cells. Previous studies have shown that the ubiquitously expressed Da protein is required for the proper expression of neuronal precursor genes and lineage identity genes in the PNS of Drosophila embryos. These genes are required for differentiation and cell fate determination in the developing PNS. These findings, however, do not explain the failure of the nascent PNS precursors to undergo a normal cell cycle and divide in da mutants. Four genes whose products are required for various stages of the cell cycle are misexpressed in the PNS of da mutant embryos. Cyclin A, barren, disc proliferation abnormal and Histone H1 transcripts are significantly reduced or undetectable in the precursors of the PNS at stages 11 and 12. Precursors are still present at these stages in da mutants. This suggests that all aspects of PNS precursor differentiation examined so far are under the transcriptional control of da. Sensory organ precursors lacking Da may fail to express and/or accumulate other factors, such as critical differentiation genes, required for SOP entry into the cell cycle. It should be pointed out that these factors are unlikely to be the thus-far described neuronal precursor genes, as mutations in these genes do not result in any obvious cell cycle defects (Hassan, 1997).

Mutations have been characterized in the Drosophila Tsc1 and Tsc2/gigas genes. Inactivating mutations in either gene cause an identical phenotype characterized by enhanced growth and increased cell size with no change in ploidy. Overall, mutant cells spend less time in G1. Coexpression of both Tsc1 and Tsc2 restricts tissue growth and reduces cell size and cell proliferation. This phenotype is modulated by manipulations in cyclin levels. In postmitotic mutant cells, levels of Cyclin E and Cyclin A are elevated. This correlates with a tendency for these cells to reenter the cell cycle inappropriately as is observed in the human lesions (Tapon, 2001).

Tsc1 and Tsc2 may modulate the cell cycle via changes in cyclin levels. In Tsc1 and Tsc2 mutant clones, the levels of both Cyclin E and Cyclin A are elevated. Cell growth driven by dmyc or Target of rapamycin (dTor) elevates Cyclin E levels. It has been postulated that Cyclin E may function as a 'growth sensor' in a manner analogous to CLN3 in yeast and that the translation of Cyclin E is more efficient in cells that have an increased rate of growth. The increased levels of Cyclin E may be responsible for the shortening of G1 in Tsc1 and Tsc2 mutants. It is unclear why Cyclin A and Cyclin B are also elevated in mutant cells. Cyclin A is normally expressed at high levels in G2. In Tsc1 or Tsc2 mutants, Cyclin A levels are elevated in the post-mitotic cells of the eye disc that are clearly not arrested in G2. Thus it seems likely that the increased growth in mutant cells may also lead to increased levels of mitotic cyclins. Alternatively Tsc1 and Tsc2 may function in a pathway that negatively regulates cyclin levels (Tapon, 2001).

Tsc mutant cells fail to maintain a developmentally induced G1 arrest posterior to the second mitotic wave in third instar eye imaginal discs. The establishment of this G1 arrest requires a downregulation of Cyclin E and Cyclin A expression. However, the transient cell cycle arrest in the morphogenetic furrow occurs normally in Tsc1 and Tsc2, suggesting that it is the maintenance of G1 arrest that is perturbed rather than its initial establishment. Postmitotic cells continue to grow abnormally in Tsc1 and Tsc2 mutants and express elevated levels of Cyclin E and Cyclin A. A likely model is that inappropriate and continued growth in postmitotic cells leads to an accumulation of Cyclin E and the mitotic cyclins. This would eventually force cells to overcome a developmentally regulated cell cycle arrest and to reenter the cell cycle. Indeed, many of the lesions in patients with TSC occur in organs that consist predominantly of postmitotic cells such as the heart and brain. A successful therapeutic strategy in tuberous sclerosis is likely to be one that can curtail the inappropriate cell growth (Tapon, 2001).

JAK/STAT controls organ size and fate specification by regulating morphogen production and signalling

A stable pool of morphogen-producing cells is critical for the development of any organ or tissue. This study presents evidence that JAK/STAT signalling in the Drosophila wing promotes the cycling and survival of Hedgehog-producing cells, thereby allowing the stable localization of the nearby BMP/Dpp-organizing centre in the developing wing appendage. The inhibitor of apoptosis dIAP1 and Cyclin A were identified as two critical genes regulated by JAK/STAT and contributing to the growth of the Hedgehog-expressing cell population. JAK/STAT was found to have an early role in guaranteeing Wingless-mediated appendage specification, and a later one in restricting the Dpp-organizing activity to the appendage itself. These results unveil a fundamental role of the conserved JAK/STAT pathway in limb specification and growth by regulating morphogen production and signalling, and a function of pro-survival cues and mitogenic signals in the regulation of the pool of morphogen-producing cells in a developing organ (Recasens-Alvarez, 2017).

Morphogens of the Wnt/Wg, Shh/Hh and BMP/Dpp families regulate tissue growth and pattern formation in vertebrate and invertebrate limbs. This study has unraveled a fundamental role of the secreted Upd ligand and the JAK/STAT pathway in facilitating the activities of these three morphogens in exerting their fate- and growth-promoting activities in the Drosophila wing primordium. Early in wing development, two distinct mechanisms ensure the spatial segregation of two alternative cell fates. First, the proximal-distal subdivision of the wing primordium into the wing and the body wall relies on the antagonistic activities of the Wg and Vn signalling molecules. While Wg inhibits the expression of Vn and induces the expression of the wing-determining genes, Vn, through the EGFR pathway, inhibits the cellular response to Wg and instructs cells to acquire body wall fate. Second, growth promoted by Notch pulls the sources of expression of these two morphogens apart, alleviates the repression of wing fate by Vn/EGFR, and contributes to Wg-mediated appendage specification. Expression of Vn is reinforced by a positive amplification feedback loop through the activation of the EGFR pathway. This existing loop predicts that, in the absence of additional repressors, the distal expansion of Vn/EGFR and its targets would potentially impair wing development. The current results indicate that Upd and JAK/STAT restrict the expression of EGFR target genes and Vn to the most proximal part of the wing primordium, thereby interfering with the loop and allowing Wg to correctly trigger wing development. Evidence is presented that JAK/STAT restricts the expression pattern and levels of its own ligand Upd and that ectopic expression of Upd is able to bypass EGFR-mediated repression and trigger wing development de novo. This negative feedback loop between JAK/STAT and its ligand is of biological relevance, since it prevents high levels of JAK/STAT signalling in proximal territories that would otherwise impair the development of the notum or cause the induction of supernumerary wings, as shown by the effects of ectopic activation of the JAK/STAT pathway in the proximal territories. Thus, while Wg plays an instructive role in wing fate specification, the Notch and JAK/STAT pathways play a permissive role in this process by restricting the activity range of the antagonizing signalling molecule Vn to the body wall region (Recasens-Alvarez, 2017).

Later in development, once the wing field is specified, restricted expression of Dpp at the AP compartment boundary organizes the growth and patterning of the whole developing appendage. Dpp expression is induced in A cells by the activity of Hh coming from P cells, which express the En transcriptional repressor. This study shows that JAK/STAT controls overall organ size by maintaining the pool of Hh-producing cells to ensure the stable and localized expression of the Dpp organizer. JAK/STAT does so by promoting the cycling and survival of P cells through the regulation of dIAP1 and CycA, counteracting the negative effects of En on these two genes. Since the initial demonstration of the role of the AP compartment boundary in organizing, through Hh and Dpp, tissue growth and patterning, it was noted that high levels of En interfered with wing development by inducing the loss of the P compartment. The capacity of En to negatively regulate its own expression was subsequently shown to be mediated by the Polycomb-group genes and proposed to be used to finely modulate physiological En expression levels. Consistent with this proposal, an increase was observed in the expression levels of the en-gal4 driver, which is inserted in the en locus and behaves as a transcriptional reporter, in enRNAi-expressing wing discs. The negative effects of En on cell cycling and survival reported in this work might also contribute to the observed loss of the P compartment caused by high levels of En. As is it often the case in development, a discrete number of genes is recurrently used to specify cell fate and regulate gene expression in a context-dependent manner. It is proposed that the capacity of En to block cell cycle and promote cell death might be required in another developmental context and that this capacity is specifically suppressed in the developing Drosophila limbs by JAK/STAT, and is modulated by the negative autoregulation of En, thus allowing En-dependent induction of Hh expression and promoting Dpp-mediated appendage growth. It is interesting to note in this context that En-expressing territories in the embryonic ectoderm are highly enriched in apoptotic cells. Whether this apoptosis plays a biological role and relies on En activity requires further study (Recasens-Alvarez, 2017).

Specific cell cycle checkpoints appear to be recurrently regulated by morphogens and signalling pathways, and this regulation has been unveiled to play a major role in development. Whereas Notch-mediated regulation of CycE in the Drosophila eye and wing primordia is critical to coordinate tissue growth and fate specification by pulling the sources of two antagonistic morphogens apart, the current results indicate that JAK/STAT-mediated regulation of CycA is critical to maintain the pool of Hh-producing cells in the developing wing and to induce stable Dpp expression. The development of the wing hinge region, which connects the developing appendage to the surrounding body wall and depends on JAK/STAT activity, has been previously shown to restrict the Wg organizer and thus delimit the size and position of the developing appendage. The current results support the notion that JAK/STAT and the hinge region are also essential to restrict the organizing activity of the Dpp morphogen to the developing appendage. Taken together, these results reveal a fundamental role of JAK/STAT in promoting appendage specification and growth through the regulation of morphogen production and activity, and a role of pro-survival cues and mitotic cyclins in regulating the pool of morphogen-producing cells in a developing organ. The striking parallelisms in the molecules and mechanisms underlying limb development in vertebrates and invertebrates have contributed to the proposal that an ancient patterning system is being recurrently used to generate body wall outgrowths. Whether the conserved JAK/STAT pathway plays a developmental role also in the specification or growth of vertebrate limbs by regulating morphogen production or activity is a tempting question that remains to be elucidated (Recasens-Alvarez, 2017).

Targets of Activity

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 CDC25^String 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 16^th 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 16^th 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).

twin, a CCR4 homolog, regulates cyclin poly(A) tail length to permit Drosophila oogenesis: Twin regulates Cyclin A translation

Cyclins regulate progression through the cell cycle. Control of cyclin levels is essential in Drosophila oogenesis for the four synchronous divisions that generate the 16 cell germ line cyst and for ensuring that one cell in each cyst, the oocyte, is arrested in meiosis, while the remaining fifteen cells become polyploid nurse cells. Changes in cyclin levels could be achieved by regulating transcription, translation or protein stability. The proteasome limits cyclin protein levels in the Drosophila ovary, but the mechanisms regulating RNA turnover or translation remain largely unclear. This study reports the identification of twin, a homolog of the yeast CCR4 deadenylase. twin is important for the number and synchrony of cyst divisions and oocyte fate. Consistent with the deadenylase activity of CCR4 in yeast, these data suggest that Twin controls germ line cyst development by regulating poly(A) tail lengths of several targets including Cyclin A (CycA) RNA. twin mutants exhibit very low expression of Bag-of-marbles (Bam), a regulator of cyst division, indicating that Twin/Ccr4 activity is necessary for wild-type Bam expression. Lowering the levels of CycA or increasing the levels of Bam suppresses the defects observed in twin ovaries, implicating CycA and Bam as downstream effectors of Twin. It is proposed that Twin/Ccr4 functions during early oogenesis to coordinate cyst division, oocyte fate specification and egg chamber maturation (Morris, 2005).

twin encodes the Drosophila homolog of the yeast ccr4 gene. ccr4 (carbon-catabolite-repression) was first identified in S. cerevisiae as a regulator of RNA levels of the alcohol-dehydrogenase II gene. Although CCR4 protein was previously shown to associate with basal transcription machinery, recent data demonstrate that CCR4 catalyzes the degradation of poly(A) tails in yeast and flies (Temme, 2004; Morris, 2005 and references therein).

It has been unclear whether mutations in CCR4 have specific developmental defects and whether these defects might reveal specific targets sensitive to CCR4 function. twin mutant cysts divide asynchronously and less than four times; oocyte specification is defective and many egg chambers die and degrade. The mitotic cyclins, CycA and CycB, are misexpressed in twin, and reducing the gene copy number of cycA partially suppresses the twin egg chamber degradation phenotype. Furthermore, the poly(A) tails of cycA, cycE and, to a lesser extent, cycB, are longer in twin extracts, suggesting that Twin/Ccr4 deadenylase activity directly controls the RNA levels of these cell cycle regulators. By contrast, cytoplasmic Bam staining is reduced in twin. Induction of extra bam expression suppresses the cyst division and oocyte fate specification defects in twin mutants, implicating low Bam levels as one of the causes of these twin phenotypes (Morris, 2005).

The twin alleles are viable and specifically affect the female germline. In S. cerevesiae, ccr4 mutations are not lethal, although CCR4 is thought to be the main cytoplasmic deadenylase. It is possible that angel and Dnocturnin (CG4796), two other genes with extensive homology to the ccr4 catalytic domain but lacking the crucial LRR repeats, can partially compensate for loss of Twin function. Alternatively, since the mutations are probably not complete nulls, oogenesis may be more sensitive than the soma to decreased Twin function. Like the ovary, the early embryo relies on precise post-transcriptional gene regulation. The mature egg contains high levels of maternally loaded twin, consistent with a role for Twin in deadenylation, and probably explaining why twin mutants carry out embryogenesis normally (Morris, 2005).

Mitotic cells regulate cyclin levels in order to progress through the cell cycle. At the protein level, Drosophila regulates CycA, CycB and CycE, via proteasome-mediated degradation. In the Drosophila ovary, the novel protein Encore has been proposed to localize components of the proteasome complex to the fusome to regulate CycE. encore mutant cysts undergo an extra cell division and contain 32 cells, probably as a consequence of misexpressing not only CycE, but also CycA. Other experiments have shown that cyst divisions are sensitive to CycA levels. Adding a brief pulse of CycA by inducing a heat-shock construct can lead to an extra round of cyst division, suggesting that downregulation of CycA is crucial for cell cycle progression. Only a small number of cysts respond to such a CycA pulse, suggesting that in the wild type not all germ cells are in a susceptible phase of the cell cycle (G2) during which they can respond to CycA (Morris, 2005).

Cyclin RNA levels are regulated by control of poly(A) tail length. In Xenopus and mouse oocytes, cycB RNA is not translated in the absence of CPEB-mediated poly(A) tail lengthening. Longer poly(A) tails also enhance cyclin translation in Drosophila embryos. In the Drosophila ovary, Orb, the CPEB homolog, regulates poly(A) tail length and expression of its own RNA and oskar RNA. Consistent with a role for Orb in cyclin regulation and cyst division, orb mutant cysts frequently contain eight germ cells (Morris, 2005).

The data suggest that Twin-mediated deadenylation of cyclin RNA regulates cyst divisions. Cyclin polyadenylation has been well studied, but much less is known about cyclin RNA deadenylation. In Drosophila, Nanos and Pumilio have been shown to control deadenylation of cycB mRNA in primordial germ cells. Furthermore, Xenopus Pumilio interacts with CPEB, and Nanos, Pumilio and Orb/CPEB are all expressed early in Drosophila oogenesis. It is intriguing to speculate that Twin may regulate the poly(A) tail lengths in the dividing cyst in conjunction with Nanos, Pumilio and/or Orb (Morris, 2005).

Cytoplasmic Bam expression is reduced in twin germaria; a phenotype that would not be predicted if Twin directly regulated Bam expression via deadenylation. Indeed, no substantial change was detected in bam poly(A) tail length in twin ovaries. It is therefore proposed that bam is an indirect target of Twin/Ccr4 (Morris, 2005).

Although bam is known to control the differentiation of the cystoblast and to promote cyst division, the biochemical role of Bam is unknown. Removing one copy of bam suppresses the extra division in cysts lacking encore or overexpressing CycA. The results further implicate Bam in the events of early oogenesis. Increased bam expression suppresses not only the cyst division defects observed in twin mutants, but also the twin oocyte specification defects. Because Twin regulates cycA directly and may regulate Bam indirectly, the simplest model would posit that high levels of CycA are sufficient to suppress Bam expression. Two pieces of evidence argue against this model: Bam and CycA are both present at high levels in the dividing cyst; and Bam is required for the fifth cyst division induced by high levels of CycA. In addition, hs-bam induces stem cells to develop into normal cysts, indicating that high Bam levels do not disrupt CycA expression. A model is favored by which Bam and CycA act in parallel to each other, downstream of Twin (Morris, 2005).

Although several models could explain the data, it is proposed that increased mitotic cyclin levels together with low Bam expression cause many of the twin phenotypes. If Bam expression were normal, overexpressing cyclins could lead to extra cyst divisions. The low level of Bam in twin germaria does not permit continued cell division, yet cyclin levels remain high, delaying cell cycle progression and probably causing the egg chamber degradation observed in twin. This model is consistent with the fact that reducing the copy number of bam suppresses the extra cyst division phenotype of encore and of hs-cycA. Corroborating evidence comes from the observation that reducing the gene dose of cycA or increasing the dose of bam can partially suppress the degradation phenotype. However, there are likely to be other, unidentified targets of twin that also contribute to the twin phenotype (Morris, 2005).

twin and Hu Li Tai Shao mutants disrupt the number and synchrony of cyst divisions and oocyte specification. This array of defects is not shared by the cell cycle mutants described above or by other mutants such as orb, the M-phase inhibitor tribbles or the M-phase activator string, which affect the number but not the synchrony of cyst divisions. Comparison of twin and hts may therefore be instructive. hts cysts have no fusome, and are thought consequently not to coordinate the cyst divisions. By contrast, cysts in twin mutants contain branched fusomes that are capable of colocalizing with CycA, suggesting the possibility that Twin/Ccr4 gene regulation may mediate the coordination of the cyst divisions with oocyte specification downstream of the fusome (Morris, 2005).

Regulation of cap-dependent translation by eIF4E inhibitory proteins

During spermatogenesis, cells coordinate differentiation with the meiotic cell cycle to generate functional gametes. The gene off-schedule (ofs) was identified as being essential for this coordinated control. During the meiotic G₂ phase, Drosophila ofs mutant germ cells do not reach their proper size and fail to execute meiosis or significant differentiation. The accumulation of four cell cycle regulators -- Cyclin A, Boule, Twine and Roughex -- is altered in these mutants, indicating that ofs reveals a novel branch of the pathway controlling meiosis and differentiation. Ofs is homologous to eukaryotic translation initiation factor eIF4G. The level of ofs expression in spermatocytes is much higher than for the known eIF4G ortholog (known as eIF-4G or eIF4G), suggesting that Ofs substitutes for this protein. Consistent with this, assays for association with mRNA cap complexes, as well as RNA-interference and phenotypic-rescue experiments, demonstrate that Ofs has eIF4G activity. Based on these studies, it is speculated that spermatocytes monitor G₂ growth as one means to coordinate the initiation of meiotic division and differentiation (Franklin-Dumont, 2007). A second studied, co-published with the Franklin-Dumont paper, see Baker (2007) below, has reported similar findings.

Initiation is the rate-limiting step in translation and is the most common target of translational control. The mRNA 5' cap is bound by eIF4F, a heterotrimeric protein complex that is the focal point for initiation. eIF4G is the backbone of this complex; it interacts not only with eIF4E, but also with eIF4A, an RNA helicase that facilitates ribosome binding and its passage along the 5' untranslated region (UTR) towards the initiation codon. eIF4G also associates with eIF3, a multisubunit factor that bridges the proteins bound to the mRNA's 5' end with the 40S ribosomal subunit. This ribosomal subunit comes 'pre-charged' as a ternary complex composed of eIF2, GTP and the initiator methionine-transfer RNA. With the aid of eIF4 initiation factor as well as ATP, this agglomeration of RNA and protein is thought to scan the mRNA in the 5' to 3' direction. When it encounters an AUG start codon in an optimal context, other factors as well as the 60S ribosomal subunit are recruited and polypeptide chain elongation begins (Richter, 2005).

The eIF4E-eIF4G interface is an important target for translational control. The core portion of eIF4G that interacts with eIF4E is small -- about 15 amino-acid residues (Mader, 1995). Strikingly, several other proteins contain similar peptide motifs, and it is this region that competes with eIF4G for binding to eIF4E; in this manner they control the rate of 40S ribosomal subunit association with mRNA, and hence translation initiation. A clear demonstration of why the competition between eIF4G and other proteins for interaction with eIF4E is so effective in preventing translation comes from X-ray crystallographic analysis. Peptides derived from the regions of eIF4G and an eIF4E inhibitory protein called 4E-BP (for 4E-binding proteins, also known as PHAS-I for phosphorylated heat and acid soluble protein stimulated by insulin; see Drosophila Thor) form nearly identical α-helical structures that lie along the same convex region of eIF4E, some distance from this protein's cap binding site (Marcotrigiano, 1997; Matsuo, 1997). Peptides with the general sequence YXXXXLphi, where phi is any hydrophobic amino acid, would probably form similar α-helical structures, implying that other proteins containing this peptide motif could control translation initiation (Richter, 2005).

The original three eIF4E inhibitory proteins, the 4E-BPs, prevent eIF4F complex formation by sequestering available eIF4E. This sequestration results in the inhibition of translation of certain mRNAs that normally require high levels of available eIF4E (Gingras, 1999). eIF4E-binding proteins interact with the eIF4E on only specific mRNAs, and do so either because they also interact with certain RNA elements directly, or do so through affiliations with RNA binding proteins (Richter, 2005).

In spermatogenesis, progenitor cells must execute the meiotic divisions in coordination with acquiring the specialized morphology and functionality of sperm. This conserved process is particularly amenable to analysis in Drosophila. The fly testis is a blind-ended tube organized as an assembly line for spermatogenesis. Germline stem cells at the blind end give rise to gonialblasts, which divide mitotically four times with incomplete cytokinesis to produce a cyst of 16 interconnected spermatogonia. These cells exit the mitotic cycle and enter meiosis as spermatocytes, exhibiting an extended G₂ phase characterized by a significant increase in cell mass and robust transcription. At the end of G₂, the spermatocytes undergo the meiotic divisions and begin the conversion from round spermatids to specialized spermatozoa (Franklin-Dumont, 2007).

Ten 'spermatocyte arrest' genes are required for both meiosis and differentiation and are sorted into two classes according to their molecular targets and specific role in promoting transcription. The always early (aly) class affects the transcription of meiotic genes such boule, twine and cyclin B, as well as that of differentiation genes such as fuzzy onions (fzo) and don juan. Notably, these mutations do not effect transcription of other spermatocyte genes, such as pelota, cyclin A and roughex. The Aly class proteins are thought to alter chromatin structure to permit the high levels of transcription necessary in spermatocytes. The cannonball (can) class affects boule and twine expression post-transcriptionally only and has no effect on cyclin B. The post-transcriptional effects must be indirect, because all can class loci encode testis-specific components of the general transcriptional machinery. Together, the spermatocyte arrest genes reveal how a diverse set of genes is selectively transcribed in spermatocytes (Franklin-Dumont, 2007).

The transcriptional regulatory pathway does not address the timing of meiotic entry and differentiation, however. Although transcripts necessary for these processes accumulate in early spermatocytes, the corresponding proteins do not appear until much later. Because there is little, if any, transcription after the G₂-M transition in flies, spermatocytes must delay meiotic division until all the necessary transcripts have accumulated. A similar dilemma exists during the mitotic cycle in yeast. For cells to maintain the same average size over several divisions, control points act during the gap phases and allow cell cycle progression only when the cell has reached a threshold size, with G₁ predominating in budding yeast and G₂ in fission yeast. Cell growth rates also feed back on mitotic cell cycle progression in Drosophila cells. Less is known about how growth might affect the specialized meiotic cell cycle (Franklin-Dumont, 2007).

Identification and characterization of off-schedule provides evidence that cell growth is linked to the coordination of meiosis and differentiation. Spermatocytes in ofs mutant males fail to execute the G₂-M transition of meiosis or substantive post-meiotic differentiation and have a significant cell size defect. The Off-schedule protein resembles the eukaryotic initiation factor 4G (eIF4G), which is a member of the eIF4F translation initiation complex and bridges mature mRNAs and the ribosome. The eIF4G activity of Ofs is apparent in its ability to associate with mRNA caps and to functionally replace canonical eIF4G in cell culture. Because translation is primarily regulated at initiation, eIF4G is instrumental in determining the translational capacity of a cell and thus its ability to accumulate mass. Thus, the ofs mutant phenotype suggests that sufficient cell mass must accumulate before spermatocytes execute meiosis and differentiation (Franklin-Dumont, 2007).

Alignment among eIF4G sequences suggests that Ofs would be part of the eIF4F complex with eIF4A and eIF4E, and demonstration of its association with 7-methyl GTP Sepharose strongly supports this. Although binding of Ofs directly to eIF4A was not measured, alignment of human and fly eIF4G proteins shows conservation of three out of four sets of amino acids necessary to bind eIF4A (Imataka, 1997). Of 12 crucial residues, ten were identical in Ofs, one was a conservative (L>I) change, and the twelfth diverged in Drosophila eIF4G as well. With regard to eIF4E binding, the putative binding site in Ofs has an arginine substituted for the usual hydrophobic residue. However, a similar substitution is tolerated in Drosophila eIF4E binding protein 1, and Baker (2007) presents evidence for interaction with Drosophila eIF4E1. Taken together, it is quite likely that Ofs participates in cap-dependent translation initiation (Franklin-Dumont, 2007).

eIF4G (CG10811) and Ofs (CG10192) appear to be the only two eIF4G proteins encoded in the fly genome. One other candidate, l(2)01424, is more related to the proposed translational inhibitor, NAT1/p97 (Rpn1)/DAP5, than to eIF4G proteins. Although the novel N-terminus of Ofs raised the possibility that it would play a role distinct from eIF4G, the data suggest that Ofs can act as the only eIF4G in cultured cells. Whether these two proteins always act redundantly in vivo cannot be assessed without mutations in eIF4G. Nevertheless, eIF4G, at its endogenous level, cannot substitute for Ofs in spermatocytes. Perhaps this is simply due to a relatively lower level of eIF4G compared with Ofs. Alternatively, Ofs might uniquely aid in the translation of a special class of mRNAs, specific to spermatocyte development. Perhaps sequences in its novel N-terminus assist in such a role. Although further experiments are needed to distinguish between these possibilities, one reason for a distinction between spermatocytes and other cells might be in their respective mode of growth control. In cultured eIF4G-deficient mitotic cells, the cell cycle effect observed was on G₁, whereas the defect in spermatocyte progression is in G₂. Although the G₁-S transition is the major control point for growth sensing in mitotic cells of the fly, G₂ might make more sense as the control point for meiosis, because it is during this phase of the cycle that spermatocytes need to prepare not just for division, but for differentiation. Furthermore, spermatocytes might commit to the meiotic cycle, versus returning to the mitotic cycle, during G₂, as is the case for the yeast Saccharomyces cerevisiae. Perhaps expressing a unique eIF4G (Ofs) in spermatocytes helps serve this role. Given the functional role for ofs, it is proposed that ofs henceforth be known as eIF4G2 (Franklin-Dumont, 2007).

