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J. Biol. Chem., Vol. 281, Issue 33, 24036-24047, August 18, 2006

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Analysis of Tetramethylrhodamine-labeled Actin Polymerization and Interaction with Actin Regulatory Proteins^*

Andrea Pelikan Conchaudron, Dominique Didry, Kim Ho Diep Le, Eric Larquet, Nicolas Boisset, Dominique Pantaloni, and Marie-France Carlier¹

From the Dynamique du Cytosquelette, Laboratoire d'Enzymologie et Biochimie Structurale, CNRS, avenue de la Terrasse, 91198 Gif-sur-Yvette, France and the Institut de Minéralogie et de Physique de la Matière Condensée, Université Pierre et Marie Curie, 75252 Paris, France

The hydrolysis of ATP accompanying actin polymerization destabilizes the filament, controls actin assembly dynamics in motile processes, and allows the specific binding of regulatory proteins to ATP- or ADP-actin. However, the relationship between the structural changes linked to ATP hydrolysis and the functional properties of actin is not understood. Labeling of actin Cys³⁷⁴ by tetramethylrhodamine (TMR) has been reported to make actin non-polymerizable and enabled the crystal structures of ADP-actin and 5'-adenylyl ,-imidodiphosphate-actin to be solved. TMR-actin has also been used to solve the structure of actin in complex with the formin homology 2 domain of mammalian Dia1. To understand how the covalent modification of actin by TMR may affect the structural changes linked to ATP hydrolysis and to evaluate the functional relevance of crystal structures of TMR-actin in complex with actin-binding proteins, we have analyzed the assembly properties of TMR-actin and its interaction with regulatory proteins. We show that TMR-actin polymerized in very short filaments that were destabilized by ATP hydrolysis. The critical concentrations for assembly of TMR-actin in ATP and ADP were only an order of magnitude higher than those for unlabeled actin. The functional interactions of actin with capping proteins, formin, actin-depolymerizing factor/cofilin, and the VCA-Arp2/3 filament branching machinery were profoundly altered by TMR labeling. The data suggest that TMR labeling hinders the intramolecular movements of actin that allow its specific adaptative recognition by regulatory proteins and that determine its function in the ATP- or ADP-bound state.

Received for publication, March 23, 2006 , and in revised form, May 25, 2006.

^* This work was supported by the Ligue Nationale contre le Cancer (Équipe Labellisée Ligue 2003-2006), Human Frontiers in Science Program Grant RGP00712/2003-C, and the European Union Specific Targeted Research Project "Biomics." The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 33-1-6982-3465; Fax: 33-1-6982-3129; E-mail: carlier{at}lebs.cnrs-gif.fr.

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