Aurora/Ipl1-related kinases are a conserved family of enzymes that have multiple functions during mitotic progression (Giet, 1999). Although it has been possible to use conventional genetic analysis to dissect the function of aurora, the founding family member in Drosophila, the lack of mutations in a second aurora-like kinase gene, IplI-aurora-like kinase (here referred to as aurora B), precluded this approach. Depleting Aurora B kinase using double-stranded RNA interference in cultured Drosophila cells results in polyploidy (Giet, 2001 and Adams, 2001a). aurora B encodes a passenger protein that associates first with condensing chromatin, concentrates at centromeres, and then relocates onto the central spindle at anaphase. Cells depleted of the Aurora B kinase show only partial chromosome condensation at mitosis. This is associated with a reduction in levels of the serine 10 phosphorylated form of Histone H3 and a failure to recruit the Barren condensin protein onto chromosomes. These defects are associated with abnormal segregation resulting from lagging chromatids and extensive chromatin bridging at anaphase, similar to the phenotype of barren mutants (Bhat, 1996). The majority of treated cells also fail to undertake cytokinesis and show a reduced density of microtubules in the central region of the spindle (Giet, 2001). RNAi for either Aurora B or Inner centromere protein (Incenp) dramatically inhibits the ability of cells to achieve a normal metaphase chromosome alignment. 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).

The segregation of chromosomes with high fidelity requires exquisite coordination of cellular processes. The mechanisms that coordinate the cycle of chromosome condensation and decondensation with the assembly, function, and subsequent disassembly of the mitotic spindle are poorly understood. Highly conserved genes essential for chromosome condensation have been found through genetic screens in yeasts and Drosophila. For example, five members of a protein complex known as condensin, have been identified that are functionally and structurally conserved. Mutants exhibit incomplete chromosome condensation associated with failure of segregation and the stretching of chromatin upon the spindle. Biochemical approaches also identified the protein complex in Xenopus and showed that it can promote chromatin condensation by directing the supercoiling of the DNA in an ATP-dependent manner. Chromosome condensation is also accompanied by phosphorylation of histones H1 and H3. Indeed, mutation of the mitotic phosphorylation site of histone H3 of Tetrahymena leads to both chromosome condensation and segregation defects. A direct link between histone H3 phosphorylation and condensin recruitment onto chromosomes has recently been suggested by the colocalization of members of the condensin complex with phosphorylated histone H3 during the early stages of mitotic chromosome condensation. However, the generality of the requirement for the phosphorylation of histone H3 for chromosome condensation and segregation must be questioned by the finding that budding yeast cells in which serine 10 of histone H3 is replaced with alanine show no apparent defects in cell cycle progression or chromosome transmission. Nevertheless, maximal chromosome condensation in meiosis does correlate with maximal levels of phospho-histone H3 in wild-type cells. The enzyme required for histone H3 phosphorylation in Saccharomyces cerevisiae is the aurora-related protein kinase Ipl1p (Hsu, 2000). Moreover, one of its two counterparts from Caenorhabditis elegans, the air-2 protein kinase, has been shown to have the same function (Giet, 2001 and references therein).

The Aurora- and Ipl1-like protein kinases form a conserved family of enzymes, the founding members of which are encoded by the S. cerevisiae and Drosophila genes, IPL1 and aurora, respectively. While the yeast genome encodes only one such kinase required for accurate chromosome segregation, metazoan genomes have at least two subfamilies of aurora-like kinases. One is associated with centrosomes and is activated in early mitosis, and a second is associated with chromosomes and the spindle midbody and is activated later. These families are referred to as Aurora-like kinases A and B, respectively. The precise effects of loss of function of either of these enzymes varies a little between different organisms and cell types. Broadly speaking, however, the A-type enzymes are required to maintain the separation of centrosomes to give normal bipolar spindle structure. This is shown, for example, in Drosophila from the phenotype of aurora mutants (termed here aurora A); or in Xenopus, where the corresponding pEg2 kinase can be eliminated using antibodies or inactive mutants. In contrast, the B-type Aurora-like kinases appear to be required for cytokinesis, as shown, for example, by transfection of an inactive kinase mutant into cultured mammalian cells (Tatsuka, 1998; Terada, 1998). An affect on cytokinesis has also been reported in mutants of the gene for the C. elegans B-type enzyme, Air-2, or after RNA interference (Schumacher, 1998; Kaitna, 2000; Severson, 2000). The air-2 encoded kinase is required for the positioning of Zen-4, a kinesin-like protein required at the midzone of the late central spindle for cytokinesis. Abnormal chromosome segregation is also observed after reduction of air-2 function (Giet, 2001 and references therein).

The dynamics of the localization of the Aurora B class of enzymes can be partially explained by recent findings showing they exist in a complex with an inner centromere protein (Incenp) (Adams, 2000; Kaitna, 2000). Incenps are one example of so-called 'passenger proteins' that localize to the centromeric regions of chromosomes at metaphase and are then redistributed to the central spindle during cytokinesis. Defects in Incenp function lead to failure of chromosome congression and cytokinesis defects. These findings, and the fact that B-type Aurora kinase becomes incorrectly localized in human cells expressing mutant Incenps that fail to localize, has led to the idea that Incenp functions to target the B-type kinases, first to chromosomes and then to the spindle midzone (Adams, 2000). A physical interaction is also seen between the Air-2 kinase and the counterpart of Incenp in C. elegans, ICP-1. Moreover, the disruption of icp-1 function by RNAi leads to the same phenotype as air-2 RNAi (Kaitna, 2000). This direct functional interaction between the Aurora-like kinases and Incenp occurs not only in metazoan cells, but also in budding yeast where the counterpart of Incenp, Sli15p (Kim, 1999), was identified through a screen for genes that interact with Ipl1 (Giet, 2001 and references therein).

Although a B-type Aurora kinase gene has been identified in Drosophila, the lack of mutants at this locus has prevented any analysis of its potential mitotic function (Reich, 1999). Levels of the Aurora B kinase can be reduced by RNAi in cultured Drosophila S2 cells. This leads to cytokinesis failure, together with chromosome condensation and segregation defects strikingly similar to those that have been described for mutations in the condensin gene barren (Bhat, 1996). The segregation defects are accompanied by aberrant chromatin condensation, a reduction in the phosphorylated form of histone H3, and a failure to recruit the Barren protein onto condensed chromosomes (Giet, 2001 and references therein).

Using double stranded RNA interference it has been shown that the Aurora B kinase is required for mitotic chromosome condensation and segregation, and subsequently for cytokinesis. The Aurora B enzyme becomes perfectly positioned to execute these processes as mitosis proceeds. It is distributed throughout the chromatin as it condenses at prophase, then becomes concentrated around the centromeric regions of the condensed chromosomes at metaphase, and finally leaves for the company of the central spindle region during anaphase. As such it behaves as a so-called passenger protein. It appears from recent studies to be in an intimate relationship with a travelling companion Incenp (Adams, 2000; Kaitna, 2000). The interaction of Incenp, or its yeast counterpart Sli5p, with Aurora-like kinases in yeast, C. elegans, and Xenopus suggests that this interaction is universal. The dynamic association of Incenp with chromosome arms at prometaphase, the centromeric region at metaphase, and then the spindle midzone at anaphase makes it an attractive candidate for targeting the Aurora B kinase to these regions. Indeed, dominant mutants of Incenp in human cells disrupt the localization of the Aurora B-like kinase AIL2 (Adams, 2000). The finding of abnormal chromosome segregation and cytokinesis after depletion of either the C. elegans Incenp, Icp-1, or its Aurora B-like kinase, Air-2 (Schumacher, 1998; Woolard, 1999; Kaitna, 2000), suggests the two passengers perform similar functions (Giet, 2001).

One striking effect of aurB RNAi is to permit progression through mitosis with improperly condensed chromosomes. It was possible to account for these condensation defects by a diminution of the phosphorylation of serine 10 of histone H3 and a failure to localize condensin on the chromosomes. The former finding is consistent with several studies that now implicate a requirement for the phosphorylation of the NH₂-terminal region of histone H3 at this residue for chromosome condensation. Not only does the formation of mitotic chromosomes in a Xenopus cell-free extract by a nucleosome-associated kinase correlate with histone H3 phosphorylation, but when the serine 10 residue is mutated to alanine it results in abnormal segregation and chromosome loss during mitosis and meiosis in Tetrahymena. One enzyme credited with the ability to phosphorylate histone H3 at mitosis is the NIMA kinase of Aspergillus. However, the finding that levels of histone H3 phosphorylation are reduced after aurB RNAi in Drosophila cells is more in keeping with the report that the Aurora-like kinase homologs, Ipl1 of yeast and Air-2 (but not Air-1) of C. elegans, are required for histone H3 phosphorylation in these organisms (Hsu, 2000). The finding of some residual histone H3 phosphorylation either could reflect the incomplete elimination of Aurora B by RNAi, or could indicate that an alternative kinase has this capability, offering an explanation of the partial chromosome condensation seen in the RNAi-treated cells. The current data are important in emphasizing the importance of histone H3 phosphorylation for chromosome transmission and as such are in line with the findings in Tetrahymena. This differs from the effects seen in budding yeast cells that continue through division cycles in the absence of histone H3 phosphorylation without showing defects in chromosome transmission. As an explanation, it has been suggested that other histones could be phosphorylated in addition to the histone H3 in the yeast cell and that such phosphorylation events could be sufficient to ensure normal chromosome dynamics. A major role of the yeast enzyme Ipl1p is to regulate the function of the kinetochore-associated protein Ndc10p through its phosphorylation (Biggins, 1999; Sassoon, 1999). Therefore, the increase in ploidy reported in ipl1 mutant cells has been attributed more to inappropriate kinetochore function, and consequently the effects of Air-2 depletion upon chromosome condensation in C. elegans have been a little overshadowed. It seems likely that the abnormal chromosome segregation in Drosophila cells after aurB RNAi is due to incomplete condensation, since a similar phenotype is seen in mutants of the condensin subunit Barren (Bhat, 1996). Of course, this does not exclude the possibility that defects in the organization of the centromeric regions and kinetochores arise directly as a result of aurB RNAi or as either a direct or indirect consequence of condensation defects. The increase in ploidy seen after aurora B RNAi is reminiscent of the Ipl1 phenotype in budding yeast, but differs in that it arises from both chromosome segregation and cytokinesis defects (Giet, 2001).

The resemblance of the mitotic phenotype of cells after RNAi with aurB to that previously reported for Drosophila barren mutants (Bhat, 1996) can be further explained by the failure of Barren protein to be recruited to the mitotic chromosomes after aurB RNAi. Originally recognized through this mutant defect, it was later realized that Barren is the fly homolog of a member of the pentameric complex, condensin, first shown to be required for mitotic chromosome condensation in Xenopus. It is possible that Barren or other members of the condensin complex could themselves be directly phosphorylated by Aurora B during chromosome condensation. However, the process seems likely to involve a plethora of phosphorylation events: the nuclear A-kinase anchoring protein (AKAP95) appears to target the human hCAP-D2 condensin to chromosomes and phosphorylation of condensin subunits by cdk1 has been associated both with their nuclear accumulation and activation. It has been proposed that phosphorylation of the NH₂ terminus of histone H3 leads to the recruitment or the activation of the condensin complex to the chromosome, where it can modify DNA topology. The data presented here indicate that phosphorylation of histone H3 by the Aurora B kinase and the localization of Barren onto chromosomes are associated events in mitosis. They support and extend a recent observation that human condensin proteins hCAP-E, hCAP-C, and hCAP-D2 colocalize with phosphorylated histone H3 in clusters in partially condensed regions of chromosomes in early prophase. The similarity of the effects seen on chromosome condensation resulting from loss of either aurora B or barren function is striking and points to the value of studying these processes in a single model organism amenable to both genetic manipulation and RNAi. It is perhaps surprising that in both cases partial chromosome condensation is achieved and that there can be some degree of segregation of chromatin to the poles (Giet, 2001 and references therein).

The second major mitotic abnormality observed after aurB RNAi in Drosophila cells is a failure of cytokinesis. Thus, like its mammalian and nematode counterparts AIM-1 and AIR-2, the enzyme encoded by aurora B appears essential for this process. Two proteins that play a role in cytokinesis have recently been shown to associate with the Aurora B-like kinases: Incenp, as discussed above (Adams, 2000; Kaitna, 2000), and the Zen-4 kinesin-like protein of C. elegans (Kaitna, 2000; Severson, 2000). The localization of the latter is disrupted after disruption of air-2 function using RNAi or conditional mutant alleles. Zen-4 is the C. elegans homolog of the Pavarotti KLP of Drosophila, which likewise is mislocalized on the central spindle from anaphase onwards after aurB RNAi. Pav-KLP also cooperates with Polo kinase to achieve its localization and function in Drosophila, suggesting that multiple mitotic kinases may be required to coordinate central spindle formation before cytokinesis, just as several kinases appear to be required for centrosome maturation and separation and chromosome condensation (Giet, 2001).

It is striking that aurB RNAi cells are not arrested by a mitotic checkpoint, given the abnormalities that they show in chromosome alignment at metaphase and the subsequent disorganization of the later mitotic spindle. However, the treated cells do undergo multiple cell cycles, as is clearly demonstrated in this cell culture system in which one can monitor the shift in ploidy by FACS analysis and the increase in chromosome and centrosome complements by immunocytology. It is possible that these abnormalities arise too late in the mitotic cycle to trigger checkpoint arrest, although this seems unlikely for the chromosome segregation defect. Although it is possible that Aurora B is itself required for checkpoint functions, it could also be that the kinetochore regions of chromosomes are insufficiently well organized after aurB RNAi to promote the checkpoint activity of the complex of Bub/Mad proteins that associate with unaligned centromeres. It is noteworthy that the C. elegans baculovirus inhibitor of apoptosis (IAP)-related repeat protein Bir-1 appears to be required for the localization of Air-2. Bir-1 localizes to chromosomes and then the spindle midzone and Air-2 fails to localize to these same sites in the absence of Bir-1 (Speliotes, 2000). These IAP proteins, also known as survivin, are caspase inhibitors and as such counteract apoptosis. Is it possible that B-type Aurora kinases might play a role alongside survivin in an apoptotic checkpoint to promote mitosis (Giet, 2001)?

It is of considerable interest to know the multiple substrates of Aurora B kinase and to understand its mode of regulation in mitotic progression. It seems that subcellular localization of the enzyme could be one critical means of controlling access to its substrates. The enzyme localizes throughout condensing chromosomes when phosphorylation of histone H3 is required. Aurora B's subsequent concentration at centromeres could direct enzyme activity toward specific chromosomal proteins at these sites, but may be instrumental in its movement onto the central spindle at anaphase, thereby providing an effective way of removing the enzyme from the chromatin to facilitate chromosome decondensation at telophase. Understanding the intricacies of these processes will be a future challenge (Giet, 2001).

Additional conclusions were reached by Adams (2001a) in their analysis of Aurora B function. Adams concludes that 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, 2001a).

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, 2001a).

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, 2001a).

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, 2001a).

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, 2001a).

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, 2001a).

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, 2001a).

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, 2001a).

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, 2001a).

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, 2001a).

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, 2001a).

Endoreplication is a cell cycle variant that entails cell growth and periodic genome duplication without cell division, and results in large, polyploid cells. Cells switch from mitotic cycles to endoreplication cycles during development, and also in response to conditional stimuli during wound healing, regeneration, aging, and cancer. This study used integrated approaches in Drosophila to determine how mitotic cycles are remodeled into endoreplication cycles, and how similar this remodeling is between induced and developmental endoreplicating cells (iECs and devECs). The evidence suggests that Cyclin A / CDK directly activates the Myb-MuvB (MMB) complex to induce transcription of a battery of genes required for mitosis, and that repression of CDK activity dampens this MMB mitotic transcriptome to promote endoreplication in both iECs and devECs. iECs and devECs differed, however, in that devECs had reduced expression of E2f1-dependent genes that function in S phase, whereas repression of the MMB transcriptome in iECs was sufficient to induce endoreplication without a reduction in S phase gene expression. Among the MMB regulated genes, knockdown of AurB protein and other subunits of the chromosomal passenger complex (CPC) induced endoreplication, as did knockdown of CPC-regulated cytokinetic, but not kinetochore, proteins. Together, these results indicate that the status of a CycA-Myb-MuvB-AurB network determines the decision to commit to mitosis or switch to endoreplication in both iECs and devECs, and suggest that regulation of different steps of this network may explain the known diversity of polyploid cycle types in development and disease (Rotelli, 2019).

Endoreplication is a common cell cycle variant that entails periodic genome duplication without cell division and results in large polyploid cells. Two variations on endoreplication are the endocycle, a repeated G/S cycle that completely skips mitosis, and endomitosis, wherein cells enter but do not complete mitosis and / or cytokinesis before duplicating their genome again. In a wide array of organisms, specific cell types switch from mitotic cycles to endoreplication cycles as part of normal tissue growth during development. Cells also can switch to endoreplication in response to conditional inputs, for example during wound healing, tissue regeneration, aging, and cancer. It is still not fully understood, however, how the cell cycle is remodeled when cells switch from mitotic cycles to endoreplication (Rotelli, 2019).

There are common themes across plants and animals for how cells switch to endoreplication during development. One common theme is that developmental signaling pathways induce endoreplication by inhibiting the mitotic cyclin dependent kinase 1 (CDK1). After CDK1 activity is repressed, repeated G / S cell cycle phases are controlled by alternating activity of the ubiquitin ligase APC/CCDH1 and Cyclin E / CDK2. Work in Drosophila has defined mechanisms by which APC/CCDH1 and CycE / Cdk2 regulate G / S progression, and ensure that the genome is duplicated only once per cycle. Despite these conserved themes, how endoreplication is regulated can vary among organisms, as well as tissues within an organism. These variations include the identity of the signaling pathways that induce endoreplication, the mechanism of CDK1 inhibition, and the downstream effects on cell cycle remodeling into either an endomitotic cycle (partial mitosis) or endocycle (skip mitosis). In many cases, however, the identity of the developmental signals and the molecular mechanisms of cell cycle remodeling are not known (Rotelli, 2019).

To gain insight into the regulation of variant polyploid cell cycles, two-color microarrays have been used to compare the transcriptomes of endocycling and mitotic cycling cells in Drosophila tissues (Maqbool, 2010). Endocycling cells of larval fat body and salivary gland have been shown to have dampened expression of genes that are normally induced by E2F1, a surprising result for these highly polyploid cells given that many of these genes are required for DNA synthesis. Nonetheless, subsequent studies showed that the expression of the E2F-regulated mouse orthologs of these genes is reduced in endoreplicating cells of mouse liver, megakaryocytes, and trophoblast giant cells. Drosophila endocycling cells also had dampened expression of genes regulated by the Myb transcription factor, the ortholog of the human B-Myb oncogene (MYBL2). Myb is part of a larger complex called Myb-MuvB (MMB), whose subunit composition and functions are mostly conserved from flies to humans. One conserved function of the MMB is the induction of periodic transcription of genes that are required for mitosis and cytokinesis. It was these mitotic and cytokinetic genes whose expression was dampened in Drosophila endocycles, suggesting that this repressed MMB transcriptome may promote the switch to endocycles that skip mitosis. It is not known, however, how E2F1 and Myb activity are repressed during endocycles, nor which of the downregulated genes are key for the remodeling of mitotic cycles into endocycles (Rotelli, 2019).

In addition to endoreplication during development, there are a growing number of examples of cells switching to endoreplication cycles in response to conditional stresses and environmental inputs. These cells will be called induced endoreplicating cells (iECs) to distinguish them from developmental endoreplicating cells (devECs). For example, iECs contribute to tissue regeneration after injury in flies, mice, humans, and the zebrafish heart, and evidence suggests that a transient switch to endoreplication contributes to genome instability in cancer. Cardiovascular hypertension stress also promotes an endoreplication that increases the size and ploidy of heart muscle cells, and this hypertrophy contributes to cardiac disease. It remains little understood how similar the cell cycles of iECs are to devECs (Rotelli, 2019).

Similar to the developmental repression of CDK1 activity to promote endocycles, it has been shown that experimental inhibition of CDK1 activity is sufficient to induce endoreplication in flies, mouse, and human cells. These experimental iECs in Drosophila are similar to devECs in that they skip mitosis, have oscillating CycE / Cdk2 activity, periodically duplicate their genome during G / S cycles, and repress the apoptotic response to genotoxic stress. This study uses these experimental iECs to determine how the cell cycle is remodeled when cells switch from mitotic cycles to endoreplication cycles, and how similar this remodeling is between iECs and devECs. The findings indicate that the status of a CycA-Myb-AurB network determines the choice between mitotic cycles and endoreplication cycles in both iECs and devECs (Rotelli, 2019).

This study has investigated how the cell cycle is remodeled when mitotic cycling cells switch into endoreplication cycles, and how similar this remodeling is between devECs and experimental iECs. Repression of a CycA-Myb-AurB mitotic network promotes a switch to endoreplication in both devECs and iECs. Although a dampened E2F1 transcriptome of S phase genes is a common property of devECs in flies and mice, this study found that repression of the Myb transcriptome is sufficient to induce endoreplication in the absence of reduced expression of the E2F1 transcriptome. Knockdown of different components of the CycA-Myb-AurB network resulted in endoreplication cycles that repressed mitosis to different extents, which suggests that regulation of different steps of this pathway may explain the known diversity of endoreplication cycles in vivo. Overall, these findings define how cells either commit to mitosis or switch to different types of endoreplication cycles, with broader relevance to understanding the regulation of these variant cell cycles and their contribution to development, tissue regeneration, and cancer (Rotelli, 2019).

