The current study focuses on the molecular mechanisms responsible for actin assembly on a defined membrane surface: the phagosome. Mature phagosomes were surrounded by filamentous actin in vivo in two different cell types. Fluorescence microscopy was used to study in vitro actin nucleation/polymerization (assembly) on the surface of phagosomes isolated from J774 mouse macrophages. In order to prevent non-specific actin polymerization during the assay, fluorescent G-actin was mixed with thymosin beta4. The cytoplasmic side of phagosomes induced de novo assembly and barbed end growth of actin filaments. This activity varied cyclically with the maturation state of phagosomes, both in vivo and in vitro. Peripheral membrane proteins are crucial components of this actin assembly machinery, and we demonstrate a role for ezrin and/or moesin in this process. We propose that this actin assembly process facilitates phagosome/endosome aggregation prior to membrane fusion.

Human cofilin possesses the tendency for self-association, as indicated by the rapid formation of dimers and oligomers when reacted with water-soluble carbodiimide, Ellman's reagent, or glutathione disulfide. Intermolecular disulfide bonds involve Cys 39 and probably Cys 147 of two adjacent cofilin units. The disulfide-linked dimers and oligomers exhibit a biological activity distinct from the monomer. While monomeric cofilin decreased viscosity and light-scattering of F-actin solutions, dimers and oligomers caused an increase in viscosity and light scattering. Electron microscopy revealed that cofilin oligomers induce the formation of highly ordered actin bundles with occasionally blunt ends similar to actin-cofilin rods observed in cells under oxidative stress. Bundling activity of the disulfidelinked oligomers could be completely reversed into severing activity by dithiothreitol. Formation of cofilin oligomers occurred also in the presence of actin at pH 8, but not at pH 6.6, and was significantly enhanced in the presence of phosphatidylinositol 4,5-bisphosphate. Our data are consistent with the idea that cofilin exists in two forms in vivo also: as monomers exhibiting the known severing activity and as oligomers exhibiting actin bundling activity. However, stabilization of cofilin oligomers in cytoplasm is probably achieved not by disulfide bonds but by a local increase in cofilin concentration and/or binding of regulatory proteins.The actin cytoskeleton plays an essential role in many cellular processes such as cytokinesis, changing of cell shape, endocytosis, and exocytosis. All of these processes are dependent on the correct spatial and temporal organization of the actin cytoskeleton (i.e. its regulated polymerization and depolymerization). These reorganizations of the actin cytoskeleton are regulated by a variety of actin-binding proteins, which in turn are regulated by external stimuli such as Ca 2ϩ levels, phosphoinositides, pH, or reversible phosphorylation (1-3). The turnover rates of actin filaments in living cells (reviewed in Ref. 4) are 100 -200 times faster than the rate constants determined for pure actin (5). This discrepancy is mainly due to the activity of ADF/cofilins (AC), 1 which are able to increase the filament turnover in vitro up to levels found in living cells (6).The essential role of AC in enhancing the turnover of actin filaments has been demonstrated in Listeria motility assays (6, 7) and in living yeast cells (8).The AC are a class of small (13-19 kDa) actin-binding proteins, which are ubiquitous in eukaryotes (reviewed in Refs. 4 and 9). They typically localize to regions of rapid actin assembly like neural growth cones (10), developing skeletal muscle (11), cell cortex (12, 13), ruffling membranes (14), and cleavage furrow (13,15). AC have been shown to be essential for the viability of Saccharomyces cerevesiae (12, 16), Dictyostelium discoideum (17), Caenorhabtidis elegans (18), Xenopus laevis (13), and Drosophila melanogaster (19). In contrast to other actin-binding proteins...

A series of thiol-specific cross-linking reagents were prepared for studying the kinetics of cross-linking between SH1 (Cys(707)) and SH2 (Cys(697)) in rabbit skeletal muscle myosin subfragment 1. The reagents were of the type RSS(CH(2))(n)()SSR, with R = 3-carboxy-4-nitrophenyl and n = 3, 6, 7, 8, 9, 10, and 12, spanning distances from 9 to 20 A. The reactions were monitored spectrophotometrically by measuring the release of 2-nitro-5-thiobenzoate. Reaction rates for modification of SH1 (k(1)) and for cross-linking (k(2)) were measured by the decrease of the K(+)(EDTA)-ATPase activity and the decrease of the Ca(2+)-ATPase activity, respectively, and corrected for the different reactivities of C(n). Cross-linking rates in the presence and absence of MgADP showed similar dependence on the length of the reagents: While the cross-linking rates for n = 3 or n = 6 were close to those for n = 0 (Ellman's reagent), those for n = 7 and 8 were significantly increased. Thus the distance between SH1 and SH2 appears to be equal in both states and can be estimated as >/=15 A, based on the length of the reagent with n = 8 in stretched conformation. Under rigor conditions, reactivity of SH1 differed significantly from that in the presence of MgADP, presumably because of shielding through a lipophilic domain. Similarly, the cross-linking rates k(2) for C(3), C(6), and C(7) in the absence of MgADP were ca. 15 times lower than in the presence of MgADP, suggesting a change in the structure of the SH2 region that depends on nucleotide binding. The results are discussed in terms of recent X-ray structures of S1 and S1-MgADP [Rayment et al. (1993) Science 261, 50-58; Gulick et al. (1997) Biochemistry 36, 11619-11628].