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.     

A new level of spindle assembly checkpoint inactivation that functions without mitotic spindles

Comment on: De Souza CP, et al. EMBO J 2011; 30:2648-61.     

Aberrantly segregating centromeres activate the spindle assembly checkpoint in budding yeast

The spindle assembly checkpoint is the mechanism or set of mechanisms that prevents cells with defects in chromosome alignment or spindle assembly from passing through mitosis. We have investigated the effects of mini-chromosomes on this checkpoint in budding yeast by performing pedigree analysis. This method allowed us to observe the frequency and duration of cell cycle delays in individual cells. Short, centromeric linear mini-chromosomes, which have a low fidelity of segregation, cause frequent delays in mitosis. Their circular counterparts and longer linear mini-chromosomes, which segregate more efficiently, show a much lower frequency of mitotic delays, but these delays occur much more frequently in divisions where the mini-chromosome segregates to only one of the two daughter cells. Using a conditional centromere to increase the copy number of a circular mini-chromosome greatly increases the frequency of delayed divisions. In all cases the division delays are completely abolished by the mad mutants that inactivate the spindle assembly checkpoint, demonstrating that the Mad gene products are required to detect the subtle defects in chromosome behavior that have been observed to arrest higher eukaryotic cells in mitosis.     

Regulated inactivation of the spindle assembly checkpoint without functional mitotic spindles

The spindle assembly checkpoint (SAC) arrests mitosis until bipolar attachment of spindle microtubules to all chromosomes is accomplished. However, when spindle formation is prevented and the SAC cannot be satisfied, mammalian cells can eventually overcome the mitotic arrest while the checkpoint is still activated. We find that Aspergillus nidulans cells, which are unable to satisfy the SAC, inactivate the checkpoint after a defined period of mitotic arrest. Such SAC inactivation allows normal nuclear reassembly and mitotic exit without DNA segregation. We demonstrate that the mechanisms, which govern such SAC inactivation, require protein synthesis and can occur independently of inactivation of the major mitotic regulator Cdk1/Cyclin B or mitotic exit. Moreover, in the continued absence of spindle function cells transit multiple cell cycles in which the SAC is reactivated each mitosis before again being inactivated. Such cyclic activation and inactivation of the SAC suggests that it is subject to cell-cycle regulation that is independent of bipolar spindle function.     

Micronuclei from misaligned chromosomes that satisfy the spindle assembly checkpoint in cancer cells

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Monitoring the fidelity of mitotic chromosome segregation by the spindle assembly checkpoint

Accurate chromosome segregation relies on activity of the spindle assembly checkpoint, a surveillance mechanism that prevents premature anaphase onset until all chromosomes are properly attached to the mitotic spindle apparatus and aligned at the metaphase plate. Defects in this mechanism contribute to chromosome instability and aneuploidy, a hallmark of malignant cells. Here, we review the molecular mechanisms of activation and silencing of the spindle assembly checkpoint and its relationship to tumourigenesis.     

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.     

Multiple assembly mechanisms anchor the KMN spindle checkpoint platform at human mitotic kinetochores

During mitosis, the spindle checkpoint senses kinetochores not properly attached to spindle microtubules and prevents precocious sister-chromatid separation and aneuploidy. The constitutive centromere-associated network (CCAN) at inner kinetochores anchors the KMN network consisting of Knl1, the Mis12 complex (Mis12C), and the Ndc80 complex (Ndc80C) at outer kinetochores. KMN is a critical kinetochore receptor for both microtubules and checkpoint proteins. Here, we show that nearly complete inactivation of KMN in human cells through multiple strategies produced strong checkpoint defects even when all kinetochores lacked microtubule attachment. These KMN-inactivating strategies reveal multiple KMN assembly mechanisms at human mitotic kinetochores. In one mechanism, the centromeric kinase Aurora B phosphorylates Mis12C and strengthens its binding to the CCAN subunit CENP-C. In another, CENP-T contributes to KMN attachment in a CENP-H-I-K–dependent manner. Our study provides insights into the mechanisms of mitosis-specific assembly of the checkpoint platform KMN at human kinetochores.     

