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.     

Domain structure in actin-binding proteins: expression and functional characterization of truncated severin

Severin from Dictyostelium discoideum is a Ca2(+)-activated actin-binding protein that severs actin filaments, nucleates actin assembly, and caps the fast growing ends of actin filaments. Sequence comparison with functionally related proteins, such as gelsolin, villin, or fragmin revealed highly conserved domains which are thought to be of functional significance. To attribute the different activities of the severin molecule to defined regions, progressively truncated severin polypeptides were constructed. The complete cDNA coding for 362 (DS362) amino acids and five 3' deletions coding for 277 (DS277), 177 (DS177), 151 (DS151), 117 (DS117), or 111 (DS111) amino acids were expressed in Escherichia coli. The proteins were purified to homogeneity and then characterized with respect to their effects on the polymerization or depolymerization kinetics of G- or F-actin solutions and their binding to G-actin. Furthermore, the Ca2+ binding of these proteins was investigated with a 45Ca-overlay assay and by monitoring Ca2(+)-dependent changes in tryptophan fluorescence. Bacterially expressed DS362 showed the same Ca2(+)-dependent activities as native severin. DS277, missing the 85 COOH-terminal amino acids of severin, had lost its strict Ca2+ regulation and displayed a Ca2(+)-independent capping activity, but was still Ca2+ dependent in its severing and nucleating activities. DS151 which corresponded to the first domain of gelsolin or villin had completely lost severing and nucleating properties. However, a residual severing activity of approximately 2% was detectable if 26 amino acids more were present at the COOH-terminal end (DS177). This locates similar to gelsolin the second actin-binding site to the border region between the first and second domain. Measuring the fluorescence enhancement of pyrene-labeled G-actin in the presence of DS111 showed that the first actin-binding site was present in the NH2-terminal 111 amino acids. Extension by six or more amino acids stabilized this actin-binding site in such a way that DS117 and even more pronounced DS151 became Ca2(+)-independent capping proteins. In comparison to many reports on gelsolin we draw the following conclusions. Among the three active actin-binding sites in gelsolin the closely neighboured sites one and two share the F-actin fragmenting function, whereas the actin-binding sites two and three, which are located in far distant domains, collaborate for nucleation. In contrast, severin contains two active actin-binding sites which are next to each other and are responsible for the severing as well as the nucleating function. The single actin-binding site near the NH2-terminus is sufficient for capping of actin filaments.     

Purification and expression of gCap39. An intracellular and secreted Ca2(+)-dependent actin-binding protein enriched in mononuclear phagocytes

A protein of approximately 40 kDa was the major Ca2(+)-binding protein purified by Ca2(+)-dependent hydrophobic affinity chromatography from the cell lysates and conditioned media of RAW macrophages. Other Ca2(+)-binding proteins, including several annexins (calelectrins), S100-like proteins, and calmodulin, were less abundant and preferentially found in the cell lysates. Amino acid sequences of tryptic fragments from the purified 40-kDa protein revealed its identity to gCap39, an actin-binding protein encoded by a cDNA isolated on the basis of its homology with gelsolin. When an expression vector containing the gCap39 coding region was transfected into COS cells, high levels of gCap39 were found in both the cells and conditioned media, whereas annexins were only present in the cells. gCap39 could also be purified from human plasma where it appeared to be a minor component. No signal sequence was detected in the primary structure of gCap39 and the secreted and intracellular forms of gCap39 are of identical size, suggesting that unlike gelsolin, the mechanism of gCap39 secretion may not depend on a signal sequence. The high concentration of gCap39 in macrophages and its constitutive secretion as well as intracellular retention suggest that this protein may have a dual role in macrophage function, namely that of a Ca2(+)- and polyphosphoinositide-regulated intracellular modulator of the cytoskeleton as well as that of a secreted protein involved in the clearance of actin from the extracellular environment.     

