Gelsolin as a calcium-regulated actin filament-capping protein.

Various concentrations of gelsolin (25-100 nM) were added to 2 microM polymerized actin. The concentrations of free calcium were adjusted to 0.05-1.5 microM by EGTA/Ca2+ buffer. Following addition of gelsolin actin depolymerization was observed that was caused by dissociation of actin subunits from the pointed ends of treadmilling actin filaments and inhibition by gelsolin of polymerization at barbed ends. The time course of depolymerization revealed an initial lag phase that was followed by slow decrease of the concentration of polymeric actin to reach the final steady state polymer and monomer concentration. The initial lag phase was pronounced at low free calcium and low gelsolin concentrations. On the basis of quantitative analysis the kinetics of depolymerization could be interpreted as capping, i.e. binding of gelsolin to the barbed ends of actin filaments and subsequent inhibition of polymerization, rather than severing. The main argument for this conclusion was that even gelsolin concentrations (100 nM) that exceed the concentration of filament ends ( approximately 2 nM), cause the filaments to depolymerize at a rate that is similar to the rate of depolymerization of the concentration of pointed ends existing before addition of gelsolin. The rate of capping is directly proportional to the free calcium concentration. These experiments demonstrate that at micromolar and submicromolar free calcium concentrations gelsolin acts as a calcium-regulated capping protein but not as an actin filament severing protein, and that the calcium binding sites of gelsolin which regulate the various functions of gelsolin (capping, severing and monomer binding), differ in their calcium affinity.     

WIP: a multifunctional protein involved in actin cytoskeleton regulation

Knowledge of the dynamics of actin-based structures is a major key to understanding how cells move and respond to their environment. The ability to reorganize actin filaments in a spatial and temporal manner to integrate extracellular signals is at the core of cell adhesion and cell migration. Several proteins have been described as regulators of actin polymerization: this review will focus on the role of WASP-interacting protein (WIP), an actin-binding protein that participates in actin polymerization regulation and signal transduction. WIP is widely expressed and interacts with Wiskott-Aldrich syndrome protein (WASP) (a hematopoietic-specific protein) and its more widely expressed homologue neural WASP (N-WASP), to regulate WASP/N-WASP function in Arp2/3-mediated actin polymerization. WIP also interacts with profilin, globular and filamentous actin (G- and F-actin, respectively) and stabilizes actin filaments. In vivo WIP participates in filopodia and lamellipodia formation, in T and B lymphocyte activation, in mast cell degranulation and signaling through the Fcepsilon receptor (FcepsilonR), in microbial motility and in Syk protein stability.     

Gelsolin binds to polymeric actin at a low rate.

Association of gelsolin with actin filament subunits was investigated by the decrease of the fluorescence intensity of a 7-nitro-2-oxa-1,3-diazole (NBD) label covalently linked to gelsolin. The rate constant of this reaction was found to be 4 x 10(3) M-1 s-1. Binding of NBD-labeled gelsolin to monomeric actin proceeds at a similar low rate. The rate of association of gelsolin that was unmodified to actin filament subunits was estimated too. Unmodified gelsolin was added to a mixture of actin filaments and actin-DNase I complex. The fractions of gelsolin that bound to actin filament subunits or to actin-DNase I complex depended on the relative rates of these two competing reactions. In this way it was possible to estimate the rate constant of association of unmodified gelsolin with actin filament subunits (2 x 10(4) M-1 s-1). Thus, gelsolin associates with actin filament subunits at a rate that is considerably slower than diffusion-controlled and similar to the rate of binding of gelsolin to monomeric actin.     

Gamma-Actinin, a new regulatory protein from rabbit skeletal muscle. II. Action on actin.

The interaction of gamma-actinin and actin was investigated under various conditions. It has been shown that gamma-actinin affects the G-F transformation of actin, causing an increase in the number of actin monomers required to form a nucleus in the initial step of polymerization. Sonicated fragments of F-actin and heavy meromyosin caused the immediate polymerization of actin under the influence of gamma-actinin. Therefore, it can be concluded that gamma-actinin inhibits the nucleation step of G-F transformation. Actin filaments which were formed in the presence of gamma-actinin (F-actin) were shown to possess certain characteristic properties when compared with control F-actin. These were as follows: F-actin solution had a high critical concentration; F-actin showed a high rate of depolymerization; the flow birefringence of F-actin decreased with time upon incubation in the absence of free ATP; finally, F-actin was demonstrated to have ATP-splitting activity. These dynamic features of F-actin were accounted for in terms of an increase in the rate constant of depolymerization in F-actin under the influence of gamma-actinin.     

