Microtubule Dynamics Reconstituted In Vitro and Imaged by Single-Molecule Fluorescence Microscopy
MICROTUBULES, IN VITRO: MICROTUBULES, IN VITRO, 95, BPF84 (2010)
In vitro assays that reconstitute the dynamic behavior of microtubules provide insight into the roles of rnicrotubule-associated proteins (MAPs) in regulating the growth, shrinkage, and catastrophe of microtubules. The use of total internal reflection fluorescence microscopy with fluorescently labeled tubulin and MAPs has allowed us to study microtubule dynamics at the resolution of single molecules. In this chapter we present a practical overview of how these assays are performed in our laboratory: fluorescent labeling methods, strategies to prolong the time to photo-bleaching, preparation of stabilized microtubules, flow-cells, microtubule immobilization, and finally an overview of the workflow that we follow when performing the experiments. At all stages, we focus on practical tips and highlight potential stumbling blocks.