Because ofs (eIF4G2) encodes the predominant eIF4G in spermatocytes, one might expect that mutant cells would exhibit decreased translation of many mRNAs. Just as a striking delay was found in in Boule accumulation, other proteins would be expected to be similarly affected. Such a global deficit could account for the delayed development of these cells, and would be predicted to influence cell size, because the translational capacity of a cell predicts its ability to accumulate mass. Indeed, one of the earliest phenotypes in eIF4G2 spermatocytes was their small size. Yet, Aly accumulation appeared normal and Rux protein appeared to accumulate to an excess degree in early spermatocytes. These data demonstrate that some mRNAs are not affected by the translational deficit, and raise an alternative scenario wherein spermatocytes actively monitor their size. If they do not achieve proper growth, a checkpoint is induced to prohibit meiosis and differentiation. Because meiosis involves two cell divisions with little intervening interphase, size monitoring would be especially important before these cells commit to divide (Franklin-Dumont, 2007).

Circumstantial support for a growth checkpoint includes the accumulation of the Cdk inhibitor Rux, which leads to aberrant behavior of Cyclin A. In this model, the postulated checkpoint causes the striking delay in the accumulation of Boule, which, in turn, explains the delay in Twine accumulation. Eventually, Boule does accumulate to reasonable levels, perhaps as cells leak through the checkpoint, just as eventually occurs in mitotic checkpoints. However, by then, Cyclin A has been degraded, and without it, the eventual accumulation of Twine cannot trigger meiosis, so the checkpoint has succeeded (Franklin-Dumont, 2007).

To establish that a checkpoint exists, one would need to identify the sensor, which detects the problem, and effectors, which execute inhibitory functions until the cell resolves the problem. No candidate is available for the sensor that detects growth at this time, nor for effectors controlling differentiation. However, it can be speculated that Rux is one effector regulating the meiosis branch, where it could serve to inhibit Cyclin A-driven Cdc2 kinase activity. Rux is not the only effector regulating meiosis, however. Previous work showed that directly increasing the level of Rux only blocked entry into the second meiotic division. Consequently, the accumulation of Rux that is observed in eIF4G2 mutants cannot fully explain the absence of the first meiotic division or the defect in differentiation. As would be typical for cell cycle regulation, several effectors must be activated at once to completely block the G₂-M transition (Franklin-Dumont, 2007).

The existence of other effectors could explain why forcing early Twine accumulation failed to restore meiotic entry to eIF4G2 mutants in a rux background. Alternatively, there might be additional positive factors necessary for G₂-M transition that have not accumulated in eIF4G2 spermatocytes. Consistent with this, prior work driving expression of another Cdc25, string (stg), in early spermatocytes directed a normal rather than a precocious G₂-M transition. Thus, advancing Cdc25 activity is insufficient to trigger a precocious G₂-M even in the absence of a growth defect. Perhaps early spermatocytes have not had enough time to accumulate an essential component, such as Cyclin B, for the meiotic divisions. It was found that eIF4G2 mutant clones exhibit Cyclin B levels comparable to neighboring heterozygous cells. However, there is a peak in Cyclin B accumulation just prior to meiosis I, and Baker (2007) describes a deficit of this Cyclin B peak in eIF4G2 mutants. Thus, Cyclin B remains a candidate factor (Franklin-Dumont, 2007).

Whether a growth checkpoint exists or not, mass accumulation could be used to time the G₂-M transition by coupling rate-limiting cell cycle proteins to the translational capacity of the cell. In the budding yeast, S. cerevisiae, cyclin CLN3 (also known as YHC3) contains an upstream open reading frame in the 5' UTR that slows its translation in G₁ under poor growth conditions. Similarly, during G₂ in the fission yeast, Schizosaccharomyces pombe, accumulation of CDC25 is disproportionately affected by defects in translation. Perhaps the translation of Boule, along with a few other meiotic cell cycle regulators, is disproportionately affected when translation is compromised in spermatocytes. Although this should be investigated, this simpler model does not explain the aberrant accumulation of Rux and the nuclear sequestration of Cyclin A that was observed (Franklin-Dumont, 2007 and references therein).

The defects in differentiation in eIF4G2 mutants are not secondary to the meiotic block, because several cell cycle mutants fail to divide but still undergo substantial post-meiotic differentiation. Several spermatid differentiation genes, such as don juan and fuzzy onions, are transcribed in primary spermatocytes under the control of spermatocyte arrest genes. Translational control delays the accumulation of their protein products. This delay is functionally relevant, because precocious don juan accumulation leads to sterility. In principle, then, the lack of significant differentiation in eIF4G2 mutants could simply be due to a more pronounced translational delay for key differentiation genes. Alternatively, the block in differentiation might reflect a direct effect of the proposed growth checkpoint. Consistent with either model, the accumulation of the mitochondrial fusion protein Fuzzy onions is delayed, although this was not timed precisely. It is expected that other differentiation targets will also be abnormally delayed in eIF4G2 mutants (Franklin-Dumont, 2007).

There are striking parallels to the role of eIF4G2 during spermatogenesis in other organisms. For instance, there are also two major isoforms of eIF4G in Caenorhabditis elegans, encoded by ifg-1. When the longest isoform was depleted from the germ line, oocytes arrested in meiosis I (B. D. Keiper, personal communication to Franklin-Dumont, 2007). The requirement for ifg-1 in spermatogenesis has not yet been examined. However, one of the five isoforms of eIF4E in the worm, IFE-1, is clearly essential for spermatogenesis. RNA interference against ife-1 results in delayed meiotic progression, and in defective sperm, in both hermaphrodites and males. Furthermore, mouse testes carrying the Y chromosome deletion Spy (also known as Eif2s3y-Mouse Genome Informatics) have a meiotic arrest phenotype due to a lack of EIF2 (also known as EIF2S2-Mouse Genome Informatics) function. Taken together, these examples suggest that translational control, and therefore possibly growth control, is a common theme for meiotic cycle cells (Franklin-Dumont, 2007).

Translational control of meiotic cell cycle progression and spermatid differentiation in male germ cells by a novel eIF4G homolog

Translational control is crucial for proper timing of developmental events that take place in the absence of transcription, as in meiotic activation in oocytes, early embryogenesis in many organisms, and spermatogenesis. Drosophila eIF4G2 is required specifically for male germ cells to undergo meiotic division and proper spermatid differentiation. Flies mutant for eIF4G2 are viable and female fertile but male sterile. Spermatocytes form, but the germ cells in mutant males skip the major events of the meiotic divisions and form aberrant spermatids with large nuclei. Consistent with the failure to undergo the meiotic divisions, function of eIF4G2 is required post-transcriptionally for normal accumulation of the core cell cycle regulatory proteins Twine and CycB in mature spermatocytes. Loss of eIF4G2 function also causes widespread defects in spermatid differentiation. Although differentiation markers Dj and Fzo are expressed in late-stage eIF4G2 mutant germ cells, several key steps of spermatid differentiation fail, including formation of a compact mitochondrial derivative and full elongation. These results suggest that an alternate form of the translation initiation machinery may be required for regulation and execution of key steps in male germ cell differentiation (Baker, 2007).

Although precedent for developmentally regulated translation initiation factor components comes from data on the cap binding protein eIF4E, such as Caenorhabditis elegans IFE-1 and IFE-4, and various eIF4Es from Drosophila, zebrafish and mammals, less is known about the potential for the core eIF4G subunit to show such tissue specificity. In a human hematopoetic stem cell line, eIF4GII is specifically recruited to 5' cap structures of mRNAs upon thrombopoietin-mediated induction of megakaryocyte differentiation, whereas levels of eIF4GI at the cap remain constant. However, this recruitment of eIF4GII could represent an overall increase in active initiation factor complex within differentiating megakaryocytes, rather than intrinsic transcript specificity on the part of eIF4GII (Baker, 2007).

Function of Drosophila eIF4G2 is required for both meiotic cell cycle progression and for many aspects of spermatid differentiation. However, loss of eIF4G2 does not cause meiotic arrest. The eIF4G2 loss-of-function phenotype in testes is different from the phenotype of mutations in the testis TAFs (tTAFs). In tTAF mutant males, spermatocytes arrest at the G2/M transition, fail to undergo meiotic division and show a complete absence of spermatid differentiation. By contrast, in eIF4G2 mutant males, germ cells appear to skip the major events of meiotic division but initiate spermatid differentiation. Germ cells in males mutant for the cell cycle phosphatase Twine, or cdc2^ts mutant males shifted to the non-permissive temperature, also skip the major events of meiotic division but proceed to execute spermatid differentiation. These data show that initiation and execution of the spermatid differentiation program can proceed even when male germ cells fail to execute the meiotic divisions (Baker, 2007).

The failure to undergo the meiotic divisions in eIF4G2 is likely to be due, at least in part, to failure to upregulate twine and cycB translation as spermatocytes mature. Although eIF4G2 is a homolog of a known translation initiation factor, and eIF4G2 mutant spermatocytes have defects in translation of cycB and twine, it is formally possible that eIF4G2 does not act directly on these transcripts, but rather on an upstream regulator of their translation. Future experiments will address whether eIF4G2 binds these two mRNAs, to determine whether its effect on their translation is likely to be direct or indirect (Baker, 2007).

Function of eIF4G2 also appears to be required for many aspects of spermatid differentiation. Although early spermatids form in eIF4G2 mutant males, the mitochondrial cloud fails to condense and form a compact mitochondrial derivative, and very little spermatid elongation takes place. The defects in spermatid differentiation in eIF4G2 mutant males are more severe than the defects observed in males mutant for the RNA-binding protein Boule, homolog of human BOULE and DAZL. These observations suggest that although both Boule and eIF4G2 are required for normal translation of twine, the requirement for eIF4G2 is more widespread. A broad requirement for eIF4G2 for timing or execution of many events during male germ cell differentiation is reflected in the pleiotropic nature of the eIF4G2 mutant phenotype in testes. Loss-of-function of eIF4G2 also affects spermatocyte growth as well as timing of events of the meiotic program in primary spermatocytes (Baker, 2007).

Given the broad defects observed in male germ cells, the predicted role of eIF4G2 in translation initiation, and the apparent reduction in transcript levels for the canonical eIF4G, it was surprising that Fzo and Dj proteins were expressed in spermatids from eIF4G2 mutant males. These findings suggest that eIF4G2 is not required (directly or indirectly) for translation of all mRNAs in mature spermatocytes and post-meiotic germ cells. It is possible that some of the canonical eIF4G protein persists from earlier germ cell stages, sufficient for translation of fzo and dj. However, if so, this is not sufficient for robust translation of cell cycle regulators twine and cycB in late spermatocytes, or for sufficient translation of additional mRNAs required for proper spermatid differentiation (Baker, 2007).

Binding of Drosophila ORC proteins to anaphase chromosomes requires cessation of mitotic cyclin-dependent kinase activity

The initial step in the acquisition of replication competence by eukaryotic chromosomes is the binding of the multisubunit origin recognition complex, ORC. A transgenic Drosophila model is described which enables dynamic imaging of a green fluorescent protein (GFP)-tagged Drosophila melanogaster ORC subunit, DmOrc2-GFP. It is functional in genetic complementation, expressed at physiological levels, and participates quantitatively in complex formation. This fusion protein is therefore able to depict both the holocomplex DmOrc1-6 and the core complex DmOrc2-6 formed by the Drosophila initiator proteins. Its localization can be monitored in vivo along the cell cycle and development. DmOrc2-GFP is not detected on metaphase chromosomes but binds rapidly to anaphase chromatin in Drosophila embryos. Expression of either stable cyclin A, B, or B3 prevents this reassociation, suggesting that cessation of mitotic cyclin-dependent kinase activity is essential for binding of the DmOrc proteins to chromosomes (Baldinger, 2009).

DmOrc2 was chosen for two reasons. First, it constitutes an essential part of the ORC core. As such, it was expected to better reflect the localization of the complex in its origin-defining function compared to peripheral ORC subunits. Second, several null alleles of k43, the DmOrc2 gene, have been identified, allowing pursuit of a genetic complementation strategy to verify transgene functionality. Rescue transgenes consisted of genomic copies of the DmOrc2 gene in which GFP was inserted in frame, coding for either an N- or C-terminal fusion of the ORC subunit. Using complementation as the most conclusive genetic criterion, both fusions proved functional. This indicates DmORC's flexibility to accommodate substantial heterologous protein moieties, exemplified in this study by GFP attached in two independent positions within the complex structure. In particular for a protein like DmOrc2, functioning as an integral part of a multiprotein complex, it was essential to avoid an overexpression situation, as in the absence of authentic binding partners its fluorescence signal is expected to mislocalize. Thus, fly lines with physiological DmOrc2 expression levels and the quantitative participation of the fusion protein in the holo- or the core complex were a prerequisite to address the cell cycle events governing DmORC's interaction with chromosomes by a biologically meaningful experimental approach. Both criteria were met in the DmOrc2-GFP transgenic as well as the rescue lines. Thus, for all parameters tested, the Drosophila model reflected faithfully the behavior of endogenous DmOrc2, allowing the visual tracing of DmORC-GFP. The use of the term 'DmORC-GFP' therefore refers to both the DmORC core and the holocomplex formed upon DmOrc1 association, which cannot be distinguished by the chosen imaging approach (Baldinger, 2009).

Aside from ensuring the congruence between transgenic and endogenous ORC subunits, this experimental strategy of tracking ORC during the cell cycle also avoids ambiguities sometimes associated with the fixation or physiological stressing of cells and organisms. Notwithstanding such methodological issues, the observed dynamics of ORC-chromatin interactions reported previously suggested a significant divergence between different biological systems. In yeast, ORC binds chromatin throughout the cell cycle, including metaphase, as has also been reported for embryonic Drosophila and mammalian ORC core subunits. Other studies came to the conclusion that members of the mammalian core complex are mostly excluded from metaphase chromatin, similar to Xenopus and also Drosophila larval neuroblasts. Based on immunolocalization studies, the latter analysis came also to the conclusion that DmOrc2 accumulates on late anaphase/telophase chromosomes, similar to the current findings. Differences in the reported localization patterns of ORC can possibly be explained by cell-type-dependent or, in particular, by interspecies variations in the control mechanism of the cell division cycl (Baldinger, 2009).

From the imaging analysis in this in vivo model, it is concluded that the majority of DmORC-GFP is displaced from the chromosomes in early mitosis and diffusely distributed throughout the cell without any recognizable localization pattern. Therefore, current models of the embryonic Drosophila ORC cycle should be scrutinized when they place the core DmORC on mitotic chromosomes. Toward the end of mitosis, DmORC-GFP is chromatin bound again, and this relocalization seems to be quantitative within the detection limits of the methodology employed. No principal differences were observed in this dynamic behavior of DmORC-GFP between syncytial and cellularized stages of embryonal development (Baldinger, 2009).

Proteolytic control of ORC core subunits has not been reported so far. In line with this lack of evidence, this study does not indicate that DmORC-GFP levels are subject to mitosis-specific protein degradation (as are other regulators of cell cycle progression), with the fluorescence signal of DmORC-GFP clearly visible in early mitosis, before gradually refocusing on late mitotic chromosomes. This entire process might be completely attributed to control over intracellular localization of DmORC-GFP during the cell cycle. However, while a substantial resynthesis of DmORC core subunits appears unlikely given the observed timing of this process, in particular with the additional requirements for complex assembly and chromophore maturation, a partial destruction of core DmORC subunits, followed by chromosomal recruitment of DmORC from cytoplasmic pools at the onset of a new round of pre-RC formation, cannot be ruled out (Baldinger, 2009).

Origin specification and pre-RC assembly in eukaryotes start with the chromatin binding of ORC. This study showed the cell-cycle-dependent changes of DmORC-GFP localization in embryos. Its rapid accumulation on chromosomes is detectable by late anaphase when CDK activity drops to the low levels observed in the late M and early G1 phases. The dependence of DmORC-GFP chromosome binding on low CDK activity was established by following the fluorescence signal upon cell cycle arrest in response to the expression of stable mitotic cyclins A, B, and B3, which are not subject to proteasomal degradation. Their presence prevented chromatin binding of DmORC-GFP. Previous reports describing the reloading of ORC to late mitotic chromatin in various cellular systems of metazoan origin have implicated mitotic CDKs in this process, supported by corresponding biochemical analyses. In Drosophila, it is known that the expression of individual stable cyclins does not interfere with the cell-cycle-controlled degradation of the endogenous cyclins. Thus, this in vivo analysis allows extension of the general assumption of a role for mitotic CDK involvement in triggering the start of pre-RC assembly to specifically conclude that all mitotic CDK/cyclin activities have to cease for DmORC-GFP to become chromatin bound (Baldinger, 2009).

How can this dynamic behavior of DmOrc2 be interpreted in the light of previous observations regarding the APC-dependent degradation of DmOrc1 in late mitosis, only to reemerge in late G1? Even when considering that metazoan Orc1 often shows expression, localization, and turnover patterns independent of other ORC subunits, reflecting temporal events in the control over ORC activity, the almost converse mitotic shuttling patterns of DmORC subunits are somewhat surprising. It should be noted, however, that DmOrc1-GFP could also be detected on telophase chromosomes before being degraded. Most studies of metazoan ORC concur that Orc1 is essential to establish initial DNA binding of ORC and subsequent steps of pre-RC formation, supported by the recent finding that elevated Orc1 levels can actually promote binding of endogenous Orc proteins during late mitosis. It is conceivable that in Drosophila this process takes place during a brief time window in late mitosis and could be sufficient to trigger the recruitment of other pre-RC proteins, which according to most analyses occurs prior to late G1. Alternatively, the remaining chromatin-bound DmORC core might be sufficient to promote completion of the pre-RC. From these lines of reasoning, it is already obvious that further experiments, in directly comparable settings for both the experimental protocols followed as well as for the cell types and developmental stages analyzed, will be required to resolve this issue. This will be facilitated by the availability of Drosophila orc1^-/- lines (Baldinger, 2009).

After this initial step in pre-RC assembly, other replication initiation proteins have to be loaded on chromosomes for them to become licensed for replication. Among these factors is the heteromultimeric minichromosome maintenance (MCM) complex, associated with a DNA helicase activity. Previous immunolocalization studies of the association/dissociation cycles of Drosophila MCM demonstrated their binding to mitotic chromatin upon cell cycle arrest by expression of stable cyclin B, corresponding to early anaphase stages. Assuming an unconditional requirement for prebound ORC for MCM chromatin binding, the current data would predict MCM binding at later cell cycle stages, after cessation of mitotic CDK activity. At first glance, these results on the timing of MCM-chromatin association might not be easy to reconcile with the current findings but can be explained by (1) the influence of the imaging methodology as describe for DmORC localization, (2) different sensitivity thresholds of the detection systems, or (3) potential uncharacterized effects of the stable cyclin-CDK complexes used in different studies. In any case, no real discrepancy is seen, since chromatin loading of MCM proteins in unperturbed cell cycles has only been evident in late anaphase/telophase, fully compatible with the current results for DmORC in both perturbed (i.e., stalled by stable cyclin expression) and unperturbed cell cycles (Baldinger, 2009).

It will be interesting to determine if this binding is partly responsive to potential changes in chromosome structure occurring as mitotic chromosomes pass toward telophase or whether DmORC responds directly or indirectly to changes in the kinase environment of late mitotic cells. The latter possibility would argue that a decrease in DmORC's phosphorylation state results in its increased affinity to chromatin. This scenario appears attractive since it has been demonstrated that in vitro binding of DmORC to DNA is strongly diminished whenever it is phosphorylated by various CDK/cyclin activities. Combined with the current cytological studies, these findings make mitotic CDKs attractive candidate kinases for actively suppressing DmORC binding to chromatin. This view is also in line with the localized cyclin destruction in syncytial cell cycles of Drosophila. The resulting abrogation of CDK1 activity in the vicinity of the mitotic spindle can be monitored by the distribution of phospho-histones, akin to the observed gradual rebinding of DmORC, starting from the centromeric regions of anaphase chromosomes (Baldinger, 2009).

In summary, this study report the spatial and temporal dynamics of the initiator protein ORC in a live metazoan organism. Along with the cell cycle, ORC periodically associates with and dissociates from chromatin. The initial interaction in preparation for the next chromosome cycle occurs in late anaphase. This binding of ORC to chromatin depends critically on the cessation of mitotic cyclin activity, linking this first step of replication licensing to the CDK-driven control pathways of cell cycle progression. Finally, it is obvious that different mechanisms evolved between species controlling the activities of ORC. While all of them are compatible with the general requirements for origin definition, pre-RC assembly, and the prevention of rereplication, it cautions against the extrapolation of findings from one experimental system to another. This underscores the value of multipurpose in vivo models like the one described in this study, allowing a comprehensive approach for probing ORC functions. Its use should not be restricted to further exploring ORC in DNA replication initiation, but it should also be useful to study ORC's role in proliferation and in the development of an organism (Baldinger, 2009).

Genome-wide analysis reveals a cell cycle-dependent mechanism controlling centromere propagation

Centromeres are the structural and functional foundation for kinetochore formation, spindle attachment, and chromosome segregation. In this study, factors required for centromere propagation were isolated using genome-wide RNA interference screening for defects in centromere protein A (CENP-A; centromere identifier [CID]) localization in Drosophila. The proteins CAL1 and CENP-C were identified as essential factors for CID assembly at the centromere. CID, CAL1, and CENP-C coimmunoprecipitate and are mutually dependent for centromere localization and function. The mitotic cyclin A (CYCA) and the anaphase-promoting complex (APC) inhibitor RCA1/Emi1 were identified as regulators of centromere propagation. CYCA was shown to be centromere localized, and CYCA and RCA1/Emi1 were shown to couple centromere assembly to the cell cycle through regulation of the fizzy-related/CDH1 subunit of the APC. These findings identify essential components of the epigenetic machinery that ensures proper specification and propagation of the centromere and suggest a mechanism for coordinating centromere inheritance with cell division (Erhardt, 2008).

This is the first example of a genome-wide RNAi screen for mislocalization of an endogenous chromosomal protein and provides the distinct advantage that the primary screen output is a direct readout of the phenotype of interest. This approach identified novel and known factors that control the assembly of centromeric chromatin and link centromere assembly and propagation to the cell cycle (Erhardt, 2008).

Although centromere assembly has been described as a hierarchical process directed by CENP-A, the data show that CID, CENP-C, and CAL1 are interdependent for centromere propagation, which is consistent with experiments in vertebrate cells showing interdependence between the CENP-H-CENP-I complex and CENP-A. However, studies in C. elegans and vertebrates have not detected a role for CENP-C in CENP-A chromatin assembly, suggesting that CENP-C plays a more prominent role in regulating centromere propagation in flies. Collectively, these results suggest that CENPs that depend on CENP-A for their localization may 'feed back' to control CENP-A assembly. Histone variants are assembled into chromatin both by histone chaperones (e.g., the histone H3.3-specific chaperone HIRA [histone regulatory A] that provides specificity to the CHD1 chromatin-remodeling ATPase) and by histone variant-specific ATPases (e.g., Swr1 that can use the general chaperone Nap1 or the specific chaperone Chz1 to assemble H2A.Z). CENP-C or CAL1 might facilitate centromere-specific CID localization by providing centromere specificity to a chromatin-remodeling ATPase in a manner analogous to HIRA or might direct the localization of chromatin assembly factors to the centromere. It will be interesting to determine what factors associate with CAL1 and CENP-C as a route to elucidating the mechanisms of centromere assembly and propagation (Erhardt, 2008).

The loading of CENP-A in human somatic cells and in Drosophila embryos occurs after anaphase initiation when APC^FZR/CDH1 activity is high. Ubiquitin-mediated proteolysis facilitates formation of a single centromere by degrading noncentromeric CENP-A, and subunits of the APC are localized to kinetochores. The results demonstrate that normal regulation of APC^FZR/CDH1 activity is required for centromere propagation, providing a link between centromere assembly and cell cycle regulation (Erhardt, 2008).

Two alternative models are proposed for the role of APC^FZR/CDH1 in centromere function. The first model is that CYCA is the relevant substrate of APC^FZR/CDH1 and that the kinase activity of the CYCA-Cdk1 complex is required for the localization of CID, CENP-C, and CAL1 to the centromere. CYCA is normally degraded as cells proceed through mitosis, suggesting that CYCA-Cdk1 would likely act during G2 or early M to phosphorylate a substrate involved in centromere assembly. The CID and CENP-C localization defect caused by CYCA depletion was rescued by the simultaneous depletion of FZR/CDH1 even though the levels of CYCA protein remained low in the double depletion. The rescue of the CID and CENP-C localization defect in cells with low CYCA protein suggests that maintaining high levels of CYCA-Cdk1 activity is not required for centromere propagation, but it cannot be ruled out that the residual CYCA protein in these cells is sufficient to rescue the centromeric phenotype when APC activity is compromised by FZR/CDH1 depletion (Erhardt, 2008).

The second model that is consistent with these observations is that one or more APC^FZR/CDH1 substrates (“X”) regulate the interdependent localization of CID, CENP-C, and CAL1 to the centromere. RCA1 and CYCA inhibit the APC in G2 to allow mitotic cyclin accumulation. An APC^FZR/CDH1 substrate could repress centromere assembly until anaphase/G1, when proteolysis would remove the repression in a manner analogous to replication licensing. If an APC^FZR/CDH1 substrate acted solely as a negative regulator of centromere assembly, FZR/CDH1 depletion should prevent CID assembly at centromeres, and premature APC^FZR/CDH1 activation by CYCA or RCA1 depletion might cause an increase of CID at centromeres as a result of premature assembly. It was observed that neither CDH1 nor CDC20 depletion alone impacted CID, CAL1, or CENP-C assembly at centromeres or the overall levels of these proteins but that premature APC activation resulted in failed centromere assembly (Erhardt, 2008).

A simple interpretation of the results is that CYCA-Cdk1 or another APC^FZR/CDH1 substrate acts during G2/metaphase before APC^FZR/CDH1 activation to make centromeres competent for assembly during anaphase and/or G1. Premature removal of the APC^FZR/CDH1 substrate would cause failure to relicense the centromeres for assembly in the next G1 phase. When compared with the process of replication licensing, in which the positive regulator CDC6 and the negative regulators geminin and CYCA are all substrates of APC^FZR/CDH1, the model of a single APC^FZR/CDH1 substrate that controls centromere licensing or propagation may be oversimplified. This study observed that defective centromere localization of CID and CENP-C after CYCA or RCA1 depletion was not rescued by CDC20 depletion, but a role for APC^FZY/CDC20 in centromere propagation cannot be ruled out because premature APC^FZR/CDH1 activation could mask a subsequent role for FZY/CDC20, which is activated at the metaphase/anaphase transition (Erhardt, 2008).

