UNDERSTANDING THE SPINDLE ASSEMBLY CHECKPOINT AND THE ERROR CORRECTION MACHINERY WITHIN THE KINETOCHORE FRAMEWORK

Kinetochores are large protein assemblies built on chromosomal loci named centromeres. Four distinct modules accomplish the main functions of kinetochores. The first module, in the inner kinetochore, contributes a sturdy interface with centromeric chromatin. The second module, the outer kinetochore, contributes a microtubule-binding interface. The third module, the spindle assembly checkpoint, is a feedback control mechanism that monitors the state of kinetochore-microtubule attachment to control progression of the cell cycle. The fourth module discerns correct from improper attachments, preventing the stabilization of the latter and allowing the selective stabilization of the former. The catalytic activity of the MPS1 kinase is crucial for the spindle assembly checkpoint and for chromosome bi-orientation on the mitotic spindle. In this thesis, I report work showing that the small-molecule Reversine is a potent mitotic inhibitor of MPS1. Reversine inhibits the spindle assembly checkpoint in a dose-dependent manner. Its addition to mitotic HeLa cells causes the ejection of Mad1 and the RZZ complex, both of which are important for the spindle checkpoint, from unattached kinetochores. By using Reversine, I also demonstrated that MPS1 is required for the correction of tensionless chromosome-microtubule attachments. An important conclusion from this work is that MPS1 acts downstream from the AURORA B kinase, another crucial component of the error correction pathway. My studies describe a very useful tool to interfere with MPS1 activity in human cells. They also shed light on the relationship between the error correction pathway and the spindle assembly checkpoint, and suggest that these processes are co-regulated and are likely to involve the same catalytic machinery.