Regulation of gelsolin to plant actin filaments and its distribution in pollen

The effect of plasma gelsolin on plant microfilaments and its localization in plant cells were investigated. The results by using ultracentrifugation and electron microscopy showed that plant microfilaments could be severed into shorter fragments by gelsolin in a Ca2+-dependent manner. By measuring the binding ability of plasma gelsolin to pollen actin using the method of immunoprecipitation, it was shown that pollen actin could bind gelsolin at a ratio of 2.0±0.21 in the presence of Ca2+. Addition of EGTA could disassociate the actin-gelsolin complexes, reducing the ratio to 1.2±0.23, and the addition of PIP2 could further reduce the ratio to 0.8±0.1. The results indicate that plant actin has similar binding properties with plasma gelsolin as that of animal actin. By Western blotting we identified the existence of gelsolin in lily pollen. The results of immunolo-calization of gelsolin in pollen and pollen tube showed that gelsolin was mainly localized at the germinal furrow in pollen grains and at the cytoplasm in pollen tube, especially in the tip region.     

Regulation of gelsolin to plant actin filaments and its distribution in pollen

The dynamics of the actin cytoskeleton depends upon the unique constellation of ac- tin-binding proteins (ABPs), as well as their spatial distribution and local activation. However, the identification and characterization of actin-binding proteins in plant cells are still limited. At pre- sent, only a few plant ABPs have been identified in plant tissues, including profilin, ADF/cofilin, fimbrin, villin and several myosins. Compared with that in animals, there is still a long way for us …     

Molecular biology of gelsolin, a calcium-regulated actin filament severing protein

Gelsolin is a Ca2+-binding protein of mammalian leukocytes, platelets and other cells which has multiple and closely regulated powerful effects on actin. In the presence of micromolar Ca2+, gelsolin severs actin filaments, causing profound changes in the consistency of actin polymer networks. A variant of gelsolin containing a 25-amino acid extension at the NH2-terminus is present in plasma where it may be involved in the clearance of actin filaments released during tissue damage. Gelsolin has two sites which bind actin cooperatively. These sites have been localized using proteolytic cleavage and monoclonal antibody mapping techniques. The NH2-terminal half of the molecule contains a Ca2+-insensitive actin severing domain while the COOH-terminal half contains a Ca2+-sensitive actin binding domain which does not sever filaments. These data suggest that the NH2-terminal severing domain in intact gelsolin is influenced by the Ca2+-regulated COOH-terminal half of the molecule. The primary structure of gelsolin, deduced from human plasma gelsolin cDNA clones, supports the existence of actin binding domains and suggests that these may have arisen from a gene duplication event, and diverged subsequently to adopt their respective unique functions. The plasma and cytoplasmic forms of gelsolin are encoded by a single gene, and preliminary results indicate that separate mRNAs code for the two forms. Further application of molecular biological techniques will allow exploration into the structural basis for the multifunctionality of gelsolin, as well as the molecular basis for the genesis of the cytoplasmic and secreted forms of gelsolin.     

The actin filament-severing domain of plasma gelsolin

Gelsolin, a multifunctional actin-modulating protein, has two actin-binding sites which may interact cooperatively. Native gelsolin requires micromolar Ca2+ for optimal binding of actin to both sites, and for expression of its actin filament-severing function. Recent work has shown that an NH2-terminal chymotryptic 17-kD fragment of human plasma gelsolin contains one of the actin-binding sites, and that this fragment binds to and severs actin filaments weakly irrespective of whether Ca2+ is present. The other binding site is Ca2+ sensitive, and is found in a chymotryptic peptide derived from the COOH-terminal two-thirds of plasma gelsolin; this fragment does not sever F-actin or accelerate the polymerization of actin. This paper documents that larger thermolysin-derived fragments encompassing the NH2-terminal half of gelsolin sever actin filaments as effectively as native plasma gelsolin, although in a Ca2+-insensitive manner. This result indicates that the NH2-terminal half of gelsolin is the actin-severing domain. The stringent Ca2+ requirement for actin severing found in intact gelsolin is not due to a direct effect of Ca2+ on the severing domain, but indirectly through an effect on domains in the COOH-terminal half of the molecule to allow exposure of both actin-binding sites.     

