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 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.
Spitz
Jean-Alexis
JA
LBPA, Institut d'Alembert, ENS Cachan, CNRS, UniverSud, 61 av President Wilson, F-94230 Cachan, France.
Polard
Valérie
V
Maksimenko
Andréi
A
Subra
Frédéric
F
Baratti-Elbaz
Catherine
C
Méallet-Renault
Rachel
R
Pansu
Robert B
RB
Tauc
Patrick
P
Auclair
Christian
C
D000199:Actins / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D002413:Cations, Divalent / Q000378:metabolism; D002471:Cell Transformation, Neoplastic; D002474:Cell-Free System; D002614:Chelating Agents; D003600:Cytosol / Q000378:metabolism; D047630:Depsipeptides / Q000494:pharmacology; D005454:Fluorescence Polarization / Q000379:methods*; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D051379:Mice; D041681:NIH 3T3 Cells; D011817:Rabbits; D013388:Succinimides; D013816:Thermodynamics
D000199:Actins; C492694:Alexa Fluor 488 carboxylic acid succinimidyl ester; D002413:Cations, Divalent; D002614:Chelating Agents; D047630:Depsipeptides; D005456:Fluorescent Dyes; D013388:Succinimides; C422265:dolastatin 11; C057531:jasplakinolide