Calcium-sensitive activity and conformation of Caenorhabditis elegans gelsolin-like protein 1 are altered by mutations in the first gelsolin-like domain

The gelsolin family of actin regulatory proteins is activated by Ca(2+) to sever and cap actin filaments. Gelsolin has six homologous gelsolin-like domains (G1-G6), and Ca(2+)-dependent conformational changes regulate its accessibility to actin. Caenorhabditis elegans gelsolin-like protein-1 (GSNL-1) has only four gelsolin-like domains (G1-G4) and still exhibits Ca(2+)-dependent actin filament-severing and -capping activities. We found that acidic residues (Asp-83 and Asp-84) in G1 of GSNL-1 are important for its Ca(2+) activation. These residues are conserved in GSNL-1 and gelsolin and previously implicated in actin-severing activity of the gelsolin family. We found that alanine mutations at Asp-83 and Asp-84 (D83A/D84A mutation) did not disrupt actin-severing or -capping activity. Instead, the mutants exhibited altered Ca(2+) sensitivity when compared with wild-type GSNL-1. The D83A/D84A mutation enhanced Ca(2+) sensitivity for actin severing and capping and its susceptibility to proteolytic digestion, suggesting a conformational change. Single mutations caused minimal changes in its activity, whereas Asp-83 and Asp-84 were required to stabilize Ca(2+)-free and Ca(2+)-bound conformations, respectively. On the other hand, the D83A/D84A mutation suppressed sensitivity of GSNL-1 to phosphatidylinositol 4,5-bisphosphate inhibition. The structure of an inactive form of gelsolin shows that the equivalent acidic residues are in close contact with G3, which may maintain an inactive conformation of the gelsolin family.     

Direct observation of actin filament severing by gelsolin and binding by gCap39 and CapZ

Dynamic behavior of actin filaments in cells is the basis of many different cellular activities. Remodeling of the actin filament network involves polymerization and depolymerization of the filaments. Proteins that regulate these behaviors include proteins that sever and/or cap actin filaments. This report presents direct observation of severing of fluorescently-labeled actin filaments. Coverslips coated with gelsolin, a multi-domain, calcium-dependent capping and severing protein, bound rhodamine-phalloidin-saturated filaments along their length in the presence of EGTA. Upon addition of calcium, attached filaments bent as they broke. Actophorin, a low molecular weight, monomer sequestering, calcium-independent severing protein did not sever phalloidin-saturated filaments. Both gCap 39, a gelsolin-like, calcium-dependent capping protein that does not sever filaments, and CapZ, a heterodimeric, non-calcium-dependent capping protein, bound the filaments by one end to the coverslip. Visualization of individual filaments also revealed severing activity present in mixtures of actin-binding proteins isolated by filamentous actin affinity chromatography from early Drosophila embryos. This activity was different from either gelsolin or actophorin because it was not inhibited by phalloidin, but was calcium independent. The results of these studies provide new information about the molecular mechanisms of severing and capping by well-characterized proteins as well as definition of a novel type of severing activity.     

Arabidopsis VILLIN1 generates actin filament cables that are resistant to depolymerization

Dynamic cytoplasmic streaming, organelle positioning, and nuclear migration use molecular tracks generated from actin filaments arrayed into higher-order structures like actin cables and bundles. How these arrays are formed and stabilized against cellular depolymerizing forces remains an open question. Villin and fimbrin are the best characterized actin-filament bundling or cross-linking proteins in plants and each is encoded by a multigene family of five members in Arabidopsis thaliana. The related villins and gelsolins are conserved proteins that are constructed from a core of six homologous gelsolin domains. Gelsolin is a calcium-regulated actin filament severing, nucleating and barbed end capping factor. Villin has a seventh domain at its C terminus, the villin headpiece, which can bind to an actin filament, conferring the ability to crosslink or bundle actin filaments. Many, but not all, villins retain the ability to sever, nucleate, and cap filaments. Here we have identified a putative calcium-insensitive villin isoform through comparison of sequence alignments between human gelsolin and plant villins with x-ray crystallography data for vertebrate gelsolin. VILLIN1 (VLN1) has the least well-conserved type 1 and type 2 calcium binding sites among the Arabidopsis VILLIN isoforms. Recombinant VLN1 binds to actin filaments with high affinity (K(d) approximately 1 microM) and generates bundled filament networks; both properties are independent of the free Ca(2+) concentration. Unlike human plasma gelsolin, VLN1 does not nucleate the assembly of filaments from monomer, does not block the polymerization of profilin-actin onto barbed ends, and does not stimulate depolymerization or sever preexisting filaments. In kinetic assays with ADF/cofilin, villin appears to bind first to growing filaments and protects filaments against ADF-mediated depolymerization. We propose that VLN1 is a major regulator of the formation and stability of actin filament bundles in plant cells and that it functions to maintain the cable network even in the presence of stimuli that result in depolymerization of other actin arrays.     

