The yeast mitotic spindle has been intensively studied using genetics, biochemistry, cell biology and ultrastructure approaches, providing an opportunity to understand its function and regulation at a level not currently achievable in any other organism. The proposed studies are critical for attaining this goal. The 10- protein Dam1 complex provides critical functions in spindle integrity and kinetochore-spindle attachment, and it is a crucial target of the budding yeast Aurora kinase. The Dam1 complex assembles into rings on microtubules that associate preferentially with the GTP-tubulin lattice, it stabilizes microtubules against disassembly, and it remains attached to the disassembling microtubule ends. These properties are ideally suited for a mechanical linkage between chromosomes and spindle microtubules. Using a recently developed real-time fluorescence assay for Dam1 ring movement and microtubule stabilization, effects of protein phosphorylation and interacting proteins on biochemical activities of the Dam1 complex will be tested. By attaching microbeads to the Dam1 complex, forces generated on the ring complex by microtubule disassembly will be measured using laser tweezers. Cryo-EM studies of Dam1 complex-coated microtubules that are already quite advanced will be used for helical reconstructions to obtain a 3D structure of the complex that can be docked onto the known structure of the microtubule surface. This analysis promises to reveal structural determinants for the function of the complex as a dynamic microtubule-binding interface, and will be extended to post-translationally modified Dam1 complex, and to the complex with associated interacting proteins. Understanding of how the Aurora kinase Ipl1 regulates mitotic spindle functions will be increased by combining bioinformatics, mass spectrometry and chemical genetics to identify additional Ipl1 target proteins. Molecular genetics will determine how phosphorylation affects target protein functions. These studies will increase understanding of fundamental aspects of mitotic spindle function and regulation, and will provide a framework for elucidating mitotic mechanisms in humans. Chromosome instability is a key contributing factor in cancer and birth defects. Therefore, understanding principles of spindle function may suggest novel strategies for prevention, detection and treatment of human diseases.