In Vitro Reconstitution of Microtubule Dynamics and Severing Imaged by Label-Free Interference-Reflection Microscopy.
Methods Mol Biol
; 2430: 73-91, 2022.
Article
en En
| MEDLINE
| ID: mdl-35476326
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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Tubulina (Proteína)
/
Microtúbulos
Tipo de estudio:
Guideline
Idioma:
En
Revista:
Methods Mol Biol
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2022
Tipo del documento:
Article
País de afiliación:
Estados Unidos