Cyclin B
Cyclin B expression is not detected in E2F-deficient clones of the eye disc, while elav is expressed normally in E2F-deficient clones (Brook, 1996)
The DPP requirement for cell fate specification and cell cycle synchronization in the developing Drosophila eye was examined by determining whether cells defective for thickveins, saxophone or schnurri show abnormalities in cell division or differentiation. Clones mutant for a null allele of tkv that are anterior or posterior to the morphogenetic furrow have amounts of cyclin B that are indistinguisable from those in surrounding cells. In contrast, tkv clones that span the MF maintain cyclin B expression in the anterior part of the furrow, even though the surrounding cells arrested in G1 have no detectable cyclin B. Maintenance of cyclin B is thought to indicate a failure of cell cycle progression, as cyclin B levels decline in M phase. In addition, mitotic figures are not observed in clones in the anterior half of the MF. The phenotype observed in the clones is similar to defects caused by mutations in division abnormally delayed (dally), which is required for G2-M progression ahead of the furrow. Mutations in dally and dpp display genetic interactions in development of the eye, antenna, and genitalia, which suggests that dally augments dpp function. The behavior of DPP-receptor mutant clones supports a role for DPP in controlling progression through G2-M as a means of synchronizing the divisions that accompany differentiation of the eye disc. Cell fate, however, is unaffected by receptor mutation as revealed by expression of atonal, a proneural gene required for retinal precursor cell 8 (R8) determination. Because atonal expression is maintained in tkv clones, hh must not act through dpp to induce its expression, and thus dpp mediates a subset of hh functions in the MF (Penton, 1997).
The c-myb proto-oncogene product (c-Myb) is a transcriptional activator. Vertebrate c-Myb is a key regulator of the G1/S transition in
cell cycle, while Drosophila Myb (dMyb) is important for the G2/M transition. dMyb induces expression of cyclin B (a critical regulator of the G2/M transition) in Drosophila eye imaginal disc. In the wild-type eye disc, dmyb mRNA is expressed in the
stripes both anterior and posterior to the morphogenetic furrow. Ectopic expression of C-terminal-truncated dMyb in the eye disc causes
ectopic expression of cyclin B and the rough eye phenotype. This rough eye phenotype correlates with prolonged M phase, caused by
overexpression of cyclin B. Cyclin B expression is lost in dmyb-deficient clones. In Schneider cells, the activity of the cyclin B promoter is dramatically reduced by loss of dMyb using the RNA interference method. Mutations of the multiple AACNG sequences in the cyclin B promoter also abolish the promoter activity.
These results indicate that dMyb regulates the G2/M transition by inducing cyclin B expression via binding to its promoter (Okada, 2002).
To investigate the effect of overexpression of dmyb in Drosophila, transgenic flies were generated carrying a transgene encoding wild-type dMyb expressed from the eye-specific expression vector, Glass multimer reporter (pGMR). The pGMR P-element vector contains a pentamer of the Glass-binding site derived from the Drosophila Rh1 promoter. Since Glass is expressed in the morphogenetic furrow (MF) and the whole region posterior to the MF of the third instar eye imaginal disc, expression of dMyb from this transgene was expected in the MF and the posterior region of the eye disc. Forty independent transgenic lines expressing wild-type dMyb were generated, but none of them exhibited any morphological abnormality in the adult eye. The C-terminal portion of vertebrate c-Myb negatively regulates c-Myb activity. However, it remains unclear whether dMyb also contains a negative regulatory domain (NRD) in its C-terminal region, since a dMyb protein lacking the C-terminal 241 amino acids activates a promoter containing tandem repeats of the Myb-binding site to almost the same extent as wild-type dMyb in co-transfection assays using Drosophila cultured cells. In spite of this, it might be possible that the C-terminal portion of dMyb negatively regulates the dMyb activity in the eye disc. Therefore, transgenic flies were generated carrying a transgene encoding a C-terminal-truncated dMyb lacking the C-terminal 241 amino acids (dMybDeltaC) expressed from the pGMR vector. In contrast to the transgenic flies overexpressing wild-type dMyb, 32 independent transgenic lines expressing the C-truncated dMyb showed a variety of dominant morphological disorders of the adult compound eye (rough eye phenotypes), probably due to the position effect. These results suggest that the C-truncated dMyb has a stronger activity than wild-type dMyb in eye imaginal disc cells. In the present study, two of the transgenic lines were used, GMR–dMybDeltaC-F25 and GMR–dMybDeltaC-F54, which exhibited severe and mild rough eye phenotypes, respectively. As expected, in the transgenic flies (GMR–dMybDeltaC-F25), dmyb mRNA was ectopically expressed in the MF and the whole posterior region to the MF in the developing eye imaginal disc (Okada, 2002).
Since it has been demonstrated that dMyb plays an important role in the G2/M cell cycle transition, the expression of regulators of cell-cycle control was investigated. Ectopic expression of cyclin B, which is a key regulator of the G2/M transition, is seen in the transgenic flies expressing the C-truncated dMyb. Imaginal disc cells in the developing wild-type eye become synchronized at the G1 phase of the cell cycle within the MF and cyclin B is strongly expressed in the stripe posterior to the MF. The width of the cyclin B-expressing region in the GMR–dMybDeltaC-F25 eye imaginal discs is broader than in wild-type control discs. Since the level of cyclin B is tightly regulated by protein degradation, in situ hybridization was used to confirm that ectopic expression of cyclin B in the GMR–dMybDeltaC-F25 eye imaginal discs takes place at the transcriptional level. In the wild-type eye disc, the cyclin B (cycB) mRNA is expressed in the whole region anterior to the MF at a high level and the whole region posterior to the MF at a low level, suggesting that the high expression of cyclin B protein in the stripe posterior to the MF of the wild-type eye disc may be due to protein stabilization. In contrast, cycB mRNA is strongly expressed in the broad stripe posterior to the MF of GMR–dMybDeltaC-F25 eye imaginal discs. To confirm the upregulation of the cycB gene in the GMR–dMybDeltaC-F25 eye imaginal discs, RT–PCR analysis was performed using a series of decreasing amount of RNA prepared from the wild-type and the GMR–dMybDeltaC-F25 eye imaginal discs. The results indicate that the level of cycB mRNA in the GMR–dMybDeltaC-F25 eye imaginal discs is~2.2-fold higher than that in wild-type discs. Since the whole eye disc was used as a source of RNA, the degree of increase in cycB mRNA levels in the region posterior to the MF may be higher than 2.2-fold. Thus, overexpression of C-truncated dMyb in the whole region posterior to the MF of eye imaginal discs causes the rough eye phenotype and ectopic expression of cycB mRNA in the stripe posterior to the MF. No induction of cycB mRNA in the posterior region other than in the stripe suggests that activation of the cycB promoter requires not only dMyb but also other transcription factor(s) expressed in the stripe posterior to the MF (Okada, 2002).
The kinase activity of cyclin B–Cdk1 complex promotes mitosis, whereas the destruction of cyclin B and loss of kinase activity is associated with and required for exit from mitosis. Therefore, ectopic expression of cyclin B in the posterior region of GMR–dMybDeltaC eye imaginal discs might be expected to slow down progression through M phase. To investigate whether the M-phase population in the posterior region of GMR–dMybDeltaC eye imaginal discs is in fact higher than that in wild-type discs, immunostaining was performed using an antibody specific for phosphorylated histone H3. Phosphorylation of histone H3 is induced at M phase and phosphorylated histone H3 can be used as a marker of M-phase cells. The entire region in the anterior and posterior portion of MF was investigated. In order to count the stained cells that are located both at the apical and basal surface, discs were visualized with a fluorescent microscope at the lower magnification. The numbers of cells stained by anti-phospho-histone H3 antibody in the whole region posterior to the MF of the GMR–dMybDeltaC-F25 and the wild-type eye discs were 93 and 59 (average of 11 discs), respectively. Thus, the population of M-phase cells in the posterior region of GMR–dMybDeltaC-F25 eye discs was 58% larger than the corresponding population in wild-type discs. In contrast, the numbers of cells stained by anti-phospho-histone H3 antibody in the whole region anterior to the MF of the GMR–dMybDeltaC-F25 and the wild-type eye discs were 44 and 49 (average of 11 discs), respectively, indicating that the populations of M-phase cells are similar sizes in the anterior region of GMR–dMybDeltaC-F25 and wild-type eye discs (Okada, 2002).
dMyb-deficient clones are not tiny, suggesting that the dmyb-deficient cells keep proliferating for a while to produce clones of reasonable
size. This may suggest that loss of dMyb function slows down cell-cycle progression through mitosis but does not stop the cell cycle. In fact, the
presence of M-phase cells stained with anti-phospho-histone H3 antibody was observed in the dmyb-deficient clone, suggesting that loss of dMyb does not block
the G2/M transition. This is consistent with the report that loss of either cyclin A or cyclin B does not block the cell-cycle progression in Drosophila embryos and that
loss of both cyclins blocks the cell-cycle progression. Probably, dMyb is required for expression of cyclin B but not cyclin A.
However, the possibility that the dMyb protein is relatively stable and persists in mutant cells for several generations after mitotic recombination
events so that mutant cells can continue to divide for a while cannot be excluded. Further analyses are required for understanding the precise role of dMyb in cell-cycle regulation (Okada, 2002).
The results suggest that overexpression of the C-truncated dMyb retains the cells in M phase and that the abnormal cell-cycle regulation causes apoptosis. These two
events result in the rough eye phenotype. Since R2-5 and R8 are already determined by the time that the GMR promoter is activated, these cells presumably are not
affected by overexpression of dMyb. This suggests that R1, 6 and 7 and later cell types are specifically affected in these discs. One possibility is that, since mitosis
may be prolonged, cells are unable to respond to differentiation signals in the appropriate temporal window and this may also lead in part to cell death (Okada, 2002).
The always early (aly), cannonball (can), meiosis I arrest (mia) and spermatocyte arrest (sa) genes of Drosophila are essential in males both for the G2-meiosis I transition and for onset of spermatid differentiation. Function of all four genes is required for transcription in primary spermatocytes of a suite of spermatid differentiation genes. aly is also required for transcription of the cell cycle control genes cyclin B and twine in primary spermatocytes. In wild-type testes, cyclin B transcripts are present in low levels in the mitotic cells at the apical tip of the testes where cells undergo pre-meiotic S-phase. Cyclin B message is abundant throughout the primary spermatocyte stage, accumulating to very high levels in mature primary spermatocytes. Cyclin B mRNA is detected in meiotically dividing cells, but is absent from the post-meiotic stages. In aly mutant testis Cyclin B mRNA is detected in mitotic cells at a level comparable to wild type. However Cyclin B mRNA is not detectable in aly mutant primary spermatocytes. The expression pattern of Twine mRNA in testis is similar to that of Cyclin B, except that Twine mRNA is not detected in the mitotic cells at the tip of the testis. can, mia and sa mutant testis express both twine and cyclin B mRNA at normal level. (White-Cooper, 1998).
The timing of entry into the meiotic divisions in wild type may be controlled by post-transcriptionally regulated accumulation of Cyclin B and Twine protein. Although Cyclin B mRNA is expressed at high levels in early spermatocytes, the accumulation of Cyclin B protein is delayed in wild-type testis until the late primary spermatocyte stage. Cyclin B protein begins to accumulate in the cytoplasm of late primary spermatocytes as chromosome condensation is initiated just before the entry into the first meiotic difision and ispresent at high levels in pro-metaphase I cells. Cyclin B protein is degraded at the metaphase to anaphase transition of meiosis I, and reaccumulates in preparation for the second meiotic division. In aly mutant testis Cyclin B protein is detected in the mitotic cells at the apical tip, but does not accumulate in the mutant spermatocytes. Twine protein is likewise delayed until just before the entry into the first meiotic division, days after the transcript is first detected. Neither protein nor mRNA is detected in an aly mutant background (White-Cooper, 1998).
aly, can, mia and sa are required for accumulation of Twine protein but not twine transcript in late primary spermatocytes. These three meiotic arrest genes are required for the expression of fuzzy onions, whose product is required for mitochondrial fusion in early spermatids. Similarly severe reductions in message level are observed for Male-specific RNA 87F (Mst87F), a gene normally transcribed in primary spermatocytes but not translated until mid- to late-spermatid stages, days after the completion of meiosis. gonadal, which is expressed as two differentially terminated variants in the testis, shows dramatic reduction of both variants in can, mia and sa mutant testis. It is proposed that the can, mia and sa gene products act together or in a pathway to turn on transcription of spermatid differentiation genes, and that aly acts upstream of can, mia and sa to regulate spermatid differentiation. It is also proposed that control of translation or protein stability regulates entry into the first meiotic division. It is suggested that a gene or genes transcribed under the control of can, mia and sa allow(s) accumulation of Twine protein, thus coordinating meiotic division with onset of spermatid differentiation (White-Cooper, 1998).
In spermatogenesis, a major transition occurs as the mitotically amplifying population of spermatogonia cease mitosis and develop into primary spermatocytes. These primary spermatocytes become committed to undergoing the meiotic divisions, and then differentiating into spermatozoa. This change in cell behavior is associated with a dramatic switch in the transcript profile: some genes are downregulated and many are upregulated or switched on for the first time. The 'meiotic arrest' genes of Drosophila are crucial for regulating transcription in primary spermatocytes.
The Drosophila always early (aly) gene is involved in this switch in spermatocyte transcriptional regulation. aly coordinately
regulates meiotic cell cycle progression and terminal
differentiation during male gametogenesis. aly is required
for transcription of key G2-M cell cycle control genes and
of spermatid differentiation genes, and for maintenance of
normal chromatin structure in primary spermatocytes. Although entry into spermatid differentiation is independent of progression
through the meiotic divisions, these processes are subject to coordinate
control, mediated by the meiotic arrest class of genes, including aly,
can, mia and sa. The meiotic arrest genes are essential for the
transcription of many mRNAs involved in spermatid differentiation, and thus
are required for spermatid differentiation. The meiotic arrest genes also
control accumulation of proteins involved in the meiotic divisions, e.g. the
cdc25 homolog Twine, and thus link differentiation to the cell
cycle. The meiotic arrest genes of Drosophila have been
split into two classes, based on the mechanism by which they control
accumulation of Twine. The can class (including can, mia and
sa) post-transcriptionally regulate Twine production. By contrast
aly regulates transcription of twine. Two other meiotic
regulators, cyclin B and boule, are also transcriptional
targets of aly, but not can, mia or sa (Jiang, 2002 and references therein).
Two tightly linked and nearly identical homeobox genes of the TGIF (TG-interacting factor) subclass called vismay and achintya (often referred to as TGIF) are essential for spermatogenesis in Drosophila.
'achintya' is a Sanskrit word meaning 'that which is beyond thought and contemplation', and relates to initial difficulties in interpreting the mutant
analysis; 'vismay' is a Hindi word meaning 'surprise', which described the reaction when the genome
sequence revealed the tandem duplication. In flies deficient for both genes, spermatogenesis is blocked prior to any spermatid differentiation and before the first meiotic division. This suggests that vismay and achintya function at the same step as two previously characterized meiotic arrest genes, always early and cookie monster. Consistent with this idea, both always early and cookie monster are still expressed in flies deficient in vismay and achintya. Conversely, Vismay and Achintya proteins are present in always early mutant testes. Co-immunoprecipitation experiments further suggest that Vismay and Achintya proteins exist in a complex with Always early and Cookie monster proteins. Because Vismay and Achintya are likely to be sequence-specific DNA binding factors, these results suggest that they help to specify the spermatogenesis program by recruiting or stabilizing Always early and Cookie monster to specific target genes that need to be transcriptionally regulated during testes development (Wang 2003; Ayyar, 2003).
The control pathway underlying spermatogenesis is, as yet, poorly defined
but a few 'meiotic-arrest' mutants have been identified. All the meiotic
arrest mutants have a similar phenotype -- mature primary spermatocytes arrest
development, and fail to enter either the meiotic divisions or spermatid
differentiation. The currently identified meiotic arrest genes have been
subdivided into two classes. The aly-class genes [always early (aly) and cookie monster
(comr)] appear to be higher in the control hierarchy and regulate
transcription of some genes involved in entry into meiosis (boule, twine,
Cyclin B) and also of many spermiogenesis genes (e.g. fuzzy onions,
janus B, don juan, gonadal) required for the differentiation of
functional sperm. In contrast, can-class meiotic arrest genes
(including cannonball, meiosis 1 arrest (mia) and spermatocyte
arrest) do not affect transcription of the meiosis cell-cycle genes but
are required for spermiogenesis gene transcriptional activation (Ayyar, 2003 and references therein).
To place achi/vis within this scheme the
expression of a set of meiosis-related genes and a selected set of
spermiogenesis genes were examined in Df(2R)achi1 homozygous
mutant testes by RT-PCR analysis, and in homozygous mutant males by in situ
hybridization. Both the set of spermiogenesis genes tested (fuzzy onions,
janus B, don juan, gonadal) and the meiosis-related cell-cycle genes
(boule, twine, Cyclin B) showed strongly reduced expression in the
mutant, placing achi/vis in the aly class of
meiotic arrest genes. Transcription of other genes (RP49, polo and
Cyclin A) was not affected in the mutants. To determine whether
Drosophila achi/vis is required upstream in the pathway for transcription
of other meiotic arrest genes, the expression of aly and
comr was tested in achi/vis mutant testes. In situ hybridization on
achiZ3922 visZ3922
mutant testes revealed aly and comr transcripts at
levels similar to wild type, and RT-PCR analysis on
Df(2R)achi1 demonstrated robust expression of
aly and can transcripts. In the RT-PCR analysis the levels
of aly and can actually appear somewhat higher than wild
type. This result is not interpreted, however, as indicative of a regulatory
interaction but rather as a reflection of the altered cellular composition of
the mutant testes. Similarly, aly and comr are not required
for the expression of achi/vis because normal levels of achi/vis
transcripts were found, by RT-PCR, in aly and comr
homozygous mutant testes (Ayyar, 2003).
The transcription factors of the Fos family have long been associated with the control of cell proliferation, although the molecular and cellular mechanisms that mediate this function are poorly understood. This study investigated the contributions of Fos to the cell cycle and cell growth control using Drosophila imaginal discs as a genetically accessible system. The RNA interference-mediated inhibition of Fos in proliferating cells of the wing and eye discs resulted in a specific defect in the G2-to-M-phase transition, while cell growth remained unimpaired, resulting in a marked reduction in organ size. Consistent with the conclusion that Fos is required for mitosis, cyclin B was identified as a direct transcriptional target of Fos in Drosophila, with Fos binding to a region upstream of the cyclin B gene in vivo and cyclin B mRNA being specifically reduced under Fos loss-of-function conditions (Hyun, 2006; full text of article).
A loss-of-function analysis relying mostly on RNAi technology was performed in the Drosophila imaginal-disc system to dissect the function of Fos in the growth control of an intact developing tissue. The data obtained in these studies suggest the following model. In continuously cycling cells, D-Fos is required for the propagation of the cell cycle. If D-Fos function is reduced, components that are limiting for the successful transition from the G2 phase to mitosis are not supplied in sufficient amounts. Consequently, cells cannot leave G2/M, which causes the accumulation of cells of 4N DNA content and an overall increase of cell size, as determined by forward scatter measurements. Such a block of cell cycle function in primary cells is expected to cause the activation of checkpoints leading ultimately to the apoptotic removal of the affected cells. The conclusion that the observed increased frequency of cell death results from defects in the cell cycle is supported by experiments with eye imaginal discs. The removal of D-Fos function from cells slated for transit through a developmentally defined mitotic wave results in apoptosis at a relatively sharp time point after cell division would normally have been completed. This indicates that apoptosis is a consequence of rather than a cause for the reported cell cycle defects. Analysis of eye discs in which Fos function has been depleted but apoptosis is inhibited by the expression of p35 supports this view: in such a genotype, defects of eye development are enhanced rather than suppressed, indicating that Fos is not required just for survival signaling. The identification of cyclin B as a transcriptional target of D-Fos in imaginal discs offers a molecular explanation for the G2/M phenotype observed under D-Fos loss-of-function conditions (Hyun, 2006).
It is possible that Fos has functions in other stages of the cell cycle that do not become phenotypically apparent at the levels of Fos suppression achieved by the RNAi-based approach employed in these studies. It has, for example, been suggested that Fos has a function during the G1/S transition and regulates cyclin D transcription. It is important to note, however, that, in contrast to studies of continuously growing imaginal-disc cells, these experiments were conducted on cultured cells that entered the cell cycle from a quiescent state upon serum stimulation. For this G0-to-G1 transition, the de novo synthesis of cyclin D can be expected to be limiting and require higher levels of Fos activity than in asynchronously cycling cells (Hyun, 2006).
The studies presented here show that Fos can control specific aspects of cell cycle progression, at least in Drosophila imaginal-disc cells. This observation, if it can be extended to higher organisms, might explain the oncogenic activities of Fos proteins. However, it is important to keep in mind that Fos is a protein with complex and pleiotropic functions that can interact with multiple other transcription factors and signaling pathways. Thus, efforts to unravel the contributions of Fos proteins to cancer and other pathologies will have to consider this complexity and integrate the contribution of Fos to processes other than growth control, such as the differentiation and control of cell mobility (Hyun, 2006).
Drosophila MCRS2 (dMCRS2; MCRS2/MSP58 and its splice variant MCRS1/p78 in humans) belongs to a family of forkhead-associated (FHA) domain proteins. Whereas human MCRS2 proteins have been associated with a variety of cellular processes, including RNA polymerase I transcription and cell cycle progression, dMCRS2 has been largely uncharacterized. Recent data show that MCRS2 is purified as part of a complex containing the histone acetyltransferase MOF (males absent on first) in both humans and flies. MOF mediates H4K16 acetylation and regulates the expression of a large number of genes, suggesting that MCRS2 could also have a function in transcription regulation. This study shows that dMCRS2 copurifies with RNA polymerase II (RNAP II) complexes and localizes to the 5' ends of genes. Moreover, dMCRS2 is required for optimal recruitment of RNAP II to the promoter regions of cyclin genes. In agreement with this, dMCRS2 is required for normal levels of cyclin gene expression. A model is proposed whereby dMCRS2 promotes gene transcription by facilitating the recruitment of RNAP II preinitiation complexes (PICs) to the promoter regions of target genes (Anderson, 2010).
The initiation of mRNA transcription involves the assembly of a transcription preinitiation complex (PIC), which as a minimum includes RNA polymerase II (RNAP II), Mediator, and six general transcription factors (TFIIA, -B, -D, -E, -F, and -H) at the core promoter DNA region. PIC assembly is initiated by the binding of the TATA box binding protein (TBP) subunit of TFIID to the promoter, which is stabilized in the presence of TFIIA and Mediator. Subsequently, TFIIB binds to and stabilizes the TFIIA-TFIIB-Mediator-DNA complex and functions as an adaptor that recruits the preformed RNAP II-TFIIF complex to the promoter. TFIIE and TFIIH then join to form the complete PIC (Anderson, 2010).
Once the PIC has been assembled on the promoter, transcription initiation occurs in several steps, which involve extensive phosphorylation of the C-terminal domain (CTD) of RNAP II. Early on in the transition from preinitiation to elongation, phosphorylation of Ser5s in the CTD heptapeptide repeats takes place, and this depends on the activity of the TFIIH-associated kinase cyclin-dependent kinase 7 (Cdk7; mammals)/Kin28 (yeast). Subsequently, Ser2s are phosphorylated by the elongation phase kinase Cdk9 (mammals)/CTDK-1 (yeast) to generate elongation-proficient RNAP II complexes. Another Cdk, Cdk8, can negatively regulate RNAP II transcription, partially via its inhibitory effect on Cdk7 activity. More recently, it has been suggested that Cdk11p110 regulates RNAP II transcription in humans. Thus, Cdk11p110 binds to hypo- and hyper-phosphorylated RNAP II, and antibody-mediated repression of Cdk11p110 activity results in inhibition of RNAP II transcription (Anderson, 2010 and references therein).
In addition to the phosphorylation events that control RNAP II activity, modification of the chromatin structure represents an important mechanism for regulating gene expression. When the chromatin is in its repressed state, the DNA is wrapped tightly around the histones, creating a barrier to the assembly of the RNAP II PIC at the promoter region. Activation of gene expression is associated with a number of histone modifications that loosen the chromatin structure, including acetylation, methylation, ubiquitylation, and phosphorylation. Histone H3 and H4 acetylations are particularly frequent toward the 5' ends of actively transcribed genes and presumably facilitate the initial assembly of the PICs at the promoter region. MOF (males absent on first) is a histone H4 lysine 16 (H4K16)-specific histone acetyltransferase (HAT) in both mammals and Drosophila. MOF is part of several complexes, including the Drosophila male-specific lethal (MSL) complex, which is required for X chromosome dosage compensation, the mammalian counterpart of the MSL complex, and the MOF-MSL1v1 complex, which mediates p53 acetylation at K120. In addition, MOF copurifies with a number of other proteins, such as the forkhead-associated (FHA) domain-containing protein MCRS2, NSL1-3 (for nonspecific lethal 1 to 3), and MBD-R2, as part of the NSL complex (Anderson, 2010).
This study focuses on the function of Drosophila MCRS2 (dMCRS2), the Drosophila ortholog of human MCRS2 (also known as MSP58). Whereas human MCRS1 and -2 proteins have been associated with a variety of cellular processes, including RNA polymerase I transcription and cell cycle progression, dMCRS2 is largely uncharacterized. In addition to the recent observation that human and Drosophila MCRS2s form complexes with MOF (Cai, 2010; Prestel, 2010; Mendjan, 2006; Raja, 2010), several other reports suggest that MCRS1 and -2 proteins could function in transcription regulation via interactions with the transcriptional repressor Daxx or the basic region leucine zipper factor Nrf1 (Anderson, 2010).
Drosophila MCRS2 and its human homologue, MCRS2, are 59% identical, with the highest level of homology being in the FHA domain. Whereas dMCRS2 is largely uncharacterized, MCRS1 and -2 have been linked with a variety of cellular processes, RNAP I-dependent transcription, transcriptional repression, and cell cycle control, though these functions remain poorly understood (Anderson, 2010).
This study shows that dMCRS2 is an essential nuclear protein required for cell cycle progression and growth during development. The data show that dMCRS2 physically associates with Cdk11 and RNAP II and colocalizes with RNAP II PICs on polytene chromosomes in vivo. Consistent with this, dMCRS2 is required for optimal binding of RNAP II components to the cyclin promoter regions and for normal levels of cyclin gene expression (Anderson, 2010).
The demonstration of colocalization of dMCRS2 with RNAP II on numerous sites on polytene chromosomes is in agreement with a recent ChIP-seq analysis, which revealed that dMCRS2 is present on the promoters of over 4,000 genes, correlating with 55% of active genes (Raja, 2010). Furthermore, gene expression profiling studies show that dMCRS2 depletion elicits the downregulation of over 5,000 genes. This essential function as a broad-specificity transcriptional regulator is reflected by the extreme growth defect of dMCRS2-depleted cells both in vivo and in cell culture and in the fact that dMCRS2 has been recovered as a hit in RNAi screens for diverse cellular functions such as centrosome maturation and Hedgehog signaling (Anderson, 2010).
In accordance with its pleiotropic function, dMCRS2 can be purified with a number of proteins, from NSL components to members of the RNAP II machinery. Moreover, dMCRS2 colocalizes with RNAP II PICs on polytene chromosomes in vivo, suggesting that it may regulate an early step in the recruitment and/or assembly of RNAP II PICs. This is consistent with the majority of dMCRS2 binding to the promoter regions of autosomal and X-linked genes and the fact that dMCRS2 is required for the loading of RNAP II components to cyclin gene promoters. Thus, dMCRS2 appears to be an important transcriptional regulator, and the data represent the first evidence for a physical connection between dMCRS2 and the core transcriptional machinery. While the results suggest that dMCRS2 associates with RNAP II complexes via protein-protein interactions, future studies will need to establish the exact molecular nature of this connection (Anderson, 2010).
Interestingly, MCRS2 and dMCRS2 copurify with the MOF HAT independently of the dosage compensation MSL complex. Furthermore, it was observed that dMCRS2 coimmunoprecipitates and colocalizes extensively with MOF on polytene chromosomes. MOF, as well as binding to the 3' ends of MSL targets along the male X chromosome, is also found on numerous promoter regions, both on the X chromosome and on autosomes in both sexes. Since MCRS2 also binds to promoters, it is possible that dMCRS2 and MOF could function in concert in transcriptional regulation. However, despite the evidence that MOF regulates a broad range of both X-linked and autosomal genes, no physical connection between the putative dMCRS2-MOF NSL complex and RNAP II complexes has been established so far. This study shows that both dMCRS2 and MOF associate with core RNAP II complexes in cultured cells (Anderson, 2010).
dMCRS2 may promote transcription by different mechanisms. Through its HAT activity, dMCRS2-associated MOF may create a relaxed chromatin state favorable to PIC assembly, either by inducing the physical weakening of DNA/histone or histone/histone interactions or by promoting the recruitment of bromodomain-containing factors. dMCRS2 may also induce PIC formation by recruiting the preformed RNAP II/TFIIF complex and/or promoting transcription elongation through the recruitment of CK2 and the FACT complex, which facilitates transcription elongation by remodeling chromatin. However, whether these different dMCRS2-containing complexes regulate common target genes or whether they represent distinct transcriptional regulators remains to be investigated. In summary, a model is proposed where dMCRS2 binds to multiple sites along the chromosomes and promotes the recruitment of RNAP II PICs to target genes (Anderson, 2010).
Fragile X syndrome, the most common form of inherited mental
retardation, is caused by the loss of the fragile X mental
retardation protein (FMRP). FMRP is a ubiquitously expressed,
multi-domain RNA-binding protein, but its in vivo function remains
poorly understood. Earlier studies show that FMRP participates in
cell cycle control during development. This study used Drosophila
mutants to test if FMRP plays a role in DNA damage response under
genotoxic stress. It was found that significantly fewer dfmr1
mutants survive to adulthood than wild-types following irradiation
or exposure to chemical mutagens, demonstrating that the loss of Drosophila
FMRP (dfmr1) results in hypersensitivity to genotoxic stress.