It is not yet known whether the localization of CYCA at centromeres is important for the regulation of centromere assembly. In Drosophila, it has been demonstrated that the subcellular localization of CYCA is not important for proper progression through the cell cycle; however, these experiments did not directly address whether mislocalization of CYCA prevented the association of CYCA with centromeres. It will be interesting to determine whether CID, CENP-C, and CAL1 localization require centromere-localized CYCA-Cdk1 activity or whether any of these proteins are a direct target of CYCA-Cdk1 (Erhardt, 2008).

The results suggest that CID or CAL1 levels are indirectly controlled by APC activity. Interestingly, the human M18BP1 has recently been proposed to act as a 'licensing factor' for centromere assembly. Although no clear homologues of M18BP1/KNL2 have been identified in Drosophila, both CAL1 in flies and M18BP1/KNL2 in other species are interdependent with CENP-A for centromere localization. Strikingly, levels of CAL1 and M18BP1/KNL2 are reduced on metaphase centromeres and increase coincident with CENP-A loading in late anaphase/telophase. Further analysis is required to determine whether CAL1 and M18BP1/KNL2 function analogously in centromere assembly. It will be important to determine whether fly homologues of other Mis18 complex components are associated with CAL1 and important for centromere assembly. Identifying the APC substrates involved in centromere assembly will be necessary to distinguish between these models and to determine how these proteins epigenetically regulate centromere assembly and couple this essential process to the cell cycle (Erhardt, 2008).

CycA is involved in the control of endoreplication dynamics in the Drosophila bristle lineage

Endocycles, which are characterised by repeated rounds of DNA replication without intervening mitosis, are involved in developmental processes associated with an increase in metabolic cell activity and are part of terminal differentiation. Endocycles are currently viewed as a restriction of the canonical cell cycle. As such, mitotic cyclins have been omitted from the endocycle mechanism and their role in this process has not been specifically analysed. In order to study such a role, this study focused on CycA, which has been described to function exclusively during mitosis in Drosophila. Using developing mechanosensory organs as model system and PCNA::GFP to follow endocycle dynamics, it was show that (1) CycA proteins accumulate during the last period of endoreplication, (2) both CycA loss and gain of function induce changes in endoreplication dynamics and reduce the number of endocycles, and (3) heterochromatin localisation of ORC2, a member of the Pre-RC complex, depends on CycA. These results show for the first time that CycA is involved in endocycle dynamics in Drosophila. As such, CycA controls the final ploidy that cells reached during terminal differentiation. Furthermore, the data suggest that the control of endocycles by CycA involves the subnuclear relocalisation of pre-RC complex members. This work therefore sheds new light on the mechanism underlying endocycles, implicating a process that involves remodelling of the entire cell cycle network rather than simply a restriction of the canonical cell cycle (Sallé, 2012).

The data reveal a role for CycA in the control of S phase in Drosophila. Until now, CycA has been considered exclusively as a mitotic cyclin in this organism. Both up- and downregulation of CycA levels reduces the number of endocycles. In particular, the transition between early and late S phase is either advanced or delayed after CycA up- or downregulation respectively. In addition, the duration of the late S phase is increased under both conditions. The combination of these effects leads to a reduction in ploidy. Finally, it was shown that CycA controls the localisation of the pre-replicative factor ORC2, an observation that may explain the effects of CycA on endocycle dynamics. All of these observations were performed under CycA hypomorphic conditions that do not affect mitoses. Collectively, these results show that the mechanism underlying endocycles requires a very low level expression (even undetectable) of major cell cycle regulators such as CycE and CycA (Sallé, 2012).

In Drosophila, CycA has been found in exclusive association with Cdk1, rather than with both Cdk1 and Cdk2 as in mammals. As such, CycA has been always considered as a mitotic cyclin. Several observations suggest that CycA is implicated in DNA replication, however. CycA overexpression can trigger S phase, even in a CycE mutant background. This study shows that CycA has a bona fide function that influences the progression of the S phase, and that more precisely involves the control of late S-phase timing during endocycles. Two possible mechanisms could account for these results: either CycA affects components required for overall S-phase progression or it modulates the activity of specific members of the core replication machinery. The former hypothesis is supported by data showing that in Drosophila Cdk1 and CycA cooperatively inhibit transcriptional activation by affecting the essential S-phase regulator E2F1. In addition, degradation of E2F1 is related to its CycA-dependent phosphorylation in mammalian cells. However, during endocycles in the bristle lineage, neither overexpression nor downregulation of CycA had a significant effect on the level of E2F1 accumulation. Hence, the results favour a mechanism where CycA regulates specific components of the replication machinery. Indeed, CycA was shown to be required for the relocalisation of the pre-RC protein ORC2 during the early-to-late S-phase transition (Sallé, 2012).

Classically, ORC2 is considered to be part of the pre-RC. However, the pre-RC component MCM5 and the replication factor PCNA::GFP do not undergo CycA-dependant control of their localisation, as was observed for ORC2. Although the possibility that localisation of Cdc6 or Cdt1 (or other components) might depend on CycA activity cannot be ruled out, these data suggest that ORC2 acts independently of the pre-RC. Indeed, apart from its well-known role in DNA replication, ORC2 has been shown to participate in heterochromatin maintenance. Moreover, ORC proteins localise to heterochromatin in vivo and interact directly with the essential heterochromatin factor HP1. As such, ORC2 could act as a gatekeeper between euchromatin and heterochromatin or, alternatively, it could be implicated in the establishment or the maintenance of chromatin structure. After CycA overexpression, the observed lengthening of late S-phase is probably due to stable relocalisation of ORC2 to the heterochromatin that, concomitant with the high CycA activity, induces a complete replication of heterochromatin. In addition, under conditions of CycA loss of function where ORC2 does not relocalise to heterochromatin, it was often observed that chromosomes were under-condensed. Similarly, irregularly condensed mitotic chromosomes were observed in Drosophila and in mammalian ORC2-depleted cells. In any case, the fact that heterochromatin can still be formed in the absence of ORC2 relocalisation suggests that ORC2 is necessary but not essential for heterochromatin maintenance. This dispensability of ORC2 has been also observed for endoreplication, as, in orc2 mutants, endocycles occurred but cell ploidy was reduced. These data are therefore in agreement with results showing that CycA controls ORC2 localisation and modulates endocycles dynamics. Thus, lengthening of the late S-phase observed after CycA mis-regulations probably reflects a dual function for ORC2 in both DNA replication and chromatin structure (Sallé, 2012).

These data show that the timing of the early S phase was also affected under conditions of CycA mis-regulations. Early S-phase was lengthened under the CycA downregulation condition and conversely shortened after CycA overexpression. As the data showed that CycA begins to accumulate at the end of early S phase, this suggests that CycA controls the transition between early and late S phase. Under CycA downregulation conditions, it was observed that ORC2 remained throughout the nucleus. This maintenance of ORC2 in the euchromatic regions may explain the delay in transitioning from early to late S phase as due to the stabilisation of a permissive state for euchromatin replication. Strengthening the case that CycA is important for this process, DSBs detected by gammaH2AV immunoreactivity are also induced under these conditions. These ectopic DSBs were not associated with ectopic heterochromatin foci, arguing against them being caused by heterochromatin boundaries. This suggests that DSBs reflect partial replication due to the low level of Cdk activity. As such, the results imply that CycA is involved in the control of DNA replication per se. These results are in agreement with recent data of Ding and MacAlpin (2010) on DNA re-replication, suggesting that Cdk/CycA activity specifically inhibits pre-RC assembly in the euchromatin (Sallé, 2012).

Using DSBs to assay for replication defects, it was shown that ORC2 overexpression also induced similar replication impairment as CycA depletion. These results suggest that CycA-dependent ORC2 intra-nuclear re-localisation would be a way to deplete euchromatin of ORC2, concluding replication and generating a period during which euchromatic DNA is unable to relicense. This model is also consistent with observations made in other systems, for example, in CHO hamster cells where Cdk1/CycA was able to hyper-phosphorylate ORC1 and decrease its affinity for chromatin. Moreover, it has also been shown that Cdk1/CyclinA can phosphorylate ORC2 in vitro. All these data support a model where CycA, in a dose-dependent manner, promotes ORC2 shuttling between euchromatin and heterochromatin as a mechanism to accomplish the early S phase (Sallé, 2012).

These data complete the current model of endocycle progression by incorporating the mitotic factor CycA. In this model, CycE activity oscillations, mediated in part by E2F1 activity, drive cyclic anti-parallel oscillations of APC/C-Fzr activity. Periods of low APC/C activity would permit accumulation of pre-RC complex proteins, such as ORC1 and Cdc6. As such, these oscillating activities generate S- and G-phase time windows, which ensure that the genome is properly replicated once in every cycle. It is suggest that CycA reinforces the exit from the replicative state, probably by controlling localisation and/or activity of pre-RC factors such as ORC2 (Sallé, 2012).

Finally, this work reveals a novel vision for the mechanism underlying endocycles. This mechanism appears not to be a subset of that controlling mitosis. Rather, this study show that, as the same factors participate in both processes, the mechanism involved in endocycles and mitotic cycles differs only by quantitative variations in protein levels and probably by the temporal expression of these factors (Sallé, 2012).

RNA helicase Belle (DDX3) is essential for male germline stem cell maintenance and division in Drosophila

This study showed that RNA helicase Belle (DDX3) was required intrinsically for mitotic progression and survival of germline stem cells (GSCs) and spermatogonial cells in the Drosophila melanogaster testes. Deficiency of Belle in the male germline resulted in a strong germ cell loss phenotype. Early germ cells are lost through cell death, whereas somatic hub and cyst cell populations are maintained. The observed phenotype is related to that of the human Sertoli Cell-Only Syndrome caused by the loss of DBY (DDX3) expression in the human testes and results in a complete lack of germ cells with preservation of somatic Sertoli cells. This study found the hallmarks of mitotic G2 delay in early germ cells of the larval testes of bel mutants. Both mitotic cyclins, A and B, are markedly reduced in the gonads of bel mutants. Transcription levels of cycB and cycA decrease significantly in the testes of hypomorph bel mutants. Overexpression of Cyclin B in the germline partially rescues germ cell survival, mitotic progression and fertility in the bel-RNAi knockdown testes. Taken together, these results suggest that a role of Belle in GSC maintenance and regulation of early germ cell divisions is associated with the expression control of mitotic cyclins (Kotov, 2016).

This study shows that RNA helicase Belle (DDX3) is required cell-autonomously for the survival and divisions of GSCs in Drosophila testes. In bel6/neo30 mutants as well as in the case of germline-specific RNAi belKD rapid elimination of germ cells via apoptosis occurred. But what events could trigger apoptosis? To address this issue, larval testes were analyzed. Testes of bel6/neo30 mutant larvae still contained all populations of early germ cells. This observation indicates that primordial germ cells (PGCs) correctly migrate into embryonic gonads during mid to late embryogenesis. In the mutant larval testes the wild-type hub and GSCs adjacent to the hub were clearly detected. It is known that the mechanism of capturing GSCs and CySCs to the hub involves a high level of adhesion molecule E-Cad on the hub/stem cells interface. In testes with STAT depletion the expression of E-Cad is severely disrupted accounting for the defects in hub-GSC adhesion and for the subsequent loss of GSCs. However, it was determined that STAT expression (in CySCs) and consequently upstream Upd signaling from the hub were not disrupted in the bel6/neo30 testes. Although the amount of Belle was strongly reduced in the bel6/neo30 testes, no reduction of E-Cad level was observed in CySCs. On the contrary, high ectopic expression of E-Cad was detected on the surface of CySCs surrounding the hub. The influence of Belle on the adherens junction formation in GSCs cannot be directly estimated. However, due to adhesion failures a loss of GSCs via premature differentiation could be expected followed by normal development of newly formed germline cysts. In contrast, this study detected a reduced germ cell content and morphological abnormalities of early germ cells including their giant nuclear and cellular sizes. It is assumed that these germ cells could not enter mitosis and are delayed in the G2 phase. Failure to enter mitosis after G2 delay appears to induce germ cell apoptosis in the bel testes, as previously has been shown for the how testes (Kotov, 2016).

It is known that Drosophila mitotic cyclins, Cyclin A, Cyclin B and Cyclin B3, each form complexes with Cdc2, and they appear to function synergistically to provide a progression throughout mitosis. Sufficient levels of mitotic cyclins must be accumulated at the end of G2 to ensure the onset of mitosis. To date, cell cycle regulation of GSCs and their daughter gonial cells is still poorly understood. It is known that PGCs suppress mitotic activity during their migration to embryonic gonads due to translational repression of maternal cycB mRNA via its 3′UTR by Pumilio–Nanos complex and other unidentified factors. Pumilio and Nanos are also known to be expressed in GSCs of gonads of adult flies and are found to be essential for GSC maintenance. However, factors overriding the repressive Pumilio-Nanos-dependent signal and providing expression of zygotic Cyclin B protein during normal testis development are currently unknown (Kotov, 2016).

It has been shown that Cyclin B and Cyclin A, but not Cyclin B3, are required in the gonad for the maintenance of early germ cells. A mutational depletion of Cyclin B leads to a complete missing of germ cells in the adult testes and their significant loss in the ovaries. However, the requirements for Cyclin A expression for the survival of early germ cells are currently obscure. It is known that overexpression of Cyclin A or expression of its nondegradable form leads to a rapid loss of GSCs in the ovaries (Kotov, 2016).

This study found that the previously published cycB testis phenotype mimicked that in the case of bel6/neo30 mutants and germline-specific RNAi belKD. It was determined that both of the mitotic cyclins, but not Cyclin E and Cdc2, were significantly decreased in the belEY08943/neo30mutant testes. Furthermore, a considerable decrease was found of cycA and cycB mRNA levels. These results suggest a specific contribution of Belle to the transcriptional regulation of mitotic cyclins in the germline. It was also revealed that the constitutive level of Cyclin B expression in control testes was significantly lower than in control ovaries. Assuming that Belle regulates mitotic cyclins in a similar way in the germline of both sexes, it is believed that deficiency of Belle has a more severe effect on spermatogenesis, due to a sharply reduced level of Cyclin B protein below a threshold, whereas its dose in the bel6/neo30 ovaries is still sufficient to allow mitosis to occur. In support of this hypothesis a partial rescue was achieved of the RNAi belKD testis phenotype by transgenic germline-specific expression of Cyclin B, but not by Cyclin A overexpression (Kotov, 2016).

The reduction of cycB transcription in belEY08943/neo30 testes places cycB downstream of bel. In rescue experiments a third copy of cycB was added to the system employing the germinal nos-Gal4 driver in combination with UAS-bel RNAi hairpin. It is assumed that the reduction of Belle in RNAi belKD testes would negatively affect the expression of both endogenous and transgenic cycB. In accordance with this assumption only a partial restoration of the Cyclin B protein level and only partial rescue of spermatogenesis was achieved. The data indicate that at least one crucial requirement for Belle in early germ cells is relevant to Cyclin B level maintenance for ensuring germ cell mitosis (Kotov, 2016).

To date, evidences of participation of DDX3 proteins in cell cycle control both at the level of transcription and translations have been obtained. DDX3 specifically cooperates with transcription factor Sp1 to positively regulate the transcription of p21waf gene. A temperature-sensitive mutation of ddx3 gene in golden hamster cell culture at nonpermissive temperature leads to G1 arrest, which is accompanied by a decline of cycA mRNA and rather suggests the transcriptional level of regulation for cycA. It has been shown that in human HeLa cells DDX3 interacts with the GC-rich, highly structured 5'UTR of Cyclin E1 mRNA and regulates its translation initiation and a knockdown of DDX3 delays the entry to the S phase. DED1, a Schizosaccharomyces pombe homolog of DDX3, is involved in the translational control of B-type cyclin mRNAs (Cig2 and Cdc13), which have extended and expectedly highly structured 5'UTRs. It is known that only a single cyclin-dependent kinase and two B-type cyclins regulate both the S phase and mitosis in yeasts. Indeed, temperature-sensitive mutations of ded1 gene inhibit B-type cyclin translation and arrest cell cycle at both S phase and G2/M transition, whereas both cig2 and cdc13 mRNA levels remain unchanged (Kotov, 2016 and references therein).

This study has presented experimental evidence that Belle has specific and essential functions in the male germline associated with proper transcriptional regulation of mitotic cyclin expression. The testis phenotype observed in Drosophila is similar to the SCOS phenotype in human testes, indicating a conserved function of DDX3 in spermatogenesis. Understanding the molecular basis for DBY (DDX3) functions in mammalian germ cell maintenance has proven to be challenging. The functions and regulation of A-type and B-type cyclins in mammalian spermatogenesis are not clearly understood. In this case, a study in the Drosophila model provides a useful insight into the mechanism of GSC maintenance in the male germline. The current findings support a mechanism according to which the determination of the fate of male GSCs is closely connected with the control of mitosis via the regulation of mitotic cyclin levels (Kotov, 2016).

Post-transcriptional Regulation

LOTUS domain protein MARF1 binds CCR4-NOT deadenylase complex to post-transcriptionally regulate gene expression in oocytes

Post-transcriptional regulation of gene expression plays an essential role during oocyte maturation. This study reports that Drosophila MARF1 (Meiosis Regulator And mRNA Stability Factor 1), which consists of one RNA-recognition motif and six tandem LOTUS domains with unknown molecular function, is essential for oocyte maturation. When tethered to a reporter mRNA, MARF1 post-transcriptionally silences reporter expression by shortening reporter mRNA poly-A tail length and thereby reducing reporter protein level. This activity is mediated by the MARF1 LOTUS domain, which binds the CCR4-NOT deadenylase complex. MARF1 binds cyclin A mRNA and shortens its poly-A tail to reduce Cyclin A protein level during oocyte maturation. This study identifies MARF1 as a regulator in oocyte maturation and defines the conserved LOTUS domain as a post-transcriptional effector domain that recruits CCR4-NOT deadenylase complex to shorten target mRNA poly-A tails and suppress their translation (Zhu, 2018).

RNA-binding proteins (RBPs) mediate post-transcriptional gene regulation by determining molecular fates of target RNAs. In addition to RNA-binding domains, RBPs often have additional auxiliary domains. These auxiliary domains may function as effector domains for post-transcriptional gene regulation directly through enzymatic activity or indirectly by mediating protein-protein interaction. Identifying these effector domains and their molecular functions is critical to understand the roles of RBPs in post-transcriptional gene regulatory mechanism (Zhu, 2018).

MARF1 is an RBP consisting of one RNA-recognition motif (RRM) followed by several tandem LOTUS domains (Limkain, Oskar, and Tudor containing proteins 5 and 7. Also called OST-HTH). Previous studies showed that mouse MARF1 is required for completion of meiosis in oogenesis by reducing protein and mRNA levels of retrotransposons and a few endogenous genes (Su, 2012a; Su, 2012b). However, the molecular mechanism by which MARF1 regulates gene expression remains unclear (Zhu, 2018).

LOTUS domains are conserved in bacteria, fungi, plants, and animals. In animals, LOTUS domain proteins are expressed almost exclusively in the germline and are implicated in RNA regulation. In Drosophila, these LOTUS domain proteins include Oskar, Tejas (human TDRD5), Tapas (human TDRD7), and MARF1 (Meiosis Regulator And mRNA Stability Factor 1 = GC17018. Human MARF1). However, the molecular function of the conserved LOTUS domain is not fully understood (Zhu, 2018).

This work studied the biological and molecular functions of Drosophila MARF1 and its LOTUS domains. MARF1 is essential for proper oocyte maturation by regulating cyclin protein levels. When tethered to a reporter mRNA, MARF1 caused shortening of reporter mRNA poly-A tail and reduced reporter protein level. This activity was mediated by MARF1 LOTUS domain. Consistent with this finding, it was found that MARF1 binds the CCR4-NOT deadenylase complex via its LOTUS domain. Furthermore, it was found that MARF1 binds cyclin A mRNA, shortens its poly-A tail, and reduces Cyclin A protein level during oocyte maturation. Thus, this study uncovered the biological and molecular functions of Drosophila MARF1 and defined its conserved LOTUS domains as a post-transcriptional effector domain to recruit the CCR4-NOT deadenylase complex to shorten target mRNA poly-A tails and suppress translation of the mRNAs (Zhu, 2018).

MARF1 is expressed in late-stage oocytes and is required for proper oocyte maturation by regulating cyclin protein levels. MARF1 binds the CCR4-NOT deadenylase complex via its LOTUS domain to shorten target mRNA poly-A tails and thus reducing cyclin protein levels without changing cyclin mRNA levels. Thus, MARF1 LOTUS domain is defined as a post-transcriptional effector domain that binds the CCR4-NOT deadenylase complex (Zhu, 2018).

Recent studies by others showed that the LOTUS domains of Drosophila Oskar, Tejas, and Tapas bind germline DEAD-box RNA helicase Vasa to stimulate Vasa ATPase and helicase activities. Crystallographic studies showed that the LOTUS domain of Oskar forms a homodimer and that each of the monomer subunits binds the C-terminal domain of the Vasa DEAD-box helicase core on the side opposite to the dimerization interface. In contrast, this study shows that the MARF1 LOTUS domain binds the CCR4-NOT deadenylase complex, but does not bind Vasa, Oskar, or another molecule of MARF1 (Zhu, 2018).

The LOTUS domains found in Oskar, Tejas, and Tapas, but not MARF1, have a C-terminal extension, which is required for interaction with Vasa. Hence the LOTUS domains are divided into two subclasses: (1) extended LOTUS (eLOTUS) domain that is present in Oskar, Tejas, and Tapas, has a C-terminal extension, and binds Vasa, and (2) minimal LOTUS (mLOTUS) domain that is present in MARF1, lacks a C-terminal extension, does not bind Vasa, and instead binds the CCR4-NOT deadenylase complex. Thus, although eLOTUS and mLOTUS domains share core sequence homology except for the C-terminal extension, they mediate distinct protein-protein interactions. Interestingly, eLOTUS proteins (Oskar, Tejas/TDRD5, Tapas/TDRD7) contain a single eLOTUS domain while mLOTUS proteins (MARF1) contain multiple tandem mLOTUS domains (Zhu, 2018).

This study also showed that MARF1 binds cyclin A mRNA. In MARF1^null mutant late-stage oocytes, cyclin A mRNA poly-A tail is longer and Cyclin A protein level is increased, without change in the cyclin A mRNA level. The degradation rate of Cyclin A protein was not changed in MARF1^null oocytes compared with control oocytes in vitro. These results indicate that in control late-stage oocytes, MARF1 post-transcriptionally regulates Cyclin A protein level by binding cyclin A mRNA, shortening cyclin A mRNA poly-A tail, and reducing Cyclin A protein level. In contrast, poly-A shortening of cyclin A mRNA is lost in MARF1^null oocytes, resulting in an accumulation of Cyclin A protein. Cyclin A is the only protein found that is increased in its level in MARF1 mutant oocytes (Zhu, 2018).

Based on these findings, a model is proposed for MARF1 molecular function. The MARF1 RRM binds specific target mRNAs, such as cyclin A mRNA, and the MARF1 mLOTUS domains recruit the CCR4-NOT deadenylase complex (see Models for MARF1 function in oocytes). This results in shortening of target mRNA poly-A tail and reduction of protein level produced from target mRNAs. The multiple, tandem mLOTUS domain may recruit multiple CCR4-NOT deadenylase complexes per single MARF1 molecule and single target mRNA, enabling efficient poly-A shortening (Zhu, 2018).

Using this model, it is proposed that MARF1 reduces Cyclin A protein level in stages 12-14 oocytes. This regulated reduction of Cyclin A protein level leads to expression of Cyclin B and Cyclin B3 proteins. This cyclin proteins expresson profile leads to stabilization of CDK1 protein and phosphorylation of CDK1 Tyr15 residue. Increase CDK1 and phosphorylation of CDK1 Tyr15 residue result in phosphorylation of appropriate target proteins of CDK1 in stage 14 oocytes. Proper global protein phosphorylation profile allows germplasm localization of Vasa and Aub and normal yolk distribution in stage 14 oocytes. As stage 14 oocytes traverse through the oviduct, meiotic Metaphase I arrest is released to complete meiosis and produce normal eggs (Zhu, 2018).

Consequently, it is speculated that Cyclin A is the main and/or most upstream target of MARF1. Persisted Cyclin A protein level in MARF1 mutant late-stage oocytes arrest them in an abnormal state rather than proceeding to a normal stage 14 including decreased protein levels of Cyclin B and Cyclin B3. Dysregulation of the three cyclin proteins levels results in the decreased CDK1 protein level and the decreased Tyr15 phosphorylation of CDK1. Dysregulation of cyclins and CDK1 alters global phosphorylation pattern. The altered phosphorylation pattern results in the loss of germplasm localization of Vasa and Aub. These together cause meiotic failure and complete sterility in MARF1 mutants (Zhu, 2018).

MARF1 seems to target specific mRNAs for gene silencing in diverse species. This study identified cyclin A mRNA as a target of Drosophila MARF1. Mouse MARF1 reduces protein and mRNA levels of retrotransposons and a few endogenous genes such as PPP2CB, suggesting that they are targets of the post-transcriptional silencing by mouse MARF1. Knockdown of human MARF1 causes upregulation of IFl44L mRNA, suggesting that IFl44L mRNA a target of human MARF1. Post-transcriptional gene silencing of target mRNAs in fly, mouse, and human MARF1 suggests that mLOTUS-domain directed recruitment of the CCR4-NOT deadenylase complex may be a widely conserved mechanism (Zhu, 2018).

LOTUS domains are found not only in animals but also in bacteria, fungi, and plants. LOTUS domains found in bacteria, fungi, and plants are more similar to the animal mLOTUS domains since they lack the C-terminal extension found in the animal eLOTUS domains. This suggests that the mLOTUS domain may be more ancient than the eLOTUS domain. It will be interesting to investigate the functions of these LOTUS domains found in non-animals, particularly the function of bacterial LOTUS domains, since bacteria do not have a poly-A tail in their mRNAs or CCR4-NOT deadenylase complex (Zhu, 2018).

