IplI-aurora-like kinase/aurora B
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 Inner centromere protein (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).
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 assessing 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).
Cdc37 has been shown to be required for the activity and stability of protein kinases that regulate different stages of cell cycle progression. However, little is known so far regarding interactions of Cdc37 with kinases that play a role in cell division. Loss of function of Cdc37 in Drosophila leads to defects in mitosis and male meiosis, and these phenotypes closely resemble those brought about by the inactivation of Aurora B. Evidence is provided that Aurora B interacts with and requires the Cdc37/Hsp90 complex for its stability. It is concluded that the Cdc37/Hsp90 complex modulates the function of Aurora B and that most of the phenotypes brought about by the loss of Cdc37 function can be explained by the inactivation of this kinase. These observations substantiate the role of Cdc37 as an upstream regulatory element of key cell cycle kinases (Lange, 2002).
To investigate the abnormal phenotypes brought about by mutation in Cdc37, meiosis was followed in mutant spermatocytes by time-lapse video microscopy. At anaphase I mutant chromosomes are poorly condensed and often fail to align in a proper metaphase plate. The overall distance between the two centrosomes at metaphase is shorter than in control cells and the overall shape of the mutant spindle is stumpier. During metaphase, the homologs split apart asynchronously and the first signs of splitting are observed 5.9 min before the onset of poleward movement, earlier than in control cells (1.2 min). During anaphase, segregation mistakes are obvious. Some chromosomes acquire an amphitelic orientation, i.e. with both sister kinetochores orientated to opposite poles. Premature sister chromatid separation takes place as the amphitelic chromosomes segregate their chromatids during anaphase I. Single chromatids are also observed at different positions within the anaphase spindle. After segregation the chromatin decondenses and the daughter nuclei are formed. No sign of furrow constriction is detected and cytokinesis does not occur giving rise to binucleated cells that sometimes might contain additional micronuclei (Lange, 2002).
Having shown that Cdc37 is essential for chromosome segregation and cytokinesis in meiosis, the function of Cdc37 was investigated via an independent approach in mitotic cells. To this end, Cdc37 was ablated by RNAi in Drosophila SL2 cells and the results were compared with the phenotypes produced by Aurora B RNAi inactivation in these cells. Depletion of Cdc37 inhibits cell proliferation starting 3 days after transfection, Aurora B depletion inhibits proliferation already after 2 days, while control cells followed exponential growth. Phase contrast and immunofluorescence microscopy analysis revealed that most Cdc37 dsRNA-treated cells had grown abnormally large, i.e. 3-4 times the size of control cells and Aurora B dsRNA-treated cells increased more than 4 times in size. Both Cdc37 and Aurora B dsRNA treated cells contained multiple abnormally shaped and unequally sized nuclei indicating cytokinesis failure and problems in chromosome segregation. In addition, these cells also had an increased DNA content. Propidium iodine labelling and subsequent FACS analysis revealed that the majority of Cdc37 RNAi-treated cells (75%) had a 4N DNA content, while Aurora B RNAi-treated cells exhibit an even higher degree of ploidy. Thus, Cdc37 RNAi cells accomplish one round of DNA synthesis but undergo no further rounds of synthesis as detected in the Aurora B dsRNA-treated cells. These results are consistent with cytokinesis failure and problems in chromosome segregation seen in Cdc37 mutant spermatocytes. They are also consistent with the hypothesis that inactivation of Aurora B might be a major contributing factor to the phenotypes brought about by inactivation of Cdc37. Taken together, these results indicate that Cdc37 function is required for cell cycle progression and cytokinesis in meiotic and mitotic cells (Lange, 2002).
To determine whether Cdc37 and Aurora B are part of a molecular complex, co-immunoprecipitation assays were performed in mitotic extracts from mammalian tissue culture cells and in Drosophila embryonic extracts. Cdc37 was found to co-immunoprecipitate with Aurora B and Hsp90 in a number of different cell lines: NIH 3T3, a mouse fibroblast cell line; SW480, a colorectal adenocarcinoma cell line, and A549, a lung carcinoma cell line. This association is absent in cells that are treated with geldanamycin (GA), an inhibitor of the Hsp90/Cdc37 complex, indicating a functional relationship between Cdc37/Hsp90 and Aurora B. Interestingly, Aurora B binds less Cdc37 and Hsp90 in A549 cells when compared with the two other cell lines, and moreover, this interaction seems to be independent of GA treatment (Lange, 2002).
A general cytoplasmic and perinuclear localization of Cdc37 has been described previously. The cytoplasmic and perinuclear localization was confirmed by immunofluorescence microscopy in interphasic mammalian cells (NIH 3T3, SW480 and HeLa). However, using single labelling with anti-Cdc37 antibodies or double labelling together with an anti-a-tubulin antibody, a distinct labelling could be detected in the central spindle and in the midbody. Moreover, Cdc37 co-localizes with Aurora B on the spindle microtubules and midbody. In vitro microtubule-pelleting assays were performed to test whether this localization of Cdc37 could be due to microtubule binding in mitosis. Extracts from mitotically enriched HeLa and SW480 cells were incubated at 37°C in the presence of taxol to polymerize and stabilize microtubules from endogenous tubulin. Control extracts were incubated with nocodazole to depolymerize microtubules. The microtubules and associated proteins were subsequently pelleted. Only the microtubule-containing pellets carried Cdc37 while the pellets of the nocodazole-treated extracts did not, indicating a specific association of Cdc37 with microtubules. Some of the Cdc37 protein remained in the supernatant in the taxol-treated samples indicating that not all the pool of Cdc37 associates with microtubules. Microtubule pelleting was also carried out using interphase extracts, in which Cdc37 did not pellet with microtubules (Lange, 2002).
Altogether, the molecular and cytological data established that the function of Cdc37 and the Cdc37/Hsp90 complex are essential in wild-type cells to maintain stability of Aurora B in diverse tissues and cells of Drosophila and humans. Interfering with Cdc37 function leads to lack of a central spindle, aberrant chromosome segregation and cell cycle arrest. Interestingly, the interaction between Aurora B and Cdc37 is defective in certain cancer cells (Lange, 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 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).
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).
Tri-methylation of histone H3 lysine 9 is important for recruiting heterochromatin protein 1 (HP1) to discrete regions of the genome, thereby regulating gene expression, chromatin packaging and heterochromatin formation. HP1alpha, -beta, and -gamma are released from chromatin during the M phase of the cell cycle, even though tri-methylation levels of histone H3 lysine 9 remain unchanged. However, the additional, transient modification of histone H3 by phosphorylation of serine 10 next to the more stable methyl-lysine 9 mark is sufficient to eject HP1 proteins from their binding sites. Inhibition or depletion of the mitotic kinase Aurora B, which phosphorylates serine 10 on histone H3, causes retention of HP1 proteins on mitotic chromosomes, suggesting that H3 serine 10 phosphorylation is necessary for the dissociation of HP1 from chromatin in M phase. These findings establish a regulatory mechanism of protein-protein interactions, through a combinatorial readout of two adjacent post-translational modifications: a stable methylation and a dynamic phosphorylation mark (Fischle, 2005).
