Kinetochore stretching inactivates the spindle assembly checkpoint

The spindle assembly checkpoint (SAC) monitors the attachment of microtubules to the kinetochore and inhibits anaphase when microtubule binding is incomplete. The SAC might also respond to tension; however, how cells can sense tension and whether its detection is important to satisfy the SAC remain controversial. We generated a HeLa cell line in which two components of the kinetochore, centromere protein A and Mis12, are labeled with green and red fluorophores, respectively. Live cell imaging of these cells reveals repetitive cycles of kinetochore extension and recoiling after biorientation. Under conditions in which kinetochore stretching is suppressed, cells fail to silence the SAC and enter anaphase after a delay, regardless of centromere stretching. Monitoring cyclin B levels as a readout for anaphase-promoting complex/cyclosome activity, we find that suppression of kinetochore stretching delays and decelerates cyclin B degradation. These observations suggest that the SAC monitors stretching of kinetochores rather than centromeres and that kinetochore stretching promotes silencing of the SAC signal.     

Chromosome missegregation and apoptosis in mice lacking the mitotic checkpoint protein Mad2

The initiation of chromosome segregation at anaphase is linked by the spindle assembly checkpoint to the completion of chromosome-microtubule attachment during metaphase. To determine the function of the mitotic checkpoint protein Mad2 during normal cell division and when mitosis goes awry, we have knocked out Mad2 in mice. We find that E5.5 embryonic cells lacking Mad2, like mad2 yeast, grow normally but are unable to arrest in response to spindle disruption. At E6.5, the cells of the epiblast begin rapid cell division and the absence of a checkpoint results in widespread chromosome missegregation and apoptosis. In contrast, the postmitotic trophoblast giant cells survive without Mad2. Thus, the spindle assembly checkpoint is required for accurate chromosome segregation in mitotic mouse cells, and for embryonic viability, even in the absence of spindle damage.     

Evolution and function of the mitotic checkpoint

The mitotic checkpoint evolved to prevent cell division when chromosomes have not established connections with the chromosome segregation machinery. Many of the fundamental molecular principles that underlie the checkpoint, its spatiotemporal activation, and its timely inactivation have been uncovered. Most of these are conserved in eukaryotes, but important differences between species exist. Here we review current concepts of mitotic checkpoint activation and silencing. Guided by studies in model organisms and our phylogenomics analysis of checkpoint constituents and their functional domains and motifs, we highlight ancient and taxa-specific aspects of the core checkpoint modules in the context of mitotic checkpoint function.     

Replication-compromised cells require the mitotic checkpoint to prevent tetraploidization

Replication stress often induces chromosome instability. In this study, we explore which factors in replication-compromised cells promote abnormal chromosome ploidy. We expressed mutant forms of either polymerase α (Polα) or polymerase δ (Polδ) in normal human fibroblasts to compromise DNA replication. Cells expressing the mutant Polα-protein failed to sustain mitotic arrest and, when propagated progressively, down-regulated Mad2 and BubR1 and accumulated 4N-DNA from the 2N-DNA cells. Significantly, a population of these cells became tetraploids. The Polα mutant expressing cells also exhibited elevated cellular senescence markers, suggesting as a mechanism to limit proliferation of the tetraploids. Expression of the Polδ mutant also caused cells to accumulate 4N-DNA. In contrast to the Polα mutant expressing cells, the Polδ mutant expressing cells expressed sufficient levels of Mad2, BubR1, and cyclin B1 to sustain mitotic arrest, and these cells had normal chromosome ploidy. Together, these results suggest that replication-compromised cells depend on the mitotic checkpoint to prevent mitotic slippage that could result in tetraploidization.     

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.     

