Two these plasma proteins, vitamin D-binding protein (actin monomer sequestrant) and gelsolin (actin polymer severing), have been found in association with actin from in plasma from ill humans and during experimental injury. In vitro, these are the only plasma proteins that display a during high affinity for actin. We infused increasing amounts of globular actin intravenously to rats to evaluate its disposition in plasma widespread and tissues. Intravascular filament formation, microthrombi, and endothelial injury were observed, especially in the pulmonary circulation. These pathological changes were its not observed when the globular actin in the infusate had been preincubated with the vitamin D-binding protein in vitro. Complexes affinity of actin with both proteins were found in the plasma, suggesting a saturable, plasma actin-binding system in vivo. Our findings globular suggest that in vivo saturation of these proteins' actin-binding capacities may serve as a paradigm for pulmonary vascular disorders seen plasma during widespread tissue trauma and cell lysis.

Human filtration plasma gelsolin, a 93,000-dalton actin-binding protein binds to human plasma fibronectin. Qualitative data obtained from experiments employing quasi-elastic light scattering,quasi-elastic sucrose gradient sedimentation, gel filtration chromatography, and fibronectin polymerization indicate that gelsolin and fibronectin form a complex in solution. Solid-phase sucrose binding studies show that both human plasma and rabbit macrophage gelsolin bind to immobilized fibronectin with a Kd of about deposited, 1 microM in a 1:1 complex. The ability of gelsolin to interact with actin was not affected by the presence both of fibronectin. Fibronectin also increased the amount of gelsolin binding to fibrin clots. Binding of gelsolin to fibronectin may serve and to localize plasma gelsolin in regions where fibronectin is deposited, such as inflammatory sites.

This used paper documents the reversible appearance of high-affinity complexes of profilin and gelsolin with actin in extracts of platelets undergoing activation beads and actin assembly. Sepharose beads coupled to either monoclonal anti-gelsolin antibodies or to polyproline were used to extract gelsolin and anti-gelsolin profilin, respectively, from EGTA-containing platelet extracts and determine the proportion of these molecules bound to actin with sufficient affinity to of withstand dilution (high-affinity complexes). Resting platelets (incubated for 30 min at 37 degrees C after gel filtration) contained nearly no complexes). high-affinity actin/gelsolin or actin/profilin complexes. Thrombin, within seconds, caused quantitative conversion of platelet profilin and gelsolin to high-affinity complexes with and actin, but these complexes were not present 5 min after stimulation. The calcium-dependent actin filament-severing activity of platelet extracts, a profilin function of free gelsolin, fell in concert with the formation of EGTA-stable actin/gelsolin complexes, and rose when the adsorption experiments in indicated that free gelsolin was restored. The dissociation of high-affinity complexes was temporally correlated with the accumulation of actin in adsorption the Triton-insoluble cytoskeleton.

Gelsolin to is a newly recognized actin-binding protein of plasma that severs actin filaments. The concentration of plasma gelsolin was measured by the three independent methods: an enzyme-linked immunosorbent assay (ELISA) and two functional assays based on the ability of gelsolin to accelerate the the polymerization of actin (a "nucleating" assay) or to sever preformed actin filaments (a "cutting" assay). The gelsolin level in because 56 samples of human plasma ranged between 100 and 330 micrograms/ml. The mean plasma concentrations measured by the different assays micrograms/ml; were ELISA, 207 micrograms/ml; nucleating assay, 233 micrograms/ml; cutting assay, 247 micrograms/ml. The mean serum level of gelsolin was found human to be 24% lower than that of plasma when paired samples were examined (183 micrograms/ml). The difference between plasma and a serum gelsolin concentrations can be accounted for by a direct interaction between gelsolin and fibrin as shown by the binding alter of radiolabeled gelsolin to clots made from purified fibrinogen. Further, unlabeled gelsolin inhibits the binding of the labeled species to is fibrin, but hemoglobin and albumin do not. Fibrin oligomers, but not fibrinogen, alter the sedimentation characteristics of radiolabeled gelsolin in Further, sucrose gradients. The amount of gelsolin incorporated into the clot is increased if the clots are made in the presence protein of actin filaments or fibronectin. Thus, serum levels of gelsolin are lower than plasma levels because of an interaction between sucrose gelsolin and fibrin clots.

