title: Probing the dynamic equilibrium of actin polymerization by fluorescence energy transfer.
abstract: Fluorescence resonance energy transfer was used to follow the dynamic equilibrium of actin polymerization. We prepared fluorescent analogs of actin by labeling actin covalently with fluorescein (as the donor) and with eosin (as the acceptor). The copolymer of donor- and acceptor-labeled actin exhibits a 60%-70% efficiency of energy transfer. We followed the subunit exchange among filaments both by mixing the donor-acceptor copolymer with unlabeled F actin, and by mixing donor-labeled F actin with acceptor-labeled F actin. The extent of subunit exchange is dependent on ionic conditions. In addition, different kinetics are observed in the two approaches in the presence of excess magnesium ions. The effects of cytochalasin B as a model actin-binding factor were also investigated. One micromolar cytochalasin B reduces the rates of subunit exchange, monomer incorporation and filament depolymerization. At 10 microM cytochalasin B, we detected destabilization of filaments using a morphological assay. The results are discussed in relation to existing models of actin subunit exchange, and to proposed mechanisms of actin-cytochalasin interactions.
mesh_terms: D000199:Actins / Q000737:chemistry* / Q000187:drug effects; D003571:Cytochalasin B / Q000494:pharmacology; D004735:Energy Transfer; D066298:In Vitro Techniques; D011108:Polymers / Q000737:chemistry; D013050:Spectrometry, Fluorescence
chemical_list: D000199:Actins; D011108:Polymers; D003571:Cytochalasin B