Project Summary/Abstract Accurate cell division is critical for the proliferation and development of organisms. The precise partitioning of duplicated chromosomes to daughter cells is an essential event in the cell cycle. Defects in chromosome segregation lead to aneuploidy, the state where entire chromosomes are gained or loss. Aneuploidy is a the most common chromosomal abnormality in cancer cells and has been postulated to be a major factor in the evolution of cancer. It is also the leading cause of spontaneous miscarriages and hereditary birth defects in humans. The proposed work will lead to an understanding of the mechanisms that ensure accurate chromosome segregation and thus maintain genomic stability and prevent human disease. Chromosome segregation requires forces generated by spindle microtubules that are translated into chromosome movement through interactions with kinetochores, the highly conserved structures that assemble onto centromeric chromatin. Accurate segregation requires kinetochores to maintain load-bearing attachments to the ends of microtubules that are continually growing and shrinking. Kinetochores must also biorient and attach to microtubules from opposite poles. When there is a defect in biorientation, error correction systems destabilize improper attachments. In the next funding period, this proposal will use in vitro assays to address a number of outstanding questions about kinetochore assembly and function. 1) How is kinetochore assembly regulated? 2) How do outer kinetochore proteins contribute to force-dependent kinetochore-microtubule attachments? 3) How is the Aurora B error correction pathway regulated by tension? The proposal will use budding yeast for these studies because they are amenable to biochemical, genetic and cytological studies, and the yeast kinetochore is the best characterized to date. Taken together, these studies of kinetochores in budding yeast will lead toward an understanding of the fundamental mechanisms of segregation in all eukaryotes.