Identification and characterization of a Ca2+-dependent actin filament-severing protein from lily pollen

It is well known that a tip-focused intracellular Ca2+ gradient and the meshwork of short actin filaments at the tip region are necessary for pollen tube growth. However, little is known about the connections between the two factors. Here, a novel Ca2+-dependent actin-binding protein with molecular mass of 41 kD from lily (Lilium davidii) pollen (LdABP41) was isolated and purified with DNase I chromatography. Our purification procedure yielded about 0.6 mg of LdABP41 with >98% purity from 10 g of lily pollen. At least two isoforms with isoelectric points of 5.8 and 6.0 were detected on two-dimensional gels. The results of N-terminal sequencing and mass-spectrometry analysis of LdABP41 showed that both isoforms shared substantial similarity with trumpet lily (Lilium longiflorum) villin and other members of the gelsolin superfamily. Negative-stained electron microscope images showed that LdABP41 severed in vitro-polymerized lily pollen F-actin into short actin filaments in a Ca2+-sensitive manner. Microinjection of the anti-LdABP41 antibody into germinated lily pollen demonstrated that the protein was required for pollen tube growth. The results of immunolocalization of the protein showed that it existed in the cytoplasm of the pollen tube, especially focused in the tip region. Our results suggest that LdABP41 belongs to the gelsolin superfamily and may play an important role in controlling actin organization in the pollen tube tip by responding to the oscillatory, tip-focused Ca2+ gradient.     

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.     

Ca2+ control of actin gelation. Interaction of gelsolin with actin filaments and regulation of actin gelation

We elucidated the mechanism by which gelsolin, a Ca2+-dependent regulatory protein from lung macrophages, controls the network structure of actin filaments. In the presence of micromolar Ca2+, gelsolin bound Ca2+. The Ca2+-gelsolin complex reduced the apparent viscosity and flow birefringence of F-actin and the lengths of actin filaments viewed in the electron microscope. However, concentrations of gelsolin causing these alterations did not effect proportionate changes in the turbidity of actin filament solutions or in the quantity of nonsedimentable actin as determined by a radioassay. From these findings, we conclude that gelsolin shortens actin filaments without net depolymerization. Such an effect on the distribution of actin filament lengths led to the prediction that low concentrations of gelsolin would increase the critical concentration of actin-binding protein required for incipient gelation of actin filaments in the presence of Ca2+, providing an efficient mechanism for controlling actin network structure. We verified the prediction experimentally, and we estimated that the Ca2+-gelsolin complex effectively breaks the bond between actin monomers in filaments with a stoichiometry of 1:1. The effect of Ca2+-gelsolin complex on actin solation was rapid, independent of temperature between 0 degrees and 37 degrees C, and reversed by reducing the free Ca2+ concentration.     

Comparison between the gelsolin and adseverin domain structure.

Adseverin (74-kDa protein, scinderin) is a calcium- and phospholipid-modulated actin-binding protein that promotes actin polymerization, severs actin filaments, and caps the barbed end of the actin filament, with its NH2-terminal half retaining these properties (Sakurai, T., Kurokawa, H., and Nonomura, Y. (1991) J. Biol. Chem. 266, 4581-4585). Further proteolysis of this NH2-terminal half generated five fragments, and two of them (Mr 15,000 and 31,000) showed Ca(2+)-dependent binding to monomeric actin. The Mr 31,000 fragment especially caused actin filament fragmentation, although its severing activity was also inhibited by several acidic phospholipids as was found in adseverin and its NH2-terminal half. Amino acid sequencing demonstrated that the two fragments' NH2 terminus were blocked in the same manner as the NH2 terminus of adseverin, and thus these two fragments are possibly located at the NH2-terminal of the adseverin molecule. This would then indicate that NH2-terminal fragments had a Ca(2+)-sensitive actin-binding function that relates to actin severing. The other two fragments' NH2-terminal sequencing showed a similar homology to the amino acid sequences of gelsolin and villin. Based on these observations, we propose that adseverin has a functional domain structure similar to that of the gelsolin and villin core.     

Gelsolin is expressed in early erythroid progenitor cells and negatively regulated during erythropoiesis

