PROJECT SUMMARY The accurate segregation of chromosomes by the microtubule-based mitotic spindle during mitosis is essential for the preservation of genomic integrity. Errors in mitotic spindle function lead to chromosome missegregation, aneuploidy, and the formation of micronuclei, all hallmarks of tumor cells. Molecular motors of the kinesin superfamily play important mechanical roles in controlling the movement and organization of chromosomes within the spindle, including the generation of forces on chromosome arms, regulation of spindle microtubule length changes, and the crosslinking and sliding of microtubule overlaps. Many of the molecular mechanisms underlying kinesin function in cells remain poorly understood. This proposal seeks to fill knowledge gaps in our understanding of how kinesins function at the molecular level to ensure the accuracy of mitotic chromosome segregation. How do the structures of kinesins tune their activities for particular roles in cells? How are these activities spatially and temporally regulated? What is the molecular basis for cell type specific requirements of kinesin function? What are the short and long-term consequences of abnormal kinesin activities? An interdisciplinary approach combining biophysics, quantitative live cell imaging, and structural mutagenesis will be employed to address these outstanding questions across biological scales from the single molecule to single cell, to tissue and whole organism levels. This administrative supplement application seeks funds to purchase a spinning disk microscope system essential for successfully addressing these questions.