Kinetochore protein KNL1 links kinetochore-microtubule attachment and checkpoint signaling during mitosis, The

Abstract

Mitosis is the phase of the cell cycle in which replicated chromosomes physically separate, resulting in the formation of two genetically identical daughter cells. This process is not only essential for the development of a single fertilized cell into a multicellular organism, but also for replacement of damaged and dying cells during the span life of an organism. The distribution of chromosomes during mitotic cell division requires accurate yet dynamic attachment between the plus-ends of spindle microtubules (MTs) and kinetochores, which are protein structures assembled at the centromeric region of replicated chromatids. The tightly regulated connection between kinetochores and MTs allows for chromosome congression to the metaphase plate and subsequent separation of the replicated chromosomes during anaphase. Not surprisingly, the inability of cells to resolve erroneous kinetochore-MT attachments results in missegregation of chromosomes, which is linked to uncontrolled cell proliferation and cancer. Thus, proper kinetochore-MT attachment during cell division is essential for the maintenance of genetic integrity. Despite a growing understanding of the identity of proteins that compose the kinetochore and the processes for which they are required, the precise functions of many kinetochore proteins are still unknown. KNL1, a large kinetochore scaffolding protein, contributes to several signaling pathways coordinated by the kinetochore. Yet, how KNL1 recruits its various binding partners to the kinetochore, and whether KNL1 directly or indirectly modulates protein function during mitosis are unresolved questions. In this dissertation, I examine the function of KNL1 in the regulation of kinetochore-MT attachment and determine the regions of KNL1 required for the accumulation of an array of kinetochore proteins. By loss of function analyses using a set of KNL1 mutants, combined with functional assays in cells, I demonstrate that the KNL1 N-terminus is essential for Aurora B kinase activity at kinetochores and for correct kinetochore-MT dynamics. Aurora B kinase phosphorylates kinetochore proteins during early mitosis, increasing kinetochore-microtubule (MT) turnover and preventing premature stabilization of kinetochore-MT attachments. Therefore, KNL1 is required for correct Aurora B-mediated kinetochore-MT attachment regulation during mitosis. I provide evidence that the KNL1 N-terminus influences Aurora B activity by mediating the activity of Bub1 kinase, a kinetochore protein required for the spindle assembly checkpoint (SAC). The SAC mediates amplification of an inhibitory signal to prevent mitotic exit until all chromosomes are correctly attached to MTs. Although the SAC is known to be tightly coupled to kinetochore-MT attachment, how such coupling occurs at the kinetochore is a major unanswered question. The finding that KNL1 mediates Aurora B activity through Bub1 establishes KNL1 as a key integrator of multiple signaling pathways at the kinetochore. Finally, I determine the regions of KNL1 required for the accumulation of several kinetochore proteins, providing a broad view and better understanding of kinetochore organization inside the cell. Overall, results from these studies establish KNL1 as a central organizer of kinetochore architecture and function, and demonstrate the direct influence of this scaffolding protein on kinetochore-mediated regulatory processes during mitosis.