The spindle checkpoint ensures proper chromosome segregation by monitoring kinetochore-microtubule interactions: unattached kinetochores recruit checkpoint proteins that combine to form a diffusible inhibitor which delays anaphase, thus buying cells time to fix attachment errors. Although the major checkpoint proteins were identified some 25 years ago, we have only just begun to understand how they assemble at unattached kinetochores to generate the crucial checkpoint signal. Much of this can be attributed to the difficulty associated with studying these proteins at the kinetochores, which are highly complex and thus often make clean dissection of function impossible. To circumvent this problem, a synthetic version of the spindle checkpoint was engineered on an ectopic location on a chromosome arm away from kinetochores in S. pombe. This work describes how the co-targeting of only two checkpoint components, the outer kinetochore protein Spc7 and the checkpoint kinase Mph1, was found to be sufficient to successfully generate a checkpoint-dependent metaphase arrest and how this paves the way for a clearer, more joined-up understanding of how the checkpoint works.

No description available.

The eukaryotic cell cycle is governed by molecular checkpoints that ensure genomic integrity and the faithful transmission of chromosomes to daughter cells. They inhibit the cycle until conditions prevail that guarantee accurate DNA duplication and chromosome segregation. Two major mechanisms are the ‘spindle assembly checkpoint’ and the ‘DNA damage checkpoint’. During pro-metaphase, the spindle checkpoint monitors the orientation process of chromatid pairs on the bipolar microtubule array nucleated by spindle pole bodies. In the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, six proteins are at the heart of spindle checkpoint function: Mad1, Mad2, Mad3, Bub1, Bub3 and Mph1/Mps1. The formation of spindle checkpoint complexes signals the presence of incorrect spindle microtubule attachments to kinetochores. These complexes cooperate to suppress the activity of the anaphase promoting complex (APC) and inhibit the onset of anaphase. By isolating these distinct complexes and analysing their composition by mass-spectrometry (MS) this work revealed several intriguing disparities between the two yeast species, and the way in which the Bub and Mad proteins cooperate to achieve inhibition. The ‘mitotic checkpoint complex’, which in S.cerevisiae consists of Mad2, Mad3, Bub3 and the APC activator Cdc20, was found to lack Bub3 in S.pombe. The S.pombe complex was shown to interact with the APC, but no stable interaction was found to be required in S.cerevisiae cells. And whereas Bub1 and Bub3 were found to form a complex with Mad1 in S.cerevisiae, in S.pombe they were shown to associate with Mad3 to form the ‘BUB+ spindle checkpoint complex’. In addition, MS analysis uncovered TAPAS: a novel S.pombe complex that was found to interact with the BUB+ complex and revealed to consist of Tfg3, Abo1 (gene product of SPAC31G5.19), Pob3 and Spt16. TAPAS mutant cells were shown to lose viability as a result of genotoxic stress, a phenotype that was surprisingly shared with bub1Δ and bub1kd ‘kinase dead’ mutants. Sensitivity of cells deficient in TAPAS or Bub1 did not appear to be due to the loss of DNA damage checkpoint or DNA replication checkpoint functions. Further examination revealed that Bub1 functions in the repair of DNA double strand breaks. Taken together, this work demonstrates that even though the molecular components of the spindle checkpoint pathway are conserved, their regulatory connections have to some extent diverged through molecular evolution. This process not only rewired, but entwined two molecular processes that together safeguard the genetic heritage of cells.

