Modulation of the Polymerization Kinetics of α/β-Tubulin by Osmolytes and Macromolecular Crowding

Abstract

Tubulin is one of the main components of the cytoskeleton and can be found in nearly all eukaryotic cells. In this study, we explored the effects of kosmotropic and chaotropic osmolytes, such as trimethylamine-N-oxide (TMAO) and the metabolic waste product urea, as well as the crowding agents Ficoll and sucrose on the polymerization reaction of α/β-tubulin. Time-dependent turbidimetry and fluorescence anisotropy experiments were performed to explore the kinetics of the polymerization reaction. Under different solvent conditions, diverse changes in the lag time, the half-life of the polymerization reaction, and the critical concentration of the polymerization reaction were observed. The apparent growth rate of the formation of microtubules was dramatically decreased in the presence of urea but significantly increased in the presence of TMAO. Measurements using mixtures of these two cosolvents showed that TMAO was able to counteract the deteriorating effect of urea on the polymerization reaction of tubulin. To create a more cell-like environment, Ficoll was added as a macromolecular crowding agent. The presence of 10 wt % Ficoll increased the apparent growth rate by one order of magnitude. Our results clearly show that the polymerization of tubulin is very sensitive to the surrounding solvent.

Keywords: co-solvents; crowding; kinetics; polymerization; tubulin.