Protein Interactions

The precellular mitotic divisions of the Drosophila embryo appear to provide a striking counterpoint to the demonstrated importance of cyclin degradation and Cdk1 inactivation to cell cycle progression. Drosophila embryogenesis begins with 13 metasynchronous mitotic cycles within a syncytial cytoplasm. These cycles consist only of S and M phases, rely on maternally supplied activities, and do not require zygotic gene expression. The first 10 syncytial cycles last ~9 min each; subsequently, the cycles slow gradually, leading to a transition from maternal to zygotic control of the cell cycle in cycle 14. Previous studies indicate that levels of mitotic cyclins A and B and Cdk1 activity remain high during the first eight cycles. Thereafter, oscillations of these key cell cycle regulators set in gradually, with the amplitude of the oscillation increasing in successive mitoses. Given that mitotic cyclin degradation and Cdk1 inactivation appear essential for exiting mitosis in all systems tested, how do syncytial cycles occur in the continuous presence of mitotic regulators? Injection of an established inhibitor of cyclin proteolysis, a cyclin B amino-terminal peptide, prevents exit from mitosis in syncytial embryos. Similarly, injection of a version of Drosophila cyclin B that is refractory to proteolysis results in mitotic arrest. It is inferred that proteolysis of cyclins is required for exit from syncytial mitoses. This inference can be reconciled with the failure to observe oscillations in total cyclin levels if only a small pool of cyclins is destroyed in each cycle. Antibody detection of histone H3 phosphorylation (PH3) acts as a reporter for Cdk1 activity. A gradient of PH3 along anaphase chromosomes suggests local Cdk1 inactivation near the spindle poles in syncytial embryos. This pattern of Cdk1 inactivation would be consistent with local cyclin destruction at centrosomes or kinetochores. The local loss of PH3 during anaphase is specific to the syncytial divisions and is not observed after cellularization (Su, 1998).

Therefore detailed analysis of PH3 staining upon exit from mitosis reveals an unexpected feature of PH3 loss during the syncytial cycles. As anaphase progresses, loss of PH3 begins in the kinetochore region of the chromosome. Such local gradients of PH3 are seen in syncytial mitoses from at least cycle 4 (M4; the earliest analyzed) up to and including the last syncytial mitosis, M13, although the PH3 gradient appears increasingly shallower as nuclear cycles progress. The localized loss of H3 phosphorylation during anaphase demonstrates that nonuniform conditions occur along the mitotic chromosomes during exit from syncytial mitosis. A local gradient of kinase activity or a local gradient of phosphatase activity, or a combination of both, could result in the observed gradient of PH3 staining. Given the strict correlation between PH3 and Cdk1 activity in Drosophila embryos observed in this study, it is suggested that a likely basis for the localized loss of PH3 is a localized decline in Cdk1 activity. The localized loss of PH3 is blocked by injection of the 13-110 peptide, a ubiquitin pathway inhibitor, suggesting that proteolysis contributes to local loss of Cdk1 activity and PH3. It is suggested that exit from mitosis in syncytial cycles is modified to allow nuclear autonomy within a common cytoplasm (Su, 1998).

While entry into mitosis is triggered by activation of cdc2 kinase, exit from mitosis requires inactivation of this kinase. Inactivation results from proteolytic degradation of the regulatory cyclin subunits during mitosis. At least three different cyclin types, cyclins A, B and B3, associate with cdc2 kinase in higher eukaryotes and are sequentially degraded in mitosis. The Drosophila cell cycle gene fizzy (fzy) is required for normal execution of the metaphase-anaphase transition. Mutations in fizzy (fzy) block the mitotic degradation of these cyclins. Moreover, expression of mutant cyclins (delta cyclins) lacking the destruction box motif required for mitotic degradation affects mitotic progression at distinct stages. DeltaCyclin A results in a delay in metaphase, deltacyclin B in an early anaphase arrest and deltacyclin B3 in a late anaphase arrest, suggesting that mitotic progression beyond metaphase is ordered by the sequential degradation of these different cyclins. Coexpression of deltacyclins A, B and B3 allows a delayed separation of sister chromosomes, but interferes with chromosome segregation to the poles. Mutations in fzy block both sister chromosome separation and segregation, indicating that fzy plays a crucial role in the metaphase/anaphase transition (Sigrist, 1995).

fizzy encodes a protein of 526 amino acids, the carboxy half of which has significant homology to the S. cerevisiae cell cycle gene CDC20. In early embryos fzy is expressed in all proliferating tissues; in late embryos fzy expression declines in a tissue-specific manner correlated with cessation of cell division. During interphase FZY protein is present in the cytoplasm. In mitosis, FZY is distributed throughout the cell except for the area occupied by the chromosomes. The metaphase arrest phenotype caused by fzy mutations is associated with failure to degrade both mitotic cyclins A and B, and an enrichment of spindle microtubules at the expense of astral microtubules. Thus fzy function is required for normal cell cycle-regulated proteolysis, required for successful progress through and exit from mitosis. The events during exit from mitosis appear temporally ordered by the sequential degradation of different mitotic cyclins. First to be degraded is Cyclin A prior to chromosome separation, followed by Cyclin B and Cyclin B3 (Dawson, 1995).

Drosophila fizzy related down-regulates mitotic cyclins and is required for cell proliferation arrest and entry into endocycles. In yeast, inactivation of mitotic cyclins results in acquisition of competence to initiate another round of DNA replication. The subsequent reactivation of B-type cyclin at the G1/S transition triggers initiation of DNA replication in parallel with a reorganization of protein complexes at origins of replication. fizzy-related (fzr), a conserved eukaryotic gene, negatively regulates the levels of cyclins A, B, and B3. These mitotic cyclins that bind and activate cdk1(cdc2) are rapidly degraded during exit from M and during G1. While Drosophila fizzy has previously been shown to be required for cyclin destruction during M phase, fzr is required for cyclin removal during G1 when the embryonic epidermal cell proliferation stops and during G2 preceding salivary gland endoreduplication. Loss of fzr causes progression through an extra division cycle in the epidermis and inhibition of endoreduplication in the salivary gland, in addition to failure of cyclin removal. Conversely, premature fzr overexpression down-regulates mitotic cyclins, inhibits mitosis, and transforms mitotic cycles into endoreduplication cycles. The coincidence of mitotic cyclin disappearance and cyclinE/cdk2 inactivation during G1 arrest raises the possibility that fzr activity might be inhibited by cyclinE/cdk2. fzr and fizzy encode highly similar proteins with seven WD repeats in the C-terminal region. WD repeats are found in budding yeast Cdc4p, which is required for the ubiquitin-dependent proteolysis of several cell cycle regulators. The closest yeast relative of fzr, however, is not CDC4 but HCT1, which is required for proteolysis of Clb2p, a budding yeast B-type cyclin with a characteristic destruction box. However, Drosophila fzr is unable to provide HCT1 function in yeast. Thus, fzr transcripts accumulate when cells become postmitotic and fzr is required in proliferating cells progressing through cell cycles with G1 phases and in G2 before endoreduplication, but not during mitosis (Sigrist, 1997).

Minichromosome maintenance (MCM) proteins are essential DNA replication factors conserved among eukaryotes. Three Drosophila MCM proteins have been characterized: DmMCM2, DmMCM4, and DmMCM5. MCMs cycle between chromatin bound and dissociated states during each cell cycle. Cyclin:cdks can prevent an assembly of proteins called the "prereplicative complex" on origins of DNA replication. The prereplicative complexes are thought to contain MCMs. Their absence from chromatin is thought to contribute to the inability of the post S phase nucleus to replicate DNA. Passage through mitosis restores the ability of MCMs to bind chromatin and the ability to replicate DNA. In Drosophila early embryonic cell cycles, which lack a G1 phase, MCMs reassociate with condensed chromosomes toward the end of mitosis. To explore the coupling between mitosis and MCM-chromatin interaction, a test was carried out as to whether this reassociation requires mitotic degradation of cyclins. Arrest of mitosis by induced expression of nondegradable forms of cyclins A and/or B shows that reassociation of MCMs to chromatin requires cyclin A destruction but not cyclin B destruction. In contrast to the earlier mitoses, mitosis 16 (M16) is followed by G1, and MCMs do not reassociate with chromatin at the end of M16. Thus MCM-chromosome association is delayed when mitosis is followed by a prolonged G-1 phase. dacapo mutant embryos lack an inhibitor for cyclin E, do not enter G1 quiescence after M16, and show mitotic reassociation of MCM proteins. It is proposed that cyclin E, inhibited by Dacapo in M16, promotes chromosome binding of MCMs. Thus, it is suggested that cyclins have both positive and negative roles in controlling MCM-chromatin association (Su, 1997).

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 Cdk7^ts 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).

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).

Activating the DNA damage checkpoint in a developmental context

Studies in unicellular systems have established that DNA damage by irradiation invokes a checkpoint that acts to stall cell division. During metazoan development, the modulation of cell division by checkpoints must occur in the context of gastrulation, differential gene expression and changes in cell cycle regulation. To understand the effects of checkpoint activation in a developmental context, a study was performed of the effect of X-rays on post-blastoderm Drosophila embryos. In Drosophila, DNA damage delays anaphase chromosome separation during cleavage cycles that lack a G2 phase. In post-blastoderm cycles that include a G2 phase, irradiation delays the entry into mitosis. Gastrulation and the developmental program of string (Cdc25) gene expression, which normally regulates the timing of mitosis, occurs normally after irradiation. The radiation-induced delay of mitosis accompanies the exclusion of mitotic cyclins from the nucleus. Furthermore, a mutant form of the mitotic kinase Cdk1 that cannot be inhibited by phosphorylation drives a mitotic cyclin into the nucleus and overcomes the delay of mitosis induced by irradiation. It is concluded that developmental changes in the cell cycle, for example, the introduction of a G2 phase, dictate the response to checkpoint activation, for example, delaying mitosis instead of or in addition to delaying anaphase. This unprecedented finding suggests that different mechanisms are used at different points during metazoan development to stall cell division in response to checkpoint activation. The delay of mitosis in post-blastoderm embryos is due primarily to inhibitory phosphorylation of Cdk1, whereas nuclear exclusion of a cyclin-Cdk1 complex might play a secondary role. Delaying cell division has little effect on gastrulation and developmentally regulated string gene expression, supporting the view that development generally dictates cell proliferation and not vice versa (Su, 2000).

To examine the effect of DNA damage on the progression of the cell cycle during Drosophila embryogenesis, embryos 0-4.5 hours of age were exposed to 570 rads of X-rays. At this dose, 40%-60% of cellular embryos die and fail to hatch into larvae. This dose therefore corresponds to the half-maximal lethal dose (LD50). When syncytial embryos are exposed to X-rays: nuclei enter mitosis normally but chromosome segregation is delayed. The delay is transient such that nuclei enter the next interphase without completely separating sister chromosomes, resulting in polyploid nuclei (Su, 2000).

In cellularized embryos, changes in cell cycle indicators that are consistent with a delay in the entry into mitosis are observed. In untreated embryos at these stages, cells divide in stereotypical clusters termed 'mitotic domains'. Both the location of a mitotic domain within the embryo and the time at which it goes through mitosis are invariant from embryo to embryo. The timing of morphogenetic movements that comprise gastrulation is likewise invariant from embryo to embryo. Thus, the wild-type pattern of mitotic cells at any specific time during this period, as indicated by the extent of gastrulation, is easily recognizable. In irradiated samples, embryos were found in which expected mitotic domains were not in mitosis, as judged by the absence of condensed chromosomes and mitotic figures. Antibody staining for a mitotic-specific phospho-epitope on histone H3 (PH3), and staining with wheatgerm agglutinin (WGA) to detect nuclear envelope breakdown, has confirmed the absence of mitoses in these embryos. It is inferred that irradiation delays the entry into mitosis in cellularized embryos, whereas under identical conditions, chromosome segregation is delayed in syncytial embryos. Treatment of cellularized embryos with a DNA-damaging agent, methyl methane sulfonate, results in a similar delay of mitosis. Therefore, the observed effect of irradiation on mitosis is probably due to the DNA-damaging activity of X-rays (Su, 2000).

It is an unprecedented finding that irradiation leads to two different cell cycle responses in a single organism: either the delay of anaphase chromosome segregation or the delay of mitosis. Mitotic chromosome segregation and the initiation of mitosis are regulated by different mechanisms. The former requires the proteolysis of proteins, such as PDS1 in budding yeast and cyclin A in Drosophila, whereas the latter requires the activation of mitotic cyclin-Cdk complexes. It is suggested that checkpoint activation by the same dose of radiation under identical conditions must have used different downstream mechanisms in order to delay chromosome segregation in the syncytium and mitosis in the cellularized embryos. Although mechanisms that operate in the syncytium remain elusive, the mechanisms used by cellularized embryos were addressed in this study (Su, 2000).

Despite the finding that irradiation does not interfere with String expression, it might have antagonized String activity. Cdc25Stg activates Cdk1 by removing the inhibitory phosphates on Thr14 and Tyr15. A Cdk1 mutant in which these residues have been mutated (Cdk1AF) bypasses the requirement for String. If the mechanism by which radiation delays mitosis solely involves inhibitory phosphorylation of Cdk1, Cdk1AF should bypass the radiation-induced delay. To test this hypothesis, Cdk1 or Cdk1AF, in conjunction with a mitotic cyclin, was expressed from a heat-inducible (hs) promoter during interphase 14. It was then asked whether irradiation could delay the onset of mitosis 14 in embryos expressing these transgenes. It was found that many cells of heat-shocked embryos that carried hs-Cdk1AF and hs-mitotic cyclin transgenes fail to delay mitosis after irradiation. This effect was seen with mitotic cyclins A, B or Bs -- a truncated version of cyclin B that is resistant to proteolysis. In contrast, embryos carrying hs-Cdk1, in combination with the same cyclins, behave like wild-type embryos and delay mitosis. It is concluded that Cdk1AF, and not Cdk1, can overcome the radiation-induced delay in mitosis. It is inferred that inhibitory phosphorylation on Cdk1 is required to delay mitosis in response to DNA damage, in agreement with previous results from fission yeast and vertebrates (Su, 2000).

Interestingly, the ability of Cdk1AF and cyclins to overcome the delay of mitosis in Drosophila was seen only in certain cells of the embryos, and these cells represent mitotic domains, for example, domain 4. Cells of mitotic domains are distinguished from their neighbors by their accumulation of String protein. Although further experiments are required to demonstrate the importance of String, the perfect coincidence of clusters of irradiated cells that entered mitosis in the presence of Cdk1AF as well as accumulated String, has led to the following suggestion: although cyclin-Cdk1AF activity is not present in sufficient quantities to promote mitosis by itself under these experimental conditions, this activity can induce endogenous String to activate endogenous Cdk1 and induce mitosis. A similar feedback mechanism has been proposed for human Cdk1 and Cdc25. It follows then that endogenous String and Cdk1 might be inhibited by irradiation, but that this inhibition can be overcome by a small amount of Cdk1AF activity (Su, 2000).

The same amount of Cdk1AF activity overcomes another consequence of irradiation, namely, the nuclear exclusion of a mitotic cyclin. Nuclear cyclin/Cdk1 activity is a prerequisite to mitosis and the exclusion of cyclin B1 from the nucleus appears to contribute to the delay in mitosis after irradiation in human cells. In cellular-stage Drosophila embryos, cyclins A and B remain enriched in the cytoplasm in interphase. Cyclin A accumulates in the nucleus of cells that initiate mitosis, as does cyclin B. In irradiated embryos, both cyclins A and B are excluded from nuclei although their levels remain unchanged. In cells that express Cdk1AF (with a mitotic cyclin) that enter mitosis even after irradiation, nuclear accumulation of cyclin A is evident. Thus, a low level of Cdk1 activity, provided by Cdk1AF in these experiments, leads to both the nuclear accumulation of a cyclin and the entry into mitosis (Su, 2000).

Given these two observations -- that Cdk1AF drives the nuclear accumulation of cyclin A and that nuclear accumulation of mitotic cyclins coincides with the entry into mitosis in unperturbed cell cycles -- it has been proposed that Cdk1 activity normally drives the nuclear accumulation of cyclin-Cdk1 complexes. In support of this idea, Cyclin A remains excluded from nuclei in string mutants. In accordance with this, Cdk1AF, in conjunction with endogenous String, overcomes the radiation-induced delay of mitosis because Cdk1AF can start the feedback loop that activates endogenous Cdk1 by endogenous String and Cdk1 activity can drive the nuclear accumulation of cyclin-Cdk1. These ideas help explain previous observations in human cells. In the latter, although the exclusion of cyclin B1 from nuclei appears to be of some importance to regulating mitotic entry, Cdk1AF can overcome the checkpoint-induced delay of mitosis, regardless of whether cyclin B1 or NLS-cyclin B1, which is constitutively localized to the nucleus, is co-expressed. Thus, Cdk1AF in human cells, as in Drosophila, might also drive the nuclear accumulation of cyclin-Cdk complexes and the entry into mitosis by initiating a positive feedback loop for the activation of endogenous Cdk1. Whether a similar feedback loop of Cdk1, String and cyclin-localization operates to control mitosis in other tissues, such as larval imaginal discs, remains to be seen (Su, 2000).

DNA damage leads to a Cyclin A-dependent delay in metaphase-anaphase transition in the Drosophila gastrula

In response to DNA damage, fission yeast, mammalian cells, and cells of the Drosophila gastrula inhibit Cdk1 to delay the entry into mitosis. In contrast, budding yeast delays metaphase-anaphase transition by stabilization of an anaphase inhibitor, Pds1p. A variation of the second response is seen in Drosophila cleavage embryos; when nuclei enter mitosis with damaged DNA, centrosomes lose gamma-tubulin, spindles lose astral microtubules, chromosomes fail to reach a metaphase configuration, and interphase resumes without an intervening anaphase. The resulting polyploid nuclei are eliminated. The cells of the Drosophila gastrula can also delay metaphase-anaphase transition in response to DNA damage. This delay accompanies the stabilization of Cyclin A, a known inhibitor of sister chromosome separation in Drosophila. Unlike the process in cleavage embryos, gamma-tubulin remains at the spindle poles, and anaphase always occurs after the delay. Cyclin A mutants fail to delay metaphase-anaphase transition after irradiation and show an increased frequency of chromosome breakage in the subsequent anaphase. It is concluded that DNA damage delays metaphase-anaphase transition in Drosophila by stabilizing Cyclin A. This delay may normally serve to preserve chromosomal integrity during segregation. This is the first report of a metazoan metaphase-anaphase transition being delayed in response to DNA damage. Though mitotic progression is modulated in response to DNA damage in both cleaving and gastruating embryos of Drosophila, different mechanisms operate. These differences are discussed in the context of differential cell cycle regulation in cleavage and gastrula stages (Su, 2001).

It is possible that stabilization of Cyclin A is a secondary consequence of a delay in metaphase rather than a cause for it. This possibility is not favored for two reasons. (1) Cyclin A is unstable during a spindle checkpoint arrest in metaphase in both Drosophila and other systems. Thus, a delay in metaphase alone cannot delay Cyclin A proteolysis. (2) Cyclin A appears to be necessary for the metaphase delay after irradiation and to be sufficient for a metaphase delay in general. Thus, the proposal that stabilization of Cyclin A mediates a metaphase delay after irradiation is the simplest one that fits the data. Note also that the metaphase delay reported here is mechanistically distinct from the metaphase delay that occurs in response to spindle defects. This is because Cyclin A is stable during the first response but unstable during the second (Su, 2001).

In budding yeast, the only other cell type known to delay metaphase-anaphase transition in response to DNA damage, this delay occurs by the stabilization of anaphase inhibitor Pds1p. Thus, the stabilization of molecules that normally inhibit metaphase-anaphase transition appears to be a response to damaged DNA that is in common between Drosophila and budding yeast. In addition to the delay in metaphase-anaphase transition, budding yeast can also delay the exit from mitosis (i.e., the progression beyond anaphase) in response to DNA damage. Recent data suggest the existence of a similar delay in human cells. The finding that the combined length of anaphase and telophase remain unchanged after irradiation, despite the presence of DNA defects through these phases, suggests that these phases are not affected by irradiation in Drosophila cellularized embryos under the experimental conditions used here (Su, 2001).

In S. cerevisiae, stabilization of Pds1p inhibits the proteolysis of B-type cyclins by APC and thereby ensures that exit from mitosis and cytokinesis are prevented during a delay in metaphase. In Drosophila, Cyclin B (which normally becomes degraded after metaphase-anaphase transition) persists during prolonged metaphases that follow irradiation. Thus, DNA damage in Drosophila may also lead to stabilization of B-type cyclins during the delay in metaphase, much like in budding yeast. What role Cyclin A plays, if any, in the stabilization of Cyclin B after DNA damage remains to be examined (Su, 2001).

It is formally possible that the delay in metaphase-anaphase transition is simply due to a direct physical hindrance to chromosome separation imposed by aberrant DNA structures. This explanation is not favored for three reasons. (1) Aberrant DNA structures cannot easily account for the observed inhibition of cyclin proteolysis. In addition, in Drosophila pimple mutants, sister chromosomes do not separate, but mitotic progression is otherwise normal. This indicates that a hindrance to chromosome separation alone cannot modulate other mitotic steps. (2) The observation of chromosome bridges indicates that spindles can segregate unresolved sister chromosomes, although not always to completion. (3) Irradiated cells of Cyclin A mutants fail to delay metaphase-anaphase transition even though these cells presumably suffered as much DNA damage as wild-type controls that were irradiated simultaneously. Thus, damaged DNA alone cannot prolong metaphase. Any delay in chromosome segregation, therefore, is more likely to be due to regulatory control rather than to physical hindrance (Su, 2001).

The delay in metaphase-anaphase transition in cellular embryos reported here might seem similar to the failure in chromosome segregation seen in response to damaged DNA in precellular-stage embryos. Important phenomenological and mechanistic differences, however, distinguish these responses. (1) In precellular embryos that initiate mitosis in the presence of damaged DNA, chromosomes fail to compact and reach a recognizable metaphase configuration; mitosis is aborted and interphase is resumed without an intervening anaphase. In the irradiated cellular embryos observed in this study, chromosomes clearly condense and compact onto metaphase plates, and anaphase always follows; in live analyses an abortion of mitosis is never observed under the experimental conditions used here. (2) Irradiation in precellular embryos results in the loss of gamma-tubulin ring components from the centrosome and the loss of astral microtubules. These have been proposed to account for the failure to compact and segregate chromosomes in precellular embryos. In contrast, in irradiated cellular embryos gamma-tubulin remains at the spindle poles, and the spindles appear functional. Although a diminishment of astral microtubules is observed, it is unclear what role this plays in prolonging metaphase. Instead, these data identify the stabilization of Cyclin A as a likely mechanism for the metaphase delay in cellular embryos. (3) The delay in chromosome segregation in precellular embryos seems to target these nuclei for elimination. In contrast, prolonging metaphase in cellular embryos seems to promote successful anaphase because cells that cannot delay metaphase-anaphase transition are more likely to show lagging chromosomes in anaphase (Su, 2001).

It is suggested that the above-described differences seen in cleavage and cellular embryos reflect a developmental change in how chromosome segregation is regulated in response to DNA damage in Drosophila. Prior to cellularization, nuclei share a common cytoplasm. The loss of a few nuclei at these stages may be of little consequence to the embryo. In addition, nuclear division cycles are extremely rapid (approximately 10 min per cycle). Speed, rather than fidelity, may be the goal in these cycles, which eliminate damaged nuclei rather than pausing to fix them. Mechanisms therefore exist to selectively 'cull' damaged nuclei, which subsequently 'fall into' the embryo interior and become incorporated into yolk. The centrosomes are closely associated with individual nuclei and are thought to aid in attaching nuclei to the embryo cortex. Compromising centrosomal function in precellular stages, therefore, may be a way both to abort mitosis at a single nucleus and to eliminate it. In cellular stages 'falling in' is no longer an option because a cell membrane encloses each nucleus. Therefore, a delay in metaphase-anaphase transition may allow time to resolve DNA defects and increase the chance of a successful anaphase. Note also that stabilization of an anaphase inhibitor may be an unsuitable mechanism for precellular embryos in which nuclei share a common cytoplasm; stabilization of molecules near one nucleus may affect its neighbors (Su, 2001).

Mitotic degradation of cyclin A is mediated by multiple and novel destruction signals

Exit from mitosis requires Cdk1 inactivation, with the most prominent mechanism of Cdk1 inactivation being proteolysis of mitotic cyclins. In higher eukaryotes this involves sequential destruction of A- and B-type cyclins. CycA is destroyed first, and CycA/Cdk1 inactivation is required for the metaphase-to-anaphase transition. The degradation of CycA is delayed in response to DNA damage but is not prevented when the spindle checkpoint is activated. Cyclin destruction is thought to be mediated by a conserved motif, the destruction box (D box). Like B-type cyclins, A-type cyclins contain putative destruction box sequences in their N termini. However, no detailed in vivo analysis of the sequence requirements for CycA destruction has been described so far. Several mutations in the CycA coding region have been tested for destruction in Drosophila embryos. It has been shown that D box sequences are not essential for mitotic destruction of CycA. Destruction is mediated by at least three different elements that act in an overlapping fashion to mediate its mitotic degradation (Kaspar, 2001).

The sequence elements in CycA that are responsible for its degradation are located in the N-terminal part of the molecule and consist of a KEN box, a D box and another yet-uncharacterized destruction element C-terminal to the D box. The D box is probably the target of the APC/C-fizzy/Cdc20 complex since all APC/C-fizzy/Cdc20 substrates characterized so far contain D boxes. The KEN box has been characterized as a destruction element present in proteins targeted by the APC/C-Cdh1/fizzy-related complex. Analysis of fizzy-related (fzr) mutants in Drosophila has shown that Fzr is mainly required for the degradation of mitotic cyclins in G1; degradation appears during embryogenesis after mitosis 16. However, it is possible that fzr also functions in earlier cycles. Recently, the complete sequence of the Drosophila genome revealed another fzy/fzr family member, encoded by the gene CG16783. It remains to be seen if this protein participates in cyclin destruction. The third pathway of CycA destruction does not rely on KEN or D boxes. The minimal sequences required for this degradation mechanism could not be identified. Deletions of amino acids located C-terminal to the first D box impair this destruction mechanism, but no conserved sequences could be identified in this region of CycA. The identification of molecules involved in this pathway of CycA degradation is necessary to reveal how they act in the mitotic exit program to control the metaphase/anaphase transition (Kaspar, 2001).

Protein Interactions: Cyclin A interaction with Roughex

During spermatogenesis, germ cells execute two meiotic divisions, then withdraw from the cell cycle and initiate postmeiotic differentiation. The roughex is a dose-dependent regulator of meiosis II during Drosophila spermatogenesis. rux mutant germ cells execute the two meiotic divisions, but then undergo an additional M phase resembling an extra meiosis II. Conversely, germ cells with excess rux function fail to undergo meiosis II. rux does not appear to act directly at meiosis II; instead it appears to act through Cyclin A during premeiotic G2 to regulate meiosis II. Cyclin A-cdc2 kinase at the G2 to M transition of meiosis I activates a target necessary for meiosis II, thereby coupling the two meiotic divisions (Gonczy, 1994).

