chapter · 01/01/2010
Studying Kinesin Motors by Optical 3D-Nanometry in Gliding Motility Assays
Résumé
Recent developments in optical microscopy and nanometer tracking have facilitated our understanding of microtubules and their associated proteins. Using fluorescence microscopy, dynamic interactions are now routinely observed in vitro on the level of single molecules, mainly using a geometry in which labeled motors move on surface-immobilized microtubules. Yet, we think that the historically older gliding geometry, in which motor proteins bound to a substrate surface drive the motion microtubules, offers some unique advantages. (1) Motility can be precisely followed by coupling multiple fluorophores and/or single bright labels to the surface of microtubules without disturbing the activity of the motor proteins. (2) The number of motor proteins involved in active transport can be determined by several strategies. (3) Multimotor studies can be performed over a wide range of motor densities. These advantages allow for studying cooperativity of processive as well as non-processive motors. Moreover, the gliding geometry has proven to be most promising for nanotechnological applications of motor proteins operating in synthetic environments. In this chapter we review recent methods related to gliding motility assays in conjunction with 3D-nanometry. In particular, we aim to provide practical advice on how to set up gliding assays, how to acquire high-precision data from microtubules and attached quantum dots, and how to analyze data by 3D-nanometer tracking.
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Nitzsche, B., Bormuth, V., Braeuer, C., Howard, J., Ionov, L., Kerssemakers, J., Korten, T., Leduc, C., Ruhnow, F., & Diez, S. (2010). Studying Kinesin Motors by Optical 3D-Nanometry in Gliding Motility Assays. Methods Cell Biol., 95(BPF84). https://doi.org/10.1016/S0091-679X(10)95014-0@incollection{Nitzsche2010_134,
author = {Nitzsche, Bert and Bormuth, Volker and Braeuer, Corina and Howard, Jonathon and Ionov, Leonid and Kerssemakers, Jacob and Korten, Till and Leduc, Cecile and Ruhnow, Felix and Diez, Stefan},
year = {2010},
month = {1},
title = {Studying Kinesin Motors by Optical 3D-Nanometry in Gliding Motility Assays},
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 = {Recent developments in optical microscopy and nanometer tracking have facilitated our understanding of microtubules and their associated proteins. Using fluorescence microscopy, dynamic interactions are now routinely observed in vitro on the level of single molecules, mainly using a geometry in which labeled motors move on surface-immobilized microtubules. Yet, we think that the historically older gliding geometry, in which motor proteins bound to a substrate surface drive the motion microtubules, offers some unique advantages. (1) Motility can be precisely followed by coupling multiple fluorophores and/or single bright labels to the surface of microtubules without disturbing the activity of the motor proteins. (2) The number of motor proteins involved in active transport can be determined by several strategies. (3) Multimotor studies can be performed over a wide range of motor densities. These advantages allow for studying cooperativity of processive as well as non-processive motors. Moreover, the gliding geometry has proven to be most promising for nanotechnological applications of motor proteins operating in synthetic environments. In this chapter we review recent methods related to gliding motility assays in conjunction with 3D-nanometry. In particular, we aim to provide practical advice on how to set up gliding assays, how to acquire high-precision data from microtubules and attached quantum dots, and how to analyze data by 3D-nanometer tracking.},
url = {http://www.dx.doi.org/10.1016/S0091-679X(10)95014-0},
doi = {10.1016/S0091-679X(10)95014-0},
issn = {0091-679X},
}TY - CHAP
AU - Nitzsche, Bert
AU - Bormuth, Volker
AU - Braeuer, Corina
AU - Howard, Jonathon
AU - Ionov, Leonid
AU - Kerssemakers, Jacob
AU - Korten, Till
AU - Leduc, Cecile
AU - Ruhnow, Felix
AU - Diez, Stefan
PY - 2010
DA - 2010/01/01
TI - Studying Kinesin Motors by Optical 3D-Nanometry in Gliding Motility Assays
JO - Methods Cell Biol.
BT - MICROTUBULES, IN VITRO: MICROTUBULES, IN VITRO
VL - 95
IS - BPF84
PB - ELSEVIER ACADEMIC PRESS INC
SN - 0091-679X
AB - Recent developments in optical microscopy and nanometer tracking have facilitated our understanding of microtubules and their associated proteins. Using fluorescence microscopy, dynamic interactions are now routinely observed in vitro on the level of single molecules, mainly using a geometry in which labeled motors move on surface-immobilized microtubules. Yet, we think that the historically older gliding geometry, in which motor proteins bound to a substrate surface drive the motion microtubules, offers some unique advantages. (1) Motility can be precisely followed by coupling multiple fluorophores and/or single bright labels to the surface of microtubules without disturbing the activity of the motor proteins. (2) The number of motor proteins involved in active transport can be determined by several strategies. (3) Multimotor studies can be performed over a wide range of motor densities. These advantages allow for studying cooperativity of processive as well as non-processive motors. Moreover, the gliding geometry has proven to be most promising for nanotechnological applications of motor proteins operating in synthetic environments. In this chapter we review recent methods related to gliding motility assays in conjunction with 3D-nanometry. In particular, we aim to provide practical advice on how to set up gliding assays, how to acquire high-precision data from microtubules and attached quantum dots, and how to analyze data by 3D-nanometer tracking.
DO - 10.1016/S0091-679X(10)95014-0
UR - http://www.dx.doi.org/10.1016/S0091-679X(10)95014-0
ER -