Amphiastral mitotic spindle assembly in vertebrate cells lacking centrosomes

The role of centrosomes and centrioles during mitotic spindle assembly in vertebrates remains controversial. In cell-free extracts and experimentally derived acentrosomal cells, randomly oriented microtubules (MTs) self-organize around mitotic chromosomes and assemble anastral spindles. However, vertebrate somatic cells normally assemble a connected pair of polarized, astral MT arrays--termed an amphiaster ("a star on both sides")--that is formed by the splitting and separation of the microtubule-organizing center (MTOC) well before nuclear envelope breakdown (NEB). Whether amphiaster formation requires splitting of duplicated centrosomes is not known. We found that when centrosomes were removed from living vertebrate cells early in their cell cycle, an acentriolar MTOC reassembled, and, prior to NEB, a functional amphiastral spindle formed. Cytoplasmic dynein, dynactin, and pericentrin are all recruited to the interphase aMTOC, and the activity of kinesin-5 is needed for amphiaster formation. Mitosis proceeded on time and these karyoplasts divided in two. However, ~35% of aMTOCs failed to split and separate before NEB, and these entered mitosis with persistent monastral spindles. Chromatin-associated RAN-GTP--the small GTPase Ran in its GTP bound state--could not restore bipolarity to monastral spindles, and these cells exited mitosis as single daughters. Our data reveal the novel finding that MTOC separation and amphiaster formation does not absolutely require the centrosome, but, in its absence, the fidelity of bipolar spindle assembly is highly compromised.     

Microtubules do not promote mitotic slippage when the spindle assembly checkpoint cannot be satisfied

When the spindle assembly checkpoint (SAC) cannot be satisfied, cells exit mitosis via mitotic slippage. In microtubule (MT) poisons, slippage requires cyclin B proteolysis, and it appears to be accelerated in drug concentrations that allow some MT assembly. To determine if MTs accelerate slippage, we followed mitosis in human RPE-1 cells exposed to various spindle poisons. At 37°C, the duration of mitosis in nocodazole, colcemid, or vinblastine concentrations that inhibit MT assembly varied from 20 to 30 h, revealing that different MT poisons differentially depress the cyclin B destruction rate during slippage. The duration of mitosis in Eg5 inhibitors, which induce monopolar spindles without disrupting MT dynamics, was the same as in cells lacking MTs. Thus, in the presence of numerous unattached kinetochores, MTs do not accelerate slippage. Finally, compared with cells lacking MTs, exit from mitosis is accelerated over a range of spindle poison concentrations that allow MT assembly because the SAC becomes satisfied on abnormal spindles and not because slippage is accelerated.     

The RAS GTPase RIT1 compromises mitotic fidelity through spindle assembly checkpoint suppression

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Differential spindle assembly checkpoint response in human lung adenocarcinoma cells

CI-980 is an antimicrotubule agent that binds the colchicine site on tubulin. We examined CI-980 cytotoxicity in two lung adenocarcinoma cell lines, A549 and A427. Depolymerization of microtubules following CI-980 treatment resulted in a mitotic arrest in the A549 population, but not in the A427 population. Similar responses were obtained following treatment with Taxol and nocodazole. Drug-treated A427 cells exited mitosis, generating a population dominated by multinucleated cells, while both multinucleated and apoptotic cells were present in the A549 population after extended drug treatment. CI-980-induced microtubule depolymerization was only partially reversible. However, regrowth of some microtubules in mitotic A549 cells following drug washout resulted in multinucleation of the population in the absence of apoptosis. These results show that A427 cells have a defective spindle assembly checkpoint. Levels of the MAD2 and BUB1 checkpoint proteins were similar in both A549 and A427 cells, suggesting that the checkpoint defect in the A427 cells is downstream of these proteins. In addition, induction of apoptosis in response to CI-980 correlates with the presence of a functional mitotic checkpoint and the extent of microtubule depolymerization.     