4E-BP1, a multifactor regulated multifunctional protein

Eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) is a member of a family of translation repressor proteins, and a well-known substrate of mechanistic target of rapamycin (mTOR) signaling pathway. Phosphorylation of 4E-BP1 causes its release from eIF4E to allow cap-dependent translation to proceed. Recently, 4E-BP1 was shown to be phosphorylated by other kinases besides mTOR, and overexpression of 4E-BP1 was found in different human carcinomas. In this review, we summarize the novel findings on mTOR independent 4E-BP1 phosphorylation in carcinomas. The implications of overexpression and possible multi-function of 4E-BP1 are also discussed.     

Nebulin, a helical actin binding protein.

Nebulin, a giant protein (molecular mass 800 kDa) specific for the skeletal muscle of vertebrates, has been suggested to be involved in the length regulation of the thin filament as a 'molecular ruler'. Despite its size, nebulin appears to be composed mainly of small repeats of approximately 35 amino acids. We have characterized in this study the conformational and functional properties of single repeats. Complete repeats were found to bind to F-actin while a truncated one did not. One repeat is therefore the smallest unit for nebulin--actin interaction. Circular dichroism and nuclear magnetic resonance spectra measured for the peptides in water indicated a transient helical conformation. The folded region is located for them all around the conserved sequence SDxxYK. The helical conformation is strongly stabilized by anionic detergents and trifluoroethanol while uncharged or positively charged detergents have no effect. Since the surface of the actin filament is known to contain clusters of negative charges, anionic detergents may mimic the effect of an actin environment. 3D structures were calculated for three representative peptides in SDS. In vivo, the nebulin helices should form a complex with the actin filament. Based on the assumed importance of charge interactions between nebulin and actin, we propose a model for the structure of the F-actin-nebulin complex in vivo. According to that, two nebulin molecules occupy symmetrical positions along the central cleft of the actin filament bridging the two strands of the actin two-start helix. The consistency of this model with experimental data is discussed.     

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.     

The Arp2/3 complex: a multifunctional actin organizer

The actin-related proteins (Arps) constitute a recently characterized family of proteins, many of which function as members of multiprotein complexes. The discovery that two family members, Arp2 and Arp3, act as multifunctional organizers of actin filaments in all eukaryotes has generated much excitement. Over the past two years, newly discovered properties of the Arp2/3 complex have suggested a central role in the control of actin polymerization. First, it promotes actin assembly on the surface of the motile intracellular pathogen Listeria monocytogenes. Second, it can nucleate and cross-link actin filaments in vitro. Third, it localizes with dynamic actin-rich spots of mammalian cells suggesting a role in protrusion; it is found in cortical actin patches in the budding and fission yeasts where it may control patch movement and cortical actin function. Clearly, the complex has a central role in actin cytoskeletal function and will be the subject of much research in the coming years.     

Galectin-3 as a multifunctional protein

Galectin-3 is a 31 kDa member of a growing family of beta-galactoside-binding animal lectins. This protein is expressed in a variety of tissues and cell types and is mainly found in the cytoplasm, although, depending on cell type and proliferative state, a significant amount of this lectin can also be detected in the nucleus, on the cell surface or in the extracellular environment. Galectin-3 is secreted from cells by a novel and incompletely understood mechanism that is independent of the classical secretory pathway through the endoplasmic reticulum/Golgi network. Galectin-3 exhibits pleiotropic biological function, playing a key role in many physiological and pathological processes.     

Endophilin-1: a multifunctional protein

Endophilin-1, a cytoplasmic Src homology 3 (SH3) domain-containing protein, localises in brain presynaptic nerve termini. Endophilin dimerises through its N-terminus, and participates at multiple stages in clathrin-coated endocytosis, from early membrane invagination to synaptic vesicle uncoating. Both its C-terminal SH3 domain and N-terminus are required for endocytosis. Through its SH3 domain, endophilin bound to proline-rich domains (PRDs) in other endocytic proteins, including synaptojanin and dynamin. The N-terminal region possesses unique functions affecting lipid membrane curvature, through lysophosphatidic acid acyl transferase (LPAAT) activity and direct binding and tubulating activity. In addition to synaptic vesicle formation, endophilin-1 complexes with signalling molecules, including cell surface receptors, metalloprotease disintegrins and germinal centre kinase-like kinase (GLK). Therefore, endophilin-1 may serve to couple vesicle biogenesis with intracellular signalling cascades.     

NMR assignment of actin depolymerizing and dynamics regulatory protein from Leishmania donovani

Leishmania donovani cofilin displays low sequence similarity to other mammalian cofilins and also possesses characteristic activity of its own. Determination of its solution structure would facilitate understanding of the molecular mechanism of actin dynamics regulation in this disease causing pathogen.     

