Dissecting the Mitotic Requirements for Efficient Sister Chromatid Separation and Partitioning in Anaphase

Equal division of genetic information is essential to cell proliferation. In eukaryotes, the division of genetic information is mediated by a cellular machine called the mitotic spindle. To ensure the equal division of genetic information, cells must execute three important processes: 1) they must establish and maintain mechanical links between sister chromatids (chromatid cohesion), 2) properly attach sister chromatids to the spindle apparatus (biorientation), and 3) remove mechanical links between sister chromatids coincident with elongation of the spindle. The focus of my dissertation is elucidating the mechanisms responsible for the removal of mechanical links from sister chromatid arms during budding yeast mitosis. In contrast to current models, we find the sister chromatid cohesion between chromatid arms persists into anaphase causing sister arm loci to follow a stereotypical segregation trajectory during anaphase. The segregation trajectory was defined by sister arm loci becoming juxtaposed on the spindle axis seconds prior to their separation. While investigating the mechanism responsible for this segregation trajectory, we find that proper localization of separase (ESP1 in budding yeast) to the spindle axis in anaphase contributes to the efficient separation of sister telomere-proximal loci. Spindle localization of separase is dependent on the protein SLK19, suggesting the existence of a spindle-based, multi-protein complex specifically responsible for separating sister chromatid arm loci (a cleavasome). Consistent with the novel cleavasome hypothesis, sister centromere-proximal loci separation is unaffected by perturbation of ESP1 localization. In addition to cohesion dissolution between sister chromatid arms, investigation of chromosome-spindle attachment was also performed using a conditionally functional centromere (GALCEN). Classically annotated as completely non-functional when cells are grown on galactose, we find evidence of the GALCEN's ability to signal the spindle assembly checkpoint and form weak, transient attachments to the spindle machinery capable of partitioning the GALCEN-containing chromosome between mother and daughter cells. The investigations into the removal of sister chromatid cohesion from chromatid arms and the partitioning of the GALCEN chromosome contribute valuable insight into the process of chromosome segregation, advancing the field's understanding of the mitosis