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

Abstract

Chromosome 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.