Structural analysis of an Echinococcus granulosus actin-fragmenting protein by small-angle x-ray scattering studies and molecular modeling

The Echinococcus granulosus actin filament-fragmenting protein (EgAFFP) is a three domain member of the gelsolin family of proteins, which is antigenic to human hosts. These proteins, formed by three or six conserved domains, are involved in the dynamic rearrangements of the cytoskeleton, being responsible for severing and capping actin filaments and promoting nucleation of actin monomers. Various structures of six domain gelsolin-related proteins have been investigated, but little information on the structure of three domain members is available. In this work, the solution structure of the three domain EgAFFP has been investigated through small-angle x-ray scattering (SAXS) studies. EgAFFP exhibits an elongated molecular shape. The radius of gyration and the maximum dimension obtained by SAXS were, respectively, 2.52 +/- 0.01 nm and 8.00 +/- 1.00 nm, both in the absence and presence of Ca2+. Two different molecular homology models were built for EgAFFP, but only one was validated through SAXS studies. The predicted structure for EgAFFP consists of three repeats of a central beta-sheet sandwiched between one short and one long alpha-helix. Possible implications of the structure of EgAFFP upon actin binding are discussed.     

Eps8 controls actin-based motility by capping the barbed ends of actin filaments

Actin filament barbed-end capping proteins are essential for cell motility, as they regulate the growth of actin filaments to generate propulsive force. One family of capping proteins, whose prototype is gelsolin, shares modular architecture, mechanism of action, and regulation through signalling-dependent mechanisms, such as Ca(2+) or phosphatidylinositol-4,5-phosphate binding. Here we show that proteins of another family, the Eps8 family, also show barbed-end capping activity, which resides in their conserved carboxy-terminal effector domain. The isolated effector domain of Eps8 caps barbed ends with an affinity in the nanomolar range. Conversely, full-length Eps8 is auto-inhibited in vitro, and interaction with the Abi1 protein relieves this inhibition. In vivo, Eps8 is recruited to actin dynamic sites, and its removal impairs actin-based propulsion. Eps8-family proteins do not show any similarity to gelsolin-like proteins. Thus, our results identify a new family of actin cappers, and unveil novel modalities of regulation of capping through protein-protein interactions. One established function of the Eps8-Abi1 complex is to participate in the activation of the small GTPase Rac, suggesting a multifaceted role for this complex in actin dynamics, possibly through the participation in alternative larger complexes.     

Conservation and amyloid formation: a study of the gelsolin-like family

The mechanism through which globular proteins transform into amyloid fibrils is still not understood. Here we analyze the structure and sequence conservation to assess the differential stability of segments from two structurally related protein families: the amyloidogenic gelsolin-like and its structurally related cofilin-like. The two families belong to the actin depolymerizing proteins, with a central beta-sheet stacked between 2 and 4 alpha-helices. Although sequentially remote, the two families share regions of high and low conservation and stability. Our results show a highly conserved hydrophobic and aromatic cluster, located at a central buried beta-hairpin. The geometry of the aromatic residues with respect to each other is strictly conserved, suggesting involvement in strand registering and beta-sheet stabilization. Consistent with experiment, we find a region of weak conservation and stability at one of the exposed beta-strands (strand B in the gelsolin-like family). This region was recently found to be affected by a point mutation-mediated destabilization of the human gelsolin domain 2, which facilitates the first proteolytic event in the formation of the amyloidogenic fragment. Thus, both experimental and computational conservation analyses suggest that this unstable region may constitute a first step in amyloid formation. Our analysis uses a recently developed multiple-structure comparison algorithm in which molecules are aligned simultaneously.     

Flightless I: an actin-remodelling protein and an important negative regulator of wound repair

Flightless I (FliI) is a member of the gelsolin family of actin-remodelling proteins, and has been identified as having two functional protein family domains: a leucine rich repeat (LRR) domain and a gelsolin-like domain. This unique structure allows FliI to act as an actin-remodelling protein as well as a nuclear receptor co-activator with ability to interact with various other proteins important in cellular signaling. The actin cytoskeleton is an integral component of all cells and the effect of FliI protein on actin remodelling is a vital part of cellular motility, contraction and adhesion. The product of the FliI gene is expected to provide a vital link between the molecules of yet unidentified signal transduction pathways and the actin cytoskeleton. Exact signaling pathways and mechanisms underpinning FliI effects in wound healing are yet to be fully identified however strong research evidence clearly identifies this molecule as a possible new therapeutic target whose manipulation may greatly improve wound healing and could lead to potential innovative medical applications.     

