In-Silico Modeling of the Mitotic Spindle Assembly Checkpoint

The Mitotic Spindle Assembly Checkpoint ((M)SAC) 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.We have constructed and validated for the human (M)SAC 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 (M)SAC 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.Only in the controlled case, our models show (M)SAC behaviour at meta- to anaphase transition in agreement with experimental observations. Our simulations revealed that for (M)SAC activation, Cdc20 is not fully sequestered; instead APC is inhibited by MCC binding

Targeting the major regulator of mitosis.

Mitosis-inhibiting chemotherapeutics (e.g. taxanes) are frequently used to treat multiple cancer types. Recently, there has been much concern about the limited success of these drugs due to resistance and a lack of molecular targets. Thus, there is high demand for new drugs with diverse cellular targets. Targeting the regulators of mitosis is a promising approach. The anaphase promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that controls cell cycle progression at multiple points. The interaction of ANAPC2 and ANAPC11, catalytic core subunits, is necessary for APC/C function. An in silico approach was used to identify compounds predicted to prevent assembly of ANAPC2 and ANAPC11, causing APC/C inhibition and mitotic arrest. Several of the predicted compounds possess cytotoxic properties in multiple cancer cell types. These hit compounds induce mitotic arrest and cell death in malignant, but not non-malignant cells. Additionally, hit compounds are effective in taxane-resistant cells

Systems Biology of Mitosis

In mitosis, `surveillance control mechanisms' regulate transitions across the several stages of cell division. The so called `Mitotic-Spindle-Assembly-Checkpoint (MSAC)' and `Exit-From-Mitosis (EFM)' are examples of such mechanisms. MSAC ensures the correct segregation of chromosomes by preventing cell-cycle progression until all chromosomes have made proper bipolar attachments to the mitotic spindle through their kinetochores. EFM ensures that each of the two daughter nuclei receives one copy of each chromosome. Both mechanisms are seemingly regulated by the so called `Anaphase-Promoting-Complex (APC)', bound, in turn, to either `Cdc20' or `Cdh1', which are associated regulatory proteins. APC remains inactive during metaphase. In the transition from metaphase to anaphase, and only after all chromosomes are attached, a newly formed `APC:Cdc20' complex mediates the ubiquitination and degradation of the protein `Securin'; this leads, in turn, to the activation of the protein `Separase', the dissolution of the so called `Cohesin Complex', and, eventually, to chromatid separation. `APC:Cdc20' also mediates the initial phase of `Cyclin B' proteolysis. In the transition from anaphase to telophase, APC:Cdh1 completely ubiquitinates `Cyclin B', thus inactivating a protein called `CyclinB:Cdk1-Mitotic-Kinase' and triggering the exit from mitosis. Both MSAC and EFM prevent chromosome miss-segregation and aneuploidy, and their failure eventually leads to cell death; both mechanisms have been implicated in cancer

Multi-scale stochastic organization-oriented coarse-graining exemplified on the human mitotic checkpoint

The complexity of biological models makes methods for their analysis and understanding highly desirable. Here, we demonstrate the orchestration of various novel coarse-graining methods by applying them to the mitotic spindle assembly checkpoint. We begin with a detailed fine-grained spatial model in which individual molecules are simulated moving and reacting in a three-dimensional space. A sequence of manual and automatic coarse-grainings finally leads to the coarsest deterministic and stochastic models containing only four molecular species and four states for each kinetochore, respectively. We are able to relate each more coarse-grained level to a finer one, which allows us to relate model parameters between coarse-grainings and which provides a more precise meaning for the elements of the more abstract models. Furthermore, we discuss how organizational coarse-graining can be applied to spatial dynamics by showing spatial organizations during mitotic checkpoint inactivation. We demonstrate how these models lead to insights if the model has different “meaningful” behaviors that differ in the set of (molecular) species. We conclude that understanding, modeling and analyzing complex bio-molecular systems can greatly benefit from a set of coarse-graining methods that, ideally, can be automatically applied and that allow the different levels of abstraction to be related

The Influence of Catalysis on Mad2 Activation Dynamics

Mad2 is a key component of the spindle assembly checkpoint, a safety device ensuring faithful sister chromatid separation in mitosis. The target of Mad2 is Cdc20, an activator of the anaphase-promoting complex/cyclosome (APC/C). Mad2 binding to Cdc20 is a complex reaction that entails the conformational conversion of Mad2 from an open (O-Mad2) to a closed (C-Mad2) conformer. Previously, it has been hypothesized that the conversion of O-Mad2 is accelerated by its conformational dimerization with C-Mad2. This hypothesis, known as the Mad2-template hypothesis, is based on the unproven assumption that the natural conversion of O-Mad2 required to bind Cdc20 is slow. Here, we provide evidence for this fundamental assumption and demonstrate that conformational dimerization of Mad2 accelerates the rate of Mad2 binding to Cdc20. On the basis of our measurements, we developed a set of rate equations that deliver excellent predictions of experimental binding curves under a variety of different conditions. Our results strongly suggest that the interaction of Mad2 with Cdc20 is rate limiting for activation of the spindle checkpoint. Conformational dimerization of Mad2 is essential to accelerate Cdc20 binding, but it does not modify the equilibrium of the Mad2:Cdc20 interaction, i.e., it is purely catalytic. These results surpass previously formulated objections to the Mad2-template model and predict that the release of Mad2 from Cdc20 is an energy-driven process

