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.     

Arabidopsis VILLIN5, an Actin Filament Bundling and Severing Protein, Is Necessary for Normal Pollen Tube Growth

A dynamic actin cytoskeleton is essential for pollen germination and tube growth. However, the molecular mechanisms underlying the organization and turnover of the actin cytoskeleton in pollen remain poorly understood. Villin plays a key role in the formation of higher-order structures from actin filaments and in the regulation of actin dynamics in eukaryotic cells. It belongs to the villin/gelsolin/fragmin superfamily of actin binding proteins and is composed of six gelsolin-homology domains at its core and a villin headpiece domain at its C terminus. Recently, several villin family members from plants have been shown to sever, cap, and bundle actin filaments in vitro. Here, we characterized a villin isovariant, Arabidopsis thaliana VILLIN5 (VLN5), that is highly and preferentially expressed in pollen. VLN5 loss-of-function retarded pollen tube growth and sensitized actin filaments in pollen grains and tubes to latrunculin B. In vitro biochemical analyses revealed that VLN5 is a typical member of the villin family and retains a full suite of activities, including barbed-end capping, filament bundling, and calcium-dependent severing. The severing activity was confirmed with time-lapse evanescent wave microscopy of individual actin filaments in vitro. We propose that VLN5 is a major regulator of actin filament stability and turnover that functions in concert with oscillatory calcium gradients in pollen and therefore plays an integral role in pollen germination and tube growth.     

The Villin/Gelsolin/Fragmin Superfamily Proteins in Plants

The villin/gelsolin/fragmin superfamily is a conserved Ca^2＋-dependent family of actin-regulating proteins that is widely present both in mammalian and non-mammalian organisms. They have traditionally been characterized by the same core of three or six tandem gelsolin subdomains. The study in vertebrates and lower eukaryotic cells has revealed that the villin/gelsolin/fragmin superfamily of proteins has versatile functions including severing, capping, nucleating or bundling actin filaments. In plants, encouraging progress has been made in this field of research in recent years. This review will summarize the identified plant homologs of villin/gelsolin/fragmin superfamily, thus providing a basis for reflection on their biochemical activities and functions in plants.     

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.     

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.     

The 135 kDa actin-bundling protein fromLilium longiflorum pollen is the plant homologue of villin

Summary Actin microfilaments, which are essential for cell growth and cytoplasmic streaming in pollen tubes, are closely dependent on actin-binding proteins for their organization and regulation. We have purified the plant 135 kDa actin-bundling protein (P-135-ABP) fromLilium longiflorum pollen and determined that its amino acid composition is highly similar to members of the villin-gelsolin family of proteins. We used antibodies against P-135-ABP to probe an expression cDNA library ofL. longiflorum pollen and isolated a full-length clone (ABP135) that corresponds to a 106 kDa polypeptide. The deduced amino acid sequence ofABP135 shows homology with members of the villin-gelsolin family of proteins and contains the characteristic six repeats of this family, as well as an extended carboxy-terminal domain that includes the villin headpiece preceded by a highly variable region. Using two-dimensional polyacrylamide gel electrophoresis we detected at least 5 isoforms of P-135-ABP, with isoelectric points (pI) ranging between 5.6 to 5.9. The most abundant P-135-ABP isoform has a pI of 5.8, closely approximating the pI predicted from the deducedABP135 amino acid sequence. These data, together with the partial amino acid sequence from a proteolytic peptide of the protein, indicate that P-135-ABP is a plant villin. Immuno-detection of Lilium villin in rapidly frozen pollen tubes localized it to actin bundles. Lilium villin is also ubiquitously expressed in all tissues tested. Since villins, like gelsolins, are also Ca²⁺-dependent severing, capping, and nucleating proteins, Lilium villin may participate in F-actin fragmentation and nucleation in the apex of the pollen tube where there is steep Ca²⁺ gradient.Abbreviations BMM butyl methyl-methacrylate - PPI polyphos-phoinositides - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Severin is a gelsolin prototype