凝溶胶蛋白及其生物医学作用

gelsolin(凝溶胶蛋白)有胞浆和血清二型,分别存在于哺乳动物各类细胞和血清中。该蛋白的基本生物功能是以钙依赖的方式通过切断、封端肌动蛋白丝,或使肌动蛋白聚集成核等方式控制肌动蛋白的结构。大量动物实验和临床研究均表明,gelsolin的结构、功能及调节与烧伤和急性挫伤的诊断与治疗,以及某些炎症、肿瘤和淀粉样变等多种疾病的病理过程密切相关。作为一种急症治疗药,该蛋白的工程化产品已在美国进行Ⅱ期临床实验。最近发现正常细胞经电离辐射后,gelsolin的表达水平发生显著改变,其在辐射效应中的作用和意义值得关注。     

Sequential binding of actin monomers to plasma gelsolin and its inhibition by vitamin D-binding protein

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

Microinjection of gelsolin into living cells

Gelsolins are actin-binding proteins that cap, nucleate, and sever actin filaments. Microinjection of cytoplasmic or plasma gelsolin into living fibroblasts and macrophages did not affect the shape, actin distribution, deformability, or ruffling activity of the cells. Gelsolin requires calcium for activity, but the NH2-terminal half is active without calcium. Microinjection of this proteolytic fragment had marked effects: the cells rounded up, stopped ruffling, became soft, and stress fibers disappeared. These changes are similar to those seen with cytochalasin, which also caps barbed ends of actin filaments. Attempts to raise the cytoplasmic calcium concentration and thereby activate the injected gelsolin were unsuccessful, but the increases in calcium concentration were minimal or transient and may not have been sufficient. Our interpretation of these results is that at the low calcium concentrations normally found in cells, gelsolin does not express the activities observed in vitro at higher calcium concentrations. We presume that gelsolin may be active at certain times or places if the calcium concentration is elevated to a sufficient level, but we cannot exclude the existence of another molecule that inhibits gelsolin. Microinjection of a 1:1 gelsolin/actin complex had no effect on the cells. This complex is stable in the absence of calcium and has capping activity but no severing and less nucleation activity as compared with either gelsolin in calcium or the NH2-terminal fragment. The NH2-terminal fragment-actin complex also has capping and nucleating activity but no severing activity. On microinjection it had the same effects as the fragment alone. The basis for the difference between the two complexes is unknown. The native molecular weight of rabbit plasma gelsolin is 82,500, and the extinction coefficient at 280 nm is 1.68 cm2/mg. A new simple procedure for purification of plasma gelsolin is described.     

The amino-terminal fragment of gelsolin is cross-linked to Cys-374 of actin in the EGTA-resistant actin-gelsolin complex.

It has been shown that the EGTA-resistant actin, one of the two actin molecules associated to gelsolin, can be predominantly cross-linked to gelsolin by benzophenone-4-maleimide (BPM), a photoaffinity-labeling reagent, which was conjugated to Cys-374 of actin prior to cross-linking (Doi, Y., Banba, M. and Vertut-Do?, A. (1991) Biochemistry 30, 5769-5777). When a chymotryptic digest of gelsolin containing the amino-terminal 15-kDa fragment was mixed with BPM-actin (42 kDa) and irradiated for cross-linking, a band of 58 kDa appeared on SDS-PAGE which was shown to contain actin molecule by using fluorescently labeled actin. The amino-terminal sequence of the 58-kDa complex was identical to that of gelsolin, confirming that the amino-terminal segment (residues 1-133) of pig plasma gelsolin lies closely to Cys-374 of actin in the EGTA-resistant complex.     