The findings indicate that the status of the CycA-Myb-AurB network determines the choice between mitotic or endoreplication cycles (The CycA-Myb-AurB network regulates the choice between cell cycle programs). These proteins are essential for the function of their respective protein complexes: CycA activates CDK1 to regulate mitotic entry, Myb is required for transcriptional activation of mitotic genes by the MMB transcription factor complex, and AurB is the kinase subunit of the four-subunit CPC. While each of these complexes were previously known to have important mitotic functions, the data indicate that they are key nodes of a network whose activity level determines whether cells switch to the alternative growth program of endoreplication. The results are consistent with previous evidence in several organisms that lower activity of the Myb transcription factor results in polyploidization, and further shows that repressing the function of the CPC and cytokinetic proteins downstream of Myb also promotes endoreplication. Importantly, genetic evidence indicates that not all types of mitotic inhibition result in a switch to endoreplication. For example, knockdown of the Spc25 and Spc105R kinetochore proteins or the Polo kinase resulted in a mitotic arrest, not a switch to repeated endoreplication cycles. These observations are consistent with CycA / CDK, MMB, and the CPC playing principal roles in the mitotic network hierarchy and the decision to either commit to mitosis or switch to endoreplication cycles (Rotelli, 2019).

While knockdown of different proteins in the CycA-Myb-AurB network were each sufficient to induce endoreplication cycles, these iEC populations had different fractions of cells with multiple nuclei diagnostic of an endomitotic cycle. Knockdown of cytokinetic genes pav and tum resulted in the highest fraction of endomitotic cells, followed by the CPC subunits, then Myb, and finally CycA, with knockdown of this cyclin resulting in the fewest endomitotic cells. These results suggest that knocking down genes higher in this branching mitotic network (e.g. CycA) inhibits more mitotic functions and preferentially promotes G / S endocycles that skip mitosis, whereas inhibition of functions further downstream in the network promote endomitosis. Moreover, different levels of CPC function also resulted in different subtypes of endoreplication. Strong knockdown of AurB inhibited chromosome segregation and cytokinesis resulting in cells with a single polyploid nucleus, whereas a mild knockdown resulted in successful chromosome segregation but failed cytokinesis, suggesting that cytokinesis requires more CPC function than chromosome segregation. It thus appears that different thresholds of mitotic function result in different types of endoreplication cycles. This idea that endomitosis and endocycles are points on an endoreplication continuum is consistent with evidence that treatment of human cells with low concentrations of CDK1 or AurB inhibitors induces endomitosis, whereas higher concentrations induce endocycles. The results raise the possibility that in tissues of flies and mammals both conditional and developmental inputs may repress different steps of the CycA-Myb-AurB network to induce slightly different types of endoreplication cycles that partially or completely skip mitosis. Together, these findings show that there are different paths to polyploidy depending on both the types and degree to which different mitotic functions are repressed (Rotelli, 2019).

The findings are relevant to the regulation of periodic MMB transcription factor activity during the canonical mitotic cycle. Knockdown of CycA compromised MMB transcriptional activation of mitotic gene expression, and their physical association suggests that the activation of the MMB by CycA may be direct. The MMB-regulated mitotic genes were expressed at lower levels in CycA iECs, even though Myb protein levels were not reduced. This result is consistent with the hypothesis that CycA / CDK phosphorylation of the MMB is required for its induction of mitotic gene expression. Moreover, misexpression of Myb in CycA knockdown follicle cells did not prevent the switch to endoreplication, further evidence that CycA / CDK is required for MMB activity and mitotic cycles. While the dependency of the MMB on CycA was not previously known in Drosophila, it was previously reported that in human cells CycA / CDK2 phosphorylates and activates human B-Myb in late S phase, and also triggers its degradation. While further experiments are needed to prove that CycA / CDK regulation of the MMB is direct, interrogation of the results of multiple phosphoproteome studies using iProteinDB indicated that Drosophila Myb protein is phosphorylated at three CDK consensus sites including one, S381 that is of a similar sequence and position to a CDK phosphorylated site on human B-Myb (T447). The hypothesis is favored that it is CycA complexed to CDK1 that regulates the MMB because, unlike human cells, in Drosophila CycA / CDK2 is not required for S phase, and Myb is degraded later in the cell cycle during mitosis. Moreover, it is known that mutations in CDK1, but not CDK2, induce endocycles in Drosophila, mouse, and other organisms. A cogent hypothesis is that CycA / CDK1 phosphorylates Myb, and perhaps other MMB subunits, to stimulate MMB activity as a transcriptional activator of mitotic genes, explaining how pulses of mitotic gene expression are integrated with the master cell cycle control machinery. It remains formally possible, however, that both CycA / CDK2 and CycA / CDK1 activate the MMB in Drosophila. The early reports that CycA / CDK2 activates B-Myb in human cells were before the discovery that it functions as part of the MMB and the identification of many MMB target genes, and further experiments are needed to fully define how MMB activity is coordinated with the central cell cycle oscillator in fly and human cells (Rotelli, 2019).

Endocycles were experimentally induced by knockdown of CycA to mimic the repression of CDK1 that occurs in devECs. The data revealed both similarities and differences between these experimental iECs and devECs. Both iECs and SG devECs had a repressed CycA-Myb-AurB network of mitotic genes. In contrast, only devECs had reduced expression of large numbers of E2F1-dependent S phase genes, a conserved property of devECs in fly and mouse. In CycA iECs, many of these key S phase genes were not downregulated, including Cyclin E, PCNA, and subunits of the pre-Replicative complex, among others. This difference between CycA dsRNA iECs and SG devECs indicates that repression of these S phase genes is not essential for endoreplication. In fact, none of the E2F1 -dependent S phase genes were downregulated in Myb dsRNA iEC. Instead, the 12 E2F1-dependent genes that were commonly downregulated in Myb dsRNA iEC, CycA dsRNA iEC, and SG devEC all have functions in mitosis. These 12 mitotic genes are, therefore, dependent on both Myb and E2F1 for their expression, including the cytokinetic gene tum whose knockdown induced endomitotic cycles. This observation leads to the hypothesis that downregulation of the E2F transcriptome in fly and mouse devECs may serve to repress the expression of these mitotic genes, and that the repression of S phase genes is a secondary consequence of this regulation. These genomic data, together with the genetic evidence in S2 cells and tissues, indicates that in Drosophila the repression of the Myb transcriptome is sufficient to induce endoreplication without repression of the E2F1 transcriptome. The observation that both CycAdsRNA iECs and devECs both have lower CycA / CDK activity, but differ in expression of E2F1 regulated S phase genes, also implies that there are CDK-independent mechanisms by which developmental signals repress the E2F1 transcriptome in devECs (Rotelli, 2019).

The results have broader relevance to the growing number of biological contexts that induce endoreplication. Endoreplicating cells are induced and contribute to wound healing and regeneration in a number of tissues in fly and mouse, and, depending on cell type, can either inhibit or promote regeneration of the zebrafish heart. An important remaining question is whether these iECs, like experimental iECs and devECs, have a repressed CycA-Myb-AurB network. If so, manipulation of this network may improve regenerative therapies. In the cancer cell, evidence suggests that DNA damage and mitotic stress, including that induced by cancer therapies, can switch cells into an endoreplication cycle. These therapies include CDK and AurB inhibitors, which induce human cells to polyploidize, consistent with the fly data that CycA / CDK and the CPC are key network nodes whose repression promotes the switch to endoreplication. Upon withdrawal of these inhibitors, transient cancer iECs return to an error-prone mitosis that generates aneuploid cells, which have the potential to contribute to therapy resistance and more aggressive cancer progression. The finding that the Myb transcriptome is repressed in iECs opens the possibility that these mitotic errors may be due in part to a failure to properly orchestrate a return of mitotic gene expression. Understanding how this and other networks are remodeled in polyploid cancer cells will empower development of new approaches to prevent cancer progression (Rotelli, 2019).

According to the prevailing 'clock' model, chromosome decondensation and nuclear envelope reformation when cells exit mitosis are byproducts of Cdk1 inactivation at the metaphase-anaphase transition, controlled by the spindle assembly checkpoint. However, mitotic exit was recently shown to be a function of chromosome separation during anaphase, assisted by a midzone Aurora B phosphorylation gradient - the 'ruler' model. This study found that Cdk1 remains active during anaphase due to ongoing APC/C(Cdc20)- and APC/C(Cdh1)-mediated degradation of B-type Cyclins in Drosophila and human cells. Failure to degrade B-type Cyclins during anaphase prevented mitotic exit in a Cdk1-dependent manner. Cyclin B1-Cdk1 localized at the spindle midzone in an Aurora B-dependent manner, with incompletely separated chromosomes showing the highest Cdk1 activity. Slowing down anaphase chromosome motion delayed Cyclin B1 degradation and mitotic exit in an Aurora B-dependent manner. Thus, a crosstalk between molecular 'rulers' and 'clocks' licenses mitotic exit only after proper chromosome separation (Afonso, 2019).

Taken together, this work reveals that degradation of B-type Cyclins specifically during anaphase is rate-limiting for mitotic exit among animals that diverged more than 900 million years ago. Most importantly, Cdk1 activity during anaphase was shown to be a function of chromosome separation and is spatially regulated by Aurora B localization and activity at the spindle midzone. In concert with previous work, the findings unveil an unexpected crosstalk between molecular 'rulers' (Aurora B) and 'clocks' (B-type Cyclins-Cdk1) that ensures that cells only exit mitosis after proper chromosome separation during anaphase, consistent with the previously proposed chromosome separation checkpoint hypothesis. An Aurora B-dependent spatial control mechanism regulating normal NER in human cells has been recently confirmed. However, nuclear envelope defects associated with incomplete chromosome separation during anaphase (namely, anaphase lagging chromosomes due to mitotic errors) were proposed as an inevitable pathological condition. The present work provides yet additional evidence for a molecular network operating during anaphase that promotes chromosome segregation fidelity by controlling mitotic exit in space and time. According to this model, APC/CCdc20 mediates the initial degradation of Cyclin B1 during metaphase under SAC control. The consequent decrease in Cdk1 activity as cells enter anaphase targets Aurora B to the spindle midzone (via Subito/Mklp2/kinesin-6); Aurora B at the spindle midzone (counteracted by PP1/PP2A phosphatases on chromatin establishes a phosphorylation gradient that locally delays APC/CCdc20- and APC/CCdh1-mediated degradation of residual Cyclin B1 (and possibly B3) at the spindle midzone, at least in Drosophila cells. Localization experiments in human cells suggest that Cdk1 itself might be enriched at the spindle midzone. Consequently, as chromosomes separate and move away from the spindle midzone, Cdk1 activity decreases, allowing the PP1/PP2A-mediated dephosphorylation of Cdk1 and Aurora B substrates (e.g., Lamin B and Condensin I) necessary for mitotic exit. This model is consistent with the recent demonstration that Cdk1 inactivation promotes the recruitment of PP1 phosphatase to chromosomes to locally oppose Aurora B phosphorylation and recent findings in budding yeast demonstrating equivalent phosphorylation and dephosphorylation events during mitotic exit. It is also consistent with a premature Greatwall inactivation and PP2A:B55 reactivation that would be predicted after acute Cdk1 inactivation during anaphase. Most important, this model provides an explanation for the coordinated action of two unrelated protein kinases that likely regulate multiple substrates required for mitotic exit (Afonso, 2019).

Previous landmark work has carefully monitored the kinetics of Cyclin B1 degradation in living human HeLa and rat kangaroo Ptk1 cells during mitosis, and concluded that Cyclin B1 was degraded by the end of metaphase, becoming essentially undetectable as cells entered anaphase. However, it was noticed that, consistent with the current findings, a small pool of Cyclin B1 continued to be degraded during anaphase in Ptk1 cells. Subsequent work investigating cellular response to anti-mitotic drugs has also shown that human DLD-1 cells undergoing normal mitosis entered anaphase with as much as 32% of Cyclin B1 compared to metaphase levels, suggesting that human cells enter anaphase with significant Cdk1 activity. Indeed, quantitative analysis with a FRET biosensor in human HeLa cells also revealed residual Cdk1 activity during anaphase. However, the significance of persistent Cdk1 activity for the control of anaphase duration and mitotic exit was not investigated in these original studies. Previous works also clearly demonstrated that forcing Cdk1 activity during anaphase through expression of non-degradable Cyclin B1 (and Cyclin B3 in Drosophila) prevents chromosome decondensation and NER. However, while these works suggested the existence of different Cyclin B1 thresholds that regulate distinct mitotic transitions, expression of non-degradable Cyclin B1 could be interpreted as an artificial gain of function that preserves Cdk1 activity during anaphase. For example, it was shown that expression of non-degradable Cyclin B1 during anaphase 'reactivates' the SAC, inhibiting APC/CCdc20. The current work demonstrates in five different experimental systems, from flies to humans, including primary tissues, that Cdk1 activity persists during anaphase and is rate-limiting for the control of mitotic exit. Failure to degrade B-type Cyclins during anaphase blocked cells in an anaphase-like state with separated sister chromatids that remained condensed for several hours, whereas complete Cdk1 inactivation in anaphase triggered chromosome decondensation and NER. Importantly, if a positive feedback loop imposed by phosphatases was sufficient to drive mitotic exit simply by reverting the effect of Cdk1 phosphorylation prior to anaphase, cells would exit mitosis regardless of the remaining pool of B-type Cyclins that sustains Cdk1 activity during anaphase. The main conceptual implication of these findings is that, contrary to what was previously assumed, mitotic exit is determined during anaphase and not at the metaphase-anaphase transition under SAC control (Afonso, 2019).

This model also implies that persistent Cyclin B1-Cdk1 in anaphase is spatially regulated by a midzone Aurora B gradient. Indeed, a residual pool of Cyclin B1-Cdk1 was identified enriched at the spindle midzone and midbody, this localization was dependent on Aurora B activity and localization at the spindle midzone. Interestingly, human Cyclin B2 (which contains a recognizable KEN box), as well as Cdk1, were identified at the midbody and Cdk1 inactivation during late mitosis was required for the timely completion of cytokinesis in human cells. Thus, it is possible that in human cells, Cdk1 activity during anaphase is regulated not only by Cyclin B1, but also by Cyclin B2. Importantly, this model predicted the existence of a midzone-centered Cdk1 activity gradient during anaphase, which was confirmed experimentally by targeting a FRET reporter of Cdk1 activity to chromosomes (Afonso, 2019).

Finally, the experiments indicate that Aurora B activity regulates Cyclin B1 homeostasis and consequently anaphase duration in the presence of incompletely separated chromosomes. One possibility is that direct Cyclin B1 phosphorylation by Aurora B spatially regulates Cyclin B1 degradation during anaphase, mediated by both APC/CCdc20 and APC/CCdh1. Another non-mutually exclusive possibility is that Aurora B indirectly controls Cyclin B1 during anaphase by regulating APC/CCdc20 and/or APC/CCdh1 activity, as recently shown for Cdk1. Future work will be necessary to test these hypotheses (Afonso, 2019).

In conclusion, this study has uncovered an unexpected level of regulation at the end of mitosis in metazoans and reconciled what were thought to be antagonistic models of mitotic exit relying either on molecular 'clocks' or on 'rulers'. These findings have profound implications to fundamental understanding of how tissue homeostasis is regulated, perturbation of which is a hallmark of human cancers (Afonso, 2019).

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).

In many tissues, the stem cell niche must coordinate behavior across multiple stem cell lineages. How this is achieved is largely unknown. This study has identified delayed completion of cytokinesis in germline stem cells (GSCs) as a mechanism that regulates the production of stem cell daughters in the Drosophila testis. Through live imaging, a secondary F-actin ring was shown to form through regulation of Cofilin activity to block cytokinesis progress after contractile ring disassembly. The duration of this block is controlled by Aurora B kinase. Additionally, a requirement was identified for somatic cell encystment of the germline in promoting GSC abscission. It is suggested that this non-autonomous role promotes coordination between stem cell lineages. These findings reveal the mechanisms by which cytokinesis is inhibited and reinitiated in GSCs and why such complex regulation exists within the stem cell niche (Lenhart, 2015).

This first real-time analysis of GSCs through abscission has revealed surprising complexities layered in cytokinesis. First, cytokinesis is blocked after central spindle and contractile ring disassembly and before entry to the abscission phase. This block is imposed by a secondary F-actin-ring. Second, AurB regulates the transition between phase one and phase two. That transition marks a vital step in the reinitiation of cytokinesis, permitting cytoplasmic isolation and recruitment of abscission machinery. Finally, somatic cell encystment is essential to abscission. Thus, three discrete nodes of regulation are layered on top of the canonical cytokinesis program to achieve tight temporal control over daughter cell production, and thus tissue maintenance by the resident stem cells (Lenhart, 2015).

Incomplete cytokinesis is a deeply conserved feature of germ cells that establishes the syncytium necessary for robust germline development. Differentiating germ cells appear to arrest cytokinesis immediately following contractile ring ingression because the known components of stable ring canals are identical to those of the contractile ring. It was thought that delayed cytokinesis in GSCs was simply a remnant of this conserved program. In contrast, this study found that the delay is mechanistically distinct from that occurring in differentiating germ cells. GSCs complete ingression, disassemble their contractile ring F-actin, and dissolve central spindle microtubules before engaging a ROK-LimK-Cofilin pathway to regulate a secondary F-actin ring that blocks cytokinesis progression until its disassembly at the entry to phase two (Lenhart, 2015).

Interestingly, the F-actin rings of gonial cells were not disrupted by manipulation of Cofilin activity, in contrast to their precocious disassembly in GSC-Gb pairs. This functional distinction is likely tied to the different biological goal of the stem cell versus the differentiating germ cell. One must release a differentiating daughter cell while the other must communicate syncitially for differentiation to progress normally. Ultimately, because the stem cell niche confers this functional distinction, future work will investigate whether it directly controls F-actin dynamics in the stem cell by possibly modulating Cofilin, or acts indirectly through other stem cell factors to do so (Lenhart, 2015).

These data strongly indicate that the secondary F-actin ring must be disassembled for abscission to be reinitiated. This suggests that F-actin at the IC bridge inhibits abscission, and work in other cells supports this. Inhibition of the Cofilin phosphatase, activation of AurB, depletion of phosphoinositide 5-phosphatase, or of Rab35 all lead to retention of F-actin at the IC bridge and inhibit abscission. Importantly, abscission could be restored after Rab35 depletion by forcing F-actin disassembly (Lenhart, 2015).

GSC-Gb pairs depleted for aurB fail to complete abscission prior to mitotic entry and form interconnected germ cells attached to the hub. This could suggest that AurB is normally required to promote abscission. However, expressing an activated form of Svn did not induce precocious abscission as would be expected in this model. Rather, Svn^S125E expression advanced the transition from phase one to two, while aurB depletion delayed it. These reciprocal effects suggest instead that AurB times the phase one-phase two transition. In this model, the lack of abscission in aurB mutants is an indirect consequence of spending a shorter fraction of the total cycle in phase two. For example, this study has shown that ESCRTIII is localized during phase two and in the apparent absence of central spindle microtubules. In aurB-depleted cells, there simply may not be enough time during the shortened phase two for the already compromised recruitment of ESCRTIII machinery to promote abscission prior to mitotic entry. It is also noted that the lack of a central spindle raises the issue of how ESCRTIII components are delivered to the IC bridge. Perhaps the midbody performs this role, as has been suggested for the C. elegans first cell division (Lenhart, 2015).

Recent studies have found that shrub is negatively regulated by AurB in female GSCs (Matias, 2015). Although the current results suggest that AurB activity should promote ESCRTIII function in the testis, it is compelling to speculate that AurB might control the phase one-phase two transition through shrub. Alternatively, AurB could directly control this transition by regulating disassembly of the secondary F-actin ring, as there is precedent for AurB controlling actin dynamics. For example, in the 'No Cut' pathway, maintenance of AurB activity late in cytokinesis is associated with persistence of F-actin at the IC bridge. Intriguingly, AurB can phosphorylate formin proteins and thereby regulate actin stress fiber formation. Although in this context AurB activity positively regulates actin polymerization, the interaction between AurB and formin suggests a direct link between CPC activity and actin dynamics. This connection is particularly compelling given that formins can also promote severing of actin filaments. Thus, it is intriguing to speculate that AurB phosphorylation of formins at the IC bridge in GSC-Gb pairs may promote severing of actin filaments in the secondary ring and thereby promote transition from phase one to phase two of delay (Lenhart, 2015).

Perhaps most excitingly, this study has identified non-autonomous control over GSC-Gb abscission by somatic cell encystment. This sheds light on the functional relevance of abscission delay. Encystment of spermatogonia by two somatic cells is required for proper germ cell differentiation. However, GSCs and their flanking CySCs do not coordinate daughter cell production by synchronizing their cell cycles. Linking abscission to encystment is an elegant alternative for promoting coordinated release of stem cell daughters from the niche (Lenhart, 2015).

Several questions are raised by the current observations, such as precisely when abscission is triggered relative to cyst cell engulfment of the Gb. It would be necessary to carry out live imaging simultaneously on germline and adjacent somatic cells to address this. However, imaging CySCs and cyst cells is fraught with difficulty due to their irregular morphology and small size. Thus, it has not yet been possible to image somatic cells with anywhere near the resolution achieved for GSC-Gb pairs (Lenhart, 2015).

