Tβ₄ is not a simple G-actin sequestering protein and interacts with F-actin at high concentration

Thymosin β₄ is acknowledged as a major G-actin binding protein maintaining a pool of unassembled actin in motile vertebrate cells. We have examined the function of Tβ₄ in actin assembly in the high range of concentrations (up to 300 μM) at which Tβ₄ is found in highly motile blood cells. Tβ₄ behaves as a simple G-actin sequestering protein only in a range of low concentrations (<20 μM). As the concentration of Tβ₄ increases, its ability to depolymerize F-actin decreases, due to its interaction with F-actin. The Tβ₄-actin can be incorporated, in low molar ratios, into F-actin, and can be cross-linked in F-actin using 1-ethyl-3-(3-dimethylaminopropyl)carbo-diimide. As a result of the copolymerization of actin and Tβ₄-actin complex, the critical concentration is the sum of free G-actin and Tβ₄-G-actin concentrations at steady state, and the partial critical concentration of G-actin is decreased by Tβ₄-G-actin complex. The incorporation of Tβ₄-actin in F-actin is associated to a structural change of the filaments and eventually leads to their twisting around each other. In conclusion, Tβ₄ is not a simple passive actin-sequestering agent, and at high concentrations the ability of Tβ₄-actin to copolymerize with actin reduces the sequestering activity of G-actin-binding proteins. These results question the evaluation of the unassembled actin in motile cells. They account for observations made on living fibroblasts overexpressing β-thymosins.