Identification of critical functional and regulatory domains in gelsolin

Gelsolin can sever actin filaments, nucleate actin filament assembly, and cap the fast-growing end of actin filaments. These functions are activated by Ca2+ and inhibited by polyphosphoinositides (PPI). We report here studies designed to delineate critical domains within gelsolin by deletional mutagenesis, using COS cells to secrete truncated plasma gelsolin after DNA transfection. Deletion of 11% of gelsolin from the COOH terminus resulted in a major loss of its ability to promote the nucleation step in actin filament assembly, suggesting that a COOH-terminal domain is important in this function. In contrast, derivatives with deletion of 79% of the gelsolin sequence exhibited normal PPI-regulated actin filament-severing activity. Combined with previous results using proteolytic fragments, we deduce that an 11-amino acid sequence in the COOH terminus of the smallest severing gelsolin derivative identified here mediates PPI-regulated binding of gelsolin to the sides of actin filaments before severing. Deletion of only 3% of gelsolin at the COOH terminus, including a dicarboxylic acid sequence similar to that found on the NH2 terminus of actin, resulted in a loss of Ca2+-requirement for filament severing and monomer binding. Since these residues in actin have been implicated as potential binding sites for gelsolin, our results raise the possibility that the analogous sequence at the COOH terminus of gelsolin may act as a Ca2+-regulated pseudosubstrate. However, derivatives with deletion of 69-79% of the COOH-terminal residues of gelsolin exhibited normal Ca2+ regulation of severing activity, establishing the intrinsic Ca2+ regulation of the NH2-terminal region. One or both mechanisms of Ca2+ regulation may occur in members of the gelsolin family of actin-severing proteins.     

Formation of vitamin D-binding protein-actin and binary and ternary plasma gelsolin-actin complexes in human serum

After the addition of actin to serum, the binding of actin to serum actin-binding proteins was analyzed by the method of immunoblotting using monospecific antibodies against vitamin D-binding protein (DBP) (group-specific component, Gc), human skeletal actin and human plasma gelsolin. When increasing amounts of globular actin were added to serum, actin bound to DBP preferentially. After exhausting DBP, actin began to bind to plasma gelsolin. When equally increasing amounts of filamentous actin were added to serum, actin was bound to both plasma gelsolin and DBP, and then uncomplexed DBP removed one actin molecule from gelsolin-actin 1:2 complex, resulting in a gelsolin-actin 1:1 complex. These results support the theory that the actin-depolymerizing activity of serum is due to the concerted role of plasma gelsolin and DBP.     

Isolation and properties of two actin-binding domains in gelsolin

Gelsolin is a Ca2+-sensitive 90-kDa protein which regulates actin filament length. A molecular variant of gelsolin is present in plasma as a 93-kDa protein. Functional studies have shown that gelsolin contains two actin-binding sites which are distinct in that after Ca2+-mediated binding, removal of free Ca2+ releases actin from one site but not from the other. We have partially cleaved human plasma gelsolin with alpha-chymotrypsin and identified two distinct actin-binding domains. Peptides CT17 and CT15, which contain one of the actin-binding domains, bind to actin independently of Ca2+; peptides CT54 and CT47, which contain the other domain, bind to actin reversibly in response to changes in Ca2+ concentration. These peptides sequester actin monomers inhibiting polymerization. Unlike intact gelsolin, neither group of peptides nucleates actin assembly or forms stable filament end caps. CT17 and CT15 can however sever actin filaments. Amino acid sequence analyses place CT17 at the NH2 terminus of gelsolin and CT47 at the carboxyl-terminal two-thirds of gelsolin. Circular dichroism measurements show that Ca2+ induces an increase in the alpha-helical content of CT47. These studies provide a structural basis for understanding the interaction of gelsolin with actin and allow comparison with other Ca2+-dependent actin filament severing proteins.     

Plasma gelsolin caps and severs actin filaments

Plasma gelsolin caps actin filaments at their 'barbed' ends and severs them along their length. Capping has been demonstrated both by direct visualization using gold-labeled gelsolin and by inhibition of actin polymerization onto the barbed ends of fragments of the acrosomal process of Limulus sperm. Severing activity is demonstrated by the fact that actin filaments nucleated off acrosomal fragments are shortened or removed within a few seconds by added plasma gelsolin without any obvious disruption of the actin bundles in the acrosomal processes themselves.     

