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 disjoined 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 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).
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).
The chromosomal passenger complex (CPC) is a key regulator of mitosis in many organisms, including yeast and mammals. Its components co-localise at the equator of the mitotic spindle and function interdependently to control multiple mitotic events such as assembly and stability of bipolar spindles, and faithful chromosome segregation into daughter cells. This study reports the first detailed characterisation of a CPC mutation in Drosophila, using a loss-of-function allele of borealin (borr). Like its mammalian counterpart, Borr colocalises with the CPC components Aurora B kinase and Incenp in mitotic Drosophila cells, and is required for their localisation to the mitotic spindle. borr mutant cells show multiple mitotic defects that are consistent with loss of CPC function. These include a drastic reduction of histone H3 phosphorylation at serine 10 (a target of Aurora B kinase), and a pronounced attenuation at prometaphase and multipolar spindles. The evidence suggests that borr mutant cells undergo multiple consecutive abnormal mitoses, producing large cells with giant nuclei and high ploidy that eventually apoptose. The delayed apoptosis of borr mutant cells in the developing wing disc appears to cause non-autonomous repair responses in the neighbouring wild-type epithelium. These responses involve Wingless signalling, which ultimately perturbs the tissue architecture of adult flies. Unexpectedly, during late larval development, cells survive loss of borr and develop giant bristles that may reflect their high degree of ploidy (Hanson, 2005).
One crucial role of the CPC during mitosis is to mediate the H3 phosphorylation of serine 10 (P-H3) by Aurora B, as has been demonstrated in budding yeast, C. elegans and Drosophila. The numbers of P-H3-positive (dividing) cells are reduced in the VNC of borr mutant embryos. Furthermore, the P-H3 levels of individual borr mitotic nuclei are typically reduced compared with those of wild-type nuclei. Often, they exhibit blotchy P-H3 staining rather than the more 'structured' staining outlining condensed chromosomes as observed in the wild type. A similar loss of P-H3 staining has also been observed in borr RNAi-depleted Kc167 cells. This reduction of the P-H3 levels in borr mutant cells is consistent with a loss of Aurora B kinase activity and, thus, with a disruption of CPC function (Hanson, 2005).
Despite the strong reduction of the P-H3 levels in mitotic VNC cells of borr mutant embryos, these cells display only a slight undercondensation of their chromatin, although the degree of undercondensation is somewhat variable from cell to cell. These results suggest that borr may not be essential for chromatin condensation (Hanson, 2005).
To examine the effects of borr loss on actively dividing epithelial cells, FRT-FLP-mediated recombination was used to generate borr mutant clones in imaginal discs whose cells undergo cell divisions throughout larval development. If borr mutant clones are induced during early larval stages and examined in fully grown larval discs, these clones are rare and are much smaller than the corresponding wild-type twin spots, suggesting that a large fraction of the mutant cells die. Hoechst staining revealed that many of the surviving borr mutant cells are large, with giant but well-formed nuclei that appear healthy, and well integrated into the epithelial tissue (Hanson, 2005).
Imaginal discs bearing borr mutant clones were stained with antibodies against Incenp and Aurora B, to assess the effect of borr loss on these CPC components during mitosis. Wild-type cells in metaphase show characteristic well-ordered mitotic spindles, with distinct staining of Aurora B and Incenp at specific sites along condensed chromatin. By contrast, borr mutant cells invariably show abnormal mitotic spindles, including multipolar ones. Most of these mutant spindles do not show any chromatin-associated Incenp or Aurora B staining, although occasionally patches of Incenp staining can still be observed, but they do not seem to be associated with any of the spindle components. These staining patterns suggest that these CPC components fail to localise properly to mitotic spindles in the absence of borr (and their levels may also be reduced, though the low frequency of surviving borr mutant cells does not allow assessment of this quantitatively). Therefore, as in mammalian cells, the correct localisation of Incenp and Aurora B to mitotic spindles of dividing imaginal disc cells depends on Borr. This is further evidence that Borr is a CPC protein, and that it interacts functionally with other known CPC components (Hanson, 2005).
A biochemical and double-stranded RNA-mediated interference (RNAi) analysis of the role of two chromosomal passenger proteins, inner centromere protein (INCENP) and aurora B kinase, was performed in cultured cells of Drosophila melanogaster. INCENP and aurora B function is tightly interlinked. The two proteins bind to each other in vitro, and INCENP is required for Aurora B to localize properly in mitosis and function as a histone H3 kinase. Aurora B is required for INCENP accumulation at centromeres and transfer to the spindle at anaphase. RNAi for either protein dramatically inhibits the ability of cells to achieve a normal metaphase chromosome alignment. Cells were not blocked in mitosis, however, and entered an aberrant anaphase characterized by defects in sister kinetochore disjunction and the presence of large amounts of amorphous lagging chromatin. Anaphase A chromosome movement appeared to be normal, however cytokinesis often failed. INCENP and Aurora B are not required for the correct localization of the kinesin-like protein Pavarotti to the midbody at telophase. These experiments reveal that INCENP is required for aurora B kinase function and confirm that the chromosomal passengers have essential roles in mitosis (Adams, 2001b).
