Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores

The Aurora/Ipl1 family of protein kinases plays multiple roles in mitosis and cytokinesis. Here, we describe ZM447439, a novel selective Aurora kinase inhibitor. 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, we show 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.     

Aurora B kinase cooperates with CENP-E to promote timely anaphase onset

Error-free chromosome segregation requires that all chromosomes biorient on the mitotic spindle. The motor protein Centromere-associated protein E (CENP-E) facilitates chromosome congression by mediating the lateral sliding of sister chromatids along existing K-fibers, while the mitotic kinase Aurora B detaches kinetochore–microtubule interactions that are not bioriented. Whether these activities cooperate to promote efficient chromosome biorientation and timely anaphase onset is not known. We here show that the chromosomes that fail to congress after CENP-E depletion displayed high centromeric Aurora B kinase activity. This activity destabilized spindle pole proximal kinetochore–microtubule interactions resulting in a checkpoint-dependent mitotic delay that allowed CENP-E-independent chromosome congression, thus reducing chromosome segregation errors. This shows that Aurora B keeps the mitotic checkpoint active by destabilizing kinetochore fibers of polar chromosomes to permit chromosome congression in CENP-E-compromised cells and implies that this kinase normally prevents pole proximal syntelic attachments to allow CENP-E-mediated congression of mono-oriented chromosomes.     

Kinetochore-microtubule interactions “in check” by Bub1, Bub3 and BubR1: The dual task of attaching and signalling

The spindle assembly checkpoint (SAC) prevents anaphase onset until all chromosomes accomplish proper bipolar attachments to the mitotic spindle and come under tension, thereby ensuring the fidelity of chromosome segregation. Despite significant advances in our understanding of SAC signalling, a clear link between checkpoint signalling and the molecular mechanisms underlying chromosome attachment to microtubules has not been established so far. However, independent studies from many groups have interestingly found that the bone-a-fide Bub1, BubR1 and Bub3 SAC proteins are themselves required for proper kinetochore-microtubule (K-MT) interactions. Here, we review these findings and discuss the specific contribution of each of these proteins in the regulation of K-MT attachment, taking into consideration their interdependencies for kinetochore localization as well as their relationship with other proteins with a known role in chromosome attachment and congression.     

Inhibition of aurora B kinase blocks chromosome segregation,overrides the spindle checkpoint,and perturbs microtubule dynamics in mitosis

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, we microinjected mitotic XTC cells with inhibitory antibody and found several related effects. 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. Our 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.     

BubR1 is essential for kinetochore localization of other spindle checkpoint proteins and its phosphorylation requires Mad1

The spindle checkpoint delays anaphase onset until all chromosomes have attached properly to the mitotic spindle. Checkpoint signal is generated at kinetochores that are not bound with spindle microtubules or not under tension. Unattached kinetochores associate with several checkpoint proteins, including BubR1, Bub1, Bub3, Mad1, Mad2, and CENP-E. I herein show that BubR1 is important for the spindle checkpoint in Xenopus egg extracts. The protein accumulates and becomes hyperphosphorylated at unattached kinetochores. Immunodepletion of BubR1 greatly reduces kinetochore binding of Bub1, Bub3, Mad1, Mad2, and CENP-E. Loss of BubR1 also impairs the interaction between Mad2, Bub3, and Cdc20, an anaphase activator. These defects are rescued by wild-type, kinase-dead, or a truncated BubR1 that lacks its kinase domain, indicating that the kinase activity of BubR1 is not essential for the spindle checkpoint in egg extracts. Furthermore, localization and hyperphosphorylation of BubR1 at kinetochores are dependent on Bub1 and Mad1, but not Mad2. This paper demonstrates that BubR1 plays an important role in kinetochore association of other spindle checkpoint proteins and that Mad1 facilitates BubR1 hyperphosphorylation at kinetochores.     

