PROJECT SUMMARY Many mitotic kinesins play multiple roles in mitosis—each likely requiring distinct mechanical behavior. This multi-functionality implies that cells need to be able to “fine tune” these motors. We propose that post- translational modifications (PTMs) of the motor domain (MD) of mitotic kinesins serve this role. These PTMs are found in many mitotic kinesins and they cluster in structures that form an allosteric pathway which couples nucleotide and microtubule binding to movement. In this proposal, we will apply a combination of transient kinetics, single molecule mechanics, and cell biology to examine how MD PTMs affect three multifunctional kinesins—Kif11, Kif18A, and Kif22. Results of these studies should allow us to develop an integrated model for how cells modulate their kinesins to meet diverse needs.