Real-Time Imaging of Single γTuRC-Mediated Microtubule Nucleation Events In Vitro by TIRF Microscopy.
Functionalized glass surface
Gamma–tubulin ring complex
In vitro assay
Microtubule dynamics
Microtubule nucleation
Protein immobilization
Single molecule imaging
TIRF microscopy
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2022
2022
Historique:
entrez:
27
4
2022
pubmed:
28
4
2022
medline:
30
4
2022
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 35476342
doi: 10.1007/978-1-0716-1983-4_21
doi:
Substances chimiques
Microtubule-Associated Proteins
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
315-336Subventions
Organisme : Wellcome Trust
ID : FC001163
Pays : United Kingdom
Organisme : Cancer Research UK
Pays : United Kingdom
Organisme : Wellcome Trust
ID : 100145/Z/12/Z
Pays : United Kingdom
Informations de copyright
© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
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