Induction of the polymerization of actin from the actin:thymosin beta 4 complex by phalloidin, skeletal myosin subfragment 1, chicken intestinal myosin I and free ends of filamentous actin.

Abstract

Thymosin beta 4 is able to form 1:1 complexes with monomeric (G) actin, thereby stabilizing the intracellular pool of unpolymerized actin. We have searched for factors that are able to induce the polymerization of actin from the actin:thymosin beta 4 complex. Phalloidin, subfragment 1 isolated from rabbit skeletal muscle myosin and chicken intestinal myosin I are demonstrated to be able to polymerize the actin from this complex in the presence of 1 mM MgCl2. Polymerization of actin was verified by the DNase I inhibition assay, by cosedimentation and from the fluorescence increase of pyrene-labelled actin. Actin filaments formed under the influence of subfragment 1 or phalloidin were visualized under the electron microscope after negative staining. Polymerization of skeletal muscle actin from the complex with thymosin beta 4 by phalloidin is accompanied by the hydrolysis of the actin-bound ATP to ADP. Polymerization was also induced by sonicated F-actin which possessed a high concentration of free filament ends. F-actin was severed by 0.01 M human cytoplasmic gelsolin, which is known to possess blocked+ends. Free, slowly growing-ends were unable to induce polymerization of actin from the thymosin beta 4 complex. However, when gelsolin on its own or in complex with two actin molecules was added to actin:thymosin beta 4 under nucleating conditions, it was found to be able to promote actin repolymerization provided that its concentration was close to the dissociation constant (Kd) of actin:thymosin beta 4. This Kd was found to be 0.4 microM in the presence of 1 mM MgCl2 and the absence of KCl and, thus, close to the critical concentration of actin polymerization under these conditions. The source of actin did not influence its polymerization from the thymosin beta 4 complex; rabbit skeletal muscle actin and porcine brain actin were polymerized with equal efficiency from their complexes with thymosin beta 4 by both phalloidin and myosin subfragment 1. Skeletal muscle, but not cytoplasmic actin, was found to be also polymerized in the presence of increased CaCl2 concentrations to values above 1 mM.