In the developing eye of Drosophila melanogaster, cells become synchronized in the G1 phase of the cell cycle just prior to the onset of cellular differentiation and morphogenesis. In roughex (rux) mutants, cells enter S phase precociously because of ectopic activation of a Cyclin A/Cdk complex in early G1. This leads to defects in cell fate and pattern formation, and results in abnormalities in the morphology of the adult eye. A screen for dominant suppressors of the rux eye phenotype led to the identification of mutations in cyclin A, string (cdc25), and new cell cycle genes. One of these genes, regulator of cyclin A (rca1), encodes a novel 412 amino acid protein required for both mitotic and meiotic cell cycle progression. rca1 mutants arrest in G2 of embryonic cell cycle 16, with a phenotype very similar to cyclin A loss of function mutants. Expression of rca1 transgenes in G1 or in postmitotic neurons promotes Cyclin A protein accumulation and drives cells into S phase in a Cyclin A-dependent fashion. RCA1 mRNA is present maternally. In a stage 9 embryo, transcript is found primarily in the mesoderm and the anterior and posterior midgut primordia and in stage 11 embryos, transcript is found throughout the embryo, except within the amnioserosa. By stage 13 expression is restricted primarily to proliferating cells of the CNS. Roughex appears to function by suppressing Cyclin A within the morphogenetic furrow. In contrast, RCA1 is likely to enhance Cyclin A protein stability and thus Cyclin A activity within this region. Cyclin E may facilitate the activity and/or the accumulation of RUX and/or RCA1. Cyclin E may inactivate RUX. Cyclin A accumulates at the posterior edge of the MF where it might be required for entry into S (Dong, 1997).

Because Stg activates CycA-Cdk complexes in vitro and rca1 encodes an upstream positive regulator of cycA (Dong, 1997), a test was made as to whether rux suppresses entry in S phase by preventing ectopic activation (directly or indirectly) of a CycA-Cdk complex in the G1 domain within the MF. Consistent with this interpretation, overexpression of cycA mimics the rux mutant phenotype, showing extensive induction of S-phase cells just anterior to and within the MF. Coexpression of rux results in suppression of the ectopic S phases induced by cycA in all discs assayed. Therefore, ectopic CycA expression can drive G1 cells into S phase, and coexpression of Rux inhibits this phenotype. It is concluded that rux acts as a negative regulator of CycA. This is the first demonstration of a role for CycA in regulation of G1 or S phase in Drosophila (Thomas, 1997).

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).

Roughex is a cell-cycle regulator that contributes to the establishment and maintenance of the G1 state in the fruit fly Drosophila. Genetic data show that Rux inhibits the S-phase function of the cyclin A (CycA)-cyclin-dependent kinase 1 (Cdk1) complex; in addition, it can prevent the mitotic functions of CycA and CycB when overexpressed. Rux has been shown to interact with CycA and CycB in coprecipitation experiments. Expression of Rux causes nuclear translocation of CycA and CycB, and inhibits Cdk1 but not Cdk2 kinase activity. Cdk1 inhibition by Rux does not rely on inhibitory phosphorylation, disruption of cyclin-Cdk complex formation or changes in subcellular localization. Rux inhibits Cdk1 kinase in two ways: Rux prevents the activating phosphorylation on Cdk1 and also inhibits activated Cdk1 complexes. Surprisingly, Rux has a stimulating effect on CycA-Cdk1 activity when present in low concentrations. It is concluded that Rux fulfils all the criteria for a CKI. This is the first description in a multicellular organism of a CKI that specifically inhibits mitotic cyclin-Cdk complexes. This function of Rux is required for the G1 state and male meiosis and could also be involved in mitotic regulation, while the stimulating effect of Rux might assist in any S-phase function of CycA-Cdk1 (Foley, 1999).

The interaction of a variety of proteins, including CKIs, with cyclins is mediated by RXL motifs. Rux contains three RXL motifs, starting at positions 30, 197 and 249, that could mediate the observed interaction of Rux with cyclins. An association of Rux with mitotic cyclins is supported by the observed changes in subcellular localization of cyclins upon expression of Rux. A large proportion of CycA, which is normally cytoplasmic during interphase, moves into the nucleus and overlaps with Rux. The Rux protein itself is nuclear and requires a functional bipartite NLS sequence at its carboxyl terminus for its localization. RuxDeltaNLS fails to localize into the nucleus and CycA remains in the cytoplasm. The observed nuclear accumulation of CycA after Rux expression could thus be explained by a nuclear transport of CycA-Rux complexes mediated by the NLS of Rux. Alternatively, Rux could interfere with a putative nuclear export of CycA, leading to a nuclear accumulation of CycA (Foley, 1999).

Rux can inhibit Cdk1-dependent mitosis and CycA-Cdk1-dependent S phases. Evidence is presented that the molecular basis of these effects is inhibition of CycA- and CycB-dependent Cdk1 kinase activity. Rux expression leads to a marked decrease in Cdk1 kinase activity from embryos: an inhibition of kinase activity has been demonstrated using in vitro assembled and activated Cyc-Cdk1 complexes. In the latter assays, both CycA- and CycB-dependent kinase activities are suppressed. Genetic data have already indicated the importance of Rux in downregulation of CycA-Cdk1 activity during G1. The importance of inhibiting CycB-Cdk1 kinase activity is less clear, since CycB is unable to induce S phase in Drosophila. Nevertheless, the effects of Rux on mitotic Cyc-Cdk1 complexes opens up the possibility that it may also contribute to regulating entry into or exit from mitosis. It is interesting to note that Sic1, a CKI from S. cerevisiae that inhibits S-phase-inducing activity during G1 can also contribute to exit from mitosis under certain circumstances. Rux has no effect on CycE-Cdk2 kinase activity in vitro and cannot inhibit CycE/Cdk2-dependent S phases in vivo. Thus, inhibition by Rux is specific for mitotic cyclins and, like the Sic1 inhibitor of S. cerevisiae, would help to enforce a requirement for G1 cyclins to promote S phase (Foley, 1999).

How does Rux inhibit Cdk1 activity? Activation of Cdk1 requires cyclin association, phosphorylation of Thr161 in the T-loop and dephosphorylation of inhibitory Thr14 and Tyr15 phosphorylation sites. On the basis of the following evidence it is concluded that Rux inhibition does not require modulation of the inhibitory phosphorylations: (1) Rux is able to inhibit kinase activity and induction of mitosis by Cdc2^AF, a mutant form of Cdk1 that lacks the inhibitory phosphorylation sites; (2) phosphorylation on Thr14 and Tyr15 is not observed in the in vitro assays in which Rux is able to inhibit kinase activity. The mechanism of Cdk1 inhibition by Rux also does not rely on preventing Cyc-Cdk1 complex formation. No significant change in the level of cyclins coprecipitating with Cdk1 was found in the presence of Rux. Markedly reduced levels of Thr161 phosphorylation where however found both after expression in vivo and in the in vitro experiments. Phosphorylation of Thr161 in the T-loop is carried out by a CAK. Rux could influence the level of Thr161 phosphorylation in several ways. (1) Rux could have a Thr161-dephosphorylating activity. This is unlikely as Rux is not able to change the state of Thr161 phosphorylation when added after the initial Thr161-phosphorylation event. (2) It is possible that Rux inhibits CAK activity directly. Rux prevents Thr161 phosphorylation by two very different CAKs, however. In one case, a monomeric kinase, CIV1, the in vivo CAK in S. cerevisiae was used. The other source of CAK was a crude Drosophila extract that contained CycH-Cdk7. Embryos lacking Cdk7 activity do not provide CAK activity, indicating that the CAK activity in the extracts depends on CycH-Cdk7 activity. CIV1 and CycH-Cdk7 are very different in nature; therefore, it is very unlikely that Rux can inhibit both kinase activities. (3) Should Rux function by inhibiting CAK, an inhibition of Cdk2-CycE by Rux would be seen, which is not the case in in vitro assays. Instead, Rux might prevent CAK access to the T-loop or recognition of Cyc-Cdk complexes by CAK. Rux does not act solely by preventing Thr161 phosphorylation, however, since it also is able to inhibit activated, Thr161-phosphorylated Cdk1 kinase activity. The molecular nature of this inhibition is at present not known. In summary, Rux can inhibit kinase activity by at least two mechanisms: prevention of Thr161 phosphorylation and inhibition of active Cyc-Cdk complexes. Such dual effects have previously been described for a number of CKIs (Foley, 1999 and references therein).

The inhibition of kinase activity by Rux in vitro occurs in a progressive fashion when using CycB-Cdk1, but a more complex effect on CycA-Cdk1 is observed. The addition of small amounts of Rux results in a stimulation of kinase activity and only larger amounts result in an inhibition. The increase in activity is not associated with an increase in Cyc-Cdk1 association or Thr161 phosphorylation. The seemingly contradictory ability of CKIs to enhance the activity of Cyc-Cdk complexes has previously been described for members of the CIP/KIP family. How Rux stimulates activity in this situation remains to be resolved. Several explanations are possible. Rux could have a chaperone-type function for CycA, or different stoichiometric configurations of Rux and cyclins might exist that can be either stimulatory or inhibitory. Finally, Rux might contain several binding sites with different affinities whose effect on CycA might be qualitatively different (Foley, 1999).

It has been suggested that Rux acts by targeting mitotic cyclins for destruction. CycA destruction is not a necessary component of Rux function, however. Rux prevents the S-phase-inducing activity of a non-destructible CycA (CycADelta170) in vivo and it can inhibit kinase activity stimulated by CycADelta170 in vitro. Cyclin degradation in G1 is caused by fizzy-related/HCT1-dependent anaphase-promoting complex (APC) activity. This function in turn is downregulated by Cyc-Cdk activity. Thus, by inhibiting Cdk1 kinase activity, Rux may contribute towards maintaining a G1 by keeping APC activity high and causing cyclin degradation. Disappearance of mitotic cyclins has also been described when Rux is expressed during S and G2 phases. These experiments have been repeated by expressing Rux in paired stripes in the embryo and also followed CycA disappearance after heat-shock expression of Rux. In both cases, CycA disappearance is only observed after a considerable time (3 hours after Rux expression). Embryos of this age are older than 7 hours and would normally prepare to enter G1 of cycle 17, a stage when CycE is downregulated and Fizzy-related is upregulated in the epidermis. These changes, and not the presence of Rux, most likely lead to the 'eventual disappearance' of CycA (Foley, 1999).

Inhibition by Rux also does not rely on changes in the subcellular distribution of cyclins. Although both CycA and CycB move to the nucleus upon Rux expression, mitosis could still be suppressed when a mutant form of Rux lacking the NLS is expressed: in this case, no CycA accumulation in the nucleus is observed. The presence of Rux in the nucleus would, however, be advantageous in protecting the nucleus from S-phase-inducing CycA-Cdk1 activity during G1 (Foley, 1999).

Rux is the first CKI to be reported in a multicellular organism that is specific for mitotic cyclins. Since similar CKIs have been identified in unicellular eukaryotes, such as SIC1 from S. cerevisiae and rum1 from Schizosaccharomyces pombe, there may be an evolutionarily conserved requirement for an activity that keeps mitotic cyclins in check during G1. During the G1 state, cyclin turnover is high, resulting in low mitotic cyclin levels. At this stage, even low levels of Rux are high relative to cyclins and Rux can prevent Cyc-Cdk1 kinase activity by interfering with Thr161 phosphorylation and inhibiting Cyc-Cdk1 kinase activity. As such, Rux is a typical CKI involved in control of the G1 state. As the cell progresses through G1, CycE levels rise. Rux is a substrate for CycE-Cdk2, and CycE has been shown to promote Rux turnover. Thus, while CycE levels rise, Rux levels decrease, and switching off APC activity at the G1-S transition allows CycA levels to rise. At this stage, the ability of small amounts of Rux to enhance CycA-Cdk1 kinase activity may have a physiological relevance. It is conceivable that low levels of Rux enhance any S-phase and/or mitotic functions of CycA by increasing CycA-Cdk1 kinase activity and promoting their transport to the nucleus (Foley, 1999).

Roughex mediates G1 arrest through a physical association with Cyclin A

Differentiation in the developing Drosophila eye requires synchronization of cells in the G1 phase of the cell cycle. The roughex gene product plays a key role in this synchronization by negatively regulating cyclin A protein levels in G1. Coexpressed Roughex and cyclin A physically interact in vivo. Roughex is a nuclear protein, while cyclin A has previous been shown to be exclusively cytoplasmic during interphase in the embryo. In contrast, in interphase cells in the eye imaginal disc, cyclin A has been shown to be present in both the nucleus and the cytoplasm. In the presence of ectopic Roughex, cyclin A becomes strictly nuclear and is later degraded. Nuclear targeting of both Roughex and cyclin A under these conditions is dependent on a C-terminal nuclear localization signal in Roughex. Disruption of this signal results in cytoplasmic localization of both Roughex and cyclin A, confirming a physical interaction between these molecules. Cyclin A interacts with both Cdc2 and Cdc2c, the Drosophila Cdk2 homolog, and Roughex inhibits the histone H1 kinase activities of both cyclin A-Cdc2 and cyclin A-Cdc2c complexes in whole-cell extracts. Two-hybrid experiments have suggested that the inhibition of kinase activity by Roughex results from competition with the cyclin-dependent kinase subunit for binding to cyclin A. These findings suggest that Roughex can influence the intracellular distribution of cyclin A and define Roughex as a distinct and specialized cell cycle inhibitor for cyclin A-dependent kinase activity (Avedisov, 2000).

Although genetic and immunohistochemical experiments indicate that Rux prevents CycA accumulation in early G1 in the developing Drosophila eye, an understanding of the mechanism by which Rux functions to reduce CycA protein levels has been unclear. Using two in vivo techniques, two-hybrid analysis and coimmunoprecipitation, it has been shown that Rux and CycA interact in both Drosophila and mammalian cells. Although the possibility that other as yet unidentified proteins mediate the interaction between Rux and CycA cannot be ruled out, analysis of Rux point mutations as well as in vitro experiments suggest that the interaction is direct. Binding of Rux to CycA both in vitro and in vivo is eliminated by a mutation in a motif, RXL, which has been shown in mammalian cells to mediate binding of a variety of proteins to CycA, including p107, p130, and the CKIs p21 and p27. In Rux, a single amino acid substitution in this motif is sufficient to eliminate CycA interaction in both the two-hybrid assay and Drosophila cultured cells. These data provide strong evidence that Leu-31 is part of a CycA-binding site that contains the same minimal consensus sequence seen in mammalian cell cycle inhibitors (Avedisov, 2000).

Although in vitro experiments indicate that Leu-31 is necessary for CycA binding, the phenotype resulting from overexpression of the Rux[L31A] mutant in the eye is unexpectedly complicated. In the presence of the mutant protein, CycA still localizes to the nucleus, both in the eye disc and in SL2 cells. It is possible that, although Leu-31 is critical for binding to CycA in cultured cells and in vitro, residual binding occurs via one or both of the remaining two RXL sites in the protein. However, Rux mutant proteins in which all three RXL sites are eliminated still display nuclear localization of CycA in SL2 cells. This result suggests that Rux is not directly involved in CycA nuclear import. CycA protein is stabilized in Rux[L31A] relative to expression of wild-type Rux, indicating that binding to Rux via Leu-31 may be required for degradation of CycA. Finally, mitosis does not occur in eye discs expressing Rux[L31A], a phenotype also seen in nondegradable CycA mutant proteins lacking a destruction box. However, in contrast to cells expressing nondegradable CycA mutant proteins, which arrest in metaphase, cells expressing Rux[L31A] arrest prior to chromosome condensation (Avedisov, 2000).

The simplest explanation of these data, taken together, is that the Rux[L31A] mutant protein displays residual binding to CycA in vivo. Because the Rux[L31A] mutant protein is stable in cells that reenter the cell cycle behind the MF whereas wild-type Rux is degraded, the Rux[L31A] mutant protein is expressed to much higher levels in these S-phase cells than is the wild-type protein. In addition, mutation of a second RXL motif in Rux (at position 248) showed a reduction in CycA binding in the mammalian two-hybrid system, suggesting that this second RXL site also participates in binding. It is possible that this weak residual binding coupled with the stabilization of the mutant protein in S/G2 cells leads to disruption of mitotic CycA-Cdk complexes and the observed G2 arrest. Indeed, fly transformant lines in which Rux[L31A] is expressed at lower levels than in the line analyzed in this study display a completely wild-type phenotype, indicating that extremely high levels of expression of the mutant protein are required to detect these mitotic effects (Avedisov, 2000).

The Rux-CycA interaction occurs via a motif similar to that of characterized CKIs. However, unlike other CKIs, which typically bind both cyclin and CDK subunits, Rux does not interact with either Drosophila CDK in the two-hybrid assay. In addition, coimmunoprecipitation of CDKs with Rux and CycA from SL2 cells expressing all three proteins is not observed. Instead, two-hybrid data indicate that Rux competes with CDKs for binding to CycA. Rux may do this by reducing the stability of CycA-CDK complexes or, alternatively, by preventing CDKs from binding to CycA. This conclusion is conditioned by the finding that low levels of added Rux cause a modest stimulation of CycA-CDK interaction, suggesting that the associations between these proteins may be more complex than has been suggested by a simple competition model (Avedisov, 2000).

In addition to the expected interaction between CycA and the G2 CDK Cdc2, an interaction between CycA and the G1 Cdk2 homolog Cdc2c was detected. Previous experiments using stage 11 Drosophila embryos have detected coimmunoprecipitation of only Cdc2 with CycA. Stage 11 corresponds roughly to embryonic cell cycle 16, which consists of a regulated G2 phase with no apparent G1. It is possible that CycA-Cdc2c complexes are normally present in S phase at such low levels that they cannot be detected at this stage of embryonic development. Human CycA associates with Cdk1 in G2 and with Cdk2 in S phase. These data suggest that the same may happen during larval cell divisions in Drosophila melanogaster. If such an interaction occurs, the activity of this complex may also be a target for regulation by Rux (Avedisov, 2000).

Rux is a nuclear protein both in SL2 cells and in eye imaginal discs. In Drosophila embryos, CycA is cytoplasmic during those stages of interphase when it can be detected (late S phase and G2). A different pattern of localization has been found in eye discs where CycA, as in higher eukaryotes, is also present in the nuclei of S- and G2-phase cells. A similar distribution of Drosophila CDKs in S-phase cells has been seen in the developing eye using anti-PSTAIR antibodies, indicating that active CycA-Cdk complexes may be present in both cellular compartments. As a consequence, it is suggested that some of the activities associated with CycA-dependent kinase complexes are likely to be regulated at the level of subcellular distribution. In support of this hypothesis, eye discs expressing the RuxDeltaNLS construct show an expansion in the domain of S-phase cells behind the MF, as compared with a similar domain in control discs, consistent with an increase in the length of S phase. This observation suggests that the subcellular localization of CycA is important for S-phase progression and is blocked by expression of the RuxDeltaNLS mutant protein but not by expression of wild-type Rux (Avedisov, 2000).

How does Rux function to reduce CycA levels in G1? It is suggested that CycA normally exists in an equilibrium between nuclear and cytoplasmic fractions. In support of this notion, CycA expressed from a heat-inducible promoter in a GMR-Rux background is predominantly cytoplasmic immediately after heat shock and gradually becomes localized to the nucleus when the heat shock is removed. It is suggested that in G1 cells in the MF, the level of endogenous CycA protein is very low as a consequence of the abrupt destruction of mitotic cyclins just prior to G1 arrest in the MF. In contrast, Rux is stable in these G1 cells but is absent in cells that are actively cycling. Thus, relatively high levels of Rux in G1 can shift the CycA subcellular distribution by binding to and effectively targeting CycA protein to the nucleus. Rux may then inhibit CycA-dependent kinase activity by preventing or disrupting the CycA-CDK interaction. Nuclear CycA is also targeted for destruction by binding with Rux, although proteolysis of CycA is apparently not required for inactivation of CycA-dependent functions. When cells reenter S phase behind the MF, Rux levels decline and CycA reaccumulates for its S/G2 functions. This model implies that the level of Rux relative to that of CycA must be significantly higher in G1 (where inhibition of CycA occurs) than in S phase (where Rux levels are reduced) (Avedisov, 2000).

Rux contains four consensus phosphorylation sites for CDKs, and Rux itself is a good substrate for phosphorylation by both CycE-Cdk2 and CycA-Cdc2 activities immunoprecipitated from Drosophila embryos and SL2 cells. Phosphorylation of these sites is not required for binding to CycA. The effect of ectopic Rux expression on CycA localization and stability in eye imaginal tissue can be overcome by overexpression of CycE, suggesting that Rux itself may be a target for CycE-dependent kinase activity. In both yeast and mammalian cells, phosphorylation of CKIs in G1 is absolutely required for their destruction by ubiquitin-mediated proteolysis. The sequence defined in this paper as a CycA-binding site overlaps a region predicted to be important for ubiquitin-mediated degradation, suggesting that CycA may compete with the ubiquitination apparatus for binding to Rux. Indeed, the Rux[L31A] mutant protein, in which this motif is disrupted, shows increased stability in cells that reenter S phase behind the MF. It remains to be seen, however, whether Rux is phosphorylated and/or ubiquitinated in vivo. Experiments to address the role of CycE in inhibiting Rux function are in progress (Avedisov, 2000).

Rca1 inhibits APC-Cdh1^Fzr and is required to prevent cyclin degradation in G2

Regulator of cyclin A1 (Rca1) specifically inhibits Cdh1^Fzr-dependent anaphase-promoting complex/cyclosome (APC) activity and prevents cyclin degredation in G2. The APC is a multisubunit ubiquitin ligase that targets several mitotic regulators for degradation and thereby triggers an exit from mitosis. APC activity is restricted to mitotic stages and G1. This is achieved by the cell cycle-dependent association of proteins encoded by fizzy (fzy) and fizzy-related (fzr) genes, respectively, termed here Cdc20Fzy and Cdh1Fzr, referring to their homologs Cdc20 and Cdh1, found in yeast. In the absence of rca1 function, mitotic cyclins are prematurely degraded, and cells fail to enter mitosis. This phenotype is reminiscent of the phenotype produced by overexpression of Cdh1^Fzr. Double-mutant analysis demonstrates that premature cyclin destruction in rca1 mutants is mediated by Cdh1^Fzr. Furthermore, Rca1 can block the effects of Cdh1^Fzr overexpression, supporting the notion that Rca1 inhibits Cdh1^Fzr-dependent APC activity. Coimmunoprecipitation experiments reveal that Rca1 and Cdh1^Fzr are in a complex that also contains the APC component Cdc27. Collectively, these data show that Rca1 is a negative regulator of Cdh1^Fzr-dependent APC activity. It is suggested that a similar function is required in all cells in which kinase activity is low during G2 to prevent a premature activation of the APC by Cdh1 (Grosskortenhaus, 2002).

Rca1 is an essential inhibitor of the anaphase-promoting complex/cyclosome (APC) in Drosophila. APC activity is restricted to mitotic stages and G1 by its activators Cdc20-Fizzy (Cdc20^Fzy) and Cdh1-Fizzy-related (Cdh1^Fzr), respectively. In rca1 mutants, cyclins are degraded prematurely in G2 by APC-Cdh1^Fzr-dependent proteolysis, and cells fail to execute mitosis. Overexpression of Cdh1^Fzr mimics the rca1 phenotype, and coexpression of Rca1 blocks this Cdh1^Fzr function. Previous studies have shown that phosphorylation of Cdh1 prevents its interaction with the APC. The data reveal another mode of APC regulation; this one is fulfilled by Rca1 at the G2 stage, when low Cdk activity is unable to inhibit Cdh1^Fzr interaction (Grosskortenhaus, 2002).

In rca1 mutants, levels of mitotic cyclins are reduced during interphase of the 16^th cell cycle. This premature cyclin disappearance becomes obvious only when mutant and rescued segments in a given embryo are compared and is more difficult to detect when mutant and wt embryos are compared. The lower levels of mitotic cyclins are not caused by changes in cyclin transcription or translation, since mitotic cyclins accumulate normally at the beginning of cell cycle 16. Mitotic cyclins are usually stable in interphase cells of cellularized Drosophila embryos. It is therefore concluded that their disappearance in rca1 mutants is caused by premature degradation. The remaining cyclin levels are apparently not sufficient to allow entry into mitosis. In Drosophila, CycA and CycB are cytoplasmic during interphase and accumulate in the nucleus only during prophase. It has been speculated that the nuclear accumulation of mitotic cyclins is required for certain mitotic events like DNA condensation. Rca1 is a nuclear protein and could be required to prevent degradation of mitotic cyclins, specifically in the nucleus. Another possibility is that Rca1 sequesters parts of the degradation machinery in the nucleus away from the bulk of mitotic cyclins present in the cytoplasm (Grosskortenhaus, 2002).

In rca1 mutant embryos, residual levels of cytoplasmic CycA and CycB are visible. Supplying additional CycA (but not CycB) is sufficient to rescue the mitotic failure of rca1 mutants. This demonstrates that CycA is the crucial mitotic factor missing in rca1 mutant embryos (Grosskortenhaus, 2002).

The cyclin-dependent kinase inhibitor Roughex is involved in mitotic exit in Drosophila

Exit from mitosis is a tightly regulated event. This process has been studied in greatest detail in budding yeast, where several activities have been identified that cooperate to downregulate activity of the cyclin-dependent kinase (CDK) Cdc28 and force an exit from mitosis. Cdc28 is inactivated through proteolysis of B-type cyclins by the multisubunit ubiquitin ligase termed the anaphase promoting complex/cyclosome (APC/C) and inhibition by the cyclin-dependent kinase inhibitor (CKI) Sic1. In contrast, the only mechanism known to be essential for CDK inactivation during mitosis in higher eukaryotes is cyclin destruction. Evidence is presented that the Drosophila CKI Roughex (Rux) contributes to exit from mitosis. Observations of fixed and living embryos show that metaphase is significantly longer in rux mutants than in wild-type embryos. In addition, Rux overexpression is sufficient to drive cells experimentally arrested in metaphase into interphase. Furthermore, rux mutant embryos are impaired in their ability to overcome a transient metaphase arrest induced by expression of a stable cyclin A. Rux has numerous functional similarities with Sic1. While these proteins share no sequence similarity, Sic1 inhibits mitotic Cdk1-cyclin complexes from Drosophila in vitro and in vivo. It is concluded that Rux inhibits Cdk1-cyclin A kinase activity during metaphase, thereby contributing to exit from mitosis. This is the first mitotic function ascribed to a CKI in a multicellular organism and indicates the existence of a novel regulatory mechanism for the metaphase to anaphase transition during development (Foley, 2001).