Genotoxic stress significantly reduces mitotic cells in wild-type
brains, indicating the activation of a DNA damage-induced G2/M
checkpoint, while mitosis is only moderately suppressed in dfmr1
mutants. Elevated expression of cyclin B, a protein critical for
the G2 to M transition, is observed in the larval brains of dfmr1
mutants. CycB mRNA transcripts are enriched in the
dFMRP-containing complex, suggesting that dFMRP regulates DNA
damage-induced G2/M checkpoint by repressing CycB mRNA
translation. Reducing CycB dose by half in dfmr1 mutants
rescues the defective G2/M checkpoint and reverses
hypersensitivity to genotoxic stress. In addition, dfmr1
mutants exhibit more DNA breaks and elevate p53-dependent
apoptosis following irradiation. Moreover, a
loss-of-heterozygosity assay shows decreased irradiation-induced
genome stability in dfmr1 mutants. Thus, dFMRP maintains
genome stability under genotoxic stress and regulates the G2/M DNA (Liu, 2012).
Phenotypic analysis of animal models of FXS continues to reveal
novel functions for FMRP. This study presents multiple lines of
experimental evidence demonstrating for the first time that dFMRP
is involved in DNA damage response. DNA damage responses are
executed through coordinated interplays and cross-talks of
multiple players from sensors to transducers, and finally to
effectors. There are four distinct pathways involved in the DNA
damage response: cell cycle arrest (also known as DNA damage
checkpoint), transcriptional induction, DNA repair and apoptosis;
the four pathways act independently under certain conditions, but
frequently, they interact to repair the damaged DNA or commit
apoptosis. The hypersensitivity to irradiation, G2/M checkpoint
defects, excessive apoptosis and increased number of DNA breaks in
dfmr1 mutants after irradiation all support that dfmr1
plays a critical role in DNA damage response. In addition, dfmr1
mutants show an elevated rate of loss-of-heterozygosity (LOH) upon
DNA damage, indicating reduced genome stability in dfmr1
mutants. It is well established that proteins involved in
checkpoint control and DNA repair play a critical role in
maintaining genome integrity. Thus, the decreased genome stability
in dfmr1 mutants also supports the conclusion that dfmr1
participates in DNA damage response (Liu, 2012). Loss of dfmr1 affects cell cycle progression in
different developmental processes. This study speculated that the
hypersensitivity to DNA damage in dfmr1 mutants might be
due to a defective cell cycle control. However, in the absence of
genotoxic stress, normal expression of the G1/S checkpoint
regulator CycE, normal DNA synthesis activity and normal G2/M
checkpoint were detected in dfmr150M mutants compared
with the wild-type. These results indicate that the G1/S and G2/M
checkpoints are functional in dfmr1 mutants. Following
genotoxic stress, however, significantly more mitotic cells were
found in the larval brains and wing discs of dfmr150M
mutants compared with the wild-type controls, indicating a defect
in the G2/M DNA damage checkpoint in dfmr1 mutants. In
support of this checkpoint defect, cell cycle profiling of the
larval brain cells by flow cytometry demonstrates a similar trend
of cell cycle profile between dfmr1 and mei-41
mutants. The study therefore concludes that dfmr1
primarily regulates the G2/M checkpoint in response to genotoxic
stress (Liu, 2012). Cyclins and their CDK partners play an important role in
regulating cell cycle progression. Misregulation of these
cyclin–CDK complexes causes defective cell cycle
progression, especially in the cells with damaged DNA. When DNA
damage is inflicted at the G1 stage, the G1/S checkpoint regulator
CycE–CDK2 is silenced to arrest the G1 to S transition.
Alternatively, when DNA damage occurs at the G2 stage or if DNA
damage remains unrepaired from the previous G1 or S phase, the
CycB–CDK1 complex (also known as a mitosis promoting factor)
is inhibited to arrest cells at the G2/M transition. In light of a
report demonstrating that dFMRP suppresses the expression of CycB
at the mid-blastula transition during early embryonic development,
this study speculated that the G2/M checkpoint defect observed in
dfmr1 mutants after genotoxic stress might be due to the
altered expression of CycB. Indeed, CycB protein was found to be
elevated in dfmr150M brains, while the other two G2/M
checkpoint regulators, CycA and CycB3, were unaltered, indicating
a specific suppression of CycB by dFMRP. This specific
regulation of CycB by dFMRP is further supported by the
observation that CycB mRNA is enriched in the dFMRP-mRNA
protein complex from larval brains. Moreover, reducing the dose of
CycB by half partially rescues the increased mitosis and
hypersensitivity of dfmr1 mutants to genotoxic stress.
Thus, up-regulation of CycB in dfmr1 mutants accounts,
at least partially, for the G2/M checkpoint defect in response to
DNA damage. In support of this conclusion, overexpression of the
truncated, stable form of CycB is sufficient to induce G2/M
transition defect in both eye discs and wing discs after
irradiation (Liu, 2012). The CycB level is tightly regulated during the cell cycle at both
the transcriptional and post-translational levels. Among the many
regulators of CycB, the transcription factors NF-Y, FoxM1 and
B-Myb activate transcription of CycB. These CycB regulators are
important for the G2/M progression under both normal and stress
conditions. Activation of FoxM1 is critical for the G2/M arrest,
whereas B-Myb is required for the recovery of G2/M checkpoint in
p53-negative cells. This study reveals a negative regulation of
CycB by dFMRP at the post-transcriptional level that controls the
G2/M checkpoint under genotoxic stress (Liu, 2012). In addition to the G2/M cell cycle defect in dfmr1
mutants, an obviously disrupted nucleolar structure in the mutant
salivary gland cells was found. As the nucleolus is critical for
the DNA damage-induced p53 activation and apoptosis, apoptosis in
the wing discs was examined by anti-caspase-3 staining.
Spontaneous apoptosis is undetectable in the untreated dfmr1
mutants and wild-types, while genotoxic stress evokes excessive
p53-dependent apoptosis in dfmr1 mutants. Overactivation
of p53 in the dfmr1 mutants was further confirmed by
elevated expressions of the pro-apoptotic hid-rpr-grim
genes transcriptionally regulated by p53. It is unknown at present
why dfmr1 mutants show increased p53-dependent apoptosis
after irradiation. One interpretation for the phenotype is
compromised DNA damage repair in dfmr1 mutants.
Alternatively, the increased level of CycB in dfmr1
mutants may also lead to elevated apoptosis. It is worth noting
that without genotoxic stress, dfmr1 is required for
apoptosis and clearance of developmentally transient neurons in
the adult brains. On the other hand, overexpression of dFMRP in
multiple tissues including the wings and eyes also induces
apoptosis, though a possible role of p53 in the process was not
examined. Thus, dFMRP can either promote or inhibit apoptosis
under different conditions by distinct yet uncharacterized
mechanisms (Liu, 2012). Drosophila dfmr1 null mutants also exhibit
decreased genome stability as revealed by the LOH assay. Apoptosis
is an endogenous and well conserved program to eliminate cells
with severely damaged DNA to avoid propagation of potential
mutations. It is conceivable that loss of dfmr1
decreases genome stability, presumably resulting from a DNA repair
defect, leading to increased apoptosis and lethality following DNA
damage. Further experiments are required to unravel the causal
relationships between dFMRP, DNA repair and apoptosis (Liu,
2012). It is not known if FXS patients and Fmr1 knockout mice
are also hypersensitive to genotoxic stress. It is well
established that the fragile sites of chromosomes are more prone
to DNA damage and thus more dependent on the integrity of DNA
repair mechanisms to maintain chromosomal stability. A study using
fibroblasts reported that the DNA damage response is required to
maintain the stability of the fragile X site. In addition,
mutagen-induced genome instability is observed in the cultured
lymphocytes from FXS patients. However, a subsequent study
reported that lymphocytes from FXS patients display normal genome
stability under genotoxic stress. This discrepancy remains to be
resolved. In Drosophila, there is only one FMRP homolog
instead of three FMRP family members in mammals. The redundancy of
three FMRP members in mammals may make the phenotype of single
mutants too weak to be detected. It would be of interest to test
if double or triple mouse knockouts of the three FMRP family
members exhibit the hypersensitivity to genotoxic stress observed
in the Drosophila dfmr1 mutants. Such a result
would underscore a novel role for FMRP in maintaining genome
stability and cell cycle control to allow for proper neuronal
proliferation during brain development (Liu, 2012). Messenger RNA localization is a conserved mechanism for spatial control of protein synthesis, with key roles in generating cellular and developmental asymmetry. Whereas different transcripts may be targeted to the same subcellular domain, the extent to which their localization is coordinated is unclear. Using quantitative single-molecule imaging, this study analysed the assembly of Drosophila germ plasm mRNA granules inherited by nascent germ cells. The germ-cell-destined transcripts nanos, cyclin B and polar granule component travel within the oocyte as ribonucleoprotein particles containing single mRNA molecules but co-assemble into multi-copy heterogeneous granules selectively at the posterior of the oocyte. The stoichiometry and dynamics of assembly indicate a defined stepwise sequence. The data suggest that co-packaging of these transcripts ensures their effective segregation to germ cells. In contrast, compartmentalization of the germline determinant oskar mRNA into different granules limits its entry into germ cells. This exclusion is required for proper germline development (Little, 2015).
Messenger RNA localization is a conserved mechanism for spatial control of protein synthesis, with key roles in generating cellular and developmental asymmetry. Whereas different transcripts may be targeted to the same subcellular domain, the extent to which their localization is coordinated is unclear. Using quantitative single-molecule imaging, this study analysed the assembly of Drosophila germ plasm mRNA granules inherited by nascent germ cells. The germ-cell-destined transcripts nanos, cyclin B and polar granule component travel within the oocyte as ribonucleoprotein particles containing single mRNA molecules but co-assemble into multi-copy heterogeneous granules selectively at the posterior of the oocyte. The stoichiometry and dynamics of assembly indicate a defined stepwise sequence. The data suggest that co-packaging of these transcripts ensures their effective segregation to germ cells. In contrast, compartmentalization of the germline determinant oskar mRNA into different granules limits its entry into germ cells. This exclusion is required for proper germline development (Little, 2015).
Quantitative analysis of germ plasm-localized mRNAs has revealed several intriguing features about the localization process and the coordinate regulation of their integration into the pole cells. nos, pgc and cycB mRNAs are transported within the oocyte as single mRNAs and are co-packaged into granules specifically at the posterior cortex concomitant with localization. Thus, localization serves not only to concentrate these transcripts at the posterior but also generates large, multi-copy polar granules to coordinate the efficient incorporation of these transcripts into the pole cells (Little, 2015).
Polar granules are heterogeneous with respect to both the amount of a particular mRNA and the combination of different mRNAs. Although nos mRNA content of polar granules varies over a large range, there is a tendency towards higher values fitting a log-normal distribution. Log-normal distributions are often associated with exponential growth processes, whereby the rate at which an object grows is proportional to the size of the object. Thus, a log-normal distribution suggests that large granules grow at faster rates compared with small ones as assembly is accelerated through positive feedback. In addition, this study found that for granules containing both nos and pgc or nos and cycB, the quantities of the two different mRNAs are correlated, and there is a greater fraction of granules completely lacking one species of mRNA entirely than granules containing just a few copies of that mRNA. Together, these data suggest that for each type of mRNA, cooperative interactions generate homotypic RNPs that then 1) accelerate the recruitment of additional mRNAs of the same type, and 2) facilitate granule assembly by promoting interactions with similarly sized homotypic clusters of other mRNAs. These results also predict the existence of dedicated molecular pathways, one to form homotypic clusters, and another to assemble homotypic clusters of many different transcripts into higher-order granules. These higher-order granules may form by fusion of smaller homotypic granules and indeed fusion of granules labelled with GFP-Vas is observed by live imaging. Alternatively, clusters of different mRNAs may grow alongside each other on a predefined granule scaffold. The localization of Caenorhabditis elegans germ granules -- P granules -- occurs through a phase transition in which soluble RNP components condense at the posterior of the embryo. It is interesting to consider whether formation of homotypic clusters occurs by a condensation of single transcript RNPs mediated by RNA-binding proteins (Little, 2015).
In contrast to other posteriorly localized RNAs, which travel as single molecules, osk forms oligomeric complexes beginning in the nurse cells. Previous studies indicated that reporters containing the osk 3'UTR can hitchhike on wild-type osk mRNA by 3'UTR-mediated dimerization. Hitchhiking is not required for osk transport, but co-packaging may be important for translational repression of osk before localization. Consistent with this, multi-copy RNPs are competent for localization by both kinesin-dependent transport during mid-oogenesis and diffusion/entrapment during late stages of oogenesis (Little, 2015).
Previous ISH-immuno-electron microscopy analysis of stage 10 oocytes showed co-localization of osk with Stau, but not with Osk protein in polar granules and the results indicate that osk/Stau RNPs are continuously segregated from the germ plasm granules. This physical separation has functional consequences. Whereas co-packaging of nos, pgc and cycB coordinates their transport to posterior nuclei and consequent segregation to the pole cells, osk is specifically excluded. Although it is not known why targeting of Osk to polar granules seems to alter their function, it is clearly detrimental to germline development. It will be interesting to determine whether osk/ Stau granules contain other mRNAs whose functions are required specifically during oogenesis or early embryogenesis but must be excluded from pole cells (Little, 2015).
Epithelial cancer tissues often possess polyploid giant cells, which are thought to be highly oncogenic. However, the mechanisms by which polyploid giant cells are generated in tumor tissues and how such cells contribute to tumor progression remain elusive. Cells mutant for the endocytic gene rab5 in Drosophila imaginal epithelium exhibit enlarged nuclei. This study finds that mutations in endocytic 'neoplastic tumor-suppressor' genes, such as rab5, vps25, erupted, or avalanche result in generation of polyploid giant cells. Genetic analyses on rab5-defective cells reveal that cooperative activation of JNK and Yorkie generates polyploid giant cells via endoreplication. Mechanistically, Yorkie-mediated upregulation of Diap1 cooperates with JNK to downregulate the G2/M cyclin CycB, thereby inducing endoreplication. Interestingly, malignant tumors induced by Ras activation and cell polarity defect also consist of polyploid giant cells, which are generated by JNK and Yorkie-mediated downregulation of CycB. Strikingly, elimination of polyploid giant cells from such malignant tumors by blocking endoreplication strongly suppressed tumor growth and metastatic behavior. These observations suggest that JNK and Yorkie, two oncogenic proteins activated in many types of human cancers, cooperatively drive tumor progression by generating oncogenic polyploid giant cells (Cong, 2018).
Polyploid giant cells, which contain multiples of the diploid genome equivalents, are often observed in human cancer tissues. Such polyploidy can be generated by endoreplication, a cell cycle variation that gives rise to genomic contents by replicating DNA in the absence of cell division. Polyploid giant cancer cells were shown to be more tumorigenic than normal diploid cancer cells. However, the mechanisms by which polyploid giant cells are generated in tumors and how they contribute to tumor progression remain elusive (Cong, 2018).
In Drosophila imaginal epithelia, loss-of-function mutations in the endocytic genes, such as rab5, vps25, erupted (ept), or avalanche (avl) cause neoplastic tissue overgrowth and therefore these genes are called 'neoplastic tumor-suppressors'. Previously found that cells deficient for Rab5, a small GTPase essential for generating early endosomes, induce non-autonomous overgrowth of surrounding tissue when induced as mosaic clones in the imaginal disc (Takino, 2014). Mechanistically, loss-of-Rab5 causes activation of Eiger (a tumor necrosis factor (TNF) homolog)-JNK signaling and EGFR-Ras signaling, which cooperate together to activate the Hippo pathway effector Yorkie (Yki, a YAP homolog), leading to upregulation of a secreted growth factor Unpaired (Upd, an IL-6 homolog) (Takino, 2014). Intriguingly, this study also noticed that such Rab5-deficient cells exhibited enlarged nuclei, which was suppressed by inhibition of JNK signaling, Ras signaling, or Yki activity, although the underlying mechanisms and its function have been unknown (Cong, 2018).
This study also analyzed Rab5-defective cells in detail and found that rab5 mutation generates polyploid giant cells through endoreplication. Genetic analyses reveal that JNK and a Yki-target Diap1 (Drosophila inhibitor-of-apoptosis protein 1) cooperate to induce endoreplication in Rab5-defective cells via downregulation of the G2/M cyclin CyclinB (CycB). Furthermore, this study also showed that generation of such polyploid giant cells is essential for tumor growth and metastasis in a Drosophila model of malignant tumors bearing Ras activation and cell polarity defect (Cong, 2018).
In Drosophila cells cyclin B is normally degraded in two phases: (1) destruction of the spindle-associated cyclin B initiates at centrosomes and spreads to the spindle equator, and (2) any remaining cytoplasmic cyclin B is degraded slightly later in mitosis. The APC/C regulators Fizzy (Fzy)/Cdc20 and Fzy-related (Fzr)/Cdh1 bind to microtubules in vitro and associate with spindles in vivo. Fzy/Cdc20 is concentrated at kinetochores and centrosomes early in mitosis, whereas Fzr/Cdh1 is concentrated at centrosomes throughout the cell cycle. In syncytial embryos, only Fzy/Cdc20 is present, and only the spindle-associated cyclin B is degraded at the end of mitosis. A destruction box-mutated form of cyclin B (cyclin B triple-point mutant [CBTPM]-GFP) that cannot be targeted for destruction by Fzy/Cdc20, is no longer degraded on spindles in syncytial embryos. However, CBTPM-GFP can be targeted for destruction by Fzr/Cdh1. In cellularized embryos, which normally express Fzr/Cdh1, CBTPM-GFP is degraded throughout the cell but with slowed kinetics. These findings suggest that Fzy/Cdc20 is responsible for catalyzing the first phase of cyclin B destruction that occurs on the mitotic spindle, whereas Fzr/Cdh1 is responsible for catalyzing the second phase of cyclin B destruction that occurs throughout the cell. These observations have important implications for the mechanisms of the spindle checkpoint (Raff, 2002).
This study follows the subcellular localization of Fzy/Cdc20 and Fzr/Cdh1 throughout the cell cycle in living Drosophila embryos. GFP-Fzy is concentrated on kinetochores, centrosomes, and spindles early in mitosis, and starts to disappear from these structures once the chromosomes align at the metaphase plate. This localization is similar to that reported for p55cdc20 in fixed human cells, and it fits in well with the proposed role of Fzy/Cdc20 in linking the spindle assembly checkpoint to the APC/C. In higher eukaryotes, the spindle checkpoint system consists of several proteins, including the Mad and Bub proteins as well as CenpE, Mps1, Rod, and ZW10. As cells enter mitosis, most of these proteins accumulate on unattached kinetochores, and are then lost from the kinetochores once the chromosomes align at the metaphase plate. Several of these checkpoint proteins can bind to Fzy/Cdc20, and this appears to inhibit the ability of Fzy/Cdc20 to activate the APC/C. Therefore, an unattached kinetochore is thought to continuously generate inhibitory checkpoint protein/Fzy (Cdc20) complexes, thus ensuring that the APC/C is not activated until all of the chromosomes have aligned properly at the metaphase plate (Raff, 2002).
The checkpoint proteins Mad2, BubR1, CENP-E, Rod, and ZW10 have all been shown to bind to kinetochores and then move along microtubules to the centrosomes in a dynein-dependent manner. During mitosis, the localization of GFP-Fzy to kinetochores is microtubule independent, whereas its localization at centrosomes is microtubule dependent. This is consistent with the possibility that Fzy/Cdc20 may also load onto kinetochores and then move along microtubules to the centrosomes (Raff, 2002).
In contrast to GFP-Fzy, GFP-Fzr is strongly concentrated at centrosomes throughout the cell cycle, apparently in a microtubule-independent fashion. The concentration of Fzr/Cdh1 at centrosomes was unexpected, since it had been previously proposed that Fzr/Cdh1 catalyzes the second phase of cyclin B destruction that occurs in the cytoplasm. However, the Fluorescence Redistribution After Photobleaching (FRAP) analysis suggested that Fzr/Cdh1 is rapidly turned over at centrosomes. Although the significance of this turnover is unclear, it is possible that Fzr (Cdh1)-APC/C complexes activated at centrosomes could diffuse throughout the cell to catalyze the destruction of cyclin B (Raff, 2002).
Fzy/Cdc20 protein is abundant in syncytial embryos, whereas Fzr/Cdh1 protein is virtually undetectable. Moreover, a D-box-mutated form of cyclin B (CBTPM-GFP), which cannot be targeted for destruction by Fzy/Cdc20, is not degraded on spindles in syncytial embryos. CBTPM-GFP can be targeted for destruction by Fzr/Cdh1, and, in cellularized embryos, where Fzr/Cdh1 is normally present, CBTPM-GFP is destroyed throughout the cell but with slowed kinetics. Taken together, these findings indicate that Fzy/Cdc20 alone is responsible for catalyzing the destruction of cyclin B on the spindle in syncytial embryos, whereas Fzr/Cdh1 can catalyze the destruction of cyclin B throughout the cell in cellularized embryos (Raff, 2002).
These results suggest a model of how the destruction of Drosophila cyclin B is regulated in space and time. Early in mitosis, inhibitory checkpoint protein/Fzy (Cdc20) complexes form at unattached kinetochores. It is proposed that these complexes are restricted to the spindle microtubules, and spread from the kinetochore to the centrosome, and then throughout the spindle. As the kinetochores align at the metaphase plate, inhibitory complexes no longer form, and this leads to the activation of Fzy (Cdc20)-APC/C complexes. Exactly where and how this activation occurs is unclear, but it is proposed that only the specific pool of Fzy/Cdc20 that has passed through the kinetochore (and so is restricted to the spindle) is activated to degrade cyclin B. The destruction of cyclin B on the spindle then initiates the second phase of cyclin B destruction by activating Fzr/Cdh1-APC/C complexes. Unlike the Fzy/Cdc20 complexes, activated Fzr/Cdh1 complexes are not restricted to spindle microtubules, and can target cyclin B for destruction throughout the cell (Raff, 2002).
Since the destruction of cyclin B appears to initiate at centrosomes, it is suspected that the Fzy (Cdc20)-APC/C complexes initially become activated to degrade cyclin B at centrosomes. Presumably, the activated complexes then spread along the microtubules toward the spindle equator. This would explain why, in syncytial Drosophila embryos where only Fzy/Cdc20 is present, the attachment between centrosomes and spindles appears to be essential for the destruction of the spindle-associated cyclin B. Why Fzy/Cdc20 might initially be activated at centrosomes is unclear. Perhaps the disassembly of the inhibitory checkpoint protein/Fzy (Cdc20) oligomers that form at the unattached kinetochores requires some activity that is concentrated at centrosomes (Raff, 2002).
It is stressed that this model applies only to the destruction of cyclin B. For example, cyclin A is also targeted for destruction by Fzy (Cdc20)-APC/C complexes, but it is not concentrated on spindles. It seems unlikely that Fzy/Cdc20 also catalyzes the destruction of cyclin A only on the spindle. Therefore, it is speculated that there must be separate pools of Fzy/Cdc20 that are responsible for degrading cyclin A and B. An attractive aspect of this model is that it explains how these different pools are generated. Only the pool of Fzy/Cdc20 that passes through the kinetochore is inhibited from activating the APC/C by the spindle checkpoint system, and only this pool of Fzy/Cdc20 is competent to catalyze the destruction of cyclin B. In this way, the destruction of cyclin B is inhibited by the spindle checkpoint system, whereas the destruction of cyclin A is not (Raff, 2002).
Could this mechanism for regulating cyclin B destruction in Drosophila embryos apply to other systems? If two vertebrate mitotic cells are fused to form a single cell, the presence of an unattached kinetochore on one spindle (spindle A) does not block the exit from mitosis on the other spindle (spindle B) once the chromosomes on spindle B have aligned. Moreover, once spindle B exits mitosis, spindle A exits mitosis soon afterwards, even if some of its kinetochores remain unattached. These observations are consistent with the model. It would be predicted that the Fzy (Cdc20)/checkpoint-protein complexes generated at the unattached kinetochores of spindle A are restricted to microtubules and so cannot inhibit the exit from mitosis on the neighboring spindle B. Moreover, the activation of Fzy/Cdc20 on spindle B would eventually activate Fzr (Cdh1)-APC/C complexes on spindle B. These complexes can then spread throughout the cell, ultimately degrading cyclin B on spindle A and forcing it to exit mitosis. The degradation of clb2 in S. cerevisiae also occurs in two phases that appear to be catalyzed sequentially by Fzy/Cdc20 and Fzr/Cdh1, although the spatial organization of this destruction has not been investigated (Raff, 2002).
However, the model cannot explain how cyclin B is degraded in early Xenopus embryo extracts. Like early Drosophila embryos, these extracts contain Fzy/Cdc20, but lack Fzr/Cdh1. Nonetheless, cyclin B is completely degraded at the end of mitosis in these extracts, even if no nuclei or spindles are present. Thus, Fzy/Cdc20 can catalyze the destruction of cyclin B that is not spindle associated in Xenopus extracts. The reason for this apparent difference is unclear. However, it is noted that early Xenopus extracts do not have a functional spindle checkpoint. The mechanisms that link the destruction of cyclin B to the spindle checkpoint may also be required to restrict Fzy/Cdc20 complexes to the mitotic spindle (Raff, 2002).
The finding that Fzy/Cdc20 and Fzr/Cdh1 are concentrated at centrosomes highlights the potential importance of this organelle in regulating the exit from mitosis. It is speculated that the concentration of these proteins at centrosomes might serve two purposes. (1) It might enhance the fidelity of their sequential activation. The inactivation of cyclin B/cdc2 triggered by Fzy/Cdc20 seems to start at centrosomes, and cyclin B levels might only have to fall below a certain threshold level at the centrosome (rather than throughout the whole cell) to trigger the activation of the centrosomal Fzr/Cdh1. (2) In budding yeast there is a second, Bub2-dependent checkpoint that monitors the positioning of the spindle between the mother and daughter cell. Bub2 is concentrated at the spindle pole body where it is thought to suppress the activation of the mitotic exit network, and so block the activation of Fzr/Cdh1 and the exit from mitosis. It is not clear if mammalian cells also have a spindle orientation checkpoint, but if they do, the concentration of Fzr/Cdh1 at centrosomes may be important for the function of this checkpoint (Raff, 2002).
In Drosophila cells, the destruction of cyclin B is spatially regulated. In cellularized embryos, cyclin B is initially degraded on the mitotic spindle and is then degraded in the cytoplasm. In syncytial embryos, only the spindle-associated cyclin B is degraded at the end of mitosis. The anaphase promoting complex/cyclosome (APC/C) targets cyclin B for destruction, but its subcellular localization remains controversial. GFP fusions of two core APC/C subunits, Cdc16 and Cdc27, were constructed. These fusion proteins were incorporated into the endogenous APC/C and were largely localized in the cytoplasm during interphase in living syncytial embryos. Both fusion proteins rapidly accumulate in the nucleus prior to nuclear envelope breakdown but only weakly associate with mitotic spindles throughout mitosis. Thus, the global activation of a spatially restricted APC/C cannot explain the spatially regulated destruction of cyclin B. Instead, different subpopulations of the APC/C must be activated at different times to degrade cyclin B. Surprisingly, it was noticed that GFP-Cdc27 associates with mitotic chromosomes, whereas GFP-Cdc16 does not. Moreover, reducing the levels of Cdc16 or Cdc27 by >90% in tissue culture cells leads to a transient mitotic arrest that is both biochemically and morphologically distinct. Taken together, these results raise the intriguing possibility that there could be multiple forms of the APC/C that are differentially localized and perform distinct functions (Huang, 2002).
To test whether Cdc16 and Cdc27 could perform distinct functions, the levels of each protein was reduced in Drosophila tissue culture cells
using RNAi. Although this procedure depletes both proteins by >90%, the
affect of depleting Cdc27 was always much more deleterious to cells than
depleting Cdc16. Moreover, cyclin A is normally undetectable on metaphase
chromosomes, and this was true in Cdc16RNAi cells but not in
Cdc27RNAi cells. This suggests that a chromosome-associated
fraction of cyclin A can be degraded when Cdc16 is depleted but not when Cdc27
is depleted, correlating with the observation that Cdc27 associates with
mitotic chromatin whereas Cdc16 does not. Intriguingly, a slower migrating
form of cyclin A was also reproducibly detectable in Western blots of
Cdc27RNAi cells but not Cdc16RNAi cells. Perhaps this
slower migrating form of cyclin A represents a chromatin-bound form of cyclin
A that is not degraded properly when Cdc27 is depleted (Huang, 2002).
It is possible, however, that the different phenotypes induced by depleting
Cdc16 and Cdc27 could be explained if depleting Cdc27 simply inactivated the
APC/C more efficiently than depleting Cdc16. This would be surprising, as
previous studies have suggested that both proteins are 'core' components of
the APC/C that are present in roughly stoichiometric amounts. And, perturbing
the function of either protein by mutation or antibody injection causes the
same phenotype -- a strong metaphase arrest.
Thus, one would not predict that depleting either protein by >90% would
produce such different affects on total APC/C activity. In addition, two lines
of evidence suggest that in these experiments depleting Cdc27 is not simply
inducing a stronger version of the same phenotype induced by depleting Cdc16.
(1) Depleting either protein weakly stabilizes cyclin B and strongly
stabilises Fzy/Cdc20 to about the same extent, suggesting that at least some
aspects of APC/C function are equally inhibited by the depletion of either
protein. (2) Cells in which both proteins are simultaneously depleted by
>90% appear to have an intermediate chromosome/spindle morphology
phenotype, arguing that the Cdc27RNAi phenotype is not simply a
more extreme version of the Cdc16RNAi phenotype (Huang, 2002).
The interpretation of this RNAi data is complicated, however, because the behavior is being studied of a population of cells that appears to only
transiently arrest in mitosis as the cells run out of Cdc16 or Cdc27. How these
cells eventually exit mitosis is unknown, but it is noted that Drosophila
tissue culture cells are notoriously difficult to arrest in mitosis, even with
microtubule destabilizing agents. This 'mitotic slippage' mechanism probably
explains why only a maximum of ~25% of RNAi treated cells
arrested in mitosis was seen. A similar failure to completely arrest cells in mitosis
has been made in Drosophila larval neuroblasts mutant in the ida/APC5
subunit of the APC/C (Bentley,
2002). Therefore the interpretation of these
experiments should be taken cautiously. Nevertheless, these data are at least consistent with the
possibility that Cdc16 and Cdc27 could exist in multiple complexes that
perform at least partially non-overlapping functions (Huang, 2002).
The APC/C has been purified from several systems, and in all cases it has
been found to contain homologs of Cdc16 and Cdc27. In human cells,
APC/C complexes are homogeneous enough that a structure has been derived from cryo-electron microscopy and angular reconstitution studies. Moreover, previous studies in several systems have shown that perturbing APC/C
activity always produces a similar phenotype - a strong mitotic arrest. How
can these findings be reconciled with the suggestion that the APC/C could
exist in several complexes (Huang, 2002)?
