In Vitro Reconstitution of Microtubule Dynamics and Severing Imaged by Label-Free Interference-Reflection Microscopy.

ABSTRACT

The dynamic architecture of the microtubule cytoskeleton is crucial for cell division, motility and morphogenesis. The dynamic properties of microtubules-growth, shrinkage, nucleation, and severing-are regulated by an arsenal of microtubule-associated proteins (MAPs). The activities of many of these MAPs have been reconstituted in vitro using microscope assays. As an alternative to fluorescence microscopy, interference-reflection microscopy (IRM) has been introduced as an easy-to-use, wide-field imaging technique that allows label-free visualization of microtubules with high contrast and speed. IRM circumvents several problems associated with fluorescence microscopy including the high concentrations of tubulin required for fluorescent labeling, the potential perturbation of function caused by the fluorophores, and the risks of photodamage. IRM can be implemented on a standard epifluorescence microscope at low cost and can be combined with fluorescence techniques like total-internal-reflection-fluorescence (TIRF) microscopy. Here we describe the experimental procedure to image microtubule dynamics and severing using IRM , providing practical tips and guidelines to resolve possible experimental hurdles.