A number of Ca2(+)-activated actin filament severing proteins have been identified in eukaryotic cells of diverse lineages. Gelsolin and villin, with molecular mass of about 80-90 kDa, and severin and fragmin, with molecular mass of about 40 kDa, have been isolated from vertebrates and invertebrates, respectively. We report here a direct comparison of the functional properties of gelsolin and severin, and the finding that the actin filament severing activity of severin, like that of gelsolin, is inhibited by polyphosphoinositides. However, severin does not nucleate actin filament assembly as well as gelsolin. These characteristics are very similar to those ascribed to the NH2-terminal half of gelsolin, supporting the idea that they are evolutionarily related. Regulation of severin by polyphospholipids raises the possibility that it may participate in agonist-stimulated regulation of the actin cytoskeleton in Dictyostelium discoideum.     

Arabidopsis VILLIN1 and VILLIN3 Have Overlapping and Distinct Activities in Actin Bundle Formation and Turnover

Actin filament bundles are higher-order cytoskeletal structures that are crucial for the maintenance of cellular architecture and cell expansion. They are generated from individual actin filaments by the actions of bundling proteins like fimbrins, LIMs, and villins. However, the molecular mechanisms of dynamic bundle formation and turnover are largely unknown. Villins belong to the villin/gelsolin/fragmin superfamily and comprise at least five isovariants in Arabidopsis thaliana. Different combinations of villin isovariants are coexpressed in various tissues and cells. It is not clear whether these isovariants function together and act redundantly or whether they have unique activities. VILLIN1 (VLN1) is a simple filament-bundling protein and is Ca²⁺ insensitive. Based on phylogenetic analyses and conservation of Ca²⁺ binding sites, we predict that VLN3 is a Ca²⁺-regulated villin capable of severing actin filaments and contributing to bundle turnover. The bundling activity of both isovariants was observed directly with time-lapse imaging and total internal reflection fluorescence (TIRF) microscopy in vitro, and the mechanism mimics the “catch and zipper” action observed in vivo. Using time-lapse TIRF microscopy, we observed and quantified the severing of individual actin filaments by VLN3 at physiological calcium concentrations. Moreover, VLN3 can sever actin filament bundles in the presence of VLN1 when calcium is elevated to micromolar levels. Collectively, these results demonstrate that two villin isovariants have overlapping and distinct activities.     

Regulation of cell structure and function by actin-binding proteins: villin's perspective

Villin is a tissue-specific actin modifying protein that is associated with actin filaments in the microvilli and terminal web of epithelial cells. It belongs to a large family of actin-binding proteins which includes actin-capping, -nucleating and/or -severing proteins such as gelsolin, severin, fragmin, adseverin/scinderin and actin crosslinking proteins such as dematin and supervillin. Studies done in epithelial cell lines and villin knock-out mice have demonstrated the function of villin in regulating actin dynamics, cell morphology, epithelial-to-mesenchymal transition, cell migration and cell survival. In addition, the ligand-binding properties of villin (F-actin, G-actin, calcium, phospholipids and phospholipase C-gamma1) are mechanistically important for the crosstalk between signaling pathways and actin reorganization in epithelial cells.     

Nucleotide sequence of pig plasma gelsolin. Comparison of protein sequence with human gelsolin and other actin-severing proteins shows strong homologies and evidence for large internal repeats

Pig plasma gelsolin (Mr = 81595; 739 residues) contains 704 identical residues out of a maximum 730 when compared to the cytoplasmic form of human gelsolin. The cDNA sequence also codes for a peptide of 33 residues N-terminal to the nine-residue plasma extension sequence previously reported: these 33 residues are highly homologous to the human signal peptide and plasma extension. Comparison of the gelsolin sequences with chicken brush border villin, severin from Dictyostelium discoideum and fragmin from Physarum polycephalum shows a strong evolutionary relationship between all these proteins. There are six large repeating segments in gelsolin and villin, and three similar segments in severin and fragmin. Although these multiple repeats cannot be related to any known function of these actin-severing proteins, this superfamily of proteins appears to have evolved from an ancestral sequence of 120 to 130 amino acid residues.     

