chapter · 01/01/2010
Microtubule Dynamics Reconstituted In Vitro and Imaged by Single-Molecule Fluorescence Microscopy
Résumé
In vitro assays that reconstitute the dynamic behavior of microtubules provide insight into the roles of rnicrotubule-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.
Citer cet article
Gell, C., Bormuth, V., Brouhard, G.-J., Cohen, D.-N., Diez, S., Friel, C.-T., Helenius, J., Nitzsche, B., Petzold, H., Ribbe, J., Schaeffer, E., Stear, J.-H., Trushko, A., Varga, V., Widlund, P.-O., Zanic, M., & Howard, J. (2010). Microtubule Dynamics Reconstituted In Vitro and Imaged by Single-Molecule Fluorescence Microscopy. Methods Cell Biol., 95(BPF84). https://doi.org/10.1016/S0091-679X(10)95013-9@incollection{Gell2010_133,
author = {Gell, Christopher and Bormuth, Volker and Brouhard, Gary J. and Cohen, Daniel N. and Diez, Stefan and Friel, Claire T. and Helenius, Jonne and Nitzsche, Bert and Petzold, Heike and Ribbe, Jan and Schaeffer, Erik and Stear, Jeffrey H. and Trushko, Anastasiya and Varga, Vladimir and Widlund, Per O. and Zanic, Marija and Howard, Jonathon},
year = {2010},
month = {1},
title = {Microtubule Dynamics Reconstituted In Vitro and Imaged by Single-Molecule Fluorescence Microscopy},
journal = {Methods Cell Biol.},
booktitle = {MICROTUBULES, IN VITRO: MICROTUBULES, IN VITRO},
publisher = {ELSEVIER ACADEMIC PRESS INC},
volume = {95},
number = {BPF84},
series = {Methods in Cell Biology},
address = {525 B STREET, SUITE 1900, SAN DIEGO, CA 92101-4495 USA},
abstract = {In vitro assays that reconstitute the dynamic behavior of microtubules provide insight into the roles of rnicrotubule-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.},
url = {http://www.dx.doi.org/10.1016/S0091-679X(10)95013-9},
doi = {10.1016/S0091-679X(10)95013-9},
issn = {0091-679X},
}TY - CHAP
AU - Gell, Christopher
AU - Bormuth, Volker
AU - Brouhard, Gary J.
AU - Cohen, Daniel N.
AU - Diez, Stefan
AU - Friel, Claire T.
AU - Helenius, Jonne
AU - Nitzsche, Bert
AU - Petzold, Heike
AU - Ribbe, Jan
AU - Schaeffer, Erik
AU - Stear, Jeffrey H.
AU - Trushko, Anastasiya
AU - Varga, Vladimir
AU - Widlund, Per O.
AU - Zanic, Marija
AU - Howard, Jonathon
PY - 2010
DA - 2010/01/01
TI - Microtubule Dynamics Reconstituted In Vitro and Imaged by Single-Molecule Fluorescence Microscopy
JO - Methods Cell Biol.
BT - MICROTUBULES, IN VITRO: MICROTUBULES, IN VITRO
VL - 95
IS - BPF84
PB - ELSEVIER ACADEMIC PRESS INC
SN - 0091-679X
AB - In vitro assays that reconstitute the dynamic behavior of microtubules provide insight into the roles of rnicrotubule-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.
DO - 10.1016/S0091-679X(10)95013-9
UR - http://www.dx.doi.org/10.1016/S0091-679X(10)95013-9
ER -