Aurora B is required for some, but not all, aspects of Incenp localization in mitosis. In the absence of Aurora B, Incenp localizes normally to chromosomes during pro-phase; however, it is subsequently unable to concentrate at centromeres and transfer to the central spindle or midbody. As predicted from previous studies, Incenp is essential for Aurora B targeting: after Incenp RNAi, Aurora B does not localize to chromosomes, midzone microtubules, or midbodies. Thus, the chromosomal passenger proteins are interdependent on one another for proper targeting during mitosis (Adams, 2001b).
This interdependence, plus the fact that the two proteins are stockpiled in an 11S complex in Xenopus eggs, suggests that they could function in vivo in a protein complex. Incenp binds microtubules in vitro and may be responsible for targeting Aurora B to the central spindle, as the kinase appears to lack microtubule binding activity of its own. However, the differences in centromere targeting in Drosophila early embryos suggest that the two proteins may not function in an obligate complex, at least during prophase (Adams, 2001b).
S. cerevisiae aurora/Ipl1p and C. elegans aurora B/AIR-2 are required for H3-serine10 phosphorylation in mitosis (Hsu, 2000). Not only is Incenp essential for the proper targeting of Aurora B in mitotic cells, but this targeting is required for normal levels of histone H3 phosphorylation on serine10. This is the first evidence that Incenp is an essential cofactor required for Aurora B kinase function in vivo (Adams, 2001b).
The availability of mitotic cells containing chromosomes with a range of levels of H3 phosphorylated on serine10 has enabled an assessment of the widely held hypothesis that H3 phosphorylation is correlated with the degree of chromatin condensation. When phospho-H3 levels and the degree of chromatin compaction were compared by quantitative fluorescence microscopy, only a weak correlation between the two values was observed. Instead, interference with Incenp and Aurora B function appears to correlate much more strongly with difficulties in assembling mitotic chromosomes of normal morphology. Mitotic chromosomes deficient in phospho-H3 have a characteristic dumpy morphology, with no evidence of resolved sister chromatids. This resembles the defects seen in Drosophila mutants in the SMC4 subunit of condensin (Steffensen, 2001) and also those of a ts mutant in C. elegans aurora B/AIR-2 when it enters mitosis at nonpermissive temperature (Severson, 2000). Phosphorylation of histone H3 or another chromosomal substrate by Aurora B might be required for the binding of condensins or other chromosomal proteins that give mitotic chromosomes their characteristic morphology (Adams, 2001b).
At later times, after addition of dsRNA, a dramatic increase is seen in the percentage of mitotic cells in prometaphase coupled with a corresponding decrease in the number of metaphase cells. This is particularly dramatic in the Incenp RNAi, where no Incenp-negative cells in metaphase are seen. Surprisingly, this did not lead to an increase in the mitotic index of the cultures. Therefore, in the absence of Incenp and/or Aurora B function, Drosophila Dmel2 cells must exit mitosis from prometaphase. Elimination of Incenp and Aurora B function does not trigger a mitotic checkpoint in Dmel2 cells. However, since these cells do not arrest in mitosis in colcemid, they appear to lack a robust metaphase checkpoint in any case (Adams, 2001b).
What is the ultimate fate of these prometaphase cells? They are not removed by cell death. An alternative explanation for the lack of an increase in mitotic index would be if the cells transit directly from prometaphase into anaphase or telophase, as is the case for budding yeast cells mutant in the essential kinetochore protein Ndc10p. Consistent with this, a variety of striking abnormalities were seen in cells either undergoing anaphase, or early in the next cell cycle. Although anaphase/telophase cells with kinetochores at opposite poles of the chromatin mass could be seen, the kinetochores were often not clustered as tightly as normal This may reflect the initiation of anaphase movement without prior alignment of the chromosomes at a metaphase plate (Adams, 2001b).
Why does abrogation of Incenp and/or Aurora B function prevent cells from attaining a stable metaphase chromosome alignment? One obvious possibility is that kinetochore function is impaired. In budding yeast, the aurora kinase Ipl1p phosphorylates the essential kinetochore component Ndc10p (Biggins, 1999). It is therefore possible that, in metazoans, one or more kinetochore components must be phosphorylated by Aurora B in order for kinetochores to function in mitosis. An obvious candidate for this is CENP-A/Cid. CENP-A retains a site homologous to serine10, which is serine5 in Cid. It will be important to determine whether CENP-A/Cid is phosphorylated in an Aurora B kinase-dependent manner (Adams, 2001b).