The accurate segregation of DNA during cell division is essential for the viability of future cellular generations. Genetic material is packaged in the form of chromosomes during cell division, and chromosomes are segregated equally into two daughter cells. Chromosome mis-distribution leads to genetic disorders (e.g. Down's syndrome), aneuploidy and cancer. The spindle checkpoint ensures proper chromosome segregation by monitoring kinetochore-microtubule interactions. Upon checkpoint activation, unattached kinetochores recruit checkpoint proteins that combine to form a diffusible inhibitor (the Mitotic Checkpoint Complex-MCC). The MCC delays anaphase, thus giving cells time to fix attachment errors. Although the major checkpoint proteins were identified several years ago, we have only just begun to understand how they assemble at unattached kinetochores to generate the checkpoint signal. Yeast genetics and proteomics have revealed that kinetochores are highly complex molecular machines with almost 50 kinetochore components and ~10 components of the spindle checkpoint machinery. Such complexity makes the separation of error correction, kinetochore bi-orientation and microtubule attachment functions very challenging. To circumvent this complexity, a synthetic version of the spindle checkpoint (SynCheck), based on tetO array was engineered at an ectopic location on a chromosome arm away from kinetochores in S. pombe. This work describes that combined targeting, initially of KNL1Spc7 with Mps1Mph1 and later of Bub1 (but not Mad1) with Mps1Mph1 fragments, was able to activate the spindle checkpoint and generate a robust arrest. The system is based on, soluble complexes, which were formed between KNL1Spc7 or Bub1 with Mps1Mph1. The synthetic checkpoint or 'Syncheck' is independent of localisation of the checkpoint components to the kinetochores, to spindle pole bodies (SPBs) and to nuclear pores. By using the synthetic tethering system a Mad1-Bub1 complex was identified for the first time in S.pombe. Bub1- Mad1 complex formation is crucial for checkpoint activation. Bub1-Mad1 gets phosphorylated itself and is thought to act as an assembly platform for MCC production and thereby generation of the "wait anaphase" signal. The ectopic tetO array is an important tool, not only for generating MCC formation and activating the spindle checkpoint, but also for providing a nice system for analysing in vivo protein-protein interactions. The ectopic array is capable of not only recruiting checkpoint components, but also recruiting them in a physiological manner (similar to the unattached kinetochores). For this reason it was decided to adopt this system to examine the role of the conserved Bub1TPR domain in the recruitment of other spindle checkpoint proteins. This work represents two novel functions for the S. pombe Bub1TPR domain. For the first time in S. pombe, both in vivo tethering and in vitro experiments with purified, recombinant proteins showed that the Bub1 has the ability to homodimerise and to form a complex with Mad3BubR1 through its TPR domain. These results revealed that complex formation of Bub1 with Mad3BubR1 is important for checkpoint signalling and that the highly conserved TPR domains in BubR1Mad3 and Bub1 have key roles to play in their interactions.

Errors in segregation of genetic materials are detrimental to all organisms. The budding yeast ensures accurate chromosome segregation by employing a system called the spindle checkpoint. The spindle checkpoint, which consists of proteins such as Mad1, Mad2, Mad3, Bub1, and Bub3, monitors the attachment of microtubules to the chromosomes and prevents cell cycle progression until all chromosomes are properly attached. To understand how the spindle checkpoint arrests cells in response to attachment errors at the chromosomes, we recruited different checkpoint proteins to an ectopic site on the chromosome by taking advantage of the binding of the lactose repressor (LacI) to the lactose operator (LacO). We found that cells expressing Bub1-LacI arrest in metaphase. The phenotype is in fact caused by dimerization of Bub1 when it is fused to LacI rather than the recruitment of Bub1 to chromosome. The cell cycle arrest by the Bub1 dimer depends on the presence of other checkpoint proteins, suggesting that the dimerization of Bub1 represents an upstream event in the spindle checkpoint pathway. The results with the Bub1 dimer inspired us to fuse checkpoint proteins to each other to mimic protein interactions that may contribute to checkpoint activation. We showed that fusing Mad2 and Mad3 arrests cells in mitosis and that this arrest is independent of other checkpoint proteins. We believe that combining Mad2 and Mad3 arrests cells because both proteins can bind weakly to Cdc20, the main target of the spindle checkpoint, and the sum of these two weak bindings creates a hybrid protein that binds tightly to Cdc20. We reasoned that if Mad3's role is to make Mad2 bind tightly, artificially tethering Mad2 directly to Cdc20 should also arrest cells and this arrest should not depend on any other checkpoint components. Our experiments confirmed these predictions, suggesting that Mad3 is required for the stable binding of Mad2 to Cdc20 in vivo, that this binding is sufficient to inhibit APC activity, and that this reaction is the most downstream event in spindle checkpoint activation. The interactions among spindle checkpoint proteins thus play an important role in cell cycle arrest and must be carefully regulated.

No description available.