We have identified an approximately 85-kD protein in chicken erythrocytes which is immunologically, structurally, and functionally related to the gelsolin found in many muscle and nonmuscle cell types. Cell fractionation reveals a Ca2+-dependent partitioning of gelsolin into the soluble cytoplasm and the membrane-associated cytoskeleton of differentiating or mature erythrocytes. Depending on either the presence of Ca2+ during cell lysis or on the preincubation of the intact cells with the Ca2+-ionophore A23187, up to 40% of the total cellular gelsolin is found associated with the membrane skeleton. Expression of gelsolin shows a strong negative regulation during erythroid differentiation. From quantitations of its steady-state molar ratio to actin, gelsolin is abundant in early progenitor cells as revealed from avian erythroblastosis virus- and S13 virus-transformed cells which are arrested at the colony forming unit erythroid (CFU-e) stage of erythroid development. In these cells, which have a rudimentary and unstable membrane skeleton, gelsolin remains quantitatively cytoplasmic, irrespective of the Ca2+ concentration. During chicken embryo development and maturation, the expression of gelsolin decreases by a factor of approximately 10(3) in erythroid cells. This down regulation is independent from that of actin, which is considerably less, and is observed also when S13-transformed erythroid progenitor cells are induced to differentiate under conditions where the actin content of these cells does not change. In mature erythrocytes of the adult the amount of gelsolin is low, and significantly less than required for potentially capping of all membrane-associated actin filaments. We suggest that the gelsolin in erythroid cells is involved in the assembly of the actin filaments present in the membrane skeleton, and that it may provide for a mechanism, by means of its severing action on actin filaments, to extend the meshwork of the spectrin-actin-based membrane skeleton in erythroid cells during erythropoiesis.     

Molecular cloning of human macrophage capping protein cDNA. A unique member of the gelsolin/villin family expressed primarily in macrophages.

Macrophage capping protein (MCP) is a Ca(2+)-sensitive protein which reversibly blocks the barbed ends of actin filaments but does not sever preformed actin filaments. The human cDNA for MCP has been cloned and sequenced. The derived amino acid sequence predicts a polypeptide of 38.4 kDa. Human MCP expressed in Escherichia coli using a pET12a vector was functionally identical to the native protein purified from rabbit alveolar macrophages with respect to Ca2+ sensitivity and ability to block monomer exchange at the barbed end of actin filaments. Sequence comparison with other actin-binding protein sequences indicates that MCP is a member of the gelsolin/villin family of barbed end blocking proteins. Unlike gelsolin, this protein has a limited tissue distribution being detected primarily in macrophages where it was abundant, representing 0.9-1% of the total cytoplasmic protein. Northern blot analysis of U937 and HL60 cells differentiated to macrophage-like cells demonstrated that MCP message increases to 2.6 and greater than 7 times initial levels, respectively. Human MCP displays a 93% amino acid sequence identity with two recently described mouse proteins, gCap39 and Mbh1. Its abundance in macrophages and the corresponding increases in mRNA levels upon promyelocyte and monocyte development into macrophages indicate that MCP may play an important role in macrophage function.     

Chromaffin cell scinderin, a novel calcium-dependent actin filament-severing protein.

Scinderin, a novel Ca2+-activated actin filament-severing protein, has been purified to homogeneity from bovine adrenal medulla using a combination of several chromatographic procedures. The protein has an apparent mol. wt of 79,600 +/- 450 daltons, three isoforms (pIs 6.0, 6.1 and 6.2) and two Ca2+ binding sites (Kd 5.85 x 10(-7) M, Bmax 0.81 mol Ca2+/mol protein and Kd 2.85 x 10(-6) M, Bmax 1.87 mol Ca2+/mol protein). Scinderin interacts with F-actin in the presence of Ca2+ and produces a decrease in the viscosity of actin gels as a result of F-actin filament severing as demonstrated by electron microscopy. Scinderin is a structurally different protein from chromaffin cell gelsolin, another actin filament-severing protein described. Scinderin and gelsolin have different mol. wts, isoelectric points, amino acid composition and yield different peptide maps after limited proteolytic digestion by either Staphylococcus V8 protease or chymotrypsin. Moreover, scinderin antibodies do not cross-react with gelsolin and gelsolin antibodies fail to recognize scinderin. Immunofluorescence with anti-scinderin demonstrated that this protein is mainly localized in the subplasmalemma region of the chromaffin cell. Immunoblotting tests with the same antibodies indicated that scinderin is also expressed in brain and anterior as well as posterior pituitary. Presence of scinderin and gelsolin, two Ca2+-dependent actin filament-severing proteins in the same tissue, suggests the possibility of synergistic functions by the two proteins in the control of cellular actin filament networks. Alternatively, the actin filament-severing activity of the two proteins might be under the control of different transduction and modulating influences.     

