Mechanical states of a motor protein in the spindle

Motor proteins perform essential roles in spindle assembly and division, but little is known about the forces that motors produce in spindles. Here, we report new tension sensors designed to measure loads across a kinesin-14 motor protein that both slides and crosslinks microtubules in spindles. The new tension sensors are motors that show active motility in vitro-they also produce fluorescence in spindles that is sensitive to loads across the motor. We find that assembling and mature spindles respond differently to increased loads caused by osmotic shock and show differences in binding by the tension-sensor motors. Binding to spindles that are still forming is dominated by rapid, transient microtubule binding and unbinding and sliding interactions. By contrast, the motors bind tightly to mature spindles, crosslinking microtubules and resisting opposing forces, bearing higher loads. Tension sensors created from motor variants or mutants that bind more tightly to microtubules than wild type bear even greater loads. The higher motor loads in mature spindles greatly exceed the forces that the wild-type motor produces-this implies that the motor in mature spindles acts primarily to oppose forces from microtubule dynamics or other motors rather than producing force as a motor. Thus, our studies define mechanical states of a spindle motor that are characterized by loads and microtubule-binding interactions and dominated by microtubule sliding or crosslinking, resisting opposing forces. These findings provide a new way of thinking about how motors create tension and contribute to forces in the spindle.