Cytokinesis is the final stage of cell division, resulting in the generation of two separate daughter cells. This stage encompasses the physical segregation of the cytoplasm between the nascent daughter cells and is driven by the constriction of the actomyosin contractile ring that bisects the mother cell and the segregated genomic material. The contractile ring progressively compacts the central spindle microtubules to form an intercellular bridge that contains a small organelle at its centre, known as the midbody. The midbody is essential for the final abscission events, acting as a platform to recruit the essential proteins. Failure in cytokinesis is associated with many human diseases, including cancer, microcephaly, infertility and blood disorders, thus understanding the mechanisms underpinning this process is crucial for the development of treatments for these pathologies.

In this thesis I investigated the function and regulation of the contractile ring component Citron-kinase (CIT-K). CIT-K is a conserved serine/threonine kinases with an evolutionary conserved role in the formation and organisation of the midbody in late cytokinesis. In the absence of CIT-K, the midbody matrix becomes scarce and detached from the equatorial cortex, highlighting the importance of CIT-K in maintaining the proper architecture of the midbody. Recent research from the host laboratory has indicated that CIT-K is regulated by other mitotic kinases, including Cdk1 and Aurora B, suggesting that its functions must be tightly controlled. Furthermore, although several lines of evidence indicate that CIT-K kinase activity is important (the most significant being the identification of mutations in the CIT-K kinase domain in microcephaly patients), only one known CIT-K substrate, INCENP, has been identified so far.

To assess the role and function of CIT-K kinase activity I employed the chemical genetic approach of generating an analogue sensitive (AS) variant capable of accepting a “bulky” ATP analogue that would selective inhibit this kinase. Through a series of in vitro phosphorylation assays of four AS variants, I identified that the AS-CIT-K M174G variant retained the highest levels of kinase activity relative to the WT levels. I then tested four inhibitors for their ability to inactivate AS-CIT-K M174G kinase and identified 3MB-PP1 as the most efficient one. Unfortunately, expression of the CIT-K M174G mutant in cultured cells failed to rescue the functions of CIT-K, most likely because this mutant did not retain sufficient kinase activity.

To investigate the regulation of CIT-K by other mitotic kinases, I focused on the phosphorylation of two serines, S440 and S699, by Cdk1 and Aurora B. I utilised phospho-specific antibodies and phospho-mimetic and non-phosphorylatable mutants to investigate the role of these two phosphorylation events. My experiments indicated that CIT-K is phosphorylated at both S440 and S699 in early mitotic stages, but whilst S440 is de-phosphorylated right after anaphase onset, S699 remains phosphorylated into anaphase and telophase. Furthermore, perturbing both phosphorylation events led to incorrect localisation of CIT-K, and in the case of S440 to abnormal midbody formation, and accumulation of midbody remnants. In vivo pull downs indicated that both phosphorylation events significantly reduced the interaction of CIT-K with its midbody partners Aurora B, KIF14 and KIF23/MKPL1. Together, these findings indicate that the coordinated regulation of CIT-K by Cdk1 and Aurora B temporally regulates the association of CIT-K with its partners in order to finely control midbody formation.