Kinase activity of fission yeast Mph1 is required for Mad2 and Mad3 to stably bind the anaphase promoting complex

Defects in chromosome segregation result in aneuploidy, which can lead to disease or cell death [1, 2]. The spindle checkpoint delays anaphase onset until all chromosomes are attached to spindle microtubules in a bipolar fashion [3, 4]. Mad2 is a key checkpoint component that undergoes conformational activation, catalyzed by a Mad1-Mad2 template enriched at unattached kinetochores [5]. Mad2 and Mad3 (BubR1) then bind and inhibit Cdc20 to form the mitotic checkpoint complex (MCC), which binds and inhibits the anaphase promoting complex (APC/C). Checkpoint kinases (Aurora, Bub1, and Mps1) are critical for checkpoint signaling, yet they have poorly defined roles and few substrates have been identified [6-8]. Here we demonstrate that a kinase-dead allele of the fission yeast MPS1 homolog (Mph1) is checkpoint defective and that levels of APC/C-associated Mad2 and Mad3 are dramatically reduced in this mutant. Thus, MCC binding to fission yeast APC/C is dependent on Mph1 kinase activity. We map and mutate several phosphorylation sites in Mad2, producing mutants that display reduced Cdc20-APC/C binding and an inability to maintain checkpoint arrest. We conclude that Mph1 kinase regulates the association of Mad2 with its binding partners and thereby mitotic arrest.     

Loss of function of the Cik1/Kar3 motor complex results in chromosomes with syntelic attachment that are sensed by the tension checkpoint

The attachment of sister kinetochores by microtubules emanating from opposite spindle poles establishes chromosome bipolar attachment, which generates tension on chromosomes and is essential for sister-chromatid segregation. Syntelic attachment occurs when both sister kinetochores are attached by microtubules from the same spindle pole and this attachment is unable to generate tension on chromosomes, but a reliable method to induce syntelic attachments is not available in budding yeast. The spindle checkpoint can sense the lack of tension on chromosomes as well as detached kinetochores to prevent anaphase onset. In budding yeast Saccharomyces cerevisiae, tension checkpoint proteins Aurora/Ipl1 kinase and centromere-localized Sgo1 are required to sense the absence of tension but are dispensable for the checkpoint response to detached kinetochores. We have found that the loss of function of a motor protein complex Cik1/Kar3 in budding yeast leads to syntelic attachments. Inactivation of either the spindle or tension checkpoint enables premature anaphase entry in cells with dysfunctional Cik1/Kar3, resulting in co-segregation of sister chromatids. Moreover, the abolished Kar3-kinetochore interaction in cik1 mutants suggests that the Cik1/Kar3 complex mediates chromosome movement along microtubules, which could facilitate bipolar attachment. Therefore, we can induce syntelic attachments in budding yeast by inactivating the Cik1/Kar3 complex, and this approach will be very useful to study the checkpoint response to syntelic attachments.     

The 'anaphase problem': how to disable the mitotic checkpoint when sisters split

Two closely connected mechanisms safeguard the fidelity of chromosome segregation in eukaryotic cells. The mitotic checkpoint monitors the attachment of kinetochores to microtubules and delays anaphase onset until all sister kinetochores have become attached to opposite poles. In addition, an error correction mechanism destabilizes erroneous attachments that do not lead to tension at sister kinetochores. Aurora B kinase, the catalytic subunit of the CPC (chromosomal passenger complex), acts as a sensor and effector in both pathways. In this review we focus on a poorly understood but important aspect of mitotic control: what prevents the mitotic checkpoint from springing into action when sister centromeres are split and tension is suddenly lost at anaphase onset? Recent work has shown that disjunction of sister chromatids, in principle, engages the mitotic checkpoint, and probably also the error correction mechanism, with potentially catastrophic consequences for cell division. Eukaryotic cells have solved this 'anaphase problem' by disabling the mitotic checkpoint at the metaphase-to-anaphase transition. Checkpoint inactivation is in part due to the reversal of Cdk1 (cyclin-dependent kinase 1) phosphorylation of the CPC component INCENP (inner centromere protein; Sli15 in budding yeast), which causes the relocation of the CPC from centromeres to the spindle midzone. These findings highlight principles of mitotic checkpoint control: when bipolar chromosome attachment is reached in mitosis, the checkpoint is satisfied, but still active and responsive to loss of tension. Mitotic checkpoint inactivation at anaphase onset is required to prevent checkpoint re-engagement when sister chromatids split.     