Although histone H3S10ph is widely seen as a hallmark of mitosis, the function of this modification during M phase has been enigmatic. The data suggest that phosphorylation of H3S10 by Aurora B disrupts the chromodomain-H3K9me3 interaction, which is important for HP1 recruitment to chromatin during interphase. This disruption causes a net shift in the dynamic HP1-chromatin binding equilibrium towards the unbound state. In this reaction sequence, dephosphorylation of H3S10 at the end of mitosis re-establishes the overall association of HP1 with chromatin (Fischle, 2005).
It is propose that this binary 'methyl/phos switching' permits dynamic control of the HP1-H3K9me interaction. Intriguingly, the mechanism for HP1 release from M-phase chromatin does not involve a temporary loss of H3K9me3, but instead requires a combination of this unchanging mark and the dynamic H3S10ph modification that is only transiently added to chromatin during mitosis. It is reasoned that stable transmission of the heterochromatin-defining H3K9me3 mark is needed to accurately convey, from one cell generation to the next, which regions of the genome are supposed to be permanently silenced. If removal of HP1 from M-phase chromatin were accomplished by H3K9me3-erasing demethylase activities, the epigenetic information underlying this mark- and effector-system would have to be accurately re-established at the end of every cell cycle (Fischle, 2005).
In addition to H3S10 phosphorylation, other mechanisms might be involved in the mitotic release of HP1 from chromatin. These might include further modifications of the H3-tail, HP1 proteins and/or their interaction partners. Nevertheless, inhibition, knockdown or depletion of Aurora B is sufficient to cause aberrant interaction of all HP1 isoforms with mitotic, condensed chromatin. Although the possibility cannot be excluded that HP1 proteins themselves might be in vivo targets of Aurora B kinase activity (for example, increased association of the xHP1aW57A mutant protein was observed with metaphase chromosomes assembled in DCPC extracts), it is known that the phosphorylation level of HP1b and HP1g does not increase during mitosis. Since phosphorylation of an H3K9me3 peptide is sufficient to dissociate HP1 from this site in vitro, it is concluded that Aurora B-mediated phosphorylation of H3S10 must be the central event in mitotic release of HP1 from chromatin (Fischle, 2005).
Notably, a fraction of HP1a, but not HP1b or HP1g, remains associated with the (peri-)centromeric chromosome region, where it performs important functions for centromere cohesion and kinetochore formation and might be required to identify and define this specialized area of heterochromatin throughout the cell cycle. This mitotic retention of HP1a at centromeres depends on a carboxy-terminal region of the protein, but is independent of the chromodomain8. It is therefore suggested that 'methyl/phos switching' uniformly disrupts HP1-chromatin interaction but that mechanisms other than chromodomain-H3K9me3 interaction are responsible for the lingering HP1a association with pericentromeric regions (Fischle, 2005).
What is the function of HP1 dissociation from chromatin during Mphase? It is tempting to speculate that removal of HP1 is important for allowing access by factors necessary for mediating proper chromatin condensation and faithful chromosome segregation during mitosis. Indeed, inhibition of Aurora B in vertebrate cells results in defects in chromosome alignment, segregation, chromatin-induced spindle assembly and cytokinesis. Furthermore, mutation of H3S10 causes faulty chromosome segregation in Tetrahymena and S. pombe, organisms that rely on HP1 and H3K9me3 for the establishment and maintenance of heterochromatin, but not in Saccharomyces cerevisiae, an organism that lacks this silencing system. Interestingly, most histone phosphorylation sites are rapidly phosphorylated early in M phase. It remains to be seen whether these bursts in histone phosphorylation are directly involved in the release of proteins bound to interphase chromatin, which might need to be removed to ensure faithful progression through mitosis. It is conceivable that similar 'methyl/phos switches' play critical roles in governing other histone-non-histone or even non-histone-nonhistone interactions (Fischle, 2005).
The chromosomal passenger complex protein INCENP is required in mitosis for chromosome condensation, spindle attachment and function, and cytokinesis. INCENP has an essential function in the specialized behavior of centromeres in meiosis. Mutations affecting Drosophila incenp profoundly affect chromosome segregation in both meiosis I and II, due, at least in part, to premature sister chromatid separation in meiosis I. INCENP binds to the cohesion protector protein MEI-S332, which is also an excellent in vitro substrate for Aurora B kinase. A MEI-S332 mutant that is only poorly phosphorylated by Aurora B is defective in localization to centromeres. These results implicate the chromosomal passenger complex in directly regulating MEI-S332 localization and, therefore, the control of sister chromatid cohesion in meiosis (Resnick, 2006).
This analysis of Drosophila incenp mutants reveals for the first time a crucial role for INCENP in regulating centromeric cohesion during the reductional division of meiosis. INCENP influences the localization and/or function of MEI-S332: precocious sister chromatid separation is observed at the centromeres in the mutants, the distribution of MEI-S332 is abnormal when INCENP levels are decreased, INCENP can bind MEI-S332 in vitro, the protein is phosphorylated in vitro by Aurora B, and MEI-S332 localization to centromeres in mitosis is perturbed when its preferred Aurora B phosphorylation site is mutated (Resnick, 2006).
The QA26 incenp mutation perturbs chromosome condensation and causes precocious separation of the sister chromatids in spermatocytes. Quantitative genetic nondisjunction tests showed that chromosome segregation fails in both meiosis I and II, and that these nondisjunction events are consistent with premature separation of sister chromatids and random segregation in both meiotic divisions. This genetic analysis is likely to underestimate the true rates of nondisjunction because many of the defects caused by loss of passenger function (e.g., defective spindle organization or cytokinesis) would not yield functional gametes, thereby preventing the scoring all of the nondisjunction events. Although the aberrant condensation in prophase and prometaphase I made direct visualization of the onset of loss of cohesion difficult, completely separated sister chromatids could unambiguously be seen in mutant anaphase I cells, confirming one mechanism that contributes to the genetic nondisjunction phenotype (Resnick, 2006).