Endogenous rux is expressed at low levels and cannot be detected by in situ hybridization, so RT-PCR was used to determine the stages of rux expression. rux is zygotically expressed through most of Drosophila development. Interestingly, rux is already expressed in 2-4 hr embryos, which are in the 14^th and 15^th cell cycles. These are the first two cellular divisions during embryogenesis and occur without any G1 phase. S phases in cycles 14 and 15 immediately follow the preceding mitoses and a prolonged interphase is established during a G2 state. Transition from G2 to mitosis is determined by the temporally controlled transcription of cdc25^stg, the Drosophila homolog of Cdc25. rux mutants were analyzed for cell cycle alterations to determine whether rux is required during the 14^th cell cycle. Initially, focus was placed on rux³ mutants. The wild-type Rux protein has 335 amino acids and the rux³ allele carries a frameshift mutation that encodes a protein with 21 out of frame amino acids after amino acid 320. Although the rux³ mutants have rough eyes, they are not male sterile and can be maintained as a homozygous stock. The pattern of mitosis 14 was compared in rux³ mutant embryos and wild-type embryos. Mitosis 14 is the first zygotically controlled division and occurs in a spatiotemporal pattern of domains. Additionally, cells in individual domains proceed through mitosis in a stereotyped sequence. For example, domain 4 starts as a thin wisp of cells, expands laterally and assumes defined contours. The central cells are the first to exit mitosis, followed by the peripheral cells (Foley, 2001).

Germband extension is a morphological process which occurs independent of cell cycle progression. The extent of germband extension was used as a marker for developmental stage. No differences were found in the timing of entry into mitosis between wild-type files and rux³ mutants for individual domains. However, changes in the overall pattern of mitotic domains were noticed. To study this difference the rate of progression through mitosis 14 for wild-type and rux mutant embryos was compared by selecting two embryos in which a chosen domain in both embryo types appeared to be identical; progression of the other domains was then compared. When embryos in which domain 4 was set at the same stage for wild-type and mutant embryos, nearly all cells of domain 1 in wild-type embryos had completed mitosis and only a few telophase cells of the peripheral region of this domain were present. In contrast, domain 1 in rux mutants lags behind with most central cells in telophase and all peripheral cells of this domain in metaphase. Similarly, when embryos in which domain 1 appeared identical when wild-type and rux mutant embryos were compared, it was observed that mitosis lags behind in mutant embryos when compared with other domains, such as domain 4. Thus, patterns of mitotic domains co-exist in rux mutants that are temporally separated in wild-type embryos, suggesting that individual mitotic domains persist longer in mutant embryos (Foley, 2001).

The effect described above is not specific to the rux³ allele and is not the result of a background mutation in the rux³ genotype. The same observations were made for rux² mutants and a heteroallelic combination of rux⁸ and rux³: rux⁸ is a null allele and rux² bears a mutation in the rux promoter region. At least 10 embryos of the individual genotypes were compared with wild-type embryos of the same stage. Each mutant genotype showed a similar deviation from the wild-type pattern as that observed for rux³ (Foley, 2001).

To analyze the basis of this phenotype, mitoses were followed in living wild-type and rux³ mutant embryos using time-lapse video microscopy where DNA had been marked with a green fluorescent protein (GFP)-tagged histone transgene (His2AvD). Mitosis 14 was compared in single cells of domains 2 and 5 from 10 living wild-type and rux³ embryos to determine the duration of the individual phases of mitosis. Sister chromatid separation was set as time point 0. Metaphase was defined as the time of maximal DNA alignment on the metaphase plate. Cells in which chromosomes were observed outside the metaphase plate were still considered to be in prophase. The duration of prophase, metaphase, anaphase and telophase was determined for a representative population of cells for both genotypes. It was found that metaphase is on average 79% longer in rux³ mutants than wild-type embryos. The other stages of mitosis are not significantly altered in rux mutant embryos. Whereas prophase, anaphase and telophase are the same length in both embryos, metaphase is twice as long in the rux mutant embryo (metaphase lasts ~60 s in the wild-type and 120 s in the mutant embryos) (Foley, 2001).

Therefore, rux mutant embryos appear to be compromised in their ability to execute the metaphase to anaphase transition. Since Rux is a CKI that specifically inhibits Cdk1-CycA and interacts both genetically and physically with CycA, it was inferred from these data that Rux is required to inhibit Cdk1-CycA kinase activity during metaphase, thereby facilitating the transition to anaphase (Foley, 2001).

To test whether Rux can downregulate Cdk1-CycA activity during metaphase, an indestructible form of CycA (CycAdelta170) was expressed in segmental stripes of the embryonic epidermis by crossing UAS-CycAdelta170 flies with prd-GAL4 flies (prd-GAL4 X UAS-CycAdelta170). This led to an accumulation of cells in metaphase during mitosis 15 in CycAdelta170-expressing stripes of the epidermis. In a parallel experiment, embryos were examined that carried a heat-inducible rux transgene in addition to UAS-CycAdelta170 (prd-GAL4 X UAS-CycAdelta170; hs-rux). Overexpression of Rux does not induce a decrease in the levels of CycAdelta170 expressed from a second transgene. Rux expression was induced by a 5 min heat pulse after cells had arrested in metaphase. Administration of a mild heat pulse resulted in rux expression throughout the embryo 10 min after induction. Rux protein was detected 5 min later and after an additional 5 min, numerous cells were observed that exited mitosis in the UAS-CycAdelta170-expressing stripes. Concomitant with the reduction in the number of metaphase cells in these stripes, a number of cells were observed in anaphase or telophase. After 1 hr, all cells were in interphase. Thus, Rux expression is sufficient to induce a mitotic exit in CycAdelta170-arrested cells (Foley, 2001).

Cyclins A, B and B3 are degraded in a sequential manner as cells progress through mitosis. It is not known whether this sequential destruction is a prerequisite for the chromosome movements that accompany progression through mitosis. Since anaphase and telophase chromosome structures were observed in prd-GAL4; UAS-CycAdelta170; hs-rux embryos, these embryos were examined for the presence of mitotic cyclins at the point Rux induces an exit from metaphase. Expression of Rux does not affect the levels of CycAdelta170, indicating that Rux induces an exit from mitosis independent of CycAdelta170 proteolysis. CycB protein is detected in cells in which the metaphase to anaphase transition has not yet occurred, indicating that destruction of endogenous cyclins has not proceeded to completion at the time when Rux is expressed and an exit from mitosis is observed (Foley, 2001).

One of the initial pieces of evidence that demonstrates that Sic1 is involved in exit from mitosis in Saccharomyces cerevisiae is that low-level expression of a stable cyclin in yeast does not induce a permanent metaphase arrest. The arrest is transitory since Sic1 eventually inhibits Cdc28 to an extent that cells exit mitosis. prd-GAL4 X UAS-CycAdelta170 embryos were examined at later developmental stages and it was observed that the metaphase arrest induced by expression of CycAdelta170 is transitory. The DNA of most cells in prd-GAL4 X UAS-CycAdelta170 embryos of stage 12 was in a decondensed state. The nuclear density of CycAdelta170-expressing stripes was half of that in interstripes, suggesting that cells expressing CycAdelta170 do not segregate sister chromatids before entering interphase. When rux³; prd-GAL4 X rux³; UAS-CycAdelta170 embryos of the same developmental stage were examined, it was noticed that numerous cells expressing stable CycA in a rux mutant embryo remain trapped in metaphase. Whereas almost all cells in the prd-GAL4 X UAS-CycAdelta170 embryo are in interphase, approximately half the cells in a rux mutant are still in metaphase. Therefore, Rux is required to downregulate Cdk1-CycAdelta170 activity and allow a metaphase exit in these embryos (Foley, 2001).

In contrast to the Rux-induced metaphase exit, the CycAdelta170 expressing cells exit mitosis without segregation of sister chromatids. These cells exit mitosis at a much later stage. The possibility was therefore considered that all endogenous CycB has been destroyed prior to metaphase exit and that the absence of CycB at the time of metaphase exit prevents anaphase movements. To address this question CycB protein levels were examined in stage 12 embryos expressing CycAdelta170. Whereas the CycAdelta170 protein persists in metaphase-arrested cells of embryos of stage 12, no CycB protein above background levels was detected. Endogenous CycB is degraded in embryos of this stage as a result of the developmentally controlled transcription of the APC/C component fizzy-related (fzr). In the absence of CycB protein, cells apparently do not execute anaphase and instead decondense their chromosomes without segregation of sister chromatids (Foley, 2001).

The data suggest a role for Rux in mitotic exit. Progression through mitosis, as determined by the pattern of domains in mitosis 14 is prolonged in all rux mutants. Live observations reveal that the length of metaphase is almost doubled in rux mutants. In addition, rux expression induces an exit from a metaphase imposed by CycAdelta170 and rux mutants expressing CycAdelta170 are compromised in their ability to exit metaphase. Thus, Rux appears to perform a similar function in Drosophila as that performed by Sic1 in S. cerevisiae. There is no obvious sequence homology between Rux and Sic1. However, Sic1 and the CKI rum1 from Schizosaccharomyces pombe can functionally replace one another, even though the sequence similarity between the two proteins is minimal. This raises the possibility that the sequence requirements for CKIs specific for mitotic cyclins are not very stringent, making it difficult to identify them on the basis of the primary amino acid sequence (Foley, 2001).

An understanding of mitotic exit in yeast has advanced rapidly in the last decade and a picture of multiple intrinsic cellular activities controlling the process has emerged. The only aspect known so far to be conserved in multicellular organisms is proteolytic destruction of cyclins. It is thought that the more complex nature of metazoans necessitates an equal if not more rigorous regulation of exit from mitosis. It is proposed that Rux performs functions similar to Sic1 from S. cerevisiae and that Rux cooperates with other mechanisms to trigger exit from mitosis. Removal of Rux function does not abrogate the ability of a cell to leave mitosis; the process is delayed, however. This delay is specific to metaphase, although Rux can inhibit both Cdk1-CycA and Cdk1-CycB, at least in vitro. Apparently, Rux functions during mitosis mainly as a negative regulator of Cdk1-CycA, which must be downregulated to exit metaphase. It appears that Rux-dependent inhibition of CycB is not limiting for anaphase (Foley, 2001).

Rux is not transcribed during the first 2 hr of embryogenesis, which corresponds to the period of nuclear divisions. These cell cycles are extremely rapid and aberrant divisions at this stage are not repaired; instead, the resulting nuclei are destroyed. During the cellular cycles this option is no longer available, since cellular loss at such a critical developmental period is potentially deleterious to the entire organism. In a situation where a metaphase plate has correctly formed, it is advantageous to the cell to complete metaphase promptly and thereby ensure a faithful segregation of chromatids to two sister cells. Rux is transcribed during the cellular cycles and it is proposed that Rux functions during these cycles by contributing to Cdk1-CycA kinase inactivation (Foley, 2001).

The transition from metaphase to anaphase is tightly regulated: DNA must align properly on the metaphase plate and CycA-dependent kinase activity must be downregulated. DNA damage or incorrectly oriented spindles induce metaphase arrest. No misaligned chromosomes, delay of entry into mitosis, and no abnormal spindles or lagging chromosomes were detected in rux mutants, suggesting that the metaphase delay is not caused by activation of a checkpoint. An additional requirement for the metaphase to anaphase transition is inactivation of Cdk1-CycA. In Drosophila, CycA function is required for metaphase execution and expression of an indestructible form of CycA prevents the metaphase to anaphase transition. Rux interacts genetically and physically with CycA and inhibits the in vitro kinase activity of Cdk1-CycA. Therefore, the most likely explanation of the results above is that Rux contributes to the inactivation of Cdk1-CycA during metaphase. In the absence of Rux function, CycA-Cdk1 activity is only downregulated by cyclin proteolysis and this leads to an extension of metaphase (Foley, 2001).

Rux is expressed at levels that are insufficient to completely inactivate Cdk1-CycA at the beginning of mitosis. However, CycA levels drop rapidly during metaphase after the initiation of cyclin proteolysis and even low levels of Rux become significant for the inhibition of the residual CycA-Cdk1 complexes and for the transition into anaphase. When higher levels of Rux are expressed, Cdk1 can be quickly inhibited in a manner independent of cyclin proteolysis and cells exit mitosis in a normal fashion (Foley, 2001).

Overexpression of Rux in cells that had been arrested in metaphase by a stable form of CycA is sufficient to induce an exit from mitosis. Cells exit mitosis by proceeding though anaphase and telophase and segregating sister chromatids into two distinct cells. rux mutants expressing stable CycA are impaired in their ability to exit mitosis. When these cells eventually exit mitosis they do so without a separation of sister chromatids. It is thought that the presence or absence of endogenous cyclins is the cause of the two different forms of mitotic exit. Expression of an indestructible cyclin does not inhibit destruction of endogenous cyclins. However, Rux was induced in cells at a time when endogenous CycB was still present in the case of prd-GAL4; UAS-CycAdelta170; hs-rux embryos. It is thought that this endogenous CycB then contributes to execution of anaphase. In the case of rux^-/-; prd-GAL4; UAS-CycAdelta170 mutants, cells exit mitosis at a much later stage; ~3.5 hr later. At this point in development the APC/C component Fzr is active and all endogenous CycB is destroyed. In the absence of CycB, cells exit metaphase without separation of sister chromatids. These observations also support a model in which sequential destruction of mitotic cyclins is a prerequisite for the chromosome movements that occur during mitosis (Foley, 2001).

The only other CKI known to perform a mitotic function is Sic1. Rux and Sic1 have many similarities. Both specifically inhibit mitotic cyclin-Cdk1 complexes. Sic1 interacts with cyclin molecules via a classic RXL motif; in single letter amino-acid code where X is any amino acid), and Rux-CycA interactions also rely on an RXL motifs in Rux. Both are non-essential because they cooperate with other mechanisms such as cyclin proteolysis. However, while Sic1 acts as a late step during mitosis, Rux is involved in the metaphase-anaphase transition. Both genes are then also required to establish a G1 phase. Sic1 is stable during G1 and is destroyed at the G1-S transition by the proteasome. Rux is also stable during G1 and is destroyed at the G1-S transition by the proteasome. The analogy between Sic1 and Rux is also strengthened by the data that Sic1 is able to inhibit mitotic cyclin-Cdk1 complexes from Drosophila, both in vivo and in vitro. Because the SIC1 and rux genes have evolved separately there appears to be an evolutionarily conserved advantage behind such gene products, raising the possibility that such CKIs remain to be discovered in other eukaryotes (Foley, 2001).

Bub3 protein is required for the mitotic checkpoint and for normal accumulation of cyclins during G2 and early stages of mitosis

During mitosis, a checkpoint mechanism delays metaphase-anaphase transition in the presence of unattached and/or unaligned chromosomes. This delay is achieved through inhibition of the anaphase promoting complex/cyclosome (APC/C) preventing sister chromatid separation and cyclin degradation. Bub3 is an essential protein required during normal mitotic progression to prevent premature sister chromatid separation, missegreation and aneuploidy. Bub3 is required during G2 and early stages of mitosis to promote normal mitotic entry. Loss of Bub3 function by mutation or RNAi depletion causes cells to progress slowly through prophase, a delay that appears to result from a failure to accumulate mitotic cyclins A and B. Defective accumulation of mitotic cyclins results from inappropriate APC/C activity, since mutations in the gene encoding the APC/C subunit Cdc27 (see Drosophila Cdc27) partially rescue this phenotype. Furthermore, analysis of mitotic progression in cells carrying mutations for cdc27 and bub3 suggests the existence of differentially activated APC/C complexes. Altogether, these data support the hypothesis that the mitotic checkpoint protein Bub3 is also required to regulate entry and progression through early stages of mitosis (Lopez, 2005).

These results suggest that Bub3 is required for normal accumulation of cyclin B before and during mitosis. However, cyclin A is also required to promote mitotic entry in Drosophila. Cyclin A accumulates during S phase and G2 and it is degraded by the APC/C prior to the degradation of cyclin B as cells progress through early stages of mitosis. However, in contrast to cyclin B, cyclin A levels are not stabilized by the spindle damage-associated checkpoint response. In order to determine if Bub3 is also required to promote accumulation of cyclin A, its levels were analysed during cell cycle progression in Bub3-depleted cells. The centrosomal marker gamma-tubulin was used to distinguish cells in G1/S or G2. The results show that although cyclin A accumulates from G1 to G2 in control cells and can still be detected in some early mitotic cells, cyclin A fails to accumulate and can hardly be detected in cells depleted for Bub3. Analysis of cyclin A accumulation in bub3¹ homozygous mutant neuroblasts gave very similar results. These observations further support the role of Bub3 as a negative regulator of the APC/C during G2 and mitosis (Lopez, 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 SCF^Skp2 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, 2005b).

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. Cks30A^KO 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 APC^Cortex 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 APC^Fzy. Cyclin A is destroyed first in prometaphase, dependent on APC^Fzy activity. Although the APC^Fzy is active, the spindle checkpoint is thought to inhibit its activity on Cyclin B. Upon spindle assembly, the checkpoint is relieved and APC^Fzy 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 APC^Cortex-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 APC^Fzy 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 APC^Cortex and if the APC^Cortex 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 APC^Cortex 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).

Terminal mitoses require negative regulation of Fzr/Cdh1 by Cyclin A, preventing premature degradation of mitotic cyclins and String/Cdc25

Cyclin A expression is only required for particular cell divisions during Drosophila embryogenesis. In the epidermis, Cyclin A is strictly required for progression through mitosis 16 in cells that become post-mitotic after this division. By contrast, Cyclin A is not absolutely required in epidermal cells that are developmentally programmed for continuation of cell cycle progression after mitosis 16. These analyses suggest the following explanation for the special Cyclin A requirement during terminal division cycles. Cyclin E is known to be downregulated during terminal division cycles to allow a timely cell cycle exit after the final mitosis. Cyclin E is therefore no longer available before terminal mitoses to prevent premature Fizzy-related/Cdh1 activation. As a consequence, Cyclin A, which can also function as a negative regulator of Fizzy-related/Cdh1, becomes essential to provide this inhibition before terminal mitoses. In the absence of Cyclin A, premature Fizzy-related/Cdh1 activity results in the premature degradation of the Cdk1 activators Cyclin B and Cyclin B3, and apparently of String/Cdc25 phosphatase as well. Without these activators, entry into terminal mitoses is not possible. However, entry into terminal mitoses can be restored by the simultaneous expression of versions of Cyclin B and Cyclin B3 without destruction boxes, along with a Cdk1 mutant that escapes inhibitory phosphorylation on T14 and Y15. Moreover, terminal mitoses are also restored in Cyclin A mutants by either the elimination of Fizzy-related/Cdh1 function or Cyclin E overexpression (Reber, 2006).

Mitotic cyclins accumulate during the S and G2 phases of the cell cycle. Their C-terminal cyclin boxes mediate binding to cyclin-dependent kinase 1 (Cdk1). Their rapid degradation during late M and G1 phase depends on the D- and KEN-boxes in their N-terminal domains. These degradation signals are recognized by Fizzy/Cdc20 (Fzy) and Fizzy-related/Cdh1 (Fzr), which recruit the mitotic cyclins to the anaphase-promoting complex/cyclosome (APC/C) during M and G1, respectively. The ubiquitin ligase activity of the APC/C allows cyclin poly-ubiquitination and consequential proteolysis (Reber, 2006).

Metazoan species express three different types of mitotic cyclins: A, B and B3. The specific functions of these different cyclins are not understood in detail. The presence of single genes coding for either Cyclin A (CycA), Cyclin B (CycB) or Cyclin B3 (CycB3) has facilitated a genetic dissection of their functional specificity in Drosophila melanogaster. In this organism, development to the adult stage requires the zygotic function of CycA, but not of CycB or CycB3. Initial analysis of the embryonic cell proliferation program in CycA mutants revealed that epidermal cells fail to progress through the sixteenth round of mitosis. Cyclin A is also required for mitosis 16 in the epidermis of dup/Cdt1 mutant embryos, in which mitosis 16 is no longer dependent upon completion of the preceding S phase. The failure of mitosis 16 in CycA mutants therefore does not simply result from the activation of a DNA replication or damage checkpoint -- a possibility suggested by evidence obtained in vertebrate cells in which Cyclin A binds not only to Cdk1 but also to Cdk2, and provides crucial functions during S phase (Reber, 2006 and references therein).

The accumulation of Cyclin B and Cyclin B3 during cycle 16, which also occurs in CycA mutants, complicates the explanation of why mitosis 16 in the epidermis requires Cyclin A. In Xenopus egg extracts, Cyclin B can trigger entry into mitosis in the absence of Cyclin A. Conversely, mitosis is clearly inhibited in cultured human cells after the microinjection of antibodies against cyclin A. Cyclin A-Cdk1 complexes are thought to have special properties, important for starting up a positive-feedback loop that confers a switch-like behavior on the Cdk1 activation process. In this feedback loop, Cdk1 activity results in phosphorylation and suppression of the inhibitory Wee1 kinase, as well as in phosphorylation and activation of the String/Cdc25 phosphatase, which removes the inhibitory phosphate modifications from Cdk1. However, the analyses described in this study indicate that the Cyclin A requirement in Drosophila is not linked to this positive-feedback loop. Rather, it is linked to the fact that the sixteenth round of mitosis during embryogenesis is the last cell division for the great majority of the epidermal cells (Reber, 2006).

After mitosis 16, most epidermal cells enter a G1 phase and become mitotically quiescent. By contrast, all the previous embryonic divisions (mitoses 1-15) are followed by an immediate onset of S phase. The G1 phase after mitosis 16 is therefore the first G1 phase during development. Entry into this G1 phase is dependent upon a complete, developmentally controlled inactivation of Cyclin E-Cdk2 and Cyclin A-Cdk1, because both complexes can trigger entry into S phase. Cyclin E-Cdk2 inactivation results from transcriptional CycE downregulation and concomitant upregulation of dacapo, which encodes the single Drosophila CIP/KIP-type inhibitor specific for Cyclin E-Cdk2. Cyclin A-Cdk1 inactivation is dependent on Fzr, which is also transcriptionally upregulated. Moreover, Fzr is activated as a consequence of Cyclin E-Cdk2 inactivation. Importantly, this cell cycle exit program is initiated already during G2 of the final division cycle (Reber, 2006).

Although cycle 16 is the final division cycle for most epidermal cells, some defined regions do not activate the cell cycle exit program during cycle 16. Instead, they maintain CycE expression, enter S phase immediately after mitosis 16 and complete an additional division cycle 17. In these regions, mitosis 16 is not fully inhibited in CycA mutants. Cyclin A is therefore especially important for terminal mitoses preceding G1 and cell cycle exit. This study shows that the downregulation of Cyclin E-Cdk2 before terminal divisions, in preparation for the imminent cell cycle exit, converts Cyclin A from a redundant into an indispensable, negative regulator of Fizzy-related/Cdh1, preventing premature degradation of the mitotic inducers String/Cdc25 and the mitotic cyclins. The significance of the basic cell cycle regulator Cyclin A therefore depends on the developmental context (Reber, 2006).

The phenotypical characterization of mutations in the Drosophila genes encoding the A-, B- and B3-type cyclins have indicated that Cyclin A is the most crucial of these co-expressed mitotic cyclins. Although zygotic CycB or CycB3 function is not essential for cell proliferation and development to the adult stage, null mutations in CycA result in embryonic lethality. This study has clarified the molecular basis of the distinct importance of Cyclin A. The results indicate that the crucial role of Cyclin A is linked to its ability to inhibit Fzr-APC/C-mediated degradation. Moreover, this Cyclin A-dependent negative regulation of the Fzr-APC/C-degradation pathway is of particular importance for progression through the very last mitotic division preceding cell cycle exit and the proliferative quiescence of epidermal cells during embryogenesis. This particular Cyclin A requirement during terminal divisions is caused by a cell cycle exit program that is initiated already before the terminal mitosis. The cell cycle exit program includes downregulation of Cyclin E-Cdk2, which has a comparable ability to inhibit the Fzr-APC/C-degradation pathway to Cyclin A. The downregulation of Cyclin E-Cdk2 by the cell cycle exit program turns Cyclin A into an indispensable inhibitor of the premature degradation of mitotic cyclins and String/Cdc25 via Fzr-APC/C before the terminal mitosis. Accordingly, the terminal mitosis in the epidermis of CycA mutants can be restored by overexpression of Cyclin E, by genetic elimination of Fzr, or by simultaneous expression of the String/Cdc25-independent Cdk1^AF mutant and B-type cyclin versions that are no longer Fzr-APC/C substrates (Reber, 2006).

The fact that Cyclin A is also a substrate of Fzr-APC/C-mediated degradation complicates the interpretation of the results. Two findings, however, strongly suggest that Cyclin A functions not just downstream of Fzr, but also upstream as a negative regulator. The observed premature loss of B-type cyclins in CycA mutants is readily explained by a negative effect of Cyclin A on Fzr-APC/C activity and is difficult to explain if Cyclin A was only a Fzr-APC/C substrate. Moreover, the suppression of the UAS-fzr overexpression phenotype by co-expression of UAS-CycA, which is described here, includes the re-accumulation of B-type cyclins and not just the restoration of terminal mitosis 16 (Reber, 2006).

Work in mammalian cells has clearly established that Cyclin A functions as a negative regulator of Fzr/Cdh1. Human Cyclin A can bind directly to Cdh1. Moreover, Cyclin A-dependent Cdk activity phosphorylates Cdh1, resulting in the dissociation of Cdh1 from APC/C. Conversely, mutations in Cdk consensus phosphorylation sites of human CDH1 were reported to abolish inhibition by Cyclin A. The current findings point to alternative modes of Fzr-APC/C-inhibition by Cyclin A. Fzr^psm variant no longer contains canonical Cdk consensus phosphorylation sites (S/T P) and yet its activity is still suppressed by CycA overexpression. Fzr inhibition by CyclinA-dependent phosphorylation of non-consensus sites remains a possibility in Drosophila. However, it is pointed out that, apart from a potential control by Cdk phosphorylation, Fzr is inhibited by the Emi1-like Drosophila protein Rca1. Rca1 overexpression has been shown to prevent premature Cyclin B degradation and restore mitosis 16 in the epidermis of CycA mutant embryos. Based on these observations, the failure of mitosis 16 in CycA mutants was proposed to reflect premature Fzr activation, a suggestion fully confirmed by the current work. It is conceivable, therefore, that the Cyclin A-mediated suppression of Fzr^psm activity involves Rca1 or other unknown targets. The fact that not only Cyclin A, but also Cyclin E, effectively suppresses Drosophila Fzr and Fzr^psm provides further support of additional regulatory complexity. In vertebrate systems, only Cyclin A and not Cyclin E was shown to bind and inhibit Cdh1 (Reber, 2006).

