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).
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 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 expr