In C. elegans meiosis, the chromosome passenger complex is necessary for chiasma resolution. If chromosomal passengers were to participate both in regulation of centromeric cohesion as well as processing of chiasmata in C. elegans, essential roles in the latter might obscure roles in the former. In Drosophila male meiosis, there is no synapsis of homologs or recombination. Rather, segregation of homologous chromosomes is regulated via specific pairing sites. The analysis of passenger function was therefore simplified in Drosophila males, where chiasmata do not form (Resnick, 2006).
The MEI-S332-related yeast Shugoshin proteins are critical for the maintenance of the meiotic-specific cohesin subunit Rec8 at centromeres during anaphase Interestingly, no Rec8 homolog has yet been found in Drosophila. The only Drosophila meiotic kleisin, C(2)M, is a component of the synaptonemal complex and has been shown to have an earlier role in female and male meiosis. Thus, what MEI-S332 protects at centromeres in meiosis remains unclear. In mitosis, ablation of MEI-S332 does not lead to premature loss of the mitotic cohesin Rad21 (Resnick, 2006).
In both incenp mutants, impaired INCENP function results in a failure of MEI-S332 localization to centromeres in meiosis. This presumably leads to defects in the protection of cohesion at sister centromeres and contributes to the observed increase in meiotic nondisjunction. The failure to localize MEI-S332 in the incenp mutants is not a general secondary effect of prophase I condensation defects or of premature sister chromatid separation prior to the onset of anaphase I: ord mutants, which display both of those phenotypes, localize MEI-S332 normally. Although the data support a role for MEI-S332 in the increased nondisjunction in incenp mutants, mei-S332 mutants predominantly lead to meiosis II nondisjunction, whereas the incenp alleles show defects in both meiotic divisions. Thus, INCENP must be required for additional functions beyond its role in MEI-S332 regulation described in this study (Resnick, 2006).
One mechanism by which INCENP could promote MEI-S332 function is through its role in establishing or maintaining the specialized chromatin structure around centromeres. The chromosomal passenger complex is involved in regulation of chromatin remodeling complexes like ISWI, and it interacts with histone and nonhistone proteins from the pericentric heterochromatin. Recent studies show a direct link between Aurora B activity and regulation of HP1 localization in mitosis, suggesting a possible role in the regulation of heterochromatin structure. Since heterochromatin is important for cohesin binding to centromeres, it is possible that modifications of both MEI-S332 and the underlying heterochromatin are important for stabilizing centromeric cohesion during meiosis I (Resnick, 2006).
Alternatively, INCENP could act as a platform for the regulation of MEI-S332 at centromeres. The direct binding between INCENP and MEI-S332 could target MEI-S332 to heterochromatin, or it could help to direct its regulation by protein kinases. MEI-S332 binds better in vitro to a mixture of INCENP and Aurora B than to INCENP alone, suggesting that the interaction is strengthened by phosphorylation of either INCENP or MEI-S332. In addition to its role in binding and activating Aurora B, INCENP that has been phosphorylated by CDK1 can bind to Plk1, the human homolog of POLO kinase (Resnick, 2006).
Binding to phosphorylated INCENP is required to target Plk1 to centromeres in mitosis. Thus, INCENP could potentially coordinate the functions of POLO and Aurora B, both of which have been implicated in the regulation of cohesin (and also in the regulation of MEI-S332 in the case of POLO). These kinases have been shown to cooperate in the release of arm cohesion in chromosomes assembled in Xenopus extract. In contrast to Aurora B, however, POLO promotes the dissociation of MEI-S332 from centromeres during mitosis and meiosis. In polo mutants, MEI-S332 persists on the centromere, and mutation of two POLO box domains disrupts POLO binding and phosphorylation of MEI-S332 in vitro, as well as MEI-S332 dissociation from the centromeres (Resnick, 2006).
Together, these observations suggest that INCENP may act to integrate the various pathways controlling MEI-S332 function in meiosis I. Early in meiosis I, INCENP/Aurora B complexes may stabilize centromeric MEI-S332 through direct binding or modification of the underlying chromatin as described above. Similar to what happens in mitosis, CDK1 could phosphorylate INCENP at the POLO binding site, and phosphorylation-dependent binding of POLO to INCENP could target the kinase to the centromere. This binding might also render the kinase unavailable to phosphorylate MEI-S332. During the metaphase-anaphase I transition, INCENP remains on the centromeres and might therefore prevent MEI-S332 from being phosphorylated by POLO. At the onset of anaphase II, however, as INCENP transitions off the centromere, POLO may be free to phosphorylate MEI-S332, thereby releasing it from centromeres, allowing the release of sister chromatid cohesion (Resnick, 2006).
INCENP is emerging as a key regulator of kinase signaling pathways in mitosis. The present study reveals that this versatile protein may have a similar role in meiosis and may use its interactions with Aurora B and POLO to coordinate the specialized behavior of sister chromatids in meiosis I (Resnick, 2006).
To study the localization of the Aurora B protein kinase in Drosophila cells, an antipeptide antibody was raised against its specific 15 COOH-terminal amino acids. This antibody does not recognize recombinant Aurora A protein or endogenous Aurora A protein of 50 kD, but does stain a single band of ~40 kD in Western blots of extracts of S2 cells that is greatly reduced when cells are treated with the aurora B dsRNA. This staining, together with the immunostaining of mitotic cells, can be competed out by the peptide used to raise the antibody. Aurora B cannot be detected by this antibody in interphase cells, but it is readily apparent with a punctate distribution throughout all regions of condensing chromosomes in prophase cells. By metaphase, the concentration of the protein has strongly increased in the centromeric regions. Some of this centromeric staining persists in early anaphase, at which time the enzyme appears to become relocated on the central region of the spindle. During anaphase B, when the poles start to move apart, labeling of the central spindle has become predominant and the enzyme is essentially confined to the midbody during cytokinesis. Proteins showing this dynamic pattern of localization during mitosis have been termed 'passengers'; they ride upon the chromosomes until metaphase, whereupon they alight to the platform of the central spindle (Giet, 2001).
To demonstrate the distribution of Aurora B's interactive partner Incenp, antibodies were raised to two nonoverlapping regions of the protein. Both sera recognized a single protein of 110 kD on immunoblots of embryo extract. This is larger than the predicted molecular mass of 83.5 kD but consistent with the behavior of Incenps from other species, which also migrate anomalously on protein gels (Adams, 2001a).
In syncytial embryos, Incenp associates with condensing chromatin during prophase, before becoming focussed to the centromeric regions of metaphase chromosomes. Upon entry into anaphase, the protein leaves the chromosomes to form a ring of spots between the segregating chromatids. Each spot seems to be at the converging focus of bundles of microtubules. As telophase progresses, the Drosophila Incenp ring decreases in diameter until it becomes a single midbody-like structure between the central spindle microtubule bundles. A similar distribution of Incenp was also observed in cellularized embryos and Dmel2 cultured cells (Adams, 2001a).
