Nucleate Actin Assembly Research Articles

Gelsolin superfamily proteins: key regulators of cellular functions

Cytoskeletal rearrangement occurs in a variety of cellular processes and involves a wide spectrum of proteins. Among these, the gelsolin superfamily proteins control actin organization by severing filaments, capping filament ends and nucleating actin assembly [1]. Gelsolin is the founding member of this family, which now contains at least another six members: villin, adseverin, capG, advillin, supervillin and flightless I. In addition to their respective role in actin filament remodeling, these proteins have some specific and apparently non-overlapping particular roles in several cellular processes, including cell motility, control of apoptosis and regulation of phagocytosis (summarized in table 1). Evidence suggests that proteins belonging to the gelsolin superfamily may be involved in other processes, including gene expression regulation. This review will focus on some of the known functions of the gelsolin superfamily proteins, thus providing a basis for reflection on other possible and as yet incompletely understood roles for these proteins.

Actin binding proteins: regulation of cytoskeletal microfilaments.

The actin cytoskeleton is a complex structure that performs a wide range of cellular functions. In 2001, significant advances were made to our understanding of the structure and function of actin monomers. Many of these are likely to help us understand and distinguish between the structural models of actin microfilaments. In particular, 1) the structure of actin was resolved from crystals in the absence of cocrystallized actin binding proteins (ABPs), 2) the prokaryotic ancestral gene of actin was crystallized and its function as a bacterial cytoskeleton was revealed, and 3) the structure of the Arp2/3 complex was described for the first time. In this review we selected several ABPs (ADF/cofilin, profilin, gelsolin, thymosin beta4, DNase I, CapZ, tropomodulin, and Arp2/3) that regulate actin-driven assembly, i.e., movement that is independent of motor proteins. They were chosen because 1) they represent a family of related proteins, 2) they are widely distributed in nature, 3) an atomic structure (or at least a plausible model) is available for each of them, and 4) each is expressed in significant quantities in cells. These ABPs perform the following cellular functions: 1) they maintain the population of unassembled but assembly-ready actin monomers (profilin), 2) they regulate the state of polymerization of filaments (ADF/cofilin, profilin), 3) they bind to and block the growing ends of actin filaments (gelsolin), 4) they nucleate actin assembly (gelsolin, Arp2/3, cofilin), 5) they sever actin filaments (gelsolin, ADF/cofilin), 6) they bind to the sides of actin filaments (gelsolin, Arp2/3), and 7) they cross-link actin filaments (Arp2/3). Some of these ABPs are essential, whereas others may form regulatory ternary complexes. Some play crucial roles in human disorders, and for all of them, there are good reasons why investigations into their structures and functions should continue.

Structure of an F-actin Trimer Disrupted by Gelsolin and Implications for the Mechanism of Severing

Stable oligomers of filamentous actin were obtained by cross-linking F-actin with 1,4-N,N'-phenylenedimaleimide and depolymerization with excess segment-1 of gelsolin. Segment-1-bound and cross-linked actin oligomers containing either two or three actin subunits were purified and shown to nucleate actin assembly. Kinetic assembly data from mixtures of monomeric actin and the actin oligomers fit a nucleation model where cross-linked actin dimer or trimer reacts with an actin monomer to produce a competent nucleus for filament assembly. We report the three-dimensional structure of the segment-1-actin hexamer containing three actin subunits, each with a tightly bound ATP. Comparative analysis of this structure with twelve other actin structures provides an atomic level explanation for the preferential binding of ATP by the segment-1-complexed actin. Although the structure of segment-1-bound actin trimer is topologically similar to the helical model of F-actin (1), it has a distorted symmetry compared with that of the helical model. This distortion results from intercalation of segment-1 between actin protomers that increase the rise per subunit and rotate each of the actin subunits relative to their positions in F-actin. We also show that segment-1 of gelsolin is able to sever actin filaments, although the severing activity of segment-1 is significantly lower than full-length gelsolin.

Actin-dependent propulsion of endosomes and lysosomes by recruitment of N-WASP.