Encystment could promote abscission through contact-dependent signaling, where CySCs or cyst cells produce the ligand. Alternatively, the abscission trigger might be mechanical, because tension has been suggested to regulate abscission in cultured cells. Here, as daughter cells migrated apart in culture following mitosis, tension along the bridge connecting them increased and this lengthened the time to abscission. Experimentally decreasing bridge tension triggered earlier abscission. In the current system, most Gbs are displaced some distance from the hub during phase two, with a consequent elongation of the IC bridge connecting those cells to the GSC. Perhaps movement of the Gb away from its mother GSC generates increased tension along the bridge. Symmetric encystment might relieve that tension by providing equalizing forces on both sides of the IC bridge, inducing abscission while ensuring that the Gb is properly associated with two somatic cells. In culture, increased tension delayed abscission by disrupting assembly of functional ESCRTIII complexes at the IC bridge. Therefore, it will be interesting to address whether ESCRTIII complexes in GSCs are temporally regulated by encystment. Whatever the mechanism, the cyst cells are clearly poised for intimate contact at the appropriate time, because the midbody remnant is sometimes taken up byÊencysting somatic cells after abscission (Lenhart, 2015).

This work has clarified the mechanism by which cytokinesis is delayed in GSCs, identifying three distinct regulatory events layered on top of the traditional program of cytokinesis. These events impose an appropriate delay, a timed reinitiation, and a regulated abscission in the GSCs. This stem cell-specific program assists in the coordinate release of differentiating daughter cells from the resident stem cell populations in this niche. Because similar requirements for synchronized daughter cell production between multiple stem cell populations exist in other tissues, it is enticing to speculate that regulated abscission might be used to promote coordination in other niches. Membrane scission is difficult to demonstrate in vivo in many systems, so it is not yet known if stem cells other than the germline exhibit abscission delay. As higher resolution methods are developed to visualize stem cell dynamics within endogenous niches, it will be interesting to see if abscission delay emerges as a conserved mechanism of niche-dependent control over stem cell proliferation (Lenhart, 2015).

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-α-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-B^S) 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-B^S arrest. Finally, the normal anaphase release of AuroraB/INCENP from kinetochores is blocked by CYC-B^S 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-B^S 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-B^S 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-B3^S, 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-B^S and not CYC-B3^S 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-B^S, 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-B^S (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-B^S-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-B^S 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-B^S arrest. Furthermore, despite the presence of only a single kinetochore, many of the chromosomes congress to a pseudometaphase plate in double parked and CYC-B^S 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-B^S-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-B^S. 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-B^S-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-B^S 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-B^S 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).

During meiosis in the females of many species, spindle assembly occurs in the absence of the microtubule-organizing centers called centrosomes. In the absence of centrosomes, the nature of the chromosome-based signal that recruits microtubules to promote spindle assembly as well as how spindle bipolarity is established and the chromosomes orient correctly toward the poles is not known. To address these questions, this study focused on the chromosomal passenger complex (CPC). The CPC localizes in a ring around the meiotic chromosomes that is aligned with the axis of the spindle at all stages. Using new methods that dramatically increase the effectiveness of RNA interference in the germline, it was shown that the CPC interacts with Drosophila oocyte chromosomes and is required for the assembly of spindle microtubules. Furthermore, chromosome biorientation and the localization of the central spindle kinesin-6 protein Subito, which is required for spindle bipolarity, depend on the CPC components Aurora B and Incenp. Based on these data it is proposed that the ring of CPC around the chromosomes regulates multiple aspects of meiotic cell division including spindle assembly, the establishment of bipolarity, the recruitment of important spindle organization factors, and the biorientation of homologous chromosomes (Radford, 2012).

Previous work using Xenopus egg extracts demonstrated that both RanGTP and the CPC are required for chromatin-induced spindle assembly. In contrast, RanGTP appears not to be required for acentrosomal spindle assembly in Drosophila (Cesario, 2011) and mouse oocytes. This study has shown that the CPC is essential for the accumulation of microtubules around the chromosomes in Drosophila oocytes, suggesting that in vivo the CPC is the critical factor for regulating acentrosomal spindle assembly. A model is presented for acentrosomal spindle assembly with implications for how the CPC simultaneously promotes bipolarity and homolog bi-orientation (Radford, 2012).

The results support a model in which the primary step in the establishment of meiotic spindle bipolarity is the accumulation of the CPC in a ring encircling the chromosomes. The enrichment of CPC proteins in a ring around the karyosome may provide the increased local concentration of Aurora B that has been postulated to be necessary to activate the Aurora B kinase for chromosome-based spindle assembly in Xenopus egg extracts. It is proposed that the CPC has two critical functions in Drosophila oocytes: it promotes microtubule accumulation near the chromosomes and also constrains microtubule growth into two poles by establishing the spindle axis. This replaces two functions of the centrosomes: recruitment of microtubules and organizing a bipolar spindle. Previous studies have suggested that the CPC promotes spindle assembly by suppressing the microtubule-depolymerizing activity of a kinesin-13 protein near the chromosomes. In contrast, this study has shown that down-regulating KLP10A, a Drosophila kinesin-13 protein known to regulate spindle length, is not a sufficient explanation for the activity of the CPC. While a role for the CPC in regulating two additional kinesin-13s encoded by the Drosophila genome, KLP59C and KLP59D cannot be ruled out, during acentrosomal spindle assembly, evidence summarized below suggests that the CPC positively regulates spindle assembly factors (Radford, 2012).

For the second function, constraining microtubule assembly towards two poles, a simple model is suggested by the shape of the ring: the ring may act like a tube that restricts microtubules to assemble in only two directions. Additionally, the CPC ring establishes the location for recruitment of other spindle assembly factors that regulate bipolarity, including Subito. A direct physical interaction between Subito and Incenp would be consistent with results showing that the mammalian Subito ortholog MKLP2 physically interacts with Aurora B and Incenp (Gruneberg, 2004). This must depend on Aurora B activity since no Subito localization was observed in plI-aurora-like kinase RNAi oocytes even though Incenp was associated with the chromatin. It is suggested that the CPC interacts with chromosomes in a ring, promotes microtubule accumulation, and recruits proteins like Subito to these microtubules, which results in the establishment or stabilization of antiparallel microtubules, spindle bipolarity, and the formation of two poles (Radford, 2012).

Subito and the CPC appear to have a mutual dependency. It has been shown previously that the meiotic central spindle localization of the CPC depended on Subito (Jang, 2005). To explain these results, it is suggested that the CPC is first recruited to the chromosomes, and then moves to the central spindle microtubules. In the absence of Subito and the central spindle microtubules, the interaction of Incenp with the chromosomes persists and the CPC does not move to the microtubules. While interacting with the chromosomes the CPC can apparently promote spindle assembly, but not bi-orientation (Radford, 2012).

What controls the localization of the CPC ring and how it gets targeted to the region between bi-oriented centromeres remains to be uncovered. In the absence of Aurora B, the localization pattern of Incenp within the karyosome is disorganized, suggesting that the kinase activity of the CPC may play a role in shaping the ring, but underlying features of the chromosomes may also be important. It is intriguing that the passenger proteins are not detected in the centromere regions as they are in mitotic and centrosomal meiotic cells. The results are consistent with data from C. elegans oocytes, showing that the CPC interacts with non-centromeric chromatin at metaphase of meiosis I. In C. elegans, the CPC forms a ring at the center of each bivalent that colocalizes with cohesion proteins distal to chiasmata. The C. elegans CPC ring is a complex structure which, like in Drosophila, contains motor proteins (Klp-19) and is required for segregation of homologs at meiosis I. The importance of non-centromeric CPC in a variety of organisms suggests that the unique demands of acentrosomal meiosis have resulted in a meiosis-specific CPC/central spindle localization pattern with a conserved role in spindle assembly and chromosome segregation. Finding out the identity or structural features of the chromosome locations to which the CPC ring localizes will be critical to understanding how the chromosomes organize acentrosomal spindles (Radford, 2012).

Centromeres are paired in Drosophila oocytes prior to NEB. Based on examination of oocytes depleted of the CPC and spindle assembly motors Subito and NCD, the following pathway leading to homolog bi-orientation is proposed. First, the CPC binds in a ring to the chromosomes and recruits spindle assembly factors such as Subito. This stage is defined by the observation that the CPC can bind chromosomes independent of microtubules and, in its absence, the microtubules and Subito fail to accumulate around the chromosomes. Second, microtubules with attachments to the chromosomes provide a poleward force on the centromeres. This stage is defined by the observation that, in the absence of the CPC, and consequently the absence of microtubules, the homologous centromeres fail to separate. Third, the homologs bi-orient through interactions with the central spindle microtubules. This stage is defined by the observation that, in sub mutants, the central spindle is absent but microtubules with attachments to the chromosomes still form and the homologous centromeres separate but fail to bi-orient (Radford, 2012).

The nature of the microtubule attachments to the chromosomes that lead to centromere separation is not known. Some previous studies have suggested that chromosome alignment depends on lateral interactions during acentrosomal meiosis. However, an alternative model incorporates an important role for kinetochore microtubules). Kinetochore microtubules in oocytes have been inferred by Hughes (2011) and could be the cold-resistant karyosome-associated microtubules observed in previous studies. Whether the microtubules connect to the chromosomes though traditional end-on kinetochore attachments or lateral attachments, it is proposed that these microtubules are bundled with central spindle microtubules to achieve bi-orientation. Interactions between central spindle microtubules and the microtubules with attachments to the chromosomes could be mediated by the kinesin-5 KLP61F or the kinesin-14 NCD. Indeed, this study has shown that NCD is required for homolog bi-orientation. The frayed spindles that are typical of ncd mutants could be explained by the loss of bundling between chromosome and central spindle microtubules (Radford, 2012).

A possible mechanism for how the CPC ring may facilitate bi-orientation at meiosis is suggested by two recent studies in mammalian mitotic and meiotic cells (Kitajima, 2011; Magidson, 2011). In both systems, prometaphase chromosomes move towards the outside edges of the developing spindle and then congress via lateral interactions to a ring around the central part of the spindle. This 'prometaphase belt' facilitates and enhances the rate of bi-orientation by bringing kinetochores into the vicinity of a high density of microtubules, which leads to stable kinetochore-microtubule attachments. It is proposed that the ring of CPC protein promotes a prometaphase belt-like organization to enhance the interaction of centromeres with a high density of microtubules in Drosophila oocytes (Radford, 2012).

Chromosome-based spindle assembly is a well described phenomenon, but the responsible chromatin-based factors in intact oocytes have not been previously identified. The current data suggests that the CPC interacts with noncentromeric chromatin and not only promotes the accumulation of microtubules around the chromosomes, but also regulates multiple aspects of spindle function, including the establishment of bipolarity and bi-orientation of homologs. Indeed, the localization to a central spindle ring and not centromeres may be critical for these functions. At this location, the CPC could regulate several different types of target protein that organize microtubules. One type is represented by Subito, which is required for spindle bipolarity, perhaps through the stabilization of antiparallel microtubules in the central spindle. Another type of target protein may function to promote microtubule attachment to the chromosomes. Indeed, these results provide the starting point for investigating what controls the localization of the CPC and what are its critical targets during acentrosomal meiosis (Radford, 2012).

The coordinated activities at centromeres of two key cell cycle kinases, Polo and Aurora B, are critical for ensuring that the two sister kinetochores of each chromosome are attached to microtubules from opposite spindle poles prior to chromosome segregation at anaphase. Initial attachments of chromosomes to the spindle involve random interactions between kinetochores and dynamic microtubules, and errors occur frequently during early stages of the process. The balance between microtubule binding and error correction (e.g., release of bound microtubules) requires the activities of Polo and Aurora B kinases, with Polo promoting stable attachments and Aurora B promoting detachment. This study concerns the coordination of the activities of these two kinases in vivo. INCENP, a key scaffolding subunit of the chromosomal passenger complex (CPC), which consists of Aurora B kinase, INCENP, Survivin, and Borealin/Dasra B, also interacts with Polo kinase in Drosophila cells. It was known that Aurora A/Bora activates Polo at centrosomes during late G2. However, the kinase that activates Polo on chromosomes for its critical functions at kinetochores was not known. This study shows that Aurora B kinase phosphorylates Polo on its activation loop at the centromere in early mitosis. This phosphorylation requires both INCENP and Aurora B activity (but not Aurora A activity) and is critical for Polo function at kinetochores. The results demonstrate clearly that Polo kinase is regulated differently at centrosomes and centromeres and suggest that INCENP acts as a platform for kinase crosstalk at the centromere. This crosstalk may enable Polo and Aurora B to achieve a balance wherein microtubule mis-attachments are corrected, but proper attachments are stabilized allowing proper chromosome segregation (Carmena, 2012).

Coordination of Polo and Aurora B activity at kinetochores is critical in early mitosis, as the two kinases play potentially antagonistic but complementary roles in regulating kinetochore-microtubule interactions. Aurora B is essential for the correction of aberrant attachments, and indeed, tethering Aurora B too close to kinetochores interferes with the formation of stable attachments. In contrast, Plk1 activity is required for initial stabilisation of microtubule attachments to kinetochores. It is suggested that interactions with INCENP may provide a mechanism to coordinate the activities of these two essential kinases during early mitosis (Carmena, 2012).

Recent studies suggest that Plk1 is activated at centrosomes when its T-loop (T210) is phosphorylated by Aurora A kinase-Bora, and that this promotes the G2/M transition upstream of Cdk1, although Polo activity is not required for mitotic entry. How Plk1 is activated at kinetochores remained an important unsolved question. The present results show that Aurora B and INCENP, which are concentrated at inner centromeres, function there to activate Polo by phosphorylating its T-loop (Carmena, 2012).

Plk1 recruitment to centromeres in late G2 has been variously proposed to be mediated by Bub1, INCENP, and BubR1. Another report implicated the self-primed interaction of Plk1 with PBIP1/CENP-U. This could potentially explain why Plk1 activity is reportedly required for its localisation to kinetochores in human cells (Carmena, 2012).

The current RNAi studies confirmed that Plk1 is partially dependent on the CPC for its centromeric localization in human cells. However, this appears not to be the case in Drosophila, where Polo is present at centromeres before NEB, at a time when INCENP is not yet concentrated at inner centromeres and before Polo^T182ph, the active form of the kinase, is detected there. Indeed, no significant decrease was observed in kinetochore-associated Polo levels after INCENP RNAi in Drosophila cells (Carmena, 2012).

Although Polo targeting to kinetochores is independent of the CPC in Drosophila, its activation there does require the CPC with active Aurora B. The data suggest that INCENP binding to Polo facilitates its subsequent activation by Aurora B kinase. Indeed, INCENP and Polo interact physically in vitro and co-immunoprecipitate in mitotic cell extracts. Although most centromeric Polo kinase is concentrated in the outer kinetochore in prophase and prometaphase, active Polo (Polo^T182ph) is also found in inner centromeres, where it overlaps with INCENP as confirmed by a proximity ligation assay (PLA)(Carmena, 2012).

A range of evidence presented in this study suggests that Aurora B is the upstream kinase responsible for Polo kinase activation at centromeres. Firstly, Aurora B phosphorylates Polo at Thr182 in vitro. Secondly, RNAi depletion of INCENP or Aurora B, but not Aurora A, reduces levels of active Polo^T182ph at kinetochores. Thirdly, tissue culture cells and third larval instar neuroblasts treated with a specific inhibitor of Drosophila Aurora B kinase show decreased levels of Polo^T182ph at kinetochores. In all of the preceding experiments, Polo^T182ph levels are affected at kinetochores but not at centrosomes, where Polo is presumably activated by Aurora A. Importantly, this involvement of Aurora B in Polo activation at centromeres discovered in Drosophila is conserved for Plk1 in human cells (Carmena, 2012).

The current results suggest a model for interactions between Polo kinase and the CPC at centromeres (see Model for the interactions between the CPC and Polo kinase at the centromere/kinetochore). In Drosophila cells, Polo targets to centromeres before the CPC is recruited by Survivin binding to histone H3T3ph (Yamagishi, 2010: see Schematic depiction of the pathways that regulate CPC targeting to centromeres). At inner centromeres of chromosomes whose kinetochores are not under tension, Polo now binds to INCENP. This promotes Polo kinase activation, as Aurora B phosphorylates Polo^T182. It is suggested that interactions with INCENP link the two complementary kinase activities, thereby potentially creating a microtubule attachment/detachment cycle at kinetochores. Such a cycle would not be possible without a balancing phosphatase activity, and PP2A-B56 has recently been shown to oppose both Aurora B and Plk1 activities at kinetochores to promote stable attachments (Carmena, 2012).

At metaphase, when chromosomes are bioriented and under tension, the CPC and Polo kinase exhibit only a partial overlap. A weakening of the INCENP/Polo PLA signals in metaphase suggests that Polo may be released from INCENP after its activation—possibly moving to the outer kinetochore. During metaphase, the CPC localizes in the inner centromere, stretching between sister kinetochores, whereas Polo and Polo^T182ph concentrate mainly at the kinetochores. This separation may be necessary to allow Polo-mediated stabilisation of kinetochore-microtubule attachments. The coordinated activities of both kinases at kinetochores and their tension-mediated separation might facilitate a dynamic equilibrium between attached and unattached kinetochores, selectively stabilizing proper chromosome attachments (Carmena, 2012).

In summary, the results reveal that INCENP and Aurora B are responsible for Polo kinase activation at centromeres but not at centrosomes during mitosis. These experiments support the hypothesis that INCENP acts as a scaffold integrating the cross-talk between these two important mitotic kinases (Carmena, 2012).

Cell cycle progression is regulated by members of the cyclin-dependent kinase (CDK), Polo and Aurora families of protein kinases. The levels of expression and localization of the key regulatory kinases are themselves subject to very tight control. There is increasing evidence that crosstalk between the mitotic kinases provides for an additional level of regulation. Previous work has shown that Aurora B activates Polo kinase at the centromere in mitosis, and that the interaction between Polo and the chromosomal passenger complex (CPC) component INCENP is essential in this activation. This report shows that Polo kinase is required for the correct localization and activity of the CPC in meiosis and mitosis. Study of the phenotype of different polo allele combinations compared to the effect of chemical inhibition revealed significant differences in the localization and activity of the CPC in diploid tissues. These results shed new light on the mechanisms that control the activity of Aurora B in meiosis and mitosis (Carmena, 2014).

Drosophila Polo and its human orthologue Polo-like kinase 1 fulfill essential roles during cell division. Members of the Polo-like kinase (Plk) family contain an N-terminal kinase domain (KD) and a C-terminal Polo-Box domain (PBD), which mediates protein interactions. How Plks are regulated in cytokinesis is poorly understood. This study shows that phosphorylation of Polo by Aurora B is required for cytokinesis. This phosphorylation in the activation loop of the KD promotes the dissociation of Polo from the PBD-bound microtubule-associated protein Map205, which acts as an allosteric inhibitor of Polo kinase activity. This mechanism allows the release of active Polo from microtubules of the central spindle and its recruitment to the site of cytokinesis. Failure in Polo phosphorylation results in both early and late cytokinesis defects. Importantly, the antagonistic regulation of Polo by Aurora B and Map205 in cytokinesis reveals that interdomain allosteric mechanisms can play important roles in controlling the cellular functions of Plks (Kachaner, 2014).

Accurate chromosome segregation during mitosis requires the physical separation of sister chromatids before nuclear envelope reassembly (NER). However, how these two processes are coordinated remains unknown. This study, carried out in Drosophila S2 cells, identified a conserved feedback control mechanism that delays chromosome decondensation and NER in response to incomplete chromosome separation during anaphase. A midzone-associated Aurora B gradient was found to monitor chromosome position along the division axis and to prevent premature chromosome decondensation by retaining Condensin I. PP1/PP2A phosphatases (see Twins) counteracted this gradient and promoted chromosome decondensation and NER. Thus, an Aurora B gradient appears to mediate a surveillance mechanism that prevents chromosome decondensation and NER until effective separation of sister chromatids is achieved. This allows the correction and reintegration of lagging chromosomes in the main nuclei before completion of NER (Afonso, 2014).

The Lethal giant larvae (Lgl) protein was discovered in Drosophila as a tumor suppressor in both neural stem cells (neuroblasts) and epithelia. In neuroblasts, Lgl relocalizes to the cytoplasm at mitosis, an event attributed to phosphorylation by mitotically activated aPKC kinase and thought to promote asymmetric cell division. This study shows that Lgl also relocalizes to the cytoplasm at mitosis in epithelial cells, which divide symmetrically. The Aurora A and Aurora B kinases directly phosphorylate Lgl to promote its mitotic relocalization, whereas aPKC kinase activity is required only for polarization of Lgl. A form of Lgl that is a substrate for aPKC, but not Aurora kinases, can restore cell polarity in lgl mutants but reveals defects in mitotic spindle orientation in epithelia. It is proposed that removal of Lgl from the plasma membrane at mitosis allows Pins/LGN to bind Dlg and thus orient the spindle in the plane of the epithelium. These findings suggest a revised model for Lgl regulation and function in both symmetric and asymmetric cell divisions (Bell, 2014).

Trimethylation at histone H3K27 is central to the polycomb repression system. Juxtaposed to H3K27 is a widely conserved phosphorylatable serine residue (H3S28) whose function is unclear. To assess the importance of H3S28, a Drosophila H3 histone mutant was generated with a serine-to-alanine mutation at position 28. H3S28A mutant cells lack H3S28ph on mitotic chromosomes but support normal mitosis. Strikingly, all methylation states of H3K27 drop in H3S28A cells, leading to Hox gene derepression and to homeotic transformations in adult tissues. These defects are not caused by active H3K27 demethylation nor by the loss of H3S28ph. Biochemical assays show that H3S28A nucleosomes are a suboptimal substrate for PRC2 (containing Esc, Su(z)12, E(z) and Nurf55), suggesting that the unphosphorylated state of serine 28 is important for assisting in the function of polycomb complexes. Collectively, these data indicate that the conserved H3S28 residue in metazoans has a role in supporting PRC2 catalysis (Yung, 2015).

