Chapter 10 Assay of Microtubule Movement Driven by Single Kinesin Molecules

Abstract

Low-density motility assays are largely used to explore the mechanism of force generation by kinesin. This chapter describes the assay of microtubule movement driven by single kinesin molecule. The assay confers advantages over the standard high-density assays because the movement of a microtubule by a single motor molecule can be observed. In addition, the assay is extremely sensitive: movement at densities as low as one motor molecule per square micrometer can be observed. Fluorescence microscopy for rhodamine-labeled microtubules and dark-field microscopy for unlabeled microtubules are used in the assay. The Diastar upright microscope has a fixed stage and binocular tube. To analyze motility, a personal computer-based board that projects cross-hairs onto the monitor, the position of which is controlled by the mouse is used. The advantage of the “low-density” assay is that movement occurs at kinesin densities as low as 1 μ m -2 , some 3 to 4 orders of magnitude lower than the minimum density required in the standard assay in which the kinesin is adsorbed directly to the glass.