Significance of mitotic spindle checkpoint genes in leukemia

Leukemia is a clonal proliferative disorder of a multipotent hematopoietic stem cell that leads to abnormal cell growth and/or differentiation. The hallmark of the disease is the presence of oncogene expression in bone marrow or peripheral blood resulting from chromosome translocations. The development of leukemia and the progression of the disease are multistage processes implicated in a series of molecular changes preceeded chromosomal instability and aneuploidy. The most likely of the above-mentioned changes include the following: the appearance of additional chromosome translocations, the activation of other oncogenes not expressed previously, the loss of tumor suppressor genes, abnormal centrosome duplication, and the dysfunction of genes that coordinate accurate chromosome alignment and chromosome segregation during mitosis. The last two molecular events controlled by mitotic spindle checkpoint genes play a role in leukemogenesis and are probably involved in apoptosis.     

Mutual antagonism between the anaphase promoting complex and the spindle assembly checkpoint contributes to mitotic timing in Caenorhabditis elegans

The anaphase promoting complex/cyclosome (APC/C) triggers the separation of sister chromatids and exit from mitosis across eukaryotic evolution. The APC/C is inhibited by the spindle assembly checkpoint (SAC) until all chromosomes have achieved bipolar attachment, but whether the APC/C reciprocally regulates the SAC is less understood. Here, we report the characterization of a novel allele of the APC5 component SUCH-1 in Caenorhabditis elegans. We find that some such-1(t1668) embryos lack paternally contributed DNA and centrioles and assemble a monopolar spindle in the one-cell stage. Importantly, we show that mitosis is drastically prolonged in these embryos, as well as in embryos that are otherwise compromised for APC/C function and assemble a monopolar spindle. This increased duration of mitosis is dependent on the SAC, since inactivation of the SAC components MDF-1/MAD1 or MDF-2/MAD2 rescues proper timing in these embryos. Moreover, partial depletion of the E1 enzyme uba-1 significantly increases mitosis duration upon monopolar spindle assembly. Taken together, our findings raise the possibility that the APC/C negatively regulates the SAC and, therefore, that the SAC and the APC/C have a mutual antagonistic relationship in C. elegans embryos.     

Kinetochore "memory" of spindle checkpoint signaling in lysed mitotic cells

The spindle checkpoint prevents errors in mitosis. Cells respond to the presence of kinetochores that are improperly attached to the mitotic spindle by delaying anaphase onset. Evidence suggests that phosphorylations recognized by the 3F3/2 anti-phosphoepitope antibody may be involved in the kinetochore signaling of the spindle checkpoint. Mitotic cells lysed in detergent in the absence of phosphatase inhibitors rapidly lose expression of the 3F3/2 phosphoepitope. However, when ATP is added to lysed and rinsed mitotic cytoskeletons, kinetochores become rephosphorylated by an endogenous, bound kinase. Kinetochore rephosphorylation in vitro produced the same differential phosphorylation seen in appropriately fixed living cells. In chromosomes not yet aligned at the metaphase plate, kinetochores undergo rapid rephosphorylation, while those of fully congressed chromosomes are under-phosphorylated. However, latent 3F3/2 kinase activity is retained at kinetochores of cells at all stages of mitosis including anaphase. This latent activity is revealed when rephosphorylation reactions are carried out for extended times. The endogenous, kinetochore-bound kinase can be chemically inactivated. Remarkably, a soluble kinase activity extracted from mitotic cells also caused differential rephosphorylation of kinetochores whose endogenous kinase had been chemically inactivated. We suggest that, in vivo, microtubule attachment alters the kinetochore 3F3/2 phosphoprotein, causing it to resist phosphorylation. This kinetochore modification is retained after cell lysis, producing a "memory" of the in vivo phosphorylation state.     

Releasing the spindle assembly checkpoint without tension

Proteins of the Bcl-2 family are critical regulators of apoptosis, but how its BH3-only members activate the essential effectors Bax and Bak remains controversial. The indirect activation model suggests that they simply must neutralize all of the prosurvival Bcl-2 family members, whereas the direct activation model proposes that Bim and Bid must activate Bax and Bak directly. As numerous in vitro studies have not resolved this issue, we have investigated Bim''s activity in vivo by a genetic approach. Because the BH3 domain determines binding specificity for Bcl-2 relatives, we generated mice having the Bim BH3 domain replaced by that of Bad, Noxa, or Puma. The mutants bound the expected subsets of prosurvival relatives but lost interaction with Bax. Analysis of the mice showed that Bim''s proapoptotic activity is not solely caused by its ability to engage its prosurvival relatives or solely to its binding to Bax. Thus, initiation of apoptosis in vivo appears to require features of both models.     

