Cyclin A
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).
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).
Cyclin A (CycA), the only essential mitotic cyclin in Drosophila,
is cytoplasmic during interphase and accumulates in the nucleus
during prophase. Interphase localization is mediated by Leptomycin B
(LMB)-sensitive nuclear export. This is a feature shared with human
CyclinB1, and it is assumed that nuclear accumulation is necessary
for mitotic entry. Whether the unique mitotic function of CycA
requires nuclear accumulation has been tested. Subcellular
localization signals were fused to CycA and their mitotic capability
was tested. Surprisingly, nuclear accumulation was not required, and
even a membrane-tethered form of CycA is able to induce mitosis. It
was noted that Cyclin B (CycB) protein disappears prematurely in CycA
mutants, reminiscent of rca1 mutants. Rca1 is an inhibitor of
Fizzy-related-APC/C activity, and in rca1 mutants, mitotic
cyclins are degraded in G2 of the 16th embryonic cell cycle.
Overexpression of Rca1 can restore mitosis in CycA mutants,
indicating that the mitotic failure of CycA mutants is caused by
premature activation of the APC/C. The essential mitotic function of
CycA is therefore not the activation of numerous mitotic substrates
by Cdk1-dependent phosphorylation. Rather, CycA-dependent kinase
activity is required to inhibit one inhibitor of mitosis, the Fzr
protein (Dienemann, 2004).
Drosophila CycA displays a striking change in its subcellular
localization at the onset of mitosis. This is true for a HA-tagged
version of CycA (HA-CycA), whose localization and destruction is
indistinguishable from the endogenous CycA. HA-CycA is cytoplasmic in
interphase and accumulates in the nucleus at prophase. This nuclear
accumulation of HA-CycA correlates with chromosome condensation in
prophase cells. Therefore, nuclear CycA-Cdk1 activity might trigger
mitotic events in the nucleus, and this might be the essential
function of CycA for mitosis (Dienemann, 2004).
Human Cyclin B1 (CycB1) displays a similar subcellular
distribution throughout the cell cycle caused by a dynamic shuttling
between nucleus and cytoplasm. During interphase, export from the
nucleus prevails and results in cytoplasmic localization of CycB1.
Preventing nuclear export by using LMB, which is a well-established
inhibitor of nuclear export, results in nuclear accumulation even in
interphase. To test if CycA localization is mediated by nuclear
export, Drosophila embryos were treated with LMB and nuclear
accumulation of CycA was observed in interphase cells. Thus
localization of CycA is mediated by a controlled balance between
nuclear import and export, similar to what has been shown for CycB1
(Dienemann, 2004).
To test the functional significance of the subcellular dynamics of
CycA, its localization was changed by forcing constitutive nuclear
accumulation or preventing prophase accumulation. This was realized
by the fusion of heterologous localization signals onto the N
terminus of CycA. Such N-terminal fusions did not impair the ability
of these constructs to activate Cdk1. The expression of the
constructs was accomplished by the UAS-Gal4 system. Expression levels
were comparable and below that of endogenous CycA. Thus, any effects
caused by these constructs are not due to overexpression artifacts
(Dienemann, 2004).
To achieve a constitutive nuclear localization, CycA was fused to
the nuclear localization sequence (NLS) of the SV 40 large T antigen.
This results in constitutive nuclear CycA even during interphase.
Yet, no premature mitotic events were observed. Obviously, nuclear
accumulation of CycA is not sufficient to induce mitosis since
inhibitory phosphorylations are present on Cdk1 and expression of the
phosphatase CDC25String is limiting for Cdk1 activation
(Dienemann, 2004).
To enhance nuclear export of CycA even during prophase, the
nuclear export signal (NES) of human PKI was fused to CycA. This
construct delayed nuclear prophase accumulation. In comparison with
the endogenous CycA, HA-NES-CycA was nuclear only in an advanced
state of prophase probably after nuclear envelope breakdown. This
shows that nuclear export is not globally shut down during prophase,
allowing the continuing export of HA-NES-CycA at early prophase
stages. Thus, nuclear export of wild-type CycA must be regulated to
allow nuclear accumulation during prophase, which can be counteracted
by the fusion of an exogenous export signal that is apparently not
subject to this regulation (Dienemann, 2004).
In order to completely exclude CycA from the nucleus, CycA was
tethered to the membrane. The Torso receptor tyrosine kinase was used
and the cytoplasmic region with the HA-CycA coding region was
replaced. Initial localization studies were carried out by a
transient expression assay in which mRNA encoding Tor-HA-CycA was
injected into Drosophila embryos. The distribution of Tor-HA-CycA in
such embryos displays the expected plasma membrane localization
pattern. Importantly, even mitotic cells show membrane localized HA
staining of the Tor-HA-CycA construct. Similarly, in older embryos in
which expression was induced by the UAS-Gal4 system, Tor-HA-CycA was
never found to accumulate in the nucleus. However, this construct is
not restricted to the outer rim of the plasma membrane. Extracts from
embryos expressing Tor-HA-CycA were fractionated into membrane and
cytoplasmic fractions and Tor-HA-CycA was found specifically enriched
in the membrane fraction. This indicates that Tor-HA-CycA is confined
to the membrane compartment within the cell, and the observed
staining in older embryos likely reflects the presence of this
construct in the endomembrane system. These localization data show
that the used heterologous localization signals are functional in
Drosophila, redirecting the CycA constructs to the desired locations
(Dienemann, 2004).