The finding that anti-GFP antibodies can immunoprecipitate Cdc16 from
extracts expressing GFP-Cdc16 and can immunoprecipitate Cdc27 from extracts
expressing GFP-Cdc27 may give a clue to this apparent paradox. This finding
suggests that the APC/C either contains multiple copies of both proteins or
that multiple APC/Cs can bind to each other during purification. If the APC/C
contains multiple copies of Cdc16 and Cdc27, then different forms of the APC/C
could vary in their ratio of Cdc27 to Cdc16. Perhaps a form with a high ratio
of Cdc27 to Cdc16 might interact with mitotic chromatin, whereas a form with a
low Cdc27 to Cdc16 ratio might not. In this study, Cdc16 reproducibly migrates
at a slightly smaller size on gel filtration columns than Cdc27 (and the same
is true of GFP-Cdc16 compared with GFP-Cdc27), supporting the idea that the
two proteins may not always exist in identical complexes. Such subtly
different complexes, however, might be difficult to detect in purified APC/C
preparations. Similarly, if multiple APC/Cs can bind to each other during
purification, this might obscure the existence of several related complexes in
purified preparations. Interestingly, Cdc16, Cdc27 and another APC/C
component, Cdc23, all contain TPR repeats and can bind to themselves and to
each other. This could explain how the APC/C can contain multiple
copies of Cdc16 and Cdc27 or how different APC/C complexes might bind to each
other during purification (Huang, 2002).
In summary, it has widely been assumed that the APC/C exists as a single
complex, although there is little direct evidence to support this assumption.
The data raise the possibility that the APC/C may exist as several related
complexes that perform at least partially non-overlapping functions. These
observations suggest that there must be subpopulations of the APC/C that are
independently activated to degrade cyclin B at different times and at
different places. A requirement to regulate overall APC/C activity in a
temporally and spatially co-ordinated fashion could explain why the APC/C is
so structurally complex (Huang, 2002).
In the endo cell cycle, rounds of DNA replication occur in the absence of mitosis, giving rise to polyploid or polytene cells. The Drosophila morula gene is essential to maintain the absence of mitosis during the endo cycle. During oogenesis in wild-type Drosophila, nurse cells become polyploid and do not contain cyclin B protein. Nurse cells in female-sterile alleles of morula begin to become polyploid but revert to a mitotic-like state, condensing the chromosomes and forming spindles. In strong, larval lethal alleles of morula, the polytene ring gland cells also inappropriately regress into mitosis and form spindles. In addition to its role in the endo cycle, morula function is necessary for dividing cells to exit mitosis. Embryonic S-M cycles and the archetypal (G1-S-G2-M) cell cycle are both arrested in metaphase in different morula mutants. These phenotypes suggest that morula acts to block mitosis-promoting activity in both the endo cycle and at the metaphase/anaphase transition of the mitotic cycle. Consistent with this, cyclin B protein was found to be inappropriately present in morula mutant nurse cells. Thus morula serves a dual function as a cell cycle regulator that promotes exit from mitosis and maintains the absence of mitosis during the endo cycle, possibly by activating the cyclin destruction machinery (Reed, 1997).
The identification of Morula (Mr) as the APC2 component of the anaphase promoting complex readily explains the metaphase arrest
observed in proliferating tissues from mr mutants and establishes that APC2 is essential for APC/C activity. This
identification is significant also for demonstrating that the APC/C
is necessary during endo cycles to inhibit mitotic functions and is
consistent with the previous observation that levels of cyclin B are
inappropriately high in mr mutant nurse cells (Reed, 1997). The
finding that APC/C is required for endo cycles raised the question of
whether increased levels of cyclin B were responsible, at least in
part, for the larval mr mutant phenotypes. To address this
question, whether increased levels of cyclin B could enhance
mr phenotypes was examined. The transheterozygous combination of the
mr1/mr3
mutant alleles provided a sensitized test because these
transheterozygotes produce viable adults, though at only 50% the
number predicted for a fully viable combination. The copy number of
wild-type cyclin B genes was increased by two, thereby increasing the
level of cyclin B protein. Increased cyclin
B enhances the lethal phenotype such that in the presence of extra
copies of the cyclin B gene, no viable
mr1/mr3
adults were recovered. These results provide in
vivo confirmation that levels of cyclin B affect the mr
phenotype and contribute to the lethality of strong mr mutants (Kashevsky, 2002).
Tests were also performed for enhancement of the female-sterile phenotype of the
mr1/mr2 alleles by increased levels of cyclin B to examine the requirements for
APC/C function during specific differentiation aspects of the nurse
cell endo cycle. The five initial endo
cycles of the nurse cells produce polytene chromosomes in which the
replicated sister chromatids remain in tight association. After cycle
5, the chromosomes condense, and then the replicated copies partially
disperse so that in subsequent endo cycles the chromosomes appear
polyploid rather than polytene. A striking feature of the
mr1/mr2
phenotype is that the first five nurse cell endo cycles appear normal. The mr defect is not manifested until the
polytene/polyploid transition, when in mr mutant nurse
cells the chromosomes condense more fully than in wild type; spindles
are formed, and the condensed chromosomes remain arrested in a
metaphase-like state. This phenotype shows the same time of onset in
nurse cells mutant for the lethal mr5 mutation,
generated by germline clones. This finding raises the possibility
that the polyteny/polyploidy transition involves a cell cycle change
to a transient mitotic state and that, at this point, mr
mutant nurse cells are vulnerable to reenter mitosis fully (Kashevsky, 2002).
Consistent with the proposal that the onset of the mr
phenotype reflects cell cycle changes in the nurse cells at the
polytene/polyploid transition, it was found that increased levels of
cyclin B protein do not cause an earlier appearance of mitosis in the
mr mutant nurse cells. No increase in the
number of later stage egg chambers with pycnotic or degenerating nurse
cells is observed in the presence of increased cyclin B. Elevation of cyclin B protein in a wild-type background is
insufficient to cause nurse cells to revert to mitosis. It remains possible that increasing the
levels of other APC/C substrates would cause an earlier endo cycle defect (Kashevsky, 2002).
The initial step in the acquisition of replication competence by eukaryotic chromosomes is the binding of the multisubunit origin recognition complex, ORC. A transgenic Drosophila model is described which enables dynamic imaging of a green fluorescent protein (GFP)-tagged Drosophila melanogaster ORC subunit, DmOrc2-GFP. It is functional in genetic complementation, expressed at physiological levels, and participates quantitatively in complex formation. This fusion protein is therefore able to depict both the holocomplex DmOrc1-6 and the core complex DmOrc2-6 formed by the Drosophila initiator proteins. Its localization can be monitored in vivo along the cell cycle and development. DmOrc2-GFP is not detected on metaphase chromosomes but binds rapidly to anaphase chromatin in Drosophila embryos. Expression of either stable cyclin A, B, or B3 prevents this reassociation, suggesting that cessation of mitotic cyclin-dependent kinase activity is essential for binding of the DmOrc proteins to chromosomes (Baldinger, 2009).
DmOrc2 was chosen for two reasons. First, it constitutes an essential part of the ORC core. As such, it was expected to better reflect the localization of the complex in its origin-defining function compared to peripheral ORC subunits. Second, several null alleles of k43, the DmOrc2 gene, have been identified, allowing pursuit of a genetic complementation strategy to verify transgene functionality. Rescue transgenes consisted of genomic copies of the DmOrc2 gene in which GFP was inserted in frame, coding for either an N- or C-terminal fusion of the ORC subunit. Using complementation as the most conclusive genetic criterion, both fusions proved functional. This indicates DmORC's flexibility to accommodate substantial heterologous protein moieties, exemplified in this study by GFP attached in two independent positions within the complex structure. In particular for a protein like DmOrc2, functioning as an integral part of a multiprotein complex, it was essential to avoid an overexpression situation, as in the absence of authentic binding partners its fluorescence signal is expected to mislocalize. Thus, fly lines with physiological DmOrc2 expression levels and the quantitative participation of the fusion protein in the holo- or the core complex were a prerequisite to address the cell cycle events governing DmORC's interaction with chromosomes by a biologically meaningful experimental approach. Both criteria were met in the DmOrc2-GFP transgenic as well as the rescue lines. Thus, for all parameters tested, the Drosophila model reflected faithfully the behavior of endogenous DmOrc2, allowing the visual tracing of DmORC-GFP. The use of the term 'DmORC-GFP' therefore refers to both the DmORC core and the holocomplex formed upon DmOrc1 association, which cannot be distinguished by the chosen imaging approach (Baldinger, 2009).
Aside from ensuring the congruence between transgenic and endogenous ORC subunits, this experimental strategy of tracking ORC during the cell cycle also avoids ambiguities sometimes associated with the fixation or physiological stressing of cells and organisms. Notwithstanding such methodological issues, the observed dynamics of ORC-chromatin interactions reported previously suggested a significant divergence between different biological systems. In yeast, ORC binds chromatin throughout the cell cycle, including metaphase, as has also been reported for embryonic Drosophila and mammalian ORC core subunits. Other studies came to the conclusion that members of the mammalian core complex are mostly excluded from metaphase chromatin, similar to Xenopus and also Drosophila larval neuroblasts. Based on immunolocalization studies, the latter analysis came also to the conclusion that DmOrc2 accumulates on late anaphase/telophase chromosomes, similar to the current findings. Differences in the reported localization patterns of ORC can possibly be explained by cell-type-dependent or, in particular, by interspecies variations in the control mechanism of the cell division cycl (Baldinger, 2009).
From the imaging analysis in this in vivo model, it is concluded that the majority of DmORC-GFP is displaced from the chromosomes in early mitosis and diffusely distributed throughout the cell without any recognizable localization pattern. Therefore, current models of the embryonic Drosophila ORC cycle should be scrutinized when they place the core DmORC on mitotic chromosomes. Toward the end of mitosis, DmORC-GFP is chromatin bound again, and this relocalization seems to be quantitative within the detection limits of the methodology employed. No principal differences were observed in this dynamic behavior of DmORC-GFP between syncytial and cellularized stages of embryonal development (Baldinger, 2009).
Proteolytic control of ORC core subunits has not been reported so far. In line with this lack of evidence, this study does not indicate that DmORC-GFP levels are subject to mitosis-specific protein degradation (as are other regulators of cell cycle progression), with the fluorescence signal of DmORC-GFP clearly visible in early mitosis, before gradually refocusing on late mitotic chromosomes. This entire process might be completely attributed to control over intracellular localization of DmORC-GFP during the cell cycle. However, while a substantial resynthesis of DmORC core subunits appears unlikely given the observed timing of this process, in particular with the additional requirements for complex assembly and chromophore maturation, a partial destruction of core DmORC subunits, followed by chromosomal recruitment of DmORC from cytoplasmic pools at the onset of a new round of pre-RC formation, cannot be ruled out (Baldinger, 2009).
Origin specification and pre-RC assembly in eukaryotes start with the chromatin binding of ORC. This study showed the cell-cycle-dependent changes of DmORC-GFP localization in embryos. Its rapid accumulation on chromosomes is detectable by late anaphase when CDK activity drops to the low levels observed in the late M and early G1 phases. The dependence of DmORC-GFP chromosome binding on low CDK activity was established by following the fluorescence signal upon cell cycle arrest in response to the expression of stable mitotic cyclins A, B, and B3, which are not subject to proteasomal degradation. Their presence prevented chromatin binding of DmORC-GFP. Previous reports describing the reloading of ORC to late mitotic chromatin in various cellular systems of metazoan origin have implicated mitotic CDKs in this process, supported by corresponding biochemical analyses. In Drosophila, it is known that the expression of individual stable cyclins does not interfere with the cell-cycle-controlled degradation of the endogenous cyclins. Thus, this in vivo analysis allows extension of the general assumption of a role for mitotic CDK involvement in triggering the start of pre-RC assembly to specifically conclude that all mitotic CDK/cyclin activities have to cease for DmORC-GFP to become chromatin bound (Baldinger, 2009).
How can this dynamic behavior of DmOrc2 be interpreted in the light of previous observations regarding the APC-dependent degradation of DmOrc1 in late mitosis, only to reemerge in late G1? Even when considering that metazoan Orc1 often shows expression, localization, and turnover patterns independent of other ORC subunits, reflecting temporal events in the control over ORC activity, the almost converse mitotic shuttling patterns of DmORC subunits are somewhat surprising. It should be noted, however, that DmOrc1-GFP could also be detected on telophase chromosomes before being degraded. Most studies of metazoan ORC concur that Orc1 is essential to establish initial DNA binding of ORC and subsequent steps of pre-RC formation, supported by the recent finding that elevated Orc1 levels can actually promote binding of endogenous Orc proteins during late mitosis. It is conceivable that in Drosophila this process takes place during a brief time window in late mitosis and could be sufficient to trigger the recruitment of other pre-RC proteins, which according to most analyses occurs prior to late G1. Alternatively, the remaining chromatin-bound DmORC core might be sufficient to promote completion of the pre-RC. From these lines of reasoning, it is already obvious that further experiments, in directly comparable settings for both the experimental protocols followed as well as for the cell types and developmental stages analyzed, will be required to resolve this issue. This will be facilitated by the availability of Drosophila orc1-/- lines (Baldinger, 2009).
After this initial step in pre-RC assembly, other replication initiation proteins have to be loaded on chromosomes for them to become licensed for replication. Among these factors is the heteromultimeric minichromosome maintenance (MCM) complex, associated with a DNA helicase activity. Previous immunolocalization studies of the association/dissociation cycles of Drosophila MCM demonstrated their binding to mitotic chromatin upon cell cycle arrest by expression of stable cyclin B, corresponding to early anaphase stages. Assuming an unconditional requirement for prebound ORC for MCM chromatin binding, the current data would predict MCM binding at later cell cycle stages, after cessation of mitotic CDK activity. At first glance, these results on the timing of MCM-chromatin association might not be easy to reconcile with the current findings but can be explained by (1) the influence of the imaging methodology as describe for DmORC localization, (2) different sensitivity thresholds of the detection systems, or (3) potential uncharacterized effects of the stable cyclin-CDK complexes used in different studies. In any case, no real discrepancy is seen, since chromatin loading of MCM proteins in unperturbed cell cycles has only been evident in late anaphase/telophase, fully compatible with the current results for DmORC in both perturbed (i.e., stalled by stable cyclin expression) and unperturbed cell cycles (Baldinger, 2009).
It will be interesting to determine if this binding is partly responsive to potential changes in chromosome structure occurring as mitotic chromosomes pass toward telophase or whether DmORC responds directly or indirectly to changes in the kinase environment of late mitotic cells. The latter possibility would argue that a decrease in DmORC's phosphorylation state results in its increased affinity to chromatin. This scenario appears attractive since it has been demonstrated that in vitro binding of DmORC to DNA is strongly diminished whenever it is phosphorylated by various CDK/cyclin activities. Combined with the current cytological studies, these findings make mitotic CDKs attractive candidate kinases for actively suppressing DmORC binding to chromatin. This view is also in line with the localized cyclin destruction in syncytial cell cycles of Drosophila. The resulting abrogation of CDK1 activity in the vicinity of the mitotic spindle can be monitored by the distribution of phospho-histones, akin to the observed gradual rebinding of DmORC, starting from the centromeric regions of anaphase chromosomes (Baldinger, 2009).
In summary, this study report the spatial and temporal dynamics of the initiator protein ORC in a live metazoan organism. Along with the cell cycle, ORC periodically associates with and dissociates from chromatin. The initial interaction in preparation for the next chromosome cycle occurs in late anaphase. This binding of ORC to chromatin depends critically on the cessation of mitotic cyclin activity, linking this first step of replication licensing to the CDK-driven control pathways of cell cycle progression. Finally, it is obvious that different mechanisms evolved between species controlling the activities of ORC. While all of them are compatible with the general requirements for origin definition, pre-RC assembly, and the prevention of rereplication, it cautions against the extrapolation of findings from one experimental system to another. This underscores the value of multipurpose in vivo models like the one described in this study, allowing a comprehensive approach for probing ORC functions. Its use should not be restricted to further exploring ORC in DNA replication initiation, but it should also be useful to study ORC's role in proliferation and in the development of an organism (Baldinger, 2009).
Within the mammalian ovary, oocytes remain arrested at G2 for several years. Then a peri-ovulatory hormonal cue triggers meiotic resumption by releasing an inhibitory phosphorylation on the kinase Cdk1. G2 arrest, however, also requires control in the concentrations of the Cdk1-binding partner cyclin B1, a process achieved by anaphase-promoting complex (APCCdh1) activity, which ubiquitylates and so targets cyclin B1 for degradation. Thus, APCCdh1 activity prevents precocious meiotic entry by promoting cyclin B1 degradation. However, it remains unresolved how cyclin B1 levels are suppressed sufficiently to maintain arrest but not so low that they make oocytes hormonally insensitive. This study examined spatial control of this process by determining the intracellular location of the proteins involved and using nuclear-targeted cyclin B1. It was found that raising nuclear cyclin B1 concentrations, an event normally observed in the minutes before nuclear envelope breakdown, was a very effective method of inducing the G2/M transition. Oocytes expressed only the alpha-isoform of Cdh1, which was predominantly nuclear, as were Cdc27 and Psmd11, core components of the APC and the 26S proteasome, respectively. Furthermore, APCCdh1 activity appeared higher in the nucleus, as nuclear-targeted cyclin B1 was degraded at twice the rate of wild-type cyclin B1. A simple spatial model of G2 arrest is proposed in which nuclear APCCdh1-proteasomal activity guards against any cyclin B1 accumulation mediated by nuclear import (Holt, 2010).
The Imaginal discs arrested (ida) gene that is required for proliferation of imaginal disc cells during Drosophila development has been cloned and characterized. Ida is homologous to APC5, a subunit of the anaphase-promoting complex (APC/cyclosome). ida mRNA is detected in most cell types throughout development, but it accumulates to its highest levels during early embryogenesis. A maternal component of Ida is required for the production of eggs and viable embryos. Homozygous ida mutants display mitotic defects: they die during prepupal development, lack all mature imaginal disc structures, and have abnormally small optic lobes. Cytological observations show that ida mutant brains have a high mitotic index and many imaginal cells contain an aneuploid number of aberrant overcondensed chromosomes. However, cells are not stalled in metaphase, as evidenced by the observation that mitotic stages in which chromosomes are oriented at the equatorial plate are never observed. Interestingly, some APC/C-target substrates such as cyclin B are not degraded in ida mutants, whereas others controlling sister-chromatid separation appear to be turned over. Taken together, these results suggest a model in which IDA/APC5 controls regulatory subfunctions of the anaphase-promoting complex (Bentley, 2002).
In Drosophila the APC/C complex is estimated to consist of 11 proteins. However the biochemical function and requirement of so many subunits is unclear. One hypothesis proposes that the large number of subunits reflects the need to identify and target a large number of substrates. The model is supported by the recent characterization of the 3D structure of the human APC/C. The structure has an asymmetric morphology with a large inner cavity surrounded by an outer protein wall. The complexity of the structure suggests that discrete subunits may guide substrates into the inner cavity, where ubiquitination could take place. Thus the removal of a single subunit would disrupt the ubiquitination of only a fraction of substrates. Interestingly, the data suggests that Ida may be involved in the degradation of cyclin B but is not essential for the degradation of Securins. It should be noted that in this model not all subunits need play a role in substrate identification, since some are required for core stability and catalyzing the ubiquitination events. For example, Cdc27 and Cdc16 play critical roles in core stability, and Apc11 is required for the ubiquitination of substrates (Bentley, 2002 and references therein).
Another consideration for the role of APC/C subunits concerns the possibility that they specifically interact with regulators of APC/C activity during the cell cycle. Perhaps the most actively studied regulators of APC/C activity are the components of the spindle checkpoint pathway. Upon detection of DNA damage or unattached kinetochores, the spindle checkpoint pathway will send a 'wait' signal. In response to this signal, Mad2 will bind the APC/C, preventing its activity and halt progression of all mitotic events until the checkpoint has been fulfilled. Positive regulators play an equally important role in driving the cell through coordinated mitotic events. In Drosophila, the WD40-repeat protein, Fizzy (Fzy), binds to and drives APC/C-dependent ubiquitin-ligase activity in vitro. The Fzy homolog in yeast, Cdc20p, positively regulates the destruction of Pds1p, and Fzy is thought to serve a comparable role in Drosophila because Fzy is required for Pimples (Securin) degradation during mitosis. Consistent with these predictions, loss-of-function mutations in fzy prohibit cells from progressing through metaphase, and demonstrate that Fzy is required for metaphase exit and completion of mitosis in Drosophila. Fzy is highly unstable and present only at late S phase and during mitosis, further ensuring that Fzy-dependent APC/C events are temporally regulated. Finally, Fzy degradation is dependent on APC/C subunits, demonstrating that Fzy is also a substrate of the APC/C. An additional WD40-repeat protein, Fizzy-related (Fzy), is also believed to be required for the degradation of B-type cyclins during M and G1 phases, but differs from Fzy in that it is stable throughout the cell cycle (Bentley, 2002 and references therein).
In ida mutants, cyclin B levels are not properly degraded during anaphase. Thus it is possible that the function of Ida, alone or in concert with other subunits, is to direct cyclin B to the APC/C for degradation. However, other defects observed in ida cells are thought to be distinct from those observed in cells expressing a non-degradable cyclin B transgene. Therefore, it is proposed that there are additional regulatory functions for the IDA protein to help explain the lack of metaphase figures, the observed sister-chromatid separation, the high levels of Bub1 staining during anaphase, and the resulting aneuploidy that is observed in ida mutant cells (Bentley, 2002).
In one model, Ida functions as a part of the APC/C that receives a spindle checkpoint 'wait' signal. Thus when IDA is missing, the spindle checkpoint signal is not received, but the cell initiates sister-chromatid separation and anaphase onset prematurely. Presumably, this could occur even in the absence of proper chromosome attachment and alignment at the metaphase plate. Thus, metaphase figures would not be observed in ida mutants, but aberrant anaphases containing lagging chromosomes with high Bub1 staining (equal to signal checkpoint firing) would be detected. The missegregation of the unattached chromatids would also lead to cells containing an aneuploid number of chromosomes (Bentley, 2002).
In an alternative model, IDA plays a role in targeting Fzy for ubiquitin-dependent degradation. In this case, the removal of Ida would result in ectopic levels of Fzy, which would prematurely activate sister-chromatid separation and progression through mitosis. Consistent with this model, mutations in ida suppress the embryonic lethality associated with a fizzy null mutation (Bentley, 2002).
It should be noted that neither model directly address the high mitotic index -- a hallmark of cell cycle stall -- observed in squashes of ida cells. However, it is proposed that as cells become more and more aneuploid, alternative pathways, including the DNA replication checkpoint, may eventually cause a prometaphase stall or arrest (Bentley, 2002).
Successful mitosis requires that anaphase chromosomes sustain a commitment to move to their assigned spindle poles. This requires stable spindle attachment of anaphase kinetochores. Prior to anaphase, stable spindle attachment depends on tension created by opposing forces on sister kinetochores. Because tension is lost when kinetochores disjoin, stable attachment in anaphase must have a different basis. After expression of nondegradable cyclin B (CYC-BS) in Drosophila embryos, sister chromosomes disjoin normally but their anaphase behavior is abnormal. Chromosomes exhibit cycles of reorientation from one pole to the other. Additionally, the unpaired kinetochores accumulate attachments to both poles (merotelic attachments), congress (again) to a pseudometaphase plate, and reacquire associations with checkpoint proteins more characteristic of prometaphase kinetochores. Unpaired prometaphase kinetochores, which occur in a mutant entering mitosis with unreplicated (unpaired) chromosomes, behave just like the anaphase kinetochores at the CYC-BS arrest. Finally, the normal anaphase release of AuroraB/INCENP (see Inner centromere protein) from kinetochores is blocked by CYC-BS expression and, reciprocally, is advanced in a CycB mutant. Given its established role in destabilizing kinetochore-microtubule interactions, Aurora B dissociation is likely to be key to the change in kinetochore behavior. These findings show that, in addition to loss of sister chromosome cohesion, successful anaphase requires a kinetochore behavioral transition triggered by CYC-B destruction (Parry, 2003).
Stable cyclins have been shown to block mitotic exit in numerous systems, and detailed analyses of the cytological consequence of stabilization of each of the cognate mitotic cyclins of Drosophila have begun to reveal regulatory features that were not evident in other experimental systems. A group of chromosomal 'passenger proteins' that are localized between paired kinetochores at metaphase usually relocalizes to the central spindle upon onset of anaphase. This relocalization is blocked upon expression of stable sea urchin cyclin B in mammalian cells. In agreement with this, expression of Drosophila CYC-BS in Drosophila embryos blocks relocalization of two interacting passenger proteins, INCENP and Aurora B. Normal metaphase foci of INCENP split in two at anaphase, half segregating with each sister kinetochore without relocalization to the spindle. Failure to release kinetochore-localized AuroraB/INCENP and a slowing of anaphase A chromosome movements are the earliest perturbations of mitotic progression observed upon CYC-BS expression. The onset of these defects immediately follows or overlaps the time of destruction of normal CYC-B (Parry, 2003).
Embryos expressing a different stabilized mitotic cyclin, CYC-B3S, arrest with chromosomes at the spindle poles after normal anaphase movements and normal redistribution of AuroraB/INCENP from the kinetochore to the spindle midzone. Thus, CYC-BS and not CYC-B3S maintains kinetochore localization of AuroraB/INCENP (Parry, 2003).
As a result of partial redundancy among Drosophila cyclins, CycB null mutants undergo mitosis. As in wild-type, AuroraB/INCENP is associated with kinetochores in metaphase cells lacking CYC-B; however, its anaphase relocalization occurs prematurely. Thus, the endogenous CYC-B in the wild-type inhibits AuroraB/INCENP relocalization, and relocalization appears to await its destruction. Together, precocious relocalization in the CycB mutant, coincidence in the onset of relocalization and the time of CYC-B destruction, and the block to relocalization by persistent CYC-B lead to the conclusion that CYC-B destruction times AuroraB/INCENP relocalization (Parry, 2003).
The dramatic transition in kinetochore-protein interactions upon destruction of CYC-B might serve only to release the sequestered passenger proteins to play their important function at the spindle midzone in cytokinesis. However, elegant studies of Ipl1, the Aurora B kinase homolog of yeast, suggest that Ipl1 can destabilize kinetochore interactions with the spindle. These studies, as well as supporting work in vertebrate cells, suggest that loss of Aurora B function upon CYC-B destruction might alter kinetochore behavior. Indeed, the current results suggest that CYC-B destruction does have an important influence on anaphase chromosome behavior (Parry, 2003 and references therein).
When Drosophila cells enter anaphase in the presence of CYC-BS, poleward movement of unpaired chromosomes is abortive and chromosome behavior is unusual. It has been suggested that this chromosome behavior might represent an extension of prometaphase/metaphase behavior, differing only in so far as the loss of kinetochore pairing at metaphase/anaphase alters the behavior. The behavior of unpaired prometaphase kinetochores has been examined in a mutant in maize, exhibiting premature loss of chromosome pairing and after microsurgical production of single kinetochore chromosomes in mammalian cells. In these experiments, single-kinetochore chromosomes behaved much as the chromosomes of Drosophila cells that progress to anaphase (to produce unpaired kinetochores) in the presence of CYC-BS (Parry, 2003 and references therein).
To further test this parallel, the Drosophila mutant, double parked was examined, in which unpaired chromosomes exist in prometaphase. Double Parked is an essential replication protein that is also required for a checkpoint function that ordinarily prevents cells from entering mitosis with unreplicated DNA, and like analogous mutants in S. cerevisiae (e.g., cdc6), Drosophila cells lacking Double Parked enter mitosis with unreplicated DNA. When a maternal supply of Double Parked is depleted, replication fails in double parked embryos and cells accumulate in mitosis. The mitotic arrest occurs because unpaired chromosomes are incapable of normal bipolar alignment and consequently induce the spindle checkpoint (Parry, 2003).
In fixed images of the double parked arrest, most chromosomes are scattered along the spindle, with some clustered in a central pseudometaphase plate, just as in CYC-BS-arrested cells. Real-time analysis shows that this is a dynamic situation, with chromosomes making oscillatory movements between the poles. This chromosome movement between the poles resembles that observed during the CYC-BS block and is consistent with reorientation of the kinetochore from one pole to the other, as occurs for prometaphase chromosomes (Parry, 2003).
Despite the absence of prior replication, INCENP and Aurora B localize to the unpaired kinetochores in the double parked arrest, as in the CYC-BS arrest. Furthermore, despite the presence of only a single kinetochore, many of the chromosomes congress to a pseudometaphase plate in double parked and CYC-BS arrests. It is concluded that, when CYC-B persists, unpaired chromosomes behave similarly before and after the metaphase/anaphase transition (Parry, 2003).
Although it was somewhat puzzling that some chromosomes congressed to a pseudometaphase plate in double parked embryos, a similar observation was made when single kinetochore chromosomes were present in prometaphase in mammals. These congressed single kinetochore chromosomes have attachments to both poles (merotelic attachment). Robust kinetochore fibers are observed in double parked spindles, and in cases that are not confounded by the clustering of chromosomes in the middle, it is apparent that kinetochore fibers from both poles impinge on single kinetochores. These observations are interpreted as an indication of frequent merotelic attachment in the double parked arrest; similar findings have been noted in the CYC-BS-arrested cells (Parry, 2003).
The finding that merotelic attachments accumulate in the double parked arrest suggests that kinetochore pairing normally helps to prevent merotelic attachments under prometaphase conditions. It is suggested that such an effect could be explained by an extension of the idea that trial and error processes contribute to bipolar attachment of paired kinetochores in prometaphase. Because kinetochore-spindle interactions are unstable in prometaphase, all modes of attachment can be sampled, at least transiently, but the most stable mode ultimately predominates. Consequently, the most stable (correct bipolar attachment) precludes less stable and incorrect attachments. Spindle tension stabilizes attachment, and it has been suggested that, upon bipolar arrangement, tension deforms the paired kinetochore, effectively 'pulling' the attachment sites away from a centrally localized destabilizing activity. Although tension also deforms a merotelically attached kinetochore, it is suggested that the distortion is not as orderly as in bipolar attachment and that the separation from the destabilizing activity is less effective. Consequently, when kinetochores are paired, bipolar attachments will accumulate as the most stable outcome and hence exclude merotelic attachments. When kinetochores are unpaired, the dynamics of formation and decay of merotelic attachments appears to favor their accumulation (Parry, 2003).