Microtubule capture by CENP-E silences BubR1-dependent mitotic checkpoint signaling

The mitotic checkpoint is the major cell cycle control mechanism for maintaining chromosome content in multicellular organisms. Prevention of premature onset of anaphase requires activation at unattached kinetochores of the BubR1 kinase, which acts with other components to generate a diffusible "stop anaphase" inhibitor. Not only does direct binding of BubR1 to the centromere-associated kinesin family member CENP-E activate its essential kinase, binding of a motorless fragment of CENP-E is shown here to constitutively activate BubR1 bound at kinetochores, producing checkpoint signaling that is not silenced either by spindle microtubule capture or the tension developed at those kinetochores by other components. Using purified BubR1, microtubules, and CENP-E, microtubule capture by the CENP-E motor domain is shown to silence BubR1 kinase activity in a ternary complex of BubR1-CENP-E-microtubule. Together, this reveals that CENP-E is the signal transducing linker responsible for silencing BubR1-dependent mitotic checkpoint signaling through its capture at kinetochores of spindle microtubules.     

Detection and Correction of Merotelic Kinetochore Orientation by Aurora B and its Partners

Merotelic kinetochore orientation is a kinetochore-microtubule mis-attachment in which a single kinetochore binds microtubules to both spindle poles, rather than just one. Merotelic attachments occur frequently in early mitosis and can induce anaphase lagging chromosomes and aneuploidy if not corrected before anaphase onset. Merotelic kinetochore orientation does not interfere with chromosome alignment at the metaphase plate and does not activate the mitotic spindle checkpoint. However, a correction mechanism for merotelic attachment reduces the number of merotelic kinetochores entering anaphase, thus preventing chromosome mis-segregation. Result from many different studies support the idea that Aurora B kinase plays a critical role in this merotelic correction mechanism by phosphorylating key substrates at the kinetochore and promoting turnover of kinetochore microtubules. In addition, recent studies are starting to identify the possible ‘sensors’ of the system that would be able to detect the mis-attachment and communicate this to Aurora B. Here, I review these studies and discuss a model for how merotelic kinetochore orientation could be detected and corrected before anaphase onset.     

The human spindle assembly checkpoint protein Bub3 is required for the establishment of efficient kinetochore-microtubule attachments

The spindle assembly checkpoint monitors the status of kinetochore-microtubule (K-MT) attachments and delays anaphase onset until full metaphase alignment is achieved. Recently, the role of spindle assembly checkpoint proteins was expanded with the discovery that BubR1 and Bub1 are implicated in the regulation of K-MT attachments. One unsolved question is whether Bub3, known to form cell cycle constitutive complexes with both BubR1 and Bub1, is also required for proper chromosome-to-spindle attachments. Using RNA interference and high-resolution microscopy, we analyzed K-MT interactions in Bub3-depleted cells and compared them to those in Bub1- or BubR1-depleted cells. We found that Bub3 is essential for the establishment of correct K-MT attachments. In contrast to BubR1 depletion, which severely compromises chromosome attachment and alignment, we found Bub3 and Bub1 depletions to produce defective K-MT attachments that, however, still account for significant chromosome congression. After Aurora B inhibition, alignment defects become severer in Bub3- and Bub1-depleted cells, while partially rescued in BubR1-depleted cells, suggesting that Bub3 and Bub1 depletions perturb K-MT attachments distinctly from BubR1. Interestingly, misaligned chromosomes in Bub3- and Bub1-depleted cells were found to be predominantly bound in a side-on configuration. We propose that Bub3 promotes the formation of stable end-on bipolar attachments.     

Timing and checkpoints in the regulation of mitotic progression

Accurate chromosome segregation relies on the precise regulation of mitotic progression. Regulation involves control over the timing of mitosis and a spindle assembly checkpoint that links anaphase onset to the completion of chromosome-microtubule attachment. In this paper, we combine live-cell imaging of HeLa cells and protein depletion by RNA interference to examine the functions of the Mad, Bub, and kinetochore proteins in mitotic timing and checkpoint control. We show that the depletion of any one of these proteins abolishes the mitotic arrest provoked by depolymerizing microtubules or blocking chromosome-microtubule attachment with RNAi. However, the normal progress of mitosis is accelerated only when Mad2 or BubR1, but not other Mad and Bub proteins, are inactivated. Moreover, whereas checkpoint control requires kinetochores, the regulation of mitotic timing by Mad2 and BubR1 is kinetochore-independent in fashion. We propose that cytosolic Mad2-BubR1 is essential to restrain anaphase onset early in mitosis when kinetochores are still assembling.     