To localize Aurora B in embryos and tissue culture cells, antibodies were raised against the NH2-terminal 58 amino acids of the protein fused to GST. Although neither of the two sera raised detected a protein on immunoblots of embryo extract, both recognized the recombinant protein expressed in bacteria. The affinity-purified antibodies worked well for indirect immunofluorescence in both embryos and cells. The distribution of Aurora B resembles that of Incenp throughout mitosis. However, interphase nuclei showed no detectable staining for Aurora B (they did for Incenp), and as nuclei entered prophase, Aurora B appears first at the centromere: no staining was observed along the chromosome arms. Nuclei just entering mitosis (i.e., adjacent to interphase nuclei) accumulate Aurora B only at the centromere. Aurora B remains at the centromere until the metaphase to anaphase transition, when it transfers to the central spindle and subsequently to the midbody. This distribution is also observed in cellularized embryos and in cultured cells. It is concluded that Incenp and Aurora B are chromosomal passenger proteins whose distribution in mitosis resembles their vertebrate counterparts (Adams, 2001a).
To determine whether decrease in expression levels of the aurora B gene would affect the progression of Drosophila Schneider S2 cells through their division cycle, such cells were treated with dsRNA synthesized from the aurora B cDNA. FACS analysis of control cells showed two predominant peaks of G1 (2N) and G2/M (4N) cells. 3 d after transfection of aurB dsRNA, the number of G1 cells with a 2N complement of DNA is strongly reduced and the profile shows prominent peaks at 4N and 8N. This indicates a doubling in ploidy of a substantial proportion of the population of cells, such that G1 cells now fall within the 4N peak and G2/M cells in the 8N peak. This was confirmed by staining the cells to reveal DNA, whereupon it could be seen that most of the transfected interphase cells were significantly larger than the controls and that they contained two or more nuclei or a single large nucleus. Although the level of Aurora B protein within the population of cells is reduced by ~90% after aurB RNAi, amounts of total cellular protein appear unchanged, as do levels of alpha-tubulin or the mitotically labile protein cyclin B. This suggests that the cells are capable of progressing through S phase, but that defects occur in either or both chromosome segregation at mitosis or during cytokinesis (Giet, 2001).
To assess the nature of the defects in cell cycle progression, cells were stained to reveal DNA and microtubules and defects were quantified in the mitotic cells within the asynchronous population 3 d after treatment with aurB dsRNA. Whereas >90% of control interphase cells appear to have normal DNA content, as judged by the size of their nuclei, 70% of cells become polyploid after aurB RNAi. Of these, 19% had a single abnormally large nucleus and 52% were multinucleate. The mitotic index of the population of aurB dsRNA-treated cells (5%) was not significantly different from control cells, indicating that in spite of the mitotic defects, cell cycle progression was not affected. Within the population of aurB RNAi cells a proportion of cells showed mitotic figures comparable to control cells. However, the proportion of these cells undergoing apparently normal mitosis was greatly reduced. These apparently normal cells may not have taken up the dsRNA and their proportion relative to abnormal mitoses is comparable to the reduction in level of Aurora B kinase detected by Western blotting. The most striking feature is that incomplete chromosome condensation is seen in essentially all mitotic cells, albeit to varying extents. No obvious defects were detected at the spindle poles in prophase and microtubules are well nucleated by the centrosomes at this and other mitotic stages. There were also failures in the alignment of chromosomes on the metaphase plate. The extent to which chromatin can segregate to the spindle poles at anaphase varies dramatically, from situations in which there appear to be lagging chromatids, to cases in which massive chromatin bridges are formed. Such bridges fail to resolve at telophase and are presumably one means by which cells can arise that have a single polyploid nucleus. In other cases, lagging chromatids fail to resolve and will eventually form micronuclei, and cytokinesis appears to be blocked. The proportion of binucleate cells at these late mitotic stages is elevated ~15-fold over control cells. The density of microtubules in the central region of the mitotic spindle in these cells appears much lower than in control cells able to undergo cytokinesis. Thus, defects in chromosome condensation after aurB RNAi are accompanied with abnormalities in chromosome segregation and failure of cytokinesis. Interestingly, the cells are not subject to checkpoint arrest and are able to undertake at least two to three rounds of polyploidization within the time frame of the experiment (Giet, 2001).
The defects in chromosome segregation after aurB RNAi have suggested that the centromeric regions of chromosomes may not form correct attachments to be able to move along the spindle microtubules. As centromeric heterochromatin is difficult to identify within these poorly condensed chromosomes, the region was immunolabelled using antibodies to Prod, the product of the gene proliferation disrupter. In control cells, Prod localizes to centromeric regions of congressed metaphase chromosomes, and in late anaphase this centromeric marker is clearly seen near the poles of the spindle. In aurB RNAi cells, Prod shows only a slight tendency to be associated with the spindle poles at anaphase and there is conspicuous punctate staining given by anti-Prod throughout the poorly condensed chromatin mass. These observations indicate that in addition to being required for cytokinesis, Aurora B also functions to regulate chromatin dynamics during mitosis, in particular in directing the organization and function of centromeric regions (Giet, 2001).
Aurora-related kinases direct the phosphorylation of histone H3 in meiosis and mitosis (Hsu, 2000). Since the Ipl1 aurora-like kinase is required for accurate chromosome transmission in budding yeast, and since phospho-histone H3 is found on mitotic chromosomes, the phosphorylation state of this histone was examined in aurB RNAi cells that show defects in chromosome condensation and segregation. To this end, immunostaining and Western blotting was carried out using an antibody specific to this phosphoepitope. The immunostaining of control cells indicates that histone H3 begins to become phosphorylated at the onset of prophase and increases to give intense signals at metaphase and anaphase. Staining levels decrease during telophase and then disappear during cytokinesis. Western blotting of asynchronous cultures of control or RNAi-treated cells indicates a pronounced decrease in levels of phosphorylated histone H3 in parallel with reductions in the level of Aurora B kinase. Immunostaining indicates that phosphorylation of histone H3 in mitotic cells is dramatically reduced after aurB RNAi but not completely extinguished. It remains present in foci distributed throughout the poorly condensed chromatin mass, rather than being uniformly associated with the chromosomes (Giet, 2001).
