The yeast mitotic spindle is less complex than its counterparts in larger eukaryotes and has been intensively studied using genetics, biochemistry, cell biology and ultrastructure approaches, providing an opportunity to develop an understanding of its function and regulation at a level that is not currently achievable in any other organism. The studies proposed here are critical for attaining these goals. The understanding of mitotic mechanisms that will result from studies in yeast will provide a framework for elucidating mitotic mechanisms in humans where mitotic errors contribute to cancer and birth defects. The following aims will be pursued: 1. Phospho-regulation of microtubule (MT) dynamics and nucleation Protein kinases direct every phase of mitosis. Because the Chromosome Passenger Complex (CPC) undergoes programed cellular localization changes central to its mediation of sundry mitotic events, the hypothesis that CPC phosphorylation by Ipl1 and Cdk1 regulates interactions with specific docking proteins will be tested biochemically and genetically. Having identified the MT-nucleating -TuSC as a Hrr25 target, Hrr25's role in -TuSC assembly and nucleation regulation will be investigated biochemically. 2. Spindle disassembly pathways and control of MT dynamics as cells exit mitosis, the mitotic spindle must be disassembled rapidly. Synthetic-lethal screens and real-time, live cell imaging of spindle disassembly in mutants identified three mechanistically distinct subprocesses necessary for efficient disassembly. Understanding spindle disassembly and the CPC's role in this process will be increased by investigating microtubule dynamics regulation biochemically. Leveraging the ability to purify biochemical quantities of assembly-competent yeast tubulin, CPC, Ipl1, Hrr25 and She1, MT regulation will be reconstituted and a newly developed yeast extract system will enable complementary studies in the full complexity of the cytoplasm. Since She1 is a key CPC target for spindle disassembly, an in-depth analysis of She1's biochemical activities on MTs will be performed. 3. Spatiodynamics of checkpoint activation/inactivation Errors in kinetochore-MT attachments are detected by the spindle checkpoint. An innovative, image-based approach to monitor spindle checkpoint activation and inactivation at single kinetochores will determine whether the checkpoint is active in every normal cell cycle, how and when the PPI protein phosphatase Glc7 is recruited for checkpoint inactivation, and why kinetochores do not detach during their retrieval to the spindle due to lack of tension. In total, these studies will advance understanding of fundamental aspects of mitotic spindle function and regulation. The principles established are expected to apply generally across diverse phyla.