Real-Time Imaging of Single γTuRC-Mediated Microtubule Nucleation Events In Vitro by TIRF Microscopy

Abstract

The γ-tubulin ring complex (γTuRC) is the major microtubule nucleator in cells. How γTuRC nucleates microtubules, and how nucleation is regulated is not understood. To gain an understanding of γTuRC activity and regulation at the molecular level, it is important to measure quantitatively how γTuRC interacts with tubulin and potential regulators in space and time. Here, we describe a total internal reflection fluorescence microscopy-based assay on chemically functionalized glass slides for the in vitro study of surface immobilized purified γTuRC. The assay allows to measure microtubule nucleation by γTuRC in real time and at a single molecule level over a wide variety of assay conditions, in the absence and presence of potential regulators. This setup provides a previously unavailable opportunity for quantitative studies of the kinetics of microtubule nucleation by γTuRC.

Keywords: Functionalized glass surface; Gamma–tubulin ring complex; In vitro assay; Microtubule dynamics; Microtubule nucleation; Protein immobilization; Single molecule imaging; TIRF microscopy.