It has been proposed that the modification of histone H3 by phosphorylation on serine 10 could lead to the recruitment of condensation factors on the DNA (Wei, 1999). To test the feasibility of this model, the dynamics by which condensin would associate with mitotic chromosomes in Drosophila cells were followed using Barren protein as a marker. The Drosophila Barren protein is a component of the condensin complex, homologous to the XCAPH protein of Xenopus. Mutations in barren are characterized by an absence of chromosome condensation and the formation of chromatin bridges during anaphase that are very similar to defects seen in aurB RNAi cells (Bhat, 1996). Barren protein appears on the chromosomes with the same timing and dynamics as phosphorylation of histone H3. It is first detected on condensing chromosomes at prophase as multiple foci; it is maximal during metaphase and anaphase A; and it disappears from chromosomes as they decondense at late anaphase/telophase. In aurB RNAi cells, a dramatic decrease is found of Barren protein associated with chromosomes, although Western blotting indicates that there is no diminution in levels of the protein. Thus, Aurora B activity is essential to recruit Barren protein to chromosomes during mitosis and the dynamics of the process are consistent with a model in which the phosphorylation of histone H3 is required for this process (Giet, 2001).
Examination of spindle microtubules in aurB RNAi cells indicated abnormalities in the organization of the central spindle. A kinesin-like protein encoded by the gene pavarotti (Pav-KLP) is required for this aspect of spindle organization before cytokinesis. Since the C. elegans ortholog of Pav-KLP, Zen4-klp, is not recruited to the spindle in conditional mutants for the Aurora B-like kinase Air-2, it was of interest to determine whether the localization of Pav-KLP would be affected in aurB RNAi cells. Pav-KLP normally localizes to the central spindle during anaphase and to the midbody during telophase at the onset of cytokinesis. However, when S2 cells were subjected to aurB RNAi, a marked decrease of Pav-KLP immunostaining was seen from the diminished central spindle and in many cells localized Pav-KLP could not be detected. Total Pav-KLP levels are not affected by aurB RNAi. This strongly suggests that in normal mitosis the presence of Aurora B kinase on the central spindle is essential for its correct organization and the recruitment of Pav-KLP in order for cytokinesis to take place (Giet, 2001).
A biochemical and double-stranded RNA-mediated interference (RNAi) analysis has been performed of the role of two chromosomal passenger proteins, inner centromere protein (Incenp) and Aurora B kinase, in cultured cells of Drosophila. Incenp and Aurora B function is tightly interlinked. RNAi for either Aurora B or Incenp dramatically inhibits the ability of cells to achieve a normal metaphase chromosome alignment. Cells were not blocked in mitosis, however, and enter an aberrant anaphase characterized by defects in sister kinetochore disjunction and the presence of large amounts of amorphous lagging chromatin. Anaphase A chromosome movement appear to be normal, however cytokinesis often fails. Drosophila Incenp and Aurora B are not required for the correct localization of the kinesin-like protein Pavarotti (ZEN-4/CHO1/MKLP1) 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, 2001a).
A candidate Incenp cDNA was identified by querying the Berkeley Drosophila Genome Project database with the conserved COOH-terminal 90 amino acids of vertebrate Incenp. The cDNA sequence was mapped onto genomic a P1 clone. The candidate Incenp gene maps to cytological region 43B1, contains six exons, and encodes a 2,441-bp cDNA with a continuous ORF of 2,265 bp. Both sequence and exon/intron analysis agree exactly with that described by Celera Genomics for the hypothetical gene CG12165 (Adams, 2001a).
Incenp has a predicted molecular mass of 83.5 kD and a calculated isoelectric point of 9.63. Between residues 540 and 660, a coiled coil-forming region is predicted. The major region of homology with vertebrate Incenps is in the COOH-terminal IN-BOX (Adams, 2000), a 40-50-amino acid domain that defines the Incenp family from yeasts to humans. The NH2-terminal 540 amino acids are poorly conserved relative to vertebrate Incenps, however it is this region that contains all the previously known functional domains for Incenp, such as the heterochromatin protein 1 (HP-1) and ß-tubulin binding domains, the centromere targeting region and the spindle targeting domain (Adams, 2001a).
During anaphase, Incenp colocalizes with microtubules of the central spindle. Furthermore, Incenp overexpression in cultured vertebrate cells or disruption of murine Incenp leads to a dramatic remodeling of the microtubule network. To test whether Incenp binds microtubules directly, soluble full-length GST-Incenp was expressed in bacteria, purified, and incubated with taxol-stabilized microtubules prepared from purified tubulin. The reaction mixture was then sedimented through a sucrose cushion. GST-Incenp cosediments with the microtubules. This suggests that the binding of Incenp to microtubules in mitosis is likely to be direct (Adams, 2001a).
Incenp is stockpiled in Xenopus eggs in a complex with aurora B kinase (Adams, 2000). Drosophila contains two recognizable aurora-like protein kinases: the founder member of the family, Aurora (Aurora A), which is required for centrosome separation, and the recently described Aurora B/ial (Reich, 1999), whose function and localization are unknown. To determine whether Aurora B can associate with Incenp, Aurora B expressed in bacteria was incubated with beads laden with GST-Incenp. As a control, the kinase was incubated with beads carrying GST alone. Under these conditions, Aurora B binds specifically to GST-Incenp but not to GST alone (Adams, 2001a).
RNAi was added to eliminate Incenp and Aurora B/ial from cultured cells. dsRNA was added to exponentially growing cultures of Dmel-2 tissue culture cells, and at different time points samples were taken for analysis by immunoblotting and indirect immunofluorescence. Analysis of RNAi experiments is complicated by the fact that this technique causes a gradual depletion of the proteins under study, and that proteins are not necessarily lost from all cells in the population at the same rate. Furthermore, inhibition of Incenp or Aurora B function causes cultures to become polyploid, and it is difficult to exclude that some of the aspects of aberrant mitosis seen in these experiments are caused by complications arising during polyploid mitosis. To minimize these complications, the phenotypes described here were examined at various times after the onset of RNAi treatment; certain phenotypic aspects, such as defects in histone H3 phosphorylation and mitotic chromosome assembly, are observed in cells in some cases within the first cell cycle, before cultures become highly polyploid. In addition, where possible, phenotypic conclusions were limited to cells that were demonstrably lacking Incenp or Aurora B, detectable by indirect immunofluorescence (Adams, 2001a).
Immunoblotting analysis of cells treated with Incenp dsRNA showed that the levels of Incenp in the culture became greatly decreased 36-48 h after the addition of dsRNA to the culture. In the best experiments, ~95% of the protein was lost. The RNAi treatment for Aurora B takes effect more rapidly, the protein becoming undetectable by indirect immunofluorescence in most mitotic cells by 24 h. In both cases, the loss of protein is transient, with levels beginning to recover at later times (Adams, 2001a).
