Data from affinity chromatography, analytical ultracentrifugation, covalent cross-linking, and fluorescence anisotropy show that profilin, thymosin  4 , and actin form a ternary complex. In contrast, steady-state assays measuring F-actin concentration are insensitive to the formation of such a complex. Experiments using a peptide that corresponds to the N terminus of thymosin  4 (residues 6 -22) confirm the presence of an extensive binding surface between actin and thymosin  4 , and explain why thymosin  4 and profilin can bind simultaneously to actin. Surprisingly, despite much lower affinity, the N-terminal thymosin  4 peptide has a very slow dissociation rate constant relative to the intact protein, consistent with a catalytic effect of the C terminus on conformational change occurring at the N terminus of thymosin  4 . Intracellular concentrations of thymosin  4 and profilin may greatly exceed the equilibrium dissociation constant of the ternary complex, inconsistent with models showing sequential formation of complexes of profilin-actin or thymosin  4 -actin during dynamic remodeling of the actin cytoskeleton. The formation of a ternary complex results in a very large amplification mechanism by which profilin and thymosin  4 can sequester much more actin than is possible for either protein acting alone, providing an explanation for significant sequestration even if molecular crowding results in a very low critical concentration of actin in vivo.The amount of unpolymerized actin in many cells is large. Several actin-monomer sequestering proteins have been identified that are responsible for maintaining this pool, and attempts have been made to account for the quantity of unpolymerized actin by calculation of the sum of sequestered actin in cells (1-3). These calculations depend not only on the concentration of each sequestering protein and its equilibrium dissociation constant for actin, but also on several other parameters, including an estimate of the critical concentration of actin (i.e. the amount of unpolymerized, unsequestered actin), the stoichiometry with which the sequestering proteins bind to actin, the potential qualitative and quantitative effects should different sequestering proteins interact simultaneously with a single actin subunit, and on assumptions regarding the effects of cytoplasmic molecular crowding on equilibrium association constants. Not surprisingly, with so many parameters to evaluate, even small quantitative errors in measurement or qualitative errors in mechanism result in a wide range of plausible estimates of total sequestered actin. In this report we investigate assumptions that have very significant effects on predictions related to the amount of sequestered actin, finding significant discrepancies with previously reported results, and discuss the expected consequences of these observations. Based on assays measuring steady-state F-actin levels and the failure to obtain a covalently cross-linked ternary complex, thymosin  4 and profilin have been reported to bind ...
Loss of the actin filament capping protein CapG has no apparent effect on the phenotype of mice maintained under sterile conditions; however, bone marrow-derived macrophages from CapG ؊/؊ mice exhibited distinct motility defects. We examined the ability of CapG ؊/؊ mice to clear two intracellular bacteria, Listeria monocytogenes and Salmonella enterica serovar Typhimurium. The 50% lethal dose of Listeria was 10-fold lower for CapG ؊/؊ mice than for CapG ؉/؉ mice (6 ؋ 10 3 CFU for CapG ؊/؊ mice and 6 ؋ 10 4 CFU for CapG ؉/؉ mice), while no difference was observed for Salmonella. The numbers of Listeria cells in the spleens and livers were significantly higher in CapG ؊/؊ mice than in CapG ؉/؉ mice at days 5 to 9, while the bacterial counts were identical on day 5 for Salmonella-infected mice. Microscopic analysis revealed qualitatively similar inflammatory responses in the spleens and livers of the two types of mice. Specific immunofluorescence staining analyzed by fluorescence-activated cell sorting revealed similar numbers of macrophages and dendritic cells in infected CapG ؊/؊ and CapG ؉/؉ spleens. However, analysis of bone marrow-derived macrophages revealed a 50% reduction in the rate of phagocytosis of Listeria in CapG ؊/؊ cells but a normal rate of phagocytosis of Salmonella. Stimulation of bone marrow-derived dendritic cells with granulocyte-macrophage colony-stimulating factor resulted in a reduction in the ruffling response of CapG ؊/؊ cells compared to the response of CapG ؉/؉ cells, and CapG ؊/؊ bone-marrowed derived neutrophils migrated at a mean speed that was nearly 50% lower than the mean speed of CapG ؉/؉ neutrophils. Our findings suggest that specific motility deficits in macrophages, dendritic cells, and neutrophils render CapG ؊/؊ mice more susceptible than CapG ؉/؉ mice to Listeria infection.Macrophages and neutrophils play a critical role in protecting the host against invading pathogens. These cells are able to crawl to the site of infection and ingest and kill invading pathogens. Chemotaxis and phagocytosis require rearrangement of the actin cytoskeleton, and a myriad of actin regulatory proteins orchestrate this rearrangement (23). One way to regulate actin filament length and concentration is to cap or block actin monomer exchange at the fast-growing or barbed (as defined by electron micrographs of heavy meromyosin-decorated filaments) ends of actin filaments. The barbed end is the site of new actin filament growth in living cells. One actin regulatory protein that can serve this function is CapG, a 38-kDa protein (21) that is particularly abundant in macrophages (1% of the total cytoplasmic protein) and neutrophils (0.5% of the total cytoplasmic protein) (5). CapG is the only member of the gelsolin-villin family that caps the barbed ends of actin filaments but does not sever them. Like other members of the gelsolin-villin family, CapG requires micromolar Ca 2ϩ concentrations to function, and its activity is inhibited by the phosphotidylinositol-4,5-bisphosphate (30). The ability of CapG...
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