The Effects of Nanomolar Concentrations of Taxol on Microtubule Polymerization

Abstract

Microtubules are intracellular polymers that assemble from heterodimeric ab-tubulin subunits. Polymerizing microtubules exhibit dynamic instability, a GTP-hydrolysis-driven phenomenon in which they alternate abruptly between phases of growth and relatively rapid shortening. The kinetics of tubulin subunit exchange at the microtubule tip, which are crucial to processes such as mitosis, are affected by the chemotherapeutic drug taxol, but the precise mechanism of action at therapeutic concentrations remains unclear. An understanding of how taxol alters exchange kinetics may assist in the development of new chemotherapeutic drugs. We observe microtubules polymerized at 4.5 micromolar tubulin and at 1-480 nanomolar taxol using total internal reflection fluorescence microscopy (TIRFM), achieving improvements in spatial and temporal resolution of, respectively, 1 and 2 orders of magnitude compared to previous taxol studies. We measure microtubule length changes to within 25 nm at 8 Hz, and we detect changes in the structure of the microtubule tip. We find that taxol potently affects microtubule growth at concentrations as low as 10 nM. At these concentrations we observed higher variability in growth rate than seen under control conditions, especially on long timescales (on the order of 100 seconds). In some cases, microtubules switched between normal growth and periods of very slow growth. Further, we have found that 10 nM taxol almost completely suppresses rapid shortening, and beginning at 100 nM taxol, the tubulin on and off rates gradually decrease, though the microtubule net growth rate (excluding periods of very slow growth in taxol-microtubules) remains constant.