Mechanisms underlying spatial control of exit from mitosis

Abstract

During mitosis, cells must accurately segregate their genome in order to produce healthy daughter cells. In budding yeast, cells align their anaphase spindle along a predetermined axis of division in order to partition their genome into the daughter cells. In the event that the spindle becomes mispositioned, cells will prevent exit from mitosis by inhibiting the mitotic exit network (MEN). The MEN functions to regulate the localization of the essential phosphatase Cdc 4. Control of the MEN by spindle position is established through MEN inhibitory signals in the mother cell compartment (such as the kinase Kin4), MEN promoting signals in the bud (such as Ltel) and a GTPase sensor (Tem1) that moves between them. While the molecular functions of Kin4 and Tem1 are well defined, the function of Ltel has remained unclear. In the first part of this thesis I attribute a function to Ltel in promoting exit from mitosis. I show that Ltel functions to prevent Kin4 from inappropriately localizing to SPBs (spindle pole bodies) in the bud cell compartment. I find that these two proteins interact and that the Nterminus of Kin4 mediates this interaction. This work highlights the importance of spatial restriction of Ltel in the bud and Kin4 in the mother for the proper execution of chromosome segregation in anaphase. In the second part of this thesis I investigate the role of cytoplasmic microtubules in spatial regulation of the MEN. It has been proposed that spatial regulation of the MEN functions as a checkpoint that requires contact between cytoplasmic microtubules (cMTs) and the budneck to arrest cells in anaphase. Loss of cMT-budneck contact was reported to lead to checkpoint failure resulting in anucleate and multinucleate cells. In contradiction to these results, I find that Cdc14 release is responsible for the loss of cMT-budneck interactions that precede inappropriate exit from mitosis. Lastly, through the generation of cells with two nuclei, I show that the coupling of spindle position to exit from mitosis is established in a dual manner through both inhibitory signals in mother cell compartment and through activating signals in the bud.