Coarse graining particle simulations of biological systems: exemplified on PML nuclear bodies and the SAC

Simulating biological systems in-silico has become a common method to provide deeper insights into the regarding systems than in-vitro investigations. Classic approaches like differential equations, Boolean networks or Markov chains are performant, but usually cannot express often desired spatial features. For that reason particle based simulators like Smoldyn, GFRD or ReaDDy came up that are able to examine a reaction network in space, but usually not on a large timescale. Due to methodological and instrumental restrictions it still is elusive to simulate complex systems in full detail over a large period of time. Coarse-graining methods allow the reduction of a highdetailed system into a little-detail system, whereby the qualitative behavior of the simulated systems is conserved. The aim of this thesis is to derive techniques that allow a detailed simulation on a large timescale. For that reason, firstly, a pathway is developed that translates a reaction network between a set of species into space using properties from literature, like mass and diffusion coefficient of all species. Secondly, coarse-graining methods are developed that are automatized applicable to real biological systems. These methods allow the simulation of the generated particle based model in a feasible amount of time, whereby the focus lies on the reduction of the simulation complexity rather than the models complexity. Thirdly, a novel simulation tool is established that allows a simplified study of self assembling processes by coarse-graining the diffusion. To study these methods at work, two models of biological systems emerged in the scope of this thesis, namely the spindle assembly checkpoint and PML nuclear bodies. The spindle assembly checkpoint (SAC) guards proper cell division by prolonging the metaphase until all 92 sister chromatids are aligned properly. Even a single unattached kinetochore keeps the SAC active, which is rapidly inactivated after the last attachment

To Divide or Not to Divide; MicroRNAs and Small Compounds as Modulators of Mitosis

Mitosis is under the stringent quality control of the spindle assembly checkpoint (SAC). However, in cancer cells this control can fail, leading to excessive cellular proliferation and ultimately to the formation of a tumor. Novel cancer cell selective therapies are needed to stop the uncontrolled cell proliferation and tumor growth. The aim of the research presented in this thesis was to identify microRNAs (miRNAs) that could play a role in cancer cell proliferation as well as low molecular weight (LMW) compounds that could interfere with cell division. The findings could be used to develop better cancer diagnostics and therapies in the future. First, a high-throughput screen (HTS) was performed to identify LMW compounds that possess a similar chemical interaction field as rigosertib, an anti-cancer compound undergoing clinical trials. A compound termed Centmitor-1 was discovered that phenocopied the cellular impact of rigosertib by affecting the microtubule dynamics. Next, another HTS aimed at identifying compounds that would target the Hec1 protein, which mediates the interaction between spindle microtubules and chromosomes. Perturbation of this connection should prevent cell division and induce cell death. A compound termed VTT-006 was discovered that abrogated mitosis in several cell line models and exhibited binding to Hec1 in vitro. Lastly, using a cell-based HTS two miRNAs were identified that affected cancer cell proliferation via Aurora B kinase, which is an important mitotic regulator. MiR-378a-5p was found to indirectly suppress the production of the kinase whereas let-7b showed direct binding to the 3’UTR of Aurora B mRNA and repressed its translation. The miRNA-mediated perturbation of Aurora B induced defects in mitosis leading to abnormal chromosome segregation and induction of aneuploidy. The results of this thesis provide new information on miRNA signaling in cancer, which could be utilized for diagnostic purposes. Moreover, the thesis introduces two small compounds that may benefit future drug research.Siirretty Doriast

New Roads to Genomic Imbalance: Microrna- and Protein Phosphatase-Mediated Regulation of Mitosis

