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Title: Reconstituting the spindle assembly checkpoint and the signalling roles of Mad1
Author: Amin, Priya
ISNI:       0000 0004 7969 4577
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2019
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Cell division allows the passage of genetic information to a new cell. During this process, maintaining chromosome transmission fidelity is important in preventing diseases such as cancer and Down's syndrome. To ensure accurate chromosome segregation, eukaryotes have developed a cell cycle control mechanism that monitors kinetochore-microtubule attachments, known as the spindle assembly checkpoint (SAC). The SAC is active in metaphase and is able to sense a lack of tension and incorrect attachments between kinetochores and microtubules. This leads to a metaphase arrest, allowing time for error correction to take place before anaphase onset. The Mad and Bub proteins, along with Mps1 kinase are central to this signalling pathway which leads to the formation of the mitotic checkpoint complex (MCC) - the key inhibitor of the anaphase promoting complex/cyclosome (APC/C). APC/C inhibition prevents proteolytic degradation of Securin and Cyclin B, blocking cells in metaphase. Although we are familiar with the components of the SAC pathway, the mechanism by which they interact to form the MCC remains unclear. It is well established that SAC signalling is initiated at kinetochores. These are complex structures that are involved in key mitotic functions such as microtubule attachment and biorientation of sister chromatids. To study the checkpoint without interfering with kinetochore function, we have devised a minimalist approach. This study describes an ectopic reconstitution of SAC activation and silencing in S. pombe. Using abscisic acid induced dimerisation, we are able to control the co-recruitment of the checkpoint proteins KNL1 and Mps1 to recapitulate robust SAC arrest and silencing. Additionally, we provide insight into how S. pombe and HeLa cells respond to a prolonged ectopic arrest. It is widely accepted that Mad1 recruits Mad2 to unattached kinetochores, enabling MCC formation. However, recent findings point towards a more active role of Mad1 in checkpoint activation. This study shows that Mad1 interacts with Bub1 in S. pombe to form a scaffold complex that is essential for SAC function. We also investigate Mad1 C-terminal mutants to further dissect the roles of Mad1 and find that it forms a complex with the APC/C coactivator Cdc20. As a result, this study provides evidence in support of the hypothesis that the C-terminus of Mad1 has additional roles in SAC signalling aside from Mad2 kinetochore recruitment.
Supervisor: Hardwick, Kevin ; Sawin, Ken Sponsor: Medical Research Council (MRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: spindle assembly checkpoint ; Mad1 ; kinetochores ; S. pombe