roughex

Targets of Activity

A test was made as to whether rux suppresses entry in S phase by preventing ectopic activation (directly or indirectly) of a Cyclin A-Cdk complex in the G1 domain 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. It is though that Rux controls CycA levels by promoting its nuclear localization and thereby its rapid degradation (Thomas, 1997).

Protein Interactions

Rux can be shown to physically interact with CycE, but does not inhibit the kinase activity of Drosophila CycE-Cdk complexes. Rux overexpression also fails to inhibit CycE-induced S phase. Therefore, rux does not appear to inhibit CycE-dependent processes in vivo. Conversely, CycE inhibits Rux accumulation. Rux rapidly disappears from cycling cells at the posterior edge of the MF where the level of CycE increases. It can be shown that overexpression of CycE, but not of CycA inhibits Rux protein accumulation. These results suggest that CycE targets Rux for destruction in cells that re-enter S phase behind the MF. Therefore, during normal eye development, accumulation of CycE in late G1 cells may down-regulate Rux protein. This, in turn, facilitates the accumulation of CycA, which is required in subsequent cell cycle stages (Thomas, 1997). P>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). >

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

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).

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).

DEVELOPMENTAL BIOLOGY

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Larval

Theye imaginal disc can be divided into five cell cycle domains:

1. Domain I consists of the unpatterned region anterior to the morphogenetic furrow (MF). In this region many S phase cells are seen and many cells express the S and G2 phase cycA and cycB. Mitotic cells are also seen in this region and they transiently express String mRNA. These cells are cycling asynchronously and are in various stages of the cell cycle.
2. Domain II is defined by expression of STG mRNA. stg is expressed to very high levels in a roughly 5- 6-cell diameter band, just anterior to the MF. stg appears to be expressed in all cells in this region. S phases in domain I stop at the anterior boundary of stg expression, although normally some S phase cells are occasionally seen overlapping the anterior edge of stg. This suggests that cells entering S phase at the posterior edge of domain I will complete S phase within the anterior edge of the stg domain.
3. Just anterior to the MF (domain III), expression of both cycA and cycB abruptly terminates, with cycA expression ending slightly earlier than cycB. Degradation of CycB is known to be associated with passage through the G2/M transition. Similarly, in the eye disc, the posterior edge of CycB staining in front of the MF is associated with the domain of increased mitosis. cycB expression terminates within the domain of stg expression. Since none of these markers are expressed in the cells within the MF, it is concluded that these cells are in G1. In the MF, cells within this G1 domain begin to segregate into groups that later form the precluster.
4. The remaining cells between the preclusters enter S phase synchronously (domain IV).
5. More posteriorly (domain V), mitoses are seen extending over a wide interval (Thomas 1994).

Aoughex mRNA concentration is to low to be detected in the eye disc, examination of the mutant phenotype is necessary to reveal the probable expression domain of roughex. roughex mutant discs show four defects in cell cycle regulation in the MF:

1. The G1 domain normally seen in wild-type discs is absent in the mutant.
2. All cells enter S phase beginning in the stg domain anterior to the MF, including cells that would normally exit the cell cycle within the MF and differentiate.
3. The domain of S phase cells is expanded relative to wild type.
4. Many DNA synthesizing, string positive, and cyclin-positive cells are detected in more posterior regions of the disc.

Cells do not normally reenter the cell cycle in this region. DNA synthesis, the expression of cycA and cycB, and the distribution of mitotic cells in domain I appear normal in roughex mutant discs. It is concluded that rux is required in domain II just anterior to the MF to establish G1 (Thomas, 1994).

Subsuent studies used a Rux-lacZ fusion protein to study the expression pattern of Rux. High levels of expression are observed in the nuclei of cells in a band just anterior to the MF. Lower and variable levels are seen within the MF, whereas behind the MF, expression increases. Therefore, Rux is ubiquitously expressed in early G1 cells just anterior to the MF. In rux mutants, it is these cells that accumulate CycA and enter S phase precociously. Rux protein is observed initially in the nuclei of most cells at the posterior edge of the MF. Just behind the MF, Rux protein remains at high levels within the nuclei of differentiating photoreceptor cells but is rapidly down-regulated in the nuclei of S/G2 cells. Rux protein becomes localized to a stripe of cells anterior to the MF and in differentiating neurons behind the MF. Therefore, high levels of Rux correlate with low levels and CycA and conversely, low levels of Rux correlate with high levels of CycA (Thomas 1997)

Effects of mutation or overexpression

Rough, a novel protein required for establishment of G1 in eye morphogenesis, is thought to be required either to negatively regulate string, Cyclin A or both. roughex mutants, which circumvent G1 and enter S, are suppressed by reduced gene dosage of CycA or string (Thomas, 1994).