The Spindle Assembly Checkpoint (SAC) works to maintain the genomic balance by monitoring the correctness of attachments between the chromosomes and microtubules during early cell division (mitosis). Importantly, chromosome missegregation and genomic instability is caused by defects in SAC function, which can lead to cell transformation and cancer. Moreover, malfunction of SAC can confer resistance to microtubule-targeting drugs (MTAs) such as paclitaxel. The identification and functional characterization of novel SAC regulating biomolecules and analysis of their expression profiles in tumor cells may in the future facilitate improved cancer diagnosis and predict patient’s response to MTA therapy. The microRNA- (miRNA) and protein phosphatase-mediated regulation of mitotic signaling were investigated in my thesis. First, to identify new miRNAs that regulate SAC signaling, a cell-based high-throughput screen (HTS) was performed to test the ability of 810 different pre-miRNAs to override a drug imposed M phase arrest. The HTS led to the discovery of miR-378a-5p and miR-493-3p as suppressors of MTA action in vitro. Further molecular biology experiments revealed that miR-378a-5p and miR-493-3p negatively regulated the expression of AURKB and MAD2L1, respectively. Retrospective analysis of ovarian and breast cancer samples in vivo indicated deregulated expression of both miRNAs in different tumor grades and subtypes compared to healthy tissues. Moreover, high expression of miR-493-3p was found to associate with reduced survival of ovarian and breast cancer patients with aggressive tumors, especially if the patients were treated with paclitaxel chemotherapy in comparison to epirubicin. Secondly, the functions of the Dual specificity protein phosphatase 3 (Dusp3) in dividing cells were explored. The maintenance of bipolar spindle architecture during mitosis was found to be dependent on Dusp3. In summary, the previously unknown mitotic functions for three biomolecules, namely miR-378a-5p, miR-493-3p and Dusp3 were identified. The determination of miR-378a-5p and miR-493-3p expression profiles, in tumor samples can in the future assist in cancer subtyping and selecting the most effective chemotherapy for ovarian and breast cancer patients with advanced disease.Siirretty Doriast

Regulation of spindle assembly checkpoint (SAC) by phosphorylation and protein-protein interactions in Drosophila melanogaster

PhD ThesisChromosome segregation is a complex, but subsequently error- prone process, who’s accuracy is essential to prevent uneven DNA distribution between mother and daughter cells. Such unequal chromosome segregation can often result in aneuploidy, which is a prevalent phenotype of cancer cells, and so surveillance mechanisms must exist within the cell cycle to detect and correct the cause of such chromosome division errors, before allowing the cell to divide. The Spindle Assembly Checkpoint (SAC) has evolved to monitor the interaction between microtubules, and the point at which they attach sister chromosomes, the kinetochore. By detecting attachment and resulting tension abnormalities, the SAC halts the metaphase to anaphase transition if chromosomes are not aligned correctly at the metaphase plate. By disallowing cell division to occur in the absence of proper chromosome alignment, the SAC minimises the frequency of uneven DNA distribution and the consequent problems this can incur. Silencing of the SAC, and normal cell progression is not promoted until correction mechanisms have achieved proper bioriented chromosome attachments. The target of the SAC is widely accepted to be Cell Division Cycle 20 (Cdc20), which is the activator of the Anaphase Promoting Complex or Cyclosome (APC/C), the E3 ubiquitin ligase that drives cells into anaphase. By inhibiting Cdc20, the activity of the APC/C is halted, and cells are arrested at metaphase. A number of key proteins are believed to be involved in the sequestration of Cdc20, by incorporating it into an inhibitory Mitotic Checkpoint Complex (MCC). This MCC complex is believed to comprise of Cdc20, BubR1, Bub1 and Mad2, although there is speculation as to whether Mad2 is part of the complex, or merely promotes its formation. The proteins involved in the MCC all localise to kinetochores with activation of the SAC, although it remains unclear as to whether the MCC forms at the kinetochore upon localisation of the various components, or can form in part or as a whole, moving to kinetochores upon SAC activation. Sub-complexes of the MCC have been detected outside of mitosis, which provide evidence in favour of a kinetochore-independent MCC formation. However, if this were the case, it could be assumed that modification (such as phosphorylation) to either MCC components or the APC/C itself would need to occur in mitosis or with SAC activation, allowing for APC/C inhibition only with SAC activation, and to prevent IV non-specific inhibition of APC/C by the MCC elsewhere in the cell cycle. These issues still remain unclear. In order to investigate further, the requirement of direct kinetochore localisation of MCC components in the formation of the complex, this thesis aims to provide evidence of the effect of disrupting such kinetochore localisation upon checkpoint function, as well as the impact of removal of Cdc20 modifications on MCC formation. In addition to this, the protein-protein interaction domains between Cdc20 and BubR1, proven essential for SAC function, are investigated within Drosophila melanogaster. Collectively, the data in this thesis provides an insight into the regulation of SAC in Drosophila. The Cdk1/Cyclin B phosphorylation of Fizzy (the Drosophila homologue of Cdc20) is confirmed to have an effect on MCC formation, and can be mapped to three specific sites on the N-terminal of Fizzy, which are conserved across various species. In addition to the effect of Cdk1/Cyclin B phosphorylation on the interaction between Fizzy and other SAC proteins, the importance of the BubR1 KEN box motif on the Fizzy-BubR1-Mad2 interaction is confirmed, implicating another essential domain for MCC formation in Drosophila. With regard to kinetochore localisation of SAC components, a model is achieved in which a dramatic reduction of Mps1, previously shown to disturb kinetochore localisation of Mad1, Mad2 and BubR1 in Xenopus, confirms a role for Mad2 kinetochore localisation in SAC activation, even though Fizzy localisation is unperturbed. Overall, these findings may provide a useful insight into the complex relationships, kinetochore localisation requirements and inter-protein dependencies within the regulation of MCC formation and SAC signalling in Drosophila melanogaster