Project Summary During chromosome segregation, each daughter cell receives a complete complement of the genome, and this is repeated for every cell division. Therefore, chromosome segregation must be extraordinarily accurate and robust to ensure the health of an adult human; otherwise, disasters like cancers can occur. Cancer cells exploit and rewire their chromosome segregation machinery to meet their insatiable need of uncontrolled cell division. Successful chemotherapeutic drugs kill cancer cells through disrupting this obligate need. Thus, understanding mechanisms of chromosome segregation has far-reaching implications to human health. Kinetochores execute chromosome segregation by connecting chromosomal centromeres to spindle microtubules. This connection must be flexible to accommodate the fleeting passage of the DNA polymerases that replicate centromeres during the S phase; it must also be strong to withstand the pulling force of spindle microtubules during mitosis. Cells coordinate these opposing attributes of kinetochores temporally and regulate the transition between them. Because kinetochores and their associated regulators are highly conserved among the eukaryotic kingdoms of life, we will use the yeast Saccharomyces cerevisiae as a primary research organism to study how kinetochores are assembled. The central hypothesis is that kinetochore assembly is a highly cooperative process that involves multiple protein-protein and protein-DNA contacts, which are controlled by cell cycle signals. To understand how kinetochores are assembled, Specific Aim 1 will apply a quantitative proteomics platform to define the steps of kinetochore assembly; making use of stable isotope based mass spectrometry (MS) to analyze native kinetochores as well as reconstituted kinetochores assembled from concentrated cell extracts. Two key interfaces govern kinetochore assembly: the first one is between centromeres and inner kinetochores, while the second one is between inner and outer kinetochores. Specific Aim 2 will probe the centromere-inner kinetochore interface and focus on how phosphorylation of specific inner kinetochore components may regulate it. Specific Aim 3 will dissect the inner-outer kinetochore interface and study its cell cycle control with an ultimate goal of reconstituting the kinetochore that retains its physiological properties. All together, these studies are aimed at understanding how kinetochores are assembled. Understanding kinetochore assembly has broad relevance, because the rules and methods of study apply to all systems in which signals are integrated to control macromolecular assemblies. Our collaborative team, equipped with interdisciplinary expertise and shared interest in kinetochore biology, is uniquely qualified to carry out the proposed projects and to make impactful advance in this area of considerable biomedical significance.