The current findings demonstrate that the Cyclin A requirement in epidermal cells is maximal for progression through the last mitosis of Drosophila embryogenesis, which precedes cell cycle exit and proliferative quiescence. A prominent Cyclin A requirement for terminal mitoses appears to exist in neuroblast lineages during development of the embryonic CNS, although definitive proof will require further work. On the basis of this analysis in epidermal cells, a high Cyclin A requirement for entry into mitosis is expected whenever Fzr levels are high and Cyclin E levels low. During the comparatively slow postembryonic cell cycles of imaginal cells, the periodicity of Cyclin E expression is presumably far more pronounced than during the rapid embryonic cycles in which the persistent presence of maternally contributed Cyclin E eliminates G1 phases. In imaginal cell cycles, which have a G1 phase, Cyclin E expression might therefore be low before each mitosis, and not just before terminal divisions. In combination with Fzr expression, every imaginal mitosis might therefore be strongly dependent upon Cyclin A. By contrast, in the absence of Fzr, progression through mitosis appears to be almost completely independent of Cyclin A, as is evidenced by the observation that the epidermal cells in fzr CycA double mutant embryos not only progress successfully through mitosis 16, but also complete an extra division cycle 17. Nevertheless, 10% of the late mitosis 17 figures in these double mutants displayed lagging chromosomes, indicating that cell cycle progression is not entirely normal in the absence of Cyclin A (Reber, 2006).

The cell cycle exit program, which is activated during the final division cycle in the embryonic epidermis, includes the strong transcriptional upregulation of the CIP/KIP-type Cyclin E-Cdk2 inhibitor Dacapo, apart from the downregulation of Cyclin E and the upregulation of Fzr. Accordingly, genetic elimination of dacapo function should also restore progression through terminal mitosis 16 in CycA mutants. However, mitosis 16 was not observed in the epidermis of dacapo CycA double mutants. The contribution of Dacapo to Cyclin E-Cdk2 inhibition appears to be insignificant before mitosis 16. After the stage of mitosis 16, however, the epidermal cells in these double mutants entered an endoreduplication cycle, a behavior that is also displayed by some cells in the prospective anterior spiracle region of CycA single mutants. This region does not downregulate Cyclin E during cycle 16 in the wild type, it does not upregulate Dacapo, and it progresses through an additional cycle 17 instead of becoming postmitotic after mitosis 16, in contrast to the great majority of the other epidermal cells. The premature activation of Fzr in CycA mutants, therefore, appears to result in DNA replication origin re-licensing, perhaps as a result of B-type cyclin and geminin degradation. Cyclin E-Cdk2 activity might subsequently trigger endoreduplication in cells in which it is not effectively eliminated by both Cyclin E downregulation and Dacapo upregulation. Importantly, not all cells in the anterior spiracle region of CycA mutants endoreduplicate, some of the cells still manage to divide. This variability could reflect minor differences in the onset and strength of the zygotic Cyclin E expression. The outcome of insufficient Cyclin A levels appears to be highly dependent on the levels of Cyclin E and Fzr, which, in turn, are subject to developmental regulation, in particular during cell cycle exit. The significance of basic cell cycle regulators in vivo is therefore different in various tissues and developmental stages, and most likely in various cultured mammalian cell types as well (Reber, 2006).

The Cdc20 (Fzy)/Cdh1-related protein, Cort, cooperates with Fzy in cyclin destruction and anaphase progression in meiosis I and II in Drosophila

Meiosis is a highly specialized cell division that requires significant reorganization of the canonical cell-cycle machinery and the use of meiosis-specific cell-cycle regulators. The anaphase-promoting complex (APC, a machine for degrading proteins; see APC subunits Cdc27 and morula; for review see Acquaviva, 2006) and a conserved APC adaptor/activator, Cdc20 (also known as Fizzy), are required for anaphase progression in mitotic cells. The APC has also been implicated in meiosis, although it is not yet understood how it mediates these non-canonical divisions. Cortex (Cort) is a diverged Fzy homologue that is expressed in the female germline of Drosophila, where it functions with the Cdk1-interacting protein Cks30A to drive anaphase in meiosis II. This study shows that Cort functions together with the canonical mitotic APC adaptor Fzy to target the three mitotic cyclins (A, B and B3) for destruction in the egg and drive anaphase progression in both meiotic divisions. In addition to controlling cyclin destruction globally in the egg, Cort and Fzy appear to both be required for the local destruction of cyclin B on spindles. Cyclin B associates with spindle microtubules throughout meiosis I and meiosis II, and dissociates from the meiotic spindle in anaphase II. Fzy and Cort are required for this loss of cyclin B from the meiotic spindle. These results lead to a model in which the germline-specific APC^Cort cooperates with the more general APC^Fzy, both locally on the meiotic spindle and globally in the egg cytoplasm, to target cyclins for destruction and drive progression through the two meiotic divisions (Swan, 2007).

The cell divisions of female meiosis and the ensuing mitotic cycles of early embryogenesis represent two examples of non-canonical cell cycles. Meiosis differs from the typical mitotic cycle in several respects. Most notably, two divisions occur in sequence without an intervening S-phase, resulting in the production of four haploid gametes. Additionally, the first meiotic division involves the segregation of homologous chromosomes and occurs without sister chromatid segregation, whereas the second meiotic division involves the segregation of sister chromatids, as occurs in mitosis. The regulation of meiosis requires a significant reorganization of the canonical cell-cycle machinery and the use of a number of meiosis-specific cell-cycle regulators. One example is in the regulation of anaphase - the coordinated series of events that results in the segregation of chromosomes to produce two daughter nuclei. In mitotically dividing cells, anaphase progression crucially depends on the inactivation of the mitotic kinase Cdk1 (also known as Cdc2) and on the subsequent release of sister chromatid cohesion through the destruction of cohesin complexes. These events are controlled by an E3 ubiquitin ligase -- the anaphase-promoting complex (APC) -- in association with an adaptor protein, Fzy, and this complex targets mitotic cyclins and securin (potential Drosophila homolog; Pimples) for destruction. The role of the APC in meiosis appears to be more complex than in mitotic cells. For example, the APC only partially inhibits Cdk1 activity between meiotic divisions and sister chromatid cohesion persists at centromeres through anaphase I. It is not yet clear how the activity of the APC is modified in these specialized cell divisions (Swan, 2007).

In most eukaryotes, the meiotic cell cycle is followed by another atypical cell cycle -- the cleavage divisions of early embryogenesis. In Drosophila, these cleavage cycles occur as a series of synchronized, rapid nuclear divisions and are referred to as syncytial divisions. The female meiotic cell cycle is not only closely linked to the syncytial mitotic cell cycle in time, but it also occurs within a shared cytoplasm -- that of the egg. Therefore, these two distinct cell cycles share a common pool of cell-cycle regulators, and may share common strategies for spatially and temporally regulating cell-cycle progression within a syncytium (Swan, 2007).

One way in which the syncytial cell cycle is modified appears to be in the limited destruction of mitotic cyclins in each cell cycle, apparently by restricting their destruction to the area of the mitotic nuclei. Although there is evidence that cyclin destruction is spatially regulated in somatic cells, this strategy appears to be of particular importance in the syncytial embryo of Drosophila as a means to conserve mitotic cyclins for the duration of the rapid syncytial divisions. Several lines of evidence suggest that at least one cyclin, cyclin B, undergoes limited local destruction on mitotic spindles in the syncytial embryo. It is not yet known what mediates this local cyclin B destruction, and it is also not known whether this is unique to the syncytial mitotic cell cycle or if it occurs in the preceding meiotic divisions (Swan, 2007).

Drosophila represents an excellent model system for understanding how the canonical cell-cycle machinery is developmentally modified, and how novel cell-cycle regulators are used to control meiosis and syncytial divisions. cortex (cort) encodes a Cdc20/Cdh1 (Cdh1 is also known as Fzr and Rap)-related protein, that appears to be required specifically in female meiosis and functions with a germline-specific Cks gene, Cks30A, to mediate the destruction of cyclin A. This study shows that the canonical APC adaptor Fzy functions together with Cort to target mitotic cyclins for destruction, and to drive anaphase in both meiosis I and meiosis II. Female meiosis, like the subsequent syncytial mitotic cell cycles, appears to involve the local destruction of cyclin B, and both Cort and Fzy were found to be required for this process (Swan, 2007).

In most cell types, in both Drosophila and in other metazoans, the APC^Fzy drives anaphase progression by targeting mitotic cyclins and other mitotic proteins for destruction. This study shows that the female germline is an exception in that the APC^Fzy is not sufficient. A germline-specific APC adaptor, Cort, cooperates with Fzy to mediate cyclin destruction in meiosis (Swan, 2007).

The cort gene encodes a diverged member of the Fzy/Cdh1 family. Fzy/Cdh1 homologues interact with the APC and with specific sequences (D-box, KEN box or A-box) found on cyclins and on other APC targets. As such, Fzy/Cdh1 proteins act as specificity factors to target proteins for ubiquitination and eventual destruction. Cort protein, like all Fzy/Cdh1-family proteins, contains seven WD domains in the C-terminal-half of the protein, implicated in substrate recognition. Cort has an N-terminal C-box (amino acids 482, 483) and a C-terminal IR tail (amino acids 54-60), both implicated in binding to the APC. In addition to containing these conserved functional domains, Cort displays a conserved ability to mediate cyclin destruction. cort mutations result in the overaccumulation of cyclin A, cyclin B and cyclin B3 in the egg, whereas the ectopic expression of Cort in the wing disc leads to a reduction in the levels of these mitotic cyclins. Taken together, these results indicate that Cortex encodes a functional member of the Fzy/Cdh1 family (Swan, 2007).

Although the Drosophila genome has four genes that encode Fzy/Cdh1 proteins, only two of these proteins, Fzy and Cort, are expressed at detectable levels in the female germline. The role of these two APC adaptors has been studied both individually and in double mutants, and it was found that they function together to promote anaphase in both the first and second meiotic divisions of female meiosis. In most cell types in Drosophila and other eukaryotes, a single APC complex, APC^Fzy, is responsible for cyclin destruction and anaphase progression. It is therefore surprising that, in the female germline of Drosophila, two APC adaptors are necessary for meiotic progression. In the case of meiosis I, Cort and Fzy appear to play largely redundant roles, since only removing both genes results in a significant block in meiosis I. The two APC complexes may also be functionally redundant with respect to global cyclin levels. Mutations in either fzy or cort result in an increase in the levels of cyclin A, cyclin B and cyclin B3, whereas mutation in both genes results in even-further increases in cyclin levels (Swan, 2007).

Although Cort and Fzy have overlapping roles in promoting anaphase I, both are essential for meiosis II. This could simply reflect a greater quantitative requirement for APC activity in meiosis II. Alternatively, the two APC complexes could have distinct roles in the second meiotic division. Consistent with this latter possibility, mutations in either cort or fzy both result in arrest at different stages of meiosis II: cort mutants arrest with the sister chromatids associated, and therefore in metaphase, whereas fzy mutants almost invariably arrest with separated sister chromatids, and are therefore in anaphase. cort and fzy also result in different patterns of cyclin B stabilization on the arrested spindles, suggesting roles in metaphase and anaphase, respectively. Therefore, Cort may function to initiate sister chromatid separation at the onset of anaphase II and Fzy may primarily function later, in anaphase II. Alternatively, the later arrest observed in fzy could simply reflect the fact that the fzy alleles that have been used are not nulls, and it is possible that a complete loss of Fzy activity would also result in a metaphase arrest, as seen in cort. However, comparing the meiosis II phenotypes of fzy with Cks30A-null mutants suggests that the later arrest in fzy is not simply due to residual activity. Cks30A-null mutants have a weaker meiotic arrest than fzy; they complete meiosis at high frequency, but they display a higher frequency of metaphase arrest or delay. The fact that fzy does not similarly cause a delay in metaphase of meiosis II suggests that it is only required at anaphase. Therefore, it is possible that Fzy is crucial at anaphase, whereas Cort is necessary for the metaphase to anaphase transition (Swan, 2007).

The different temporal requirements for Cort and Fzy prior to and after sister chromatid separation, respectively, could be related to their apparent differences in substrate specificity. Western analysis reveals that Cort is more important for the destruction of cyclin A and cyclin B3, whereas Fzy appears to play a greater role in cyclin B destruction in the egg. In mitotic cells, cyclin destruction occurs sequentially. Cyclin A is destroyed first, in prometaphase, and this is a prerequisite for sister chromatid separation. Cyclin B destruction occurs at anaphase onset and is necessary for later anaphase events, subsequent to sister chromatid separation. Therefore, it is possible that Cort promotes the early stages of meiotic anaphase by targeting cyclin A for destruction, whereas Fzy is more crucial later, through its targeting of cyclin B for destruction (Swan, 2007).

The meiotic cell cycle differs in many respects from the standard mitotic cycle. Whereas APC-mediated destruction of mitotic regulators appears to be required for anaphase progression in most or all mitotic cells, the role of the APC and cyclin destruction in meiosis is not as well-understood. This analysis of the two APC adaptors Cort and Fzy has permitted an evaluation of the role of the APC complex in female meiosis in Drosophila. The APC is required for anaphase progression in both meiotic divisions. Correlating with its requirement for the completion of meiosis, the APC is required for the destruction of mitotic cyclins. At least one of these cyclins, cyclin B, is a crucial substrate in meiosis, because the expression of a stabilized form of cyclin B disrupts this process. Therefore, APC activity and cyclin destruction are required for anaphase progression in both meiotic divisions, in addition to in mitosis. APC activity has been implicated in both meiotic divisions in C. elegans and in the mouse, and in the second, but not the first, meiotic division in Xenopus. In yeast, two APC complexes, the mitotic APC^Fzy and a meiosis-specific complex (APC^Ama1 in S. cerevisiae and APC^Mfr1 in S. pombe) function together to mediate protein destruction in meiosis. It now appears that Drosophila also uses two APC complexes in female meiosis, and this may turn out to be a common strategy in other eukaryotes (Swan, 2007).

Cks30A belongs to a highly conserved family of proteins that bind to and stimulate the activity of the mitotic kinase Cdk1. In Xenopus, the Cks30A homologue Xep9 stimulates the Cdk-dependent phosphorylation of APC subunits, and thereby promotes the activation of the APC^Fzy complex (Patra, 1998). The current results suggest that Cks30A may have a similar role in stimulating both the APC^Fzy and APC^Cort in female meiosis in Drosophila. (1) Cks30A, like cort and fzy, is required for the completion of meiosis II and, like fzy, it is required for the completion of the first mitotic division of embryogenesis. (2) Cks30A, as are Cort and Fzy, is necessary for global cyclin destruction in the Drosophila egg and for local cyclin B destruction on the meiotic spindle. Global levels of cyclin A and cyclin B3 are elevated to a greater extent in Cks30A mutants than in single mutants for cort or fzy, consistent with the idea of Cks30A activating both Cort and Fzy. (3) Cks30A is necessary for the activity of ectopically expressed Cort in the adult wing. Cks30A may also play a role in activating APC^Fzy in mitotic cells. the temperature-sensitive fzy⁶ allele is lethal at all temperatures in a Cks30A-null background, suggesting that the Cks30A-dependent activation of APC^Fzy becomes essential when Fzy activity is compromised (Swan, 2007).

Although Cks30A appears to promote the activity of the APC^Cort and the APC^Fzy, these complexes seems to retain some activity in the absence of Cks30A. Whereas cort and fzy cause an arrest in meiosis II, Cks30A-null mutants are typically delayed only in meiosis II. Also, although cyclin A and cyclin B3 levels are elevated more in Cks30A eggs than in either fzy or cort, their levels are still not as high as in fzy; cort double mutants, indicating that Fzy and Cort can destroy cyclin A and cyclin B3 to some degree in the absence of Cks30A. Cyclin B destruction is even less dependent on Cks30A, because cyclin B levels are affected less in Cks30A mutants than in either cort or fzy single mutants. Therefore, Cks30A may be more crucial for the activity of APC^Cort and APC^Fzy complexes on cyclin A and cyclin B3, and less crucial for their activity on cyclin B. The relatively weaker meiotic arrest in Cks30A mutants compared to fzy; cort double mutants may also indicate that the APC has other meiotic targets that can be destroyed in the absence of Cks30A (Swan, 2007).

Cyclin B undergoes local oscillations in its association with mitotic spindles in syncytial embryos, appearing transiently along the full length of the mitotic spindle in early metaphase and gradually disappearing from the spindle starting at the centrosomes and ending at the kinetochores. The timing of this loss of cyclin B from the spindle, at the onset of anaphase, corresponds with the timing of cyclin B destruction in other cell types, suggesting the possibility that cyclin B is locally destroyed on the spindle in anaphase. This study shows that cyclin B is subject to similar local oscillations in the female meiotic cycles, and that cyclin B destruction is necessary for the completion of female meiosis. Importantly, the local loss of cyclin B from the spindle in meiosis is dependent on the APC adaptors Cort and Fzy, and that the local loss of cyclin B from the spindle in mitosis depends on Fzy. These results strongly suggest that the local loss of cyclin B from the spindle in anaphase of meiosis II and anaphase of mitosis is actually due to its local destruction (Swan, 2007).

The pattern of accumulation and loss of cyclin B from the spindle in meiosis differs in some respects compared to syncytial mitotic cycles. (1) In metaphase of mitosis, cyclin B initially accumulates throughout the spindle microtubules, whereas, in metaphase of the meiotic divisions, cyclin B first appears exclusively at the spindle mid-zone. This difference may reflect the fact that the meiotic spindle does not contain centrosomes and cyclin B may, therefore, not load onto spindles from centrosomes and progress along the spindles to the kinetochores, as has been proposed for mitosis. (2) The timing of cyclin B destruction appears to be different between the meiotic and mitotic cycles. Most strikingly, there is no loss of cyclin B from the spindle in anaphase of meiosis I, implying that local cyclin B destruction is not necessary for the completion of the first meiotic division. In addition, the loss of cyclin B from the spindle following meiosis II occurs only late in anaphase rather than at the onset of anaphase, as occurs in the syncytial mitotic cycles. It is not yet known how cyclin B destruction is prevented in anaphase I and early in anaphase of meiosis II. One possibility is that the spindle-assembly checkpoint is locally active during these stages. This checkpoint is required for the proper completion of female meiosis in Drosophila, and it will be interesting to see if this requirement reflects a role in inhibiting either APC^Fzy or APC^Cort activity (Swan, 2007).

The specific accumulation of cyclin B at the spindle mid-zone in meiosis may reflect the unique properties of the meiotic spindle. The mid-zone microtubules or central spindle microtubules are a subset of spindle microtubules that do not end in kinetochores, but instead overlap at the mid-zone with microtubules from the other pole. In dividing cells, the central spindle is crucial for cytokinesis, but, in female meiosis, it appears to have a role in spindle assembly. Along with cyclin B, the chromosomal passenger proteins Aurora B and Incenp are recruited to the spindle mid-zone. It will be of great interest to determine what these proteins do at the mid-zone and how cyclin B destruction at this site may be important for anaphase in meiosis. It will also be important to determine how the APC^Cort targets cyclin B at the spindle mid-zone. It has not been possible to detect any specific localization of GFP or HA-tagged Cortex in meiosis or in the syncytial embryo, but it is possible that its activity is spatially regulated (Swan, 2007).

In conclusion, these results support a model in which two APC complexes, APC^Fzy and APC^Cort, cooperate to mediate the destruction of meiotic cyclins and allow progression through female meiosis (Swan, 2007).

APC/C^Fzr/Cdh1 promotes cell cycle progression during the Drosophila endocycle

The endocycle is a commonly observed variant cell cycle in which cells undergo repeated rounds of DNA replication with no intervening mitosis. How the cell cycle machinery is modified to transform a mitotic cycle into endocycle has long been a matter of interest. In both plants and animals, the transition from the mitotic cycle to the endocycle requires Fzr/Cdh1, a positive regulator of the Anaphase-Promoting Complex/Cyclosome (APC/C). However, because many of its targets are transcriptionally downregulated upon entry into the endocycle, it remains unclear whether the APC/C functions beyond the mitotic/endocycle boundary. This study reports that APC/C^Fzr/Cdh1 activity is required to promote the G/S oscillation of the Drosophila endocycle. Compromising APC/C activity, after cells have entered the endocycle, inhibits DNA replication and results in the accumulation of multiple APC/C targets, including the mitotic cyclins and Geminin. Notably, the data suggest that the activity of APC/CFzr/Cdh1 during the endocycle is not continuous but is cyclic, as demonstrated by the APC/C-dependent oscillation of the pre-replication complex component Orc1. Taken together, these data suggest a model in which the cyclic activity of APC/C^Fzr/Cdh1 during the Drosophila endocycle is driven by the periodic inhibition of Fzr/Cdh1 by Cyclin E/Cdk2. It is proposed that, as is observed in mitotic cycles, during endocycles, APC/C^Fzr/Cdh1 functions to reduce the levels of the mitotic cyclins and Geminin in order to facilitate the relicensing of DNA replication origins and cell cycle progression (Narbonne-Reveau, 2008).

The endocycle provides a useful model for determining the minimum cell cycle inputs required to achieve a G/S oscillation and the once-per-cell-cycle replication of the genome. This study demonstrates that APC/C activity is required for endocycle progression. During the endocycle, mitotic activities are repressed. This is accomplished, at least in part, by preventing the accumulation of the mitotic activators Cyclin A, Cyclin B and Cdc25, which function to activate the mitotic kinase Cdk1. During the mitotic cycle, the mitotic cyclins are periodically targeted for regulated proteolysis by the E3-Ubiquintin ligase the APC/C. Yet the transcriptional downregulation of several APC/C targets at the mitotic/endocycle boundary, including the mitotic cyclins and String/Cdc25, suggested that the proteolytic activity of the APC/C might not be necessary during endocycles. However, this study found that compromising APC/C activity, after cells have entered the endocycle, results in the accumulation of Geminin and the mitotic cyclins, and in a block of DNA replication. Thus, the transcriptional downregulation of APC/C targets observed at the mitotic/endocycle transition is either downstream of APC/C activity and/or not sufficient to maintain low levels of these proteins. Taken together, these data suggest a model in which APC/C promotes the G/S oscillation of the endocycle by preventing the unscheduled accumulation of Geminin and the mitotic cyclins (Narbonne-Reveau, 2008).

During endocycles, APC/C activity prevents the inappropriate accumulation of Geminin, an inhibitor of the DNA replication-licensing factor Cdt1/Dup. When directly expressed in endocycling cells, Geminin efficiently inhibits DNA replication. These results strongly suggest that an essential function of the APC/C during the endocycle is to prevent the unregulated accumulation of Geminin. A similar role has been proposed for the APC/C during endoreplicative cycles of mouse trophoblasts (Gonzalez, 2006). However, the current data indicate that Geminin is not the only essential target of the APC/C during endocycles. A candidate for a second important target of the APC/C during endocycles is Cyclin A. Previous studies have shown that the overexpression of Cyclin A in the salivary gland, between the first and second endocycle, results in variable inhibitory effects on endoreplication. Although the majority of salivary gland cells that overexpress Cyclin A appear to be unaffected, a small percentage show a marked decrease in ploidy values. The reason for this variability is not clear. However, if the inhibitory influence of Cyclin A is mediated through binding and activation of Cdk1, this effect may be greatly amplified in the presence of high levels of String/Cdc25, which removes an inhibitory phosphate from Cdk1. Recent studies indicate that String/Cdc25, which contains both a consensus Ken box and D-box, is a target of the APC/C (Barbara Thomas, personal communication to Narbonne-Reveau, 2008). Therefore, an essential function of the APC/C during endocycles may involve restricting the activity of the mitotic kinase Cdk1, by preventing the accumulation of both Cyclin A and String/Cdc25. Finally, it is noted that the APC/C may have additional essential targets during the endocycle, which have yet to be identified (Narbonne-Reveau, 2008).

The periodic accumulation of the Orc1 protein during endocycles strongly suggests that the activity of the APC/C^Fzy/Cdh1 may not be continuous but cyclical. Previous work indicates that in Drosophila Cyclin E/Cdk2 inhibits the activity of APC/C^Fzy/Cdh1. These data are consistent with the observation that phosphorylation of Fzr/Cdh1 by Cdks inhibits the ability of Fzr/Cdh1 to bind and activate the APC/C in yeast, Xenopus and mammals. During the endocycle, the levels of Cyclin E oscillate. Taken together, these observations suggest a model in which APC/C^Fzy/Cdh1 is regulated by the periodicity of Cyclin E/Cdk2 activity, with high levels of Cyclin E resulting in the inhibition of APC/C^Fzy/Cdh1 activity and low levels of Cyclin E permitting full APC/C^Fzy/Cdh1 activity. The current data support this hypothesis. First, it was found that the periodicity of Orc1 levels during the endocycle requires a functional O-box, consistent with the cyclic destruction of Orc1 by APC/C^Fzy/Cdh1. Second, the levels of Orc1 are sensitive to Cyclin E. Specifically, overexpressing Cyclin E after cells have entered the endocycle results in the accumulation of APC/C^Fzy/Cdh1 targets, including Orc1, Cyclin A, Cyclin B and Geminin. Thus, the regulatory relationship observed between Cyclin E/Cdk2 and Fzr/Cdh1 that has been reported during mitotic cycles is conserved during endocycles. Finally, in endocycling cells the accumulation of Orc1 occurs during periods of high Cyclin E/Cdk2 activity, when APC/C^Fzy/Cdh1 dependent proteolysis would be predicted to be low. These data support the idea that the oscillations of Cyclin E/Cdk2 activity drive the periodicity of APC/C^Fzy/Cdh1 activity during the endocycle (Narbonne-Reveau, 2008).

Although a requirement for the oscillation of APC/C^Fzy/Cdh1 activity during the Drosophila endocycle has not been formally demonstrated, it is interesting to speculate on how the cyclic, rather than the continuous, activity of the APC/C might serve to facilitate endocycle progression. The data indicate that a period of high APC/C^Fzy/Cdh1 activity is required during the G phase of the endocycle in order to degrade the mitotic cyclins and Geminin, which can function to inhibit the formation of pre-RCs. However, a period of low APC/C activity may also be important. The continuous activation of APC/CCdh1 significantly slows DNA replication in mouse tissue culture cells. This inhibition may reflect the inability of a cell to accumulate adequate levels of proteins required for DNA replication, such as the APC/CCdh1 target and pre-replication complex component CDC6, in the presence of a constitutively active APC/CCdh1. In Drosophila, continuous APC/C^Fzy/Cdh1 activity might prevent the accumulation of two pre-RC components, CDC6 and Orc1. Intriguingly, APC/C activity also appears to oscillate during mammalian endocycles. In endocycling mouse trophoblasts, the levels of Cyclin A oscillate, consistent with the regulated destruction of the Cyclin A protein by the APC/C. Additionally, the inhibition of APC/C activity in endocycling trophoblasts results in the accumulation of the APC/C targets Cyclin A and Geminin. Taken together, these observations support a model in which the oscillation of APC/C^Fzy/Cdh1 activity, which is driven by the regulatory influences of Cdks, promotes efficient cell cycle progression during the endocycle (Narbonne-Reveau, 2008).

