Analysis of Tetramethylrhodamine-labeled Actin Polymerization and Interaction with Actin Regulatory Proteins
Autor: | Dominique Didry, Nicolas Boisset, Eric Larquet, Marie-France Carlier, Kim Ho Diep Le, Andrea Pelikan Conchaudron, Dominique Pantaloni |
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Přispěvatelé: | Laboratoire d'Enzymologie et Biochimie Structurale, CNRS, Laboratoire d'Enzymologie et Biochimie Structurale, Institut de minéralogie et de physique des milieux condensés (IMPMC), Université Pierre et Marie Curie - Paris 6 (UPMC)-IPG PARIS-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Diderot - Paris 7 (UPD7)-Institut de Physique du Globe de Paris (IPG Paris)-Centre National de la Recherche Scientifique (CNRS) |
Rok vydání: | 2006 |
Předmět: |
Fetal Proteins
Actin Capping Proteins Formins Arp2/3 complex macromolecular substances Microfilament Biochemistry 03 medical and health sciences 0302 clinical medicine Animals Humans [SDV.BBM]Life Sciences [q-bio]/Biochemistry Molecular Biology Actin-binding protein Molecular Biology 030304 developmental biology 0303 health sciences biology Rhodamines Microfilament Proteins Nuclear Proteins Actin remodeling Cell Biology Cofilin Actins Protein Structure Tertiary Cell biology Adenosine Diphosphate Actin Cytoskeleton Destrin Actin Depolymerizing Factors Profilin Actin-Related Protein 3 Multiprotein Complexes Actin-Related Protein 2 biology.protein Rabbits MDia1 030217 neurology & neurosurgery Protein Binding |
Zdroj: | Journal of Biological Chemistry Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2006, 281, pp.24036-47. ⟨10.1074/jbc.M602747200⟩ Journal of Biological Chemistry, 2006, 281, pp.24036-47. ⟨10.1074/jbc.M602747200⟩ |
ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.m602747200 |
Popis: | 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 beta,gamma-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. |
Databáze: | OpenAIRE |
Externí odkaz: |
Abstrakt: | 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 beta,gamma-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. |
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ISSN: | 00219258 1083351X |
DOI: | 10.1074/jbc.m602747200 |