Arguing against this model is the observation that kinetochores assemble correctly, at least as far as CENP-A/Cid binding is concerned, and move to the spindle poles at anaphase/telophase. This implies that the ability of kinetochores to bind microtubules and to undergo anaphase A movement are preserved after abrogation of Incenp and Aurora B function. However, other aspects of kinetochore function, namely the ability to form bipolar spindle attachments and disjoin at anaphase, appear to be defective. How RNAi of Incenp or Aurora B leads to defects in chromosome biorientation is unknown, but this is unlikely to be a result of interference with binding of the condensin subunit barren, since barren mutants successfully complete metaphase chromosome alignment. Furthermore, the possibility that some of the abnormal aspects of chromosome behavior reflect an impairment of microtubule and/or spindle function cannot be excluded. The detailed behavior of the mitotic spindle after RNAi of Incenp and Aurora B requires further analysis (Adams, 2001b).
Anaphase/telophase cells after RNAi for Incenp or Aurora B exhibit three highly unusual chromosomal phenotypes: (1) they often have one or more pairs of sister kinetochores located in the central spindle or flanking the midbody; (2) the foci of CENP-A/Cid staining at or near the spindle poles is often present as pairs, suggesting that sister kinetochores remain paired despite having undergone anaphase A-like poleward movement; (3) separated masses of chromatin are often connected by a mass of lagging chromatin. This is referred to as chromatin and not chromosomes because the material is amorphous, and little or no evidence of a condensed mitotic chromosome morphology can be observed (Adams, 2001b).
The first two phenotypes can be explained if centromeres fail to disjoin at anaphase onset. Under these circumstances, centromeres of bioriented chromosomes would tend to accumulate near the spindle equator -- later, near the midbody -- and be stretched apart by the spindle forces. Mono-oriented kinetochores would move as pairs to one or the other spindle pole. If this occurred in cells that had attained metaphase, then the bulk of kinetochores would remain as pairs in the spindle midzone. However, abrogation of Incenp and/or Aurora B function prevents cells from reaching metaphase and would therefore be expected to lead to the observed phenotype, with most centromeres at the poles and only a few remaining in the midzone. Defects in sister kinetochore disjunction could arise if Incenp and/or Aurora B were involved in regulation of the cohesin complex at centromeres; experiments are under way to determine whether cohesin components are substrates for Aurora B (Adams, 2001b).
The presence of massive amounts of lagging chromatin is highly characteristic of anaphase/telophase after loss of Incenp and/or Aurora B function. This lagging chromatin might arise from difficulties in sister chromatid disjunction, but it is more likely that it represents a failure of the chromosomes to move as integral units under the physical stress of anaphase movement. If the dumpy chromosomes observed in prometaphase cells lack an organized infrastructure then when centromeres begin to move polewards, the chromatin of the arms might simply unravel and be left behind as a smear of amorphous chromatin. This would be consistent with the observation that interference with Aurora B function in Drosophila cells interferes with the binding of the condensin subunit barren to mitotic chromosomes (Giet, 2001). Indeed, barren mutants exhibit dramatic chromatin bridges during syncytial mitosis, however such a dramatic defect was not seen in mutants affecting the condensin subunit SMC4 in Drosophila (Steffensen, 2001). It is possible that action of Incenp/aurora B on other chromosomal components, in addition to condensin subunits, contributes to a loss of chromosomal integrity during anaphase (Adams, 2001b).
In the oocytes of many species, bipolar spindles form in the absence of centrosomes. Drosophila oocyte chromosomes have a major role in nucleating microtubules, a process that precedes the bundling and assembly of these microtubules into a bipolar spindle. Evidence is presented that a region similar to the anaphase central spindle functions to organize acentrosomal spindles. subito mutants are characterized by the formation of tripolar or monopolar spindles and nondisjunction of homologous chromosomes at meiosis I. subito encodes a kinesinlike protein and associates with the meiotic central spindle, consistent with its classification in the Kinesin 6/MKLP1 family. This class of proteins is known to be required for cytokinesis, but the current results suggest a new function in spindle formation. The meiotic central spindle appears during prometaphase and includes passenger complex proteins such as AurB and Incenp. Unlike mitotic cells, the passenger proteins do not associate with centromeres before anaphase. In the absence of Subito, central spindle formation is defective and AurB and Incenp fail to properly localize. It is proposed that Subito is required for establishing and/or maintaining the central spindle in Drosophila oocytes, and this substitutes for the role of centrosomes in organizing the bipolar spindle (Jang, 2005).
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