Identification and purification of calcium ion dependent modulators of actin polymerization from bovine thyroid

We describe the purification of Ca2+-dependent actin modulator proteins from bovine thyroid using DNase I affinity chromatography and diethylaminoethylcellulose chromatography. The 40K actin modulator has been purified to 98% homogeneity. It is a single polypeptide chain with a molecular weight of approximately 40 000 and an isoelectric point of 8.1. Its amino acid composition is different from previously described actin-associated proteins and thyroid actin. On the basis of the centrifugation assay and the DNase I inhibition assay, the actin complexed with the 40K protein is G-actin in its conformation rather than F-actin oligomers. Substoichiometric concentrations of the 40K protein rapidly inhibit actin polymerization in the presence of physiological concentrations of Ca2+ and Mg2+. An 80K actin modulator also has been purified to 98% homogeneity. It is a single polypeptide chain with a molecular weight of approximately 80 000 and an isoelectric point of 6.35-7.0. Its amino acid composition is different from those of villin, gelsolin, and leukocyte actin polymerization inhibitor. On the basis of the DNase inhibition assay and the centrifugation assay, the nonprecipitable actin associated with the 80K protein was F-actin in its conformation. The 80K protein acts very efficiently as a Ca2+-dependent nucleator for actin assembly and reduces its viscosity. In addition to the 40K and 80K actin modulators, 91K and 95K actin-associated proteins were partially purified. The 91K-95K fraction has similar activity to the 80K protein regarding precipitation of F-actin. The 125I-G-actin polyacrylamide gel overlay technique [Snabes, M. C., Boyd, A.E., & Bryan, J. (1981) J. Cell Biol. 90, 809-812] revealed that both the 91K and 95K proteins bind 125I-actin after sodium dodecyl sulfate (NaDodSO4) electrophoresis while the 80K and 40K proteins do not. Thyroid 91K protein comigrated with a human platelet 91K actin binding protein on NaDodSO4 gels and may be similar to macrophage gelsolin. The 95K protein may be similar to villin, the intestinal cytoskeletal protein.     

Functional comparison of villin and gelsolin. Effects of Ca2+, KCl, and polyphosphoinositides

A family of homologous actin-binding proteins sever and cap actin filaments and accelerate actin filament assembly. The functions of two of these proteins, villin and gelsolin, and of their proteolytically derived actin binding domains were compared directly by measuring their effects, under various ionic conditions, on the rates and extents of polymerization of pyrene-labeled actin. In 1 mM Ca2+ and 150 mM KCl, villin and gelsolin have similar severing and polymerization-accelerating properties. Decreasing [Ca2+] to 25 microM greatly reduces severing by villin but not gelsolin. Decreasing [KCl] from 150 to 10 mM at 25 microM Ca2+ increases severing by villin, but not gelsolin, over 10-fold. The C-terminal half domains of both proteins have Ca2+-sensitive actin monomer-binding properties, but neither severs filaments nor accelerates polymerization. The N-terminal halves of villin and gelsolin contain all the filament-severing activity of the intact proteins. Severing by gelsolin's N-terminal half is Ca2+-independent, but that of villin has the same Ca2+ requirement as intact villin. The difference in Ca2+ sensitivity extends to 14-kDa N-terminal fragments which bind actin monomers and filament ends, requiring Ca2+ in the case of villin but not gelsolin. Severing of filaments by villin and its N-terminal half is shown to be inhibited by phosphatidylinositol 4,5-bisphosphate, as shown previously for gelsolin (Janmey, P.A., and Stossel, T.P. (1987) Nature 325, 362-364). The functional similarities of villin and gelsolin correlate with known structural features, and the greater functional dependence of villin on Ca2+ compared to gelsolin is traced to differences in their N-terminal domains.     

Distribution of gelsolin in human testis

Gelsolin, an actin-binding and severing protein present in many mammalian cells, was characterized in human testis. Although abundant in testicular extracts, gelsolin was not detected in purified spermatogenic cells by immunoblot analysis. Immunofluorescence studies of testis sections showed that gelsolin has two main localizations: peritubular cells and the seminiferous epithelium. In peritubular cells, gelsolin was present together with α-SM actin, in agreement with the myoid cell characteristics of these cells. In a large proportion of the tubules, gelsolin was found mainly, together with actin, in the apical part of the seminiferous epithelium. This localization of gelsolin also was observed in seminiferous tubules with a partial or complete absence of germinal cells, which evokes a presence of gelsolin at the apex of Sertoli cells. However, in normal testis, a complex pattern of gelsolin labeling was also present, mostly in the apical third of the epithelium, around cells or groups of cells, mainly spermatids, and, less frequently, in various other localizations from the apical to the basal part of the seminiferous epithelium. Taken together, these observations suggest that gelsolin may play different functions in the seminiferous epithelium: (1) regulation of the dynamic alterations of the actin cytoskeleton in the apical cytoplasm of Sertoli cells, and (2) modification of actin filaments assemblies in specific structures at germ cell-Sertoli cell contacts. Thereby, the actin-modulating properties of gelsolin are probably involved in reorganization of the seminiferous epithelium related to germ cell differentiation. Mol. Reprod. Dev. 48:63–70, 1997. © 1997 Wiley-Liss, Inc.     