Prior to the time at which CYC-B is usually degraded, no defects are seen in mitotic progression in cells expressing CYC-BS. Sister chromatids separate from one another, and other substrates of the APC/C are degraded. The dissociation of BubR1 from kinetochores marks the release of checkpoint control. CYC-BS-expressing cells having an anaphase configuration (prior to final arrest) have a greatly decreased level of kinetochore staining. However, at the final arrest point, BubR1 again localizes to the kinetochores. BubR1 staining does not completely disappear during anaphase, and levels at final arrest do not match the highest levels at prometaphase. Nevertheless, since a return of BubR1 to the kinetochore after sister chromatid separation is never observed in wild-type cells, there appears to be some reactivation of the checkpoint at the CYC-BS arrest (Parry, 2003).
Looking for additional reporters of checkpoint activity, probes were carried out for Rough deal (Rod) and ZW10, components of a mitotic checkpoint that relocalize in a manner suggesting a role in sensing tension. In prometaphase cells, a Rod/ZW10 complex localizes tightly to kinetochores. During the time chromosomes develop bipolar attachment, the kinetochore staining for Rod/ZW10 is reduced, and staining appears on kinetochore fibers. Upon disjunction of sister chromosomes, the staining returns to the kinetochores. All of these events appear to occur normally in CYC-BS- and CYC-B3S-expressing cells as they progress toward an arrest. However, during the course of arrest with CYC-BS, but not with CYC-B3S, spindle microtubules once again stain for Rod/ZW10. The spindle localization of Rod/ZW10 in the CYC-BS arrest is another example of checkpoint components that have reverted to their characteristic preanaphase localization and further suggests that some aspects of the checkpoint have been reactivated, perhaps in response to defective chromosome-spindle interactions (Parry, 2003).
Spindle staining of ROD/ZW10 during metaphase has been shown to require bipolar attachment and perhaps tension across the kinetochores. The spindle staining in cells at the CYC-BS arrest could imply some level of bipolar attachment. Initially, this seemed unlikely because the anaphase chromosomes are unpaired and contain only one kinetochore when at the arrest point. However, staining for kinetochores and microtubules shows robust kinetochore fibers extending from both poles to the pseudometaphase plate, suggesting merotelic attachment (Parry, 2003).
Merotelic attachments are obvious when only one or a few chromosomes remain near the middle of the spindle, as occurs frequently early after the transition to anaphase in the presence of CYC-BS and when the arrest is less complete. At the level of CYC-BS expression in these experiments, some cells are not fully arrested. Live observations reveal occasional cells with slow mitotic progress but without the full complement of arrest behaviors. These prolonged mitoses show a high frequency of chromosome segregation anomalies. Kinetochores successfully retained at the pole after CYC-BS expression lack INCENP and BubR1, whereas chromosomes localized to the middle of the spindle display merotelic attachments and stain strongly for INCENP and BubR1. The presence of two categories of kinetochore, one having and one lacking INCENP, suggests that there is a switch-like event at individual kinetochores and that the cells with an incomplete arrest are near the threshold of the switch (Parry, 2003).
These findings show that CYC-BS promotes merotelic attachments, which accumulate after the initially successful chromosome disjunction at the transition to anaphase. Furthermore, the results are consistent with proposals that merotelic attachments underlie congression of chromosomes with a single kinetochore. It is suggested that the accumulation of merotelic attachments at the CYC-BS arrest is the consequence of persistence of the dynamic phase of kinetochore spindle attachment beyond the time of sister kinetochore disjunction. Rather than preserving the established monopolar orientation of the anaphase kinetochores, persistence of dynamic exchange favors change toward the arrangements that are most stable for unpaired kinetochores, and one such arrangement is merotelic attachment (Parry, 2003).
In conclusion, these results show that a change in kinetochore composition and behavior accompanies the metaphase/anaphase transition and that a change in kinetochore behavior is essential for the unerring commitment of chromosomes to their assigned poles. Because the success of mitosis depends on this change, the transition is thought of as an integral part of the metaphase/anaphase transition. Destruction of CYC-B triggers and times the kinetochore transition at the onset of anaphase. The kinetochore transition is coordinated with the disjunction of sister chromosomes as a result of their common regulation by APC/C, which promotes the destruction of CYC-B as well as the sister cohesion regulators, securin and cyclin A. The change in kinetochore behavior can be understood as a change from dynamically exchanging tension-stabilized attachment to fixed stable attachment. The striking coupling of this change with the release of Aurora B/INCENP from the kinetochore, and the identified role of Aurora B kinase in destabilizing kinetochore spindle attachments, suggests a plausible mechanism in which the dissociation of Aurora B stabilizes spindle attachments. However, a stable derivative of the sea urchin cyclin B does not produce similar modifications of chromosome behavior in mammalian cells despite blocking the release of GFP-Aurora B from the kinetochores. Clearly, further work is required to elucidate the regulatory paths connecting kinetochore behavior with CYC-B destruction (Parry, 2003).
It was found that unpaired chromosomes developed merotelic attachments whenever AuroraB/INCENP was associated with unpaired kinetochores, whether this occured in anaphase as a result of CYC-BS expression or in prophase as a result of a failure in DNA replication (in the double parked arrest). It is suggested that kinetochore pairing influences the outcome of dynamic reassortment of kinetochore attachments. Evidently, it is important to stabilize kinetochore-spindle attachments upon disjunction of sisters; otherwise attachments reequilibrate to the most stable states available to unpaired kinetochores, including merotelic attachments (Parry, 2003).
Proteolytic degradation of mitotic regulatory proteins first requires these targets to be ubiquitinated. This is regulated at the level of conjugation of ubiquitin to substrates by the anaphase-promoting complex/cyclosome (APC/C) ubiquitin-protein ligase. Substrate specificity and temporal activity of the APC/C has been thought to lie primarily with its two activators, Cdc20/Fizzy and Cdh1/Fizzy-related.
Reduction in the E2 ubiquitin-conjugating enzyme (UBC) of the E2-C family that is encoded by the Drosophila gene vihar (vih), by either mutation or RNAi, leads to an accumulation of cells in a metaphase-like state. Cyclin B accumulates to high levels in all mitotic vih cells, particularly at the spindle poles. Vihar E2-C is present in the cytoplasm of mitotic cells but also associates with centrosomes, and its own degradation is initiated at the metaphase-anaphase transition. Expression of destruction D box mutants of vihar in the syncytial embryo results in mitotic arrest at late anaphase. In contrast to hypomorphic mutants, Cyclin B is degraded at the spindle poles and accumulates in the equatorial region of the spindle.
It is concluded that in Drosophila, the Vihar E2 UBC contributes to the spatiotemporal control of Cyclin B degradation that first occurs at the spindle poles. APC/C-mediated proteolysis of Vihar E2-C autoinactivates the APC/C at the centrosome before a second wave of proteolysis to degrade Cyclin B on the rest of the spindle and elsewhere in the cell (Mathe, 2004).
The ordered progression of cells through the division cycle is brought about by periodic series of protein modification events that involve cycles of phosphorylation that are mediated by multiple protein kinases and cycles of ubiquitination that lead to the periodic degradation of specific regulatory proteins. The ubiquitination of target proteins is achieved by three enzymes. The first ATP-dependent step is the activation of ubiquitin by the formation of a thio-ester bond between its C terminus and a cysteine residue in the activating enzyme E1 itself. In the second step, the ubiquitin is transferred as a thio-ester to a cysteine residue in a ubiquitin-conjugating enzyme (UBC) E2. In cooperation with an ubiquitin protein ligase, E3, the ubiquitin residue is then transferred to a lysine residue in the target protein. Polyubiquitinated proteins are targeted to the proteasome for their destruction. Proteolytic degradation is controlled in the cell cycle by two major classes of E3 enzyme: the Skp1 protein, Cullin, and F box (SCF) complex, which is required for the G1-S transition, and the anaphase-promoting complex/cyclosome (APC/C), which is functional during mitosis and G1. The APC/C catalyzes the ubiquitination of securin, an inhibitor of the protease that cleaves chromosome cohesion proteins. It is also responsible for degradation of the mitotic cyclins and of other regulatory molecules. The complex is comprised of 12 to 13 protein subunits and targets proteins that contain either of two types of destruction motif, the D box or the KEN box. APC/C activity is regulated in part by two related classes of WD40 repeat-containing proteins named after the Drosophila and budding yeast orthologs: Fizzy (Fzy) or Cdc20, which is required at the metaphase-anaphase transition, and Fizzy-related (Fzr) or Cdh1, which is effective toward the end of mitosisand into G1 to maintain mitotic cyclins at a low level. Activation of many APC/C functions may be delayed by the spindle assembly checkpoint that monitors microtubule attachment of and tension at kinetochores and signals Mad2 to complex with and inhibit the APC/C (Mathe, 2004 and references therein).
The mitotic cyclins are a major target of the APC/C, and in normal mitotic
progression A-type cyclins are degraded ahead of the B-type cyclins. Experiments with stable forms of B-type cyclins in several organisms have shown their degradation to be required not at the time of chromatid separation but at later stages of mitosis. This is supported by real-time studies in Drosophila embryos that show stable Cyclin B1 (hereafter referred to simply as Cyclin B) functions to block spindle elongation at anaphase B, resulting in the oscillation of disjoined chromatids. Stable Cyclin B3 gave a late arrest in which anaphase and cytokinesis were completed, but chromosomes failed to decondense (Parry, 2001). Parry (2003) subsequently showed that the oscillation of disjoined chromatids resulted from the establishment of merotelic attachments of their kinetochores to both poles and that this was likely to be a consequence of the failure to release the Aurora B kinase from the kinetochore. It also had the consequence of blocking cytokinesis (Mathe, 2004 and references therein).
Early studies of Cyclin B behavior in syncytial Drosophila embryos had shown degradation to be incomplete, but these were complicated by the use of fixed preparations of embryos where the pattern of immunostaining depended upon fixation conditions. The use of GFP-tagged Cyclin B, however, has permitted time-lapse studies in both Drosophila and mammalian cells that have suggested that Cyclin B degradation begins first on the mitotic apparatus and then occurs subsequently in the cytoplasm (Huang, 1999; Clute, 1999). In Drosophila, Cyclin B is degraded on the spindle in a wave that spreads from the poles and then subsequently in the cytoplasm. Consistently, in mutant embryos derived from centrosome fall off (cfo) mothers, Cyclin B is degraded on the detached centrosomes but not on the acentrosomal spindles, as though the physical detachment presents a barrier to the wave of cyclin destruction (Wakefield, 2000). Degradation of spindle-associated Cyclin B has been attributed to APC/C associated with Fzy/Cdc20, and degradation of the cytoplasmic Cyclin B to Fzr/Cdh1, a protein that only appears to be active after cellularization (Raff, 2002). This led to the hypothesis that Fzy/Cdc20, localized on the kinetochores and centrosome prior to the metaphase-anaphase transition, might mediate the degradation of cyclin throughout the spindle once the metaphase checkpoint has been relieved at the kinetochore (Mathe, 2004 and references therein).
However, the above hypothesis does not satisfactorily account for how Fzy/Cdc20 might direct Cyclin B degradation to begin at the spindle poles rather than elsewhere on the spindle. The possibility that components of the ubiquitination pathway other than the APC/C and its associated proteins may contribute to determining the specificity of proteolytic degradation of proteins in mitosis by the APC/C was first raised by the finding of E2 enzymes that were specific for the mitotic cyclins. However, when the catalytic cysteine of the clam enzyme E2-C was changed to serine, this resulted in a dominant-negative form of the enzyme that was able to arrest mammalian cells in metaphase and inhibit destruction of both Cyclin A and Cyclin B (Townsley, 1997). Elimination of E2-C function through either mutation or RNA interference in Drosophila cells results in the accumulation of Cyclin B principally at the centrosomes and a characteristic delay of cells in a metaphase-anaphase-like state. Metazoan E2-C enzymes themselves contain putative destruction D boxes. It is now shown directly that Drosophila E2-C is concentrated at the centrosome and that it is itself subject to cyclical degradation. Expression of a D box mutant form of the Vihar E2-C enzyme leads to mitotic defects in which Cyclin B is degraded at the spindle poles but not in the equatorial region of the spindle (Mathe, 2004).
This gene has been named vihar (Hungarian for storm) after the characteristic mutant phenotype in which chromosomes are scattered throughout the mitotic spindle. Similar reductions are observed in the levels of Vihar E2-C protein both in syncytial embryos derived from vih mutant mothers and S2 cells subjected to vih RNAi; both these treatments lead to comparable mitotic abnormalities. The majority of mitoses are delayed as metaphase figures in which chromosomes have undergone congression to the equator of the spindle. As seen with other mitotic mutants in Drosophila, many of these mitotic spindles lack centrosomes from one or both poles. Thus, this aspect of the phenotype cannot be attributed directly to reduction of the Vihar protein. The scattered chromosomes have the BubR1 checkpoint protein at their kinetochores, and the Aurora B passenger protein kinase appears not to have been transferred to the spindle, which does not adopted its characteristic late anaphase morphology. Such features have also been reported in cells arrested in mitosis due to the presence of nondegradable Cyclin B. Indeed, cells with reduced Vihar levels show pronounced increases in Cyclin B levels (Mathe, 2004).
The accumulation of Cyclin B at the centrosomes of vihar arrested cells is particularly striking and is an exaggeration of the association of the cdk1-Cyclin B complex with spindle poles that has been described in a wide number of organisms. The extent of degradation of Cyclin B varies during the embryonic development of Drosophila. Although the mitotic cyclins undergo extensive degradation at the metaphase-anaphase transition in cellularized Drosophila embryos and in tissues at later stages, they appear to persist throughout mitosis in the syncytial Drosophila embryo. There are successively increasing levels of Cyclin B degradation throughout the early syncytial cycles. This has been suggested to occur in restricted areas around the spindles as a result of observations of a gradient of the dephosphorylation of phospho-histone H3 along anaphase chromosomes, which is maximal near the spindle poles. Such a gradient has been interpreted as reflecting reduction of cdk1 activity near the spindle poles or centromere. Although this finding would probably now be interpreted as the activation of the protein phosphatase that opposes the B-type Aurora kinase that phosphorylates Histone H3, it nevertheless reflects a gradient of the activities of several enzymes associated with mitotic exit, which is initiated at the spindle poles. Support for this idea was provided by real-time imaging of GFP-tagged Cyclin B (Huang, 1999) that shows that its degradation begins at the spindle poles in cellularized embryos (Mathe, 2004).
Some indication of how Cyclin B degradation might be propagated along the spindle comes from studies of the localization of the Vihar E2-C protein and its own pattern of proteolysis. Immunolocalization experiments show not only that a considerable proportion of Vihar E2-C is associated with the centrosome, but also that it too undergoes degradation following the metaphase-anaphase transition. This was confirmed by the rapid destruction of accumulated Vihar E2-C protein following release of cells from a nocodazole block and also by the stabilization of the protein following treatment of embryos with a proteasome inhibitor. The Vihar centrosomal associated enzyme either dissociates or is directly degraded during anaphase, leaving a diffuse distribution of protein in the central part of the cell that is largely degraded upon mitotic exit. Thus, the spatiotemporal pattern of Vihar distribution is reminiscent of that described by Huang (1999) for Cyclin B. It suggests that degradation of the two proteins might be mediated by activated APC/C in similar subcellular compartments. However, Vihar E2-C appears to be more tightly localized to the centrosome in the syncytial embryo than Cyclin B, which has a more punctate distribution over astral microtubules and is more strongly associated with other regions of the spindle. Vihar also has a tighter distribution on the centrosome than the APC/C component Apc11 that clusters in a much larger 'cloud' of staining around the spindle poles as if associated with astral microtubules. Thus, the focus for APC/C activity may initially be the centrosome itself, where Vihar E2-C would not be rate limiting (Mathe, 2004).
Both of the Vihar D boxes appear to be required for efficient destruction of the Vihar protein, because mutations in either box alone result in defects in embryonic development, whereas expression of Vihar E2-C having mutations in both boxes gives a dominant mitotic phenotype. Previous studies with the mammalian counterpart of Vihar had shown that mutation of these sequences stabilized the protein in an in vitro assay, but it had not been possible to study effects in vivo (Yamanaka, 2000). Expression of the double D box mutant of Vihar leads to the preferential degradation of Cyclin B at the spindle poles and its continued presence in the equatorial region of the spindle, in contrast to the phenotype seen following downregulation of the enzyme when Cyclin B accumulates at the poles. How can this dominant effect be explained, and what is its relevance to wild-type Vihar function? Because the double D box mutant shows some reduction in catalytic activity (albeit measured in vitro against a nonphysiological substrate, a fragment of human Cyclin B), it is possible that the effects of downregulating Vihar function are being observed. However, it is noted that when a truly catalytically dead version of Vihar (mutated at its catalytic cysteine residue) is expressed in mitotic cells, the phenotype is similar to loss of Vihar function, metaphase arrest with accumulation of Cyclin B at the centrosomes. Nevertheless, the dominant effect of the Vihar double D box mutant is seen only at reduced concentration of the wild-type protein. In the whole organism, this is seen following reduction of the gene dosage of the wild-type allele, and in mitosis only once the wild-type protein has been partially destroyed. Thus, it may be that at this critical ratio of wild-type to D box mutant Vihar protein the dominant-negative effect comes into action to prevent the late pattern of Cyclin B degradation. However, it is felt that this explanation does not fully take into account the spatial allocation of Vihar to different subcellular compartments. There is no reason to suspect that at the onset of mitosis the ratio of wild-type to D box forms should vary whether associated with the centrosome, spindle, or cytoplasm. Thus, even though the ratio of wild-type to D box forms would first reduce at the poles, it should remain unchanged in other parts of the cell, where there should still be sufficient wild-type Vihar to give the potential for APC/C activity. Thus, the dominant-negative effect could be viewed from two perspectives, being a result either of reduced E2 activity or of continued E2 activity albeit at a reduced level, but in either event the mutant protein would persist at the spindle poles. The outcome of either of these viewpoints of the dominant effect might be similar: namely, the observed effect of preventing subsequent waves of Cyclin B proteolysis on the central part of the spindle and in the cytoplasm. Thus, the preferred hypothesis is that the continued presence of this nondegradable form of Vihar at the spindle poles could block mitotic progression, irrespective of its own level of E2 activity, by sequestering other rate-limiting components of the ubiquitination machinery. Conversely, in the wild-type situation, when the APC/C becomes competent at the metaphase-anaphase transition, the concentration of Vihar at the poles would direct ubiquitination of APC/C targets at this site, and its own polar destruction would be required to permit subsequent waves of proteolysis elsewhere in the cell (Mathe, 2004).
Perhaps the key to such a spatially regulated system lies in processes of microtubule-mediated transport. Raff (2002) has proposed that the degradation of spindle-associated Cyclin B might be mediated by APC/C associated with Fzy/Cdc20 and that Fizzy-related/Cdh1 directs more the degradation of Cyclin B in the cytoplasm. It is further suggested that once the metaphase-anaphase checkpoint is satisfied, flux of Fzy/Cdc20 from the kinetochore to the poles first mediates cyclin degradation. The finding that the Vihar E2-C is concentrated at the poles provides an explanation of why the Cyclin B degradation is initiated at the poles. The spatiotemporal pattern by which Vihar E2-C is then degraded suggests that it is ubiquitinated for degradation alongside its Cyclin B target once the APC/C has been activated in the vicinity of the spindle poles. Such autoregulatory inactivation of APC/C activity at the spindle poles might also release the APC/C or other regulatory components to mediate Cyclin B degradation within the central part of the spindle. This may be linked to a general influx of late mitotic regulators to the central spindle region. Indeed, plus end-directed motor proteins such as Pavarotti-KLP and several other regulators of cytokinesis begin to accumulate in the central spindle structure that forms at this time in preparation for cytokinesis. Once the ubiquitination process has been shut down at the poles, then similar autoinhibition could later operate upon Vihar E2-C in other regions of the cell following the subsequent wave of APC/C activity (Mathe, 2004).
Finally, it is noted that in vitro studies using Xenopus and clam extracts indicate that APC-mediated ubiquitination reactions are supported equally well by the Ubc4 and UBCx/E2-C enzymes. However, it has remained unclear whether these enzymes are redundant in vivo. A recent study from Seino (2004) in fission yeast, however, suggests that these two classes of E2 ubiquitin-conjugating enzymes are not functionally equivalent and have distinct roles in degrading the mitotic cyclin Cdc13. The phenotypes of cells deficient for Vihar E2-C enzyme indicate that it cannot be redundant with the Ubc4/5 family of E2 enzymes that remain functional in these cells. Thus, it remains of considerable interest to understand how different E2 family members cooperate in the APC/C-mediated destruction of mitotic cyclins and other mitotic regulatory proteins (Mathe, 2004).
It is concluded that the Vihar E2 UBC is concentrated at the spindle poles to facilitate the degradation of Cyclin B that first occurs at this site during the metaphase-anaphase transition. Vihar E2 UBC is also subject to ubiquitination and degradation by the APC/C. This results in the autoinactivation of APC/C-mediated ubiquitination at the spindle poles. Cyclin B degradation then takes place in the central spindle and other parts of the cell (Mathe, 2004).
During mitosis, a checkpoint mechanism delays metaphase-anaphase transition in the presence of unattached and/or unaligned chromosomes. This delay is achieved through inhibition of the anaphase promoting complex/cyclosome (APC/C) preventing sister chromatid separation and cyclin degradation. Bub3 is an essential protein required during normal mitotic progression to prevent premature sister chromatid separation, missegreation and aneuploidy. Bub3 is required during G2 and early stages of mitosis to promote normal mitotic entry. Loss of Bub3 function by mutation or RNAi depletion causes cells to progress slowly through prophase, a delay that appears to result from a failure to accumulate mitotic cyclins A and B. Defective accumulation of mitotic cyclins results from inappropriate APC/C activity, since mutations in the gene encoding the APC/C subunit Cdc27 (see Drosophila Cdc27) partially rescue this phenotype. Furthermore, analysis of mitotic progression in cells carrying mutations for cdc27 and bub3 suggests the existence of differentially activated APC/C complexes. Altogether, these data support the hypothesis that the mitotic checkpoint protein Bub3 is also required to regulate entry and progression through early stages of mitosis (Lopez, 2005).
Tests were performed to see whether the slower progression through early mitotic stages observed after Bub3 depletion could result from abnormal accumulation of mitotic cyclins. Cyclin B levels were measured by immunofluorescence and Western blot analysis in cells that had been depleted of Bub3 by RNAi. Control and RNAi-treated cells were incubated in colchicine, to promote high levels of cyclin accumulation and increase the number of cells in mitosis, and cyclin B levels were measured by immunofluorescence. The centrosomal marker gamma-tubulin was used to distinguish between different cell cycle stages. Interphase cells without gamma-tubulin staining were classified as G1/S, whereas those with centrosomal staining but without centrosome separation were classified as G2. Cells in mitosis (prophase and prometaphase) had gamma-tubulin staining, well-separated centrosomes and clear chromosome condensation. The fluorescence intensity values obtained for G1/S cells did not differ between control and RNAi-treated cells, allowing a direct comparison between the two samples. In agreement with the expected pattern for cyclin B accumulation, S2 control cells start to accumulate cyclin B during G2 and attain their highest levels of cyclin B during mitosis. However, cyclin B accumulation in Bub3-depleted cells is surprisingly different. These cells already show significantly lower levels of cyclin B in G2 and also during mitosis. The failure to maintain high levels of cyclin B during mitosis is in agreement with the predicted function of Bub3 in the mitotic checkpoint response (similar results were also observed in bub31 mutant neuroblasts). However, these results also indicate that Bub3 might be required during G2 to ensure accumulation of cyclin B. To eliminate the possibility that colchicine treatment could indirectly affect cyclin B accumulation during G2, cyclin B levels were measured in control and Bub3-depleted cells in the absence of the drug. Quantification of cyclin B levels shows that in contrast to control cells, Bub3-depleted cells fail to accumulate cyclin B during G2. In agreement with the immunofluorescence data, western blot analysis of total protein extracts shows that after Bub3 depletion, accumulation of cyclin B is significantly reduced reaching only 50% of the levels observed in untreated cells. In addition, although treatment with colchicine leads to accumulation of cyclin B in control cells, the same is not observed after Bub3 depletion, where accumulation of cyclin B is severely compromized. These results show that Bub3 does indeed appear to be required during G2 to promote accumulation of cyclin B, strongly suggesting that Bub3 might have a function in G2 before its well-established role in the mitotic checkpoint during prometaphase (Lopez, 2005).
Accumulation of cyclins during G2 in somatic cells depends primarily upon transcriptional activation and the fact that the anaphase promoting complex/cyclosome (APC/C) is inhibited at this stage by early mitotic regulators. To test whether APC/C activity might be required to mediate the reduction in cyclin B levels after depletion of Bub3, the effect of removing the APC/C subunit cdc27 in bub3 mutant cells was analysed. It has been shown that in Drosophila, the APC/C subunit cdc27 is required for the degradation of cyclin B but not of securin (another APC/C substrate), since mutations in cdc27 mutant neuroblasts or depletion of cdc27 by RNAi in S2 cells, results in cells with high levels of cyclin B and separated sister chromatids. Accordingly, mutations in the bub3 and cdc27 genes would be predicted to have opposite effects on the accumulation of cyclin B and the combination of the two mutations might even lead to normal mitotic entry. Therefore cyclin B levels were measured in single (bub31 or cdc27) and double (bub31;cdc27) mutant cells after incubation in colchicine. The results show that cdc27 mutant cells have significantly higher levels of cyclin B both in G2 and mitosis than do control wild-type cells, in agreement with the phenotype previously described for this mutant. As expected, accumulation of cyclin B in double mutant cells (bub31; cdc27) during G2 is not significantly different from wild-type controls and it is higher than in bub31 cells. These results support the hypothesis that the inability to accumulate normal levels of cyclin B after depletion of Bub3 is mediated by the APC/C (Lopez, 2005).
Next, several mitotic parameters in single (bub31 or cdc27) and double (bub31; cdc27) mutant cells were analysed after spindle damage induced by incubating the cells in colchicine. Under these conditions, mutant cdc27 neuroblasts arrest in mitosis with well-condensed chromosomes and unseparated sister chromatids resulting in an increased mitotic index when compared to the wild-type control. This behaviour is due to the additive effect of the mutation in the APC/C subunit and the checkpoint-dependent arrest induced by spindle damage. In neuroblasts mutant for both bub31 and cdc27, the percentage of cells in mitosis is increased relative to bub31 alone. This result shows that the premature mitotic exit characteristic of bub31 mutant cells is dependent on APC/C activity. Nevertheless, the mitotic index of double mutant cells (bub31 and cdc27) is significantly lower than that seen in wild-type cells. This difference relative to control cells is most likely explained by premature mitotic exit due to the absence of an effective checkpoint response when Bub3 activity is absent, coupled with the fact that cdc27 does not seem to be required for APC/C-mediated securin degradation. Finally, the frequency of cells in prophase in double mutant cells was observed to be indistinguishable from wild-type controls, suggesting that the double mutant transits normally through this early stage of mitosis. To rule out the possibility that cyclin B levels could be regulated by a mechanism other than APC/C-driven proteolysis, a non-degradable form of cyclin B was overexpressed in bub31 mutant neuroblasts using the GAL4/UAS system. The results show that stabilization of cyclin B in bub31 mutant cells results in an increase in the mitotic index to values similar to those observed in control cells. Taken together, these results indicate that Bub3 appears to negatively regulate APC/C activity during the G2-M transition, allowing the accumulation of sufficient cyclin B for timely progression through the early stages of mitosis (Lopez, 2005).
These results suggest that Bub3 is required for normal accumulation of cyclin B before and during mitosis. However, cyclin A is also required to promote mitotic entry in Drosophila. Cyclin A accumulates during S phase and G2 and it is degraded by the APC/C prior to the degradation of cyclin B as cells progress through early stages of mitosis. However, in contrast to cyclin B, cyclin A levels are not stabilized by the spindle damage-associated checkpoint response. In order to determine if Bub3 is also required to promote accumulation of cyclin A, its levels were analysed during cell cycle progression in Bub3-depleted cells. The centrosomal marker gamma-tubulin was used to distinguish cells in G1/S or G2. The results show that although cyclin A accumulates from G1 to G2 in control cells and can still be detected in some early mitotic cells, cyclin A fails to accumulate and can hardly be detected in cells depleted for Bub3. Analysis of cyclin A accumulation in bub31 homozygous mutant neuroblasts gave very similar results. These observations further support the role of Bub3 as a negative regulator of the APC/C during G2 and mitosis (Lopez, 2005).
Unlike other checkpoint proteins like Mad2 or BubR1, Bub3 has never been found to interact directly with the APC/C or with its activator Cdc20. Thus, it was of interest to determine if other proteins that interact simultaneously with Bub3 and Cdc20 or APC/C subunits could mediate Bub3-dependent APC/C inhibition. BubR1 is, at first glance, a good candidate to perform this function as it has been found in an interphase high molecular weight complex. Accordingly, tests were performed to see whether mutations in bubR1 could affect cyclin B accumulation in G2 or mitosis. Wild-type or bubR11 third larval neuroblasts were incubated in colchicine and immunostained to reveal the level of chromatin condensation and cyclin B. Analysis of control cells shows that cells with no cyclin B and no chromosome condensation (classified as G1/S) could be identified; cells with cyclin B levels but no visible chromatin condensation (classified as G2), and cells with high levels of cyclin B and well-condensed chromosomes (classified as mitotic), were identified as expected for normal cyclin B accumulation. Similarly, bubR11 mutant neuroblasts were detected in G1 and also in G2 with normal levels of cyclin B. However, mitotic cells showed significantly lower levels of cyclin B consistent with its known function in the mitotic checkpoint response. These results show that the pattern of cyclin B accumulation in the absence of bubR11 is significantly different from that of bub31 mutant cells. Therefore, the data suggest that accumulation of cyclin B during G2 and early mitosis requires Bub3, independent of its interaction with BubR1 (Lopez, 2005).
During mitosis, cyclin B is extremely dynamic and although it is concentrated at the centrosomes and spindle microtubules (MTs) in organisms ranging from yeast to humans, the mechanisms that determine its localisation are poorly understood. To understand how cyclin B is targeted to different locations in the cell, proteins were isolated that interact with cyclin B in Drosophila embryo extracts. Cyclin B interacts with the molecular chaperone Hsp90 and with the MT-associated protein (MAP) Mini spindles. Both Hsp90 and Msps are concentrated at centrosomes and spindles, and Hsp90, but not Msps, is required for the efficient localisation of cyclin B to these structures. Unlike what happens with other cell cycle proteins, Hsp90 is not required to stabilise cyclin B or Msps during mitosis. Thus, it is proposed that Hsp90 plays a novel role in regulating the localisation of cyclin B and Msps during mitosis (Basto, 2007; full text of article).