ZW10 links mitotic checkpoint signaling to the structural kinetochore

The mitotic checkpoint ensures that chromosomes are divided equally between daughter cells and is a primary mechanism preventing the chromosome instability often seen in aneuploid human tumors. ZW10 and Rod play an essential role in this checkpoint. We show that in mitotic human cells ZW10 resides in a complex with Rod and Zwilch, whereas another ZW10 partner, Zwint-1, is part of a separate complex of structural kinetochore components including Mis12 and Ndc80-Hec1. Zwint-1 is critical for recruiting ZW10 to unattached kinetochores. Depletion from human cells or Xenopus egg extracts is used to demonstrate that the ZW10 complex is essential for stable binding of a Mad1-Mad2 complex to unattached kinetochores. Thus, ZW10 functions as a linker between the core structural elements of the outer kinetochore and components that catalyze generation of the mitotic checkpoint-derived "stop anaphase" inhibitor.     

Centromeres and kinetochores: from epigenetics to mitotic checkpoint signaling

The centromere is a chromosomal locus that ensures delivery of one copy of each chromosome to each daughter at cell division. Efforts to understand the nature and specification of the centromere have demonstrated that this central element for ensuring inheritance is itself epigenetically determined. The kinetochore, the protein complex assembled at each centromere, serves as the attachment site for spindle microtubules and the site at which motors generate forces to power chromosome movement. Unattached kinetochores are also the signal generators for the mitotic checkpoint, which arrests mitosis until all kinetochores have correctly attached to spindle microtubules, thereby representing the major cell cycle control mechanism protecting against loss of a chromosome (aneuploidy).     

Acute ablation of survivin uncovers p53-dependent mitotic checkpoint functions and control of mitochondrial apoptosis

Survivin is a member of the Inhibitor of Apoptosis gene family that has been implicated in cell division and suppression of apoptosis. Here, we show that preferential ablation of the nuclear pool of survivin by RNA interference produces a mitotic arrest followed by re-entry into the cell cycle and polyploidy. Survivin ablation causes multiple centrosomal defects, aberrant multipolar spindle formation, and chromatin missegregation, and these phenotypes are exacerbated by loss of the cell cycle regulator, p21(Waf1/Cip1) in p21(-/-) cells. The mitotic checkpoint activated by loss of survivin is mediated by induction of p53 and associated with increased expression of its downstream target, p21(Waf1/Cip1). Accordingly, p53(-/-) cells exhibit reduced mitotic arrest and enhanced polyploidy upon survivin ablation as compared with their p53(+/+) counterparts. Partial reduction of the cytosolic pool of survivin by RNA interference sensitizes cells to ultraviolet B-mediated apoptosis and results in enhanced caspase-9 proteolytic cleavage, whereas complete ablation of cytosolic survivin causes loss of mitochondrial membrane potential and spontaneous apoptosis. These data demonstrate that survivin has separable checkpoint functions at multiple phases of mitosis and in the control of mitochondrial-dependent apoptosis.     

In-silico modeling of the mitotic spindle assembly checkpoint

Background

The Mitotic Spindle Assembly Checkpoint (^MSAC) is an evolutionary conserved mechanism that ensures the correct segregation of chromosomes by restraining cell cycle progression from entering anaphase until all chromosomes have made proper bipolar attachments to the mitotic spindle. Its malfunction can lead to cancer.

Principle Findings

We have constructed and validated for the human ^MSAC mechanism an in silico dynamical model, integrating 11 proteins and complexes. The model incorporates the perspectives of three central control pathways, namely Mad1/Mad2 induced Cdc20 sequestering based on the Template Model, MCC formation, and APC inhibition. Originating from the biochemical reactions for the underlying molecular processes, non-linear ordinary differential equations for the concentrations of 11 proteins and complexes of the ^MSAC are derived. Most of the kinetic constants are taken from literature, the remaining four unknown parameters are derived by an evolutionary optimization procedure for an objective function describing the dynamics of the APC:Cdc20 complex. MCC:APC dissociation is described by two alternatives, namely the “Dissociation” and the “Convey” model variants. The attachment of the kinetochore to microtubuli is simulated by a switching parameter silencing those reactions which are stopped by the attachment. For both, the Dissociation and the Convey variants, we compare two different scenarios concerning the microtubule attachment dependent control of the dissociation reaction. Our model is validated by simulation of ten perturbation experiments.

