Abstract
Thymosin beta 4 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 Tbeta 4 in actin assembly in the high range of concentrations (up to 300 micron) at which Tbeta 4 is found in highly motile blood cells. Tbeta 4 behaves as a simple G-actin sequestering protein only in a range of low concentrations (<20 micron). As the concentration of Tbeta 4 increases, its ability to depolymerize F-actin decreases, due to its interaction with F-actin. The Tbeta 4-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)carbodiimide. As a result of the copolymerization of actin and Tbeta 4-actin complex, the critical concentration is the sum of free G-actin and Tbeta 4-G-actin concentrations at steady state, and the partial critical concentration of G-actin is decreased by Tbeta 4-G-actin complex. The incorporation of Tbeta 4-actin in F-actin is associated to a structural change of the filaments and eventually leads to their twisting around each other. In conclusion, Tbeta 4 is not a simple passive actin-sequestering agent, and at high concentrations the ability of Tbeta 4-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 beta-thymosins.
Highlights
In the presence of ATP, capping proteins block the dynamics at the barbed ends of actin filaments and establish the high critical concentration of the pointed ends; when barbed ends are uncapped, the effective critical concentration is close to the critical concentration of the barbed end
T4 Binds G-actin Selectively at Low Concentration—In living cells, the physiological ionic conditions are such that Factin is assembled at steady state, i.e. filaments coexist with G-actin at the critical concentration
The present results demonstrate that the function of T4 in the regulation of actin assembly is more complex than previously thought
Summary
T4 is not a simple passive actin-sequestering agent, and at high concentrations the ability of T4-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. The role of T4 appears more complex than previously thought, because actin filaments fail to totally depolymerize in the presence of high concentrations (100 –200 M) of T4, due to incorporation of very low amounts of T4-actin in the filaments. The consequences of this property of T4 on the structure of filaments and on the regulation of actin assembly in living cells is examined. These changes, are largely amplified by G-actin binding proteins which maintain a pool of unassembled
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