The data raises important questions. Why do levels of some APC/C^Fzy/Cdh1 targets, such as Cyclin A, Cyclin B and Geminin, remain below the level of detection while the levels of Orc1 protein oscillate? What might account for these different modes of regulation? Currently, there is no definitive explanation. However, at least three possibilities, which are not mutually exclusive, are envisaged, that may contribute to this differential behavior. First, it was found that relative to the Cyclin A and geminin, the levels of Orc1 transcript are only minimally downregulated upon entry into the endocycle. Transcriptional downregulation, or changes in transcript stability, may help contribute to the low levels of Geminin and Cyclin A proteins observed during the endocycle. Second, the translational efficiency of a subset of transcripts may be reduced upon entry into the endocycle. Finally, it is possible that the Orc1 protein is not as efficiently targeted by the APC/C^Fzy/Cdh1 as the mitotic cyclins or Geminin. Indeed the cis-acting sequences that target these proteins for destruction show considerable variability. Orc1 is targeted for APC/C^Fzy/Cdh1 destruction via a novel motif called the O-box (Araki, 2005). By contrast, Cyclin B and Geminin are targeted by a similar but unique sequence called the destruction-box (D-box), while Drosophila Cyclin A is targeted for destruction by a large complex N-terminal degradation sequence. There is precedence for post-translational regulation of APC/C^Fzy/Cdh1 targets, resulting in differential expression. In mammalian cells the pre-RC component CDC6, which is structurally related to Orc1, is protected from APC/CFzr/Cdh1 degradation by phosphorylation by Cyclin E/Cdk2. One or all of these potential mechanisms may contribute to the differential expression of various APC/CFzr/Cdh1 targets during the endocycle (Narbonne-Reveau, 2008).

Recent evidence from mice indicates that the depletion of the APC/C inhibitor Emi1/Rca1, results in both a strong decrease in E2F target mRNAs, such as geminin and Cyclin A, as well as APC/C activation. This study suggested that the regulation of APC/C activity, by the inhibitor Emi1/Rca1, drives a positive feedback circuit that controls both protein stability and mRNA expression. Thus, the observed decrease in the levels of at least some APC/C targets that occurs upon depletion of Emi1/Rca1, including Geminin and Cyclin A, are controlled at the levels of transcription and protein stability. Developmentally programmed endocycles may provide a natural example where cell cycle progression occurs in the context of increased APC/CFzr/Cdh1 activity. Thus, a similar positive-feedback circuit may be operating during Drosophila endocycles to downregulate the transcription of E2F target genes. Determining the precise regulatory relationships between the upregulation of APC/CFzr/Cdh1 activity and the transcriptional downregulation of genes such as Cyclin A and geminin, during the Drosophila endocycle represents an exciting area for future research (Narbonne-Reveau, 2008).

The requirement for APC/C activity to promote endocycle progression may help answer several longstanding questions concerning the regulation of the Drosophila endocycle. For example, why does the continuous expression of Cyclin E inhibit cell cycle progression during the endocycle but not the mitotic cycle? Several models have been proposed to explain this difference. First, the breakdown of the nuclear envelope that occurs during the mitotic cycle, but not the endocycle, may allow for a transient decrease in local Cyclin E/Cdk2 activity, thus allowing for the relicensing of DNA replication origins. Alternatively, there may be differences in the machinery required to produce a functional pre-RC in mitotic versus endocycling cells. The current results suggest an alternative model for why endocycles are unusually sensitive to continuous Cyclin E expression. This model is based on the demonstration that endocycle progression requires APC/C activity. Both Fzy/Cdc20 and Fzr/Cdh1 function as activators of the APC/C. However, the regulation of these APC/C activators is very distinct. During the mitotic cycle, the binding of Fzy/Cdc20 to the APC/C is dependent on the phosphorylation of several APC/C subunits by the mitotic kinase Cdk1. By contrast, a Cdk-dependent inhibitory phosphorylation on Fzr/Cdh1 relegates APC/CFzr/Cdh1 activity to late M phase and G1. Because of its requirement for Cdk1 activity, APC/C^Fzy/Cdc20 is unlikely to be active during most endocycles. Indeed, Drosophila endocycles proceed normally in fzy mutants. Thus, the only available activator of the APC/C during the endocycle is Fzr/Cdh1. As previously discussed, Fzr/Cdh1 is inhibited by Cyclin E/Cdk2 activity. Therefore, it is proposed that during the endocycle, continuous Cyclin E/Cdk2 activity results in the permanent inhibition of the only available activator of the APC/C, Fzr/Cdh1. This leads to the accumulation of Geminin, Cyclin A and other potential targets, which act to block cell cycle progression. Thus, the ability of continuous Cyclin E to inhibit DNA replication during the endocycle may reflect differences in the available activators of the APC/C present in mitotic versus endocycling cells (Narbonne-Reveau, 2008).

Effete-mediated degradation of Cyclin A is essential for the maintenance of germline stem cells in Drosophila

Increasing evidence supports the idea that the regulation of stem cells requires both extrinsic and intrinsic mechanisms. However, much less is known about how intrinsic signals regulate the fate of stem cells. Studies on germline stem cells (GSCs) in the Drosophila ovary have provided novel insights into the regulatory mechanisms of stem cell maintenance. This study demonstrates that a ubiquitin-dependent pathway mediated by the Drosophila eff gene, which encodes the E2 ubiquitin-conjugating enzyme. Effete (Eff), plays an essential role in GSC maintenance. Eff both physically and genetically interacts with dAPC2, a key component of the anaphase-promoting complex (APC), which acts as a multisubunit E3 ligase and plays an essential role in targeting mitotic regulators for degradation during exit from mitosis. This interaction indicates that Eff regulates the APC/C-mediated proteolysis pathway in GSCs. Moreover, expression of a stable form of Cyclin A, but not full-length Cyclin A, results in GSC loss. Finally it was shown that, in common with APC2, Eff is required for the ubiquitylation of Cyclin A, and overexpression of full-length Cyclin A accelerates the loss of GSCs in the eff mutant background. Collectively, these data support the idea that Effete/APC-mediated degradation of Cyclin A is essential for the maintenance of germline stem cells in Drosophila. Given that the regulation of mitotic Cyclins is evolutionarily conserved between flies and mammals, this study also implies that a similar mechanism may be conserved in mammals (Chen, 2009).

Germline stem cells (GSCs) of the Drosophila ovary provide an excellent model system for studying the molecular mechanisms of stem cell regulation in vivo. In adult Drosophila females, two to three GSCs are easily recognized by their molecular markers (either a spherical spectrosome, or an extending fusome when GSCs are dividing) and their location at the apical region of the germarium in close contact with surrounding somatic cells, the terminal filament and cap cells, which together generate a specific micro-environment, or niche, for GSC regulation. The GSC divisions take place along the anteroposterior axis of the ovary to produce an anterior GSC, which remains attached to the niche cells, and a posterior cystoblast (Cb). The Cb divides precisely four times by incomplete cytokinesis to generate 16 interconnected cells that form the germline cyst of the follicle and sustain oogenesis (Chen, 2009).

Genetic analyses have revealed that the stem cell state of GSCs is maintained by both extrinsic and intrinsic mechanisms that repress their differentiation. BMP ligands (Dpp, Gbb) from the niche cells maintain GSCs by suppressing Cb differentiation in the anteriormost cells. This is achieved by silencing the transcription of the bam gene, which encodes a GSC/Cb differentiation-promoting factor. In the GSCs, BMP signaling activates cytoplasmic Mad and Medea, the Drosophila Smads, and promotes their nuclear translocation. In the nucleus, the Smads complex physically interacts with both the bam silencer element and nuclear lamin-associated protein (Ote), resulting in bam transcriptional silencing. Thus, BMP/Dpp-dependent bam transcriptional control serves as the primary pathway for the regulation of GSC fate. Independent of the niche-based regulation of the bam silencing mechanism, the fate of GSCs is also intrinsically controlled by other GSC maintenance factors that repress their differentiation. It has been demonstrated that Pum/Nos-mediated and microRNA-mediated translational repression pathways are not required for bam silencing, suggesting that these pathways act either downstream of or parallel to bam action. Although it is proposed that the Pum/Nos-mediated and microRNA-mediated pathways repress the translation of key differentiation factors to prevent GSC differentiation, the targets of these translational pathways in GSCs are not identified. Thus, the intrinsic mechanisms that repress GSC differentiation are still poorly understood. In addition, as loss of function of the components in these translational repression pathways is not sufficient to completely cause bam mutant germ-cell differentiation, it is speculated that the repression of GSC differentiation may also be controlled by other unknown intrinsic mechanisms (Chen, 2009).

Ubiquitin-mediated protein degradation plays a variety of roles in the regulation of many developmental processes. The enzymatic reaction of protein ubiquitylation is a coordinated three-step process involving three classes of enzymes known as E1 (Uba1 -- FlyBase), E2 (UbcD4 -- FlyBase) and E3. Firstly, E1 (Ubiquitin activating enzyme 1) catalyzes the formation of a thiolester bond linkage between the active-site cysteine residue on E1 and the C terminus of ubiquitin. Secondly, the activated ubiquitin (E1-Ub) is then transferred to E2 (Ubiquitin conjugating enzyme 4) via formation of an E2-Ub thiolester. Thirdly, E3 (ubiquitin ligase) promotes the transfer of the ubiquitin from E2-Ub to a lysine residue of the target protein through an isopeptide bond. Repeated cycles of this reaction can result in polyubiquitylation of the target protein, which is finally targeted for degradation by the 26S proteasome. The Drosophila effete (eff) gene encodes a class I ubiquitin-conjugating enzyme that was first shown to be required for proper telomere behavior (Cenci, 1997). Early studies also showed that eff is required for proper cyst formation in ovary (Lilly, 2000). However, whether eff is involved in the regulation of GSC fate remains unknown (Chen, 2009). Mitotic cyclins, in concert with their Cyclin-dependent kinase (CDK) partners, play important roles in the promotion of mitosis, such as entry into mitosis and the alignment of chromosomes at metaphase. The degradation of mitotic cyclins by the anaphase-promoting complex/cyclosome (APC/C), a large multisubunit E3 ligase, leads to inactivation of CDKs during the metaphase-anaphase transition and is essential for the completion of mitosis in all eukaryotes. In Drosophila, it has been shown that induction of stable versions of the three mitotic cyclins, Cyclin A, B and B3, arrests mitosis with different phenotypes, suggesting that degradation of distinct mitotic cyclins is required for specific mitotic events. It has been shown that the asymmetric division of GSCs requires the activity of Cyclin B; however, it remains unknown whether the regulation of mitotic cyclins also contributes to fate determination in GSCs. This study demonstrates that the eff gene, which encodes an E2 ubiquitin-conjugating enzyme (Eff), is intrinsic to and essential for Drosophila GSC maintenance. Moreover, Eff both genetically and physically interacts with dAPC2, a key component of APC/C complexes. dCDC20/Fzy, a key regulator of APC/C complexes, is shown to be involved in GSC regulation. Finally, constant expression of a stable form of Cyclin A results in the loss of GSCs. Notably, in common with APC2, Eff is required for the ubiquitylation of Cyclin A, and overexpression of full-length Cyclin A accelerates the loss of GSCs in the eff mutant background. Thus, these data support the idea that Effete/APC-mediated degradation of Cyclin A is essential for the maintenance of GSCs in Drosophila (Chen, 2009).

To identify new factors that regulate the self-renewal or differentiation of GSCs in the Drosophila ovary, female sterile lines, or weak fertile lines with P-element insertion, available from Bloomington Stock Center, were screened. The typical characteristic of GSC maintenance defects is a reduction in germ-cell number. This eventually results in an empty germarium lacking germ cells, and a decline in the production of egg chambers. Based on these criteria, a line with a P-element insertion in the third chromosome, P{PZ}eff⁸ was identified, that exhibits severe defects in germline development, including the loss of germ cells. To systematically study the behavior of GSCs and early germ cells in the eff⁸ mutant, anti-Vasa and anti-Hts antibodies were used to visualize germ cells and fusomes, respectively. In the tip of wild-type germarium, two or three GSCs were readily recognized by anti-Vasa antibody, and fusomes were morphologically spherical and anchored between the GSCs and cap cells. In addition, a normal germline lineage with sequentially differentiated cells marked by branched fusomes was also observed. However, in the 7-day-old ovaries from eff⁸ homozygous females, about 30% of mutant ovarioles contained either empty or abnormal germaria. Furthermore, the germ-cell defect phenotype became much more severe with age. These findings suggest that the loss of eff may affect the maintenance of GSCs. To determine whether the GSC maintenance defect associated with eff⁸ was indeed due to the loss of eff function rather than other genetic backgrounds, the phenotypes resulting from removal of eff were analyzed in several allelic combinations, eff⁸/eff^s1782, eff⁸/eff^mer1 and eff⁸/eff^mer4. The number of GSCs in the available eff allelic combinations were quantified at days 1, 7 and 14 after eclosion. Compared to wild type, the average number of GSCs in all eff mutants either rapidly or progressively declined during the testing period, indicating that the loss of eff resulted in the loss of GSCs. To further confirm this observation, a transgene, P{effP-eff}, was generated in which an eff cDNA was placed under the control of a 5.8 kb eff promoter. The GSC loss phenotype in different eff allelic backgrounds was fully rescued by the transgene line, P{effP-eff}. Taken together, these findings indicate that the eff gene plays an essential role in the maintenance of GSCs (Chen, 2009).

Attempts were made to understand the molecular mechanism underlying the action of Eff in GSCs by searching for Eff-interacting partners. Given that Eff functions as an E2 ubiquitin-conjugating enzyme, an E2/E3-based small-scale candidate screen was carried out by performing yeast two-hybrid experiments in which Eff was used as the bait to screen Eff-interacting E3. Notably, it was found that, among the candidates, dAPC2, encoded by the Drosophila morula (mr) gene, can strongly interact with Eff protein. To confirm this yeast two-hybrid interaction, whether Eff interacts with dAPC2 in Drosophila S2 cells was investigated by performing immunoprecipitation experiments. It was shown that Eff and dAPC2 can co-immunoprecipitate each other in transfected S2 cells, suggesting that Eff and dAPC2 are physically associated. To test whether dAPC2 physically associates with endogenous Eff in germ cells, a transgene, P{nosP-myc:dAPC2}, was generated. Results from co-immunoprecipitation showed that endogenous Eff physically associated with Myc:dAPC2, supporting further the argument that Eff interacts with dAPC2 in germ cells. In mitosis, the anaphase-promoting complex/cyclosome APC/C), a multisubunit complex that functions as an E3 ligase, plays important roles in ubiquitylating mitotic regulators such as mitotic cyclins and thus targets them for degradation by 26S proteasome. During this process, APC2, a cullin domain-containing protein, has been shown to function as a key mediator of APC/C complex activity. To test whether eff genetically interacts with mr (dAPC2) in the regulation of GSCs, the number of GSCs in both eff single-mutant and mr; eff double-mutant backgrounds were quantified at different time points. A weak allelic combination of mr (mr¹/mr²) exhibited no apparent defect in GSC maintenance. However, mr; eff double-mutant ovaries showed more rapid GSC loss than the eff mutant alone, suggesting that dAPC2/mr enhances the phenotype of GSC loss in eff. Together, these results demonstrate that Eff interacts both physically and genetically with dAPC2 (Chen, 2009).

The ubiquitin-mediated proteolysis mechanism, which is evolutionarily conserved for the regulation of protein turnover, has been shown to play important roles in numerous biological processes, such as the cell cycle, pattern formation and tissue homeostasis. Drosophila Eff, which was initially identified as a class I E2 ubiquitin-conjugating enzyme encoded by the eff gene, has been shown to be involved in several cellular and developmental processes, including chromosome segregation, chromatin remodeling and protection against cell death. This study found that loss of eff function results in the depletion of GSCs, revealing a new role for eff in the regulation of GSC fate. Using germline clonal analysis and rescue experiments, it was further defined that the eff gene is an intrinsic, rather than extrinsic, factor for the maintenance of GSCs. Previous studies have shown that Eff is involved in the rpr-induced apoptosis pathway through a physical interaction with DIAP1 that stimulates DIAP1 auto-ubiquitylation. Therefore, it is possible that loss of GSCs in eff mutants may be due to reduced viability of GSCs. The results clearly show that eff^-/- GSCs undergo differentiation rather than apoptosis, thus supporting the idea that the role of eff is to repress the premature differentiation of GSCs (Chen, 2009).

In the ubiquitin pathway, E2 conjugating enzymes have much lower specificity compared with E3 ligases. Certain E2s are known to function together with distinct type E3 ligases for substrate ubiquitylation and degradation. Eff is involved in protein degradation mediated by various RING finger-containing E3 ligases [e.g., Sina, Neur and DIAP (Iap2 -- FlyBase)], and regulates several signaling transduction pathways (Kuo, 2006; Ryoo, 2002; Tang, 1997). Since Eff plays a role downstream of, or parallel to, bam function, it is important to know what biochemical functions Eff performs in the regulation of GSCs. This work provided biochemical evidence showing that Eff not only physically interacts with the dAPC2 protein, but is also crucial for the ubiquitylation and degradation of Cyclin A. Moreover, genetic analyses revealed that eff interacts with both dAPC2 and cyclin A with respect to the regulation of GSCs. In addition, this study shows that dCDC20/Fzy, a key regulator of APC/C complexes, is involved in GSC regulation. Together, these data strongly support a model in which Eff facilitates the E3 ligase function of APC/CDC20 to ensure the self-renewal of GSCs (Chen, 2009).

Early studies in Xenopus and clam extracts demonstrated that both UBC4, a homolog of Eff, and UBCx/E2-C equally supported APC-mediated ubiquitylation reactions in vitro (. However, an in vivo study showed that these two classes of E2 are not functionally equivalent but exhibit distinct functions in mitotic cyclin degradation, suggesting that different E2 family members probably execute distinct functions (Seino, 2003). The Drosophila ortholog of UBCx/E2-C, Vihar E2, has been reported to be involved in Cyclin B degradation during the metaphase-anaphase transition. This work presents both genetic and biochemical evidence that Eff, the Drosophila homolog of UBC4, is essential for Cyclin A degradation in GSCs. Because the mitosis-related ubiquitin-conjugating enzyme, Vihar E2, is involved in APC/C-mediated ubiquitylation that potentially regulates Cyclin A degradation, it would be interesting to determine whether and/or how different E2 family members (e.g. Eff and Vihar E2) coordinately support specific APC-mediated mitotic cyclin destruction with respect to GSC regulation (Chen, 2009).

It has been shown that APC/C activity is required for the asymmetric localization of Miranda and its cargo proteins during neuroblast division. In Drosophila ovary, previous studies have demonstrated that Cyclin B plays important roles in GSC division and is essential role for GSC maintenance. However, it still remains unexplored whether the tight regulation of cyclins is also required for the fate determination of GSCs. The regulatory roles of mitotic cyclins at the cellular level during mitosis have been explored in detail. It has been reported that the sequential degradation of Cyclin A, Cyclin B and Cyclin B3 completes mitotic exit, which is mediated by APC/CDC20 in early M phase and by APC/Cdh1 during late M phase (Zachariae, 1999). Interestingly, the expression of stable forms of each cyclin leads to distinct mitosis defects, suggesting that the degradation of distinct mitotic cyclins is responsible for specific steps of mitosis. However, the biological basis for the control of the cyclin destruction remains elusive. Given that the APC-mediated pathway plays important roles in the proper cell mitosis, as loss of function of components in the pathway results in upregulation of mitotic cyclins that cause mitosis delay/or arrest, the question becomes whether the maintenance of GSCs requires the proper cell mitosis mediated by the regulation of mitotic cyclins. This study has shown that the forced expression of a stable form of Cyclin A leads to defects in GSC maintenance, suggesting that blocking mitotic progression may force germline stem cells to precociously differentiate, essentially altering their fate. Although the forced expression of a stable form of Cyclin B or Cyclin B3 does not give rise to any apparent defect in GSCs, one explanation is that stabilized Cyclin A may block cell-cycle progression more severely than the other stabilized Cyclins and prolonged M phase might be unfavorable for stem cell maintenance (Chen, 2009).

Taken together, these findings support a mechanism underlying the fate determination of stem cells that is linked to the control of the proper cell mitosis. Since the control of degradation of mitotic cyclins is evolutionarily conserved between flies and mammals, it would be interesting to also determine whether the control of proper cell mitosis is important for the maintenance of stem cells from other organisms, including mammals (Chen, 2009).

Regulation of cyclin A localization downstream of Par-1 function is critical for the centrosome orientation checkpoint in Drosophila male germline stem cells.

Male germline stem cells (GSCs) in Drosophila melanogaster divide asymmetrically by orienting the mitotic spindle with respect to the niche, a microenvironment that specifies stem cell identity. The spindle orientation is prepared during interphase through stereotypical positioning of the centrosomes. It has been demonstrated that GSCs possess a checkpoint ('the centrosome orientation checkpoint') that monitors correct centrosome orientation prior to mitosis to ensure an oriented spindle and thus asymmetric outcome of the division. This study shows that Par-1, a serine/threonine kinase that regulates polarity in many systems, is involved in this checkpoint. Par-1 shows a cell cycle-dependent localization to the spectrosome, a germline-specific, endoplasmic reticulum-like organelle. Furthermore, the localization of cyclin A, which is normally localized to the spectrosome, is perturbed in par-1 mutant GSCs. Interestingly, overexpression of mutant cyclin A that does not localize to the spectrosome and mutation in hts, a core component of the spectrosome, both lead to defects in the centrosome orientation checkpoint. It is proposed that the regulation of cyclin A localization via Par-1 function plays a critical role in the centrosome orientation checkpoint (Yuan, 2012).

Thus Par-1 acts as a component of the centrosome orientation checkpoint, probably through its ability to influence cyclin A localization. This checkpoint ensures the asymmetric outcome of GSC division by delaying cell cycle progression when centrosomes are not correctly oriented. Such a checkpoint would provide an additional layer of accuracy in oriented stem cell division. This study highlights the importance of cyclin A localization in the centrosome orientation checkpoint. Intriguingly, it was reported that in cultured mammalian cells, cyclin A is confined to the endoplasmic reticulum (ER) via its interaction with a protein called SCAPER (Tsang, 2007). The spectrosome/fusome has been shown to be a part of the ER (Snapp, 2004), therefore, regulation of cyclin A through its localization is likely evolutionarily conserved (Yuan, 2012).

The fact that the wild type misoriented GSCs tend to have lower/non-detectable cyclin A levels suggests that GSCs degrade cyclin A or that the arrest point of the centrosome orientation checkpoint is before cyclin A accumulation. It is possible that distinct mechanisms stall the cell cycle, depending on when the centrosome misorientation is sensed. For example, when the centrosome misorientation is detected earlier in the cell cycle (i.e., before cyclin A accumulation), the cell cycle would be stalled before cyclin A protein synthesis/accumulation. In contrast, when the centrosome misorientation is detected later in the cell cycle (i.e., after cyclin A accumulation), the cell cycle would be stalled by preventing translocation of cyclin A from the spectrosome to the cytoplasm/nucleus. Further studies are required to dissect the detailed mechanisms that monitor centrosome orientation, possibly depending on the cell cycle stage (Yuan, 2012).

It is currently unclear how Par-1 might regulate cyclin A localization in response to centrosome misorientation. Direct interaction between Par-1 and cyclin A was not detected in immunoprecipitation experiments, thus the molecular mechanism by which Par-1 regulates cyclin A localization to the spectrosome/fusome remains to be determined. It is formally possible that cyclin A mislocalization and the defective checkpoint response are two unrelated consequences of par-1 mutation. However, considering that the expression of cyclin A mutant proteins defective in spectrosome localization is sufficient to perturb the centrosome orientation checkpoint, the possibility is favored that cyclin A is indeed part of a Par-1-dependent checkpoint response to centrosome misorientation. Future identification of proteins that recruit/anchor cyclin A to the spectrosome will provide further insight into this process (Yuan, 2012).

This study has shown that the mother centrosome is consistently located at the hub–GSC interface, while the daughter centrosome migrates to the opposite side. Whether the centrosome orientation checkpoint monitors the correct positioning of the mother centrosome or any centrosome is currently unknown. However, given that dedifferentiated GSCs, which must have lost their 'original' mother centrosome (generated earlier during development) when they committed to differentiation, still retain the centrosome orientation checkpoint, the centrosome orientation checkpoint does not appear to monitor the presence of 'original' mother centrosomes. It is still possible that the centrosome orientation checkpoint monitors the presence of 'mature' centrosomes (not necessarily from earlier in development, but > 2 cell cycle-old centrosomes) at the hub–GSC interface. Interestingly, it was recently shown that the daughter centrosome is consistently inherited by stem cells during the divisions of Drosophila neuroblast. Given the precise inheritance of mother or daughter centrosomes depending on the context/stem cell system, it is tempting to speculate that the centrosome orientation checkpoint monitors the presence of the mother centrosome in male GSCs, and possibly an equivalent mechanism monitors the daughter centrosome inheritance in neuroblasts (Yuan, 2012).

In developing embryos, cyclin A localization was reported to be dispensable for its activity. Even the plasma membrane-bound form of cyclin A was shown to be able to fulfill its function to promote mitosis. Indeed, the mutant forms of cyclin A protein used in this study (NLS-CycA and Cyclin AγC) are 'functional' in that they can promote the cell cycle progression into mitosis. Instead, it is proposed that these cyclin A mutant proteins cannot be subjected to a negative regulation by Par-1. It is possible that the embryonic cell cycle has minimal negative regulation as in embryonic stem cells, while male GSCs have an additional regulatory step (i.e., the centrosome orientation checkpoint) that negatively regulates mitotic entry (Yuan, 2012).

The lack of spindle misorientation in Dsas-4 mutant male GSCs is intriguing. In the complete absence of the centrosome, the spindle was correctly oriented in dividing GSCs, while defective centrosome function in cnn mutant leads to abrogation of the centrosome orientation checkpoint. Dsas-4 mutant male GSCs apparently orient the mitotic spindle via anchorage of spindle pole to the apically-localized spectrosome, which is highly reminiscent to the spindle orientation mechanism in female GSCs. The prediction would be that the spindle orientation is randomized in Dsas-4 hts double mutant male GSCs, which lacks both the centrosome and spectrosome. Unfortunately, the analysis of the double mutant was technically very challenging; Dsas-4 single mutant flies die as pharate adult, and the survival of the double mutant was worse. Furthermore, it was never possible to observe any mitotic GSCs from those pharate adult double mutants that were recovered and analyzed. Thus, future studies will be required to test this prediction (Yuan, 2012).

This study illuminates the importance of stem cell-specific regulators of the general cell cycle machinery such as cyclin A. We propose that stem cells have developed elaborate mechanisms to ensure an asymmetric outcome of the stem cell division, the failure of which can lead to tumorigenesis or tissue degeneration (Yuan, 2012).

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).

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