How might Hsp90 function in recruiting cyclin B to centrosomes and spindles? As Hsp90 is itself located at centrosomes and can bind to tubulin, it is possible that Hsp90 binds cyclin B and directly targets it to these locations. It is suspected that this is not how Hsp90 targets cyclin B to MTs, since only a small fraction of the total cyclin B is bound to Hsp90 in embryo extracts. Virtually all of the cyclin B in an embryo extract is capable of binding to MTs in MT spin-down experiments. Thus, it seems unlikely that Hsp90 could act as an essential co-factor that directly mediates the interaction between cyclin B and MTs. Similarly, it is suspected that Hsp90 does not directly target cyclin B to centrosomes, since the initial recruitment of cyclin B to centrosomes during prophase is only mildly disrupted in Drosophila cells (in HeLa prophase recruitment is not disrupted) when Hsp90 has been perturbed. Rather, Hsp90 seems to be involved in maintaining the centrosomal localisation of cyclin B during prometaphase and metaphase. Perhaps Hsp90 is essential for the proper folding or function of a specific domain of cyclin B that is required for the localisation of cyclin B on centrosomes and spindles. In such a scenario Hsp90 could even act indirectly to allow cyclin B to associate with other proteins that target it to centrosomes and MTs (Basto, 2007).
If the assumption that Hsp90 does not directly target cyclin B to centrosomes or MTs is correct, it raises the intriguing question of what targets cyclin B to these locations? It has previously been shown that cyclin B can interact with XMAP215 and it has been proposed that this interaction could target cyclin B to centrosomes and MTs. It was also found that cyclin B can interact with Msps/XMAP215 in Drosophila embryo extracts, suggesting that this interaction is conserved between frogs and flies. In msps mutant cells, or in human cells partially depleted of ch-TOG, however, it was found that cyclin B was still localised to centrosomes and MTs. Thus, it is concluded that Msps is not directly responsible for targeting cyclin B to centrosomes or MTs. Msps family members play an important role in regulating MT dynamics during the cell cycle so the interaction between cyclin B and Msps may simply reflect the fact that cyclin B/Cdc2 regulates Msps activity during the cell cycle. Indeed, it has been shown that Msps/XMAP215 is phosphorylated by cyclin B/Cdc2 in vitro (Vasquez, 1999; Basto, 2007 and references therein).
If Msps and Hsp90 do not directly target cyclin B to centrosomes and MTs, it remains unclear what does. A priori, it is expected that cyclin B in Drosophila embryo extracts would exist in a tight complex with any factor that would target it to MTs, since the vast majority of cyclin B binds to MTs in embryo extracts. Since cyclin B appears to localise at centrosomes and MTs in virtually all systems, it remains possible that cyclin B can directly bind to centrosomes and MTs. Indeed, bacterially expressed MBP-CBFL interacts strongly with purified MTs in MT-pelleting assays. In light of these results it seems that cyclin B is capable of interacting directly with MTs, although caution should b taken in interpretation of this in vitro experiment since fusion proteins containing cyclin B could have a tendency to aggregate in solution (Basto, 2007).
Nevertheless, the fact that no one has identified a factor that directly mediates the interaction between cyclin B and MTs or centrosomes, despite many years of effort in identifying cyclin B interacting proteins, suggests that there may be no other protein directly required for these interactions. In the favoured hypothesis, Hsp90 would serve simply to ensure that cyclin B was correctly folded to allow it to directly interact with MTs and with centrosomes. Interestingly it has been proposed that Hsp90 also contributes to increasing the association efficiency of Tau with MTs. Tau is a MAP with an important role in Alzheimer's disease. In the absence of Hsp90, Tau tends to aggregate and therefore less soluble Tau is available to bind to MTs. In this study, although it was also found that cyclin B and Msps require Hsp90 for their efficient recruitment to the spindle it is not thought that their activity, outside the spindle, is compromised (Basto, 2007).
Finally, it was found that Hsp90 was not only required to allow cyclin B to localise efficiently to centrosomes and MTs, it was also required to allow Msps to localise properly, and it was shown that the endogenous Hsp90 can interact with the endogenous Msps. Importantly, Hsp90 is not required for the localisation of several other proteins to centrosomes or MTs, demonstrating that its function in localising cyclin B and Msps is specific. Like cyclin B, the levels of Msps protein were not decreased in cells where Hsp90 function had been perturbed, suggesting that Hsp90 is not simply required to stabilise Msps protein. Thus, it is proposed that Hsp90 may act on several MT-associated proteins to ensure that specific domains of these proteins are in the correct conformation to allow these proteins to be targeted to different locations within the cell (Basto, 2007).
The Drosophila Pan Gu (Png) kinase complex regulates the developmental translation of cyclin B. cyclin B mRNA becomes unmasked during oogenesis independent of Png activity, but Png is required for translation from egg activation. Although polyadenylation of cyclin B augments translation, it is not essential, and a fully elongated poly(A) is not required for translation to proceed. In fact, changes in poly(A) tail length are not sufficient to account for Png-mediated control of cyclin B translation and of the early embryonic cell cycles. Evidence is presented that Png functions instead as an antagonist of Pumilio-dependent translational repression. The data argue that changes in poly(A) tail length are not a universal mechanism governing embryonic cell cycles, and that Png-mediated derepression of translation is an important alternative mechanism in Drosophila (Vardy, 2007).
These data indicate that in Drosophila translation of cyclin B can proceed in the absence of polyadenylation in ovaries and syncytial embryos. Polyadenylation is not required for the unmasking of the mRNA, but it may play a role in fine-tuning translation. A model is proposed in which Png has dual roles—poly(A) dependent and independent—in promoting cyclin B translation. At egg activation, Png is needed for full-length poly(A) tails, and this may augment translation. The data indicate, however, that Png also promotes translation independently of poly(A) tail length, most likely by overcoming repressive action by Pum. Removal of Pum in a png mutant therefore allows translation of the target mRNA even if the poly(A) tails are not fully elongated (Vardy, 2007).
SMAUG (SMG) protein levels and the poly(A) tail are decreased in the png mutant, and restoration of poly(A) tail length by overexpressing poly(A) polymerase is not enough to promote smaug translation in a png mutant. Thus, Png also appears to regulate smaug translation in a poly(A)-dependent and -independent manner. This indicates that multiple levels of translational regulation may be a common strategy in development (Vardy, 2007).
If the Png kinase complex functions ultimately to regulate the activity of Pum, one of the key questions concerns the nature of this regulation. Since NANOS is found predominantly at the posterior pole, Pum may be able to recruit different factors to the target mRNA. The defect seen in png is likely due to a failure to relieve translational repression as opposed to a failure in activation, because injection of excess amounts (1 μg/μl) of the 5′FF3′−cycB transcript into png mutant embryos allows translation to proceed at much stronger levels. This suggests that the repressor is limiting in this reaction, thus allowing translation to proceed, and is consistent with Pum being downstream of Png. It will be interesting to determine the nature of the Png kinase substrates and how they relate to Pum function (Vardy, 2007).
Png likely has multiple targets, because removal of Pum in a png mutant does not completely restore the embryonic divisions. While the early syncytial cycles can progress in a png;pum double mutant, mitosis still fails in the later S-M cycles. Smg protein levels are decreased in the png mutant due to a failure in translation. Smg protein levels are not restored in a png;pum double mutant, and given Smg's role in progression through cycle 11/12, it provides an explanation for why the embryos fail at this stage. An independent role for the Png kinase complex later in embryogenesis has been described in the maternal mRNA degradation pathway. Although these pathways appear to be independent, in each case translational regulation of a key component appears to be the mechanism: Cyclin B to control the S-M cycles and SMG to control maternal transcript destruction (Vardy, 2007).
This study has established a role for the Png kinase complex in the translational regulation of cyclin B in the early syncytial cycles of Drosophila. A role for the Png kinase complex in protein stability has been suggested, and it is possible that it could act to regulate Cyclin B levels by multiple mechanisms. These data, however, reveal it to be a key regulator of cyclin B translation, ensuring coordinated passage through these early cycles (Vardy, 2007).
Meiosis is a highly specialized cell division that requires significant
reorganization of the canonical cell-cycle machinery and the use of
meiosis-specific cell-cycle regulators. The anaphase-promoting complex (APC, a machine for degrading proteins; see APC subunits Cdc27 and morula; for review see Acquaviva, 2006)
and a conserved APC adaptor/activator, Cdc20 (also known as Fizzy), are required for
anaphase progression in mitotic cells. The APC has also been implicated in
meiosis, although it is not yet understood how it mediates these non-canonical divisions. Cortex (Cort) is a diverged Fzy homologue that is expressed in the female germline of Drosophila, where it functions with the Cdk1-interacting protein Cks30A to drive anaphase in meiosis II. This study shows
that Cort functions together with the canonical mitotic APC adaptor Fzy to
target the three mitotic cyclins (A, B and B3) for destruction in the egg and
drive anaphase progression in both meiotic divisions. In addition to
controlling cyclin destruction globally in the egg, Cort and Fzy appear to
both be required for the local destruction of cyclin B on spindles. Cyclin B associates with spindle microtubules throughout meiosis I and
meiosis II, and dissociates from the meiotic spindle in anaphase II. Fzy and
Cort are required for this loss of cyclin B from the meiotic spindle. These
results lead to a model in which the germline-specific APCCort
cooperates with the more general APCFzy, both locally on the
meiotic spindle and globally in the egg cytoplasm, to target cyclins for
destruction and drive progression through the two meiotic divisions (Swan, 2007).
The cell divisions of female meiosis and the ensuing mitotic cycles of
early embryogenesis represent two examples of non-canonical cell cycles.
Meiosis differs from the typical mitotic cycle in several respects. Most
notably, two divisions occur in sequence without an intervening S-phase,
resulting in the production of four haploid gametes. Additionally, the first
meiotic division involves the segregation of homologous chromosomes and occurs
without sister chromatid segregation, whereas the second meiotic division
involves the segregation of sister chromatids, as occurs in mitosis. The
regulation of meiosis requires a significant reorganization of the canonical
cell-cycle machinery and the use of a number of meiosis-specific cell-cycle
regulators. One example is in the regulation of anaphase - the
coordinated series of events that results in the segregation of chromosomes to
produce two daughter nuclei. In mitotically dividing cells, anaphase
progression crucially depends on the inactivation of the mitotic kinase Cdk1
(also known as Cdc2) and on the subsequent release of sister chromatid
cohesion through the destruction of cohesin complexes. These events are
controlled by an E3 ubiquitin ligase -- the anaphase-promoting complex (APC) --
in association with an adaptor protein, Fzy, and this complex targets mitotic
cyclins and securin (potential Drosophila homolog; Pimples) for destruction. The role of the
APC in meiosis appears to be more complex than in mitotic cells. For example,
the APC only partially inhibits Cdk1 activity between meiotic divisions
and sister chromatid cohesion persists at centromeres through anaphase I. It is not yet clear how the activity of the APC is modified in these specialized
cell divisions (Swan, 2007).
In most eukaryotes, the meiotic cell cycle is followed by another atypical
cell cycle -- the cleavage divisions of early embryogenesis. In
Drosophila, these cleavage cycles occur as a series of synchronized,
rapid nuclear divisions and are referred to as syncytial divisions. The female
meiotic cell cycle is not only closely linked to the syncytial mitotic cell
cycle in time, but it also occurs within a shared cytoplasm -- that of the egg.
Therefore, these two distinct cell cycles share a common pool of cell-cycle
regulators, and may share common strategies for spatially and temporally
regulating cell-cycle progression within a syncytium (Swan, 2007).
One way in which the syncytial cell cycle is modified appears to be in the
limited destruction of mitotic cyclins in each cell cycle, apparently by
restricting their destruction to the area of the mitotic nuclei. Although
there is evidence that cyclin destruction is spatially regulated in somatic
cells, this strategy appears to be of particular importance in the syncytial embryo of Drosophila as a means to conserve mitotic cyclins for the duration of the rapid syncytial divisions. Several lines of evidence suggest that at least one cyclin, cyclin B, undergoes limited local destruction on mitotic spindles in the syncytial embryo. It is not yet known what mediates this local cyclin B destruction, and it is also not known whether this is unique to the syncytial mitotic cell cycle or if it occurs in the preceding meiotic divisions (Swan, 2007).
Drosophila represents an excellent model system for understanding
how the canonical cell-cycle machinery is developmentally modified, and how
novel cell-cycle regulators are used to control meiosis and syncytial
divisions. cortex (cort) encodes a Cdc20/Cdh1 (Cdh1 is also
known as Fzr and Rap)-related protein, that appears to be required specifically in female meiosis and functions with a germline-specific Cks gene, Cks30A, to mediate the destruction of cyclin A. This study shows that the canonical APC adaptor Fzy functions together with Cort to target mitotic cyclins for destruction, and to drive anaphase in both meiosis I and meiosis II. Female meiosis, like the subsequent syncytial mitotic cell cycles, appears to involve the local destruction of cyclin B, and both Cort and Fzy were found to be required for this process (Swan, 2007).
In most cell types, in both Drosophila and in other metazoans, the
APCFzy drives anaphase progression by targeting mitotic cyclins and other mitotic proteins for destruction. This study shows that the female germline is an exception in that the APCFzy is not sufficient. A germline-specific APC adaptor, Cort, cooperates with Fzy to mediate cyclin destruction in meiosis (Swan, 2007).
The cort gene encodes a diverged member of the Fzy/Cdh1 family. Fzy/Cdh1
homologues interact with the APC and with specific sequences (D-box, KEN box
or A-box) found on cyclins and on other APC targets. As such, Fzy/Cdh1
proteins act as specificity factors to target proteins for ubiquitination and
eventual destruction. Cort protein, like all Fzy/Cdh1-family proteins,
contains seven WD domains in the C-terminal-half of the protein, implicated in substrate recognition. Cort has an N-terminal C-box (amino
acids 482, 483) and a C-terminal IR tail (amino acids 54-60), both implicated
in binding to the APC. In addition to containing these conserved functional
domains, Cort displays a conserved ability to mediate cyclin destruction.
cort mutations result in the overaccumulation of cyclin A, cyclin B
and cyclin B3 in the egg, whereas the ectopic expression of Cort in the wing disc leads to a reduction in the levels of these mitotic cyclins. Taken together, these results indicate that Cortex encodes a functional member of the Fzy/Cdh1 family (Swan, 2007).
Although the Drosophila genome has four genes that encode Fzy/Cdh1
proteins, only two of these proteins, Fzy and Cort, are expressed at
detectable levels in the female germline. The role of these two APC adaptors has been studied both individually and in double
mutants, and it was found that they function together to promote anaphase in
both the first and second meiotic divisions of female meiosis. In most cell
types in Drosophila and other eukaryotes, a single APC complex,
APCFzy, is responsible for cyclin destruction and anaphase
progression. It is therefore surprising that, in the female germline of
Drosophila, two APC adaptors are necessary for meiotic progression.
In the case of meiosis I, Cort and Fzy appear to play largely redundant roles, since only removing both genes results in a significant block in meiosis I. The
two APC complexes may also be functionally redundant with respect to global
cyclin levels. Mutations in either fzy or cort result in an
increase in the levels of cyclin A, cyclin B and cyclin B3, whereas mutation
in both genes results in even-further increases in cyclin levels (Swan, 2007).
Although Cort and Fzy have overlapping roles in promoting anaphase I, both
are essential for meiosis II. This could simply reflect a greater quantitative
requirement for APC activity in meiosis II. Alternatively, the two APC
complexes could have distinct roles in the second meiotic division. Consistent
with this latter possibility, mutations in either cort or
fzy both result in arrest at different stages of meiosis II:
cort mutants arrest with the sister chromatids associated, and
therefore in metaphase, whereas fzy mutants almost invariably arrest
with separated sister chromatids, and are therefore in anaphase. cort
and fzy also result in different patterns of cyclin B stabilization
on the arrested spindles, suggesting roles in metaphase and anaphase,
respectively. Therefore, Cort may function to initiate sister chromatid
separation at the onset of anaphase II and Fzy may primarily function later,
in anaphase II. Alternatively, the later arrest observed in fzy could
simply reflect the fact that the fzy alleles that have been used are
not nulls, and it is possible that a complete loss of Fzy activity would also
result in a metaphase arrest, as seen in cort. However, comparing the
meiosis II phenotypes of fzy with Cks30A-null mutants
suggests that the later arrest in fzy is not simply due to residual
activity. Cks30A-null mutants have a weaker meiotic arrest than
fzy; they complete meiosis at high frequency, but they
display a higher frequency of metaphase arrest or delay. The fact that
fzy does not similarly cause a delay in metaphase of meiosis II
suggests that it is only required at anaphase. Therefore, it is possible that
Fzy is crucial at anaphase, whereas Cort is necessary for the metaphase to
anaphase transition (Swan, 2007).
The different temporal requirements for Cort and Fzy prior to and after
sister chromatid separation, respectively, could be related to their apparent
differences in substrate specificity. Western analysis
reveals that Cort is
more important for the destruction of cyclin A and cyclin B3, whereas Fzy
appears to play a greater role in cyclin B destruction in the egg. In mitotic
cells, cyclin destruction occurs sequentially. Cyclin A is destroyed first, in
prometaphase, and this is a prerequisite for sister chromatid separation.
Cyclin B destruction occurs at anaphase onset and is necessary for later
anaphase events, subsequent to sister chromatid separation.
Therefore, it is possible that Cort promotes the early stages of meiotic
anaphase by targeting cyclin A for destruction, whereas Fzy is more crucial
later, through its targeting of cyclin B for destruction (Swan, 2007).
The meiotic cell cycle differs in many respects from the standard mitotic
cycle. Whereas APC-mediated destruction of mitotic regulators appears to be
required for anaphase progression in most or all mitotic cells, the role of
the APC and cyclin destruction in meiosis is not as well-understood. This
analysis of the two APC adaptors Cort and Fzy has permitted an evaluation of
the role of the APC complex in female meiosis in Drosophila. The APC is required for anaphase progression in both meiotic divisions.
Correlating with its requirement for the completion of meiosis, the APC is
required for the destruction of mitotic cyclins. At least one of these
cyclins, cyclin B, is a crucial substrate in meiosis, because the expression
of a stabilized form of cyclin B disrupts this process. Therefore, APC
activity and cyclin destruction are required for anaphase progression in both
meiotic divisions, in addition to in mitosis. APC activity has been implicated
in both meiotic divisions in C. elegans and in
the mouse, and in the second, but not the first, meiotic division in
Xenopus. In yeast, two APC complexes, the mitotic APCFzy
and a meiosis-specific complex (APCAma1 in S. cerevisiae
and APCMfr1 in S. pombe) function together to mediate
protein destruction in meiosis. It now appears that Drosophila also uses two APC complexes in female meiosis, and this may turn out to be a common strategy in other eukaryotes (Swan, 2007).
Cks30A belongs to a highly conserved family of proteins that bind to and
stimulate the activity of the mitotic kinase Cdk1. In Xenopus, the
Cks30A homologue Xep9 stimulates the Cdk-dependent phosphorylation of APC
subunits, and thereby promotes the activation of the APCFzy complex (Patra, 1998). The current
results suggest that Cks30A may have a similar role in stimulating both the
APCFzy and APCCort in female meiosis in
Drosophila. (1) Cks30A, like cort and
fzy, is required for the completion of meiosis II and, like
fzy, it is required for the completion of the first mitotic division
of embryogenesis. (2) Cks30A, as are Cort and Fzy, is necessary for
global cyclin destruction in the Drosophila egg and for local cyclin
B destruction on the meiotic spindle. Global levels of cyclin A
and cyclin B3 are elevated to a greater extent in Cks30A mutants than
in single mutants for cort or fzy, consistent with the idea
of Cks30A activating both Cort and Fzy. (3) Cks30A is
necessary for the activity of ectopically expressed Cort in the adult wing. Cks30A may also play a role in activating APCFzy in mitotic cells.
the temperature-sensitive fzy6 allele is lethal at all
temperatures in a Cks30A-null background, suggesting that the Cks30A-dependent activation of APCFzy becomes essential when Fzy activity is compromised (Swan, 2007).
Although Cks30A appears to promote the activity of the APCCort
and the APCFzy, these complexes seems to retain some activity in
the absence of Cks30A. Whereas cort and fzy cause an arrest
in meiosis II, Cks30A-null mutants are typically delayed only in
meiosis II. Also, although cyclin A and cyclin B3 levels are elevated
more in Cks30A eggs than in either fzy or cort, their levels
are still not as high as in fzy; cort double mutants, indicating that
Fzy and Cort can destroy cyclin A and cyclin B3 to some degree in the absence
of Cks30A. Cyclin B destruction is even less dependent on Cks30A, because
cyclin B levels are affected less in Cks30A mutants than in either
cort or fzy single mutants. Therefore, Cks30A may be more
crucial for the activity of APCCort and APCFzy complexes
on cyclin A and cyclin B3, and less crucial for their activity on cyclin B.
The relatively weaker meiotic arrest in Cks30A mutants compared to
fzy; cort double mutants may also indicate that the APC has other
meiotic targets that can be destroyed in the absence of Cks30A (Swan, 2007).
Cyclin B undergoes local oscillations in its association with mitotic
spindles in syncytial embryos, appearing transiently along the full length of
the mitotic spindle in early metaphase and gradually disappearing from the
spindle starting at the centrosomes and ending at the kinetochores. The
timing of this loss of cyclin B from the spindle, at the onset of anaphase,
corresponds with the timing of cyclin B destruction in other cell types,
suggesting the possibility that cyclin B is locally destroyed on the spindle
in anaphase. This study shows that cyclin B is subject to similar local
oscillations in the female meiotic cycles, and that cyclin B
destruction is necessary for the completion of female meiosis. Importantly, the local loss of cyclin B from the spindle in meiosis is
dependent on the APC adaptors Cort and Fzy, and that the local loss of cyclin
B from the spindle in mitosis depends on Fzy. These results
strongly suggest that the local loss of cyclin B from the spindle in anaphase
of meiosis II and anaphase of mitosis is actually due to its local
destruction (Swan, 2007).
The pattern of accumulation and loss of cyclin B from the spindle in
meiosis differs in some respects compared to syncytial mitotic cycles. (1) In metaphase of mitosis, cyclin B initially accumulates throughout the spindle microtubules, whereas, in metaphase of the meiotic divisions, cyclin B first appears exclusively at the spindle mid-zone. This difference may reflect the fact that the meiotic spindle does not contain centrosomes and cyclin B may, therefore, not load onto spindles from centrosomes and progress along the spindles to the kinetochores, as has been proposed for mitosis.
(2) The timing of cyclin B destruction appears to be different between the meiotic and mitotic cycles. Most strikingly, there is no loss of cyclin B from the spindle in anaphase of meiosis I, implying that local cyclin B destruction is not necessary for the completion of the first meiotic division. In addition, the loss of cyclin B from the spindle following meiosis II occurs only late in anaphase rather than at the onset of anaphase, as occurs in the syncytial mitotic cycles. It is not yet known how cyclin B destruction is prevented in anaphase I and early in anaphase of meiosis II. One possibility is that the spindle-assembly checkpoint is locally active during these stages. This checkpoint is required for the proper completion of female meiosis in Drosophila, and it will be interesting to see if this requirement reflects a role in inhibiting either APCFzy or APCCort activity (Swan, 2007).
The specific accumulation of cyclin B at the spindle mid-zone in meiosis
may reflect the unique properties of the meiotic spindle. The mid-zone
microtubules or central spindle microtubules are a subset of spindle
microtubules that do not end in kinetochores, but instead overlap at the
mid-zone with microtubules from the other pole. In dividing cells, the central spindle is crucial for cytokinesis, but, in female meiosis, it appears to have a role in spindle assembly. Along with cyclin B, the chromosomal passenger proteins
Aurora B and Incenp are recruited to the spindle mid-zone. It will be of great interest to determine what these proteins do at the mid-zone and how cyclin B destruction at this site may be important for anaphase in meiosis. It will also be important to determine how the APCCort targets cyclin B at the spindle mid-zone. It has not been possible to detect any specific localization of GFP or HA-tagged Cortex in meiosis or in the syncytial embryo, but it is possible that its activity is
spatially regulated (Swan, 2007).
In conclusion, these results support a model in which two APC complexes,
APCFzy and APCCort, cooperate to mediate the destruction of meiotic cyclins and allow progression through female meiosis (Swan, 2007).
Translational control of maternal mRNA through regulation of poly(A) tail length is crucial during early development. The nuclear poly(A) binding protein, PABP2, was identified biochemically from its role in nuclear polyadenylation. This study analyzed the in vivo function of PABP2 in Drosophila. PABP2 is required in vivo for polyadenylation, and Pabp2 function, including poly(A) polymerase stimulation, is essential for viability. An unanticipated cytoplasmic function is demonstrated for PABP2 during early development. In contrast to its role in nuclear polyadenylation, cytoplasmic PABP2 acts to shorten the poly(A) tails of specific mRNAs. PABP2, together with the deadenylase CCR4, regulates the poly(A) tails of oskar and cyclin B mRNAs, both of which are also controlled by cytoplasmic polyadenylation. Both Cyclin B protein levels and embryonic development depend upon this regulation. These results identify a regulator of maternal mRNA poly(A) tail length and highlight the importance of this mode of translational control (Benoit, 2005).
During early development in most species, regulation of gene expression is strictly posttranscriptional. One major posttranscriptional regulatory mechanism involves variations in poly(A) tail length, which regulate mRNA expression by affecting both mRNA stability and translation. Cytoplasmic changes in mRNA poly(A) tail length by deadenylation and polyadenylation play, thus, an essential role in controlling the production of key proteins during early development. While cytoplasmic polyadenylation has been studied extensively, the mechanisms underlying the control of poly(A) tail length in the cytoplasm are unknown (Benoit, 2005).
In Xenopus oocytes, cytoplasmic poly(A) tail elongation requires cis elements, including the cytoplasmic polyadenylation element (CPE) located in the 3' UTR of mRNAs and the nuclear polyadenylation signal, AAUAAA. CPEs are bound by the CPE binding protein (CPEB; see Drosophila CPEB, Orb), a primary factor in cytoplasmic polyadenylation, which also requires a poly(A) polymerase (PAP) and a complex that binds the AAUAAA element, called the Cleavage and Polyadenylation Specificity Factor (CPSF). Cytoplasmic elongation of the poly(A) tail leads to translational activation by remodeling the mRNP: in the repressed state, a translational repressor called Maskin binds to CPEB and eIF4E, the cap binding initiation factor, and precludes the eIF4E-eIF4G interaction that is required for translation initiation. When polyadenylation occurs, the elongated poly(A) tail is bound by the cytoplasmic poly(A) binding protein, PABP, which then interacts with eIF4G. This promotes the association between eIF4E and eIF4G, thereby allowing translation initiation (Benoit, 2005).
The role of the poly(A) tail in translational control in Drosophila is more controversial. In early embryos, a long poly(A) tail is both necessary and sufficient to induce translation of bicoid and Toll mRNAs, which encode the anterior morphogen and a determinant of dorsoventral polarity, respectively). Translation of the posterior determinant oskar (osk) mRNA, is also highly regulated during oogenesis. Translation is repressed until mid-oogenesis, and subsequently in the oocyte, as osk mRNA is being transported to the posterior pole. During this transport, a major translational repressor is Bruno, whose mechanism of action was found to be independent of the 5' cap and the poly(A) tail in vitro. A recent study, however, has identified a new translational repressor of osk mRNA, called Cup, which interacts with both Bruno and eIF4E. This strongly suggests that Cup/Bruno-mediated translational repression is cap-dependent, acting to prevent the eIF4E-eIF4G interaction in a manner similar to Maskin. While the mechanism underlying the release of Bruno repression at the posterior pole is unknown, accumulation of Osk protein at the posterior of the oocyte requires osk mRNA cytoplasmic polyadenylation involving Orb, the Drosophila homolog of CPEB and Drosophila PAP (Benoit, 2005 and references therein).
Before cytoplasmic regulation can occur, poly(A) tails are added to mRNAs in the nucleus in a cotranscriptional reaction involving endonucleolytic cleavage followed by polyadenylation. In mammals, the reaction involves two signals flanking the cleavage site, the upstream polyadenylation signal, AAUAAA, and a downstream GU-rich element. Cleavage requires several cleavage factors, including CPSF, and polyadenylation of cleaved RNAs can be recapitulated in vitro with CPSF, PAP, and the nuclear poly(A) binding protein, PABP2 (PABPN1 in mammals). While PAP has a very low affinity for, and binds aspecifically to, RNA, specificity is achieved through the recognition of the AAUAAA element by CPSF, which then tethers PAP to the RNA by direct protein-protein interaction. While CPSF thus stimulates PAP, complete stimulation occurs only in the additional presence of PABP2, which binds the poly(A) tail when it has reached ten residues in size. At this point, the reaction becomes processive, and a complete poly(A) tail is synthesized very rapidly and without dissociation of PAP from the RNA. PABP2 has a second function in nuclear polyadenylation, namely, to control poly(A) tail length: once the poly(A) tail has reached full length (250 residues in mammalian cells), the reaction becomes slow and distributive. These two functions of PABP2 in nuclear polyadenylation are carried out by different domains of the protein, and they can be uncoupled by point mutations. These data have led to a model in which multiple PABP2 proteins coat the growing poly(A) tail, with only one of them directly interacting with PAP (Benoit, 2005).
Although PABP2 is nuclear at steady-state levels in somatic cells, it shuttles from nuclear to cytoplasmic compartments. While a possible role for PABP2 in mRNA export has not been investigated, PABP2 has been found to be associated with an mRNA during its docking at the nuclear pore, and it was present on the cytoplasmic side of the nuclear envelope. This suggests that the exchange between nuclear PABP2 and cytoplasmic PABP on poly(A) tails occurs in the cytoplasm (Benoit, 2005).