Expression of human plasma gelsolin in Escherichia coli and dissection of actin binding sites by segmental deletion mutagenesis

Human plasma gelsolin has been expressed in high yield and soluble form in Escherichia coli. The protein has nucleating and severing activities identical to those of plasma gelsolin and is fully calcium sensitive in its interactions with monomeric actin. A number of deletion mutants have been expressed to explore the function of the three actin binding sites. Their design is based on the sixfold segmental repeat in the protein sequence. (These sites are located in segment 1, segments 2-3, and segments 4-6). Two mutants, S1-3 and S4-6, are equivalent to the NH2- and COOH-terminal halves of the molecule obtained by limited proteolysis. S1-3 binds two actin monomers in the presence or absence of calcium, it severs and caps filaments but does not nucleate polymerization. S4-6 binds a single actin monomer but only in calcium. These observations confirm and extend current knowledge on the properties of the two halves of gelsolin. Two novel constructs have also been studied that provide a different pairwise juxtaposition of the three sites. S2-6, which lacks the high affinity site of segment 1 (equivalent to the 14,000-Mr proteolytic fragment) and S1,4-6, which lacks segments 2-3 (the actin filament binding domain previously identified using the 28,000-Mr proteolytic fragment). S2-6 binds two actin monomers in calcium and nucleates polymerization; it associates laterally with filaments in the presence or absence of calcium and has a weak calcium-dependent fragmenting activity. S1,4-6 also binds two actin monomers in calcium and one in EGTA, has weak severing activity but does not nucleate polymerization. A model is presented for the involvement of the three binding sites in the various activities of gelsolin.     

Interaction Between a Gelsolin from <Emphasis Type="Italic">Dendrorhynchus zhejiangensis</Emphasis> with Three Gelsolin-Like Domains and Actin In Vitro

The gelsolin family proteins are best known for involvement in cytoskeletal rearrangement by controlling actin organization during a variety of cellular processes. Previously, a 1962 bp cDNA encoding a 41.7 kDa protein with three gelsolin-like domains (G domains) from Dendrorhynchus zhejiangensis was identified and named as DzGSN. In this study, the sequence and function of a novel member of the gelsolin family proteins from D. zhejiangensis have been analyzed. Sequence alignment indicates that DzGSN is highly homologous to human gelsolin (35% identity) and human CapG (36% identity). The important functional motifs and critical amino acids were identified. The nucleating- and severing-actin activities of recombinant DzGSN (rDzGSN) were then investigated by using atomic force microscopy in vitro. After incubation with rDzGSN in the presence of Ca²⁺, global actin (G-actin) was observed to aggregate into a ring structure, while filament actin (F-actin) was observed to be shortened. Additionally, the yeast two-hybrid system also verified that DzGSN can interact with actin. The results provide new insight into functional diversity and evolution of gelsolin family proteins.     

Phototactic Migration of Dictyostelium Cells Is Linked to a New Type of Gelsolin-related Protein

The molecular and functional characterization of a 125-kDa Ca²⁺-extractable protein of the Triton X-100–insoluble fraction of Dictyostelium cells identified a new type of a gelsolin-related molecule. In addition to its five gelsolin segments, this gelsolin-related protein of 125 kDa (GRP125) reveals a number of unique domains, two of which are predicted to form coiled-coil regions. Another distinct attribute of GRP125 concerns the lack of sequence elements known to be essential for characteristic activities of gelsolin-like proteins, i.e. the severing, capping, or nucleation of actin filaments. The subcellular distribution of GRP125 to vesicular compartments suggests an activity of GRP125 different from actin-binding, gelsolin-related proteins. GRP125 expression is tightly regulated and peaks at the transition to the multicellular pseudoplasmodial stage of Dictyostelium development. GRP125 was found indispensable for slug phototaxis, because slugs fail to correctly readjust their orientation in the absence of GRP125. Analysis of the GRP125-deficient mutant showed that GRP125 is required for coupling photodetection to the locomotory machinery of slugs. We propose that GRP125 is essential in the natural environment for the propagation of Dictyostelium spores. We also present evidence for further representatives of the GRP125 type in Dictyostelium, as well as in heterologous cells from lower to higher eukaryotes.     