This report has established a H3S28A histone mutant in Drosophila. In theory, this mutation could have two different effects on the polycomb system. (1) It could be that PcG proteins are not evicted from H3K27me3-binding sites in the absence of H3S28ph, and thus, PcG target genes might become ectopically repressed or (2) the mutation at H3S28 or the absence of H3S28ph could compromise PcG functions, resulting in derepression of PcG target genes. No evidence was found for the first possibility, although it is formally possible that H3S28 is phosphorylated under certain developmental conditions or in response to particular stimuli to counteract polycomb silencing. Instead, the data point to an inhibition of PRC2 activity by the H3S28A mutation. This inhibition is independent of active H3K27 demethylation by dUtx. Besides, RNAi against Aurora B kinase and hence depletion of H3S28ph did not hamper polycomb silencing. On the other hand, H3S28A nucleosomes proved to be a suboptimal substrate for in vitro PRC2 HMT activity. Although a 3D structure of the human Ezh2 SET domain is available, the exact contribution of the hydroxyl group of H3S28 for H3K27 methylation is difficult to deduce from the available data. vSET, the only other protein capable of H3K27 methylation in the absence of PRC2 subunits, does not require H3S28 for catalysis, whereas it does use H3A29 to define substrate specificity. Clearly, more work will be required to determine the exact structural and biochemical role of H3S28 in PRC2 catalysis. Consistent with the in vitro HMT assays, in vivo the H3S28A mutant exhibits defects in H3K27 methylation and shows similar, though milder, Hox derepression profiles and transformation phenotypes to those observed in H3K27R mutant flies (Yung, 2015).

Interestingly, the 'KS' module is frequently found in Ezh2 substrates other than K27S28 of histone H3. These include K26S27 of human histone H1 variant H1b (H1.4), K38S39 of the nuclear orphan receptor RORα, and K180S181 of STAT3. Whether these serine residues act similarly to H3S28 to support methylation of the adjacent lysine residue remains unknown. Of note, some other Ezh2 substrates can be methylated despite the lack of a 'KS' module. These include K26 of mouse histone H1 variant H1e, K49 of STAT3, and K116 of Jarid2, where the lysine residue is followed by an alanine, glutamate, and phenylalanine, respectively. Moreover, the link between peptide sequence and enzymology of Ezh2 was shown to differ in non-histone substrates. Hence, the role of serine following the Ezh2 methylation target amino acid might not be extrapolated to all other Ezh2 substrates and should be tested individually (Yung, 2015).

Previous reports revealed discrepancies in Drosophila PcG protein localization on mitotic chromosomes depending on staining protocols and tissue types. Nonetheless, live imaging of Pc-GFP, Ph-GFP, and E(z)-GFP in early Drosophila embryos has suggested that the majority of these PcG components are dissociated from mitotic chromosomes. Because stress-induced H3S28ph evicts PcG complexes during interphase, one might expect rebinding of PcG proteins on mitotic chromosomes depleted of H3S28ph. Whereas loss of Ph from mitotic chromosomes was observed in WT background, significant Ph association was not observed in H3S28A mutant condition. The reduced levels of H3K27me3 in the H3S28A mutant could contribute to this observation. Alternatively, other mechanisms might operate to dissociate the majority of PcG proteins during mitosis (Yung, 2015).

The establishment of the histone replacement system in Drosophila has proven to be an important tool to complement functional characterization of chromatin modifiers. Whereas depletion of H3K27 methylation, either by mutation of the histone mark writer E(z) or by mutation of the histone itself in the H3K27R mutant, leads to similar loss of polycomb-dependent silencing, other histone mutations revealed different phenotypes than the loss of their corresponding histone mark writers. For example, H3K4R mutations in both H3.2 and H3.3, hence a complete loss of H3K4 methylation, did not hamper active transcription. Also, the loss of H4K20 methylation upon H4K20R mutation unexpectedly supports development and does not phenocopy cell cycle and gene silencing defects reported upon the loss of the H4K20 methylase PR-Set7. In this study, by comparing the phenotype of Aurora B knockdown and H3S28A mutation in vivo, together with in vitro HMT assay, the requirement of the unmodified H3S28 residue is specifically attributed to supporting PRC2 deposition of H3K27 methylation (Yung, 2015).

Whereas the published data suggest that H3S28 phosphorylation might be important for eviction of PcG components for derepression of PcG target genes upon stimulatory cues, the data reveal a so far unacknowledged function of the unphosphorylated state of H3S28. This study shows that serine 28 is required to enable proper methylation of H3K27 by PRC2 and thus to establish polycomb-dependent gene silencing. Serine 28 of histone H3 is universally conserved in species that display canonical PRC2-dependent silencing mechanisms. Given the fact that no major mitotic defects are found upon its mutation, it is proposed that the major role of this residue is to ensure optimal PRC2 function while facilitating the removal of polycomb proteins in response to signals that induce phosphorylation (Yung, 2015).

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;polo^16-1/+ 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 polo^16-1/+ 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).

Chromosome segregation depends on kinetochores, the structures that mediate chromosome attachment to the mitotic spindle. Mutants in IPL1, which encodes a protein kinase, were isolated in a screen for budding yeast mutants that have defects in sister chromatid separation and segregation. Cytological tests show that ipl1 mutants can separate sister chromatids but are defective in chromosome segregation. Kinetochores assembled in extracts from ipl1 mutants show altered binding to microtubules. Ipl1p phosphorylates the kinetochore component Ndc10p in vitro and it is proposed that Ipl1p regulates kinetochore function via Ndc10p phosphorylation. Ipl1p localizes to the mitotic spindle and its levels are regulated during the cell cycle. This pattern of localization and regulation is similar to that of Ipl1p homologs in higher eukaryotes, such as the human aurora2 protein. Because aurora2 has been implicated in oncogenesis, defects in kinetochore function may contribute to genetic instability in human tumors (Biggins, 1999).

The conserved Ipl1 protein kinase is essential for proper chromosome segregation and thus cell viability in the budding yeast Saccharomyces cerevisiae. Sister chromatids that have separated from each other are not properly segregated to opposite poles of ipl1-2 cells. Failures in chromosome segregation are often associated with abnormal distribution of the spindle pole-associated Nuf2-GFP protein, thus suggesting a link between potential spindle pole defects and chromosome missegregation in ipl1 mutant cells. A small fraction of ipl1-2 cells also appears to be defective in nuclear migration or bipolar spindle formation. Ipl1 associates, probably directly, with the novel and essential Sli15 protein in vivo, and both proteins are localized to the mitotic spindle. Conditional sli15 mutant cells have cytological phenotypes very similar to those of ipl1 cells, and the ipl1-2 mutation exhibits synthetic lethal genetic interaction with sli15 mutations. sli15 mutant phenotype, like ipl1 mutant phenotype, is partially suppressed by perturbations that reduce protein phosphatase 1 function. These genetic and biochemical studies indicate that Sli15 associates with Ipl1 to promote its function in chromosome segregation (Kim, 1999).

Ipl1 and Sli15 are required for chromosome segregation in Saccharomyces cerevisiae. Sli15 associates directly with the Ipl1 protein kinase and these two proteins colocalize to the mitotic spindle. Sli15 stimulates the in vitro, and likely in vivo, kinase activity of Ipl1, and Sli15 facilitates the association of Ipl1 with the mitotic spindle. The Ipl1-binding and -stimulating activities of Sli15 both reside within a region containing homology to the metazoan inner centromere protein (INCENP; see Drosophila Inner centromere protein). Ipl1 and Sli15 also bind to Dam1, a microtubule-binding protein required for mitotic spindle integrity and kinetochore function. Sli15 and Dam1 are most likely physiological targets of Ipl1 since Ipl1 can phosphorylate both proteins efficiently in vitro, and the in vivo phosphorylation of both proteins is reduced in ipl1 mutants. Some dam1 mutations exacerbate the phenotype of ipl1 and sli15 mutants, thus providing evidence that Dam1 interactions with Ipl1-Sli15 are functionally important in vivo. Similar to Dam1, Ipl1 and Sli15 each bind to microtubules directly in vitro, and they are associated with yeast centromeric DNA in vivo. Given their dual association with microtubules and kinetochores, Ipl1, Sli15, and Dam1 may play crucial roles in regulating chromosome-spindle interactions or in the movement of kinetochores along microtubules (Kang, 2001).

The spindle checkpoint prevents cell cycle progression in cells that have mitotic spindle defects. Although several spindle defects activate the spindle checkpoint, the exact nature of the primary signal is unknown. The budding yeast member of the Aurora protein kinase family, Ipl1p, is required to maintain a subset of spindle checkpoint arrests. Ipl1p is required to maintain the spindle checkpoint that is induced by overexpression of the protein kinase Mps1. Inactivating Ipl1p allows cells overexpressing Mps1p to escape from mitosis and segregate their chromosomes normally. Therefore, the requirement for Ipl1p in the spindle checkpoint is not a consequence of kinetochore and/or spindle defects. The requirement for Ipl1p distinguishes two different activators of the spindle checkpoint: Ipl1p function is required for the delay triggered by chromosomes whose kinetochores are not under tension, but is not required for arrest induced by spindle depolymerization. Ipl1p localizes at or near kinetochores during mitosis, and it is proposed that Ipl1p is required to monitor tension at the kinetochore (Biggins, 2001).

Metazoans contain three aurora-related kinases. Aurora A is required for spindle formation while aurora B is required for chromosome condensation and cytokinesis. Less is known about the function of aurora C. S. pombe contains a single aurora-related kinase, Ark1. Although Ark1 protein levels remain constant as cells progress through the mitotic cell cycle, its distribution alters during mitosis and meiosis. Throughout G2 Ark1 is concentrated in one to three nuclear foci that are not associated with the spindle pole body/centromere complex. Following commitment to mitosis, Ark1 associates with chromatin and is particularly concentrated at several sites, including kinetochores/centromeres. Kinetochore/centromere association diminishes during anaphase A, after which it is distributed along the spindle. The protein becomes restricted to a small central zone that transiently enlarges as the spindle extends. As in many other systems mitotic fission yeast cells exhibit a much greater degree of phosphorylation of serine 10 of histone H3 than interphase cells. A number of studies have linked this modification with chromosome condensation. Ark1 immuno-precipitates phosphorylate serine 10 of histone H3 in vitro. This activity is highest in mitotic extracts. The following all suggest that Ark1 phosphorylates serine 10 of histone H3 in vivo: the absence of the histone H3 phospho-serine 10 epitope from mitotic cells in which the ark1(+) gene has been deleted (ark1.Delta1); the inability of these cells to resolve their chromosomes during anaphase, and the co-localization of this phospho-epitope with Ark1 early in mitosis. ark1.Delta1 cells also exhibit a reduction in kinetochore activity and a minor defect in spindle formation. Thus the enzyme activity, localization and phenotype arising from manipulations of this single fission yeast aurora kinase family member suggest that this single kinase is executing functions that are separately implemented by distinct aurora A and aurora B kinases in higher systems (Petersen, 2002).

How sister kinetochores attach to microtubules from opposite spindle poles during mitosis (bi-orientation) remains poorly understood. In yeast, the ortholog of the Aurora B-INCENP protein kinase complex (Ipl1-Sli15) may have a role in this crucial process, because it is necessary to prevent attachment of sister kinetochores to microtubules from the same spindle pole. IPL1 function was investigated in cells that cannot replicate their chromosomes but nevertheless duplicate their spindle pole bodies (SPBs). Kinetochores detach from old SPBs and reattach to old and new SPBs with equal frequency in IPL1+ cells, but remain attached to old SPBs in ipl1 mutants. This raises the possibility that Ipl1-Sli15 facilitates bi-orientation by promoting turnover of kinetochore-SPB connections until traction of sister kinetochores toward opposite spindle poles creates tension in the surrounding chromatin (Tanaka, 2002).

The spindle checkpoint inhibits anaphase until all chromosomes have established bipolar attachment. Two kinetochore states trigger this checkpoint. The absence of microtubules activates the attachment response, while the inability of attached microtubules to generate tension triggers the tension/orientation response. The processes regulated by the single aurora kinase of fission yeast, Ark1, represent a combination of the events that are regulated by aurora-A and aurora-B kinases in higher systems. The aurora kinase of budding yeast, Ipl1, is required for the tension/orientation, but not attachment, response. In contrast, the single aurora kinase of fission yeast, Ark1, is required for the attachment response. Having established that the initiator codon assigned to ark1⁺ was incorrect and that Ark1-associated kinase activity depends upon survivin function and phosphorylation, it was found that the loss of Ark1 from kinetochores by either depletion or use of a survivin mutant overides the checkpoint response to microtubule depolymerization. Ark1/survivin function is not required for the association of Bub1 (see Drosophila Bub1) or Mad3 with the kinetochores. However, it is required for two aspects of Mad2 function that accompany checkpoint activation: full-scale association with kinetochores and formation of a complex with Mad3. Neither the phosphorylation of histone H3 that accompanies chromosome condensation nor condensin recruitment to mitotic chromatin is seen when Ark1 function is compromised. Cytokinesis is not affected by Ark1 depletion or expression of the 'kinase dead' ark1.K118R mutant (Petersen, 2003).

At anaphase onset, the protease separase triggers chromosome segregation by cleaving the chromosomal cohesin complex. Cohesin destruction in metaphase was shown to be sufficient for segregation of much of the budding yeast genome, but not of the long arm of chromosome XII that contains the rDNA repeats. rDNA in metaphase, unlike most other sequences, remains in an undercondensed and topologically entangled state. Separase, concomitantly with cleaving cohesin, activates the phosphatase Cdc14. Cdc14 exerts two effects on rDNA, both mediated by the condensin complex. Lengthwise condensation of rDNA shortens the chromosome XII arm sufficiently for segregation. This condensation depends on the aurora B kinase complex. Independently of condensation, Cdc14 induces condensin-dependent resolution of cohesin-independent rDNA linkage. Cdc14-dependent sister chromatid resolution at the rDNA could introduce a temporal order to chromosome segregation (Sullivan, 2004).

A balance in the activities of the Ipl1 Aurora kinase and the Glc7 phosphatase is essential for normal chromosome segregation in yeast. This balance is modulated by the Set1 methyltransferase. Deletion of SET1 suppresses chromosome loss in ipl1-2 cells. Conversely, combination of SET1 and GLC7 mutations is lethal. Strikingly, these effects are independent of previously defined functions for Set1 in transcription initiation and histone H3 methylation. Set1 is required for methylation of conserved lysines in a kinetochore protein, Dam1. Biochemical and genetic experiments indicate that Dam1 methylation inhibits Ipl1-mediated phosphorylation of flanking serines. These studies demonstrate that Set1 has important, unexpected functions in mitosis. Moreover, these findings suggest that antagonism between lysine methylation and serine phosphorylation is a fundamental mechanism for controlling protein function (Zhang, 2005).

The data reveal unexpected functional connections between the Set1 methyltransferase and phosphorylation events governed by the Ipl1 kinase and the Glc7 phosphatase. Loss of Set1 suppresses chromosome segregation defects caused by the ipl1-2 allele and is synthetic lethal with the glc7-127 allele. The mitotic functions of Set1 require Bre2, Swd1, and Sdc1, indicating that Set1 functions in the context of the COMPASS complex to modulate Ipl1-Glc7 functions in chromosome segregation (Zhang, 2005).

Previous studies have revealed a role for Set1 and COMPASS (Complex Proteins Associated with Set1) in gene transcription that requires Paf1 and ubiquitylation of histone H2B at K123. However, the data demonstrate that deletion of PAF1 or mutation of H2B K123 cannot suppress ipl1-2. Therefore, the suppression of ipl1-2 upon deletion of SET1 is independent of COMPASS functions in transcription initiation and early elongation (Zhang, 2005).

Prior to these studies, histone H3 K4 was the only known substrate of Set1. However, loss of H3 K4 methylation is not likely the molecular basis for the observed genetic interactions between SET1, IPL1, and GLC7: (1) mutations in SET1, PAF1, or histone H2B K123 all globally diminish H3 K4 methylation, yet only SET1 deletion suppresses ipl1-2; (2) no correlation was found between the effects of deletion of other COMPASS components on H3 K4 methylation and suppression of ipl1-2; (3) mutation of H3 K4 to R suppresses ipl1-2 more weakly than does deletion or mutation of SET1, and the H3 K4R mutation is not synthetic lethal with glc7-127; (4) chromatin immunoprecipitation results indicate that little or no H3 K4 methylation occurs at centromeres in S. cerevisiae, consistent with the replacement of H3 with Cse4 in centromeric nucleosomes (Zhang, 2005).

Unlike centromeres in S. pombe and most other organisms, centromeres in S. cerevisiae are not flanked by heterochromatic repeat elements, and this yeast does not contain HP1-like proteins or Suv39 methyltransferases. H3 K9 is not methylated in S. cerevisiae, and mutations in H3 S10 do not affect chromosome segregation. Moreover, no evidence was found of global changes in phosphorylation of S10 in the absence of Set1. Therefore, the effects of Set1 loss on Ipl1 functions do not likely reflect indirect effects on modifications at S10 or K9 in H3. Rather, the results indicate that these effects are mediated through Set1-mediated methylation of at least one nonhistone substrate, Dam1 (Zhang, 2005).

How might Dam1 methylation at histone H2B K233 or K194 contribute to proper chromosome segregation? By analogy to the effects of histone methylation on the occurrence of other posttranslational modifications of the histones, K233 methylation might directly affect phosphorylation of neighboring serines. This model is consistent with the observation that Ipl1-mediated phosphorylation of methylated Dam1 peptides is inhibited in vitro, as well as genetic data that reveal functional connections between K233 and S232, S234, and S235. The data indicate that prevention of K233 methylation by set1Δ allows improved phosphorylation of Dam1 by the cripples ipl1-2 kinase, as reflected by suppression of the ipl1-2 phenotype, but might allow too much or too persistent phosphorylation by wild-type Ipl1. Conversely, the suppression of the lethality of the DAM1 K233A allele by flanking S to A mutations or by the ipl1-2 mutation (but not by S to D mutations) strongly suggests that negative effects associated with loss of Dam1 methylation can be countered by decreased phosphorylation at these sites. Several previous findings that indicate a balance in the phosphorylation and dephosphorylation of IPL1 and GLC7 substrates is essential for normal cell growth and chromosome segregation. These strongly suggest that the region between K194 and S235 is a critical module in Dam1 that is regulated by both phosphorylation and methylation (Zhang, 2005).

Several protein kinases collaborate to orchestrate and integrate cellular and chromosomal events at the G2/M transition in both mitotic and meiotic cells. During the G2/M transition in meiosis, this includes the completion of crossover recombination, spindle formation, and synaptonemal complex (SC) breakdown. Ipl1/Aurora B kinase was identified as the main regulator of SC disassembly. Mutants lacking Ipl1 or its kinase activity assemble SCs with normal timing, but fail to dissociate the central element component Zip1, as well as its binding partner, Smt3/SUMO (see Drosophila Smt3), from chromosomes in a timely fashion. Moreover, lack of Ipl1 activity causes delayed SC disassembly in a cdc5 as well as a CDC5-inducible ndt80 mutant. Crossover levels in the ipl1 mutant are similar to those observed in wild type, indicating that full SC disassembly is not a prerequisite for joint molecule resolution and subsequent crossover formation. Moreover, expression of meiosis I and meiosis II-specific B-type cyclins occur normally in ipl1 mutants, despite delayed formation of anaphase I spindles. These observations suggest that Ipl1 coordinates changes to meiotic chromosome structure with resolution of crossovers and cell cycle progression at the end of meiotic prophase (Jordan, 2009).

During mitosis and meiosis, sister chromatid cohesion resists the pulling forces of microtubules, enabling the generation of tension at kinetochores upon chromosome biorientation. How tension is read to signal the bioriented state remains unclear. Shugoshins form a pericentromeric platform that integrates multiple functions to ensure proper chromosome biorientation. This study shows that budding yeast shugoshin Sgo1 (see Drosophila Mei-S332) dissociates from the pericentromere reversibly in response to tension. The antagonistic activities of the kinetochore-associated Bub1 kinase and the Sgo1-bound phosphatase protein phosphatase 2A (PP2A)-Rts1 (see Drosophila Twins) underlie a tension-dependent circuitry that enables Sgo1 removal upon sister kinetochore biorientation. Sgo1 dissociation from the pericentromere triggers dissociation of condensin and Aurora B from the centromere, thereby stabilizing the bioriented state. Conversely, forcing sister kinetochores to be under tension during meiosis I leads to premature Sgo1 removal and precocious loss of pericentromeric cohesion. Overall, this study shows that the pivotal role of shugoshin is to build a platform at the pericentromere that attracts activities that respond to the absence of tension between sister kinetochores. Disassembly of this platform in response to intersister kinetochore tension signals the bioriented state. Therefore, tension sensing by shugoshin is a central mechanism by which the bioriented state is read (Nerusheva, 2014).

The Shugoshin/Aurora circuitry that controls the timely release of cohesins from sister chromatids in meiosis and mitosis is widely conserved among eukaryotes, although little is known about its function in organisms whose chromosomes lack a localized centromere. This study shows that C. elegans chromosomes rely on an alternative mechanism to protect meiotic cohesin that is shugoshin-independent and instead involves the activity of a new chromosome-associated protein named LAB-1 (Long Arm of the Bivalent). LAB-1 preserves meiotic sister chromatid cohesion by restricting the localization of the C. elegans Aurora B kinase, AIR-2, to the interface between homologs via the activity of the PP1/Glc7 phosphatase GSP-2. The localization of LAB-1 to chromosomes of dividing embryos and the suppression of mitotic-specific defects in air-2 mutant embryos with reduced LAB-1 activity support a global role of LAB-1 in antagonizing AIR-2 in both meiosis and mitosis. Although the localization of a GFP fusion and the analysis of mutants and RNAi-mediated knockdowns downplay a role for the C. elegans shugoshin protein in cohesin protection, shugoshin nevertheless helps to ensure the high fidelity of chromosome segregation at metaphase I. It is proposed that, in C. elegans, a LAB-1-mediated mechanism evolved to offset the challenges of providing protection against separase activity throughout a larger chromosome area (de Carvalho, 2009).

