Polarized fluorescence microscopy of individual and many kinesin motors bound to axonemal microtubules

Biophysical Journal
Erwin J G PetermanW E Moerner

Abstract

Kinesin is a molecular motor that interacts with microtubules and uses the energy of ATP hydrolysis to produce force and movement in cells. To investigate the conformational changes associated with this mechanochemical energy conversion, we developed a fluorescence polarization microscope that allows us to obtain information on the orientation of single as well as many fluorophores. We attached either monofunctional or bifunctional fluorescent probes to the kinesin motor domain. Both types of labeled kinesins show anisotropic fluorescence signals when bound to axonemal microtubules, but the bifunctional probe is less mobile resulting in higher anisotropy. From the polarization experiments with the bifunctional probe, we determined the orientation of kinesin bound to microtubules in the presence of AMP-PNP and found close agreement with previous models derived from cryo-electron microscopy. We also compared the polarization anisotropy of monomeric and dimeric kinesin constructs bound to microtubules in the presence of AMP-PNP. Our results support models of mechanochemistry that require a state in which both motor domains of a kinesin dimer bind simultaneously with similar orientation with respect to the microtubule.

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Citations

Mar 23, 2005·Nature Methods·Matthew J LangSteven M Block
Jun 20, 2006·Nature Structural & Molecular Biology·Ana B AsenjoHernando Sosa
May 16, 2006·Proceedings of the National Academy of Sciences of the United States of America·Nicholas R Guydosh, Steven M Block
Mar 27, 2009·Proceedings of the National Academy of Sciences of the United States of America·Ana B Asenjo, Hernando Sosa
May 1, 2004·Annual Review of Physical Chemistry·Erwin J G PetermanW E Moerner
Apr 14, 2016·Biophysical Journal·Chandrima ChatterjeeHernando Sosa
Apr 23, 2015·ELife·Johan O L AndreassonSteven M Block
Feb 25, 2003·Annual Review of Biophysics and Biomolecular Structure·Xavier MichaletShimon Weiss
Nov 7, 2012·Proceedings of the National Academy of Sciences of the United States of America·Mikael P BacklundW E Moerner
Jan 3, 2014·Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry·Mikael P BacklundW E Moerner
Dec 17, 2008·Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry·Christophe JungChristoph Bräuchle
Sep 13, 2003·Nature Structural Biology·Ana B AsenjoHernando Sosa
Dec 7, 2005·Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry·Karl Otto Greulich
Aug 25, 2005·The Journal of Biological Chemistry·Scott D Auerbach, Kenneth A Johnson
Mar 17, 2011·Physical Chemistry Chemical Physics : PCCP·H Peter Lu
Sep 15, 2015·Optics Express·Per Niklas HeddeEnrico Gratton
Sep 6, 2011·The Journal of Physical Chemistry. B·Adam Muschielok, Jens Michaelis
Oct 13, 2021·Nature Communications·Christiaan N HullemanBernd Rieger

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