Conclusion

Only in the controlled case, our models show ^MSAC behaviour at meta- to anaphase transition in agreement with experimental observations. Our simulations revealed that for ^MSAC activation, Cdc20 is not fully sequestered; instead APC is inhibited by MCC binding.     

PP1 inactivates Greatwall to release PP2A-B55 from mitotic confinement

Entry into and exit from mitosis are brought about by the increase and decrease, respectively, in the activity of cyclin‐dependent kinases (CDKs). Many examples are known of how the properties of particular proteins can be altered by phosphorylation, promoting processes like nuclear envelope breakdown or assembly of the mitotic spindle. The regulation of protein phosphatases is shedding new light on how this quantitative change of protein phosphorylation is achieved by a tight linkage between CDK activity and CDK‐antagonizing phosphatases. On entering mitosis, increasing CDK activity ignites a repressive pathway that acts on PP2A‐B55, one of the major phosphatases for CDK substrates in higher eukaryotes. This repression allows rapid and near complete substrate phosphorylation. But this raises a serious bootstrapping problem at mitotic exit. Because the phosphatase responsible for CDK substrates has been shut off, how can the repression pathway, which was activated by CDK, be reversed? In the current issue, Heim and colleagues propose an answer to this question 1 . Their data show that dephosphorylation of Greatwall kinase (Gwl) at its auto‐phosphorylation site(s) is targeted by PP1, which leads to significant decrease in Gwl kinase activity. This early action by PP1 seems to be a prerequisite for PP2A‐B55 to escape from repression and to return Gwl back to its inactive hypophosphorylated interphase state. This study provides an important piece of evidence for how the repression mechanism of PP2A‐B55 is made reversible, and offers a solution to the bootstrap problem.     

Intra-nuclear microtubules and a mitotic spindle orientation checkpoint

Cells of the fission yeast Schizosaccharomyces pombe have a checkpoint mechanism that reportedly monitors the orientation of the mitotic spindle. Astral microtubules in pre-anaphase spindles are thought to contact the contractile actin ring at the plasma membrane in order to rotate the spindle and to sense spindle orientation. Here, we show that these microtubules are actually inside the nuclear envelope.     

Human diploid fibroblast cells in senescence; Cycling through polyploidy to mitotic cells

Summary Previously, it was found that senescent cells can undergo a modified cell cycle with mitotic cells as the end results. The major cycling events started with polyploidization, followed by depolyploidization to multinucleated cells (MNCs). These latter cells produced mononuclear offspring cells that could express mitotic cell divisions. In this report the emphasis is on late senescent fibroblasts that exhibited the senescence-associated change in cell morphology to large flat cells. Prior to live cell photography, flat cell cultures were maintained for months in the same culture flasks and therefore judged to be in a late senescent phase. All of the cellular events outlined above were present in these old cell cultures. Time lapse pictures showed movements of mitotic daughter cells away from each other and alignment of the chromosomes on the metaphase plate was visible in other mitotic cells. These data challenge the common view that cell senescence is irreversible and, therefore, an antitumor mechanism. A new finding was that the spike in polyploid cells in the near senescent phase consisted of cells with pairs of sister chromosomes from endoreduplication of DNA (two rounds of DNA synthesis and no mitosis). The lack of cells with 92 single chromosomes (e.g., G2 tetraploid cells) suggested that these polyploid cells also went through a changed cell cycle. The question now is whether these atypical polyploid cells are a subpopulation in senescence that can undergo the cycling from polyploidy to genome-reduced mitotic cells.     