The phenotypes observed after Incenp and Aurora B RNAi were complex, revealing defects at multiple stages of the mitotic cycle. To follow the appearance of the various phenotypes after the onset of RNAi, cultures were harvested at 24, 36, 48, and 72 h after exposure to dsRNA and assessed for the following parameters: cell number, frequency of dead (Trypan blue-positive) cells, frequency of overtly polyploid cells, mitotic index, percentage of mitotic cells negative for Incenp or Aurora B by indirect immunofluorescence, and for the cells in mitosis, the distribution of the various mitotic phases (Adams, 2001a).
RNAi treatment causes an increase in the cell doubling time from 21 h in Dmel2 cells (21.6 h in cells after exposure to control dsRNA) to 36.1 and 27.5 h in cultures after exposure to dsRNA to Aurora B and Incenp, respectively. This was accompanied by an increase in polyploid cells starting at 24 h in the Aurora B experiment (36 h for Incenp). These correspond to the first times when significant numbers of mitotic cells were observed to be lacking Aurora B and Incenp, respectively (Adams, 2001a).
Strikingly, RNAi of Incenp abolishes the ability of cells to achieve a metaphase chromosome alignment. A similar phenotype was observed in the Aurora B RNAi. Instead, the population of mitotic cells comes to be dominated by cells with a prometaphase-like chromosome arrangement. Importantly, this increase in the percentage of prometaphase cells does not reflect an arrest in mitosis, as the mitotic index of the culture remains constant at the control level of ~5% throughout the entire experiment. It is thought that many cells in these cultures exit mitosis directly from prometaphase without achieving a metaphase chromosome alignment (Adams, 2001a).
Incenp is required for the correct localization of Aurora B kinases in human cells and C. elegans early embryos (Adams, 2000; Kaitna, 2000). However, it was not known whether Aurora B kinases have a role in Incenp localization. Elimination of Incenp by RNAi completely abolishes Aurora B localization throughout mitosis: the protein is not detected on chromosomes, central spindle microtubules, or midbodies. In contrast, Aurora B RNAi does not block Incenp association with the chromosome arms during prometaphase but impairs its ability to concentrate at centromeres and eliminates the transfer to the midbody. Thus, Aurora B function is not required for the initial stages of Incenp targeting to chromosomes during prophase, but it is necessary for Incenp behavior later in mitosis (Adams, 2001a).
In untreated and control cultures, mitotic chromosomes invariably showed high levels of histone H3 phosphorylation on serine10, detected with a specific antibody. Inhibition of Aurora B or Incenp function leads to both a decrease in the levels of detectable histone H3 phosphorylation and an increase in the incidence of malformed chromosomes starting as early as 24 h after exposure to dsRNA. The phospho-H3 staining varied from cell to cell, but by 24 h after Aurora B RNAi, the level of H3 phosphorylation was significantly reduced in 79% of the aurora-null prometaphase cells (74% of the Incenp-null cells at 36 h after Incenp RNAi). This result suggests that, as in C. elegans (Hsu, 2000), Aurora B is at least partially responsible for the histone H3 kinase activity in Drosophila cells (Adams, 2001a).
Importantly, the level of histone H3-serine10 phosphorylation shows only a weak correlation with the overall degree of chromatin condensation. Although levels of histone H3 phosphorylation on serine10 do tend to increase with increasing chromatin condensation, it is evident that there is a huge variation in the data from cell to cell. In other studies, chromosomes completely lacking detectable Aurora B kinase were observed; these showed an apparently normal level of condensation (Adams, 2001a).
An aberrant dumpy prometaphase chromosome morphology was seen in 46% of Incenp-negative and 60% of aurora B-negative cells after RNAi. These dumpy chromosomes had a 28-fold lower level of phospho-H3 staining, as detected with specific antibody, than did the chromosomes with a normal morphology. Dumpy chromosomes had an amorphous shape, and defined sister chromatids were not seen. In many cases, the dumpy chromosomes appeared to correspond to an abnormal prometaphase arrangement, characterized by a disassembled nuclear lamina and persistent high levels of cyclin B protein. Although they initially appeared less condensed than normal mitotic chromosomes, in fact, their level of condensation is normal. Instead, it appears that other aspects of chromosome higher order structure and behavior are aberrant. This may be due to defects in condensin binding (Adams, 2001a).
Dumpy chromosomes have kinetochores, as defined by the presence of double dots of CENP-A/Cid staining. Cid is the Drosophila CENP-A ortholog, and provides a marker for the kinetochore inner plate (Adams, 2001a).
As expected, given the lack of normal metaphase cells, few if any normal anaphase cells were seen that were negative for Incenp or aurora B. Instead, the anaphase/telophase cells had a range of abnormalities, including anaphase-like spindles with chromosomes distributed along their length, cells in various states of attempted cytokinesis with large amounts of amorphous lagging chromatin draped out behind the segregating chromatin, and bizarre cells in which banana-shaped nuclei were surrounded by a mitotic-like bipolar microtubule array (Adams, 2001a).
In cells with chromosomes distributed along the spindle or with banana-shaped nuclei, centromeres were seen to cluster either near opposite poles or at the opposing pointed ends of the elongate nuclei. This strongly suggests that kinetochores had attached to microtubules and that anaphase A movement of chromosomes had occurred (Adams, 2001a).
In cells that appeared to be in telophase, one or more pairs of centromeres were often noticed that appeared to be stalled midway between the spindle poles. This organization is what would be predicted if these centromeres had successfully become bioriented but were then unable to disjoin at the onset of anaphase chromosome movement. This was never seen in normal anaphases where the centromeres are typically grouped in a tight cluster at the leading edge of the segregating chromatids. Consistent with difficulties in disjunction of sister kinetochores, numerous paired kinetochore spots were seen near the spindle poles, as though nondisjoined chromatid pairs had moved together to a single pole (Adams, 2001a).
With increasing time after RNAi treatment, a dramatic increase was seen in the number of polyploid cells in both the Incenp and Aurora B RNAi so that by 72 h most of the cell population had become highly polyploid. The simplest explanation for the origin of the many binucleate cells that were observed is that chromosome segregation and nuclear reassembly occur, but that cytokinesis is then defective. Cells with one giant nucleus were also observed. These are likely to have arisen as a consequence of repeated failures in chromatid segregation. In addition to the chromosomal defects, spindle abnormalities were also observed in Incenp and Aurora B RNAi (Adams, 2001a).
Together, these observations suggest that Incenp and Aurora B might be essential for a variety of anaphase/telophase events, including sister chromatid and kinetochore disjunction, chromosome structure during anaphase, and mitotic spindle architecture (Adams, 2001a).