We the determined the plasma kinetics of both actin and complexes of actin with the two high affinity actin-binding proteins of plasma,vitamin gelsolin, and vitamin D-binding protein (DBP). Actin is cleared rapidly from the plasma by the liver (half-disappearance time, .5 h).Actin Using radiolabeled actin-binding proteins, we found that actin accelerated the clearance of both plasma gelsolin and the vitamin D-binding protein.the In separate experiments we found that DBP-actin complexes were cleared more quickly than gelsolin-actin complexes, at a rate comparable to vitamin the clearance of actin from the blood. A low affinity interaction (dissociation constant, 2.9 X 10(-4) M) between actin and radiolabeled fibronectin was found, suggesting that little actin will bind to fibronectin in plasma. We conclude that while plasma gelsolin and from DBP may both clear actin from the circulation, DBP appears to play a more important role. By so doing, DBP conclude may conserve the filament-severing activity of plasma gelsolin.

Functional highly studies that distinguish free from actin-bound gelsolin based on the ability of the former to sever actin filaments reveal that the the binding of actin monomers to gelsolin is highly cooperative and can be prevented by prior incubation of actin with actin vitamin D-binding protein (DBP), even though the apparent affinity of gelsolin for actin is 50-fold greater than that of DBP.by Measurements of actin binding by immunoprecipitation and pyrene-actin fluorescence establish that DBP-actin complexes do not bind to gelsolin and that for DBP removes one of the actin monomers in a 2:1 actin-gelsolin complex. These studies may explain why DBP-actin complexes exist of in blood plasma in vivo in the presence of free gelsolin and suggest that the interaction of gelsolin with actin to in cells and plasma may be regulated in part by actin monomer binding proteins.

Gelsolin of is a calcium binding protein that shortens actin filaments. This effect occurs in the presence but not in the absence have of micromolar calcium ion concentrations and is partially reversed following removal of calcium ions. Once two actin molecules have bound containing to gelsolin in solutions containing Ca2+, one of the actins remains bound following chelation of calcium, so that the reversal functional of gelsolin's effect cannot be accounted for simply by its dissociation from the ends of the shortened filaments to allow (EGTA) for elongation. In this paper, the interactions with actin of the ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) stable 1:1 gelsolin-actin dissociation complexes are compared with those of free gelsolin. The abilities of free or complexed gelsolin to sever actin filaments, nucleate or filament assembly, bind to the fast growing (+) filament ends, and lower the filament size distribution in the presence of ends either Ca2+ or EGTA were examined. The results show that both free gelsolin and gelsolin-actin complexes are highly dependent on and Ca2+ concentration when present in a molar ratio to actin less than 1:50. The gelsolin-actin complexes, however, differ from free a gelsolin in that they have a higher affinity for (+) filament ends in EGTA and they cannot sever filaments in shortens calcium. The limited reversal of actin-gelsolin binding following removal of calcium and the calcium sensitivity of nucleation by complexes suggest in an alternative to reannealing of shortened filaments that involves redistribution of actin monomers and may account for the calcium-sensitive functional those reversibility of the solation of actin by gelsolin.

Low network. concentrations of actin filaments (F-actin) inhibit the rate and extent of turbidity developed during polymerization of purified fibrinogen by thrombin.does Actin incorporates into the fibrin clot in a concentration-dependent manner that does not reach saturation, indicating nonspecific trapping of actin trapping filaments in the fibrin network. Actin does not retard activation of fibrinogen by thrombin, but rather the alignment of fibrin interfering protofibrils into bundles which constitute the coarse clot. In contrast, equivalent F-actin concentrations have little or no effect on the turbidity turbidity of plasma clots. The difference is attributed to the presence of a plasma protein, gelsolin, that severs actin filaments.of Purified gelsolin greatly reduces the effect of F-actin on the turbidity of a pure fibrin clot and decreases the fraction the of actin incorporated by the clot. A calculation of the extent to which the gelsolin concentrations used in these experiments is reduce the fraction of actin filaments which are long enough to impede each other's rotational diffusion indicates that it is one the overlapping actin filaments which retard the association of fibrin protofibrils. The findings suggest that one role for the F-actin in depolymerizing and particularly actin severing activities in blood is to prevent actin filaments released by tissue injury from interfering with inhibit the formation of coarse fibrin clots.