We examined the spatial and temporal control of actin assembly in living Xenopus eggs. Within minutes of egg activation, dynamic actin-rich comet tails appeared on a subset of cytoplasmic vesicles that were enriched in protein kinase C (PKC), causing the vesicles to move through the cytoplasm. Actin comet tail formation in vivo was stimulated by the PKC activator phorbol myristate acetate (PMA), and this process could be reconstituted in a cell-free system. We used this system to define the characteristics that distinguish vesicles associated with actin comet tails from other vesicles in the extract. We found that the protein, N-WASP, was recruited to the surface of every vesicle associated with an actin comet tail, suggesting that vesicle movement results from actin assembly nucleated by the Arp2/3 complex, the immediate downstream target of N-WASP. The motile vesicles accumulated the dye acridine orange, a marker for endosomes and lysosomes. Furthermore, vesicles associated with actin comet tails had the morphological features of multivesicular endosomes as revealed by electron microscopy. Endosomes and lysosomes from mammalian cells preferentially nucleated actin assembly and moved in the Xenopus egg extract system. These results define endosomes and lysosomes as recruitment sites for the actin nucleation machinery and demonstrate that actin assembly contributes to organelle movement. Conversely, by nucleating actin assembly, intracellular membranes may contribute to the dynamic organization of the actin cytoskeleton.

Localization by Segmental Deletion Analysis and Functional Characterization of a Third Actin-binding Site in Domain 5 of Scinderin

Scinderin is a Ca2+-dependent actin filament severing protein present in a variety of secretory cells. Previous work suggests that scinderin-evoked cortical F-actin disassembly is required for secretion because local disassembly of cortical cytoskeleton allows secretory vesicle exocytosis (Vitale, M. L., Rodríguez Del Castillo, A., Tchakarov, L., and Trifaró, J.-M. (1991) J. Cell Biol. 113, 1057-1067). Scinderin has six domains each containing three internal sequence motifs, two actin, and two phosphatidylinositol disphosphate-binding sites in domains 1 and 2. In this paper we report the presence of another actin-binding site at the NH2-terminal of domain 5 (Sc511-518). This site binds actin in a Ca2+-independent manner and a recombinant fragment (Sc5-6 or Sc502-715) containing this site binds to actin-DNase-I-Sepharose 4B beads, co-sediments with actin and is able to nucleate actin assembly. Recombinant ScL5-6, a fusion protein devoid of the actin-binding site (Sc519-715), did not exhibit these properties. Moreover, Sc-ABP3, a peptide constructed with sequence (RLFQVRRNLASIT) identical to Sc511-523 blocked the binding of Sc5-6 to actin. Sc5-6 and Sc-ABP3 also prevented the actin severing activity of recombinant full-length scinderin (r-Sc) and inhibited the potentiation by r-Sc of Ca2+-evoked release of serotonin from permeabilized platelets. On the other hand, ScL5-6 failed to block the effect of r-Sc on platelet serotonin release. Sc1-4,6, a construct devoid of domain 5, was able to sever but unable to nucleate actin, indicating that an actin nucleation site of scinderin was in domain 5. The results suggest that scinderin, in addition to binding actin on sites present in domains 1 and 2, must bind actin on a third site in domain 5 to sever and nucleate actin effectively.

Purification and properties of a Ca(2+)-independent barbed-end actin filament capping protein, CapZ, from human polymorphonuclear leukocytes.

In human polymorphonuclear leukocytes (PMN), changes in the actin architecture are critical for the shape changes required for chemotaxis and phagocytosis. Barbed-end capping proteins are likely to regulate actin assembly in PMN. The previously identified barbed-end blocking proteins in PMN, gelsolin and CapG, require Ca(2+) to initiate capping of actin filaments. Because chemoattractants can stimulate PMN actin assembly by a calcium-independent signal transduction pathway, we sought to purify a calcium-independent barbed-end capping activity from PMN cytoplasmic extracts. A Ca(2+) -insensitive actin polymerization inhibitory activity was partially purified from human PMN [Southwick & Stossel (1981) J. Biol. Chem 256, 3030]. Using five column chromatography steps, we purified the protein to homogeneity as assessed by silver staining. Purification was associated with an increase in specific activity of greater than 40 X. Western blot analysis identified the protein as the nonmuscle isoform of the heterodimeric capping protein capZ. Human PMN capZ has an apparent disassociation constant of 3 nM for capping in the presence or absence of micromolar Ca(2+), as assessed by both pyrenylactin elongation and depolymerization assays. Similar to the activity reported for the actin polymerization inhibitor, activity of PMN capZ was inhibited by increasing the KC1 concentration from 0.1 M to 0.6 M. The capping function was also inhibited by phosphatidylinositol 4,5-bisphosphate (PIP(2)) micelles, with half-maximal inhibition occurring at 5.5 micrograms mL(-1). PMN capZ did not nucleate actin assembly, sequester actin monomers, or sever actin filaments. Quantitative Western blot analysis revealed that capZ levels corresponded to 0.7-1.0% of the total human PMN cytoplasmic protein. Given its abundance and high affinity for barbed filament ends, capZ is likely to play an important role in the calcium-independent regulation of actin filament assembly associated with PMN chemotaxis.