During HREF="../aimorph/spermat.htm">spermatogenesis, germ cells execute two meiotic divisions, then withdraw from the cell cycle and initiate postmeiotic differentiation. 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).

Cells in the eye imaginal disc become synchronized in the G1 phase of the cell cycle just prior to the onset of cellular differentiation and morphogenesis. In roughex 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; 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).

The onset of pattern formation in the developing Drosophila eye is marked by the simultaneous synchronization of all cells in the G1 phase of the cell cycle. These cells will then either commit to another round of cell division or differentiate into neurons. Although cell cycle synchronization occurs in roughex mutants, cells circumvent G1 and all cells enter S phase, including cells that would normally differentiate. This leads to defects in early steps of pattern formation and cell fate determination. rux is suppressed by mutations in genes that promote cell cycle progression (i.e., Cyclin A and string) and enhanced by mutations in genes that promote differentiation (i.e., Ras1 and Star). A deficiency involving CycB does not suppress rux, a gene that encodes a novel protein of 335 amino acids. rux functions as a negative regulator of G1 progression in the developing eye (Thomas, 1994).

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

The r gene encodes a novel Cdk1 inhibitor that controls the onset of S phase during embryogenesis, eye development, and spermatogenesis. rux also plays a novel role in mitosis, by an unknown mechanism. Rux and Wee1 both negatively regulate Cdk1 activity; thus the observation that coexpression of these genes generates more extreme rough eye phenotypes than seen with either alone is consistent with known functions for these genes. Surprisingly, flies lacking both zygotic Dwee1 and rux functions show nearly complete synthetic lethality, with rare escapers exhibiting extensive adult bristle phenotypes. This interaction suggests that rux and Dwee1 may also cooperate in some other, as yet undefined regulatory mechanism. The extensive bristle phenotypes seen in rux;Dwee1 double mutant escapers could indicate disruption of cell cycle timing or abrogation of genome integrity checkpoints, similar to the phenotypes seen in mus304 mutants exposed to ionizing radiation, which are associated with increased genome instability (Price, 2002).

When mosiisbpassed, as in some cancer cells or in natural endocycles, sister chromosomes remain paired and produce four-stranded diplochromosomes or polytene chromosomes. Cyclin/Cdk1 inactivation blocks entry into mitosis and can reset G2 cells to G1, allowing another round of replication. Reciprocally, persistent expression of Cyclin A/Cdk1 or Cyclin E/Cdk2 blocks Drosophila endocycles. Inactivation of Cyclin A/Cdk1 by mutation or overexpression of the Cyclin/Cdk1 inhibitor, Roughex (Rux), converts the 16th embryonic mitotic cycle to an endocycle; however, Rux expression fails to convert earlier cell cycles unless Cyclin E is also downregulated. Following induction of a Rux transgene in Cyclin E mutant embryos during G2 of cell cycle 14 (G214), Cyclins A, B, and B3 disappear and cells reenter S phase. This rereplication produced diplochromosomes that segregated abnormally at a subsequent mitosis. Thus, like the yeast CKIs Rum1 and Sic1, Drosophila Rux can reset G2 cells to G1. The observed cyclin destruction suggests that cell cycle resetting by Rux is associated with activation of the anaphase-promoting complex (APC), while the presence of diplochromosomes implies that this activation of APC outside of mitosis is not sufficient to trigger sister disjunction (Vidwans, 2002).

The abrupt disaearance of mitotic cyclins is generally taken as an indication of activation of the mitotic ubiquitin ligase APC and subsequent degradation. Since Rux-mediated resetting from G2 to G1 was accompanied by cyclin disappearance, it is speculated that APC activation occurs during resetting but that it is insufficient to promote sister disjunction in this context. Consistent with this, studies in Saccharomyces cerevisiae have suggested that mitotic phosphorylation acts in conjunction with APC-targeted degradation to promote chromatid disjunction. The widespread occurrence of polytene and diplochromosomes suggests that disjunction can be bypassed in diverse systems. The variety of cell cycle defects and polyploid phenotypes in cancer cell lines has suggested that production of diplochromosomes involves the complete bypass of mitosis. Perhaps the dependence of disjunction on mitotic events is general (Vidwans, 2002).

date revised: 20 December 2024