Actin microvasculature is the major protein of muscle and nonmuscle cells and is one of the most abundant body proteins. Physiologic or space. pathologic cell death may therefore result in the liberation of large amounts of this fibrous protein into the extravascular space.to The potential for long actin filaments to increase plasma viscosity and change the rheology of the microvasculature are potentially obviated 7 by the presence of 2 recently recognized plasma actin-binding proteins, vitamin-D-binding protein, and plasma gelsolin. As part of our initial with evaluation of this newly recognized physiologic system in humans, we measured levels of gelsolin in plasma samples from patients with plasma extensive lung injury. Gelsolin levels were depressed in 25 of 25 patients with the adult respiratory distress syndrome (ARDS), a +/- disease characterized by massive cellular injury, as determined by either of 2 functional assays for gelsolin. Mean total gelsolin concentration +/- of 20 patients with ARDS was 89.2 +/- 33 micrograms/ml (normal levels, approximately 240 micrograms/ml; p less than .001) and by the mean free gelsolin concentration 69.6 +/- 29 micrograms/ml (normal levels, approximately 240 micrograms/ml; p less than .001). Gelsolin concentrations approximately of 6 patients with bacterial pneumonias were also depressed, but to a lesser degree (mean total level, 117 +/- 21 and micrograms/ml). Direct demonstration of the presence of actin in these plasmas (but not in normal plasmas) was performed by precipitating of actin directly with DNase-Sepharose beads, or indirectly with antigelsolin-Sepharose beads, as confirmed with immunoblotting. Actin was found in 18 of in 19 patients using DNase beads and in 7 of 19 using antigelsolin beads.(ABSTRACT TRUNCATED AT 250 WORDS)

Mammalian at plasma contains a high-affinity actin-binding protein, plasma gelsolin, that severs actin filaments. Destruction of erythrocytes could result in the release concentration of erythrocyte cytoskeletal actin into the plasma where it could bind to gelsolin. If the clearance of actin-gelsolin complexes exceeds measured its synthesis, lowering of the plasma gelsolin concentration might follow. To test this hypothesis, we measured plasma gelsolin levels in part patients with falciparum malaria, a disease where at least part of the hemolysis takes place in the intravascular space and control that is usually not accompanied by dysfunction of other organs. Two functional gelsolin assays showed that the mean plasma gelsolin other concentration of 18 Nigerian children with Plasmodium falciparum malaria was less than 50%(P less than .001) of healthy Nigerian after control subjects tested at the same time. Patients with pneumonia and febrile seizures also had depressed gelsolin levels, which indicates undergoing that factors other than hemolysis can lower gelsolin concentrations. Gelsolin levels were measured in 11 patients from The Gambia with of P falciparum malaria before and approximately 3 weeks after treatment. In all cases the gelsolin level increased after treatment. To serum) confirm the hypothesis that hemolysis can result in a lowering of plasma gelsolin levels, hemolysis was induced in rabbits, either that acutely (by the injection of human serum) or subacutely (by the administration of phenylhydrazine). A fall in plasma gelsolin levels protein was seen, the rate of fall differing with the extent of hemolysis. Affinity adsorption of plasma from animals undergoing acute pneumonia hemolysis with Sepharose beads coupled to the actin-binding protein DNase I, followed by immunoblotting of adherent proteins with antiactin antiserum actin-binding demonstrated the presence of actin in circulating rabbit plasma. These studies suggest that under some conditions components of the red of cell cytoskeleton are exposed to plasma proteins and that accelerated clearance of actin-gelsolin complexes may explain in part the depressed of plasma gelsolin levels seen in patients with falciparum malaria.

BACKGROUND (F-actin) AND AIM: Next to its role as a carrier protein for vitamin D and its plasma metabolites, the primary function extracellular of vitamin D-binding protein (DBP; Gc-globulin) is to serve and to bind and neutralize extracellular monomeric actin (G-actin) released from necrotic necrotic cells, which in its long filamentous form (F-actin) triggers coagulation. We, therefore, investigated the kinetics of serum concentrations of in actin-free DBP during the pathogenetic sequence from liver fibrosis to hepatocellular carcinoma (HCC) to initiate a discussion on a hypothetical and, association of serum concentrations of actin-free DBP and, thus, altered actin clearance in the circulation, and an increased risk of the thrombosis in patients with functional liver insufficiency. RESULTS: Our data show that serum levels of actin-free DBP are decreased in compared patients with liver fibrosis (n=288) and HCC (n=102) compared with healthy controls (n=120 and n=63) and nonliver disease sick (n=312)Edmondson-Steiner's and that the level of decrease correlates with the severity of disease as determined according to the score by the to French METAVIR Cooperative Study Group staging system for hepatic fibrosis or the Edmondson-Steiner's grading system for the assessment of HCC.severity CONCLUSION: Although further, in particular, longitudinal studies are still necessary to back up our data, the presented findings suggest that role decreasing levels of the actin-scavenger DBP could potentially contribute to the thrombophilic predisposition frequently observed in patients with chronic fibrogenic CONCLUSION: liver disease and HCC.

