Measurements of motor protein forces using the centrifuge microscope

In this work we report the use of a centrifuge microscope to investigate the force-velocity curves for kinesin-driven motility, as well as the effects of nucleotide concentration on the force required to separate kinesin from microtubules ("pull-off" force for kinesin molecules). We present a hypothesis based on the notion that the centrifuge microscope measures an average force generated by kinesin molecules over many stroke cycles working against a constant repulsive force and not the individual forces, as is measured using some laser tweezers techniques.; The centrifuge microscope is a light microscope equipped with a rotating stage. The rotation of the stage at predetermined velocities applies known centrifugal forces on relatively dense objects which are in the assay cell.; Using the centrifuge microscope we apply a constant repulsive centrifugal force to counter molecular forces generated by one or more kinesin molecules. From these measurements, taken as averages over intervals which allow for multiple stroke cycles, we generate a force-velocity curve. In order to better compare our results with those of other laboratories we have measured the forces necessary to stop kinesin-generated demembranated sperm movement generated by a laser trap in our motility assay. We have determined that stall forces generated by a laser trap are considerably larger than pull-off forces measured using a centrifugal microscope.; We conclude that due to the apparent intermittent nature of motor protein force generation, the magnitude of the measured force will depend on the details of the method used. Any method measuring the effect of a constant force on a motor molecule will measure different aspects of the processes compared to a method (such as the laser trap interferometer) which is sensitive to the peak force that is developed. The centrifuge microscope measures a force near the lowest force generated in a measurement interval because the centrifugal force is present even when the motor(s) are not generating any force.