There are multiple condensin-related complexes in early C. elegans embryos. In addition to the condensin complex, whose associated subunits have not yet been defined, embryos also contain the dosage compensation complex that also has two SMC proteins as core components. MIX-1 is present in both the condensin complex and the dosage compensation complex, whereas DPY-27 (a SMC-4-related protein) and DPY-26 (a protein with limited homology to Dm Barren) are solely involved in dosage compensation. Localization of MIX-1 to mitotic chromatin is independent of DPY-26, yet its localization to X chromosomes in hermaphrodites is DPY-26 dependent. The existence of multiple modes of MIX-1 binding to chromatin is compatible with the finding that condensin appears to have both AIR-2-independent and AIR-2-dependent interactions with chromatin (Kaitna, 2002).

Surprisingly, condensin function prior to nuclear envelope breakdown is AIR-2 independent, even though the mitotic recruitment of condensin to chromatin is AIR-2 dependent. The tools are currently unavailable to test if the mitotic accumulation of condensin is of physiological significance. Importantly, it is also observed that chromatin condensation occurs after nuclear envelope breakdown in condensin-depleted embryos. While it is possible that residual condensin remains after RNAi depletion and mediates this condensation, this seems unlikely, since depletion of either subunit alone or both together caused the same fully penetrant phenotype. It appears more likely that condensin-independent pathways contribute to chromosome condensation (Kaitna, 2002).

Several lines of evidence suggest that AIR-2 kinase activity promotes resolution of sister chromatid cohesion during meiosis I. This includes the remarkable concordance between AIR-2 localization and the sites where sister chromatid cohesion is lost. In addition, a phosphatase, GLC7, that is antagonistic to AIR-2 is required to prevent precocious separation of bivalents into chromatids. While the mechanism that targets AIR-2 to this discrete site is not yet clear, AIR-2 localization in diakinesis is strictly dependent upon the presence of sister chromatid cohesion, and its discrete localization requires recombination between homologs. It will be important to decipher why AIR-2 localizes to the region distal to the recombination event and why GLC-7 activity appears to predominate elsewhere. The meiotic function of AIR-2 contrasts sharply with its mitotic function, where it is dispensable for resolution of sister chromatid cohesion. The fact that the air-2(or207ts) allele exhibits penetrant defects in mitotic divisions yet no defects during meiosis provides additional evidence for mechanistic differences in AIR-2 function during mitosis and meiosis (Kaitna, 2002).

Although AIR-2 is required for proper congression of mitotic chromosomes to the metaphase plate, it is not required for this process during meiosis. However, there are significant differences in the organization of mitotic and meiotic kinetochores in C. elegans. Whereas mitotic chromosomes are holocentric, meiotic chromosomes are functionally monocentric in both meiotic divisions. The kinetic end of the chromosome is not predetermined -- rather, it is thought to be positioned at the end most distant from the crossover. The other end adopts the function of the centromere at meiosis II. Several organisms that have holocentric mitotic chromosomes have functionally monocentric meiotic chromosomes, including a variety of nematodes and arthropods. Recent ultrastructural studies have revealed a common appearance of the kinetic faces of chromosomes during meiosis and mitosis in C. elegans. However, earlier studies using different methods demonstrated a distinct kinetochore structure at the ultrastructural level during mitosis, yet, during meiosis, microtubules appear to insert directly into chromatin. Functional evidence also indicates differences between mitotic and meiotic kinetochores in C. elegans, since some factors that are critical for mitotic chromosome segregation are not critical for meiotic chromosome segregation. As shown in this study, the condensin complex does not appear to be required for meiosis, although it is essential for mitosis. Similarly, HCP-3 (the CENP-A homolog) and HCP-4 (the CENP-C homolog) are essential during mitotic chromosome segregation but only exhibit weak meiotic phenotypes. Although HCP-3 is present on the entire bivalent at diakinesis/metaphase I, this localization does not necessarily imply that it is functional, since, in mammalian cells, ectopic localization of CENP-A is insufficient to generate a functional kinetochore (Kaitna, 2002).

During meiosis, AIR-2 marks the region of the chromosome in which cohesion is lost during the first meiotic division. This region is defined by the position of the crossover that occurs during the pachytene stage. In C. elegans, usually one crossover event occurs per chromosome, regardless of the length of the chromosome or the length of recombinogenic region. Interestingly, several organisms that are holocentric during mitosis and functionally monocentric during meiosis share the additional property that only one crossover usually occurs per bivalent. One consequence of the single crossover event is that there is a single discrete region on each bivalent in which sister chromatid cohesion must be released to allow homologs to separate at meiosis I. This is in stark contrast to the situation in organisms with multiple crossover events in which sister chromatid cohesion must be released in several noncontiguous regions to allow homologs to separate (Kaitna, 2002).

In principle, separation of homologs in meiosis I could proceed by two mechanistically distinct pathways. The challenge is to destabilize sister chromatid cohesion in a regional manner so that homologs can segregate during meiosis I, while maintaining a connection between sister chromatids so that they can segregate from each other in meiosis II. One solution is to selectively destroy sister chromatid cohesion distal to the chiasmata. The data suggest that, in C. elegans, AIR-2 may regulate this selective destruction. Whereas this subset of cohesion is well defined in C. elegans, this is not the case in organisms with multiple chiasmata, and cohesion must be destroyed in numerous regions dispersed throughout the chromosome. The alternative solution is to selectively protect cohesion in the vicinity of the kinetochore. Indeed, there is evidence for centromeric protection of cohesion in yeast and Drosophila (Kaitna, 2002).

In conclusion, these results indicate that aurora B kinase, AIR-2, mediates both meiotic and mitotic chromosome segregation. Interestingly, AIR-2 acts differently in these two processes. In mitosis, AIR-2 prevents merotelic attachments and perhaps promotes a subset of condensin-dependent processes; this function is indicated by the failure of chromosomes to properly congress to form an ordered metaphase plate in air-2(or207ts) embryos. During mitosis, AIR-2 is not required for separation of sister chromatids. In contrast, during meiosis I, AIR-2 appears to be involved in the resolution of cohesion, whereas it is not obviously required for proper positioning of the bivalents on the meiotic plate. It is speculated that the role defined in this study for aurora B in mitotic chromosome segregation may apply to many organisms. It is further speculated that the meiotic function defined for AIR-2 may be restricted to organisms that have a single crossover event per chromosome during meiotic prophase and in which the kinetic end of the meiotic chromosome is not invariant. Finally, these data suggest that, while condensin plays an important role in chromosome organization, there are likely additional factors that also contribute to the condensation of mitotic chromosomes (Kaitna, 2002).

The Aurora kinases control multiple aspects of mitosis, among them centrosome maturation, spindle assembly, chromosome segregation, and cytokinesis. Aurora activity is regulated in part by a subset of Aurora substrates that, once phosphorylated, can enhance Aurora kinase activity. Aurora A substrate activators include TPX2 and Ajuba, whereas the only known Aurora B substrate activator is the chromosomal passenger INCENP. The C. elegans Tousled kinase TLK-1 is a second substrate activator of the Aurora B kinase AIR-2. Tousled kinase (Tlk) expression and activity have been linked to ongoing DNA replication, and Tlk can phosphorylate the chromatin assembly factor Asf (see Drosophila Asf). TLK-1 is phosphorylated by AIR-2 during prophase/prometaphase, and phosphorylation increases TLK-1 kinase activity in vitro. Phosphorylated TLK-1 increases AIR-2 kinase activity in a manner that is independent of TLK-1 kinase activity but depends on the presence of ICP-1/INCENP. In vivo, TLK-1 and AIR-2 cooperate to ensure proper mitotic chromosome segregation. It is concluded that the C. elegans Tousled kinase TLK-1 is a substrate and activator of the Aurora B kinase AIR-2. These results suggest that Tousled kinases have a previously unrecognized role in mitosis and that Aurora B associates with discrete regulatory complexes that may impart distinct substrate specificities and functions to the Aurora B kinase (Han, 2005).

The Aurora B kinase is the enzymatic core of the chromosomal passenger complex, which is a critical regulator of mitosis. To identify novel regulators of Aurora B, a genome-wide screen was performed for suppressors of a temperature-sensitive lethal allele of the C. elegans Aurora B kinase AIR-2. This screen uncovered a member of the Afg2/Spaf subfamily of Cdc48-like AAA ATPases as an essential inhibitor of AIR-2 stability and activity. Depletion of CDC-48.3 restores viability to air-2 mutant embryos and leads to abnormally high AIR-2 levels at the late telophase/G1 transition. Furthermore, CDC-48.3 binds directly to AIR-2 and inhibits its kinase activity from metaphase through telophase. While canonical p97/Cdc48 proteins have been assigned contradictory roles in the regulation of Aurora B, these results identify a member of the Afg2/Spaf AAA ATPases as a critical in vivo inhibitor of this kinase during embryonic development (Heallen, 2008).

The Aurora A and B protein kinases are key players in mitotic control and the etiology of human cancer. Despite the near identity of amino acid sequence in the catalytic domain, monomeric Aurora B is 50 fold lower in activity than monomeric Aurora A, and previous studies have shown that TPX2 binding to the catalytic domain activates Aurora A but not Aurora B. This study identifies G205 in Xenopus Aurora A as a key determinant of both intrinsic activity and regulation by TPX2. Mutation of G205 in Aurora A to N, the equivalent residue in Aurora B, has no effect on autophosphorylation of the T-loop but leads to a 10-fold loss of specific activity, whereas mutation of N158 in Aurora B to G causes a 350-fold increase in specific activity. G205 N Aurora A is still activated by TPX2, but protection of pT295 from dephosphorylation by protein phosphatase 1 is abolished. Structural analysis of these effects suggests that the G205 forms a pivot point in the enzyme that results in movement of the N-terminal domain glycine-rich loop closer to the ATP binding site of the enzyme and also moves the C-helix slightly closer to the activation loop. Changes in these positions are comparable to those reported for other protein kinases and demonstrate that phosphorylation of the activation loop alone is not sufficient for enzyme activation. The generation of an activated mutant of Aurora B will be important for studying its role in cell cycle control and tumorigenesis (Eyers, 2005).

As a component of the 'chromosomal passenger protein complex,' the aurora B kinase is associated with centromeres during prometaphase and with midzone microtubules during anaphase and is required for both mitosis and cytokinesis. Ablation of aurora B causes defects in both prometaphase chromosomal congression and the spindle checkpoint; however, an understanding of the mechanisms underlying these defects remains unclear. To address this question, chromosomal movement, spindle organization, and microtubule motor distribution has been examined in NRK cells transfected with a kinase-inactive, dominant-negative mutant of aurora B, aurora B(K-R). In cells overexpressing aurora B(K-R) fused with GFP, centromeres moved in a synchronized and predominantly unidirectional manner, as opposed to the independent, bidirectional movement in control cells expressing a similar level of wild-type aurora B-GFP. In addition, most kinetochores became physically separated from spindle microtubules, which appeared as a striking bundle between the spindle poles. These defects were associated with a microtubule-dependent depletion of motor proteins dynein and CENP-E from kinetochores. These observations suggest that aurora B regulates the association of motor proteins with kinetochores during prometaphase. Interactions of kinetochore motors with microtubules may in turn regulate the organization of microtubules, the movement of prometaphase chromosomes, and the release of the spindle checkpoint (Murata-Hori, 2002).

It is proposed that a substrate of the aurora B kinase, which may be an adaptor protein or motor proteins themselves, is required for the stable association of dynein and CENP-E at kinetochores. In the absence of the kinase activity, these motors are released prematurely from kinetochores as soon as they come into contact with microtubules, whereas in control cells they are released only following chromosomal congression. Since dynein contributes to the poleward movement of the chromosomes while CENP-E may be involved in the attachment of chromosomes to microtubules, loss of these motor proteins may account for the defects in chromosomal movements and kinetochore-microtubule interactions. The residual, synchronized chromosomal movements, without kinetochore fibers, are likely dragged by the 'polar ejection forces' on chromosome arms; these forces sweep the chromosomes into elongated clusters along the spindle axis. In addition, without the attachment to kinetochores, the microtubule bundling activity responsible for the formation of kinetochore fibers would induce the formation of a single microtubule bundle between the spindle poles. Therefore, deactivation of aurora B causes premature dissociation of these motors from kinetochores and leads to separation of chromosomes from microtubules and release of the spindle checkpoint. The lack of kinetochore association also causes microtubules to form a single bundle instead of multiple kinetochore fibers (Murata-Hori, 2002).

How kinetochores correct improper microtubule attachments and regulate the spindle checkpoint signal is unclear. In budding yeast, kinetochores harboring mutations in the mitotic kinase Ipl1 fail to bind chromosomes in a bipolar fashion. In C. elegans and Drosophila, inhibition of the Ipl1 homolog, Aurora B kinase, induces aberrant anaphase and cytokinesis. To study Aurora B kinase in vertebrates, mitotic XTC cells were microinjected with inhibitory antibody and several related effects were found. After injection of the antibody, some chromosomes failed to congress to the metaphase plate, consistent with a conserved role for Aurora B in bipolar attachment of chromosomes. Injected cells exited mitosis with no evidence of anaphase or cytokinesis. Injection of anti-Xaurora B antibody also altered the microtubule network in mitotic cells with an extension of the astral microtubules and a reduction of kinetochore microtubules. Finally, inhibition of Aurora B in cultured cells and in cycling Xenopus egg extracts caused escape from the spindle checkpoint arrest induced by microtubule drugs. These findings implicate Aurora B as a critical coordinator relating changes in microtubule dynamics in mitosis, chromosome movement in prometaphase and anaphase, signaling of the spindle checkpoint, and cytokinesis (Kallio, 2002).

The proper segregation of sister chromatids in mitosis depends on bipolar attachment of all chromosomes to the mitotic spindle. The small molecule Hesperadin has been identified as an inhibitor of chromosome alignment and segregation. The data imply that Hesperadin causes this phenotype by inhibiting the function of the mitotic kinase Aurora B. Mammalian cells treated with Hesperadin enter anaphase in the presence of numerous monooriented chromosomes, many of which may have both sister kinetochores attached to one spindle pole (syntelic attachment). Hesperadin also causes cells arrested by taxol or monastrol to enter anaphase within <1 h, whereas cells in nocodazole stay arrested for 3-5 h. Together, these data suggest that Aurora B is required to generate unattached kinetochores on monooriented chromosomes, which in turn could promote bipolar attachment as well as maintain checkpoint signaling (Hauf, 2003).

The spindle assembly checkpoint very faithfully ensures that anaphase is not initiated before all chromosomes have achieved bipolar attachment. In striking contrast, cells treated with Hesperadin readily enter anaphase in the presence of monooriented chromosomes. Likewise, Hesperadin is able to override the checkpoint arrest caused by taxol and monastrol, but Aurora B function is not required for several hours to maintain a checkpoint arrest induced by nocodazole. This situation is reminiscent of the role of Ipl1 in budding yeast in that Aurora B appears to be required for checkpoint signaling in the absence of tension but not in the absence of kinetochore attachments. It is possible that Aurora B is required to directly activate checkpoint proteins in the absence of tension, independent of its role in the correction of syntelic attachment. But because the correction function is thought to be activated by the lack of tension, it is also conceivable that Aurora B is indirectly required for checkpoint signaling. According to this model, Aurora B would destabilize microtubule-kinetochore interactions at kinetochores that are not under proper tension or that impinge on the kinetochore at too acute an angle, and the resulting kinetochores that are either unattached or only occupied with low numbers of microtubules would then generate the primary signal for checkpoint signaling. Hesperadin-treated cells would enter anaphase only once all kinetochores had been fully attached, which would then abolish checkpoint signaling. Because kinetochore attachment is a stochastic process, this model predicts that cells enter anaphase at different times after nuclear envelope breakdown (NEB). Indeed, a high intercell variability is observed between NEB and the onset of anaphase in Hesperadin-treated cells (Hauf, 2003).

This model also fits well with the observation that cells arrested with either taxol or monastrol always contain at least one kinetochore that appears to be unattached. Taxol- and monastrol-treated cells may therefore be arrested by the spindle checkpoint because Aurora B maintains a dynamic equilibrium between attached and unattached kinetochores. Inhibition of Aurora B would overcome this arrest because all kinetochores would eventually become fully attached. As predicted by this model, Hesperadin addition to monastrol-treated Ptk1 cells decreases the number of kinetochores staining with Mad2, suggesting that the monotelic chromosomes that existed in monastrol-arrested cells had been converted into syntelic chromosomes once Aurora B was inhibited (Hauf, 2003).

The stabilization of improper microtubule attachments is sufficient to explain the precocious exit from mitosis that Hesperadin induces in monastrol- and taxol-treated cells. However, even under conditions where none of the kinetochores are attached, Aurora B function is required to maintain checkpoint signaling over prolonged periods of time, indicating that it might also have a direct role in the spindle assembly checkpoint. Consistent with this notion, kinetochore localization of the checkpoint kinases BubR1 and Bub1 was found to be impaired in Hesperadin-treated cells. It is conceivable that Mad2, which is still present at kinetochores in cells treated with nocodazole and Hesperadin, is sufficient to sustain the transient mitotic delay that is observed. In contrast, the low levels of Mad2 at kinetochores in taxol-arrested cells might not be sufficient to delay cells in mitosis when BubR1 is depleted from kinetochores by Hesperadin (Hauf, 2003).

In summary, the data suggest that Aurora B has a dual role. It acts in the destabilization of improper microtubule attachments, which indirectly keeps checkpoint signaling active, but it also could have a more direct role in the spindle assembly checkpoint. This is consistent with data from budding yeast, where it was found that Ipl1 is required for the spindle assembly checkpoint in a kinetochore-dependent, but probably also in a kinetochore-independent, manner. Likewise, Aurora B antibodies have been shown to overcome a nocodazole-induced arrest both in Xenopus egg extracts and cultured cells, also suggesting a direct role of Aurora B in the spindle assembly checkpoint (Hauf, 2003).

BubR1 and Bub1 could be Aurora B substrates that play a role in either of these pathways, or in both. It is conceivable that BubR1 and Bub1 themselves have dual roles. Both proteins have been shown to be required for checkpoint signaling in the presence of unattached kinetochores. Interestingly, in some experimental settings, their kinase activity is not required for checkpoint function, but Bub1's kinase activity is essential for a genetically separable function that may be required for microtubule-kinetochore attachments. It will therefore be interesting to test if the kinase activity of both Bub1 and BubR1 and their Aurora B-dependent recruitment to kinetochores is required to regulate kinetochore attachments (Hauf, 2003).

Sister kinetochores must bind microtubules in a bipolar fashion to equally segregate chromosomes during mitosis. The molecular mechanisms underlying this process remain unclear. Aurora B likely promotes chromosome biorientation by regulating kinetochore-microtubule attachments. MCAK (mitotic centromere-associated kinesin) is a Kin I kinesin that can depolymerize microtubules. These two proteins both localize to mitotic centromeres and have overlapping mitotic functions, including regulation of microtubule dynamics, proper chromosome congression, and correction of improper kinetochore-microtubule attachments. Aurora B is shown to phosphorylate and regulate MCAK both in vitro and in vivo. Specifically, six Aurora B phosphorylation sites map on MCAK in both the centromere-targeting domain and the neck region. Aurora B activity was required to localize MCAK to centromeres, but not to spindle poles. Aurora B phosphorylation of serine 196 in the neck region of MCAK inhibits its microtubule depolymerization activity. This key site is phosphorylated at centromeres and anaphase spindle midzones in vivo. However, within the inner centromere there are pockets of both phosphorylated and unphosphorylated MCAK protein, suggesting that phosphate turnover is crucial in the regulation of MCAK activity. Addition of anti-p-S196 antibodies to Xenopus egg extracts or injection of anti-p-S196 antibodies into cells causes defects in chromosome positioning and/or segregation. Thus there is a direct link between the microtubule depolymerase MCAK and Aurora B kinase. These data suggest that Aurora B both positively and negatively regulates MCAK during mitosis. It is proposed that Aurora B biorients chromosomes by directing MCAK to depolymerize incorrectly oriented kinetochore microtubules (Lan, 2004).

Chromosome biorientation or bipolar kinetochore attachment is essential for equal segregation of duplicated genomes. Bipolar attachment of sister kinetochores to microtubules from opposite poles (amphitelic attachment) is a key event to allow accurate chromosome segregation during anaphase. Failure of this process is the predominant cause of aneuploidy in cultured mammalian cells. Kinetochores that are bound by microtubules from both poles (merotelic attachment) generate lagging chromosomes in anaphase that are often missegregated. Chromosomes with both kinetochores bound by microtubules from a single pole (syntelic) often have difficulty in congression, and both sisters segregate to one daughter cell. How do kinetochores resolve merotelic and syntelic attachments? A model is presented for the regulation of MCAK activity by Aurora B phosphorylation. Aurora B localizes to centromeres phosphorylates MCAK on multiple sites to facilitate centromere loading of MCAK. This ensures that MCAK is placed on the centromere in an inactive state that allows kinetochores to bind microtubules during prometaphase. Syntelic or merotelic attachment would lead to the local activation of MCAK to depolymerize the incorrectly attached microtubule. This cycle repeats until proper amphitelic attachment is achieved (Lan, 2004).

