Microtubule dynamics reconstituted in vitro and imaged by single-molecule fluorescence microscopy.
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.
Gell
Christopher
C
Max Planck Institute of Molecular Cell Biology & Genetics, 01307 Dresden, Germany.
Bormuth
Volker
V
Brouhard
Gary J
GJ
Cohen
Daniel N
DN
Diez
Stefan
S
Friel
Claire T
CT
Helenius
Jonne
J
Nitzsche
Bert
B
Petzold
Heike
H
Ribbe
Jan
J
Schäffer
Erik
E
Stear
Jeffrey H
JH
Trushko
Anastasiya
A
Varga
Vladimir
V
Widlund
Per O
PO
Zanic
Marija
M
Howard
Jonathon
J
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
D005456:Fluorescent Dyes; D014404:Tubulin