Abstract During division, chromosomes segregate on the spindle, a large array of overlapping, crosslinked microtubules that transduces mechanical forces. The forces are thought to be produced primarily by motor proteins and microtubule dynamics – rapid microtubule growth and shrinking. Despite decades of work on motors and more than a century of work on division, the motor mechanism is still not fully understood and the critical load- bearing elements of the spindle have not been identified. The molecules involved in bearing loads in the spindle are probably motors and other spindle proteins, but the forces across these molecules have not been probed – how the forces change spatially and temporally during division is not known. The proposed studies will begin to fill this gap by identifying the force-producing spring-like element of the kinesin motors and by measuring loads across a motor protein in the spindle. Kinesin-14 Ncd is essential for division in Drosophila – the motor produces force to slide microtubules and resists forces through its crosslinking activity. New Ncd mutants will be designed and tested, and structural changes that decouple the motor mechanical and chemical cycles, altering motor mechanical output, will be analyzed. New TsNcd FRET tension sensors have been created and will be assayed in mitotic spindles to measure loads borne by Ncd during mitosis and determine effects of uncoupling mutants and mutants that affect other spindle proteins. The proposed studies will yield information about the structural changes in the kinesin motors that produce force, the loads borne by a motor in the spindle, and how changes in force and microtubule crosslinking produced by the motor affect the loads. We will test the hypothesis that the Ncd motor produces tension in spindles primarily by crosslinking microtubules, mechanically resisting oppositely-directed sliding forces, rather than by its minus-end motility. Specific aims are to 1) Identify the spring-like element of the kinesins essential for force production by testing the hypothesis that bending or distortion of the central ß-sheet stores and releases free energy during the mechanochemical cycle, functioning as the elusive spring-like element of the motor, and 2) Measure motor loads in spindles due to force production and resistance to other forces using new TsNcd tension sensors created from the kinesin-14 Ncd motor and a previously reported FRET tension sensor, and by assaying mutants that increase Ncd crosslinking or both crosslinking and sliding. Mutants in other spindle proteins, including oppositely-directed motors, will be tested to identify other load-bearing spindle molecules. These studies will provide new information about how kinesin motors produce force and contribute to mechanical forces in the mitotic spindle, preventing division errors that lead to birth defects.