Ponticulin is an atypical membrane protein.

We have cloned and sequenced ponticulin, a 17,000-dalton integral membrane glycoprotein that binds F-actin and nucleates actin assembly. A single copy gene encodes a developmentally regulated message that is high during growth and early development, but drops precipitously during cell streaming at approximately 8 h of development. The deduced amino acid sequence predicts a protein with a cleaved NH2-terminal signal sequence and a COOH-terminal glycosyl anchor. These predictions are supported by amino acid sequencing of mature ponticulin and metabolic labeling with glycosyl anchor components. Although no alpha-helical membrane-spanning domains are apparent, several hydrophobic and/or sided beta-strands, each long enough to traverse the membrane, are predicted. Although its location on the primary sequence is unclear, an intracellular domain is indicated by the existence of a discontinuous epitope that is accessible to antibody in plasma membranes and permeabilized cells, but not in intact cells. Such a cytoplasmically oriented domain also is required for the demonstrated role of ponticulin in binding actin to the plasma membrane in vivo and in vitro (Hitt, A. L., J. H. Hartwig, and E. J. Luna. 1994. Ponticulin is the major high affinity link between the plasma membrane and the cortical actin network in Dictyostelium. J. Cell Biol. 126:1433-1444). Thus, ponticulin apparently represents a new category of integral membrane proteins that consists of proteins with both a glycosyl anchor and membrane-spanning peptide domain(s).

Calcium regulation of actin filament capping and monomer binding by macrophage capping protein.

Macrophage capping protein (MCP) is a unique member of the gelsolin-villin family of calcium-activated barbed end capping proteins which in micromolar Ca2+ also binds actin monomers and nucleates actin assembly. Unlike gelsolin, MCP cannot sever actin filaments, and its Ca(2+)-dependent interaction with actin is completely reversible. The Ca2+ binding properties of MCP and its Ca(2+)-dependent functions were studied quantitatively. MCP undergoes a Ca(2+)-induced conformational change as evidenced by different chymotryptic digest patterns in 0.2 mM CaCl2 compared with 2 mM EGTA. MCP has a single low affinity Ca2+ binding site (KD = 37 microM). Binding of MCP to actin monomers requires a similar Ca2+ concentration ([Ca2+]0.5 = 62 microM) suggesting that MCP.Ca2+ complex formation promotes monomer binding. In contrast, filament capping by MCP requires 1/60th of the Ca2+ concentration required for monomer binding, half-maximal capping occurring at 1 microM Ca2+. The marked difference in the Ca2+ sensitivity of these two functions indicate that MCP's primary actin regulatory role in the macrophage is likely to be capping of the barbed ends of actin filaments.

The integral membrane protein, ponticulin, acts as a monomer in nucleating actin assembly.