The inhibition of Aurora A abrogates the mitotic delay induced by microtubule perturbing agents

The spindle assembly checkpoint functions during mitosis to ensure that chromosomes are properly aligned in mitotic cells prior to the onset of anaphase, thereby ensuring an equal segregation of genetic material to each daughter cell. Defects in the function of this checkpoint lead to aneuploidy, and eventually to cell death or senescence. The Aurora-related kinases, and in particular Aurora B, have been shown to play a role in regulating the spindle assembly checkpoint. In this study, we demonstrate that Aurora A activity is required for maintainance of the spindle assembly checkpoint mediated-mitotic delay induced by microtubule perturbing agents. Inhibition of Aurora A using MLN8054, a selective small-molecule inhibitor of Aurora A, in paclitaxel- or nocodazole-treated cells induces cells to become multinucleated. Using time-lapse microscopy, we demonstrate that the multinucleation phenotype arises via mitotic slippage, which is significantly accelerated upon Aurora A inhibition. Under these conditions, the spindle assembly checkpoint protein BubR1 remains localized to kinetochores prior to mitotic slippage. Moreover, we demonstrate that Aurora B remains active in these mitotic cells, indicating that the mitotic slippage induced by MLN8054 is most likely due to the inhibition of Aurora A. This finding was corroborated by demonstrating that Aurora A depletion using RNA interference in paclitaxel-treated cells also induces multinucleation. Taken together, these results suggest that Aurora A is necessary for the maintenance of the mitotic delay induced in response to microtubule-perturbing agents.     

Chk1 is required for spindle checkpoint function

The spindle checkpoint delays anaphase onset in cells with mitotic spindle defects. Here, we show that 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. We propose 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.     

CENP-E--dependent BubR1 autophosphorylation enhances chromosome alignment and the mitotic checkpoint

How the state of spindle microtubule capture at the kinetochore is translated into mitotic checkpoint signaling remains largely unknown. In this paper, we demonstrate that the kinetochore-associated mitotic kinase BubR1 phosphorylates itself in human cells and that this autophosphorylation is dependent on its binding partner, the kinetochore motor CENP-E. This CENP-E-dependent BubR1 autophosphorylation at unattached kinetochores is important for a full-strength mitotic checkpoint to prevent single chromosome loss. Replacing endogenous BubR1 with a nonphosphorylatable BubR1 mutant, as well as depletion of CENP-E, the BubR1 kinase activator, results in metaphase chromosome misalignment and a decrease of Aurora B-mediated Ndc80 phosphorylation at kinetochores. Furthermore, expressing a phosphomimetic BubR1 mutant substantially reduces the incidence of polar chromosomes in CENP-E-depleted cells. Thus, the state of CENP-E-dependent BubR1 autophosphorylation in response to spindle microtubule capture by CENP-E is important for kinetochore function in achieving accurate chromosome segregation.     

Sds22 and Repo-Man stabilize chromosome segregation by counteracting Aurora B on anaphase kinetochores

During mitotic spindle assembly, Aurora B kinase is part of an error correction mechanism that detaches microtubules from kinetochores that are under low mechanical tension. During anaphase, however, kinetochore-microtubule attachments must be maintained despite a drop of tension after removal of sister chromatid cohesion. Consistent with this requirement, Aurora B relocates away from chromosomes to the central spindle at the metaphase-anaphase transition. By ribonucleic acid interference screening using a phosphorylation biosensor, we identified two PP1-targeting subunits, Sds22 and Repo-Man, which counteracted Aurora B-dependent phosphorylation of the outer kinetochore component Dsn1 during anaphase. Sds22 or Repo-Man depletion induced transient pauses during poleward chromosome movement and a high incidence of chromosome missegregation. Thus, our study identifies PP1-targeting subunits that regulate the microtubule-kinetochore interface during anaphase for faithful chromosome segregation.     

