Functional role of CCAN histone fold proteins in mitosis

Centromeres are chromosomal loci that direct segregation of the genome by specifying kinetochore assembly in mitosis and meiosis. Kinetochores are built upon centromeric chromatin, which contains the histone H3 variant CENP-A nucleosomes and a group of over 15 associated proteins comprising the constitutive centromere-associated network (CCAN). The CCAN contains two novel histone fold domain complexes, CENP-T/W and CENP-S/X, which are essential for error-free mitotic progression. The CCAN nucleates both kinetochore formation and spindle assembly checkpoint (SAC) activity. Together, these activities both attach chromosomes to the spindle and signal the presence of improperly attached chromosomes. Failure to correct kinetochore-spindle attachments leads to aneuploidy, a characteristic of cancer. Chronic checkpoint activation can result in mitotic slippage and consequent aneuploidy or polyploidy, cell death via apoptosis, after failed mitotic exit, or execution of a death pathway directly from mitosis. All these outcomes from prolonged mitosis are associated with increased caspase-3 activity, suggesting its involvement in a normal mitosis. Chapter 2 of this thesis, investigates the alleged link between caspase-3 and mitosis. We found no evidence that active caspase-3 is required for mitotic checkpoint activity in a normal mitosis, supported by no observable influence of caspase-3 depletion on mitotic kinetics. Chapter 3 examines the functional role of the CENP-T/W complex in mitosis. Both proteins were required for a robust mitosis in each cell cycle as well as proper mitotic checkpoint function. These results are consistent with the unique role that the CENP-T/W complex plays in specifying an early step of kinetochore assembly late in the cell cycle. Chapter 4 examines the mechanism of multipolar spindle formation following CENP-W depletion. Our analysis revealed that multipolar spindle formation occurs during mitosis. Evidence is presented for a physical model in which unbalanced spindle forces lead to pole fragmentation. These studies provide novel insight into kinetochore assembly and its role in mitotic spindle structure and function and identify the CENP-T/W complex as a potential target for anti-cancer drug development