The human mitotic checkpoint protein BubR1 regulates chromosome-spindle attachments

Loss or gain of whole chromosomes, the form of chromosomal instability (CIN) most commonly associated with human cancers, is expected to arise from the failure to accurately segregate chromosomes in mitosis. The mitotic checkpoint is one pathway that prevents segregation errors by blocking the onset of anaphase until all chromosomes make proper attachments to the spindle. Another process that prevents errors is stabilization and destabilization of connections between chromosomes and spindle microtubules. An outstanding question is how these two pathways are coordinated to ensure accurate chromosome segregation. Here we show that in human cells depleted of BubR1 - a critical component of the mitotic checkpoint that can directly regulate the onset of anaphase - chromosomes do not form stable attachments to spindle microtubules. Attachments in these cells are restored by inhibition of Aurora kinase, which is known to stabilize kinetochore-microtubule attachments. Loss of BubR1 function thus perturbs regulation of attachments rather than the ability of kinetochores to bind to microtubules. Consistent with this finding, depletion of BubR1 increases phosphorylation of CENP-A, a kinetochore-specific Aurora kinase substrate. We propose that BubR1 links regulation of chromosome-spindle attachment to mitotic checkpoint signalling.     

The role of localization in the operation of the mitotic spindle assembly checkpoint

The mitotic spindle assembly checkpoint (^MSAC) is an important regulatory mechanism of the cell cycle, ensuring proper chromosome segregation in mitosis. It delays the transition to anaphase until all chromosomes are properly attached to the mitotic spindle by emitting a diffusible “wait anaphase”-signal from unattached kinetochores. Current models of the checkpoint disregard important spatial properties like localization, diffusion and realistic numbers of kinetochores. To allow for in silico studies of the dynamics of these models in a more realistic environment, we introduce a mathematical framework for quasi-spatial simulation of localized biochemical processes that are typically observed during checkpoint activation and maintenance. The “emitted inhibition” model of the ^MSAC by Doncic et al. (Proc Natl Acad Sci USA 2005; 102:6332–7) assumes instantaneous activation of the diffusible “wait anaphase”-signal upon kinetochore encounter. We modify this model to account for binding kinetics with finite rates and use the developed framework to determine the feasible range of the binding parameters. We find that for proper activation, the binding rate constant has to be fast and above a critical value. Furthermore, this critical value depends significantly on the amount of local binding sites at each kinetochore. The critical values lie in a physiological realistic regime (10⁴–10⁶ M^-1s^-1). We also determine the feasible parameter range for fast checkpoint activation of the “Mad2 template” model, for which the kinetic parameters have recently been studied in vitro by Simonetta et al. (PLoS Biology 2009; 7:1000010). We find critical values for binding and catalysis rate constants, both significantly higher than the measured values. Our results suggest that yet unknown mechanisms at the kinetochores facilitate binding and catalysis in vivo. We conclude that quantitative models of the ^MSAC have to account for the limited availability of binding sites at kinetochores.     

Recruitment of Mad1 to metaphase kinetochores is sufficient to reactivate the mitotic checkpoint

The mitotic checkpoint monitors kinetochore–microtubule attachment and prevents anaphase until all kinetochores are stably attached. Checkpoint regulation hinges on the dynamic localization of checkpoint proteins to kinetochores. Unattached, checkpoint-active kinetochores accumulate multiple checkpoint proteins, which are depleted from kinetochores upon stable attachment, allowing checkpoint silencing. Because multiple proteins are recruited simultaneously to unattached kinetochores, it is not known what changes at kinetochores are essential for anaphase promoting complex/cyclosome (APC/C) inhibition. Using chemically induced dimerization to manipulate protein localization with temporal control, we show that recruiting the checkpoint protein Mad1 to metaphase kinetochores is sufficient to reactivate the checkpoint without a concomitant increase in kinetochore levels of Mps1 or BubR1. Furthermore, Mad2 binding is necessary but not sufficient for Mad1 to activate the checkpoint; a conserved C-terminal motif is also required. The results of our checkpoint reactivation assay suggest that Mad1, in addition to converting Mad2 to its active conformation, scaffolds formation of a higher-order mitotic checkpoint complex at kinetochores.     