The Human Actin-Regulatory Protein Cap G: Gene Structure and Chromosome Location

Cap G (formerly called macrophage capping protein or gCap39) is a member of the gelsolin/villin fanlily of actin-regulatory proteins. Unlike all other members of this family, Cap G caps the barbed ends of actin filaments, but does not sever them. This protein is half the molecular weight and contains half the number of repeat subunits (3 vs 6) of gelsolin and villin, suggesting that these two proteins may have arisen by gene duplication of the Cap G gene. To investigate this possibility we have cloned and sequenced the human Cap G gene (gene symbol CAPG). The gene is 16.6 kb in size, contains 10 exons and 9 introns and is located on the proximal short arm of chromosome 2. The open reading frame is 6.9 kb, having 9 exons and 8 introns. This region contains 3 splice sites that are nearly identical to the human gelsolin gene, but shares only one with villin, indicating that CAPG is more closely related to gelsolin. Further comparisons of these three genes, however, indicate that the evolutionary steps resulting in human gelsolin and villin are likely to have been more complex than a simple tandem duplication of the Cap G gene.     

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.     

The gelsolin/fragmin family protein identified in the higher plant Mimosa pudica

Mimosa pudica L. rapidly closes its leaves and bends its petioles downward when mechanically stimulated. It has been suggested that the actin cytoskeleton is involved in the bending motion since both cytochalasin B and phalloidin inhibit the motion. In order to clarify the mechanism by which the actin cytoskeleton functions in the motion, we attempted to find actin-modulating proteins in the M. pudica plant by DNase I-affinity column chromatography. The EGTA-eluate from the DNase I column contained proteins with apparent molecular masses of 90- and 42-kDa. The 42-kDa band consisted of two closely migrating components: the slower migrating component was actin while the faster migrating components was a distinct protein. The eluate showed an activity to sever actin filaments and to enhance the rate of polymerization of actin, both in a Ca(2+)-dependent manner. Microsequencing of the faster migrating 42-kDa protein revealed its similarity to proteins in the gelsolin/fragmin family. Our results provide the first biochemical evidence for the presence in a higher plant of a gelsolin/fragmin family actin-modulating protein that severs actin filament in a Ca(2+)-dependent manner.     

Gelsolin: calcium- and polyphosphoinositide-regulated actin-modulating protein

Receptor-mediated stimulation induces massive actin polymerization and cyto-skeletal reorganization. The activity of a potent actin-modulating protein, gelsolin, is regulated both by Ca²⁺ and polyphos-phoinositides, and it may have a pivotal role in restructuring the actin cytoskeleton in response to agonist stimulation. Structure-function analysis of gelsolin has (1) indicated that its NH₂-terminal half is primarily responsible for modulating actin filament length and polymerization; and (2) elucidated mechanisms by which Ca²⁺ and phospholipids may regulate such functions. Gelsolin is functionally and structurally similar to villin, another Ca²⁺-activated actin-severing protein found in microvilli, suggesting that gelsolin may be a prototype of this family of actin-modulating proteins. A molecular variant of gelsolin is secreted and may be involved in the clearance of actin filaments released during tissue damage. The two forms of gelsolin are encoded by a single gene, and distinct messages are derived by alternative message splicing.     