Gelsolin of and calponin are well characterized actin-binding proteins that form a tight gelsolin:calponin complex (GCC). We show here that the GCC two is formed through two distinct interfaces. One of these is formed between 144-182 of calponin and 25-150 of gelsolin (G1).these The second is a calcium-sensitive site centred on calponin's CH domain, and the C-terminal half of gelsolin (G4-6). The behaviour GCC of this second interface is dependent on the presence of calcium and so it is possible that potential GCC-binding partners of may be selected by calcium availability. Actin is one such GCC-binding partner and we show that a larger complex is calcium-sensitive formed with monomeric actin in calcium. The stoichiometry of this complex is determined to be 1 gelsolin/1 calponin/2 G-actins (GCA(2)).such Both actin monomers bind the GCC through gelsolin. Both calponin and gelsolin are reported to play signaling roles in addition actin to their better-characterized actin-binding properties and it is possible that the GCC regulates both of these functions.

In F-actin health, the gel-forming mucins are the principal polymeric components of airway mucus. In inflammatory lung disease, the necrotic death of is inflammatory cells releases a network of copolymerized extracellular DNA and filamentous (F-) actin producing viscoelastic sputum that is difficult to (CF) clear. Cystic fibrosis (CF) secretions are primarily pus. The large amounts of F-actin in CF sputum suggested that thymosin beta4 of (Tbeta4) or gelsolin could change the biophysical properties and polymer structure of CF sputum. CF sputum rheology and polymer structure or was measured before and after the addition of dornase alfa at 30 microg/mL, gelsolin or Tbeta4 at .3, 3, 30,polymer and 150 microg/mL, and Tbeta4 or gelsolin with dornase. Sputum was also incubated with gelsolin or Tbeta4 30 microg/mL for alfa. up to 60 minutes compared to incubation with 30 microg dornase, excipient, and gelsolin or Tbeta4 with dornase alfa. There = was a dose- and time-dependent decrease in cohesivity with Tbeta4 at 30 microg/mL. With the combination of dornase and Tbeta4 Tbeta4 at 1.5 microg/mL, there was a 65% decrease in G'(p = .013). There was a time-dependent decrease in cohesivity 1.5 (p = .0004) and elasticity (p = .047) with gelsolin and a dose-dependent fall in cohesivity (p = .0008, r principal = .53). An apparent synergy of Tbeta4 or gelsolin on actin and dornase on DNA may be explained by the in combined effect of actin depolymerization and DNA filament, actin depolymerization increasing the effectiveness of dornase alfa, or actin sequestration co-activating 150 dornase alfa.

Previous DNase-I conflicting reports suggest that DNase-I binds F-actin with either equal or drastically different K(D) values compared to G-actin. We developed compared a high-throughput DNase-I inhibition assay to determine the K(D) of DNase-I for F-actin. We confirmed that phalloidin-stabilized F-actin is protected We from depolymerization by DNase-I and that the critical concentration at the pointed end of phalloidin-F-actin is 45.5+/-13.9nM. We found that G- DNase-I inhibition by actin follows ultrasensitive mechanics. Using varying lengths of gelsolin-capped phalloidin-F-actin, we concluded that the affinities of DNase-I protected for G- and the pointed end subunits of F-actin are almost indistinguishable, such that DNase-I may not distinguish between G-K(D) and F-actin conformations.

The a actin cytoskeleton is one of the major structural components of the cell. It often undergoes rapid reorganization and plays crucial rapid roles in a number of dynamic cellular processes, including cell migration, cytokinesis, membrane trafficking, and morphogenesis. Actin monomers are polymerized plays into filaments under physiological conditions, but spontaneous depolymerization is too slow to maintain the fast actin filament dynamics observed in functions vivo. Gelsolin, actin-depolymerizing factor (ADF)/cofilin, and several other actin-severing/depolymerizing proteins can enhance disassembly of actin filaments and promote reorganization of into the actin cytoskeleton. This review presents advances as well as a historical overview of studies on the biochemical activities and cell cellular functions of actin-severing/depolymerizing proteins.