Preventing aneuploidy: the contribution of mitotic checkpoint proteins

Aneuploidy, an abnormal number of chromosomes, is a trait shared by most solid tumors. Chromosomal instability (CIN) manifested as aneuploidy might promote tumorigenesis and cause increased resistance to anti-cancer therapies. The mitotic checkpoint or spindle assembly checkpoint is a major signaling pathway involved in the prevention of CIN. We review current knowledge on the contribution of misregulation of mitotic checkpoint proteins to tumor formation and will address to what extent this contribution is due to chromosome segregation errors directly. We propose that both checkpoint and non-checkpoint functions of these proteins contribute to the wide array of oncogenic phenotypes seen upon their misregulation.     

CDK-dependent potentiation of MPS1 kinase activity is essential to the mitotic checkpoint

Accurate chromosome segregation relies upon a mitotic checkpoint that monitors kinetochore attachment toward opposite spindle poles before enabling chromosome disjunction [1]. The MPS1/TTK protein kinase is a core component of the mitotic checkpoint that lies upstream of MAD2 and BubR1 both at the kinetochore and in the cytoplasm [2, 3]. To gain insight into the mechanisms underlying the regulation of MPS1 kinase, we undertook the identification of Xenopus MPS1 phosphorylation sites by mass spectrometry. We mapped several phosphorylation sites onto MPS1 and we show that phosphorylation of S283 in the noncatalytic region of MPS1 is required for full kinase activity. This phosphorylation potentiates MPS1 catalytic efficiency without impairing its affinity for the substrates. By using Xenopus egg extracts depleted of endogenous MPS1 and reconstituted with single point mutants, we show that phosphorylation of S283 is essential to activate the mitotic checkpoint. This phosphorylation does not regulate the localization of MPS1 to the kinetochore but is required for the recruitment of MAD1/MAD2, demonstrating its role at the kinetochore. Constitutive phosphorylation of S283 lowers the number of kinetochores required to hold the checkpoint, which suggests that CDK-dependent phosphorylation of MPS1 is essential to sustain the mitotic checkpoint when few kinetochores remain unattached.     

Regulation of mitotic inhibitor Mik1 helps to enforce the DNA damage checkpoint

The protein kinase Chk1 enforces the DNA damage checkpoint. This checkpoint delays mitosis until damaged DNA is repaired. Chk1 regulates the activity and localization of Cdc25, the tyrosine phosphatase that activates the cdk Cdc2. Here we report that Mik1, a tyrosine kinase that inhibits Cdc2, is positively regulated by the DNA damage checkpoint. Mik1 is required for checkpoint response in strains that lack Cdc25. Long-term DNA damage checkpoint arrest fails in Δmik1 cells. DNA damage increases Mik1 abundance in a Chk1-dependent manner. Ubiquitinated Mik1 accumulates in a proteasome mutant, which indicates that Mik1 normally has a short half-life. Thus, the DNA damage checkpoint might regulate Mik1 degradation. Mik1 protein and mRNA oscillate during the unperturbed cell cycle, with peak amounts detected around S phase. These data indicate that regulation of Mik1 abundance helps to couple mitotic onset to the completion of DNA replication and repair. Coordinated negative regulation of Cdc25 and positive regulation of Mik1 ensure the effective operation of the DNA damage checkpoint.     

The vertebrate mitotic checkpoint protein BUBR1 is an unusual pseudokinase

Chromosomal stability is safeguarded by a mitotic checkpoint, of which BUB1 and Mad3/BUBR1 are core components. These paralogs have similar, but not identical, domain organization. We show that Mad3/BUBR1 and BUB1 paralogous pairs arose by nine independent gene duplications throughout evolution, followed by parallel subfunctionalization in which preservation of the ancestral, amino-terminal KEN box or kinase domain was mutually exclusive. In one exception, vertebrate BUBR1-defined by the KEN box-preserved the kinase domain but allowed nonconserved degeneration of catalytic motifs. Although BUBR1 evolved to a typical pseudokinase in some vertebrates, it retained the catalytic triad in humans. However, we show that putative catalysis by human BUBR1 is dispensable for error-free chromosome segregation. Instead, residues that interact with ATP in conventional kinases are essential for conformational stability in BUBR1. We propose that parallel evolution of BUBR1 orthologs rendered its kinase function dispensable in vertebrates, producing an unusual, triad-containing pseudokinase.