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Abstract

In Saccharomyces cerevisiae, the position of the mitotic spindle is surveyed by the spindle position checkpoint (SPOC). SPOC ensures that cells do not exit mitosis if their anaphase spindle is misaligned thereby ensuring faithful chromosomal segregation. Defects in SPOC can cause multiploidy and decrease cell survival. The SPOC kinase, Kin4, activates the Bfa1-Bub2 GTPase activating protein (GAP) complex via Bfa1 phosphorylation which in turn inhibits the mitotic exit network (MEN), a pathway that contributes to mitotic exit. Recent work has prompted Kin4- independent mechanisms that engage SPOC. Here, I have investigated the role of glycogen synthase kinase (GSK)-3 homolog, Mck1, as a novel SPOC component that activates SPOC independently of the Kin4 pathway. I show that, Mck1 and Kin4 work in parallel to stop the Cdc14 early anaphase release (FEAR) dependent activation of MEN to promote SPOC in cells with misaligned spindles. The data also indicates that Mck1 prohibits cells with compromised MEN from exiting mitosis. I illustrate that Mck1 executes its SPOC function by targeting Cdc6, a core component of the pre-replicative complex and a mitotic cyclin-dependent kinase (MCdk) inhibitor, for degradation before the cells enter mitosis. I also uncover that the cells overexpressing CDC6 cannot hold the SPOC arrest and exit mitosis. Moreover, this effect of overproduced Cdc6 was not evident when its N-terminal domain, which inhibits the M-Cdk activity, was lacking. In line with this, overexpression of the N terminal of Cdc6 could prompt SPOC deficiency. Additionally, the elevated levels of Cdc6 in mck1Δ cells capture more Clb2 (M phase cyclin) molecules. Altogether this denotes that the association of Cdc6 with Clb2 via the N-terminus seems to be crucial in regulating SPOC and mitotic exit. As cells enter the mitotic phase of the cell cycle, M-Cdk complexes phosphorylate MEN components like Mob1. I observe that the cells lacking Mck1, fail to adequately phosphorylate Mob1, which is an indication 11 of lower M-Cdk activity. Overall, this suggests that mck1Δ cells enter mitosis with higher Cdc6 levels and therefore, lower M-Cdk activity leading to SPOC deficiency and mitotic exit. This work has contributed to the understanding of Kin4 independent mechanisms that regulate SPOC and govern mitotic exit in the absence of FEAR. I also uncovered a novel function of GSK-3 kinase, Mck1. Given that GSK-3 kinases are highly conserved between organisms, the results obtained using budding yeast may open new directions of investigation into how spindle alignment and mitotic exit are regulated in higher eukaryotes.