<p>The DNA of a cell operates as its blueprint, providing coded information for the production of the RNA and proteins that allow the cell to function. Cells can face a myriad of insults to their genomic integrity during their lifetimes, from simple errors during growth and division to reactive oxygen species to chemotherapeutic reagents. To deal with these mutagenic insults and avoid passing them on to progeny, cells are equipped with multiple defenses. Checkpoints can sense problems and halt a cell’s progression through the cell cycle in order to allow repairs. More drastically, cells can also prevent passing on mutations to progeny by triggering apoptosis, or programmed cell death. This work will present two separate discoveries regarding the regulation of DNA damage-induced apoptosis and the regulation of the spindle checkpoint.</p><p> The protease caspase-2 has previously been shown to be an important regulator of DNA damage-induced apoptosis. In unstressed cells caspase-2 is present as an inactive monomer, but upon sensing a stress caspase-2 dimerizes and becomes catalytically active. The mechanisms that regulate this dimerization are poorly understood. The first research chapter details our development of a novel method to study dimerized caspase-2, which in turn identified TRAF2 as a direct activator of caspase-2. Specifically, we utilized the Bimolecular Fluorescence Complementation technique, wherein complementary halves of the Venus fluorophore are fused to caspase-2: when caspase-2 dimerizes, the non-fluorescent halves fold into a functional Venus fluorophore. We combined this technique with a Venus-specific immunoprecipitation that allowed the purification of caspase-2 dimers. Characterization of the caspase-2 dimer interactome by MS/MS identified several members of the TNF Receptor Associated Factor (TRAF) family, specifically TRAF1, 2, and 3. Knockdown studies revealed that TRAF2 plays a primary role in promoting caspase-2 dimerization and downstream apoptosis in response to DNA damage. Identification of a TRAF Interacting Motif (TIM) on caspase-2 indicates that TRAF2 directly acts on caspase-2 to induce its activation. TRAF2 is known to act as an E3 ubiquitin ligase as well as a scaffold for other E3 ubiquitin ligases. Indeed, we identified three lysine residues in the caspase-2 prodomain (K15, K152, and K153) important for its ubiquitination and complex formation. Together these results revealed a novel role for TRAF2 as a direct activator of caspase-2 apoptosis triggered by DNA damage.</p><p> During mitosis, when the cell prepares to divide, great care is taken to ensure that the chromosomes are properly segregated between the two daughter cells by the mitotic spindle. This is primarily accomplished through the spindle checkpoint, which becomes activated when the mitotic spindle is not properly attached to each chromosome’s kinetochore. When activated, the primary effector of the spindle checkpoint, the mitotic checkpoint complex (MCC), inhibits the anaphase-promoting complex (APC/C) by binding to the APC/C co-activator, CDC20. This prevents the APC/C from targeting critical pro-mitotic proteins, like cyclin B and securin, to promote mitotic exit. Although the function of the MCC is well understood, its regulation is not, especially in regard to protein phosphatases To investigate this, we activated the spindle checkpoint with microtubule inhibitors and then treated with a variety of phosphatase inhibitors, examining the effect on the MCC and APC/C. We found that two separate inhibitors, calyculin A and okadaic acid (1uM), were able to promote the dissociation of the MCC. This led to the activation of the APC/C, but the cells remained in mitosis as evidenced by high levels of Cdk1 activity and chromosome condensation. This is the first time that phosphatases have been shown to be essential to maintaining the MCC and an active spindle checkpoint.</p> / Dissertation

Accurate chromosome segregation is crucial as mis-segregation results in aneuploidy, which can lead to severe diseases such as cancer. The spindle checkpoint monitors sister-chromatid attachment and inhibits the onset of anaphase until all chromosomes are correctly bi-oriented on the mitotic spindle. The spindle checkpoint machinery of S.pombe is composed of many proteins, one of which is the kinase Mph1 (Mps1p-like pombe homolog). It previously has been shown that Mph1 is essential for the spindle checkpoint but not whether this is due to its kinase activity. In this study we determined the role of Mph1 kinase activity in the spindle checkpoint. To do so a kinase-dead version of Mph1, which had no detectable kinase activity, was analysed. Using this kinase-dead allele we showed that lack of Mph1 kinase activity abolished the spindle checkpoint and led to chromosome missegregation. As a result of these two defects cell viability of cells lacking Mph1 kinase activity was severely impaired. These results led to the question of how Mph1 kinase activity regulates the spindle checkpoint. Spindle checkpoint signalling is thought to mainly take place at two sites, at the kinetochore and at the anaphase promoting complex (APC). The APC is an E3 ubiquitin ligase that drives cells into anaphase by targeting the separase inhibitor securin and cyclin B for degradation by the 26 S proteasome. Upon activation of the spindle checkpoint the APC is inhibited by the mitotic checkpoint complex (MCC) composed of Slp1, Mad2 and Mad3. In this study we wanted to test whether the regulatory role of Mph1 kinase in the spindle checkpoint is via MCC binding to the APC. Using the kinase-dead version of Mph1 we showed that Mad2 and Mad3 binding to the APC is severely impaired in the absence of Mph1 kinase activity. This result led to the hypothesis that Mph1 might regulate Mad2 and Mad3 binding Using kinase assays Mad2 and Mad3 were identified as in vitro substrates of Mph1 and phosphorylation sites in Mad2 and Mad3 were determined by mass spectrometry. Phosphorylation mutants of Mad2 and Mad3 showed spindle checkpoint defects, indicating that they are important Mph1 substrates.

