PROJECT SUMMARY/ABSTRACT To ensure genome stability, the spindle assembly checkpoint (SAC) delays anaphase until all pairs of kinetochores are attached to the ends of microtubule fibers from opposite spindle poles. In addition, improper chromosome-spindle linkages are selectively destroyed before anaphase. Through genome editing and chemical genetics, we found that the SAC kinase Mps1 controls both responses. Through large-scale phosphoproteomics, we discovered novel Mps1-regulated substrates at the kinetochore, including the Ska complex (which helps the Ndc80 complex grip onto dynamic microtubule ends) and the coatomer-related RZZ complex (which integrates Mad1-Mad2 dependent SAC signaling and dynein-dependent transport). In Aim 1, we investigate how Mps1 affects the synergistic interactions of the Ska and Ndc80 complexes on dynamic microtubules, both in cells and in reconstituted in vitro systems. In Aim 2, we dissect how Mps1 activates the RZZ complex for Mad1-Mad2 recruitment, long-term SAC arrest, and structural expansion of unattached kinetochores. In Aim 3, we develop a new chemical- genetic system for Aurora A, a mitotic kinase that remains difficult to study because current inhibitors with Aurora A selectivity in vitro nonetheless cross-inhibit Aurora B at bioactive concentrations in vivo. Together these studies will reveal how kinetochore structure, microtubule attachment, and SAC signaling evolve during mitosis, in order to maximize the probability of error-free chromosome segregation. Ultimately this information will empower development of therapeutic agents that target aneuploidy- associated diseases such as cancer.