A model is favored in which there are redundant pathways in vertebrates where Aurora B regulates kinetochore-microtubule binding through MCAK and additional substrates at the kinetochore. Blocking centromeric MCAK function generates chromosome congression and segregation defects that are less severe than inhibiting Aurora B, suggesting that there are additional Aurora B targets. In budding yeast, Ipl1 phosphorylates three subunits of the Dam1 microtubule-interacting complex and Mif2 of the Mtw1 complex, but how this affects biorientation is unclear. In vertebrates, the motor proteins CENP-E and dynein depend on Aurora B for proper kinetochore localization. Both proteins are involved in chromosome movements, but neither of them has been implicated in biorientation. Identifying novel kinetochore-microtubule interacting activities that are regulated by Aurora B is crucial to decipher the mechanism of chromosome biorientation in vertebrates (Lan, 2004).

Thus vertebrate Aurora B kinase regulates the microtubule depolymerase activity of the centromeric fraction of MCAK by direct phosphorylation. These data provide a working model of how MCAK activity is regulated in mitosis and a molecular explanation of how Aurora B activity is involved in regulating the biorientation of kinetochore-microtubule attachments (Lan, 2004).

Cell division is finely controlled by various molecules including small G proteins and kinases/phosphatases. Among these, Aurora B, RhoA, and the GAP MgcRacGAP have been implicated in cytokinesis, but their underlying mechanisms of action have remained unclear. MgcRacGAP is shown to colocalize with Aurora B and RhoA, but not Rac1/Cdc42, at the midbody. Aurora B phosphorylates MgcRacGAP on serine residues and this modification induces latent GAP activity toward RhoA in vitro. Expression of a kinase-defective mutant of Aurora B disrupts cytokinesis and inhibits phosphorylation of MgcRacGAP at Ser387, but not its localization to the midbody. Overexpression of a phosphorylation-deficient MgcRacGAP-S387A mutant, but not phosphorylation-mimic MgcRacGAP-S387D mutant, arrests cytokinesis at a late stage and induces polyploidy. Together, these findings indicate that during cytokinesis, MgcRacGAP, a GAP for Rac/Cdc42, is functionally converted to a RhoGAP through phosphorylation by Aurora B (Minoshima, 2003).

Cell division is regulated by protein kinases of the Cdk, Polo, and Aurora families. Although it has long been established that temporal control is central to the coordinated action of these kinases, the importance of spatial regulation has only recently been appreciated and is still poorly understood. The kinesin-6 family motor protein MKlp1 is a key regulator of cytokinesis and an ideal substrate for studying spatially regulated protein-phosphorylation events. MKlp1 is negatively regulated by Cdk1 phosphorylation during metaphase and becomes activated in anaphase when cleavage-furrow assembly commences. Aurora B phosphorylates MKlp1 during anaphase and is required for its function in cytokinesis. Another kinesin-6 family motor, MKlp2, mediates the relocation of Aurora B from the centromeres to the central spindle at the onset of anaphase. This study demonstrates that this process is required for the phosphorylation of MKlp1 at S911, an Aurora B consensus site overlapping a bipartite nuclear localization sequence (NLS). MKlp1(S911A) targets to the central spindle but is prematurely imported into the nucleus and fails to support cytokinesis. Spatial restriction of Aurora B to the central spindle by MKlp2 therefore regulates MKlp1 during cytokinesis in human cells (Neef, 2006).

Three lines of investigation have suggested that interactions between Survivin and the chromosomal passenger proteins INCENP and Aurora-B kinase may be important for mitotic progression. (1) Interference with the function of Survivin/BIR1, INCENP, or Aurora-B kinase leads to similar defects in mitosis and cytokinesis. (2) INCENP and Aurora-B exist in a complex in Xenopus eggs and in mammalian cultured cells. (3) Interference with Survivin or INCENP function causes Aurora-B kinase to be mislocalized in mitosis in both C. elegans and vertebrates. Evidence is provided that Survivin, Aurora-B, and INCENP interact physically and functionally. Direct visualization of Survivin-GFP in mitotic cells reveals that it localizes identically to INCENP and Aurora-B. Survivin binds directly to both Aurora-B and INCENP in both yeast two-hybrid and in vitro pull-down assays. The in vitro interaction between Survivin and Aurora-B is extraordinarily stable in that it resists 3 M NaCl. Finally, Survivin and INCENP interact functionally in vivo; in cells in which INCENP localization is disrupted, Survivin adheres to the chromosomes and no longer concentrates at the centromeres or transfers to the anaphase spindle midzone. The data provide the first biochemical evidence that Survivin can interact directly with members of the chromosomal passenger complex (Wheatley, 2001).

The Aurora B kinase complex is a critical regulator of chromosome segregation and cytokinesis. In Caenorhabditis elegans, AIR-2 (Aurora B) function requires ICP-1 (Incenp) and BIR-1 (Survivin). In various systems, Aurora B binds to orthologues of these proteins. Through genetic analysis, a new subunit of the Aurora B kinase complex, CSC-1, an ortholog of Borealin/Dasra (see Droosphila Borealin-related), has been identified. C. elegans embryos depleted of CSC-1, AIR-2, ICP-1, or BIR-1 have identical phenotypes. CSC-1, BIR-1, and ICP-1 are interdependent for their localization, and all are required for AIR-2 localization. In vitro, CSC-1 binds directly to BIR-1. The CSC-1/BIR-1 complex, but not the individual subunits, associates with ICP-1. CSC-1 associates with ICP-1, BIR-1, and AIR-2 in vivo. ICP-1 dramatically stimulates AIR-2 kinase activity. This activity is not stimulated by CSC-1/BIR-1, suggesting that these two subunits function as targeting subunits for AIR-2 kinase (Romano, 2003).

The function of the Aurora B kinase at centromeres and the central spindle is crucial for chromosome segregation and cytokinesis, respectively. This study investigates regulation of human Aurora B by its complex partners, inner centromere protein (INCENP) and survivin. Overexpression of a catalytically inactive, dominant-negative mutant of Aurora B impairs the localization of the entire Aurora B/INCENP/survivin complex to centromeres and the central spindle and severely disturbs mitotic progression. Similar results were also observed after depletion, by RNA interference, of either Aurora B, INCENP, or survivin. These data suggest that Aurora B kinase activity and the formation of the Aurora B/INCENP/survivin complex both contribute to its proper localization. Using recombinant proteins, it was found that Aurora B kinase activity is stimulated by INCENP and that the C-terminal region of INCENP is sufficient for activation. Under identical assay conditions, survivin does not detectably influence kinase activity. Human INCENP is a substrate of Aurora B and mass spectrometry identified three consecutive residues (threonine 893, serine 894, and serine 895) containing at least two phosphorylation sites. A nonphosphorylatable mutant (TSS893-895AAA) is a poor activator of Aurora B, demonstrating that INCENP phosphorylation is important for kinase activation (Honda, 2003).

The spindle checkpoint prevents anaphase onset until all the chromosomes have successfully attached to the spindle microtubules. The mechanisms by which unattached kinetochores trigger and transmit a primary signal are poorly understood, although it seems to be dependent at least in part, on the kinetochore localization of the different checkpoint components. By using protein immunodepletion and mRNA translation in Xenopus egg extracts, the hierarchic sequence and the interdependent network that governs protein recruitment at the kinetochore in the spindle checkpoint pathway was studied. The results show that the first regulatory step of this cascade is defined by Aurora B/INCENP complex. Aurora B/INCENP controls the activation of a second regulatory level by inducing at the kinetochore the localization of Mps1, Bub1, Bub3 (see Drosophila Bub3), and CENP-E. This localization, in turn, promotes the recruitment to the kinetochore of Mad1/Mad2, Cdc20, and the anaphase promoting complex (APC). Unlike Aurora B/INCENP, Mps1, Bub1, and CENP-E, the downstream checkpoint protein Mad1 does not regulate the kinetochore localization of either Cdc20 or APC. Similarly, Cdc20 and APC do not require each other to be localized at these chromosome structures. Thus, at the last step of the spindle checkpoint cascade, Mad1/Mad2, Cdc20, and APC are recruited at the kinetochores independently from each other (Vigneron, 2004).

The chromosomal passenger complex of Aurora B kinase, INCENP, and Survivin has essential regulatory roles at centromeres and the central spindle in mitosis. Borealin, a novel member of the complex, is described in this study. Approximately half of Aurora B in mitotic cells is complexed with INCENP, Borealin, and Survivin. Borealin binds Survivin and INCENP in vitro. A second complex contains Aurora B and INCENP, but no Borealin or Survivin. Depletion of Borealin by RNA interference delays mitotic progression and results in kinetochore-spindle misattachments and an increase in bipolar spindles associated with ectopic asters. The extra poles, which apparently form after chromosomes achieve a bipolar orientation, severely disrupt the partitioning of chromosomes in anaphase. Borealin depletion has little effect on histone H3 serine10 phosphorylation. These results implicate the chromosomal passenger holocomplex in the maintenance of spindle integrity and suggest that histone H3 serine10 phosphorylation is performed by an Aurora B-INCENP subcomplex (Gassmann, 2004).

Proper chromosome segregation requires the attachment of sister kinetochores to microtubules from opposite spindle poles to form bi-oriented chromosomes on the metaphase spindle. The chromosome passenger complex containing Survivin and the kinase Aurora B regulates this process from the centromeres. A de-ubiquitinating enzyme, hFAM, regulates chromosome alignment and segregation by controlling both the dynamic association of Survivin with centromeres and the proper targeting of Survivin and Aurora B to centromeres. Survivin is ubiquitinated in mitosis through both Lys(48) and Lys(63) ubiquitin linkages. Lys(63) de-ubiquitination mediated by hFAM is required for the dissociation of Survivin from centromeres, whereas Lys(63) ubiquitination mediated by the ubiquitin binding protein Ufd1 is required for the association of Survivin with centromeres. Thus, ubiquitinaton regulates dynamic protein-protein interactions and chromosome segregation independently of protein degradation (Vong, 2005).

Chromatin-induced spindle assembly depends on regulation of microtubule-depolymerizing proteins by the chromosomal passenger complex (CPC), consisting of Incenp, Survivin, Dasra (Borealin), and the kinase Aurora B, but the mechanism and significance of the spatial regulation of Aurora B activity remain unclear. This study shows that the Aurora B pathway is suppressed in the cytoplasm of Xenopus egg extract by phosphatases, but that it becomes activated by chromatin via a Ran-independent mechanism. While spindle microtubule assembly normally requires Dasra-dependent chromatin binding of the CPC, this function of Dasra can be bypassed by clustering Aurora B-Incenp by using anti-Incenp antibodies, which stimulate autoactivation among bound complexes. However, such chromatin-independent Aurora B pathway activation promotes centrosomal microtubule assembly and produces aberrant achromosomal spindle-like structures. It is proposed that chromosomal enrichment of the CPC results in local kinase autoactivation, a mechanism that contributes to the spatial regulation of spindle assembly and possibly to other mitotic processes (Kelly, 2007).

How does chromatin activate Aurora B-dependent phosphorylation? Four lines of evidence support a model in which Aurora B is activated by increasing the local concentration of CPC molecules on chromatin: (1) Chromatin can bind to multiple molecules of the CPC and induce Aurora B pathway activation; (2) Antibody alone can activate Aurora B kinase activity, and this activity is dependent on having multiple binding sites; (3) The responses of the small microtubule-destabilizing protein Op18 hyperphosphorylation induced by sperm nuclei and antibodies are similar and Ran independent; (4) Op18 hyperphosphorylation induced by antibody clustering is insensitive to the geometry of attachment (Kelly, 2007).

Full activation of Aurora B requires Aurora B-mediated phosphorylation of the C-terminal TSS motif of Incenp, and structural analysis suggests that this phosphorylation must occur in trans. Thus, the simplest model is that the Incenp TSS motif is actively dephosphorylated in the cytoplasm, but chromatin increases the local concentration of the CPC, resulting in initiation of a positive feedback loop among bound CPC holocomplexes. It is worth noting other possible mechanisms: clustering may also activate Aurora B independent of phosphorylation, as is the case for kinases such as Raf and EGFR, or chromatin or its associated molecules might directly induce a non-clustering-mediated structural change in Aurora B (Kelly, 2007).

It is also possible that chromatin exerts its effect on the Aurora B pathway by inhibiting protein phosphatase activities. However, the data indicate that chromatin directly stimulates the kinase activity of Aurora B, since Dasra proteins (which are required for loading of the CPC onto chromatin) are needed for spindle assembly. Importantly, more than 90% of Dasra A is associated with Incenp and Aurora B in the cytoplasm of Xenopus egg extracts. In addition, it has been reported that recombinant human Dasra B/Borealin does not affect the in vitro kinase activity of Aurora B. Thus, it is unlikely that Dasra proteins stimulate the enzymatic activity of Aurora B simply by virtue of their interactions (Kelly, 2007).

The spatial distribution of phosphorylated substrates around chromatin can be finely regulated by the level of phosphatase activity, and substrate diffusibility and stability, whereas the amplitude of the gradient is most sensitive to kinase activity. For example, the freely diffusible Op18-tubulin interaction is abrogated in the vicinity of chromosomes (4-8 microm) by a gradient of Op18 phosphorylation, the extent of which is mainly determined by phosphatase activity/concentration and the Op18 diffusion rate. Alternatively, if the substrate is immobilized on chromosomes, kinase activity dictates the behavior of the phospho-substrate. MCAK, a protein that is bound to centromeric chromatin, is more efficiently phosphorylated at Ser196 by Aurora B on centromeres of unaligned chromosomes than on aligned chromosomes. This raises the question of whether a change in chromatin status between sister kinetochores can effectively regulate Aurora B activity by modulating its local concentration. In summary, these results illustrating that Aurora B is activated by increased local concentration have important implications for the several roles of this complex throughout mitosis (Kelly, 2007).

Phosphorylation of histone H3 at serine 10 occurs during mitosis and meiosis in a wide range of eukaryotes and has been shown to be required for proper chromosome transmission in Tetrahymena. Ipl1/aurora kinase and its genetically interacting phosphatase, Glc7/PP1, are responsible for the balance of H3 phosphorylation during mitosis in Saccharomyces cerevisiae and Caenorhabditis elegans. In these models, both enzymes are required for H3 phosphorylation and chromosome segregation, although a causal link between the two processes has not been demonstrated. Deregulation of human aurora kinases has been implicated in oncogenesis as a consequence of chromosome missegregation. These findings reveal an enzyme system that regulates chromosome dynamics and controls histone phosphorylation that is conserved among diverse eukaryotes (Hsu, 2000).

Aurora B is a mitotic protein kinase that phosphorylates histone H3, behaves as a chromosomal passenger protein, and functions in cytokinesis. A role for Aurora B with respect to human centromere protein A (CENP-A), a centromeric histone H3 homolog, has been examined. Aurora B concentrates at centromeres in early G2, associates with histone H3 and centromeres at the times when histone H3 and CENP-A are phosphorylated, and phosphorylates histone H3 and CENP-A in vitro at a similar target serine residue. Dominant negative phosphorylation site mutants of CENP-A result in a delay at the terminal stage of cytokinesis (cell separation). The only molecular defects detected in analysis of 22 chromosomal, spindle, and regulatory proteins were disruptions in localization of inner centromere protein (INCENP), Aurora B, and a putative partner phosphatase, PP1gamma1. These data support a model where CENP-A phosphorylation is involved in regulating Aurora B, INCENP, and PP1gamma1 targeting within the cell. These experiments identify an unexpected role for the kinetochore in regulation of cytokinesis (Zeitlin, 2001).

Proper chromosome condensation requires the phosphorylation of histone and nonhistone chromatin proteins. An in vitro chromosome assembly system based on Xenopus egg cytoplasmic extracts has been used to study mitotic histone H3 phosphorylation. A histone H3 Ser(10) kinase activity associated with isolated mitotic chromosomes has been identified. The histone H3 kinase is not affected by inhibitors of cyclin-dependent kinases, DNA-dependent protein kinase, p90(rsk), or cAMP-dependent protein kinase. The activity can be selectively eluted from mitotic chromosomes and immunoprecipitated by specific anti-X aurora-B/AIRK2 antibodies. This activity is regulated by phosphorylation. Treatment of X aurora-B immunoprecipitates with recombinant protein phosphatase 1 (PP1) inhibits kinase activity. The presence of PP1 on chromatin suggests that PP1 might directly regulate the X aurora-B associated kinase activity. Indeed, incubation of isolated interphase chromatin with the PP1-specific inhibitor I2 and ATP generates an H3 kinase activity that is also specifically immunoprecipitated by anti-X aurora-B antibodies. Nonetheless, stimulation of histone H3 phosphorylation in interphase cytosol does not drive chromosome condensation or targeting of 13 S condensin to chromatin. In summary, the chromosome-associated mitotic histone H3 Ser(10) kinase is associated with X aurora-B and is inhibited directly in interphase chromatin by PP1 (Murnion, 2001).

Phosphorylation at a highly conserved serine residue (Ser-10) in the histone H3 tail is considered to be a crucial event for the onset of mitosis. This modification appears early in the G(2) phase within pericentromeric heterochromatin and spreads in an ordered fashion coincident with mitotic chromosome condensation. Although mitotic H3 phosphorylation has been long recognized, the transduction routes and the identity of the protein kinases involved have been elusive. The expression of mammalian Aurora-A and Aurora-B, two kinases of the Aurora/AIK family, is tightly coordinated with H3 phosphorylation during the G(2)/M transition. During the G(2) phase, the Aurora-A kinase is coexpressed while the Aurora-B kinase colocalizes with phosphorylated histone H3. At prophase and metaphase, Aurora-A is highly localized in the centrosomic region and in the spindle poles while Aurora-B is present in the centromeric region concurrent with H3 phosphorylation, to then translocate by cytokinesis to the midbody region. Both Aurora-A and Aurora-B proteins physically interact with the H3 tail and efficiently phosphorylate Ser10 both in vitro and in vivo, even if Aurora-A appears to be a better H3 kinase than Aurora-B. Since Aurora-A and Aurora-B are known to be overexpressed in a variety of human cancers, the findings provide an attractive link between cell transformation, chromatin modifications and a specific kinase system (Crosio, 2002).

Histones are subject to numerous post-translational modifications. Some of these 'epigenetic' marks recruit proteins that modulate chromatin structure. For example, heterochromatin protein 1 (HP1) binds to histone H3 when its lysine 9 residue has been tri-methylated by the methyltransferase Suv39h. During mitosis, H3 is also phosphorylated by the kinase Aurora B. Although H3 phosphorylation is a hallmark of mitosis, its function remains mysterious. It has been proposed that histone phosphorylation controls the binding of proteins to chromatin, but any such mechanisms are unknown. This study shows that antibodies against mitotic chromosomal antigens that are associated with human autoimmune diseases specifically recognize H3 molecules that are modified by both tri-methylation of lysine 9 and phosphorylation of serine 10 (H3K9me3S10ph). The generation of H3K9me3S10ph depends on Suv39h and Aurora B, and occurs at pericentric heterochromatin during mitosis in different eukaryotes. Most HP1 typically dissociates from chromosomes during mitosis, but if phosphorylation of H3 serine 10 is inhibited, HP1 remains chromosome-bound throughout mitosis. H3 phosphorylation by Aurora B is therefore part of a 'methyl/phos switch' mechanism that displaces HP1 and perhaps other proteins from mitotic heterochromatin (Hirota, 2005).

Histone deacetylase (HDAC) inhibitors perturb the cell cycle and have great potential as anti-cancer agents, but their mechanism of action is not well established. HDACs classically function as repressors of gene expression, tethered to sequence-specific transcription factors. This study reports that HDAC3 is a critical, transcription-independent regulator of mitosis. HDAC3 forms a complex with A-Kinase-Anchoring Proteins AKAP95 and HA95, which are targeted to mitotic chromosomes. Deacetylation of H3 in mitosis requires AKAP95/HA95 and HDAC3 and provides a hypoacetylated H3 tail that is the preferred substrate for Aurora B kinase. Phosphorylation of H3S10 by Aurora B leads to dissociation of HP1 proteins from methylated H3K9 residues on mitotic heterochromatin. This transcription-independent pathway, involving interdependent changes in histone modification and protein association, is required for normal progression through mitosis and is an unexpected target of HDAC inhibitors, a class of drugs currently in clinical trials for treating cancer (Li, 2006).

The classic role of HDAC3 has been that of a transcriptional repressor of gene expression, as part of a complex tethered to sequence-specific transcription factors. This study reports the unexpected finding that HDAC3 has a critical, transcription-independent function in mitosis. In interphase cells, AKAP95/HA95 binds to the nuclear matrix and is less associated with HDAC3. HP1 proteins are recruited to methylated H3K9 in heterochromatin. When cells enter into mitosis, AKAP95/HA95 may target the HDAC3 complex to deacetylate H3, in a reaction that is blocked by HDAC inhibitors, and thereby provides a hypoacetylated H3 tail as substrate for Aurora B to phosphorylate on S10. Phosphorylation of S10 by Aurora B then dissociates HP1 proteins from methylated H3K9 residues on mitotic heterochromatin, which has been referred to as the 'meth-phos switch'. These interdependent changes in histone modification and protein association are required for normal progression through mitosis, perhaps by facilitating chromosome condensation, or by serving as the indicator for the mitotic checkpoint to control proper cell division (Li, 2006).

While the transcriptional effect of HDAC inhibitors on specific genes, such as p21 and other cell cycle-regulated genes, has been reported to contribute to their anti-tumor actions, especially in G1-phase arrest, their direct effects on histone acetylation levels may be equally important for the anti-tumor activity because of the important functions of histones in different cellular processes, including mitosis. It is increasingly clear that HDAC inhibition induces G2/M arrest in many human cell lines and causes mitotic defects in different cancer cell lines. This effect of HDAC inhibition is independent of ongoing gene transcription, suggesting direct effects of histone hyperacetylation on mitosis. These results indicate that the hyperacetylation of histones induced by HDAC inhibitors directly interfere with mitotic progression (Li, 2006).