Cells must faithfully segregate their chromosomes at division; errors in this process causes cells to inherit an incorrect number of chromosomes, a hallmark of birth defects and cancer. The machinery required to segregate chromosomes is called the spindle, a bipolar array of microtubules that attach to chromosomes through the kinetochore. Replicated chromosomes contain two sister chromatids whose kinetochores must attach to microtubules from opposite poles to ensure correct inheritance of chromosomes. The spindle checkpoint monitors the attachment to the spindle and prevents cell division until all chromatids are attached to opposite poles. Both the spindle and the checkpoint are critical for correct segregation, and we sought to understand the regulation of these two components. The spindle is assembled to a characteristic metaphase length, but it is unknown what determines this length. It has been proposed that spindle length could be regulated a balance of two forces: one generated by interaction between microtubules that elongates the spindle and a second due to interactions between kinetochores and microtubules that shortens the spindle. We tested this force-balance model which predicts that altering the number of kinetochores will alter spindle length. We manipulated the number of kinetochores and found that spindle length scales with the number of kinetochores; introducing extra kinetochores produces shorter spindles and inhibiting kinetochores produces longer spindles. Our results suggest that attachment of chromosomes to the spindle via kinetochores produces an inward force that opposes outward force. We also found that the number of microtubules in the spindle varied with the number of kinetochores. In addition to establishing a spindle, cells must also guarantee that chromosomes are correctly attached to it. Correct attachment generates tension as the chromatids are pulled toward opposite poles but held together by cohesin until anaphase. The spindle checkpoint monitors this tension which causes stretching of chromatin and kinetochores. Lack of tension on activates the checkpoint, but is unknown if the checkpoint measures stretch between kinetochores (inter-kinetochore stretch) or within kinetochores (intra-kinetochore). We tethered sister chromatids together to inhibit inter-kinetochore stretch and found that the checkpoint was not activated. Our results negate inter-kinetochore models and support intra-kinetochore models.

<p>The ultimate goal of any living cell is to pass on a complete, unaltered copy of its DNA to its daughter cell. The DNA damage response (DDR) and spindle checkpoint are two essential signaling pathways that make it possible for a cell to achieve this goal. The DDR protects genetic integrity by sensing errors in the DNA sequence and activating signaling pathways to arrest the cell cycle and repair the DNA. The spindle checkpoint protects chromosomal integrity by preventing the separation of chromosomes during mitosis until all chromosomes are correctly attached to the mitotic spindle. Proper regulation of both the DDR and the spindle checkpoint is critical for cell survival. In this dissertation I will describe our discovery of novel regulatory mechanisms involved in each of these signaling networks.</p><p>In the first research chapter of this dissertation, we describe our findings concerning how the DDR regulates cyclin F levels. Cyclin F is an F-box protein that associates with the SCF E3 ubiquitin ligase complex to target proteins for degradation. In response to DNA damage, cyclin F levels are downregulated to facilitate increased dNTP production for efficient DNA repair, but the molecular mechanisms regulating this downregulation of cyclin F are largely unknown. We discovered that cyclin F downregulation by the DDR is the combined result of increased protein degradation and decreased mRNA expression. At the level of protein regulation, cyclin F is targeted for proteasomal degradation by the SCF complex. Interestingly, we found that the half-life of cyclin F protein is significantly increased in cells treated with the phosphatase inhibitor calyculin A, which caused cyclin F to be hyper-phosphorylated. Calyculin A also partially prevented cyclin F downregulation following DNA damage. This result suggests that cyclin F phosphorylation stabilizes the protein, and dephosphorylation of cyclin F may be required for its degradation in both unperturbed and DNA damaged cells. We also found that cyclin F downregulation is dependent on the Chk1 kinase, which is predominately activated by the ATR kinase. In examining the mechanism by which Chk1 promotes cyclin F downregulation, we determined that Chk1 represses cyclin F transcription. Lastly, we investigated the role of cyclin F in cell cycle regulation and discovered that both increased and decreased cyclin F expression delay mitotic entry, indicating that an optimal level of cyclin F expression is critical for proper cell cycle progression.</p><p>The second research chapter of this dissertation details our discovery of the requirement for phosphatase activity to inhibit the APC/C E3 ubiquitin ligase during the spindle checkpoint. Early in mitosis, the mitotic checkpoint complex (MCC) inactivates the APC/C until the chromosomes are properly aligned and attached to the mitotic spindle at metaphase. Once all the chromosomes are properly attached to the spindle, the MCC dissociates, and the APC/C targets cyclin B and securin for degradation so that the cell progresses into anaphase. While phosphorylation is known to drive many of the events during the checkpoint, the precise molecular mechanisms regulating spindle checkpoint maintenance and inactivation are still poorly understood. In our studies, we sought to determine the role of mitotic phosphatases during the spindle checkpoint. To address this question, we treated spindle checkpoint-arrested cells with various phosphatase inhibitors and examined their effect on the MCC and APC/C activation. Using this approach we found that two phosphatase inhibitors, calyculin A and okadaic acid (1 µM), caused MCC dissociation and APC/C activation in spindle checkpoint-arrested cells. Although the cells were able to degrade cyclin B, they did not exit mitosis as evidenced by high levels of Cdk1 substrate phosphorylation and chromosome condensation. Our results provide the first evidence that phosphatases are essential for maintenance of the MCC during operation of the spindle checkpoint.</p> / Dissertation