Effect of Actin-binding Protein on Sedimentation Properties of Actin the SHARON ROSENBERG and ALFRED STRACHER

Actin and act in-binding protein (ABP) have recently been purif ied from human platelet cytoskeletons (S. Rosenberg, A. Stracher, and R. C. Lucas, 1981, J. Cel l BioL 91:201-211). Here, the effect of ABP on the sedimentation of actin was studied. When ABP was added to preformed F-actin filaments, it bound unti l a maximum ratio of 1:9 (ABP:actin, mol:mol) was reached. However, when actin was polymerized in the presence of ABP, two and a half times more ABP was able to bind to the act inthat is, every 3.4 actin monomers were now bound by an ABP dimer. ABP was not able to induce the sedimentation of actin under nonpolymeriz ing condit ions but was able to reduce the time and concentration of actin required for sedimentation under slow polymerizing condit ions. ABP, therefore, exerts its effect on G-actin by either nucleating polymerization or by cross-linking newly formed oligomers into a more sedimentable form. In muscle cells, a stable actin lattice is required for the generation of contractile force, whereas in nonmuscle cells, the more transitory contractile processes make a flexible actin cytoskeleton more desirable. Changes in the state of actin aggregation have been reported during cell division (30), mitosis (29), viral transformation (26), active phagocytosis (15), and adhesion to a substratum (8). These changes can either be a result of the reorganization of actin from fdament bundles ,~ single fdaments simply by a cross-linker going off and on again or by a monomer ~ polymer mechanism. In platelets, several investigators have found the majority of the cells' actin to be in a nonfdamentons form (6, 9, 10, 23, 25), whereas others found a significant amount of the actin to be in a f'damentous form (2, 28, 35, 38). There are reports of a rapid polymerization of the ceils' actin upon platelet activation (6, 9, 23, 25) which might be responsible for subsequent fdopodial formation though there is also the alternative that the preexistent actin ftlaments of the cytoskeleton are rearranged to form these ftlopodia (28). In either case, platelets, like all nonmuscle cells, must have mechanisms by which the state of assembly of their actin lattice is regulated. Various accessory proteins may be involved in this regulation. Actin-binding protein (ABP), a 260,000-dalton protein originally isolated from macrophages by Hartwig and Stossel (12, 32), is present in high concentrations in platelets (19, 28). ABP has the ability to cross-link and cause the low-speed sedimentation of F-actin (5, 28, 32). Profdin, a 16,000-dalton protein which binds to G-actin in a I:1 molar ratio rendering it polymerization-resistant has also been found in platelets (7, 11, 20). A 90,000to 95,000-dalton protein in platelet extracts which binds to G-actin in the presence of Ca 2+ has also been identified (21, 33). This protein may or may not be similar to gelsolin described by Yin and Stossel (36). Gelsolin has the ability to break actin t'flaments in the presence of Ca 2+, thereby increasing the amount of ABP required for incipient gelation of the actin 07). Platelets have also been found to contain a whole array of other proteins known to interact with actin including myosin, a-actinin, troponin, and tropomyosin (1). In 1976, Lucas et al. (18) introduced a procedure for the direct isolation of the platelets' cytoskeleton, using a solution containing 1% Triton X-100 and I0 mM EGTA. This structure, seen by negative staining in the electron microscope as branched bundles of actin ftlaments, consists mostly of actin, ABP, and a-actinin (27, 28). The individual components of the cytoskeleton have been purified and their in vitro interactions studied. We have previously reported that both ABP and t~actinin exhibit Ca2+-sensitive interactions with the actin cytoskeleton in vivo (28), though only a-actinin remains Ca 2+sensitive in its binding to actin in vitro (27). The binding of ABP to aetin in vitro is insensitive to the presence of calcium ions. In this report we describe the effect of ABP on the sedimentation properties of actin. 1"HE JOURNAL OF CELl. BIOLOGY • VOLUME 94 )Ul.Y 1982 51-55 © The Rockefeller University Press • 0021-9525/82/07/0051/05 $1.00 51 on M ay 0, 2017 D ow nladed fom Published July 1, 1982