This study used Pabp2 mutants to address the in vivo role of PABP2 in Drosophila.PABP2 has a role in poly(A) tail lengthening in somatic tissues, and that this function is essential for viability. The cytoplasmic presence of PABP2 is described in oocytes and early embryos (Benoit, 1999; full text of article), and it is shown that cytoplasmic PABP2 binds to poly(A) tails at these stages and shortens poly(A) tails of specific mRNAs, in conjunction with the deadenylase CCR4. Cytoplasmic poly(A) tail length control by PABP2 is essential for development, as embryos depleted of PABP2 show early developmental arrest, with elongated poly(A) tails of key maternal mRNAs (Benoit, 2005).
An important set of biochemical data has led to a precise description of PABP2 function in nuclear polyadenylation (Kühn, 2004). In in vitro polyadenylation assays, PABP2 has two distinct roles: it stimulates PAP to make polyadenylation processive, and it controls poly(A) tail length, with polyadenylation becoming distributive once the tail has reached 250 nucleotides. This length control involves a measurement of the poly(A) tail by PABP2. Drosophila PABP2 tested in mammalian polyadenylation-reconstituted assays also shows these two functions. Using a null Pabp2 allele, this study shows that poly(A) tails measured either on total or on individual mRNAs are shorter in Pabp2 mutants than they are in wild-type, consistent with a role for PABP2 in poly(A) tail elongation. The short poly(A) tails in mutant embryos appear to result from deadenylation of existing mRNAs and progressive reduction of new mRNA synthesis as the PABP2 level decreases. Poly(A) tails of newly synthesized Hsp70 mRNA were of similar size in the Pabp255 mutant and in wild-type, although they were present in a small amount in the mutant and were thought to be synthesized with the remaining maternal PABP2. The finding that newly synthesized mRNAs with short poly(A) tails do not accumulate when PABP2 is limiting suggests that PABP2 is absolutely required for polyadenylation and that PAP is unable to produce stable poly(A) tails in the absence of PABP2. In agreement with this, it was found that PABP2 is essential for viability, and specifically for cell viability, since Pabp255 mutant somatic or germline clones were found to not survive. Moreover, lethality in the absence of PAPB2 may be caused by a lack of PAP stimulation, since a Pabp2 transgene bearing a point mutation that prevents PAP stimulation is unable to rescue the lethality of the null allele Pabp255. Taken together, these results strongly suggest that the function of PABP2 in mRNA polyadenylation is essential, and that PAP in the absence of PABP2 is incapable of producing stable polyadenylated mRNAs (Benoit, 2005).
One important conclusion presented in this paper is the identification of an unexpected function for PABP2 in regulating poly(A) tail length of cytoplasmic mRNAs during early development. Using two hypomorphic Pabp2 alleles, it was found that a reduced amount of PABP2 leads to elongated poly(A) tails in two mRNAs regulated by cytoplasmic polyadenylation. Three sets of data indicate that this function of PABP2 is cytoplasmic: (1) the poly(A) tail elongation phenotype on the involved mRNAs is the opposite of the Pabp2 mutant phenotype on total mRNAs, also visible in the same RNA preparations on the control sop mRNA; (2) a reduced level of PABP2 restores longer poly(A) tails on osk and cyc B mRNAs, but not on sop mRNA, in orbmel ovaries in which cytoplasmic polyadenylation is impaired; (3) PABP2 is cytoplasmic in oocytes, and in early embryos prior to the onset of zygotic transcription, it binds poly(A) tails of mRNAs that are also bound by the cytoplasmic proteins Orb and PABP, and it is recruited into cytoplasmic cyc B mRNA particles (Benoit, 2005).
This cytoplasmic function of PABP2 is essential for early development. PABP2 is required to shorten poly(A) tails of, at least, osk and cyc B mRNAs, and in Pabp2 mutant germline clones the lengthening of cyc B poly(A) tails correlates with higher levels of Cyc B protein and with embryonic phenotypes similar to those produced by a high dosage of maternal Cyc B. Misregulation of other maternal mRNAs could also contribute to the lethality of embryos from these germline clones, since cytoplasmic PABP2 probably regulates several of them. The maternal-effect embryonic lethality of Pabp26 is strongly rescued by the Pabp2-I61S transgene, which lacks the nuclear function of PAP stimulation; this suggests that this lethality results from a defect in the cytoplasmic function of PABP2. In addition, the synergistic effect of the simultaneous decrease in PABP2 and CCR4 amounts in the female germline, which leads to important embryonic lethality and elongated poly(A) tails of osk and cyc B mRNAs, also indicates an essential function of cytoplasmic PABP2 in shortening poly(A) tails at these stages. Finally, consistent with PABP2 playing a major role in the cytoplasm during early development is the recent identification of a cytoplasmic PABP2 specific to embryos in Xenopus and mouse (Benoit, 2005).
Several lines of evidence suggest that PABP2 regulates poly(A) tail length in the cytoplasm by using a different mechanism than that used during nuclear polyadenylation. Termination of poly(A) tail elongation during nuclear polyadenylation is thought to result from a PABP2-dependent remodeling of the polyadenylation complex that blocks PAP stimulation. This remodeling depends on the complete coating of the poly(A) tail by PABP2. In sharp contrast, studies of cytoplasmic polyadenylation in Drosophila embryos suggest that the reaction is not processive and does not involve PAP stimulation by PABP2. Cytoplasmic polyadenylation of bicoid mRNA in embryos is slow, with poly(A) tail elongation depending on the level of PAP. Very long poly(A) tails are produced by overexpression of PAP, without poly(A) tail length control. Consistent with this, it was found that cytoplasmic PABP and PABP2 are present on the same mRNA poly(A) tails in ovary and early embryo extracts, thereby precluding complete coating of the poly(A) tail by PABP2 (Benoit, 2005).
In yeast, poly(A) tail length control involves deadenylation by the PAN (Pan2/Pan3) deadenylase, which is activated by poly(A) tail bound PABP. It is proposed that, similarly, during early Drosophila development, cytoplasmic PABP2 controls the poly(A) tail length of key mRNAs whose turnover and translatability are specifically regulated, by modulating the activity of a deadenylase. Poly(A) tail length control of these mRNAs would thus be achieved by the balance between cytoplasmic polyadenylation and deadenylation. A major deadenylation complex in Drosophila is the CCR4/NOT complex, in which CCR4 is the deadenylase (Temme, 2004). ccr4 function is essential in the female germline, where it regulates poly(A) tail lengths of cyc B mRNA and other cell cycle regulators. This study found that Pabp2 and ccr4 act in conjunction in shortening poly(A) tails of specific mRNAs, consistent with a possible role of PABP2 in stimulation of CCR4 activity. In yeast, deadenylation by the CCR4/NOT complex is inhibited in vitro by PABP. If this regulation is conserved in metazoans, the presence of PABP2 on poly(A) tails could modulate this effect of PABP (Benoit, 2005).
Zfrp8 is essential for hematopoiesis in Drosophila. Zfrp8 (Zinc finger protein RP-8) is the Drosophila ortholog of the PDCD2 (programmed cell death 2) protein of unknown function, and is highly conserved in all eukaryotes. Zfrp8 mutants present a developmental delay, lethality during larval and pupal stages and hyperplasia of the hematopoietic organ, the lymph gland. This overgrowth results from an increase in proliferation of undifferentiated hemocytes throughout development and is accompanied by abnormal differentiation of hemocytes. Furthermore, the subcellular distribution of γ-Tubulin and Cyclin B is affected. Consistent with this, the phenotype of the lymph gland of Zfpr8 heterozygous mutants is dominantly enhanced by the l(1)dd4 gene encoding Dgrip91, which is involved in anchoring γ-Tubulin to the centrosome. The overgrowth phenotype is also enhanced by a mutation in Cdc27, which encodes a component of the anaphase-promoting complex (APC) that regulates the degradation of cyclins. No evidence for an apoptotic function of Zfrp8 was found. Based on the phenotype, genetic interactions and subcellular localization of Zfrp8, it is proposed that the protein is involved in the regulation of cell proliferation from embryonic stages onward, through the function of the centrosome, and regulates the level and localization of cell-cycle components. The overproliferation of cells in the lymph gland results in abnormal hemocyte differentiation (Minakhina, 2007).
The developmental mechanisms of human and Drosophila blood systems show remarkable parallels. In humans, several blood cell types with specific functions develop from the same pluripotent stem cells. In Drosophila, only a few specialized cell types exist, with functions similar to human cells. These are thought to originate from a common set of hematopoietic precursors. The development and specification of blood cells in humans and flies are controlled by conserved signaling pathways. Because of its relative simplicity, hematopoiesis in Drosophila is frequently used as a model to investigate the genetic control of hematopoiesis in flies and humans (Minakhina, 2007).
In Drosophila, mature hemocytes arise from two distinct sources: the mature larval circulating hemocytes derive from the embryonic head mesoderm, whereas the lymph gland hemocytes are normally released into circulation at the onset of metamorphosis and perdure into the adult stage. As in vertebrate blood and vascular systems, the Drosophila lymph gland hemocytes and heart cells derive from a common progenitor, called the hemangioblast or cardiogenic mesoderm, which further splits into the lymph gland and cardiogenic progenitors (Mandal, 2004; Minakhina, 2007).
Among the earliest requirements for the specification of blood progenitors in mammals and Drosophila are the highly conserved, GATA zinc-finger transcription factors. The Drosophila GATA-factor Pannier (Pnr) is required for early specification of the hemangioblast/cardiogenic mesoderm. Another GATA-factor, Serpent (Srp), plays a central role in committing mesodermal precursors to the hemocyte fate (Minakhina, 2007).
By the end of embryogenesis, the lymph gland is fully formed and contains mostly pro-hemocytes. The third instar larval lymph gland contains a pair of primary and several secondary lobes. Each primary lobe is subdivided into (1) the medullary zone, populated by slowly proliferating pro-hemocytes; (2) the cortical zone, containing differentiated hemocytes; and (3) the posterior signaling center (PSC), first defined as a small group of cells expressing the Notch ligand Serrate (Ser). Under the control of the EBF-homolog (early B-cell factor) collier (col; knot), PSCs function as a hematopoietic niche to maintain a population of blood cell precursors. The blood cell precursors differentiate into three groups of hemocytes: plasmatocytes, crystal cells and lamellocytes. All three are released into the open circulating hemolymph during the onset of metamorphosis or as a part of an immune reaction. Differentiated plasmatocytes and crystal cells are found in both the cortical zone of the lymph gland and the larval hemolymph, but lamellocytes are rare (Minakhina, 2007).
Plasmatocytes, the predominant form of hemocytes in larvae, perform phagocytic functions and secrete extracellular matrix components and immune peptides similar to human white blood cells. Crystal cells are non-adhesive hemocytes responsible for melanization during wound healing and encapsulation of parasites. Crystal cell differentiation requires the cell-autonomous expression of the transcription factor Lozenge (Lz), homologous to the mammalian acute myeloid leukemia 1 protein (Aml1 or Runx1) (Minakhina, 2007).
Lamellocytes function in encapsulation. Their number is significantly increased at the onset of metamorphosis and in response to infection. Differentiation of lamellocytes is connected to two major pathways - the Drosophila Toll/NF-kappaB and the JAK/STAT - that regulate blood cells proliferation and activation during immune response. Constitutive activation of either pathway leads to overproliferation of circulating and lymph gland hemocytes, an increase in lamellocytes and activation of the cellular immune response (Minakhina, 2007).
A newly identified gene, Zfrp8, is essential for lymph gland growth and for the normal development of Drosophila larvae. Mutant larvae show hyperplasia of the hematopoietic organs. This phenotype is not linked to apoptosis but rather to an increase in cell proliferation. Mutant lymph glands also show a drastic increase in the number of lamellocytes (Minakhina, 2007).
These phenotypes are suppressed by mutations in the GATA factor gene pnr. Mutations in the two cell-cycle genes Cdc27 and l(1)dd4 [lethal (1) discs degenerate 4], have the opposite effect as they enhance the lymph gland overgrowth phenotype of Zfrp8/+. Cdc27 encodes a subunit of the APC complex, responsible for the turnover of cyclins, and l(1)dd4 encodes Dgrip91, a component of the centrosome involved in γ-Tubulin anchoring. In the Zfrp8 mutant lymph gland cells, both Cyclin B (CycB) and γ-Tubulin exhibit abnormal subcellular distribution, suggesting that Zfrp8 plays an important role in their regulation (Minakhina, 2007).
In the literature, the Zfrp8 vertebrate ortholog, PDCD2, is routinely referred to as an apoptotic gene solely because it was upregulated during steroid-induced programmed cell death in rat thymocytes. Subsequent studies, using different cells and assay conditions, found no connection between PDCD2 expression and programmed cell death (Minakhina, 2007 and references therein).
It is unlikely that a reduction in cell death is the cause of the lymph gland overgrowth observed in Zfrp8 mutant larvae. Very few or no apoptotic cells are detected in wild-type larval lymph glands. This study found a statistically insignificant increase in the number of apoptotic cells in Zfrp8 mutants. No other evidence of change in programmed cell death in Zfrp8 mutant animals, no increase in apoptotic gene expression, no change in caspase cleavage and no genetic interaction of Zfrp8 with known apoptotic genes were found (Minakhina, 2007).
The results are consistent with an increase in cell division in Zfrp8 mutants throughout development. This conclusion is supported by the observation that Zfrp8 lymph glands are already twice the size of their normal counterparts in late-stage embryos, and that the number of cells in mitosis is about 30% higher in the mutant glands than in wild type (Minakhina, 2007).
Detailed analysis of Zfrp8 lymph glands shows that its phenotype is different from that of Drosophila hematopoietic/immunity mutants. Unlike hematopoietic/immunity mutants, the increase in lymph gland cell numbers is much larger than the increase in circulating hemocytes. Furthermore, the blood cell overproliferation in Zfrp8-null mutants is not accompanied by constitutive activation of immunity. Zfrp8 larvae show normal induction of immune peptide genes in response to bacterial challenge and normal wound clogging and wound melanization. That the requirements are different for Zfrp8 and known hematopoiesis and immunity genes is underlined by the absence of their genetic interaction (Minakhina, 2007).
In normal lymph glands, plasmatocytes are found mostly in the cortical region and very few lamellocytes are detected. The PSC is formed at the base of each primary lobe. The presence of additional PSCs in mutant lymph glands might indicate that additional primary lobes are formed by the large number of cells (Minakhina, 2007).
PSCs are essential for maintaining the undifferentiated hemocyte population in the medullary zone and that they control lamellocyte differentiation during parasitic infection. Lack of the transcription factor collier, essential for PSC maintenance, leads to a decrease in the pro-hemocyte population and abolishes lamellocyte differentiation. Loss of Zfrp8 leads to the opposite phenotype - an increase in pro-hemocyte proliferation, beginning during embryogenesis, and an increased number of cells acquiring the lamellocyte fate. Expansion of the PSCs alone does not account for this phenotype. Ectopic expression of the homeotic gene Antennapedia results in expansion of the PSCs, and a concomitant increase of the medullar zone, but not the gland overgrowth. Therefore, it is unlikely that Zfrp8 is directly involved in the establishment of PSCs (Minakhina, 2007).
The results link the Zfrp8 overgrowth phenotype to a defect in normal cell proliferation. In mutant lymph glands, the cell-cycle markers γ-Tubulin and CycB are misregulated. Zfrp8 genetically interacts with at least two genes functioning in the cell cycle, Cdc27 encoding a subunit of the anaphase-promoting complex (APC), and l(1)dd4 encoding the Drosophila gamma-ring protein Dgrip91 (Minakhina, 2007).
Dgrip91 and γ-Tubulin are components of the γ-TuRC microtubule-nucleating complex anchored to centrosomes. Beyond the conventional role in microtubule organization, centrosomes also serve as a scaffold for anchoring a number of cell-cycle regulators. For instance, centrosome-association of Cdc27 and CycB proteins plays an important role in CycB activation, degradation and entrance into mitosis (Minakhina, 2007).
The link between the phenotypes described above and Zfrp8 function became clear when it was discovered that a proportion of Zfrp8 protein localizes adjacent to the centrosome in wild-type tissue. This subcellular localization is consistent with a function of Zfrp8 in centrosome organization and in the anchoring of proteins such as γ-Tubulin and CycB to this organelle (Minakhina, 2007).
Zfrp8 might also affect the expression of bona fide cell-cycle regulators. The protein contains a zinc-finger domain, MYND, present in a number of transcriptional regulators, that fosters protein-protein interactions and recruits co-repressors. PDCD2/Zfrp8 is known to interact with the HCF-1 transcriptional regulator, which suggests that PDCD2/Zfrp8 might be involved in regulating the cell cycle at the transcriptional level (Minakhina, 2007).
Zfrp8 might have a dual function, through its association with the centrosome and as a transcriptional regulator of the cell cycle. Several transcriptional regulators have been found to localize to the centrosome, but their centrosomal function has not been documented (Minakhina, 2007).
Zfrp8 function is essential for the control of cell proliferation already in the embryo. With this being the case, it functions upstream from most of the conserved signaling pathways involved in fly hematopoiesis and immunity. Because of the similarity of the protein in flies and vertebrates, it is possible that PDCD2 has a similar function in vertebrate hematopoiesis (Minakhina, 2007).
This study showed that RNA helicase Belle (DDX3) was required intrinsically for mitotic progression and survival of germline stem cells (GSCs) and spermatogonial cells in the Drosophila melanogaster testes. Deficiency of Belle in the male germline resulted in a strong germ cell loss phenotype. Early germ cells are lost through cell death, whereas somatic hub and cyst cell populations are maintained. The observed phenotype is related to that of the human Sertoli Cell-Only Syndrome caused by the loss of DBY (DDX3) expression in the human testes and results in a complete lack of germ cells with preservation of somatic Sertoli cells. This study found the hallmarks of mitotic G2 delay in early germ cells of the larval testes of bel mutants. Both mitotic cyclins, A and B, are markedly reduced in the gonads of bel mutants. Transcription levels of cycB and cycA decrease significantly in the testes of hypomorph bel mutants. Overexpression of Cyclin B in the germline partially rescues germ cell survival, mitotic progression and fertility in the bel-RNAi knockdown testes. Taken together, these results suggest that a role of Belle in GSC maintenance and regulation of early germ cell divisions is associated with the expression control of mitotic cyclins (Kotov, 2016).
This study shows that RNA helicase Belle (DDX3) is required cell-autonomously for the survival and divisions of GSCs in Drosophila testes. In bel6/neo30 mutants as well as in the case of germline-specific RNAi belKD rapid elimination of germ cells via apoptosis occurred. But what events could trigger apoptosis? To address this issue, larval testes were analyzed. Testes of bel6/neo30 mutant larvae still contained all populations of early germ cells. This observation indicates that primordial germ cells (PGCs) correctly migrate into embryonic gonads during mid to late embryogenesis. In the mutant larval testes the wild-type hub and GSCs adjacent to the hub were clearly detected. It is known that the mechanism of capturing GSCs and CySCs to the hub involves a high level of adhesion molecule E-Cad on the hub/stem cells interface. In testes with STAT depletion the expression of E-Cad is severely disrupted accounting for the defects in hub-GSC adhesion and for the subsequent loss of GSCs. However, it was determined that STAT expression (in CySCs) and consequently upstream Upd signaling from the hub were not disrupted in the bel6/neo30 testes. Although the amount of Belle was strongly reduced in the bel6/neo30 testes, no reduction of E-Cad level was observed in CySCs. On the contrary, high ectopic expression of E-Cad was detected on the surface of CySCs surrounding the hub. The influence of Belle on the adherens junction formation in GSCs cannot be directly estimated. However, due to adhesion failures a loss of GSCs via premature differentiation could be expected followed by normal development of newly formed germline cysts. In contrast, this study detected a reduced germ cell content and morphological abnormalities of early germ cells including their giant nuclear and cellular sizes. It is assumed that these germ cells could not enter mitosis and are delayed in the G2 phase. Failure to enter mitosis after G2 delay appears to induce germ cell apoptosis in the bel testes, as previously has been shown for the how testes (Kotov, 2016).
It is known that Drosophila mitotic cyclins, Cyclin A, Cyclin B and Cyclin B3, each form complexes with Cdc2, and they appear to function synergistically to provide a progression throughout mitosis. Sufficient levels of mitotic cyclins must be accumulated at the end of G2 to ensure the onset of mitosis. To date, cell cycle regulation of GSCs and their daughter gonial cells is still poorly understood. It is known that PGCs suppress mitotic activity during their migration to embryonic gonads due to translational repression of maternal cycB mRNA via its 3′UTR by Pumilio–Nanos complex and other unidentified factors. Pumilio and Nanos are also known to be expressed in GSCs of gonads of adult flies and are found to be essential for GSC maintenance. However, factors overriding the repressive Pumilio-Nanos-dependent signal and providing expression of zygotic Cyclin B protein during normal testis development are currently unknown (Kotov, 2016).
It has been shown that Cyclin B and Cyclin A, but not Cyclin B3, are required in the gonad for the maintenance of early germ cells. A mutational depletion of Cyclin B leads to a complete missing of germ cells in the adult testes and their significant loss in the ovaries. However, the requirements for Cyclin A expression for the survival of early germ cells are currently obscure. It is known that overexpression of Cyclin A or expression of its nondegradable form leads to a rapid loss of GSCs in the ovaries (Kotov, 2016).
This study found that the previously published cycB testis phenotype mimicked that in the case of bel6/neo30 mutants and germline-specific RNAi belKD. It was determined that both of the mitotic cyclins, but not Cyclin E and Cdc2, were significantly decreased in the belEY08943/neo30mutant testes. Furthermore, a considerable decrease was found of cycA and cycB mRNA levels. These results suggest a specific contribution of Belle to the transcriptional regulation of mitotic cyclins in the germline. It was also revealed that the constitutive level of Cyclin B expression in control testes was significantly lower than in control ovaries. Assuming that Belle regulates mitotic cyclins in a similar way in the germline of both sexes, it is believed that deficiency of Belle has a more severe effect on spermatogenesis, due to a sharply reduced level of Cyclin B protein below a threshold, whereas its dose in the bel6/neo30 ovaries is still sufficient to allow mitosis to occur. In support of this hypothesis a partial rescue was achieved of the RNAi belKD testis phenotype by transgenic germline-specific expression of Cyclin B, but not by Cyclin A overexpression (Kotov, 2016).
The reduction of cycB transcription in belEY08943/neo30 testes places cycB downstream of bel. In rescue experiments a third copy of cycB was added to the system employing the germinal nos-Gal4 driver in combination with UAS-bel RNAi hairpin. It is assumed that the reduction of Belle in RNAi belKD testes would negatively affect the expression of both endogenous and transgenic cycB. In accordance with this assumption only a partial restoration of the Cyclin B protein level and only partial rescue of spermatogenesis was achieved. The data indicate that at least one crucial requirement for Belle in early germ cells is relevant to Cyclin B level maintenance for ensuring germ cell mitosis (Kotov, 2016).
To date, evidences of participation of DDX3 proteins in cell cycle control both at the level of transcription and translations have been obtained. DDX3 specifically cooperates with transcription factor Sp1 to positively regulate the transcription of p21waf gene. A temperature-sensitive mutation of ddx3 gene in golden hamster cell culture at nonpermissive temperature leads to G1 arrest, which is accompanied by a decline of cycA mRNA and rather suggests the transcriptional level of regulation for cycA. It has been shown that in human HeLa cells DDX3 interacts with the GC-rich, highly structured 5'UTR of Cyclin E1 mRNA and regulates its translation initiation and a knockdown of DDX3 delays the entry to the S phase. DED1, a Schizosaccharomyces pombe homolog of DDX3, is involved in the translational control of B-type cyclin mRNAs (Cig2 and Cdc13), which have extended and expectedly highly structured 5'UTRs. It is known that only a single cyclin-dependent kinase and two B-type cyclins regulate both the S phase and mitosis in yeasts. Indeed, temperature-sensitive mutations of ded1 gene inhibit B-type cyclin translation and arrest cell cycle at both S phase and G2/M transition, whereas both cig2 and cdc13 mRNA levels remain unchanged (Kotov, 2016 and references therein).
This study has presented experimental evidence that Belle has specific and essential functions in the male germline associated with proper transcriptional regulation of mitotic cyclin expression. The testis phenotype observed in Drosophila is similar to the SCOS phenotype in human testes, indicating a conserved function of DDX3 in spermatogenesis. Understanding the molecular basis for DBY (DDX3) functions in mammalian germ cell maintenance has proven to be challenging. The functions and regulation of A-type and B-type cyclins in mammalian spermatogenesis are not clearly understood. In this case, a study in the Drosophila model provides a useful insight into the mechanism of GSC maintenance in the male germline. The current findings support a mechanism according to which the determination of the fate of male GSCs is closely connected with the control of mitosis via the regulation of mitotic cyclin levels (Kotov, 2016).
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).
CP190 is a microtubule-associated Drosophila protein, localized to the centrosome. One protein that interacts with CP190 is a 60-kDa protein named CP60. Like CP190, CP60 interacts with microtubules (specifically gammaTubulin) and is localized to the centrosome. The two proteins associate as part of a multiprotein complex. The amino acid sequence of CP60 contains six consensus sites for phosphorylation by cyclin-dependent kinases. CP60 is localized to the centrosome in a cell cycle-dependent manner. The amount of CP60 at the centrosome is maximal during anaphase and telophase, and then drops dramatically during late telophase-early interphase. This dramatic disappearance of CP60 may be due to specific proteolysis, because CP60 contains a sequence of amino acids similar to the "destruction box" that targets cyclins for proteolysis at the end of mitosis. Starting with nuclear cycle 12, CP60 and CP190 are both found in the nucleus during interphase. CP60 isolated from Drosophila embryos is highly phosphorylated; dephosphorylated CP60 is a good substrate for cyclin B/p34cdc2 kinase complexes. A second kinase activity capable of phosphorylating CP60 is present in the CP60/CP190 multiprotein complex. Bacterially expressed CP60 binds to purified microtubules, however this binding is blocked by CP60 phosphorylation (Kellogg, 1995c).
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. Mutations in fizzy , a Drosophila cell cycle gene, 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. This suggests 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).
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).
fizzy encodes a protein of 526 amino acids, the carboxy half of which has significant homology to the Saccharomyces 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; while in mitosis FZY becomes ubiquitously 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. fzy function is required for normal cell cycle-regulated proteolysis, a necessary step for successful progress through mitosis (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).
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 mitotic cyclins is required for certain mitotic events like DNA condensation. Rca1 is a nuclear protein and could be required to prevent degradation of mitotic cyclins, specifically in the nucleus. Another possibility is that Rca1 sequesters parts of the degradation machinery in the nucleus away from the bulk of mitotic cyclins present in the cytoplasm (Grosskortenhaus, 2002).
In rca1 mutant embryos, residual levels of cytoplasmic CycA and CycB are visible. Supplying additional CycA (but not CycB) is sufficient to rescue the mitotic failure of rca1 mutants. This demonstrates that CycA is the crucial mitotic factor missing in rca1 mutant embryos (Grosskortenhaus, 2002).
The behaviour of a cyclin B-green fluorescent protein (GFP) fusion protein has been studied in living Drosophila embryos in order to study how
the localization and destruction of cyclin B is regulated in space and time. The fusion protein accumulates at centrosomes in interphase,
in the nucleus in prophase, on the mitotic spindle in prometaphase and on the microtubules that overlap in the middle of the spindle in metaphase. In
cellularized embryos, toward the end of metaphase, the spindle-associated cyclin B-GFP disappears from the spindle in a wave that starts at the
spindle poles and spreads to the spindle equator; when the cyclin B-GFP on the spindle is almost undetectable, the chromosomes enter anaphase,
and any remaining cytoplasmic cyclin B-GFP then disappears over the next few minutes. The endogenous cyclin B protein appears to behave in a
similar manner. These findings suggest that the inactivation of cyclin B is regulated spatially in Drosophila cells. The
anaphase-promoting complex/cyclosome (APC/C) specifically interacts with microtubules in embryo extracts, but it is not confined to the spindle in
mitosis, suggesting that the spatially regulated disappearance of cyclin B may reflect the spatially regulated activation of the APC/C (Huang, 1999).
To study
the distribution of the APC/C in Drosophila embryos, a cDNA that encodes the
Drosophila homolog of the APC/C component cdc16 (Dmcdc16) was cloned. Antibodies against the Dmcdc16 protein were prepared, as well as against the
Drosophila homolog of the APC/C component cdc27 (Dmcdc27). Antibodies against two regions of the Dmcdc27 protein, and a single region of
the Dmcdc16 protein were examined. In Western blots of embryo extracts, both of the antibodies raised against the
Dmcdc27 protein recognize a prominent band of ~100 kDa, which is close to the predicted size (102 kDa) of the Dmcdc27 protein, as well as a number of other bands. Only the 100 kDa protein is co-precipitated with the anti-Dmcdc16 antibodies. The anti-Dmcdc16 antibodies recognize a prominent band of ~80 kDa, which is close to the predicted size (82 kDa) of the
Dmcdc16 protein, as well as a band of ~58 kDa. Only the ~80 kDa protein is
co-precipitated with anti-Dmcdc27 antibodies. In sucrose gradient sedimentation experiments, fractions of the Dmcdc16 and Dmcdc27 proteins appeared to co-migrate as a large complex (>19S), although a large fraction of the Dmcdc16 protein behaves as a much smaller protein. These results suggest that Dmcdc16 and Dmcdc27 are both components of the same higher molecular weight complex, presumably the
Drosophila APC/C. These proteins interact biochemically with microtubules in early embryo extracts. The majority of the
Dmcdc27, and a substantial fraction of Dmcdc16, interact with microtubules. The majority of cyclin B also interacts with microtubules in this type of experiment. The interaction of
these proteins with microtubules appeared to be specific, as none of them associated with actin filaments polymerized in similar extracts. The anti-Dmcdc16 and anti-Dmcdc27 antibodies were then used to stain syncytial Drosophila embryos, however, they only weakly label centrosomes and microtubules in methanol-fixed embryos. A weak punctate staining is also visible in the chromosomal regions during mitosis, but the bulk of the Dmcdc16 and Dmcdc27 appears to be distributed in a punctate fashion throughout the cytoplasm at all stages of the cell cycle. Thus, the APC/C appears to be located at multiple sites within the embryo (Huang, 1999).
