IN-VITRO MOTILITY ASSAYS USING MICROTUBULES TETHERED TO TETRAHYMENA PELLICLES

This chapter describes in vitro assay to initiate and grow microtubules (MTs) from purified tubulin to form a tethered array of polymers with known polar orientation. This array can be used to study both the binding and the movement of objects that interact with MTs under well-defined experimental conditions. This system is used to study two forms of motility—namely, (1) chromosome movement induced by MT depolymerization alone and (2) MT-MT sliding driven by conventional motor enzymes. The specific advantage of this in vitro system is that motility occurs along anchored MTs of uniform polarity, and therefore the geometry of motion is defined. In addition, conditions can be varied easily during these assays to control the dynamic state of the tethered MTs. The two assays described allow the study of movements of cellular objects driven by MT depolymerization and test whether a particular motor of interest has the ability to slide MTs past each other. These assays test motility in real time within a light microscope perfusion chamber, using video-enhanced differential interference contrast (DIC) optics. © 1993, Elsevier Science Publishers, B.V.