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

Erwin J G Peterman, Hernando J. Sosa, Lawrence S B Goldstein, W. E. Moerner

Research output: Contribution to journalArticle

50 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)2851-2863
Number of pages13
JournalBiophysical Journal
Volume81
Issue number5
StatePublished - 2001

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Kinesin
Fluorescence Microscopy
Microtubules
Adenylyl Imidodiphosphate
Anisotropy
Cryoelectron Microscopy
Fluorescence Polarization
Fluorescent Dyes
Cell Movement
Hydrolysis
Adenosine Triphosphate
Fluorescence

ASJC Scopus subject areas

  • Biophysics

Cite this

Polarized fluorescence microscopy of individual and many kinesin motors bound to axonemal microtubules. / Peterman, Erwin J G; Sosa, Hernando J.; Goldstein, Lawrence S B; Moerner, W. E.

In: Biophysical Journal, Vol. 81, No. 5, 2001, p. 2851-2863.

Research output: Contribution to journalArticle

Peterman, Erwin J G ; Sosa, Hernando J. ; Goldstein, Lawrence S B ; Moerner, W. E. / Polarized fluorescence microscopy of individual and many kinesin motors bound to axonemal microtubules. In: Biophysical Journal. 2001 ; Vol. 81, No. 5. pp. 2851-2863.
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