Cells lacking detectable Incenp were seen in which constriction of the cleavage furrow had advanced considerably and a midbody had formed. These cells showed an accumulation of actin at the cleavage furrow similar to that in untreated cells, although more actin was dispersed throughout the remainder of the cell than normal. In binucleate cells, there was no longer a focus of actin staining between the nuclei, indicating that the contractile ring had disassembled. In contrast, binucleate cells consistently showed an abnormally high density of tubulin between the two nuclei. This is likely to be a remnant of the central spindle (Adams, 2001a).
In aurora B/AIR-2 ts mutants of C. elegans, the kinesin-related protein ZEN-4 fails to localize properly, and a spindle midzone fails to form (Severson, 2000). As a result, cytokinesis begins, but the furrow regresses, and binucleate cells are produced. A similar phenotype is seen with the ZEN-4 ts mutant. In Drosophila, however, the ZEN-4 homolog PAV-KLP appears to act at an earlier stage, since pavarotti mutants do not form a stable contractile ring and fail to initiate cleavage (Adams, 2001a).
In untreated cells, PAV-KLP was invariably associated with the central spindle throughout cytokinesis. In Incenp depleted cells, PAV-KLP staining was present at the midbody of 94% of cells undergoing cytokinesis, however the staining was occasionally weaker than in untreated cells. To monitor the effect of the Aurora B RNAi on PAV-KLP localization, dsRNA-treated cells from the same well were split and stained for Aurora B and PAV-KLP on the same slide. In 90% of telophases, PAV-KLP was detected at the midbody, whereas Aurora B staining was absent from 80% of telophases. PAV-KLP was occasionally present in binucleate cells, where the cleavage furrow had regressed. It is concluded PAV-KLP localization is relatively unchanged after the loss of Incenp or Aurora B, at least in cells that form recognizable midbody structures (Adams, 2001a).
Drosophila Subito is a kinesin 6 family member and ortholog of mitotic kinesin-like protein (MKLP2) in mammalian cells. Based on the previously established requirement for Subito in meiotic spindle formation and for MKLP2 in cytokinesis, the function of Subito in mitosis was investigated. During metaphase, Subito localizes to microtubules at the center of the mitotic spindle, probably interpolar microtubules that originate at the poles and overlap in antiparallel orientation. Consistent with this localization pattern, subito mutants improperly assembled microtubules at metaphase, causing activation of the spindle assembly checkpoint and lagging chromosomes at anaphase. These results are the first demonstration of a kinesin 6 family member with a function in mitotic spindle assembly, possibly involving the interpolar microtubules. However, the role of Subito during mitotic anaphase resembles other kinesin 6 family members. Subito localizes to the spindle midzone at anaphase and is required for the localization of Polo, Incenp and Aurora B. Genetic evidence suggested that the effects of subito mutants are attenuated as a result of redundant mechanisms for spindle assembly and cytokinesis. For example, subito double mutants with ncd, polo, Aurora B or Incenp mutations are synthetic lethal with severe defects in microtubule organization (Cesario, 2006).
Subito is one of the two Drosophila kinesin 6 family members and probably the ortholog of MKLP2. In support of this classification, there are striking similarities between Subito and MKLP2. Both are required for localization of the passenger proteins to the midzone during anaphase. In addition, both Subito and MKLP2 interact with Polo kinase (or Plk1 in human) and are required for its localization to the midzone during anaphase. Plk1 phosphorylates MKLP2 at Ser528 and this phosphorylation promotes Plk1 binding to MKLP2. Plk1 phosphorylation negatively regulates MKLP2 microtubule bundling activity in vitro but is not required for the localization of MKLP2 to the midzone (Cesario, 2006).
Despite belonging to the same family, the two kinesin 6 family members probably have unique functions. The distinct phenotypes of sub and pav mutants indicate they have non-overlapping functions. Similarly, and despite having similar localization patterns, MKLP2 and MKLP1 have nonredundant functions in cytokinesis. MKLP2, but not MKLP1, has been shown to physically interact with Aurora B and Incenp. However, it has also been suggested that the MKLP2-dependent localization of Aurora B to the midzone is required for it to phosphorylate MKLP1. The importance of this phosphorylation on MKLP2 localization is unclear and the results are consistent with this indirect relationship between Subito and Pavarotti (Cesario, 2006).
It is possible that all members of the kinesin 6 group interact with antiparallel microtubules. Immunolocalization data is consistent with this because Subito is found on interpolar microtubules, which are characterized by an overlap of antiparallel microtubules in the midzone at mitotic anaphase in embryos, brains and testis. However, the localization of Subito to metaphase interpolar microtubules in the vicinity of the centromeres was a surprising finding. Although it is likely that Subito also associates with antiparallel microtubules at metaphase, the possibility that Subito interacts with the plus ends of the microtubules that interact with the kinetochores cannot be ruled. Surprisingly, a specific localization pattern of other kinesin 6 family members to metaphase microtubules has not been observed. This is not due to the absence of the appropriate substrate, since metaphase interpolar microtubules are present in most spindles. Either Subito is regulated differently than MKLP2, with an associated additional function in spindle assembly, or the localization pattern of MKLP2 at metaphase has not been informative with respect to its function (Cesario, 2006).
Since Subito is required to localize Polo, Aurora B and Incenp to the spindle midzone at anaphase, it is surprising that sub mutants are viable. Loss of MKLP2 causes cytokinesis defects. Drosophila mutants with strong defects in cytokinesis fall into the categories of male sterile, embryonic lethal (e.g. pav mutants) or pupal lethal. In fact, Incenp and polo mutants have embryonic lethal phenotypes that may be caused by a failure of cytokinesis. Unlike the loss of Incenp, Aurora B or Polo, sub mutants do not have any of these phenotypes and appear to complete cytokinesis most of the time in larval brains. In addition, because sub mutant males are fertile, and most mutants with strong defects in cytokinesis during spermatogenesis are male sterile, Subito does not appear to be essential for cytokinesis in the testis. A cytokinesis phenotype was also not evident in cultured Drosophila cells depleted of Subito by RNAi. These same studies did identify cytokinesis defects when Polo, Aurora B and Incenp were depleted. Thus, it seems likely that in some cell types, such as larval brains, the presence of Subito and the localization of the passenger proteins are not required for cytokinesis to occur (Cesario, 2006).