Ponticulin, an F-actin binding transmembrane glycoprotein in Dictyostelium plasma membranes, was isolated by detergent extraction from cytoskeletons and purified to homogeneity. Ponticulin is an abundant membrane protein, averaging approximately 10(6) copies/cell, with an estimated surface density of approximately 300 per microns2. Ponticulin solubilized in octylglucoside exhibited hydrodynamic properties consistent with a ponticulin monomer in a spherical or slightly ellipsoidal detergent micelle with a total molecular mass of 56 +/- 6 kD. Purified ponticulin nucleated actin polymerization when reconstituted into Dictyostelium lipid vesicles, but not when a number of commercially available lipids and lipid mixtures were substituted for the endogenous lipid. The specific activity was consistent with that expected for a protein comprising 0.7 +/- 0.4%, by mass, of the plasma membrane protein. Ponticulin in octylglucoside micelles bound F-actin but did not nucleate actin assembly. Thus, ponticulin-mediated nucleation activity was sensitive to the lipid environment, a result frequently observed with transmembrane proteins. At most concentrations of Dictyostelium lipid, nucleation activity increased linearly with increasing amounts of ponticulin, suggesting that the nucleating species is a ponticulin monomer. Consistent with previous observations of lateral interactions between actin filaments and Dictyostelium plasma membranes, both ends of ponticulin-nucleated actin filaments appeared to be free for monomer assembly and disassembly. Our results indicate that ponticulin is a major membrane protein in Dictyostelium and that, in the proper lipid matrix, it is sufficient for lateral nucleation of actin assembly. To date, ponticulin is the only integral membrane protein known to directly nucleate actin polymerization.

Talin anchors and nucleates actin filaments at lipid membranes A direct demonstration

Platelet talin nucleates actin assembly as we show here directly by using rhodamine—phalloidin labelling of actin filaments, Nucleation by talin still occurs after reconstitution into liposomal bilayers. This is also demonstrated directly after protein–lipid double labelling and light microscopic imaging. Talin, thus, is the first actin binding protein for which anchoring and nucleation of artin filament growth at lipid interfaces have been visualized.

Characterization of actin- and lipid-binding domains in severin, a Ca(2+)-dependent F-actin fragmenting protein.

Severin is a Ca(2+)-activated actin-binding protein that nucleates actin assembly and severs and caps the fast growing ends of actin filaments. It consists of three highly conserved domains. To investigate the domain structure of severin, we constructed genetically the N-terminal domain 1, the middle domain 2, and the tandem domains 2 + 3. Their interaction with actin, Ca2+, and lipids was characterized. Domain 1 contains the F-actin capping and a Ca(2+)-binding site [Eichinger, L., Noegel, A. A., & Schleicher, M. (1991) J. Cell Biol. 112, 665-676]. Binding of domain 2 to actin filaments was Ca(2+)-dependent and saturated at a 1:1 molar ratio. In the presence of Ca2+, about 1.5 mol of domains 2 + 3 bound per mole of F-actin subunit. Scatchard analysis gave a Kd of 18 microM for the interaction of domain 2 with F-actin subunits and a Kd of 1.6 microM for domains 2 + 3. Low-shear viscometry, electron microscopy, and low-speed sedimentation assays showed that domains 2 + 3 induced bundling of actin filaments. The influence of PIP2 micelles on the different activities of severin was assayed using native severin and N- and C-terminally truncated fragments. Severin contains at least two PIP2-binding sites since the activities of the two nonoverlapping severin fragments domain 1 and domains 2 + 3 were inhibited by PIP2. The specificity of severin-phospholipid interaction was investigated by studying the regulation of native severin by PIP2 and other pure or mixed phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)

Cap100, a novel phosphatidylinositol 4,5-bisphosphate-regulated protein that caps actin filaments but does not nucleate actin assembly.

The fast and transient polymerization of actin in nonmuscle cells after stimulation with chemoattractants requires strong nucleation activities but also components that inhibit this process in resting cells. In this paper, we describe the purification and characterization of a new actin-binding protein from Dictyostelium discoideum that exhibited strong F-actin capping activity but did not nucleate actin assembly independently of the Ca2+ concentration. These properties led at physiological salt conditions to an inhibition of actin polymerization at a molar ratio of capping protein to actin below 1:1,000. The protein is a monomer, with a molecular mass of approximately 100 kDa, and is present in growing and in developing amoebae. Based on its F-actin capping function and its apparent molecular weight, we designated this monomeric protein cap100. As shown by dilution-induced depolymerization and by elongation assays, cap100 capped the barbed ends of actin filaments and did not sever F-actin. In agreement with its capping activity, cap100 increased the critical concentration for actin polymerization. In excitation or emission scans of pyrene-labeled G-actin, the fluorescence was increased in the presence of cap100. This suggests a G-actin binding activity for cap100. The capping activity could be completely inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2), and bound cap100 could be removed by PIP2. The inhibition by phosphatidylinositol and the Ca(2+)-independent down-regulation of spontaneous actin polymerization indicate that cap100 plays a role in balancing the G- and F-actin pools of a resting cell. In the cytoplasm, the equilibrium would be shifted towards G-actin, but, below the membrane where F-actin is required, this activity would be inhibited by PIP2.