How might the activation of the APC/C to degrade cyclin B be regulated spatially? Recent
experiments suggest that the temporal control of APC/C activation toward specific substrates depends
on its association with members of the Fizzy (Fzy)/CDC20 and Fizzy-related (Fzr)/CDH1 family of
proteins. In Drosophila, for example, fzy is
required for the destruction of Cyclin A, Cyclin B and Cyclin B3 at around metaphase/anaphase,
whereas Fzr is required for the destruction of these proteins in late mitosis/G1. Perhaps the spatial regulation of cyclin B destruction is also regulated by the association of the
APC/C with members of this family of proteins: APC/C-Fzy complexes might accumulate on the
spindle and ubiquitinate the spindle-associated cyclin B toward the end of metaphase, for example, while APC/C-Fzr complexes might remain in the cytoplasm and ubiquitinate the cytoplasmic cyclin B later in mitosis. In syncytial embryos, Fzr might not be present or might be inactivate, explaining why only the spindle-associated cyclin B is degraded. Although Fzy and Fzr have not been seen associated with specific organelles in Drosophila embryos, a fraction of the p55CDC protein, a Fzy/CDC20 family member, appears to be located on centrosomes and spindles in mammalian cells (Huang, 1999 and references).
If this interpretation that the disappearance of cyclin B-GFP directly reflects its degradation is correct,
then the results suggest that a substantial fraction of cyclin B is being degraded while the cell is still in
metaphase. This contradicts the prevailing idea that cyclin B is degraded abruptly at the
metaphase-anaphase transition. However, recent experiments studying the disappearance of cyclin B-GFP fusion
proteins in mammalian cells (P. Clute and J .Pines, personal communication to Huang, 1999) and in S.pombe
(M.Yanagida, personal communication to Huang, 1999) have also concluded that a substantial fraction of the
cyclin B-GFP disappears from cells while they are in metaphase, and this disappearance also appears
to be regulated spatially in these systems. While the spatially regulated activation of the APC/C is an attractive way to explain the spatially
regulated disappearance of cyclin B, there are other possible explanations. The degradation of cyclin B,
for example, could be initiated in the cytoplasm, but the release of cyclin B from the spindle might
initially maintain a relatively constant level of the protein in the cytoplasm. This explanation, however,
would not easily account for the partial degradation of cyclin B that is observed in syncytial embryos.
Another possibility is that the APC/C is activated globally in the cell to target cyclin B for destruction,
but it is concentrated on the spindle in metaphase and then moves into the cytoplasm during the latter
stages of mitosis. This explanation is not consistent with observations as to the localization of
Dmcdc16 and Dmcdc27 in fixed embryos, but this localization may not reflect accurately the
distribution of these proteins in living cells. It is also possible that the targeting of cyclin B for
degradation and the degradation itself may be separable events: the protein may be polyubiquitinated by
the APC/C on the spindle, for example, but it may have to leave the spindle to be degraded by the 26S
proteosome. More experiments will be required to analyse how the spatially regulated disappearance of
cyclin B is related to its polyubiquitination and ultimate degradation. Moreover, it has
been shown recently that cyclin B is not essential for mitosis in cellularized embryos if cyclin B3 is
present (Jacobs, 1998). Cyclin B3 appears to be a nuclear protein; it will be interesting to
investigate how cyclin B3 compensates for the loss of cyclin B (Huang, 1999).
Proteolysis of mitotic regulators like securins and cyclins requires Fizzy(Fzy)/Cdc20 and Fizzy-related(Fzr)/Hct1/Cdh1 proteins. Budding yeast Cdh1 acts not only during G1, but is also required for B-type cyclin degradation during exit from mitosis when Cdh1 is a target of the mitotic exit network controlling progression through late mitosis and cytokinesis. In contrast, observations in frog and Drosophila embryos have suggested that the orthologous Fzr is not involved during exit from mitosis. However, the potential involvement of minor amounts of maternally derived Fzr was not excluded in these studies. Similarly, the reported absence of severe mitotic defects in chicken Cdh1-/- cells might be explained by the recent identification of multiple Cdh1 genes. This study carefully analyzed the Fzr requirement during exit from mitosis in Drosophila, which, apart from fzr, has only one additional homolog. This fzr2 gene, although expressed in the male germline, is not expressed during mitotic divisions. Moreover, by characterizing fzr alleles, it has been demonstrated that completion of mitosis including Cyclin B degradation does not require Fzr. However, fzr is an essential gene corresponding to the rap locus, and Fzr, which accumulates predominantly in the cytoplasm, is clearly required during G1 (Jacobs, 2002).
To evaluate whether fzr2 is a functional fzy/fzr family member, an analysis was performed to see whether its expression prevents fzy or fzr mutant phenotypes during Drosophila embryogenesis. A UAS-fzr2 transgene in combination with prd-GAL4 results in expression within alternating epidermal segments. While UAS-fzr2 expression does not prevent the characteristic metaphase arrest observed in fzy mutants, it clearly suppresses the fzr mutant phenotype. A lack of zygotic fzr function results in a failure to degrade the mitotic Cyclins A, B, and B3 during the first G1 phase occurring after mitosis 16 in the epidermis. After mitosis 16, therefore, Cyclin B is readily detectable by immunofluorescence in all epidermal cells of fzr mutant embryos, while, in fzr+ sibling embryos, Cyclin B is only observed in the nervous system, but not in the epidermis. Within the UAS-fzr2-expressing regions of fzr mutant embryos, ectopic reaccumulation of Cyclin B after mitosis 16 does not occur, demonstrating that Fzr2 can provide Fzr function. However, Fzr2 might be less active than Fzr, because prd-GAL4-mediated UAS-fzr expression already induces premature degradation of mitotic cyclins before mitosis 16, while analogous UAS-fzr2 expression had little effect before mitosis 16 (Jacobs, 2002).
The conclusion that the fraction of epidermal cells reaccumulating Cyclin B after mitosis 16 is sensitive to the extent of fzr function was further confirmed by RNA interference experiments. Injection of fzr dsRNA into early syncytial embryos at high concentration results in Cyclin B reaccumulation throughout the entire epidermis in half of the injected embryos after the terminal mitosis 16 and phenocopies fzr null mutants. A decrease in the injected dsRNA concentration is accompanied by an increase in the fraction of embryos displaying only a partial, graded fzr phenocopy. Epidermal regions in which all cells were Cyclin B-positive were always observed near the posterior injection site in these embryos. In contrast, far from the injection site, i.e., in the anterior epidermis, Cyclin B-positive cells were absent. Transition zones between anterior and posterior regions, usually 1–3 segments wide, were characterized by a mosaic of Cyclin B-negative and -positive cells (Jacobs, 2002).
Because of the correlation between the level of fzr function and the fraction of Cyclin B-positive epidermal cells after mitosis 16, a reduction of the maternal fzr contribution would be expected to increase this Cyclin B-positive fraction in hypomorphic fzr mutant embryos, if functionally relevant amounts of maternally derived Fzr protein perdure until after mitosis 16. In contrast, if the maternally derived Fzr protein does not perdure long enough to assist in the degradation of mitotic cyclins after mitosis 16, the maternal status of fzr function should be irrelevant for the severity of hypomorphic mutant phenotypes. By counting the number of Cyclin B-positive cells after mitosis 16 in embryos hemizygous for hypomorphic fzr alleles (rape2, rape4, or rape6) derived from mothers with either only a mutant fzr copy (rape2, rape4, or rape6) or with a fzr+ copy in addition to the mutant fzr copy, no effect of the maternal fzr genotype was observed. This result strongly argues that functionally effective levels of maternally derived Fzr protein do not perdure until after mitosis 16. The additional mitosis 17 that occurs in embryos hemizygous for fzr null alleles therefore cannot be supported by maternally derived Fzr protein. Because this additional mitosis 17 is completed normally, it is concluded that exit from mitosis, including degradation of B-type cyclins, is not dependent on Fzr function (Jacobs, 2002).
Analysis of mutant embryos lacking zygotic expression of both fzy and fzr has demonstrated that Fzy is required for exit from the additional mitosis 17 including mitotic cyclin degradation. Since no other fzy/fzr family member is expressed during the additional mitosis 17 in fzr mutants, it is concluded that Fzy alone is sufficient for the completion of mitosis. Sequential APC/C activation first by Fzy, promoting securin degradation and thereby the metaphase-to-anaphase transition, followed by Fzr, allowing B-type cyclin degradation, telophase, and cytokinesis, as originally proposed in budding yeast, therefore, is not obligatory for higher eukaryote mitosis. In addition, these results confirm that Fzr is essential during the G1 phase for preventing unscheduled accumulation of mitotic cyclins (Jacobs, 2002).
Protein phosphatase 2A opposes the phosphorylation activity of cyclin B associated cdc2. The 55 kDa regulatory subunit of Drosophila protein phosphatase 2A is located in the cytoplasm at all cell cycle stages. However, cell cycle function of the enzyme is suggested by the mitotic defects exhibited by two Drosophila mutants. The reduced levels of the 55 kDa subunit correlate with the loss in brain extracts of protein phosphatase 2A-like, okadaic acid-sensitive phosphatase activity against caldesmon and histone H1 phosphorylated by p34cdc2/cyclin B kinase. Thus the mitotic defects of the mutants are likely to result from the lack of dephosphorylation of specific substrates by protein phosphatase 2A (Mayer-Jaekel, 1994).
In the endo cell cycle, rounds of DNA replication occur in the absence of mitosis, giving rise to polyploid or polytene cells. The Drosophila morula gene is essential to maintain the absence of mitosis during the endo cycle. During oogenesis in wild-type Drosophila, nurse cells become polyploid and do not contain Cyclin B protein. Nurse cells in female-sterile alleles of morula begin to become polyploid but revert to a mitotic-like state, condensing the chromosomes and forming spindles. In strong, larval lethal alleles of morula, the polytene ring gland cells also inappropriately regress into mitosis and form spindles. In addition to its role in the endo cycle, morula function is necessary for dividing cells to exit mitosis. In different morula mutants, embryonic S-M cycles and the archetypal (G1-S-G2-M) cell cycle are both arrested in metaphase. These phenotypes suggest that morula acts to block mitosis-promoting activity in both the endo cycle and at the metaphase/anaphase transition of the mitotic cycle. Consistent with this, Cyclin B protein is inappropriately present in morula mutant nurse cells. Thus morula serves a dual function as a cell cycle regulator that promotes exit from mitosis and maintains the absence of mitosis during the endo cycle, possibly by activating the cyclin destruction machinery (Reed, 1997).
The disappearance of cyclin B at the end of mitosis in early Drosophila
embryos starts at centrosomes and spreads into the spindle. A novel mutation, centrosome fall off (cfo), has been used to investigate whether centrosomes are required to initiate the disappearance of cyclin B from the spindle. In embryos laid by homozygous cfo mutant mothers, the centrosomes co-ordinately detach from the mitotic spindle during mitosis, and the centrosomeless spindles arrest at anaphase. Cyclin B levels decrease on the detached centrosomes, but not on the arrested centrosomeless spindles, presumably explaining why the spindles arrest in anaphase in these embryos. The expression of a non-degradable cyclin B in embryos also causes an anaphase arrest, but most centrosomes remain attached to the arrested spindles, and non-degradable cyclin B levels remain high on both the centrosomes and spindles. These findings
suggest that the disappearance of cyclin B from centrosomes and spindles is closely linked to cyclin B's destruction, and that a connection between centrosomes and spindles is required for the proper destruction of the spindle-associated cyclin B in early Drosophila embryos. These results may have important implications for the mechanism of the spindle-assembly checkpoint, since they suggest that unattached kinetochores may arrest cells in mitosis, at least in part, by signaling to centrosomes to
block the initiation of cyclin B destruction (Wakefield, 2000).
In embryos laid by females homozygous for cfo, fewer than 10% of embryos develop normally to the blastoderm stage. The majority (>80%) of the pre-blastoderm embryos arrest at nuclear cycles 1-7 (as judged by the number of spindles in the embryo) in a mitotic-like state, with large barrel-shaped spindles surrounding clusters of mitotic chromatin. Almost all of these spindles are devoid of centrosomes, and free centrosomes are usually randomly distributed throughout the cytoplasm. Most of the chromosomes on these spindles are in an anaphase-like state, with the sister chromatids separated to some extent. This was confirmed by staining cfo embryos with antibodies against either the proliferation disruptor (prod) gene product or the GAGA transcription factor, which label centromeric heterochromatin. Since the majority of the embryos were in this anaphase-like state, it is assumed that this state represents a terminal-arrest stage. The same phenotype is seen in embryos transheterozygous for cfo and Df(3L)Ac1, a deficiency that removes the cfo gene (Wakefield, 2000).
Although most fixed cfo embryos arrest in this anaphase-like state, a small number of pre-blastoderm embryos in interphase or early mitosis look normal. Some pre-blastoderm embryos in anaphase also look normal, but in many of these anaphase embryos the centrosomes appear to be co-ordinately detaching from the mitotic spindle. Since there are no other detectable defects in these embryos, it seems likely that the primary defect is a failure to maintain the connection between centrosomes and spindles during anaphase (Wakefield, 2000).
To observe the cfo defect more directly, a fusion between the tau protein and the green fluorescent protein (tau-GFP) was expressed in cfo embryos and the behavior of microtubules was followed in living embryos using time-lapse video microscopy. In 13 of the 15 embryos followed, the centrosomes were already separated from the spindles by the time the spindles were close enough to the cortex to be seen clearly. In the other two embryos, however, several normal looking metaphase spindles were observed near the cortex. Since these spindles were in different focal planes and in different orientations, only one at a time could be followed in detail. The observations suggest that the detachment of the centrosomes from the spindles at anaphase is the primary defect in cfo embryos. Why centrosome detachment might arrest spindles in mitosis was then investigated (Wakefield, 2000).
Mitotic spindles arrest in an anaphase-like state if the normal degradation of cyclin B is prevented. This arrest looks similar to that seen in cfo embryos, except that no centrosome-detachment phenotype has been reported. To test whether cyclin B is degraded normally in cfo embryos, the embryos were stained with antibodies against cyclin B. Cyclin B is concentrated on mitotic spindles and is particularly enriched at the equator of the spindle, where the interpolar microtubules overlap. Late in metaphase, cyclin B starts to decrease at the spindle poles but remains high in the middle of the spindle. By the time the spindles enter anaphase, however, cyclin B throughout the spindle has fallen to essentially background levels. This can make the normal disappearance of cyclin B from centrosomes and spindles difficult to appreciate. The spatially regulated disappearance of cyclin B at the end of mitosis is most easily seen in movies of living embryos (Wakefield, 2000).
In cfo embryos where the chromosomes are in anaphase and the centrosomes are just detaching from the spindle, cyclin B is at background levels on the detached centrosomes but is still detectable on the spindles. This contrasts with the situation in wild-type embryos in anaphase, and this failure to properly degrade the spindle-associated cyclin B presumably explains why these spindles arrest in mitosis. In terminally arrested cfo embryos, cyclin B reaches abnormally high levels on the centrosomeless spindles, and it also reaccumulates on the detached centrosomes. Thus, cyclin B appears to be stable in the terminally arrested cfo embryos, and it can accumulate on the arrested spindles and centrosomes (Wakefield, 2000).
In principle, the failure to properly degrade cyclin B on the spindle could be the cause of the centrosome-fall-off phenotype. To test this possibility, a non-degradable form of cyclin B was expressed in early embryos using the Gal4 system. Western blotting showed that this protein is expressed in embryos at similar levels to the endogenous cyclin B protein. In 0-2hour collections, most embryos (>80%) were arrested in an anaphase-like state during nuclear cycles 1-7. In contrast to the situation in cfo embryos, most of the centrosomes appeared to remain attached to the arrested spindles. In 2-4hour collections, many embryos appeared to have been arrested in mitosis for some time (as judged by the overall disorganization of the embryo). In these embryos, some of the centrosomes had detached from the arrested spindles. Importantly, however, cyclin-arrested embryos were seen in which the majority of centrosomes were co-ordinately detaching from the spindles, as seen in cfo embryos. Thus, it seems unlikely that the centrosome-fall-off phenotype is simply a consequence of a failure to degrade cyclin B. As noted above, no centrosome-fall-off phenotype has been reported in any of the other systems where mitosis has been arrested by blocking the destruction of cyclin B (Wakefield, 2000).
Cyclin B remains high on both centrosomes and spindles in embryos expressing non-degradable cyclin B. Even in relatively normal looking anaphase spindles, however, the non-degradable cyclin B does not disappear from the spindles or the centrosomes. This suggests that the normal disappearance of cyclin B from the centrosome and spindle at the end of mitosis is closely linked to its destruction (Wakefield, 2000).
Perhaps the simplest interpretation of these findings is that the signal to degrade cyclin B in early Drosophila embryos is normally generated at centrosomes. In cfo mutant embryos, the link between centrosomes and spindles is broken: although cyclin B degradation may initiate on the centrosomes, the spindle-associated cyclin B is not degraded properly and, as a result, the spindles arrest in mitosis. One possibility is that the signal to degrade cyclin B normally travels along microtubules from the centrosome to the rest of the spindle and cannot do so when the centrosomes have detached from the spindle. Another possibility is that a specific cell-cycle checkpoint monitors the connection between the centrosomes and spindle, and blocks cyclin B degradation if the connection is lost. In any case, these results suggest that the centrosome may play a key role in controlling the destruction of cyclin B in the early Drosophila embryo (Wakefield, 2000).
These results could have important implications for the spindle-assembly checkpoint. It has been proposed that an unattached kinetochore might block the exit from mitosis, at least in part, by sending a signal to the centrosome to block the initiation of cyclin B degradation. If this putative signal travels along kinetochore microtubules, it could explain why an unattached kinetochore only blocks the exit from mitosis on the spindle to which it is directly attached. Consistent with this hypothesis, several components of the spindle checkpoint are concentrated at centrosomes, as well as on kinetochores, and one of these checkpoint components, Mad2, appears to travel along kinetochore microtubules to the centrosomes. Thus, signaling between kinetochores and centrosomes may play an important role in regulating the exit from mitosis (Wakefield, 2000).
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).
The maternal RNA-binding proteins Pumilio (Pum) and
Nanos (Nos) accumulate in pole
cells, the germline progenitors. Nos is required for pole
cells to differentiate into functional germline. Pum is also essential for germline development in
embryos. A mutation in pum causes a defect in
pole-cell migration into the gonads. In such pole
cells, the expression of a germline-specific marker (PZ198)
is initiated prematurely. pum mutation causes
premature mitosis in the migrating pole cells.
Pum inhibits pole-cell division by repressing translation of
cyclin B messenger RNA. Because these phenotypes are
indistinguishable from those produced by nos mutation,
it is concluded that Pum acts together with Nos to regulate
these germline-specific events (Asaoka-Taguchi, 1999).
Pole cells formed in embryos lacking Pum (pum embryos) were transplanted into wild-type host embryos. The transplanted pum pole cells pass normally through the midgut epithelium into the haemocoel. However, none of the transplanted pum pole cells are incorporated within the gonads of the hosts, whereas normal pole cells taken from control embryos were observed in the gonads. All of the transplanted pum pole cells remain in the haemocoel and the gut lumen. These results show that Pum is autonomously required in pole cells for their migration into the gonads. The expression of the enhancer-trap marker PZ198 was studied in pum pole cells. PZ198 expression, which is normally initiated in pole cells within the gonads, begins prematurely during pole-cell migration in embryos lacking Nos (nos embryos). Similarly, PZ198 expression begins prematurely, at stage 7, in pum mutant pole cells, as compared with stage 13 in control embryos. Thus Pum is also required to repress the premature expression of the enhancer-trap marker in pole cells. The effects of pum and nos mutations on cell-cycle arrest were studied during pole-cell migration. In normal development, pole cells remain quiescent in G2 phase of the cell cycle during stages 7-15. It is expected that Nos and Pum repress the entry of pole cells into mitosis, because pole cells initiate cell division just after Nos becomes undetectable in pole cells at stage 15. To monitor the cell cycle in pole cells, antibodies against a phosphorylated form of histone H3 (PH3) and cyclin E were used. PH3 is detectable in mitosis but is absent during interphase, whereas cyclin E is expressed specifically in S and G2 phases. The disappearance of cyclin E from pole cells is linked to cell-cycle progression from G2 to G1 phase, whereas cyclin E is not degraded during cell cycling of somatic cells. Consistent with the observation that migrating pole cells in wild-type embryos are arrested in G2 phase, almost all pole cells in stage 7-15 embryos show cyclin E staining, but not PH3 staining. In contrast, in pum and nos embryos, the percentage of pole cells expressing cyclin E gradually decreases during stages 7-15, and PH3-positive pole cells became detectable during these stages. Thus, the mutant pole cells are prematurely released from G2 arrest and enter into mitosis. Taken together, these observations show that Pum and Nos are both required for the repression of the G2/M transition in the migrating pole cells (Asaoka-Taguchi, 1999).
Since pum and nos mutations do not affect posterior localization of maternal cyclin B mRNA or its partitioning into pole cells, it is concluded that translation of Cyclin B mRNA is usually repressed by Pum and Nos in pole cells. Cyclin B mRNA contains an NRE-like sequence in its 3' UTR, called the translation-control element (TCE). Deletion of the TCE from the 3' UTR of an epitope-tagged Cyclin B mRNA results in a phenotype similar to that caused by nos and pum mutations. These observations lead to the conclusion that Pum/Nos-dependent translational repression of cyclin B mRNA is
mediated by the TCE. Given that Pum binds to the NRE in vitro, it is
reasonable to suggest that Pum binds directly to the TCE. This is the first demonstration that maternal factors regulate the translation of specific mRNA in germline progenitors (Asaoka-Taguchi, 1999).
Drosophila Brain tumor is a translational repressor that interacts with Nanos and Pumilio to regulate Hunchback translation. Analysis of mutant phenotypes has revealed that Nos and Pum are
required for a variety of processes in addition to the development of
abdominal segmentation. nos and pum are expressed in
tissues other than the female germ line. More important, nos and pum mutants are subviable, revealing an
(unknown) essential function for each factor in somatic cells. In the germ line, nos
and pum mutants exhibit a number of defects including loss of
germ-line stem cells in both sexes, failure of precursor cells to
migrate into and populate the somatic gonad, and premature
proliferation of precursor cells (pole cells) in the embryo. The
premature proliferation appears to result from the inappropriate
derepression of maternal Cyclin B (CycB) mRNA in the
pole cells; in no other case is the
molecular basis of Nos or Pum action currently understood (Sonoda, 2001 and references therein).
It was thus of interest to determine whether Nos and Pum also act in conjunction with
Brat to regulate maternal Cyclin B mRNA. Using antibodies directed against different regions of the Brat protein, it has been found that Brat is
distributed throughout the syncitial blastoderm stage embryo when
HB mRNA is repressed, and is also present in the cytoplasm of
the pole cells when maternal Cyclin B mRNA is regulated. However, Cyclin B mRNA is
repressed normally in the pole cells of bratfs
mutant embryos, but not in the pole cells of nos mutant
embryos. Thus, Brat does not appear to play a role in repression of Cyclin B, although the possibility that the residual activity of Bratfs1 is sufficient to regulate Cyclin
B but not hb cannot be ruled out (Sonoda, 2001).
The cis-acting signals that mediate Nos- and Pum-dependent
regulation of Cyclin B have not yet been defined precisely.
However, NRE-like sequences are present in the maternal isoform of Cyclin B mRNA, which is regulated. If indeed Pum, Nos,
and NRE-like sequences mediate its regulation, then why would repression of Cyclin B mRNA be Brat independent (Sonoda, 2001)?
To investigate this issue, an examination was made of the binding of Pum, Nos, and
Brat to the Cyclin B NRE-like element in vitro. The RNA used
in these experiments contains 136 nucleotides that include all of the
NRE homologous elements as well as flanking sequences. Pum binds to
this Cyclin B-derived RNA in gel mobility-shift experiments, but not to a derivative bearing mutations in the conserved NRE-like element, consistent with the idea that similar sequences in Cyclin B and HB are
recognized. Bound Pum can recruit Nos into a ternary complex on
Cyclin B RNA, much as it does on the HB NRE. However, the ternary complex assembled on Cyclin B RNA
recruits Brat at least 10-fold less efficiently than the corresponding
complex assembled on the hb NRE. This surprising
observation may in part explain the Brat independence of Cyclin
B regulation. Furthermore, it suggests
that the RNA sequence specifies the geometry of the Pum/Nos complex,
which in turn determines whether Brat is recruited or not (Sonoda, 2001).
The sex determination master switch, Sex-lethal, regulates the mitosis of early germ cells in Drosophila. Sex-lethal is an RNA binding protein that regulates splicing and translation of specific targets in the soma, but the germline targets are
unknown. In an immunoprecipitation experiment aimed at identifying targets of Sex-lethal in early germ cells, the RNA encoded by gutfeeling, the Drosophila homolog of ornithine decarboxylase (ODC) antizyme, was isolated (Vied, 2003).
Mammalian Antizyme negatively regulates ODC catalytically as well by directing the inactivated enzyme to the proteasome for degradation. This negative regulation of ODC is part of a feedback loop that controls the levels of polyamines within the cell. Translation of Antizyme is dependent on ribosomal frameshifting, which is promoted by high levels of polyamines. As polyamine levels in the cell rise, more Antizyme is synthesized, leading to the turnover of ODC. Polyamines have been implicated in many processes, including cell growth, transcription, and differentiation. In mammals Antizyme and ubiquitin are thought to be respectively two types of proteasome targeting devices that mark proteins for both ubiquitin-independent and ubiquitin-dependent degradation by the 26 S proteasome (Vied, 2003 and references therein).
Drosophila gutfeeling interacts genetically with Sex-lethal. It is not only a target of Sex-lethal, but also appears to regulate the nuclear entry and overall levels of Sex-lethal in early germ cells. This regulation of Sex-lethal by gutfeeling appears to occur downstream of the Hedgehog signal. Gutfeeling appears to regulate the nuclear entry of Cyclin B as well. Hedgehog, Gutfeeling, and Sex-lethal function to regulate Cyclin B, providing a link between Sex-lethal and mitosis (Vied, 2003).
In Drosophila, Cyclin B is not essential for viability but
is necessary for female fertility. Since Sxl has been shown to be
important for the mitosis of early germ cells,
a correlation between Sxl and Cyclin B was sought. In early germ cells,
Cyclin B colocalizes with Sxl and is downregulated concomitantly
with Sxl. Since Sxl and Cyclin B colocalize in early germ cells, the effect of Hh on Cyclin B was examined. Overexpression of
Hh results in fewer Cyclin B-expressing early germ cells,
as seen for Sxl. Furthermore, the germ cells that
express Cyclin B show reduced levels of the protein. This
similarity in response prompted a test to see whether Hh also
regulates the nuclear entry of Cyclin B. Overexpression of
Hh and treatment with LMB results in higher levels of
nuclear Cyclin B than in wild-type early germ cells treated
with LMB only. This observation suggests that
Hh promotes the nuclear entry of Cyclin B (Vied, 2003).
Since Guf appears to act downstream of Hh to regulate Sxl,
whether Cyclin B would also respond to Guf was also examined.
Ectopic expression of Guf has
the same effect on Cyclin B as on Sxl. Fewer early germ
cells with cytoplasmic Cyclin B were observed, and Sxl and
Cyclin B continued to colocalize. LMB treatment
with Guf overexpression increases the nuclear levels
of Cyclin B. As for Sxl, Cyclin B responds
more strongly to Guf than Hh (Vied, 2003).
To determine whether Guf also acts downstream of Hh in
affecting Cyclin B nuclear entry, hs-hh; guf118-3/+ ovaries treated with LMB were examined. Under these conditions,
Cyclin B was predominantly cytoplasmic,
suggesting that Guf also functions downstream of Hh in the
regulation of Cyclin B (Vied, 2003).
In vertebrates, the intracellular localization of Cyclin B is
critical to its function. An NES within the cytoplasmic
retention signal (CRS) allows Cyclin B to rapidly shuttle out
of the nucleus. Phosphorylation of the CRS results in nuclear
accumulation of Cyclin B and its associated Cyclin-dependent kinase with which Cyclin B initiates mitosis. Since Sxl and Cyclin B appear to undergo similar changes in response to Hh and
Guf, tests were made to determine whether Cyclin B is affected by changes in
Sxl.
Cyclin B was examined in ovaries with mutant Sxl
protein. Like Sxl, Cyclin B is localized to the cytoplasm
of Sxlf4 germ cells. Treatment of Sxlf4 ovaries with LMB
caused the Sxl, but little Cyclin B, protein to accumulate
in the germ cell nuclei. As overexpression of
Hh with LMB treatment increases the nuclear levels of
both Sxl and Cyclin B in wild-type ovaries,
the intracellular localization of both proteins was examined in Sxlf4
ovaries after similar treatment. The mutant Sxl protein
accumulates in the nuclei, but surprisingly, Cyclin B
remains in the cytoplasm. Consistent with
this observation, ovaries doubly homozygous for Sxlf4
and Su(fu) treated with LMB show nuclear accumulation
of Sxlf4 protein, but not Cyclin B. These
results suggest that, normally, Sxl affects the rate of
nuclear entry of Cyclin B. Interestingly, the mutations in
the Sxlfs alleles alter a region of the protein that is
proline-rich. Proline-rich domains
have been described as being involved in protein-protein
interactions (Vied, 2003).
Sxl can enter the nucleus without the concomitant
entry of Cyclin B. Cyclin B mutant ovaries were examined
to test the assumption that Sxl does not require Cyclin B
for nuclear exiting. A null mutation for cyclin B derived
from the imprecise excision of a P-element in the 5' UTR
(cycB3) is not lethal except when combined with other
cell cycle regulators. The
cycB3 mutation results in female sterility with many agametic
ovarioles. When germ cells were
present, the localization of Sxl was primarily cytoplasmic.
Treatment with LMB shows that Sxl is able to
enter the nucleus at levels comparable to those observed
in wild-type ovaries\. These results suggest
that Sxl does not require Cyclin B for nuclear exiting. Otherwise, Sxl would be primarily nuclear in the absence of Cyclin B (Vied, 2003).
Cyclin A expression is only required for particular cell divisions
during Drosophila embryogenesis. In the epidermis, Cyclin A
is strictly required for progression through mitosis 16 in cells that become
post-mitotic after this division. By contrast, Cyclin A is not
absolutely required in epidermal cells that are developmentally programmed for
continuation of cell cycle progression after mitosis 16. These analyses suggest
the following explanation for the special Cyclin A requirement during
terminal division cycles. Cyclin E is known to be downregulated
during terminal division cycles to allow a timely cell cycle exit after the
final mitosis. Cyclin E is therefore no longer available before terminal
mitoses to prevent premature Fizzy-related/Cdh1 activation. As a consequence,
Cyclin A, which can also function as a negative regulator of
Fizzy-related/Cdh1, becomes essential to provide this inhibition before
terminal mitoses. In the absence of Cyclin A, premature Fizzy-related/Cdh1
activity results in the premature degradation of the Cdk1 activators Cyclin B
and Cyclin B3, and apparently of String/Cdc25 phosphatase as well. Without
these activators, entry into terminal mitoses is not possible. However, entry
into terminal mitoses can be restored by the simultaneous expression of
versions of Cyclin B and Cyclin B3 without destruction boxes, along with a
Cdk1 mutant that escapes inhibitory phosphorylation on T14 and Y15. Moreover,
terminal mitoses are also restored in Cyclin A mutants by either the
elimination of Fizzy-related/Cdh1 function or Cyclin E
overexpression (Reber, 2006).