Gelsolin-Like Domain 3 Plays Vital Roles in Regulating the Activities of the Lily Villin/Gelsolin/Fragmin Superfamily

The villin/gelsolin/fragmin superfamily is a major group of Ca²⁺-dependent actin-binding proteins (ABPs) involved in various cellular processes. Members of this superfamily typically possess three or six tandem gelsolin-like (G) domains, and each domain plays a distinct role in actin filament dynamics. Although the activities of most G domains have been characterized, the biochemical function of the G3 domain remains poorly understood. In this study, we carefully compared the detailed biochemical activities of ABP29 (a new member of this family that contains the G1-G2 domains of lily ABP135) and ABP135_G1-G3 (which contains the G1-G3 domains of lily ABP135). In the presence of high Ca²⁺ levels in vitro (200 and 10 μM), ABP135_G1-G3 exhibited greater actin severing and/or depolymerization and nucleating activities than ABP29, and these proteins had similar actin capping activities. However, in the presence of low levels of Ca²⁺ (41 nM), ABP135_G1-G3 had a weaker capping activity than ABP29. In addition, ABP29 inhibited F-actin depolymerization, as shown by dilution-mediated depolymerization assay, differing from the typical superfamily proteins. In contrast, ABP135_G1-G3 accelerated F-actin depolymerization. All of these results demonstrate that the G3 domain plays specific roles in regulating the activities of the lily villin/gelsolin/fragmin superfamily proteins.     

Fragmin60 encodes an actin-binding protein with a C2 domain and controls actin Thr-203 phosphorylation in Physarum plasmodia and sclerotia

We report the isolation of a cDNA clone encoding a 60-kDa protein termed fragmin60 that cross-reacts with fragmin antibodies. Unlike other gelsolin-related proteins, fragmin60 contains a unique N-terminal domain that shows similarity with C2 domains of aczonin, protein kinase C, and synaptotagmins. The fragmin60 C2 domain binds three calcium ions, one with nanomolar affinity and two with micromolar affinity. Actin binding by fragmin60 requires higher calcium concentrations than does binding of actin by a fragmin60 mutant lacking the C2 domain, suggesting that the C2 domain secures the actin binding moiety in a conformation preventing actin binding at low calcium concentrations. The fragmin60 C2 domain does not bind phospholipids but interacts with the endogenous homologue of Saccharomyces cerevisiae S-phase kinase-associated protein (Skp1), as shown by pull-down assays and co-expression in mammalian cells. Recombinant fragmin60 promotes in vitro phosphorylation of actin Thr-203 by the actin-fragmin kinase. We further show that in vivo phosphorylation of actin in the fragmin60-actin complex occurs in sclerotia, a dormant stage of Physarum development, as well as in plasmodia. Our findings indicate that we have cloned a novel type of gelsolin-related actin-binding protein that is involved in controlling regulation of actin phosphorylation in vivo.     

The Ca2(+)-dependent actin filament-severing activity of 74-kDa protein (adseverin) resides in its NH2-terminal half.

Calcium sensitive actin severing protein, adseverin, with Mr 74,000, was cleaved into two fragments of Mr 42,000 and Mr 39,000 by V8 protease and trypsin, and both fragments were purified by high performance (pressure) liquid chromatography ion-exchange column chromatography. To understand how adseverin can sever actin filaments, we identified the actin-binding domains. The NH2 termini of native adseverin and the Mr 42,000 fragment were confirmed to be blocked by amino acid sequencing. Twelve amino acids of the Mr 39,000 fragment were sequenced from the NH2 terminus; the sequence of this part had a homology to the hinge region between segments 3 and 4 of gelsolin and villin. Thus, the Mr 42,000 fragment is the NH2-terminal half (N42), and the Mr 39,000 fragment is the COOH-terminal half (C39). Each fragment was examined for actin-severing, -nucleating, -capping, and phospholipid binding activities with and without calcium. N42 contained a calcium-dependent actin-severing activity regulated by phospholipid. C39 bound to G-actin in a calcium-dependent manner, but had no severing activity. The sequence homology and similar functional domain structure suggest a common structural basis for the calcium- and phospholipid-regulated actin-severing properties shared by adseverin, gelsolin, and villin.     