Global histone acetylation is reduced during mitosis. The current studies reveal that HDAC3 and its partner proteins AKAP95 and HA95 are required for global histone deacetylation during mitosis. Of note, the most dramatic change in acetylation that occurs during mitosis is hypoacetylation of Lys 5 of H4, which matches the substrate specificity of HDAC3. Moreover, the results clearly show that HDAC3 is required for normal mitotic progression. This is consistent with a recent study in which knockdown of HDAC3, but not HDAC1 or HDAC2, increased cells in G2/M phase in human colon cancer cells. Furthermore, knockdown of HDAC3 or AKAP95/HA95 also mimics the effects of nonselective HDAC inhibition on phosphorylation of H3S10 and retention of HP1β proteins on mitotic chromosomes. Inhibition of HDAC3 is therefore likely to be the mechanism by which HDAC inhibitors induce the G2/M block in the cell cycle. The transcription independence of this effect, while unexpected, is completely consistent with a direct mitotic function of HDAC3 in the context of the novel pathway that that is reported here (Li, 2006).

Specific patterns of histone modification at gene promoters regulate transcription via a 'histone code'. Notably, the transient phosphorylation of H3S10 has been reported in the promoter region of many mammalian immediate-early genes, which are rapidly induced in response to extracellular stimuli including UV radiation, growth factors, and cytokines. On these promoters, the phosphorylation of H3S10 precedes the H3K14 acetylation, resulting in multiple modifications of H3 that facilitate gene activation. On the contrary, this study found that the phosphorylation of H3S10 by Aurora B during mitosis requires the previous deacetylation of histones by HDAC3. Thus, in contrast to the phosphorylation of H3S10 by other kinases that prefer preacetylated histone tails, the mitotic phosphorylation of H3S10 by Aurora B kinase is linked to the deacetylation of H3, specifically by HDAC3. This characteristic of Aurora B may be specific to metazoans because IPL1, the yeast homolog of Aurora kinase, phosphorylated both monoacetylated and unacetylated H3. In addition to H3S10, Aurora B also phosphorylates H3S28 and other proteins including his- tone H3 variant centromere protein A (CENP-A). In human cell systems, Aurora B also seems to prefer hypoacetylated H3 and CENP-A H3 as substrate for phosphorylation of H3S28 and CENP-A Ser7, respectively. The global hypoacetylation of H3 tail lysines in mitotic cells and their proximity to the major sites of phosphorylation by Aurora B kinase suggest that deacetylation of histone substrates may be a general preference for Aurora B function. The relative importance of specific hypoacetylated lysines for phosphorylation of specific serine residues remains to be elucidated (Li, 2006).

The specificity of Aurora B toward hypoacetylated histone substrate suggests a mechanistic link between HDAC3-dependent histone deacetylation and a transcription-independent mechanism of mitotic arrest. H3S10 phosphorylation during mitosis is characteristic of many organisms, and is dependent on Aurora B kinase, which plays a central role throughout different stage of mitosis, including chromosome condensation, alignment, and segregation, spindle assembly, and cytokinesis. The recent finding that Aurora-dependent phosphorylation of H3S10 dissociates HP1 from mitotic heterochromatin provides molecular insight into the function of Aurora B. The current findings implicate AKAP95/HA95 and HDAC3 as upstream regulators of this "meth-phos switch", and provide a molecular mechanism to explain the anti-cancer effects of HDAC inhibitors. Aurora B kinase itself is overexpressed in a large number of cancers. The finding that Aurora B is present in HDAC3 complexes and that its kinase activity is dramatically greater when the H3 tail is hypoacetylated suggests that the interdependence of Aurora B and HDAC3 may be a novel and specific target for cancer therapies that would overcome the toxicity of nonspecific HDAC inhibitors (Li, 2006).

CPEB (Drosophila homolog: Orb) is an mRNA-binding protein that stimulates polyadenylation-induced translation of maternal mRNA once it is phosphorylated on Ser 174 or Thr 171 (species-dependent). Disruption of the CPEB gene in mice causes an arrest of oogenesis at embryonic day 16.5 (E16.5), when most oocytes are in pachytene of prophase I. CPEB undergoes Thr 171 phosphorylation at E16.5, but dephosphorylation at the E18.5, when most oocytes are entering diplotene. Although phosphorylation is mediated by the kinase aurora, the dephosphorylation is due to the phosphatase PP1. The temporal control of CPEB phosphorylation suggests a mechanism in which mRNA translation of CPE-containing messages is stimulated at pachytene and metaphase I (Tay, 2003).

The results presented here suggest a mechanism by which the translation of maternal mRNAs is differentially controlled during murine meiosis. As oogenesis progresses into pachytene, the kinase aurora is activated, perhaps by phosphorylation. The upstream kinase that phosphorylates aurora is unclear, although some evidence indicates that PKA is involved. Activated aurora then phosphorylates CPEB Thr 171, which stimulates the polyadenylation and translation of SCP1 and SCP3 mRNAs. The translation of other mRNAs might also be stimulated by CPEB at this time. SCP1 and SCP3 help form the synaptonemal complex, which is necessary for meiotic progression to diplotene. At diplotene, PP1 dephosphorylates and inactivates CPEB, an event that allows key CPE-containing mRNAs such as mos to accumulate but remain translationally dormant. As the fully grown (GV) oocytes begin to mature, aurora again becomes active and phosphorylates CPEB, which in turn induces the polyadenylation and translation of mos, and other mRNAs with encoded products that either stimulate maturation or lead to cytostatic factor (CSF)-mediated meta-phase II arrest (Tay, 2003).

Two additional points of upstream CPEB regulation should be considered. (1) Although aurora, in addition to CPEB, appears to be inactive in E18.5 diplotene oocytes (i.e., no T171 phosphorylation), it is plausible that the kinase is active at this time but its ability to phosphorylate CPEB is overcome by a very active PP1. To investigate this possibility, the phosphorylation experiments were perfomed except that E18.5 ovary extracts were supplemented with I-2, the PP1 inhibitor. Neither I-2-supplemented nor un-supplemented extracts supported T171 phosphorylation. Because I-2 inhibits dephosphorylation in the extracts, the lack of T171 phoshorylation can be attributed to inactive aurora rather than overriding PP1 activity. It is also interesting to note that PP1 has been suggested to inactivate aurora as well. Perhaps PP1 acts on a CPEB and aurora-containing complex to inactivate these proteins simultaneously at diplotene. (2) It is inferred that PP1, which is present in mouse oocytes, is also regulated; it is inactive during E16.5 (pachytene) when CPEB phosphorylation is robust but active at E18.5 (diplotene) to dephosphorylate CPEB. PP1 activity is regulated by a number of modulator proteins, some of which could function during oogenesis (Tay, 2003).

Cytoskeletal rearrangements during mitosis must be co-ordinated with chromosome movements. The 'chromosomal passenger' proteins, which include the inner centromere protein (INCENP), the Aurora-related serine-threonine protein kinase AIRK2 and the unidentified human autoantigen TD-60, have been suggested to integrate mitotic events. These proteins are chromosomal until metaphase but subsequently transfer to the midzone microtubule array and the equatorial cortex during anaphase. Disruption of INCENP function affects both chromosome segregation and completion of cytokinesis, whereas interference with AIRK2 function primarily affects cytokinesis. INCENP is stockpiled in Xenopus eggs in a complex with Xenopus AIRK2 (XAIRK2), and INCENP and AIRK2 kinase bind one another in vitro. This association was found to be evolutionarily conserved. Sli15p, the binding partner of yeast Aurora kinase Ipl1p, can be recognized as an INCENP family member because of the presence of a conserved carboxy-terminal sequence region, which is termed the IN box. This interaction between INCENP and Aurora kinase was found to be biologically relevant. INCENP and AIRK2 colocalize exactly in human cells, and INCENP is required to target AIRK2 correctly to centromeres and the central spindle (Adams, 2000).

The Aurora (Ipl1)-related kinases are universal regulators of mitosis. Aurora-A, in addition to Aurora-B, regulates kinetochore function in human cells. A two-hybrid screen identified the kinetochore component CENP-A as a protein that interacts with Aurora-A. Aurora-A phosphorylates CENP-A in vitro on Ser-7, a residue also known to be targeted by Aurora-B. Depletion of Aurora-A or Aurora-B by RNA interference reveals that CENP-A is initially phosphorylated in prophase in a manner dependent on Aurora-A, and that this reaction appears to be required for the subsequent Aurora-B-dependent phosphorylation of CENP-A as well as for the restriction of Aurora-B to the inner centromere in prometaphase. Prevention of CENP-A phosphorylation also led to chromosome misalignment during mitosis as a result of a defect in kinetochore attachment to microtubules. These observations suggest that phosphorylation of CENP-A on Ser-7 by Aurora-A in prophase is essential for kinetochore function (Kunitoku, 2003).

The phosphorylation of CENP-A is involved in efficient occupancy of kinetochores with spindle fibers. Concurrent with CENP-A phosphorylation at early prophase, various proteins assemble at the outer domain of the kinetochore. Given that CENP-A is essential for this assembly process in several species, the phosphorylation of CENP-A on Ser-7 might be required to initiate it during prophase, before the kinetochores begin to attach to microtubules. Such protein recruitment triggered by CENP-A phosphorylation might be important for the establishment of kinetochore-microtubule connections. However, this modification does not appear to be necessary for generation of the spindle assembly checkpoint signal, because Mad2, BubR1, and CENP-E localizes normally to kinetochores in prometaphase cells expressing CENP-A(S7A) and these cells show a marked delay in prometaphase (Kunitoku, 2003).

Given that Aurora-B plays an important role in correcting kinetochore-microtubule attachment in mammalian cells, the mislocalization of Aurora-B might contribute to the defect in chromosome alignment in cells expressing CENP-A(S7A) or in those deficient in Aurora-A. However, because Aurora-A-mediated phosphorylation of CENP-A on Ser-7 during prophase appears to be important for microtubule attachment, it was not possible to assess the possible contribution of the attachment-correcting function of Aurora-B. The many misaligned chromosomes that were found in cells in which CENP-A phosphorylation was prevented possessed either unattached or monotelic kinetochores, whereas those in Aurora-B-depleted cells exhibited syntelic attachment. Further molecular dissection of the regulation of kinetochore function by Aurora kinases could be facilitated by identification of the proteins that are recruited to the kinetochore in a manner dependent on CENP-A phosphorylation on Ser-7 (Kunitoku, 2003).

In vertebrate mitosis, cohesion between sister chromatids is lost in two stages. In prophase and prometaphase, cohesin release from chromosome arms occurs under the control of Polo-like kinase 1 and Aurora B, while Shugoshin is thought to prevent removal of centromeric cohesin until anaphase. The regulatory enzymes that act to sustain centromeric cohesion are incompletely described, however. Haspin/Gsg2 (see Drosophila Haspin), a positive regulator of centromeric cohesion, is a histone H3 threonine-3 kinase required for normal mitosis. Both H3 threonine-3 phosphorylation and cohesin are located at inner centromeres. Haspin depletion disrupts cohesin binding and sister chromatid association in mitosis, preventing normal chromosome alignment and activating the spindle assembly checkpoint, leading to arrest in a prometaphase-like state. Overexpression of Haspin hinders cohesin release and stabilizes arm cohesion. It is concluded that Haspin is required to maintain centromeric cohesion during mitosis. It is also suggested that Aurora B regulates cohesin removal through its effect on the localization of Shugoshin (Dai, 2007).

Mitosis is a highly coordinated process that assures the fidelity of chromosome segregation. Errors in this process result in aneuploidy which can lead to cell death or oncogenesis. This paper describes a putative mammalian protein kinase, AIM-1 (Aurora and Ipl1-like midbody-associated protein), related to Drosophila Aurora and Saccharomyces cerevisiae Ipl1, both of which are required for chromosome segregation. AIM-1 message and protein accumulate at G2/M phase. The protein localizes at the equator of central spindles during late anaphase and at the midbody during telophase and cytokinesis. Overexpression of kinase-inactive AIM-1 disrupts cleavage furrow formation without affecting nuclear division. Furthermore, cytokinesis frequently fails, resulting in cell polyploidy and subsequent cell death. These results strongly suggest that AIM-1 is required for proper progression of cytokinesis in mammalian cells (Terada, 1998).

Aurora- and Ipl1-like midbody-associated protein (AIM-1) is a serine/ threonine kinase that is structurally related to Drosophila aurora and Saccharomyces cerevisiae Ipl1, both of which are required for chromosome segregation. A kinase-negative form of AIM-1 inhibits the formation of cleavage furrow without affecting nuclear division, indicating that the gene controls entry into cytokinesis during M phase in mammalian cells. A human gene that encodes the protein AIM-1 was overexpressed in colorectal and other tumor cell lines. The regulation of AIM-1 expression is cell cycle dependent in normal and tumor cells, and the maximum accumulation is observed at G2-M. Exogenous overexpression of wild-type AIM-1 produces multinuclearity in human cells, suggesting that the excess amount of AIM-1 has a dominant-negative effect on the overexpressing cells. In long-term culture of AIM-1-overexpressing cells, multiple nuclei in a cell are occasionally fused, and then an increased ploidy and aneuploidy are induced. Thus, the overexpression of AIM-1 in colorectal tumor cell lines is thought to have a causal relationship with multinuclearity and increased ploidy. Cytokinesis error caused by AIM-1 overexpression is a major factor in the predisposition of tumor cells to the perturbation of chromosomal integrity that is commonly observed in human neoplasia. Thus, defects of pathways essential for mitotic regulation are important during human cancer development (Tatsuka, 1998).

The inner centromere protein (INCENP) is required for correct chromosome segregation and cytokinesis. The human INCENP gene has been idenified by library screening and reverse transcription-polymerase chain reaction (RT-PCR) and localized to chromosomal region 11q12. HsINCENP is a single-copy gene that consists of 17 exons and covers 25 kb of genomic DNA. The gene is expressed at highest levels in the colon, testis and prostate, consistent with its likely role in cell proliferation. HsINCENP encodes a highly basic protein of 915 amino acids that localizes to metaphase chromosomes and to the mitotic spindle and equatorial cortex at anaphase. It has been shown that INCENP is stockpiled in a complex with the Aurora-B/XAIRK2 kinase in Xenopus eggs. Consistent with such an interaction, the two proteins colocalize on human metaphase chromosomes. Levels of Aurora-B are increased in several human cancers, and HsINCENP protein levels are also significantly increased in several colorectal cancer cell lines (Adams, 2001b).

The Aurora/Ipl1 family of protein kinases plays multiple roles in mitosis and cytokinesis. ZM447439, a novel selective Aurora kinase inhibitor, is described. Cells treated with ZM447439 progress through interphase, enter mitosis normally, and assemble bipolar spindles. However, chromosome alignment, segregation, and cytokinesis all fail. Despite the presence of maloriented chromosomes, ZM447439-treated cells exit mitosis with normal kinetics, indicating that the spindle checkpoint is compromised. Indeed, ZM447439 prevents mitotic arrest after exposure to paclitaxel. RNA interference experiments suggest that these phenotypes are due to inhibition of Aurora B, not Aurora A or some other kinase. In the absence of Aurora B function, kinetochore localization of the spindle checkpoint components BubR1, Mad2, and Cenp-E is diminished. Furthermore, inhibition of Aurora B kinase activity prevents the rebinding of BubR1 to metaphase kinetochores after a reduction in centromeric tension. Aurora B kinase activity is also required for phosphorylation of BubR1 on entry into mitosis. Finally, it has been shown that BubR1 is not only required for spindle checkpoint function, but is also required for chromosome alignment. Together, these results suggest that by targeting checkpoint proteins to kinetochores, Aurora B couples chromosome alignment with anaphase onset (Ditchfield, 2003).

The spindle checkpoint ensures faithful chromosome segregation by linking the onset of anaphase to the establishment of bipolar kinetochore-microtubule attachment. The checkpoint is mediated by a signal transduction system comprised of conserved Mad, Bub and other proteins. Live-cell imaging coupled with RNA interference was used to investigate the functions of human Bub1. Bub1 is essential for checkpoint control and for correct chromosome congression. Bub1 depletion leads to the accumulation of misaligned chromatids in which both sister kinetochores are linked to microtubules in an abnormal fashion, a phenotype that is unique among Mad and Bub depletions. Bub1 is similar to the Aurora B/Ipl1p kinase in having roles in both the checkpoint and microtubule binding. However, human Bub1 and Aurora B are recruited to kinetochores independently of each other and have an additive effect when depleted simultaneously. Thus, Bub1 and Aurora B appear to function in parallel pathways that promote formation of stable bipolar kinetochore-microtubule attachments (Meraldi, 2005).

Mitosis is controlled by multiple kinases that drive cell cycle progression and prevent chromosome mis-segregation. Aurora kinase B interacts with survivin, borealin and incenp to form the chromosomal passenger complex (CPC), which is involved in the regulation of microtubule-kinetochore attachments and cytokinesis. Whereas genetic ablation of survivin, borealin or incenp results in early lethality at the morula stage, this study shows here that aurora B is dispensable for CPC function during early cell divisions and aurora B-null embryos are normally implanted. This is due to a crucial function of aurora C during these early embryonic cycles. Expression of aurora C decreases during late blastocyst stages resulting in post-implantation defects in aurora B-null embryos. These defects correlate with abundant prometaphase figures and apoptotic cell death of the aurora B-deficient inner cell mass. Conditional deletion of aurora B in somatic cells that do not express aurora C results in chromosomal misalignment and lack of chromosome segregation. Re-expression of wild-type, but not kinase-dead, aurora C rescues this defect, suggesting functional overlap between these two kinases. Finally, aurora B-null cells partially arrest in the presence of nocodazole, suggesting that this kinase is not essential for the spindle assembly checkpoint (Fernández-Miranda, 2011).

The central spindle regulates the formation and positioning of the contractile ring and is essential for completion of cytokinesis. Central spindle assembly begins in early anaphase with the bundling of overlapping, antiparallel, nonkinetochore microtubules, and these bundles become compacted and mature into the midbody. Prominent components of the central spindle include aurora B kinase and centralspindlin, a complex containing a Kinesin-6 protein (ZEN-4/MKLP1: Drosophila homolog - Pavarotti) and a Rho family GAP (CYK-4/MgcRacGAP) that is essential for central spindle assembly. Centralspindlin localization depends on aurora B kinase. Aurora B concentrates in the midbody and persists between daughter cells. In C. elegans embryos and in cultured human cells, respectively, ZEN-4 and MKLP1 are phosphorylated by aurora B in vitro and in vivo on conserved C-terminal serine residues. In C. elegans embryos, a nonphosphorylatable mutant of ZEN-4 localizes properly but does not efficiently support completion of cytokinesis. In mammalian cells, an inhibitor of aurora kinase acutely attenuates phosphorylation of MKLP1. Inhibition of aurora B in late anaphase causes cytokinesis defects without disrupting the central spindle. These data indicate a conserved role for aurora-B-mediated phosphorylation of ZEN-4/MKLP1 in the completion of cytokinesis (Guse, 2005 ).

Components of mitotic chromosomes assembled in Xenopus laevis egg extracts have been characterized and collectively referred to as Xenopus chromosome-associated polypeptides (XCAPs). They included five subunits of the condensin complex essential for chromosome condensation. In an effort to identify novel proteins involved in this process, XCAP-F has been isolated; it is the Xenopus ortholog of ISWI, a chromatin remodeling ATPase. ISWI exists in two major complexes in Xenopus egg extracts. The first complex contains ACF1 and two low-molecular-weight subunits, most likely corresponding to Xenopus CHRAC. The second complex is a novel one that contains the Xenopus ortholog of the human Williams syndrome transcription factor (WSTF). In the absence of the ISWI complexes, the deposition of histones onto DNA is apparently normal, but the spacing of nucleosomes is greatly disturbed. Despite the poor spacing of nucleosomes, ISWI depletion has little effect on DNA replication, chromosome condensation or sister chromatid cohesion in the cell-free extracts. The association of ISWI with chromatin is cell cycle regulated and is under the control of the INCENP-aurora B kinase complex that phosphorylates histone H3 during mitosis. Apparently contradictory to the generally accepted model, it has been found that neither chromosome condensation nor chromosomal targeting of condensin is compromised when H3 phosphorylation is drastically reduced by depletion of INCENP-aurora B (MacCallum, 2002).

Heterochromatin protein 1 (HP1 see Drosophila HP1) plays an important role in heterochromatin formation and undergoes large-scale, progressive dissociation from heterochromatin in prophase cells. However, the mechanisms regulating the dynamic behavior of HP1 are poorly understood. This study investigated the role of Aurora-B with respect to the dynamic behavior of HP1alpha. Mammalian Aurora-B, AIM-1, colocalizes with HP1alpha to the heterochromatin in G2. Depletion of Aurora-B/AIM-1 inhibits dissociation of HP1alpha from the chromosome arms at the G2-M transition. In addition, depletion of INCENP leads to aberrant cellular localization of Aurora-B/AIM-1, but it does not affect heterochromatin targeting of HP1alpha. It has been proposed in the binary switch hypothesis that phosphorylation of histone H3 at Ser-10 negatively regulates the binding of HP1alpha to the adjacent methylated Lys-9. However, Aurora-B/AIM-1-mediated phosphorylation of H3 induces dissociation of the HP1alpha chromodomain but not of the intact protein in vitro, indicating that the center and/or C-terminal domain of HP1alpha interferes with the effect of H3 phosphorylation on HP1alpha dissociation. Interestingly, Lys-9 methyltransferase SUV39H1 is abnormally localized together along the metaphase chromosome arms in Aurora-B/AIM-1-depleted cells. In conclusion, these results showed that Aurora-B/AIM-1 is necessary for regulated histone modifications involved in binding of HP1alpha by the N terminus of histone H3 during mitosis (Terada, 2006).

