Analysis of a Damaged Mitotic Spindle Checkpoint in Budding Yeast

Evidence continues to mount that aneuploidy is a contributing factor in tumor development, and that aneuploidy may serve as a target for anti-cancer therapy. Initial hypotheses posited that cancer cells have a destroyed spindle checkpoint pathway. However, the spindle checkpoint has been observed to be essential even in cancer cells. There are numerous ways the spindle checkpoint can be damaged, and two established mechanisms that occur in vivo are either by mutating core checkpoint components, or by generating an imbalance in the Mad1:Mad2 ratio, which are two core checkpoint proteins that form a hetero-tetrameric complex. The ratio of Mad1:Mad2 has been manipulated in several organisms and these manipulations lead to aberrant checkpoint function, chromosome loss, and promoted tumorigenesis in animal models. In Aim 1, I propose to use the in vitro system to investigate aberrant checkpoint function either by employing mutant forms of spindle checkpoint proteins, or manipulating the Mad1:Mad2 ratio. In Aim 2, I propose creating a yeast genetic system that will allow us to select for mutations that allow cells to live when they should die when they are forced to be aneuploid. These mutants might be informative with regard to the types of mutations cancer cells accumulate that allow them to live when they are aneuploid. Finally, I have identified phosphorylation sites within three subunits of the APC, a master cell cycle regulator at metaphase that is the target of spindle checkpoint. Some of the APC subunit phosphorylation events correlate with checkpoint activity as determined by protein purification and mass spectroscopy. In Aim 3, I propose to perform this analysis on all APC subunits and then to investigate the functional consequence of these phosphorylation events both in vivo and in vitro. In combination, I suggest that the results from executing these three Aims will contribute to a mechanistic understanding of checkpoint function that shall be necessary for us to evaluate potential consequences on cell cycle regulation when the spindle checkpoint is damaged in cancer cells.

Project ID:PA10108-0423
External Project ID:NSC101-2311-B182-001