title: Microtubule dynamics reconstituted in vitro and imaged by single-molecule fluorescence microscopy.
abstract: In vitro assays that reconstitute the dynamic behavior of microtubules provide insight into the roles of microtubule-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.
authors.affiliation: Max Planck Institute of Molecular Cell Biology & Genetics, 01307 Dresden, Germany.
authors.forename: Christopher
authors.initials: C
authors.lastname: Gell
authors.forename: Volker
authors.initials: V
authors.lastname: Bormuth
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Brouhard
authors.forename: Daniel N
authors.initials: DN
authors.lastname: Cohen
authors.forename: Stefan
authors.initials: S
authors.lastname: Diez
authors.forename: Claire T
authors.initials: CT
authors.lastname: Friel
authors.forename: Jonne
authors.initials: J
authors.lastname: Helenius
authors.forename: Bert
authors.initials: B
authors.lastname: Nitzsche
authors.forename: Heike
authors.initials: H
authors.lastname: Petzold
authors.forename: Jan
authors.initials: J
authors.lastname: Ribbe
authors.forename: Erik
authors.initials: E
authors.lastname: Schäffer
authors.forename: Jeffrey H
authors.initials: JH
authors.lastname: Stear
authors.forename: Anastasiya
authors.initials: A
authors.lastname: Trushko
authors.forename: Vladimir
authors.initials: V
authors.lastname: Varga
authors.forename: Per O
authors.initials: PO
authors.lastname: Widlund
authors.forename: Marija
authors.initials: M
authors.lastname: Zanic
authors.forename: Jonathon
authors.initials: J
authors.lastname: Howard
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D003116:Color; D005456:Fluorescent Dyes / Q000494:pharmacology; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D008870:Microtubules / Q000378:metabolism*; D008954:Models, Biological; D013194:Staining and Labeling / Q000379:methods; D014404:Tubulin / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D014404:Tubulin