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。     

Arabidopsis VILLIN2 and VILLIN3 act redundantly in sclerenchyma development via bundling of actin filaments

The organization of the actin cytoskeleton has been implicated in sclerenchyma development. However, the molecular mechanisms linking the actin cytoskeleton to this process remain poorly understood. In particular, there have been no studies showing that direct genetic manipulation of the actin cytoskeleton affects sclerenchyma development. Villins belong to the villin/gelsolin/fragmin superfamily and are versatile actin-modifying proteins. Several recent studies have implicated villins in tip growth of single cells, but how villins act in multicellular plant development remains largely unknown. Here, we found that two closely related villin isovariants from Arabidopsis, VLN2 and VLN3, act redundantly in sclerenchyma development. Detailed analysis of cross-sections from inflorescence stems of vln2 vln3 double mutant plants revealed a reduction in stem size and in the number of vascular bundles; however, no defects in synthesis of the secondary cell wall were detected. Surprisingly, the vln2 vln3 double mutation did not affect cell elongation of inter-fascicular fibers. Biochemical analyses showed that recombinant VLN2 was able to cap, sever and bundle actin filaments, similar to VLN3. Consistent with these biochemical activities, loss of function of VLN2 and VLN3 resulted in a decrease in the amount of F-actin and actin bundles in plant cells. Collectively, our findings demonstrate that VLN2 and VLN3 act redundantly in sclerenchyma development via bundling of actin filaments.     

Sequence of human villin: a large duplicated domain homologous with other actin-severing proteins and a unique small carboxy-terminal domain related to villin specificity

Villin is a calcium-regulated actin-binding protein that caps, severs, and bundles actin filaments in vitro. This 92,500-D protein is a major constituent of the actin bundles within the microvilli of the brush border surface of intestinal and kidney proximal tubule cells. Villin is a very early marker of cells involved in absorption and its expression is highly increased during intestinal cell differentiation. The amino acid sequence deduced from the cDNA sequence revealed that human villin is composed of three domains. The first two domains appear as the result of a duplication: their structural organization is similar. We can then define a basic unit in which a slightly hydrophilic motif is followed by three hydrophobic motifs, similar between themselves and regularly spaced. The duplicated domain is highly homologous to three other actin-severing proteins and this basic structure represents the whole molecule in severin and fragmin, while two basic units compose gelsolin. The third domain which is carboxy terminal is villin specific: it is unique among actin modulating proteins so far known. It could account for its actin-binding properties (dual regulation by calcium of severing and bundling activities). We propose that it may also be related to the subcellular localization of villin in different epithelial cell types.     

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.     

凝溶胶蛋白的结构与功能

凝溶胶蛋白（gelsolin）是凝溶胶蛋白超家族的成员之一,是一种重要的肌动蛋白结合蛋白,其通过切断、封端肌动蛋白丝,或使肌动蛋白聚集成核等方式来控制肌动蛋白的结构.凝溶胶蛋白除了在重组肌动蛋白丝中发挥作用以外,还在细胞运动、控制细胞程序性死亡等细胞活动中发挥重要的作用.此外,肿瘤细胞中凝溶胶蛋白的表达量也发生变化.凝溶胶蛋白的变异还是某些遗传疾病的基础.最近的研究发现,凝溶胶蛋白可以作为转录辅激活蛋白,促进雄激素受体的转录活性.本文对凝溶胶蛋白的结构特点、参与调节细胞的功能和机制及其研究现状进行概述.     

Severin, gelsolin, and villin share a homologous sequence in regions presumed to contain F-actin severing domains

cDNA clones encoding the actin filament severing protein severin from Dictyostelium discoideum were isolated from a cDNA library in lambda gt 11 using monoclonal antibodies. Comparison of the deduced amino acid sequence with the sequence of a severin peptide indicated that the complete coding region of severin is contained in the isolated clones. Severin, a 39.9-kDa protein, is encoded by one gene in D. discoideum. An mRNA of approximately 1.4 kilobases is present throughout the developmental cycle of D. discoideum. The amino acid sequence of severin contains a region highly homologous to a conserved sequence in villin and gelsolin, two proteins of similar function isolated from vertebrates. This homologous region is believed to participate in the actin filament severing activity of these proteins. Comparison of the severin sequence to the entire gelsolin sequence shows remarkable homologies pointing to a common origin from an ancestral gene from which gelsolin has been derived by a duplication.