Drosophila Polo regulates the spindle assembly checkpoint through Mps1‐dependent BubR1 phosphorylation

Maintenance of genomic stability during eukaryotic cell division relies on the spindle assembly checkpoint (SAC) that prevents mitotic exit until all chromosomes are properly attached to the spindle. Polo is a mitotic kinase proposed to be involved in SAC function, but its role has remained elusive. We demonstrate that Polo and Aurora B functional interdependency comprises a positive feedback loop that promotes Mps1 kinetochore localization and activity. Expression of constitutively active Polo restores normal Mps1 kinetochore levels even after Aurora B inhibition, highlighting a role for Polo in Mps1 recruitment to unattached kinetochores downstream of Aurora B. We also show that Mps1 kinetochore localization is required for BubR1 hyperphosphorylation and formation of the 3F3/2 phosphoepitope. This is essential to allow recruitment of Cdc20 to unattached kinetochores and the assembly of anaphase‐promoting complex/cyclosome‐inhibitory complexes to levels that ensure long‐term SAC activity. We propose a model in which Polo controls Mps1‐dependent BubR1 phosphorylation to promote Cdc20 kinetochore recruitment and sustained SAC function.     

Evidence that Aurora B is implicated in spindle checkpoint signalling independently of error correction

Fidelity of chromosome segregation is ensured by a tension-dependent error correction system that prevents stabilization of incorrect chromosome-microtubule attachments. Unattached or incorrectly attached chromosomes also activate the spindle assembly checkpoint, thus delaying mitotic exit until all chromosomes are bioriented. The Aurora B kinase is widely recognized as a component of error correction. Conversely, its role in the checkpoint is controversial. Here, we report an analysis of the role of Aurora B in the spindle checkpoint under conditions believed to uncouple the effects of Aurora B inhibition on the checkpoint from those on error correction. Partial inhibition of several checkpoint and kinetochore components, including Mps1 and Ndc80, strongly synergizes with inhibition of Aurora B activity and dramatically affects the ability of cells to arrest in mitosis in the presence of spindle poisons. Thus, Aurora B might contribute to spindle checkpoint signalling independently of error correction. Our results support a model in which Aurora B is at the apex of a signalling pyramid whose sensory apparatus promotes the concomitant activation of error correction and checkpoint signalling pathways.     

Activating and silencing the mitotic checkpoint through CENP-E-dependent activation/inactivation of BubR1

The mitotic checkpoint prevents advance to anaphase prior to successful attachment of every centromere/kinetochore to mitotic spindle microtubules. Using purified components and Xenopus egg extracts, the kinetochore-associated microtubule motor CENP-E is now shown to be the activator of the essential checkpoint kinase BubR1. Since kinase activity and the checkpoint are silenced following CENP-E-dependent microtubule attachment in extracts or binding of CENP-E antibodies that do not disrupt CENP-E association with BubR1, CENP-E mediates silencing of BubR1 signaling. Checkpoint signaling requires the normal level of BubR1 containing a functional Mad3 domain implicated in Cdc20 binding, but only a small fraction need be kinase competent. This supports bifunctional roles for BubR1 in the checkpoint: an enzymatic one requiring CENP-E-dependent activation of its kinase activity at kinetochores and a stoichiometric one as a direct inhibitor of Cdc20.     

Aurora kinase promotes turnover of kinetochore microtubules to reduce chromosome segregation errors

Merotelic kinetochore orientation is a misattachment in which a single kinetochore binds microtubules from both spindle poles rather than just one and can produce anaphase lagging chromosomes, a major source of aneuploidy. Merotelic kinetochore orientation occurs frequently in early mitosis, does not block chromosome alignment at the metaphase plate, and is not detected by the spindle checkpoint. However, microtubules to the incorrect pole are usually significantly reduced or eliminated before anaphase. We discovered that the frequency of lagging chromosomes in anaphase is very sensitive to partial inhibition of Aurora kinase activity by ZM447439 at a dose, 3 microM, that has little effect on histone phosphorylation, metaphase chromosome alignment, and cytokinesis in PtK1 cells. Partial Aurora kinase inhibition increased the frequency of merotelic kinetochores in late metaphase, and the fraction of microtubules to the incorrect pole. Measurements of fluorescence dissipation after photoactivation showed that kinetochore-microtubule turnover in prometaphase is substantially suppressed by partial Aurora kinase inhibition. Our results support a preanaphase correction mechanism for merotelic attachments in which correct plus-end attachments are pulled away from high concentrations of Aurora B at the inner centromere, and incorrect merotelic attachments are destabilized by being pulled toward the inner centromere.