PROJECT SUMMARY Cell division is a conserved process by which replicated chromosomes are equally partitioned into two daughter cells. Errors in this process often result in gains or losses of chromosomes, known as aneuploidy, which can cause and promote tumors and developmental diseases. During mitotic progression, chromosomes dynamically change their positions in a force-dependent manner via forces generated at kinetochores, macro-molecular protein structures built on centromeric chromatin that serves as platforms for microtubule assembly. While chromosome territories, regions preferentially occupied by specific chromosomes in interphase nuclei, have been established and are known to be involved in gene regulation and genomic protection, the presence and function of chromosome organization in mitosis have not been adequately explored. Our long-term goals are to characterize “mitotic chromosome territories” in mammalian cells and to uncover the function behind spatiotemporal regulation of both chromosome organization and kinetochore dynamics in ensuring faithful chromosome segregation. In this proposal, we will test the hypothesis that there exist chromosome organizations in mitosis as in interphase nuclei using a super-resolution microscopy method we recently developed, which will allow us to identify full sets of individual chromosomes and determine their spatial organization in mammalian cells. If there exist mitotic chromosome territories, we will explore how and when they are established and their evolution throughout mitosis. We also hypothesize that major mitotic defects (unaligned chromosomes, lagging chromosomes, and chromosome bridges) are associated with improper chromosome organization. We will examine this hypothesis by identifying which chromosomes are involved in each defect with increased frequency and determine their positionings. Mitotic cells have two major pathways for correcting mitotic errors, mediated by Aurora A or Aurora B kinases. Both kinases are spatially regulated and phosphorylate a highly conserved microtubule-binding kinetochore protein, Ndc80/Hec1, to destabilize improper microtubule bindings for promotion of error correction and regulation of SAC (spindle assembly checkpoint) activity. Aurora A-mediated error corrections require proximity of erroneous chromosomes to the spindle poles, where Aurora A is concentrated. On the other hand, Aurora B-mediated error corrections depend on dynamic deformations of kinetochores. These suggest that mitotic chromosome positioning, coupled with kinetochore dynamics, orchestrate the cooperation between Aurora A and Aurora B-mediated error correction machineries. We will dissect the contributions of chromosome positioning and kinetochore dynamics towards Aurora A and Aurora B error corrections using force- calibrated microneedles and a semi-automated, quantitative microscopy analysis software that we recently developed called the 3D speckle analyzer (3D-Speckler). Our proposed wo...