Inactivation of endotoxin by human plasma gelsolin

Septic shock from bacterial endotoxin, triggered by the release of lipopolysaccharide (LPS) molecules from the outer wall of Gram-negative bacteria, is a major cause of human death for which there is no effective treatment once the complex inflammatory pathways stimulated by these small amphipathic molecules are activated. Here we report that plasma gelsolin, a highly conserved human protein, binds LPS from various bacteria with high affinity. Solid-phase binding assays, fluorescence measurements, and functional assays of actin depolymerizing effects show that gelsolin binds more tightly to LPS than it does to its other known lipid ligands, phosphatidylinositol 4,5-bisphosphate and lysophosphatidic acid. Gelsolin also competes with LPS-binding protein (LBP), a high-affinity carrier for LPS. One result of gelsolin-LPS binding is inhibition of the actin binding activity of gelsolin as well as the actin depolymerizing activity of blood serum. Simultaneously, effects of LPS on cellular functions, including cytoskeletal actin remodeling, and collagen-induced platelet activation by pathways independent of toll-like receptors (TLRs) are neutralized by gelsolin and by a peptide based on gelsolin residues 160-169 (GSN160-169) which comprise part of gelsolin's phosphoinositide binding site. Additionally, TLR-dependent NF-kappaB translocation in astrocytes appears to be blocked by gelsolin. These results show a strong effect of LPS on plasma gelsolin function and suggest that some effects of endotoxin in vivo may be mediated or inhibited by plasma gelsolin.     

Characterization of plasma gelsolin as a substrate for matrix metalloproteinases

We previously showed that plasma gelsolin, a major component of the extracellular actin scavenging system, is an matrix metalloproteinase (MMP)-14 substrate. Here we confirmed that plasma gelsolin is cleaved by MMP-14 at the plasma level, and found that it was most efficiently digested by MMP-3 followed by MMP-2, MMP-1, MMP-14, and MMP-9, in that order. Plasma gelsolin (90 kDa) was cut into several fragments of 43-48 kDa by MMP-3. The MMP-3 cleavage sites in plasma gelsolin were determined by labeling the C termini generated by in-gel digestion with 50% H2 18O combined with peptide mass mapping, and sequencing of the N-terminal amino acids. Plasma gelsolin was cleaved at Asn416-Val417, Ser51-Met52, and Ala435-Gln436. Proteolytic cleavage by MMP-3 resulted in considerable loss of its actin filament-depolymerizing activity. This suggests that MMPs weaken the extracellular actin-scavenging system by cleaving plasma gelsolin and may, therefore, be involved in pathological conditions induced by extracellular actin, such as endothelial injury, respiratory distress syndrome, hepatic necrosis, and septic shock.     

凝溶胶蛋白与相关疾病的关系

凝溶胶蛋白（gelsolin,GSN）是一种在机体内普遍存在的,对细胞结构和代谢功能具有多种调节作用的蛋白。GSN作为凝溶胶蛋白超家族的成员之一,是一种重要的肌动蛋白（actin）结合蛋白,可通过切断、封闭肌动蛋白丝,或使actin聚集成核等方式来调控actin的结构与代谢功能.GSN不仅能在重组的肌动蛋白细丝（F-actin）中发挥作用,而且在细胞运动、细胞凋亡等细胞活动中也发挥着重要的作用。GSN有血浆型（plasma gelsolin,pGSN）和细胞质型（cytoplasmic gelsolin,cGSN）两种亚型,它们在淀粉样变性、炎症、癌症、心血管疾病、阿茨海默病（AD）及肾脏疾病中都起着重要的作用,GSN可能成为多种疾病的一个新的生物标记物或者治疗靶点。本文将就GSN与相关疾病的关系的研究进展做一综述。     