A close examination of sub mutants, however, revealed that anaphase did not proceed normally. In addition to the failure to accumulate Polo, Aurora B and Incenp in the midzone, the absence of Subito resulted in disorganized midzone microtubules at anaphase and a small increase in the frequency of polyploid cells. When the dosage of Incenp was reduced in sub mutants, the frequency of polyploidy was markedly increased. Therefore, Subito appears to have a similar function to MKLP2 in promoting cytokinesis, although there may be functional redundancy. Since the ability to complete cytokinesis in sub mutants depends on Incenp and Aurora B dosage, it is possible that unlocalized Incenp or Aurora B may promote cytokinesis. However, the observation that Incenp and Aurora B have a limited ability to spread along anaphase microtubules in the absence of Subito suggests an alternative; enough passenger protein activity may be present to promote cytokinesis. This model can account for the sensitivity of sub mutants to Incenp or Aurora B dosage because high levels of these proteins may be needed to promote cytokinesis if not concentrated in the midzone. It is also possible that anaphase may last longer and/or the microtubule organization improves with time in sub mutants. This would account for the relatively normal Fascetto localization and high success completing cytokinesis in sub mutants (Cesario, 2006).
Several lines of evidence suggest that Subito has a role in mitotic spindle assembly: (1) Subito initially localizes to interpolar microtubules at metaphase; (2) abnormally formed metaphase spindles were found in sub mutants more frequently than in the wild type; (3) sub mutant brains have an elevated mitotic index. Although the magnitude of the increase in sub mutants was lower than reported in some other mutants with spindle assembly defects, these mutants are lethal. Consistent with the conclusion that sub mutants have a defect in spindle assembly, the elevated mitotic index was dependent on BubR1, suggesting that the spindle assembly checkpoint is activated in the absence of Subito. (4) sub mutations exhibit synthetic lethality in combination with polo, Incenp and Aurora B mutations, and the cytological phenotype includes defects in spindle assembly and increased mitotic index. (5) RNAi of sub in Drosophila S2 cells results in frequent mitotic spindle abnormalities. These observations all point to a role for Subito in spindle assembly (Cesario, 2006).
The defects associated with sub mutants are less severe in mitotic cells than during female meiosis, possibly because of redundant spindle assembly pathways in mitosis. The double mutant studies suggest that the defects in spindle assembly or chromosome alignment in sub mutants are compensated for in two ways. First, the activation of the spindle assembly checkpoint allows defects in microtubule organization to be corrected. Second, the presence of redundant spindle assembly pathways allows microtubules to be assembled in the absence of sub. Double mutant studies support both of these mechanisms (Cesario, 2006).
The phenotype of the sub;polo16-1/+ double mutant is consistent with a redundant role for Subito in spindle assembly. Compared with the single mutants, the double mutants exhibit grossly abnormal metaphase and anaphase spindles. Similar to the results with sub, a role for Polo in spindle assembly has been shown through the analysis of polo hypomorphs that have an elevated mitotic index in larval brains, indicating that the spindle assembly checkpoint is activated. During metaphase, Polo localizes to the centromeres where it has a role in spindle formation but during anaphase it localizes to the spindle midzone where it has a role in cytokinesis. The very high mitotic index in the double mutants, however, suggests a more severe defect in spindle assembly than either single mutant. It is suggested that the abnormal spindle phenotype in sub/sub;polo/+ mutants arise from a combination of defects in two partially redundant spindle assembly pathways: improper assembly of kinetochore microtubules in polo/+ mutants and a reduction in assembling interpolar microtubules in sub mutants. Although polo mutants are recessive lethal, there is other evidence for dominant phenotypes, such as an elevated mitotic index in polo16-1/+ brains (Cesario, 2006).
The combination of these two spindle assembly defects in polo/+;sub/sub mutants might result in the severe spindle assembly phenotype and lethality in the double mutant. Similar conclusions apply for the interactions between sub and Incenp or Aurora B. Like Polo, the passenger proteins have an important role in spindle assembly. Indeed, the effects of all three mutants are strikingly similar, suggesting that Subito, Polo and the passenger proteins have important interactions during metaphase and anaphase. Interestingly, there is evidence of a direct interaction between Plk and Incenp in mammalian cells (Cesario, 2006).
Like its kinesin 6 homolog MKLP1, Subito is probably a plus-end-directed motor that crosslinks and slides interpolar antiparallel microtubules. The results suggest that this activity is important from metaphase through anaphase. Interestingly, the metaphase and anaphase interpolar microtubules have functional differences. Metaphase interpolar microtubules are observed in the absence of Subito whereas their anaphase counterparts depend on Subito. Another important difference is that Polo and the passenger proteins localize only to anaphase interpolar microtubules in the midzone. It has been suggested that the precocious appearance of anaphase-like interpolar microtubules is an important feature of acentrosomal meiotic spindle assembly in Drosophila oocytes. The passenger proteins Aurora B and Incenp localize to the interpolar microtubules at metaphase of meiosis I, rather than the centromeres, which is typical during mitotic metaphase. Therefore, the regulation of the passenger protein localization pattern is modified in oocytes to bypass the centromere localization that is characteristic of mitotic metaphase, resulting in precocious localization to interpolar microtubules (Cesario, 2006).
Despite these differences, the same biochemical activities of Subito could be used to organize both centrosomal mitotic and female acentrosomal meiotic spindles. In mitotic cells, kinetochores can initiate microtubule fiber formation, but these fibers are not directed toward either spindle pole. Failure to organize these fibers could result in disorganized and frayed spindles, as was observed in sub mutants. A function for Subito and interpolar microtubules could be to properly orient undirected kinetochore fibers. Interpolar microtubules could interact with and direct the organization of kinetochore microtubules via motors that bundle parallel microtubules. This mechanism has been proposed for organizing a bipolar spindle in the acentrosomal meiosis of Drosophila oocytes. With motor-driven sliding of antiparallel microtubules, this is an example of a centrosome-independent model for the spindle assembly pathway. This is consistent with previous conclusions that centrosome-independent mechanisms for spindle assembly are active in mitotic cells. Indeed, since bipolar spindles can form in the absence of centrosomes in neuroblasts and ganglion mother cells, it appears that centrosome-independent mechanisms for spindle assembly are active in the mitotic cells analyzed (Cesario, 2006).
Another possibility is that Subito functions as part of the centrosomal assembly pathway. For example, an array of interpolar microtubules could help channel centrosome microtubules towards the kinetochores. This activity could reduce the element of chance associated with making contacts between centrosome microtubules and kinetochores. It has also been proposed that centrosomal microtubules may capture the minus ends of kinetochore microtubules. An involvement of Subito in this process would be surprising, however, because the ability to bundle microtubules in parallel has not been described for a kinesin 6 family member. Nonetheless, if Subito was involved in the interactions of centrosomal and kinetochore microtubules, subsequent plus-end-directed movement would explain why Subito localization overlaps with centromeres. Whether or not these models are correct, the redundant nature of spindle assembly and function may explain why a role for kinesin 6 motor proteins in spindle assembly has not been described previously (Cesario, 2006).
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