Cell division involves complex interactive pathways that coordinate chromosome movements with cell cycle transitions. While many regulators that modulate spindle microtubule dynamics, chromosome behavior, and cell cycle control have been identified, complete understanding of their functions and interactions in controlling spindle structure and chromosome movement at different stages of mitosis remains incomplete. This deficit in understanding mitosis is due to the limited spatial and temporal resolution of current phenotypic analyses of manipulations such as protein deletion and mutant expression. The mitotic parts list is large, but continued discovery of novel mitotic components indicates that it is not yet complete, particularly for vertebrates. Bioinformatic guidance with the GAMMA algorithm continues to foster our identification of new mitotic regulators that were missed in previous screens including whole genome screens. Using innovative methods correlating in vivo and in vitro analysis, members of the laboratory target known and newly identified mitotic regulators, to test their roles in moving chromosomes during prometaphase, metaphase, and anaphase, in controlling spindle microtubule dynamic turnover, in generating mechanical tension between kinetochores and microtubules, and in directing mitotic cell cycle progression. The proposed projects combine many advanced strategies including high resolution, low phototoxicity, light sheet microscopy, degron tagging of endogenous genes, antibody- mediated protein degradation, and instantaneous, live cell quantitation of kinetochore-microtubule tension. These techniques are coupled with advanced image analysis. This new sophistication of phenotype analysis will vertically advance understanding of the functions of known and newly discovered mitotic regulators at all stages of mitosis. Further, to elucidate important protein interactions and molecular pathways, in vitro and in vivo contacts of known and novel mitotic regulators will be mapped through established methods (e.g. immunoprecipitation and mass spectroscopy) and through application of new innovations such as microscale thermophoresis and mammalian two-hybrid analysis. Specific projects include characterization of established and newly implicated mitotic regulators, investigation of a novel mechanism of chromosome instability in induced pluripotent stem cells, and evaluation of the causes and consequences of “cohesion fatigue,” a spontaneous, progressive loss of source of chromosome structure in cells stalled at metaphase.