Mitotic cyclins accumulate during the S and G2 phases of the cell cycle.
Their C-terminal cyclin boxes mediate binding to cyclin-dependent kinase 1
(Cdk1). Their rapid degradation during late M and G1 phase depends on the D-
and KEN-boxes in their N-terminal domains. These degradation signals are
recognized by Fizzy/Cdc20 (Fzy) and Fizzy-related/Cdh1 (Fzr), which recruit
the mitotic cyclins to the anaphase-promoting complex/cyclosome (APC/C) during
M and G1, respectively. The ubiquitin ligase activity of the APC/C allows
cyclin poly-ubiquitination and consequential proteolysis (Reber, 2006).
Metazoan species express three different types of mitotic cyclins: A, B and
B3. The specific functions of these different cyclins are not understood in
detail. The presence of single genes coding for either Cyclin A (CycA), Cyclin
B (CycB) or Cyclin B3 (CycB3) has facilitated a genetic dissection of their
functional specificity in Drosophila melanogaster. In this organism,
development to the adult stage requires the zygotic function of CycA,
but not of CycB or CycB3.
Initial analysis of the embryonic cell proliferation program in CycA
mutants revealed that epidermal cells fail to progress through the sixteenth
round of mitosis. Cyclin A is also required for mitosis 16 in the epidermis
of dup/Cdt1 mutant embryos, in which mitosis 16 is no longer
dependent upon completion of the preceding S phase. The
failure of mitosis 16 in CycA mutants therefore does not simply
result from the activation of a DNA replication or damage checkpoint -- a
possibility suggested by evidence obtained in vertebrate cells in which Cyclin
A binds not only to Cdk1 but also to Cdk2, and provides crucial functions
during S phase (Reber, 2006 and references therein).
The accumulation of Cyclin B and Cyclin B3 during cycle 16, which also
occurs in CycA mutants, complicates the explanation of
why mitosis 16 in the epidermis requires Cyclin A. In Xenopus egg
extracts, Cyclin B can trigger entry into mitosis in the absence of Cyclin A.
Conversely, mitosis is clearly inhibited in cultured human cells after the
microinjection of antibodies against cyclin A. Cyclin
A-Cdk1 complexes are thought to have special properties, important for
starting up a positive-feedback loop that confers a switch-like behavior on
the Cdk1 activation process. In this feedback loop, Cdk1 activity results in
phosphorylation and suppression of the inhibitory Wee1 kinase, as well as in
phosphorylation and activation of the String/Cdc25 phosphatase, which removes
the inhibitory phosphate modifications from Cdk1. However, the analyses described in this study indicate that the Cyclin A requirement in Drosophila is not linked to this positive-feedback loop. Rather, it is linked to the fact that the
sixteenth round of mitosis during embryogenesis is the last cell division for
the great majority of the epidermal cells (Reber, 2006).
After mitosis 16, most epidermal cells enter a G1 phase and become
mitotically quiescent. By contrast, all the previous embryonic divisions (mitoses
1-15) are followed by an immediate onset of S phase. The G1 phase after
mitosis 16 is therefore the first G1 phase during development. Entry into this
G1 phase is dependent upon a complete, developmentally controlled inactivation
of Cyclin E-Cdk2 and Cyclin A-Cdk1, because both complexes can trigger entry
into S phase. Cyclin E-Cdk2 inactivation results from transcriptional
CycE downregulation and concomitant upregulation of dacapo,
which encodes the single Drosophila CIP/KIP-type inhibitor specific
for Cyclin E-Cdk2. Cyclin A-Cdk1 inactivation is dependent on Fzr, which is also transcriptionally upregulated. Moreover, Fzr is activated as a consequence of Cyclin E-Cdk2 inactivation. Importantly, this cell cycle exit program is initiated already during G2 of the final division cycle (Reber, 2006).
Although cycle 16 is the final division cycle for most epidermal cells,
some defined regions do not activate the cell cycle exit program during cycle
16. Instead, they maintain CycE expression, enter S phase immediately
after mitosis 16 and complete an additional division cycle 17. In these
regions, mitosis 16 is not fully inhibited in CycA mutants. Cyclin A
is therefore especially important for terminal mitoses preceding G1 and cell
cycle exit. This study shows that the downregulation of Cyclin E-Cdk2 before terminal divisions, in preparation for the imminent cell cycle exit, converts Cyclin A from a redundant into an indispensable, negative regulator of
Fizzy-related/Cdh1, preventing premature degradation of the mitotic inducers
String/Cdc25 and the mitotic cyclins. The significance of the basic cell cycle
regulator Cyclin A therefore depends on the developmental context (Reber, 2006).
The phenotypical characterization of mutations in the Drosophila genes encoding the A-, B- and B3-type cyclins have indicated that Cyclin A is the most crucial of these co-expressed mitotic cyclins. Although zygotic CycB or CycB3 function is not essential for cell proliferation and development to the adult stage, null mutations in CycA result in embryonic lethality.
This study has clarified the molecular basis of the distinct importance of
Cyclin A. The results indicate that the crucial role of Cyclin A is linked to
its ability to inhibit Fzr-APC/C-mediated degradation. Moreover,
this Cyclin A-dependent negative regulation of the Fzr-APC/C-degradation
pathway is of particular importance for progression through the very last
mitotic division preceding cell cycle exit and the proliferative quiescence of
epidermal cells during embryogenesis. This particular Cyclin A requirement
during terminal divisions is caused by a cell cycle exit program that is
initiated already before the terminal mitosis. The cell cycle exit program
includes downregulation of Cyclin E-Cdk2, which has a comparable ability to
inhibit the Fzr-APC/C-degradation pathway to Cyclin A. The downregulation of
Cyclin E-Cdk2 by the cell cycle exit program turns Cyclin A into an
indispensable inhibitor of the premature degradation of mitotic cyclins and
String/Cdc25 via Fzr-APC/C before the terminal mitosis. Accordingly, the terminal mitosis in the epidermis of CycA mutants can be restored by overexpression of Cyclin E, by genetic elimination of Fzr, or by simultaneous expression of the String/Cdc25-independent Cdk1AF mutant and B-type cyclin versions that are no longer Fzr-APC/C substrates (Reber, 2006).
The fact that Cyclin A is also a substrate of Fzr-APC/C-mediated
degradation complicates the interpretation of the results. Two
findings, however, strongly suggest that Cyclin A functions not just
downstream of Fzr, but also upstream as a negative regulator. The observed
premature loss of B-type cyclins in CycA mutants is readily explained
by a negative effect of Cyclin A on Fzr-APC/C activity and is difficult to
explain if Cyclin A was only a Fzr-APC/C substrate. Moreover, the suppression
of the UAS-fzr overexpression phenotype by co-expression of
UAS-CycA, which is described here, includes the re-accumulation of
B-type cyclins and not just the restoration of terminal mitosis 16 (Reber, 2006).
Work in mammalian cells has clearly established that Cyclin A functions as
a negative regulator of Fzr/Cdh1. Human Cyclin A can bind directly to Cdh1.
Moreover, Cyclin A-dependent Cdk activity phosphorylates Cdh1, resulting in
the dissociation of Cdh1 from APC/C. Conversely, mutations in Cdk consensus phosphorylation sites of human CDH1 were reported to abolish inhibition by Cyclin A. The current findings point to alternative modes of Fzr-APC/C-inhibition by Cyclin A. Fzrpsm variant no longer contains canonical Cdk consensus
phosphorylation sites (S/T P) and yet its activity is still suppressed by
CycA overexpression. Fzr inhibition by CyclinA-dependent
phosphorylation of non-consensus sites remains a possibility in
Drosophila. However, it is pointed out that, apart from a potential
control by Cdk phosphorylation, Fzr is inhibited by the Emi1-like
Drosophila protein Rca1. Rca1 overexpression has been shown to prevent
premature Cyclin B degradation and restore mitosis 16 in the epidermis of
CycA mutant embryos. Based on these observations, the failure of
mitosis 16 in CycA mutants was proposed to reflect premature Fzr
activation, a suggestion fully confirmed by the current work. It is
conceivable, therefore, that the Cyclin A-mediated suppression of
Fzrpsm activity involves Rca1 or other unknown targets. The fact
that not only Cyclin A, but also Cyclin E, effectively suppresses
Drosophila Fzr and Fzrpsm provides further support of
additional regulatory complexity. In vertebrate systems, only Cyclin A and not
Cyclin E was shown to bind and inhibit Cdh1 (Reber, 2006).
The current findings demonstrate that the Cyclin A requirement in epidermal cells is maximal for progression through the last mitosis of Drosophila
embryogenesis, which precedes cell cycle exit and proliferative quiescence. A
prominent Cyclin A requirement for terminal mitoses appears to exist in
neuroblast lineages during development of the embryonic CNS, although
definitive proof will require further work. On the basis of this analysis in
epidermal cells, a high Cyclin A requirement for entry into mitosis is
expected whenever Fzr levels are high and Cyclin E levels low. During the
comparatively slow postembryonic cell cycles of imaginal cells, the
periodicity of Cyclin E expression is presumably far more pronounced than
during the rapid embryonic cycles in which the persistent presence of
maternally contributed Cyclin E eliminates G1 phases. In imaginal cell cycles,
which have a G1 phase, Cyclin E expression might therefore be low before each
mitosis, and not just before terminal divisions. In combination with Fzr
expression, every imaginal mitosis might therefore be strongly dependent upon
Cyclin A. By contrast, in the absence of Fzr, progression through mitosis
appears to be almost completely independent of Cyclin A, as is evidenced by
the observation that the epidermal cells in fzr CycA double mutant
embryos not only progress successfully through mitosis 16, but also complete
an extra division cycle 17. Nevertheless, 10% of the late mitosis 17 figures
in these double mutants displayed lagging chromosomes, indicating that cell
cycle progression is not entirely normal in the absence of Cyclin A (Reber, 2006).
The cell cycle exit program, which is activated during the final division
cycle in the embryonic epidermis, includes the strong transcriptional
upregulation of the CIP/KIP-type Cyclin E-Cdk2 inhibitor Dacapo, apart from
the downregulation of Cyclin E and the upregulation of Fzr.
Accordingly, genetic elimination of dacapo function should also
restore progression through terminal mitosis 16 in CycA mutants.
However, mitosis 16 was not observed in the epidermis of dacapo CycA
double mutants. The contribution of Dacapo to Cyclin E-Cdk2 inhibition appears
to be insignificant before mitosis 16. After the stage of mitosis 16, however,
the epidermal cells in these double mutants entered an endoreduplication
cycle, a behavior that is also displayed by some cells in the prospective
anterior spiracle region of CycA single mutants. This
region does not downregulate Cyclin E during cycle 16 in the wild type, it
does not upregulate Dacapo, and it progresses through an additional cycle 17
instead of becoming postmitotic after mitosis 16, in contrast to the great
majority of the other epidermal cells. The premature activation of Fzr in
CycA mutants, therefore, appears to result in DNA replication origin
re-licensing, perhaps as a result of B-type cyclin and geminin degradation.
Cyclin E-Cdk2 activity might subsequently trigger endoreduplication in cells
in which it is not effectively eliminated by both Cyclin E downregulation and
Dacapo upregulation. Importantly, not all cells in the anterior spiracle
region of CycA mutants endoreduplicate, some of the cells still
manage to divide. This variability could reflect minor differences in the
onset and strength of the zygotic Cyclin E expression. The outcome of
insufficient Cyclin A levels appears to be highly dependent on the levels of
Cyclin E and Fzr, which, in turn, are subject to developmental regulation, in
particular during cell cycle exit. The significance of basic cell cycle
regulators in vivo is therefore different in various tissues and developmental
stages, and most likely in various cultured mammalian cell types as well (Reber, 2006).
Polycomb group (PcG) proteins control development and cell proliferation through chromatin-mediated transcriptional repression. A transcription-independent function is described for PcG protein Posterior sex combs (PSC) in regulating the destruction of cyclin B (CYC-B). A substantial portion of PSC was found outside canonical PcG complexes, instead associated with CYC-B and the anaphase-promoting complex (APC). Cell-based experiments and reconstituted reactions have established that PSC and Lemming (LMG, also called APC11) associate and ubiquitylate CYC-B cooperatively, marking it for proteosomal degradation. Thus, PSC appears to mediate both developmental gene silencing and posttranslational control of mitosis. Direct regulation of cell cycle progression might be a crucial part of the PcG system's function in development and cancer (Mohd-Sarip, 2012).
Polycomb group (PcG) proteins are transcriptional repressors that maintain cell-fate decisions and control cell proliferation. They function as part of distinct multiprotein complexes that modulate chromatin structure. The RING domain protein Posterior sex combs (PSC) is a subunit of Polycomb repressive complex 1 (PRC1) and dRING-associated factors (dRAF), which mediate monoubiquitylation of histone H2A. A substantial portion of PSC is part of neither PRC1 nor dRAF, suggesting that PSC might have additional functions. The effects of depleting either Polycomb (PC), Polyhomeotic (PH), PSC, or dRING were compared by treating S2 cells with the appropriate double-stranded RNAs (dsRNAs). PC, PH, PSC, and dRING form the core of PRC1, whereas dRING, PSC, and dKDM2 are the central subunits of dRAF. Knock-down (KD) of PH or PSC decreased cell accumulation, whereas depletion of PC or dRING had no appreciable effects. Fluorescence-activated cell sorter (FACS) analysis indicated that cells lacking PSC primarily accumulated at the G2-M phase of the cell cycle. Loss of other PcG proteins did not give a clear cell cycle arrest. Therefore, PSC might function in cell cycle regulation, independent of PRC1 or dRAF (Mohd-Sarip, 2012).
Consistent with the G2-M arrest caused by loss of PSC, maternal effect mutations of Psc cause mitotic segregation defects in early Drosophila embryos. This is illustrated by the mitotic chromosome bridges, frequently detected in the progeny of Psch27 mutant mothers. Because early embryos have nonconventional checkpoint mechanisms, problems at either S phase or mitosis can lead to segregation defects. In S2 cells, which have a conventional cell cycle, depletion of PSC caused severe mitotic defects. After depletion of PSC, ~68% of mitotic cells displayed an abnormal phenotype, whereas loss of the other PcG proteins did not affect mitosis. The PcG system has been implicated in the regulation of cell cycle genes. Yet, because the integrity of PcG complexes is required for silencing, it was suspected that PSC's role in mitosis extends beyond transcription repression. Indeed, a portion of cellular PSC does not appear to be part of PRC1 nor dRAF (Mohd-Sarip, 2012).
To identify interaction partners, three distinct affinity-purified antibodies were used to isolate PSC from whole-cell extracts of 0- to 12-hour-old Drosophila embryos. Mass spectrometric analysis revealed that, in addition to PRC1 and dRAF subunits, cyclin B (CYC-B), cell division cycle 2 (CDC2, also called cyclin-dependent protein kinase 1), and key subunits of the anaphase-promoting complex (APC) associate with PSC. Although PSC was present in PC, dRING, and PH purifications, CYC-B and the APC were absent. The APC is a multisubunit E3 ubiquitin ligase that is pivotal to cell cycle regulation. CYC-B ubiquitylation by the APC, marking it for destruction by the proteasome, is required for completion of anaphase and cytokinesis. This study confirmed the selective association of PSC with CYC-B and APC by a series of immunoprecipitations (IPs) combined with protein immunoblotting. IPs of PcG proteins showed that only PSC associates with CYC-B; CDC2; and the APC subunits Morula (Mr, also called APC2), CDC23 (APC8), and Lemming (Lmg, also called APC11). Reverse IPs further established the unique association of PSC with CYC-B and the APC. Lmg is a small 85-amino acid protein, comprising mainly a RING domain, that is essential for the ubiquitin ligase activity of the APC. Many RING domain proteins are E3 ubiquitin ligases and frequently function as homo- or heterodimers. For example, PSC and its mammalian homolog BMI1 bind dRING or RING1B, respectively, and stimulate histone H2A ubiquitylation. This study found that the RING domain of PSC was necessary and sufficient to bind LMG, whereas its C-terminal region bound CYC-B. Thus, PSC appears to associate with Lmg and CYC-B directly (Mohd-Sarip, 2012).
To complement these biochemical experiments with a genetic-interaction assay, the GAL4-UAS system was used in Drosophila.The glass multimer reporter (GMR) was used to drive ectopic CYC-B expression (GMR>CYC-B) in the developing eye. Ectopic CYC-B caused a mild rough-eye phenotype, characterized by disorganized ommatidia and loss of bristles. Concomitant expression of dsRNA directed against Psc mRNA [GMR>CYC-B; GMR>PSCRNAi (RNAi, RNA interference)] enhanced the GMR>CYC-B phenotype, consistent with the notion that PSC is a negative regulator of CYC-B. In contrast, expression of dsRNA directed against Pc had no effect on the CYC-B overexpression phenotype. Alone, neither reduction of PSC levels nor PC depletion had an appreciable effect on eye development. Thus, PSC interacts both genetically and biochemically with CYC-B (Mohd-Sarip, 2012).
To test whether PSC regulates abundance of CYC-B in vivo, the Patched (Ptc) driver was used to direct the expression of dsRNA directed against Psc mRNA in a central band across the wing imaginal disc of third instar larvae. Immunostaining of CYC-B (red) and PSC (green) revealed a strong increase in CYC-B, precisely in the area of the disc where PSC was depleted. CYC-B abundance was also reported to increase in cellular clones that lack both Psc and Su(z)2, but not in Pc or dRing mutant clones. The effect of PSC on CYC-B was transcription-independent because expression of cyc-B mRNA was not affected by PSC depletion. Likewise, loss of PSC or LMG in S2 cells caused accumulation of CYC-B, which was even greater when both factors were depleted. However, the abundance of cyc-B mRNA in S2 cells was not affected by depletion of PSC or LMG. Thus, CYC-B accumulation appears to be caused by a transcription-independent mechanism, possibly involving PSC-directed ubiquitylation (Mohd-Sarip, 2012).
To investigate the role of PSC in CYC-B ubiquitylation, CYC-B was immunopurified from cells that were depleted of PSC or LMG and treated with proteasome inhibitors. Immunoblotting revealed that the loss of either PSC or LMG caused decreased levels of polyubiquitylated CYC-B (Ub-CYC-B). Almost no Ub-CYC-B was detectable in cells lacking both PSC and LMG. To test whether failed CYC-B destruction could explain the mitotic defects after the loss of PSC, CYC-B was overexpressed in S2 cells. After ectopic expression of CYC-B, ~70% of mitotic cells displayed a variety of defects. Concomitant overexpression of either PSC or LMG almost completely reversed the CYC-B misexpression phenotype. In contrast, extra PC had no effect. Collectively, these results suggest that PSC-mediated CYC-B ubiquitylation is crucial for normal mitosis (Mohd-Sarip, 2012).
Purified PSC, LMG, and CYC-B were used in a reconstituted ubiquitylation system, which was dependent on E1 and E2 enzymes, to test the ability of PSC to act as a ubiquitin E3 ligase for CYC-B. Approximately equimolar amounts of either PSC or LMG could direct CYC-B ubiquitylation. But together, PSC and LMG generated higher levels of Ub-CYC-B. Determination of the CYC-B ubiquitylation rate revealed a more-than-additive effect of combining PSC and LMG, indicating that they function cooperatively. A substitution mutation replacing a signature cysteine residue of the RING consensus with an alanine [PSC-C287A (C287A: Cys287→Ala287)] abrogated PSC's ability to ubiquitylate CYC-B. PSC-C287A blocked ubiquitylation of CYC-B by LMG, suggesting that it acts as a dominant negative. Indeed, the C287A mutation did not affect PSC binding to LMG or CYC-B. In contrast to ectopic PSC, expression of PSC-C287A caused severe mitotic defects in S2 cells. This mitotic phenotype was relieved by concomitant overexpression of LMG, suggesting that extra LMG squelches the dominant-negative PSC. Whereas ectopic expression of either PSC or LMG in S2 cells did not affect mitosis, overexpression of both PSC and LMG caused mitotic defects. These results suggest that PSC and LMG cooperate in the ubiquitylation of CYC-B, marking it for destruction by the proteasome (Mohd-Sarip, 2012).
Regulated protein destruction is fundamental to cell cycle progression. The work reported in this study shows that, in addition to transcriptional repression, PSC cooperates with LMG in the APC to direct CYC-B degradation. During mitosis, PSC (and its mammalian homologs) and key PRC1 subunits PH and PC dissociate from the chromatin, making a transcriptional function at that time unlikely. Like PSC, other chromatin regulators may also target proteins that are neither involved in chromatin dynamics nor transcription (Mohd-Sarip, 2012).
The role of Ago-1 in microRNA (miRNA) biogenesis has been thoroughly studied, but little is known about its involvement in mitotic cell cycle progression. This study establishes evidence of the regulatory role of Ago-1 in cell cycle control in association with the G2/M cyclin, cyclin B. Immunostaining of early embryos revealed that the maternal effect gene Ago-1 is essential for proper chromosome segregation, mitotic cell division, and spindle fiber assembly during early embryonic development. Ago-1 mutation resulted in the up-regulation of cyclin B-Cdk1 activity and down-regulation of p53, grp, mei-41, and wee1. The increased expression of cyclin B in Ago-1 mutants caused less stable microtubules and probably does not produce enough force to push the nuclei to the cortex, resulting in a decreased number of pole cells. The role of cyclin B in mitotic defects was further confirmed by suppressing the defects in the presence of one mutant copy of cyclin B. Involvement was establised of two novel embryonic miRNAs-miR-981 and miR-317-for spatiotemporal regulation of cyclin B. In summary, the results demonstrate that the haploinsufficiency of maternal Ago-1 disrupts mitotic chromosome segregation and spindle fiber assembly via miRNA-guided control during early embryogenesis in Drosophila. The increased expression of cyclin B-Cdk1 and decreased activity of the Cdk1 inhibitor and cell cycle checkpoint proteins (Mei-41 and Grp) in Ago-1 mutant embryos allow the nuclei to enter into mitosis prematurely, even before completion of DNA replication. Thus, these results have established a novel role of Ago-1 as a regulator of the cell cycle (Pushpavalli, 2013).
The present study identified the role of Ago-1 in
regulating cyclins, Cdk1 inhibitors, and p53 in Drosophila
embryos. In the rapidly dividing cells of the Drosophila
embryo, Ago-1 mutation led to severe mitotic disruption,
as evidenced by chromosome fragmentation, missegregation,
and abnormal mitosis during the precortical
syncytial cycles. The present results demonstrate
that Ago-1 modulated developmental arrays associated
with establishing the cell cycle control, seeing that Ago-1
mutation down-regulated Cyc A, CycB3, p53, mei-41, and
grp, but upregulated CycB transcripts. The reduction in
grp and mei-41 levels suggests that the replication and
DNA damage checkpoints are perturbed, allowing progression
of mitosis before completion of DNA replication
or DNA repair, which shows that the embryonic
lethality is associated with Ago-1 mutation. These results
are consistent with earlier findings that, in Drosophila,
DNA replication checkpoint genes are activated to
delay cell cycle progression during late cleavage stages (Pushpavalli, 2013).
The present study identified the role of Ago-1 in
regulating cyclins, Cdk1 inhibitors, and p53 in Drosophila
embryos. In the rapidly dividing cells of the Drosophila
embryo, Ago-1 mutation led to severe mitotic disruption,
as evidenced by chromosome fragmentation, missegregation,
and abnormal mitosis during the precortical
syncytial cycles. The present results demonstrate
that Ago-1 modulated developmental arrays associated
with establishing the cell cycle control, seeing that Ago-1
mutation down-regulated Cyc A, CycB3, p53, mei-41, and
grp, but upregulated CycB transcripts. The reduction in
grp and mei-41 levels suggests that the replication and
DNA damage checkpoints are perturbed, allowing progression
of mitosis before completion of DNA replication
or DNA repair, which shows that the embryonic
lethality is associated with Ago-1 mutation. These results
are consistent with earlier findings that, in Drosophila,
DNA replication checkpoint genes are activated to
delay cell cycle progression during late cleavage stages. In the syncytial blastoderm, the essential replication
checkpoint function is to prevent DNA damage
and ensure proper repair by delaying the cell cycle
(37). The reduced mei-41 or grp levels in the Drosophila
embryo due to Ago-1 mutation may cause rapid progression
from the S phase to mitosis, even before replication
is complete (Pushpavalli, 2013).
The syncytial blastoderm stage in Drosophila involves
only the S/M cycles and the expression patterns of cell
cycle proteins; for example, mitotic cyclins are necessary
for entry into and exit from mitosis. CycB is
localized to microtubules during the blastoderm stage
of Drosophila, and increased Cdk1/CycB activity
causes shorter microtubules with a decreased metaphase
and longer anaphase duration that leads to
defective mitosis. The effect of
miRNAs on CycB was also observed: Ago-1 affects the biogenesis of miRNAs
that regulate CycB, leading to the increased expression
of CycB. The elevated CycB levels found in the Ago-1
mutants showed that the microtubules were less stable
and probably did not produce enough force to push
the nuclei into the cortex, resulting in the observed
decrease in pole cell formation. Thus, Ago-1 is necessary
to ensure proper assembly of the mitotic spindle by
controlling the timing of CycB expression, a prerequisite
for proper nuclear migration during embryonic
development. Moreover, less stable microtubules require
a longer time to form proper metaphase structures. It is a well-established fact that PH3
staining indicates Cdk1 activity. In Ago-1 embryos,
the PH3 signal often persists over the entire
chromosome through the anaphase, whereas it is restricted
to the telomeric regions during the wild-type
anaphase, indicating the reminiscence of Cdk1 activity.
In Drosophila wee1, a Cdk1 inhibitory kinase, functions
downstream of mei-41 and is necessary for regulating
the activity of Cdk1. Ago-1 mutant embryos reduced
maternal wee1 transcript and hence reduced inhibitory
phosphorylation of Cdk1, leading to rapid mitosis.
Mutants with reduced maternal wee1 cause premature
entry into mitosis, spindle fiber defect, and chromosome
condensation defect (Pushpavalli, 2013).
The embryonic phenotypes such as mitotic asynchrony,
mitotic catastrophe, and disruption of the actin
cytoskeleton that are associated with Ago-1 mutation
were restored to a normal pattern in the presence of one
copy of mutant CycB, indicating the role of CycB in
mitotic progression. From these results, it as confirmed that
Ago-1 is necessary to ensure proper mitotic progression
by controlling the timing of Cdk1/CycB expression, a
prerequisite for proper microtubule assembly and nuclear
migration during embryonic development (Pushpavalli, 2013).
The cell cycle checkpoint proteins control the timing of
the regulatory pathways, such as DNA replication and
chromosome segregation, with high fidelity. As in Drosophila,
mammalian Atr and Chk1 are essential during
embryogenesis One of the reasons for the observed
segregation defects in these mutations in Drosophila
is that damaged DNA or incompletely replicated
DNA fails to trigger metaphase-to-anaphase delay. Recent data in mice indicate that depletion in the
miRNA processing factors down-regulates a large number
of cell cycle genes, including CycB1 (Ccnb1), implying
that miRNAs positively regulate cell-cycle genes. In
the current study, miRNAs, such as miR-774, miR-1186,
and miR-466d-3p, activated CycB1 and regulated the
cell cycle. Surprisingly
miRNA down-regulated CycB1 during early embryogenesis
in Drosophila was observed in the presence of wild-type Ago-1. The
data clearly indicate that Ago-1 functions as a mitotic
regulator by spatiotemporal regulation of Cdk1-CycB1,
Chk1 (grp), and mei-41 (Pushpavalli, 2013).
In Drosophila, p53 has no role in damage-induced cell
cycle arrest, but is absolutely necessary for genomic
stability, which is achieved by its apoptotic rather than
cell cycle function. It is speculated that decreased
levels of p53 in the Ago-1 mutant may be associated with
genomic instability in the early embryos when subjected
to stress. Both mei-41 and grp function in the same
genetic pathway and maternal mei-41 and grp are necessary
for wild-type cell cycle delays during the late
syncytial blastoderm stage. The reduction in
maternal mei-41 and grp caused mitotic defects during
the later syncytial divisions, indicating that gene expression
defects in the late embryos are secondary consequences
of the mitotic errors (Pushpavalli, 2013).
Recent studies have identified that noncoding miRNAs
act as regulators of gene expression in multicellular
eukaryotes and have been implicated in various
diseases. miRNAs control cell cycle progression by
regulating the cyclin-dependent kinases, cyclins, andcyclin-dependent kinase inhibitors. Mutation in
miRNA-processing factors (Ago-1 and Dcr-1) up-regulate
the levels of CycB mRNA and protein, which indicates
their involvement in CycB regulation. This study has
identified the miRNA-dependent regulatory circuit that
up-regulates CycB expression. It is therefore suggested that
expression of miR-981 in Drosophila embryo and its
ability to fine tune CycB make it an optimal mechanism
for maintaining a balanced level of CycB expression. To
date, no mammalian homologue of miR-981 has been
identified. The miRNAs miR-981 and miR-317 are also
Ago-1-associated miRNAs, with greatly reduced expression
under Ago-1 knockdown conditions in S2 cells. The in silico prediction of miR-317 in the red flour
beetle (insect class) indicates that components of cytoskeleton
are its target. This study found strong homology
between Drosophila and the red flour beetle in the
miR-317 mature sequence, and it is postulated that downregulation
of miR-317 in Drosophila might have affected
the normal functioning of the cytoskeleton, as well as
CycB, in the Ago-1 mutant embryos (Pushpavalli, 2013).
In the case of mammals, it has been reported that in
several tumor cell lines, the level of Ago-1 is significantly
lower than in nontumor cells. Wilms' tumor
exhibits the deletion of a region of human chromosome
1 that harbors the Ago-1 gene and is also associated
with neuroectodermal tumors. The haploinsufficient
maternal Ago-1 mutant, with all its mitotic
defects, survives to develop into the adult only if zygotic
transcription of Ago-1 occurs at about stage 9, in the absence
of which it dies during the late embryonic stage (Pushpavalli, 2013).
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