It was then asked if the differently localized CycA constructs
were able to fulfill the mitotic function of CycA. Epidermal cells
lacking CycA fail to execute the 16th mitosis. As
a consequence, mutant embryos have fewer but bigger cells compared to
wild-type. Expression of CycA from a transgene can overcome the
mitotic defect best seen by a comparison of CycA mutant
cells with those that express CycA. The prdGal4 driver line
was used to achieve expression in every other segment. Abdominal
segment A1, in which prd is active, was compared with
segment A2. Expression of HA-CycA results in increasing cell numbers
and reduction in cell size, indicating that HA-CycA can restore
mitosis in CycA mutant embryos (Dienemann, 2004).
Whether HA-NLS-CycA and HA-NES-CycA are able to overcome the
CycA mutant phenotype was tested in epidermal cells. Both
constructs induced cell divisions in the CycA mutant
background. HA-NES-CycA expression in segment A1 resulted in cell
numbers comparable to wild-type, and HA-NLS-CycA expression even
slightly increased cell numbers. Thus, neither cytoplasmic nor
nuclear localization during early prophase stages is required for
mitosis 16 in epidermal cells. Whether CycA localization is
important for any of the other mitoses that occur during development
was tested. Unfortunately, the endogenous CycA promoter is large and
not well characterized. Therefore, being aware that unpatterned
expression of CycA might disturb development, the ubiquitous
daGal4 driver line was used. When expression was at moderate
levels, CycA mutant flies were recovered that express
HA-CycA ubiquitously. As expected from an unpatterned CycA
expression, flies were recovered at low frequency and showed various
abnormalities, like rough eyes, bristle defects, and reduced
viability. But this experimental setup allowed a test of whether
HA-NES-CycA or HA-NLS-CycA are able to support all mitoses during
embryonic and larval life. In both cases, CycA mutant flies
were recoved at similar frequencies, indicating that they can mediate
proliferation throughout development. This shows that the normal
subcellular dynamic of CycA is not essential for proliferation
(Dienemann, 2004).
The expression of Tor-HA-CycA during embryogenesis in a
CycA mutant did result only in a limited increase in cell
number. In contrast, a high number of cells positive for the mitotic
marker PH3 was observed, indicating that this construct was able to
induce mitosis. This, and additional evidence leads to the conclusion
that a membrane-anchored form of CycA is able to induce mitosis
(Dienemann, 2004).
This raises the following question: how can nuclear mitotic events
be triggered when CycA-dependent Cdk1 activity is prevented from
entering the nucleus? The three mitotic cyclins in Drosophila, CycA,
CycB and CycB3, have partially redundant functions. However, in
CycA mutants, the presence of CycB and
CycB3 is clearly not sufficient to induce mitosis. To
further corroborate this, additional, HA-tagged CycB (HA-CycB) or a
nuclear-localized CycB (NLS-CycB) were expressed in a CycA
mutant. Both activate Cdk1 in vitro, but neither induced
proliferation in a CycA mutant background. CycB protein
distribution was analyzed in CycA mutant embryos; it was
noticed that CycB was degraded prematurely in cells that would
normally go into mitosis shortly -- i.e., in the G2 stage of cell
cycle 16. This can be seen best in CycA mutant embryos in
which every other segment is 'rescued' by HA-CycA, or even
Tor-HA-CycA. This phenotype is reminiscent of the rca1
mutant phenotype. In rca1 mutants, mitotic cyclins are
degraded prematurely in G2 during the 16th
embryonic cell cycle. Rca1 is an inhibitor of Fizzy-related
(Fzr)-dependent APC/C activity (Grosskortenhaus, 2002). As cells
prepare for the first G1 phase during embryogenesis, Fzr, which is
required for the establishment of G1, is upregulated. Several
partially redundant mechanisms prevent Fzr-APC/C activity in G2.
Besides Rca1, CycA-Cdk1 contributes to Fzr inactivation. The
disappearance of CycB in CycA mutants suggests that Fzr
becomes activated prematurely. To test if this is the case, Rca1 was
overexpressed in CycA mutants to prevent Fzr activation.