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.

Actin filament nucleation by the bacterial pathogen, Listeria monocytogenes.

Shortly after Listeria is phagocytosed by a macrophage, it dissolves the phagosomal membrane and enters the cytoplasm. 1 h later, actin filaments coat the Listeria and then become rearranged to form a tail with which the Listeria moves to the macrophage surface as a prelude to spreading. If infected macrophages are treated with cytochalasin D, all the actin filaments associated with the Listeria break down leaving a fine, fibrillar material that does not decorate with subfragment 1 of myosin. This material is associated with either the surface of the Listeria (the cloud stage) or one end (the tail stage). If the cytochalasin-treated infected macrophages are detergent extracted and then incubated in nuclei-free monomeric actin under polymerizing conditions, actin filaments assemble from the fine, fibrillar material, the result being that each Listeria has actin filaments radiating from its surface like the spokes of a wheel (cloud form) or possesses a long tail of actin filaments formed from the fine, fibrillar material located at one end of the Listeria. Evidence that the fine fibrillar material is involved in nucleating actin assembly comes from a Listeria mutant. Although the mutant replicates at a normal rate in macrophages, actin filaments do not form on its surface (cloud stage) or from one end (tail stage), nor does the bacterium spread. Furthermore it does not form the fine fibrillar material. Evidence that the nucleating material is a secretory product of Listeria and not the macrophage comes from experiments using chloramphenicol, which inhibits protein synthesis in Listeria but not in macrophages. If chloramphenicol is applied 1 h after infection, a time before actin filaments are found attached to the Listeria in untreated macrophages, actin filaments never assemble on the Listeria even when fixed 3 h later. Furthermore the fine fibrillar material is absent, although there is a coat of dense granular material.

Dictyostelium discoideum plasma membranes contain an actin-nucleating activity that requires ponticulin, an integral membrane glycoprotein.

In previous equilibrium binding studies, Dictyostelium discoideum plasma membranes have been shown to bind actin and to recruit actin into filaments at the membrane surface. However, little is known about the kinetic pathway(s) through which actin assembles at these, or other, membranes. We have used actin fluorescently labeled with N-(1-pyrenyl)iodoacetamide to examine the kinetics of actin assembly in the presence of D. discoideum plasma membranes. We find that these membranes increase the rate of actin polymerization. The rate of membrane-mediated actin polymerization is linearly dependent on membrane protein concentrations up to 20 micrograms/ml. Nucleation (the association of activated actin monomers into oligomers) appears to be the primary step of polymerization that is accelerated. A sole effect on the initial salt-induced actin conformational change (activation) is ruled out because membranes accelerate the polymerization of pre-activated actin as well as actin activated in the presence of membranes. Elongation of preexisting filaments also is not the major step of polymerization facilitated by membranes since membranes stripped of all peripheral components, including actin, increase the rate of actin assembly to about the same extent as do membranes containing small amounts of endogenous actin. Acceleration of the nucleation step by membranes also is supported by an analysis of the dependence of polymerization lag time on actin concentration. The barbed ends of membrane-induced actin nuclei are not obstructed by the membranes because the barbed end blocking agent, cytochalasin D, reduces the rate of membrane-mediated actin nucleation. Similarly, the pointed ends of the nuclei are not blocked by membranes since the depolymerization rate of gelsolin-capped actin is unchanged in the presence of membranes. These results are consistent with previous observations of lateral interactions between membranes and actin filaments. These results also are consistent with two predictions from a model based on equilibrium binding studies; i.e., that plasma membranes should nucleate actin assembly and that membrane-bound actin nuclei should have both ends free (Schwartz, M. A., and E. J. Luna. 1988. J. Cell Biol. 107:201-209). Integral membrane proteins mediate the actin nucleation activity because activity is eliminated by heat denaturation, treatment with reducing agents, or proteolysis of membranes. Activity also is abolished by solubilization with octylglucoside but is reconstituted upon removal or dilution of the detergent. Ponticulin, the major actin-binding protein in plasma membranes, appears to be necessary for nucleation activity since activity is not reconstituted from detergent extracts depleted of ponticulin.

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.

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.