The effects of osmolytes on in vitro kinesin-microtubule motility assays

Abstract

The gliding motility of microtubule filaments has been used to study the biophysical properties of kinesin motors, as well as being used in a variety of nanotechnological applications. While microtubules are generally stabilized in vitro with paclitaxel (Taxol®), osmolytes such as polyethylene glycol (PEG) and trimethylamine N-oxide (TMAO) are also able to inhibit depolymerization over extended periods of time. High concentrations of TMAO have also been reported to reversibly inhibit kinesin motility of paclitaxel-stabilized microtubules. Here, we examined the effects of the osmolytes PEG, TMAO, and glycerol on stabilizing microtubules during gliding motility on kinesin-coated substrates. As previously observed, microtubule depolymerization was inhibited in a concentration dependent manner by the addition of the different osmolytes. Kinesin-driven motility also exhibited concentration dependent effects with the addition of the osmolytes, specifically reducing the velocity, increasing rates of pinning, and altering trajectories of the microtubules. These data suggest that there is a delicate balance between the ability of osmolytes to stabilize microtubules without inhibiting motility. Overall, these findings provide a more comprehensive understanding of how osmolytes affect the dynamics of microtubules and kinesin motors, and their interactions in crowded environments.