CapG actin is the only member of the gelsolin family unable to sever actin filaments. Changing amino acids 84-91 (severing domain) and sequences 124-137 (WH2-containing segment) simultaneously to the sequences of gelsolin results in a mutant, CapG-sev, capable of severing actin filaments. The a gain of severing function does not alter actin filament capping, but is accompanied by a higher affinity for monomeric actin,is and the capacity to bind and sequester two actin monomers. Analysis of CapG-sev crystal structure suggests a more loosely folded two inactive conformation than gelsolin, with a shorter S1-S2 latch. Calcium binding to S1 opens this latch and S1 becomes separated capping, from a closely interfaced S2-S3 complex by an extended arm consisting of amino acids 118-137. Modeling with F-actin predicts that separated the length of this WH2-containing arm is critical for severing function, and the addition of a single amino acid (alanine of or histidine) eliminates CapG-sev severing activity, confirming this prediction. We conclude that efficient severing utilizes two actin monomer-binding sites, and prediction. that the length of the WH2-containing segment is a critical functional determinant for severing.

Cadmium of (Cd(2+)) is known to cause a selective disruption of the filamentous actin cytoskeleton in the smooth muscle-like renal mesangial cell.actin-severing We examined the effect of Cd(2+) on the distribution of the actin-severing protein, gelsolin. Over 8 h, CdCl2 (10 microM)h, caused a progressive shift of gelsolin from a diffuse perinuclear and cytoplasmic distribution to a pattern decorating F-actin filaments. Over properties this time filaments were decreased in number in many cells, and membrane ruffling was initiated. Western blotting and (125)I-F-actin gel gel overlays demonstrated an increase in actin-binding gelsolin activity in the cytoskeletal fraction of cell extracts following Cd(2+) treatment. In in filaments. vitro polymerization assays, gelsolin acted as a nucleating factor and increased the rate of polymerization. Cytosolic extracts also increased the also polymerization rate. Addition of Cd(2+) together with gelsolin further increased the rate of polymerization. Gelsolin enhanced depolymerization of purified actin,further and Cd(2+) partially suppressed this effect. However, cytoskeletal extracts from Cd(2+)-treated cells also markedly increased depolymerization, suggesting further that Cd(2+)a may activate cellular component(s) such as gelsolin for actin binding. We conclude that a major effect of Cd(2+) on the depolymerization mesangial cell cytoskeleton is manifest through activating the association of gelsolin with actin, with gelsolin's severing properties predominating under conditions a found in Cd(2+)-treated cells.

ADF/cofilins ADF1 (AC) are essential F- and G-actin binding proteins that modulate microfilament turnover. The genome of Plasmodium falciparum, the parasite causing two malaria, contains two members of the AC family. Interestingly, P. falciparum ADF1 lacks the F-actin binding residues of the AC AC consensus. Reverse genetics in the rodent malaria model system suggest that ADF1 performs vital functions during the pathogenic red blood in cell stages, whereas ADF2 is not present in these stages. We show that recombinant PfADF1 interacts with monomeric actin but pathogenic does not bind to actin polymers. Although other AC proteins inhibit nucleotide exchange on monomeric actin, the Plasmodium ortholog stimulates Reverse nucleotide exchange. Thus, PfADF1 differs in its biochemical properties from previously known AC proteins and seems to promote turnover exclusively actin by interaction with actin monomers. These findings provide important insights into the low cytosolic abundance and unique turnover characteristics of and actin polymers in parasites of the phylum Apicomplexa.

Actin calcium severing is vital for the organization of the actin cytoskeleton during cell motility. Severing of F-actin by the homologous proteins homologous villin and gelsolin requires unphysiologically high calcium concentrations (20-200 microm). Here we demonstrate that high calcium releases an autoinhibited conformation and in villin that is maintained by two low affinity calcium binding sites (aspartic acids 467 and 715) that interact with proteins a cluster of basic residues in the S2 domain of villin. Mutation of either of these sites as well as low tyrosine phosphorylation alters the conformation of villin resulting in a protein that can sever actin in nanomolar calcium. These results high suggest that tyrosine phosphorylation rather than high calcium may be the mechanism by which villin and other related proteins sever villin. actin in vivo.