PREPARATION OF MARKED MICROTUBULES FBR THE ASSAY OF THE POLARITY OF MICROTUBULE-BASED MOTORS BY FLUORESCENCE MICROSCOPY

This chapter describes the preparation of marked microtubules for the assay of the polarity of microtubule-based motors by fluorescence microscopy. It explores how to assemble microtubules with obvious polarity when viewed by fluorescence microscopy; the direction of movement of a motor protein with respect to such polarity-marked microtubules indicates the motor's polarity. Microtubules are polymers of the heterodimeric protein tubulin. The inherent polarity of the microtubule polymer has two major functional implications—namely, (1) the growth rates at the different ends of the microtubules are different and (2) different microtubule-based motor proteins move in opposite directions along the microtubule. Thus, the cylindrical surface of the microtubule has a different texture in the two directions, a texture that the motor proteins can detect. The knowledge of the polarity of movement of a motor protein is essential for interpreting its biological function. Microtubule nucleation will not occur below a certain critical concentration, but new microtubule growth will occur from the pre-existing ends of microtubules down to tubulin concentrations below that critical concentration. © 1993, Elsevier Science Publishers, B.V.