GENETIC CONTROL OF MITOTIC SPINDLE ASSEMBLY AND FUNCTION

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The long term goals of this project are to understand how the function of the mitotic spindle is integrated into the cell cycle in the budding yeast Saccharomyces cerevisiae. Various anti-tumor chemotherapeutic agents, like taxol, cause human cells to arrest in mitosis. Recent data from a variety of sources suggest that there is feedback control, by a "spindle checkpoint", that can detect errors in spindle function and arrest cell division. We propose that the yeast spindle checkpoint is complex, having at least two components. One is a kinetochore checkpoint that is sensitive to the state of microtubule occupancy at kinetochores. The other component is a spindle assembly checkpoint that is activated when the spindle is mis-assembled. We propose to use a number of mutations in kinetochore genes and kinetochore checkpoint genes to test the model of microtubule occupancy. We will also develop an in vitro assay based on predictions from our model. In addition, we will use novel genetic assays to gain greater insight into the nature of the kinetochore signalling pathway. We propose that there is a novel spindle assembly checkpoint is activated in cdc2O mutants, lacking an essential microtubule associated protein. Genetic and molecular analyses suggest that Cdc20p is regulated by phosphorylation and by protein turnover. We will determine the molecular basis for both of those phenomena. We will use cdc2O to identify mutants in the novel spindle assembly checkpoint. Many yeast spindle checkpoint genes are conserved among higher eucaryots, suggesting that the spindle checkpoint is evolutionarily conserved. Our experiments will identify some of the important regulatory mechanisms that control genomic stability in a variety of cell types. The spindle checkpoint genes may identify molecular targets for chemotherapeutic intervention in some cancers and birth defects.