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.     

Prevalent loss of mitotic spindle checkpoint in adult T-cell leukemia confers resistance to microtubule inhibitors.

Human T-cell leukemia virus type I (HTLV-I) is the causative agent for adult T-cell leukemia (ATL). Molecularly, ATL cells have extensive aneugenic abnormalities that occur, at least in part, from cell cycle dysregulation by the HTLV-I-encoded Tax oncoprotein. Here, we compared six HTLV-I-transformed cells to Jurkat and primary peripheral blood mononuclear cells (PBMC) in their responses to treatment with microtubule inhibitors. We found that both Jurkat and PBMCs arrested efficiently in mitosis when treated with nocodazole. By contrast, all six HTLV-I cells failed to arrest comparably in mitosis, suggesting that ATL cells have a defect in the mitotic spindle assembly checkpoint. Mechanistically, we observed that in HTLV-I Tax-expressing cells human spindle assembly checkpoint factors hsMAD1 and hsMAD2 were mislocated from the nucleus to the cytoplasm. This altered localization of hsMAD1 and hsMAD2 correlated with loss of mitotic checkpoint function and chemoresistance to microtubule inhibitors.     

Deficient spindle assembly checkpoint in multiple myeloma

Multiple myeloma (MM) is a hematological disease characterized by an abnormal accumulation of plasma cells in the bone marrow. These cells have frequent cytogenetic abnormalities including translocations of the immunoglobulin heavy chain gene and chromosomal gains and losses. In fact, a singular characteristic differentiating MM from other hematological malignancies is the presence of a high degree of aneuploidies. As chromosomal abnormalities can be generated by alterations in the spindle assembly checkpoint (SAC), the functionality of such checkpoint was tested in MM. When SAC components were analyzed in MM cell lines, the RNA levels of most of them were conserved. Nevertheless, the protein content of some key constituents was very low in several cell lines, as was the case of MAD2 or CDC20 in RPMI-8226 or RPMI-LR5 cells. The recovery of their cellular content did not substantially affect cell growth, but improved their ability to segregate chromosomes. Finally, SAC functionality was tested by challenging cells with agents disrupting microtubule dynamics. Most of the cell lines analyzed exhibited functional defects in this checkpoint. Based on the data obtained, alterations both in SAC components and their functionality have been detected in MM, pointing to this pathway as a potential target in MM treatment.     

Knockdown of B-Raf impairs spindle formation and the mitotic checkpoint in human somatic cells

It is well established that B-Raf signaling through the MAP kinase (ERK) pathways plays a prominent role in regulating cell proliferation but how it does this is not completely understood. Here, we show that B-Raf serves a physiological role during mitosis in human somatic cells. Knockdown of B-Raf using short interfering RNA (siRNA) resulted in pleiotropic spindle abnormalities and misaligned chromosomes in over 80% of the mitotic cells analyzed. A second B-Raf siRNA gave similar results suggesting these effects are specific to down-regulating B-Raf protein. In agreement with these findings, a portion of B-Raf was detected at the spindle structures including the spindle poles and kinetochores. Knockdown of C-Raf (Raf-1) had no detectable effects on spindle formation or chromosome alignment. Activation of the spindle assembly checkpoint was found to be dependent on B-Raf as evident by the inability of checkpoint proteins Bub1 and Mad2 to localize to unattached kinetochores in HeLa cells treated with B-Raf siRNA. Consistent with this, live-cell imaging microscopy showed that B-Raf-depleted cells exited mitosis earlier than control non-depleted cells. Finally, we provide evidence that B-Raf signaling promotes phosphorylation and kinetochore localization of the mitotic checkpoint kinase Mps1. Blocking B-Raf expression, ERK activity, or phosphorylation at Ser-821 residue perturbed Mps1 localization at unattached kinetochores. Thus, our data implicates a mitotic role for B-Raf in regulating spindle formation and the spindle checkpoint in human somatic cells.