Chapter 4 Microtubule and Axoneme Gliding Assays for Force Production by Microtubule Motor Proteins

Abstract

Publisher Summary This chapter describes the microtubule gliding assays, which involves the application of microtubules composed of purified brain tubulin to coverslips coated with either cytoplasmic dynein or kinesin. Chlamydomonas flagellar axonemes are used to compare the directions of force production by cytoplasmic dynein and kinesin and to demonstrate that cytoplasmic dynein is a minus end-directed motor. The gliding assay method involves adsorbtion of purified motor protein to a coverslip, adding taxol-stabilized microtubules and ATP, and monitoring the preparation by video microscopy. The diameter of the microtubule (24 nm) is below the limit of resolution of the light microscope, and the mass is low to detect reproducibly by conventional light microscopy—however, it is possible to use either differential interference contrast (DIC) microscopy coupled to computer-aided background subtraction or dark-field microscopy to detect these structures. Chlamydomonas flagella are ideally suited for use in the polarity assay because the distal (plus) end of the axoneme has a tendency to fray during isolation, creating a fork that is visible in the light microscope. For this reason, the Chlamydomonas axoneme has been used as the standard for all microtubule polarity determinations, and the frayed and compact ends have been defined as plus and minus, respectively.