Role of the N- and C-terminal actin-binding domains of gelsolin in barbed filament end capping.

Gelsolin is a bivalent Ca(2+)-modulated actin-binding protein that severs, nucleates, and caps filaments. In order to gain a better understanding of the capping mechanism we have studied N- and C-terminal gelsolin fragments, 14NT and 41CT, each of which contains a single functional actin-binding site. The very tight binding measured between gelsolin and the barbed filament end requires gelsolin to greatly decrease the dissociation rate constant of the terminal actin from this end. A mechanism that could account for the observed decrease in dissociation is one in which gelsolin links two actin monomers so that they dissociate more slowly as a dimer. This cannot be the only mechanism, however, since, as shown here, 14NT and 41CT, fragments with single actin-binding sites, decrease the dissociation rate of the capped terminal actin molecule. The observations suggest that these fragments induce a conformational change in the actin monomer that either increases the affinity or alters the kinetics of the terminal actin-actin bond. The available data argue for strengthening of the terminal actin-actin bond.     

Effect of staurosporine on the induction of actin/gelsolin in PMA-treated HL-60 cells

Staurosporine, an inhibitor of protein kinase C (PKC), has been shown to inhibit the induction of two cytosolic proteins, p42 and p91, during the differentiation of HL-60 cells initiated by PMA treatment. Differentiation of these cells into macrophages was also blocked. p42 has been identified as actin by Western Blot analysis and previous studies have demonstrated p91 to be the Ca+(+)-dependent actin-binding protein gelsolin. Increased levels of these proteins may be important for the attainment of macrophage functions, e.g. phagocytosis and secretion. These studies may therefore establish a link between PKC activation and the induction of specific cellular proteins, such as actin and gelsolin, as important monocyte maturation products without which the cells will be restricted from acquiring certain macrophage activities.     

Identification of actin-binding protein as the protein linking the membrane skeleton to glycoproteins on platelet plasma membranes

Platelets have previously been shown to contain a membrane skeleton that is composed of actin filaments, actin-binding protein, and three membrane glycoproteins (GP), GP Ib, GP Ia, and a minor glycoprotein of Mr = 250,000. The present study was designed to determine how the membrane glycoproteins were linked to actin filaments. Unstimulated platelets were lysed with Triton X-100, and the membrane skeleton was isolated on sucrose density gradients or by high-speed centrifugation. The association of the membrane glycoproteins with the actin filaments was disrupted when actin-binding protein was hydrolyzed by activity of the Ca2+-dependent protease, which was active in platelet lysates upon addition of Ca2+ in the absence of leupeptin. Similarly, activation of the Ca2+-dependent protease in intact platelets by the addition of a platelet agonist also caused the membrane glycoproteins to dissociate from the membrane skeleton. Affinity-purified actin-binding protein antibodies immunoprecipitated the membrane glycoproteins from platelet lysates in which actin filaments had been removed by DNase I-induced depolymerization and high-speed centrifugation. These results demonstrate that actin-binding protein links actin filaments of the platelet membrane skeleton to three plasma membrane glycoproteins and that filaments are released from their attachment site when actin-binding protein is hydrolyzed by the Ca2+-dependent protease within intact platelets during platelet activation.     