Characterization of horse plasma gelsolin

Gelsolin can be purified from horse blood plasma by treating the plasma sequentially with an anion-exchange medium in the presence and then the absence of free Ca2+. The purified gelsolin migrates as a 90-kilodalton protein on electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulfate. It has an absorption coefficient of 1.4 mL/(mg.cm) and is similar in amino acid composition to other plasma gelsolins. Horse plasma gelsolin has an intrinsic sedimentation coefficient of 4.8S and a Stokes' radius of 3.8 nm. Hydrodynamic calculations suggest it to be a rather globular protein of 75,000 relative mass, a value similar to those calculated for human and pig plasma gelsolins from their amino acid sequences. Horse plasma gelsolin is able to nucleate actin polymerization, i.e., to abolish the lag observed between the initiation of polymerization of monomeric actin by the addition of salts and the rapid elongation phase of actin filament growth. This nucleation activity also results in lower final viscosities of F-actin solutions, as the existence of a larger number of filaments in samples that contain gelsolin requires that their average length be shorter.     

Binding of pig plasma gelsolin to F-actin and partial fractionation into calcium-dependent and calcium-independent forms

The interaction of pig plasma gelsolin with F-actin has been studied by a sedimentation assay using 125I-gelsolin in a Beckman Airfuge. Over 90% of the gelsolin bound to F-actin in 0.1 mM CaCl2 in experiments using 24 microM actin and 2-10 nM 125I-gelsolin, but only 40-50% bound in 1 mM EGTA. Addition of more F-actin to the EGTA supernatant does not sediment this gelsolin. Demonstration of this partial calcium sensitivity depends critically on the use of F-actin that has been prepared in the absence of calcium ions. F-actin prepared from G-actin in calcium or pretreated with calcium, binds 125I-gelsolin more completely in EGTA. This suggests that gelsolin activity is influenced by transient exposure of actin to calcium. Further evidence for partial calcium sensitivity in the interactions between gelsolin and F-actin has been obtained by other methods, including viscometry and electron microscopy. The gelsolin present in the EGTA supernatant is complexed to G-actin, predominantly as binary complexes. Very low concentrations of these complexes reduce the viscosity of F-actin in calcium but not in EGTA. Whether this effect is due to severing activity, or capping with consequent depolymerization to establish the new critical concentration, is uncertain. The results suggest the presence of two types of gelsolin, one that requires micromolar concentrations of calcium for binding to F-actin and one that does not. Both bind to G-actin. Partial separation has been achieved using actin-Sepharose. Pig plasma gelsolin is heterogeneous on isoelectric focussing gels in urea, but the two types of gelsolin separated on actin-Sepharose do not correspond to specific isoelectric species.     

Gelsolin affects the migratory ability of human colon adenocarcinoma and melanoma cells

AimsFormation of different protrusive structures by migrating cells is driven by actin polymerization at the plasma membrane region. Gelsolin is an actin binding protein controlling the length of actin filaments by its severing and capping activity. The main goal of this study was to determine the effect of gelsolin expression on the migration of human colon adenocarcinoma LS180 and melanoma A375 cells.Main methodsColon adenocarcinoma cell line LS180 was stably transfected with plasmid containing human cytoplasmic gelsolin cDNA tagged to enhanced green fluorescence protein (EGFP). Melanoma A375 cells were transfected with siRNAs directed against gelsolin. Real-time PCR and Western blotting were used to determine the level of gelsolin. The ability of actin to inhibit DNase I activity was used to quantify monomeric and total actin level and calculate the state of actin polymerization. Fluorescence confocal microscopy was applied to observe gelsolin and vinculin distribution along with actin cytoskeleton organization.Key findingsIncreased level of gelsolin expression leads to its accumulation at the submembranous region of the cell accompanied by distinct changes in the state of actin polymerization and an increase in the migration of LS180 cells. In addition, LS180 cells overexpressing gelsolin form podosome-like structures as indicated by vinculin redistribution and its colocalization with gelsolin and actin. Downregulation of gelsolin expression in melanoma A375 cells significantly reduces their migratory potential.SignificanceOur experimental data indicate that alterations in the expression level of gelsolin and its subcellular distribution may be directly responsible for determining migration capacity of human cancer cells.     

Human plasma gelsolin binds to fibronectin

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