The spindle checkpoint delays anaphase onset in cells with mitotic spindle defects. Chk1, a component of the DNA damage and replication checkpoints, protects vertebrate cells against spontaneous chromosome missegregation and is required to sustain anaphase delay when spindle function is disrupted by taxol, but not when microtubules are completely depolymerized by nocodazole. Spindle checkpoint failure in Chk1-deficient cells correlates with decreased Aurora-B kinase activity and impaired phosphorylation and kinetochore localization of BubR1. Furthermore, Chk1 phosphorylates Aurora-B and enhances its catalytic activity in vitro. It is proposed that Chk1 augments spindle checkpoint signaling and is required for optimal regulation of Aurora-B and BubR1 when kinetochores produce a weakened signal. In addition, Chk1-deficient cells exhibit increased resistance to taxol. These results suggest a mechanism through which Chk1 could protect against tumorigenesis through its role in spindle checkpoint signaling (Zachos, 2007).

Fission yeast shugoshin Sgo1 is meiosis specific and cooperates with protein phosphatase 2A to protect centromeric cohesin at meiosis I. The other shugoshin-like protein Sgo2, which requires the heterochromatin protein Swi6/HP1 for full viability, plays a crucial role for proper chromosome segregation at both mitosis and meiosis; however, the underlying mechanisms are totally elusive. This study demonstrates that, unlike Sgo1, Sgo2 is dispensable for centromeric protection of cohesin. Instead, Sgo2 interacts with Bir1/Survivin and promotes Aurora kinase complex localization to the pericentromeric region, to correct erroneous attachment of kinetochores and thereby enable tension-generating attachment. Forced localization of Bir1 to centromeres partly restored the defects of sgo2^Delta. This newly identified interaction of shugoshin with Survivin is conserved between mitosis and meiosis and presumably across eukaryotes. It is proposed that ensuring bipolar attachment of kinetochores is the primary role of shugoshin and the role of cohesion protection might have codeveloped to facilitate this process (Kawashima, 2007).

This study demonstrates that human shugoshin hSgo1 associates with Survivin and Aurora and requires these components for its centromeric localization. Together with the recent finding in Drosophila that the Aurora kinase complex is required for centromeric localization of Sgo/Mei-S332 (Resnick, 2006), these studies suggest that the linkage between shugoshin and Aurora kinase complex is conserved among eukaryotes. Studies in human cells present the strongest data to date indicating the existence of a complex including shugoshin and Survivin in vivo; hSgo1 could coprecipitate with Survivin better than Aurora in extracts prepared from chromatin fraction. This result fits with the immunoprecipitation using a cross-linker in fission yeast and with genetic results indicating that Sgo2 closely interacts with Bir1/Survivin for the centromeric localization. Although the linkage between shugoshin and the Aurora kinase complex is conserved across species, the precise manner of interaction has apparently diverged. The centromeric localization of Drosophila Mei-S332 reportedly requires phosphorylation by Aurora (Resnick, 2006); however, fission yeast Sgo2 does not require it, albeit Sgo2, like Mei-S332, is a good substrate of Ark1 in vitro. Whereas fission yeast shugoshin (Sgo2) is required for the localization and function of Aurora kinase complex at centromeres, Drosophila Mei-S332 as well as human Sgo1 is not required for the localization of the Aurora kinase complex (Resnick, 2006), albeit the centromeric function of the Aurora kinase complex might nevertheless be regulated by Mei-S332 (Kawashima, 2007).

The sole shugoshin protein in budding yeast seems to play dual roles in protecting centromeric cohesin at meiosis I (but not at mitosis) as well as in establishing tension-generating attachment at mitosis. Drosophila SGO/MEI-S332 mutants show nondisjunction of homologs at meiosis I and a reduced ratio of meta/anaphase (but only slight or little defect in cohesion) in mitosis. Therefore, it is suggested that Mei-S332, the sole shugoshin of Drosophila, is also required for establishing tension-generating attachment, like fission yeast Sgo2. The localization of the Aurora kinase complex does not depend on Mei-S332; however, it is tenable that the activation of centromeric Aurora kinase complex may somewhat depend on Mei-S332 since they physically interact in vitro (Resnick, 2006). Similarly, fission yeast Sgo2 might play an additional role in activating centromeric Aurora rather than merely promoting its localization. Given that hSgo1 associates with Survivin (and Aurora) in HeLa cells, a similar functional link is conceivable also in human cells (Kawashima, 2007).

Studies in fission yeast enabled definition of two distinct shugoshin functions or pathways that are carried out by two diverged shugoshins, Sgo1 and Sgo2; the former interacts with PP2A to protect cohesin, but the latter interacts with the Aurora kinase complex to facilitate centromeric Aurora function. It is speculated that the ancestral shugoshin molecule played dual roles at kinetochores like in budding yeast or Drosophila; fission yeast shugoshin might have divided the labor to Sgo1 and Sgo2. Thus, these findings of a functional link between Sgo2 and the Aurora kinase complex open a new view that shugoshin in general may play a role in facilitating Aurora function at centromeres, thereby ensuring tension-generating kinetochore microtubule attachment. At the centromere, microtubule attachment is ensured by tension across centromeres, which is generated depending on the cohesion between sister chromatids. Therefore, cohesion and tension are two sides of a 'coin' ensuring bipolar attachment of kinetochores. It is suggested that the original role of shugoshin was to guarantee bipolar attachment rather than to protect cohesin, because fission yeast and presumably budding yeast, two primitive eukaryotes, exhibit this role only during mitosis. The protection role, once acquired, might facilitate the generation of tension by counteracting the spindle force, improving the fidelity of chromosome segregation. This function might have been modified to evolve meiosis, in which the requirement for centromeric protection is more essential and therefore has been preserved in all eukaryotes. Whatever the validity of this view, the finding of how Sgo2 acts will contribute to understand the fundamental regulation of eukaryotic chromosome segregation (Kawashima, 2007).

The mitotic checkpoint ensures proper chromosome segregation by monitoring two critical events during mitosis. One is kinetochore attachment to the mitotic spindle, and the second is the alignment of chromosomes at the metaphase plate, resulting in tension across sister kinetochores. Mitotic-checkpoint proteins are known to accumulate at unaligned chromosomes that have not achieved proper kinetochore-microtubule attachments or established an adequate level of tension across sister kinetochores. hZW10 and hROD, two components of the evolutionarily conserved RZZ complex (Chan, 2000; Scaerou, 2001), accumulate at kinetochores in response to the loss of tension. By using live-cell imaging and FRAP, it was shown that the accumulation of hZW10 at tensionless kinetochores stems from a 4-fold reduction of kinetochore turnover rate. It was also found that cells lacking hZW10 escape loss-of-tension-induced mitotic-checkpoint arrest more rapidly than those arrested in response to the lack of kinetochore-microtubule attachments. Furthermore, it was shown that pharmacological inhibition of Aurora B kinase activity with ZM447439 in the absence of tension, but not in the absence of kinetochore-microtubule attachments, results in the loss of hZW10, hROD, and hBub1 from kinetochores. It is therefore concluded that Aurora B kinase activity is required for the accumulation of tension-sensitive mitotic-checkpoint components, such as hZW10 and hROD, in order to maintain mitotic-checkpoint arrest (Famulski, 2007).

It is concluded that human ZW10 and human ROD are tension-sensitive components of the mitotic checkpoint and that their accumulation at tensionless kinetochores is regulated by their turnover dynamics in an Aurora B kinase-dependent manner. It is proposed that Aurora B phosphorylation of the RZZ complex might reduce its kinetochore turnover rate, therefore leading to the accumulation of hp50 and the RZZ complex at tensionless kinetochores. Lowering the kinetochore turnover rate of the RZZ complex might involve modification of the interaction between the RZZ complex and dynein. This could prevent dynein-mediated transport of the RZZ complex, and other essential mitotic-checkpoint components, off kinetochores. Mitotic-checkpoint arrest in response to the loss of kinetochore tension would thus be maintained by the prevention of the 'shedding' of essential checkpoint proteins from kinetochores, even though bipolar attachment of microtubules has been achieved (Famulski, 2007).

Shugoshin (Sgo) is a conserved centromeric protein. Mammalian Sgo1 collaborates with protein phosphatase 2A (PP2A) to protect mitotic cohesin from the prophase dissociation pathway. Although another shugoshin-like protein, Sgo2, is required for the centromeric protection of cohesion in germ cells, its precise molecular function remains largely elusive. This study demonstrates that hSgo2 plays a dual role in chromosome congression and centromeric protection of cohesion in HeLa cells, while the latter function is exposed only in perturbed mitosis. These functions partly overlap with those of Aurora B, a kinase setting faithful chromosome segregation. Accordingly, phosphorylation of hSgo2 by Aurora B was identified at the N-terminal coiled-coil region and the middle region, and these phosphorylations were shown to separately promote binding of hSgo2 to PP2A and MCAK, factors required for centromeric protection and chromosome congression, respectively. Furthermore, these phosphorylations are essential for localizing PP2A and MCAK to centromeres. This mechanism seems applicable to germ cells as well. Thus, this study identifies Sgo2 as a hitherto unknown crucial cellular substrate of Aurora B in mammalian cells (Tanno, 2010).

Faithful cell-cycle progression is tightly controlled by the ubiquitin-proteasome system. A human Cullin 3-based E3 ligase (Cul3) has been identified that is essential for mitotic division. In a complex with the substrate-specific adaptors KLHL9 and KLHL13, Cul3 is required for correct chromosome alignment in metaphase, proper midzone and midbody formation, and completion of cytokinesis. This Cul3-based E3 ligase removes components of the chromosomal passenger complex from mitotic chromosomes and allows their accumulation on the central spindle during anaphase. Aurora B directly binds to the substrate-recognition domain of KLHL9 and KLHL13 in vitro, and coimmunoprecipitates with the Cul3 complex during mitosis. Moreover, Aurora B is ubiquitylated in a Cul3-dependent manner in vivo, and by reconstituted Cul3/KLHL9/KLHL13 ligase in vitro. It is thus proposed that the Cul3/KLHL9/KLHL13 E3 ligase controls the dynamic behavior of Aurora B on mitotic chromosomes, and thereby coordinates faithful mitotic progression and completion of cytokinesis (Sumara, 2007).

During division of metazoan cells, the nucleus disassembles to allow chromosome segregation, and then reforms in each daughter cell. Reformation of the nucleus involves chromatin decondensation and assembly of the double-membrane nuclear envelope around the chromatin; however, regulation of the process is still poorly understood. In vitro, nucleus formation requires p97, a hexameric ATPase implicated in membrane fusion and ubiquitin-dependent processes (Drosophila homolog: TER94). However, the role and relevance of p97 in nucleus formation have remained controversial. This study shows that p97 stimulates nucleus reformation by inactivating the chromatin-associated kinase Aurora B. During mitosis, Aurora B inhibits nucleus reformation by preventing chromosome decondensation and formation of the nuclear envelope membrane. During exit from mitosis, p97 binds to Aurora B after its ubiquitylation and extracts it from chromatin. This leads to inactivation of Aurora B on chromatin, thus allowing chromatin decondensation and nuclear envelope formation. These data reveal an essential pathway that regulates reformation of the nucleus after mitosis and defines ubiquitin-dependent protein extraction as a common mechanism of Cdc48/p97 activity also during nucleus formation (Ramadan, 2007).

Proper spindle assembly and chromosome segregation rely on precise microtubule dynamics, which are governed in part by the kinesin-13 MCAK. MCAK microtubule depolymerization activity is inhibited by Aurora B-dependent phosphorylation, but the mechanism of this inhibition is not understood. This study developed the first Forster resonance energy transfer (FRET)-based biosensor for MCAK and showed that MCAK in solution exists in a closed conformation mediated by an interaction between the C-terminal domain (CT) and the neck. Using fluorescence lifetime imaging (FLIM) it was shown that MCAK bound to microtubule ends is closed relative to MCAK associated with the microtubule lattice. Aurora B phosphorylation at S196 in the neck opens MCAK conformation and diminishes the interaction between the CT and the neck. Using FLIM and TIRF imaging, it was found that changes in MCAK conformation are associated with a decrease in MCAK affinity for the microtubule. It is concluded that unlike motile kinesins, which are open when doing work, the high-affinity binding state for microtubule-depolymerizing kinesins is in a closed conformation. Phosphorylation switches MCAK conformation, which inhibits its ability to interact with microtubules and reduces its microtubule depolymerization activity. This work shows that the conformational model proposed for regulating kinesin activity is not universal and that microtubule-depolymerizing kinesins utilize a distinct conformational mode to regulate affinity for the microtubule, thus controlling their catalytic efficiency. Furthermore, this work provides a mechanism by which the robust microtubule depolymerization activity of kinesin-13s can be rapidly modulated to control cellular microtubule dynamics (Ems-McClung, 2013).

The Aurora B kinase coordinates kinetochore-microtubule attachments with spindle checkpoint signaling on each mitotic chromosome. This study found that EB1, a microtubule plus end-tracking protein, is required to enrich Aurora B at inner centromeres in a microtubule-dependent manner. This regulates phosphorylation of both kinetochore and chromatin substrates. EB1 regulates the histone phosphorylation marks (histone H2A phospho-Thr120 and histone H3 phospho-Thr3) that localize Aurora B. The chromosomal passenger complex containing Aurora B can be found on a subset of spindle microtubules that exist near prometaphase kinetochores, known as preformed K-fibers (kinetochore fibers). These data suggest that EB1 enables the spindle microtubules to regulate the phosphorylation of kinetochores through recruitment of the Aurora B kinase (Banerjee, 2014).

Incorrect attachment of kinetochore microtubules is the leading cause of chromosome missegregation in cancers. The highly conserved chromosomal passenger complex (CPC) (see Drosophila cell cycle), containing mitotic kinase Aurora B as a catalytic subunit, ensures faithful chromosome segregation through destabilizing incorrect microtubule attachments and promoting biorientation of chromosomes on the mitotic spindle. It is unknown whether CPC dysfunction affects chromosome segregation fidelity in cancers and, if so, how. This study shows that heterochromatin protein 1 (HP1) (see Drosophila Suppressor of variegation 205) is an essential CPC component required for full Aurora B activity. HP1 binding to the CPC becomes particularly important when Aurora B phosphorylates kinetochore targets to eliminate erroneous microtubule attachments. Remarkably, a reduced proportion of HP1 bound to CPC is widespread in cancers, which causes an impairment in Aurora B activity. These results indicate that HP1 is an essential modulator for CPC function and identify a molecular basis for chromosome segregation errors in cancer cells (Abe, 2016).

Search PubMed for articles about Drosophila Aurora B

Abe, Y., Sako, K., Takagaki, K., Hirayama, Y., Uchida, K.S., Herman, J.A., DeLuca, J.G. and Hirota, T. (2016). HP1-assisted Aurora B kinase activity prevents chromosome segregation errors. Dev Cell 36: 487-497. PubMed ID: 26954544

Adams, R. R., et al. (2000). Incenp binds the aurora-related kinase AIRK2 and is required to target it to chromosomes, the central spindle and cleavage furrow. Curr. Biol. 10: 1075-1078. 10996078

Adams, R. R., Maiato, H., Earnshaw, W. C. and Carmena, M. (2001a). Essential roles of Drosophila Inner centromere protein (Incenp) and Aurora B in Histone H3 phosphorylation, metaphase chromosome alignment, kinetochore disjunction, and chromosome segregation. J. Cell Biol. 153: 865-880. 11352945

Adams, R. R., et al. (2001b). Human INCENP colocalizes with the Aurora-B/AIRK2 kinase on chromosomes and is overexpressed in tumour cells. Chromosoma 110(2): 65-74. 11453556

Afonso, O., Matos, I., Pereira, A. J., Aguiar, P., Lampson, M. A. and Maiato, H. (2014). Feedback control of chromosome separation by a midzone Aurora B gradient. Science 345: 332-336. PubMed ID: 24925910

Afonso, O., Castellani, C. M., Cheeseman, L. P., Ferreira, J. G., Orr, B., Ferreira, L. T., Chambers, J. J., Morais-de-Sa, E., Maresca, T. J. and Maiato, H. (2019). Spatiotemporal control of mitotic exit during anaphase by an Aurora B-Cdk1 crosstalk. Elife 8. PubMed ID: 31424385

Bai, X., Melesse, M., Sorensen Turpin, C. G., Sloan, D. E., Chen, C. Y., Wang, W. C., Lee, P. Y., Simmons, J. R., Nebenfuehr, B., Mitchell, D., Klebanow, L. R., Mattson, N., Betzig, E., Chen, B. C., Cheerambathur, D. and Bembenek, J. N. (2020). Aurora B functions at the apical surface after specialized cytokinesis during morphogenesis in C. elegans. Development 147(1). PubMed ID: 31806662

Banerjee, B., Kestner, C. A. and Stukenberg, P. T. (2014). EB1 enables spindle microtubules to regulate centromeric recruitment of Aurora B. J Cell Biol 204: 947-963. PubMed ID: 24616220

Bell, G. P., Fletcher, G. C., Brain, R. and Thompson, B. J. (2014). Aurora kinases phosphorylate Lgl to induce mitotic spindle orientation in Drosophila epithelia. Curr Biol 25(1):61-8. PubMed ID: 25484300

Bhat, M. A., Philp, A. V., Glover, D. M. and Bellen, H. J. (1996). Chromatid segregation at anaphase requires the barren product, a novel chromosome-associated protein that interacts with Topoisomerase II. Cell 87: 1103-1114. 8978614

Biggins, S., Severin, F. F., Bhalla, N., Sassoon, I., Hyman, A. A. and Murray, A. W. (1999). The conserved protein kinase Ipl1 regulates microtubule binding to kinetochores in budding yeast. Genes Dev. 13: 532-544. 10072382

Biggins, S. and Murray, A. W. (2001). The budding yeast protein kinase Ipl1/Aurora allows the absence of tension to activate the spindle checkpoint. Genes Dev. 15: 3118-3129. 11731476

Carmena, M., Pinson, X., Platani, M., Salloum, Z., Xu, Z., Clark, A., Macisaac, F., Ogawa, H., Eggert, U., Glover, D. M., Archambault, V. and Earnshaw, W. C. (2012). The chromosomal passenger complex activates Polo kinase at centromeres. PLoS Biol 10: e1001250. PubMed ID: 22291575

Carmena, M., Lombardia, M. O., Ogawa, H. and Earnshaw, W. C. (2014). Polo kinase regulates the localization and activity of the chromosomal passenger complex in meiosis and mitosis in Drosophila melanogaster. Open Biol 4. PubMed ID: 25376909

Cesario, J. M., et al. (2006). Kinesin 6 family member Subito participates in mitotic spindle assembly and interacts with mitotic regulators. J. Cell Sci. 119: 4770-4780. PubMed citation; Online text

Cesario, J. and McKim, K. S. (2011). RanGTP is required for meiotic spindle organization and the initiation of embryonic development in Drosophila. J Cell Sci 124: 3797-3810. PubMed ID: 22100918

Chan, G. K., Jablonski, S. A., Starr, D. A., Goldberg, M. L. and Yen, T. J. (2000). Human Zw10 and ROD are mitotic checkpoint proteins that bind to kinetochores. Nat. Cell Biol. 2: 944-947. PubMed Citation: 11146660

Crosio, C., Fimia, G. M., Loury, R., Kimura, M., Okano, Y., Zhou, H., Sen, S., Allis, C. D. and Sassone-Corsi, P. (2002). Mitotic phosphorylation of histone H3: Spatio-temporal regulation by mammalian aurora kinases. Mol. Cell. Biol. 22: 874-885. 11784863

Dai, J., Sullivan, B. A. and Higgins, J. M. G. (2007). Regulation of mitotic chromosome cohesion by Haspin and Aurora B. Dev. Cell 11: 741-750. Medline abstract: 17084365

de Carvalho, C. E., et al. (2009). LAB-1 antagonizes the Aurora B kinase in C. elegans. Genes Dev. 22(20): 2869-85. PubMed Citation: 18923084

Ems-McClung, S. C., Hainline, S. G., Devare, J., Zong, H., Cai, S., Carnes, S. K., Shaw, S. L. and Walczak, C. E. (2013). Aurora B inhibits MCAK activity through a phosphoconformational switch that reduces microtubule association. Curr Biol 23: 2491-2499. PubMed ID: 24291095; Graphical Abstract

Eyers, P. A., Churchill, M. E. and Maller, J. L. (2005). The Aurora A and Aurora B protein kinases: a single amino acid difference controls intrinsic activity and activation by TPX2. Cell Cycle 4(6): 784-9. 15908779

Famulski, J. K. and Chan, G. K. (2007). Aurora B kinase-dependent recruitment of hZW10 and hROD to tensionless kinetochores. Curr. Biol. 17(24): 2143-9. PubMed Citation: 18065224

Fernández-Miranda, G., et al. (2011). Genetic disruption of aurora B uncovers an essential role for aurora C during early mammalian development. Development 138(13): 2661-72. PubMed Citation: 21613325

Fischle, W., et al. (2005). Regulation of HP1-chromatin binding by histone H3 methylation and phosphorylation. Nature 438(7071): 1116-22. 16222246

Gassmann, R., Carvalho, A., Henzing, A. J., Ruchaud, S., Hudson, D. F., Honda, R., Nigg, E. A., Gerloff, D. L. and Earnshaw, W. C. (2004). Borealin: a novel chromosomal passenger required for stability of the bipolar mitotic spindle. J. Cell Biol. 166: 179-191. 15249581

Giet, R. and Prigent, C. (1999). Aurora/Ipl1p-related kinases, a new oncogenic family of mitotic serine-threonine kinases. J. Cell Sci. 112:3591-601. 10523496

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date revised: 13 January 2020

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