Date of Award
Doctor of Philosophy (PhD)
Accurate chromosome segregation during cell division is essential for the maintenance of a cell’s genomic stability. The molecular surveillance mechanism called the spindle assembly checkpoint (SAC) inhibits chromosome segregation before all chromosomes are correctly attached (bi-oriented) on the microtubule spindle to prevent segregation errors that may lead to chromosomal instability and cell death. In this thesis, I have developed and used tools to examine the roles of different checkpoint proteins in the mechanism of SAC signaling. In the first part of the thesis, I manipulate the localization of the checkpoint protein Mad1 and show that its constitutive presence at the kinetochore is sufficient to induce checkpoint arrest. Being able to uncouple checkpoint signaling from chromosome biorientation, I use the system as a tool to examine the roles of checkpoint kinases in SAC signaling downstream of kinetochore recruitment of Mad1. I show that the kinases Mps1 and Aurora B are necessary for the maintenance of checkpoint arrest, independently of their other "upstream" checkpoint functions. I also show that localization of Mad1 at other chromosomal locations is not per se sufficient to induce checkpoint arrest. In the second part of the thesis, I collaborate in the development of a tool for the study of the protein dynein, a microtubule-associated motor with mitotic regulatory roles in spindle assembly, kinetochore-microtubule interactions and SAC silencing, as well as a variety of cargo-translocation roles in interphase. I examine the effects of the small molecule "ciliobrevin" in mitotic cells and help validate it as the first specific dynein inhibitor. Although I determine that its effects on the microtubule spindle will preclude its use in checkpoint silencing studies, ciliobrevin will still be a powerful tool for other biochemical, structural and cellular investigations of dynein function.
Maldonado, Maria, "Examining the Regulation of Cell Division by the Spindle Assembly Checkpoint" (2013). Student Theses and Dissertations. 233.