Smooth muscle gelsolin and a Ca2+-sensitive contractile cell model

Smooth muscle gelsolin, termed smooth muscle 90-kDa protein in our previous paper (Kanno et al. FEBS Lett. 1985; 184:202-206), was purified from bovine aorta. Antibody prepared against smooth muscle gelsolin was used to detect the presence of gelsolin in human lung fibroblast MRC-5 cells permeabilized with Triton X-100 (MRC-5 cell models). These cells contracted in the presence of MgATP and Ca2+ in doses over 1 microM. Immunofluorescence microscopy using phalloidin and antigelsolin antibody showed that gelsolin was distributed along the stress fibers, except for a marginal bundle of cells, when MRC-5 cells were growth-arrested in serum-depleted medium. Making use of immunoblotting and indirect immunofluorescence techniques, we demonstrated that gelsolin is not retained in the MRC-5 cell models. We used purified smooth muscle gelsolin as a specific agent to sever the actin filaments. Preincubation of MRC-5 cell models with gelsolin led to a destruction of stress fibers, in a dose- and Ca2+ -dependent manner. The contractility was also lost, in the same manner described above, thereby indicating that a continuous distribution of actin filaments within the stress fibers is required for cell contraction. Treatment of MRC-5 cells with the Ca2+ ionophore A23187 induced an extracellular Ca2+ -dependent contraction but not a massive destruction of stress fibers, thereby indicating that most of the endogenous gelsolin was inactive under these conditions. Our interpretation of these results is that increases in cytoplasmic Ca2+ concentrations are sufficient for the contraction but may be too transient to activate endogenous gelsolin and thereby disrupt the stress fibers. Indeed, the inhibition of contraction of the MRC-5 cell, as induced by smooth muscle gelsolin, required preincubation in the presence of Ca2+, before the addition of MgATP. These results suggest that destruction of the stress fibers by endogenous gelsolin, which leads to inhibition of cell contraction, may occur if the cytoplasmic Ca2+ is maintained at high concentrations for a few minutes.     

Gelsolin and non-muscle myosin IIA interact to mediate calcium-regulated collagen phagocytosis

The formation of adhesion complexes is the rate-limiting step for collagen phagocytosis by fibroblasts, but the role of Ca(2+) and the potential interactions of actin-binding proteins in regulating collagen phagocytosis are not well defined. We found that the binding of collagen beads to fibroblasts was temporally and spatially associated with actin assembly at nascent phagosomes, which was absent in gelsolin null cells. Analysis of tryptic digests isolated from gelsolin immunoprecipitates indicated that non-muscle (NM) myosin IIA may bind to gelsolin. Immunostaining and immunoprecipitation showed that gelsolin and NM myosin IIA associated at collagen adhesion sites. Gelsolin and NM myosin IIA were both required for collagen binding and internalization. Collagen binding to cells initiated a prolonged increase of [Ca(2+)](i), which was absent in cells null for gelsolin or NM myosin IIA. Collagen bead-induced increases of [Ca(2+)](i) were associated with phosphorylation of the myosin light chain, which was dependent on gelsolin. NM myosin IIA filament assembly, which was dependent on myosin light chain phosphorylation and increased [Ca(2+)](i), also required gelsolin. Ionomycin-induced increases of [Ca(2+)](i) overcame the block of myosin filament assembly in gelsolin null cells. We conclude that gelsolin and NM myosin IIA interact at collagen adhesion sites to enable NM myosin IIA filament assembly and localized, Ca(2+)-dependent remodeling of actin at the nascent phagosome and that these steps are required for collagen phagocytosis.     

Identification of a polyphosphoinositide-modulated domain in gelsolin which binds to the sides of actin filaments

Gelsolin is a Ca2+- and polyphosphoinositide-modulated actin-binding protein which severs actin filaments, nucleates actin assembly, and caps the "barbed" end of actin filaments. Proteolytic cleavage analysis of human plasma gelsolin has shown that the NH2-terminal half of the molecule severs actin filaments almost as effectively as native gelsolin in a Ca2+-insensitive but polyphosphoinositide-inhibited manner. Further proteolysis of the NH2-terminal half generates two unique fragments (CT14N and CT28N), which have minimal severing activity. Under physiological salt conditions, CT14N binds monomeric actin coupled to Sepharose but CT28N does not. In this paper, we show that CT28N binds stoichiometrically and with high affinity to actin subunits in filaments, suggesting that it preferentially recognizes the conformation of polymerized actin. Analysis of the binding data shows that actin filaments have one class of CT28N binding sites with Kd = 2.0 X 10(-7) M, which saturates at a CT28N/actin subunit ratio of 0.8. Binding of CT28N to actin filaments is inhibited by phosphatidylinositol 4,5-bisphosphate micelles. In contrast, neither CT14N nor another actin-binding domain located in the COOH-terminal half of gelsolin form stable stoichiometric complexes with actin along the filaments, and their binding to actin monomers is not inhibited by PIP2. Based on these observations, we propose that CT28N is the polyphosphoinositide-regulated actin-binding domain which allows gelsolin to bind to actin subunits within a filament before serving.