Characterization of a novel Ras-binding protein Ce-FLI-1 comprising leucine-rich repeats and gelsolin-like domains

Ras proteins are conserved from yeasts to mammals and implicated in regulation of the actin cytoskeleton. The flightless-1 (fli-1) gene of Drosophila melanogaster and its homologs in Caenorhabditis elegans and humans encode proteins (FLI-1) comprising a fusion of a leucine-rich repeats (LRRs) domain and a gelsolin-like domain. This LRRs domain is highly homologous to those of three proteins involved in Ras-mediated signaling; Saccharomyces cerevisiae adenylyl cyclase, C. elegans SUR-8, and mammalian RSP-1. Here we report that the LRRs domain of C. elegans FLI-1 (Ce-FLI-1) associates directly with Ras (Kd = 11 nM) and, when overexpressed, suppresses the heat shock sensitive phenotype of yeast cells bearing the activated RAS2 gene (RAS2(Val-19)). Further, the gelsolin-like domain of Ce-FLI-1 is shown to possess a Ca2+-independent G-actin-binding activity as well as F-actin-binding and -severing activities. FLI-1 may be involved in regulation of the actin cytoskeleton through Ras.     

Interactions of gelsolin and gelsolin-actin complexes with actin. Effects of calcium on actin nucleation, filament severing, and end blocking

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

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.     

Secretory cell actin-binding proteins: identification of a gelsolin- like protein in chromaffin cells

Chromaffin cells, secretory cells of the adrenal medulla, have been shown to contain actin and other contractile proteins, which might be involved in the secretory process. Actin and Ca++-sensitive actin-binding proteins were purified from bovine adrenal medulla on affinity columns using DNase-I as a ligand. Buffers that contained decreasing Ca++ concentrations were used to elute three major proteins of 93, 91, and 85 kD. The bulk of the actin was eluted with guanidine-HCl buffer plus some 93- and 91-kD proteins. These Ca++-sensitive regulatory proteins were shown to inhibit the gelation of actin using the low-shear falling ball viscometer and by electron microscopy. Actin filaments were found to be shortened by fragmentation. Using antibody raised against rabbit lung macrophage gelsolin, proteolytic digestion with Staphylococcus V8 protease and two-dimensional gel electrophoresis, the 91-kD actin-binding protein was shown to be a gelsolin-like protein. The 93-kD actin-binding protein also showed cross-reactivity with anti-gelsolin antibody, similar peptide maps, and a basic-shift in pHi indicating that this 93-kD protein is a brevin-like protein, derived from blood present abundantly in adrenal medulla. Purification from isolated chromaffin cells demonstrated the presence of 91- and 85-kD proteins, whereas the 93-kD protein was hardly detectable. The 85-kD protein is not a breakdown product of brevin-like or gelsolin-like proteins. It did not cross-react with anti-gelsolin antibody and showed a very different peptide map after mild digestion with V8 protease. Antibodies were raised against the 93- and 91-kD actin-binding proteins and the 85-kD actin-binding protein. Antibody against the 85-kD protein did not cross-react with 93- and 91-kD proteins and vice versa. In vivo, the cytoskeleton organization of chromaffin secretory cells is not known, but appears to be under the control of the intracellular concentration of free calcium. The ability of calcium to activate the gelsolin-like protein, and as shown elsewhere to alter fodrin localization, provides a mechanism for gel-sol transition that might be essential for granule movement and membrane-membrane interactions involved in the secretory process.     

A gelsolin-like protein from Papaver rhoeas pollen (PrABP80) stimulates calcium-regulated severing and depolymerization of actin filaments

The cytoskeleton is a key regulator of plant morphogenesis, sexual reproduction, and cellular responses to extracellular stimuli. During the self-incompatibility response of Papaver rhoeas L. (field poppy) pollen, the actin filament network is rapidly depolymerized by a flood of cytosolic free Ca2+ that results in cessation of tip growth and prevention of fertilization. Attempts to model this dramatic cytoskeletal response with known pollen actin-binding proteins (ABPs) revealed that the major G-actin-binding protein profilin can account for only a small percentage of the measured depolymerization. We have identified an 80-kDa, Ca(2+)-regulated ABP from poppy pollen (PrABP80) and characterized its biochemical properties in vitro. Sequence determination by mass spectrometry revealed that PrABP80 is related to gelsolin and villin. The molecular weight, lack of filament cross-linking activity, and a potent severing activity are all consistent with PrABP80 being a plant gelsolin. Kinetic analysis of actin assembly/disassembly reactions revealed that substoichiometric amounts of PrABP80 can nucleate actin polymerization from monomers, block the assembly of profilin-actin complex onto actin filament ends, and enhance profilin-mediated actin depolymerization. Fluorescence microscopy of individual actin filaments provided compelling, direct evidence for filament severing and confirmed the actin nucleation and barbed end capping properties. This is the first direct evidence for a plant gelsolin and the first example of efficient severing by a plant ABP. We propose that PrABP80 functions at the center of the self-incompatibility response by creating new filament pointed ends for disassembly and by blocking barbed ends from profilin-actin assembly.