Indeed, Rca1 overexpression is sufficient to prevent premature CycB
degradation and cell divisions could occur. Rca1 overexpression could
not completely restore cell numbers; indicating that CycA inhibition
of Fzr is of greater importance in this situation. This function of
CycA can apparently not be fulfilled by the endogenous CycB or even
after overexpression of CycB. Human Cyclin A can interact with Fzr
through a so-called RXL motif in Fzr and a hydrophic patch in Cyclin
A. Such a motif is also present in Drosophila Fzr, possibly causing
its CycA-Cdk1-dependent phosphorylation. Apparently, this function of
CycA is not necessary in the nucleus, in agreement with findings that
Fzr is predominantly localized to the cytoplasm. When CycA is
tethered to the membrane, inhibition of Fzr might be sufficient to
allow entry into mitosis. Presumably, the Fzr protein itself is
shuttling between the cytoplasm and the nucleus, thereby allowing
inactivation wherever CycA is localized (Dienemann, 2004).
In vertebrates as well as in Drosophila, overexpression of CycA
results in ectopic S phases. In addition, nuclear CycB1 was shown to
be able to induce S phase in vertebrates. Tests were performed to see
if the subcellular localization of CycA is important for S phase
induction by expressing the different CycA constructs during eye
imaginal disc development. The different CycA constructs were
expressed in postmitotic cells by using the sevGal4 driver.
Expression of HA-CycA as well as all other CycA constructs used in
this study but none of the CycB constructs, including the NLS-CycB
construct, did result in ectopic S phases. At present, it is not
known how membrane anchored CycA can induce S phase, which is a clear
nuclear event. Possibly, Fzr is inactivated in G1 by the Tor-HA-CycA
that is not degraded efficiently during mitosis and persists in the
G1 state. After Fzr inactivation, the half-life of endogenous CycA
during G1 would be increased and could trigger the observed S phases
(Dienemann, 2004).
In conclusion, these data show that the dynamic changes in the
subcellular localization of CycA are not essential for its mitotic
function. It is suggested that the unique function of CycA for
mitosis does not lie in the activation of specific mitotic substrates
by Cdk1-dependent phosphorylation. Rather, CycA dependent kinase
activity is required to inhibit one inhibitor of mitosis, namely the
Fzr protein. In the absence of CycA premature APC/C
activation results in the degradation of substrates that are required
for mitotic entry, like CycB. Since overexpression of Cyclin B is not
sufficient to restore mitosis, other substrates that are necessary
for mitotic entry might by degraded by Fzr-dependent APC/C activity
as well -- one candidate being Cdc25, whose levels are regulated by
the APC/C during the cell cycle. The Drosophila system allowed a test
the functional requirements for CycA in a mutant background. Such an
analysis is difficult in vertebrate cells since CycB1 mutant mice die
very early in utero and functional studies are complicated by the
fact that sites that are required for nuclear entry are also required
for CycB1 activation. While nuclear accumulation of CycA at prophase
might not be essential, whether it is important for the normal
kinetics of mitotic progression and whether its cytoplasmic location
during interphase is important in checkpoint controls as it was shown
for CycB1 in vertebrates is currently being investigated (Dienemann,
2004).
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).
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 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
cdc2ts 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).
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 compentence 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 possibliity 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 Cdk7ts mutant embryos, Cdc2 and Cyclin A fail to form a stable complex in the Cdk7 mutant. This is likely attributable to the fact that this event requires the phosphorylation of Cdc2 on Thr-161, as even in the wild type only the phosphorylated form is associated with Cyclin A. These in vivo results correlate well with the finding that human Cdc2 needs to be phosphorylated by CAK to form a stable complex with Cyclin A in vitro, whereas stable Cdc2/Cyclin B and Cdk2/Cyclin E complexes can form in the absence of Thr-161 (or 160) phosphorylation. The Cdc2/Cyclin A complex seems to be more sensitive to a reduction in CAK activity than the Cdc2/Cyclin B complex, as the loss of Cyclin A binding occurs more rapidly than the reduction of Thr-161 phosphorylation of Cyclin B-associated Cdc2 (Larochelle, 1998).
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).
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).
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).
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).
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 Cdc2AF, 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).
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).
Regulator of cyclin A1 (Rca1) specifically inhibits Cdh1Fzr-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 Cdh1Fzr. Double-mutant analysis demonstrates that premature cyclin destruction in rca1 mutants is mediated by Cdh1Fzr. Furthermore, Rca1 can block the effects of Cdh1Fzr overexpression, supporting the notion that Rca1 inhibits Cdh1Fzr-dependent APC activity. Coimmunoprecipitation experiments reveal that Rca1 and Cdh1Fzr are in a complex that also contains the APC component Cdc27. Collectively, these data show that Rca1 is a negative regulator of Cdh1Fzr-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 (Cdc20Fzy) and Cdh1-Fizzy-related (Cdh1Fzr), respectively. In rca1 mutants, cyclins are degraded prematurely in G2 by APC-Cdh1Fzr-dependent proteolysis, and cells fail to execute mitosis. Overexpression of Cdh1Fzr mimics the rca1 phenotype, and coexpression of Rca1 blocks this Cdh1Fzr 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 Cdh1Fzr interaction (Grosskortenhaus, 2002).
In rca1 mutants, levels of mitotic cyclins are reduced during interphase of the 16th 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 mit