Assessment of cellular actin dynamics by measurement of fluorescence anisotropy

To study cellular actin dynamics, a cell-free assay based on fluorescence anisotropy was developed. Using G-actin-Alexa as a probe, we found that anisotropy enhancement reflects F-actin elongation. Anisotropy enhancement varies with the concentration of magnesium and calcium cations and with ethylen...

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Bibliographic Details
Published in:Analytical biochemistry 2007-08, Vol.367 (1), p.95-103
Main Authors: Spitz, Jean-Alexis, Polard, Valérie, Maksimenko, Andréi, Subra, Frédéric, Baratti-Elbaz, Catherine, Méallet-Renault, Rachel, Pansu, Robert B., Tauc, Patrick, Auclair, Christian
Format: Article
Language:English
Subjects:
Actin cytoskeleton
Actins - chemistry
Actins - metabolism
Animals
Cations, Divalent - metabolism
Cell Transformation, Neoplastic
Cell-Free System
Chelating Agents
Chemical Physics
Cytosol - metabolism
Depsipeptides - pharmacology
Fluorescence anisotropy
Fluorescence Polarization - methods
Fluorescent Dyes
In Vitro Techniques
Mice
NIH 3T3 Cells
Physics
Polymerization
Rabbits
Succinimides
Thermodynamics
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Summary:To study cellular actin dynamics, a cell-free assay based on fluorescence anisotropy was developed. Using G-actin-Alexa as a probe, we found that anisotropy enhancement reflects F-actin elongation. Anisotropy enhancement varies with the concentration of magnesium and calcium cations and with ethylenediaminetetraacetate or well-known effectors of the polymerization. This assay gives the overall status of actin dynamics in cell extracts which are the closest conditions to in vivo, implying most of the regulating proteins that are missing in purified actin measurements. It can be used in a large-scale screening for chemical compounds which modulate actin polymerization.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2007.04.001