Protein disulfide isomerase, a multifunctional protein chaperone, shows copper-binding activity

Protein disulfide isomerase (PDI) is a 55 kDa multifunctional protein of the endoplasmic reticulum (ER) involved in protein folding and isomerization. In addition to the chaperone and catalytic functions, PDI is a major calcium-binding protein of the ER. Although the active site of PDI has a similar motif CXXC to the Cu-binding motif in Wilson and Menkes proteins and in other copper chaperones, there has been no report on any metal-binding capability of PDI other than calcium binding. We present evidence that PDI is a copper-binding protein. In the absence of reducing agent freshly reduced PDI can bind a maximum of 4 mol of Cu(II) and convert to Cu(I). These bound Cu(I) are surface exposed as they can be competed readily by BCS reagent, a Cu(I) specific chelator. However, when the binding is performed using the mixture of Cu(II) and 1mM DTT, the total number of Cu(I) bound increases to 10 mol/mol, and it is slower to react with BCS, indicating a more protected environment. In both cases, the copper-bound forms of PDI exist as tetramers while apo-protein is a monomer. These findings suggest that PDI plays a role in intracellular copper disposition.     

Gelsolin domains 4-6 in active,actin-free conformation identifies sites of regulatory calcium ions

Structural analysis of gelsolin domains 4-6 demonstrates that the two highest-affinity calcium ions that activate the molecule are in domains 5 and 6, one in each. An additional calcium site in domain 4 depends on subsequent actin binding and is seen only in the complex. The uncomplexed structure is primed to bind actin. Since the disposition of the three domains is similar in different crystal environments, either free or in complex with actin, the conformation in calcium is intrinsic to active gelsolin itself. Thus the actin-free structure shows that the structure with an actin monomer is a good model for an actin filament cap. The last 13 residues of domain 6 have been proposed to be a calcium-activated latch that, in the inhibited form only, links two halves of gelsolin. Comparison with the active structure shows that loosening of the latch contributes but is not central to activation. Calcium binding in domain 6 invokes a cascade of swapped ion-pairs. A basic residue swaps acidic binding partners to stabilise a straightened form of a helix that is kinked in inhibited gelsolin. The other end of the helix is connected by a loop to an edge beta-strand. In active gelsolin, an acidic residue in this helix breaks with its loop partner to form a new intrahelical ion-pairing, resulting in the breakage of the continuous sheet between domains 4 and 6, which is central to the inhibited conformation. A structural alignment of domain sequences provides a rationale to understand why the two calcium sites found here have the highest affinity amongst the five different candidate sites found in other gelsolin structures.     

Purification of calcium-sensitive regulatory protein of platelets which inhibits the gelation of actin

Ca²⁺-sensitive regulatory protein of human platelets which inhibits the gelation of actin was purified by DEAE-Sepharose and an affinity column using actin as a ligand. The protein was a single polypeptide chain with an average molecular weight of 90,000 and it bound to actin and inhibited its gelation at concentration from 10^?6–10^?7M of free calcium. Since the protein existed in the form of a complex with actin even though at concentration lower than 10^?7M of free calcium, binding and dissociation of actin and the protein appeared to be dependent on the concentration of free calcium, and complete dissociation was not seen.     

Some properties of Physarum actinin. A regulatory protein of actin polymerization.

A factor termed Physarum actinin was isolated and partially purified from plasmodia of a myxomycete, Physarum polycephalum. When Physarum actinin was mixed with purified Physarum or rabbit striated muscle G-actin in a weight ratio of about 1 actinin to 9 actin and then the polymerization of G-actin induced, G-actin polymerized to the ordinary F-actin on addition of 0.1 M KCl. However, it polymerized to Mg-polymer on addition of 2 mM MgCl2. The reduced viscosity (etasp/C) of the Mg-polymer was 1.2 dl/g, about one-seventh of that of the F-actin (7.4 dl/g). The sedimentation coefficient of the Mg-polymer was 22.8 S, almost the same as that of the F-actin (29.4 S). The Mg-polymer showed the specific ATPase activity of the order of 1 . 10(-3) mumol ATP/mg actin per min. It was shown that Physarum actinin copolymerized with G-actin to form Mg-polymer on addition of 2 mM MgCl2. The molecular weights of Physarum actinin were about 90 000 in salt-free or slat solutions and 43 000 in a dodecyl sulfate solution. The range of salting out with ammonium sulfate was 50--65% saturation, which was different from that of Physarum actin (15--35% saturation). Physarum actinin did not interact with Physarum myosin or muscle heavy meromyosin. When the weight ratio of actinin to actin increased, the flow birefringence of the formed Mg-polymer decreased, and it became almost zero at the weight ratio of 1 actinin to 5 actin. ATPase activity reached the maximum level (2.2 . 10(-3) mumol ATP/mg actin per min) at the same ratio. On the addition of Physarum actinin to purified Physarum F-actin which had been polymerized on addition of 2 mM MgCl2 the viscosity decreased rapidly, suggesting that the F-actin filaments were broken in the smaller fragments or that they transformed to Mg-polymers. A factor with properties similar to Physarum actinin was isolated from acetone powder of sea urchin eggs.     

Toxofilin, a novel actin-binding protein from Toxoplasma gondii, sequesters actin monomers and caps actin filaments

Toxoplasma gondii relies on its actin cytoskeleton to glide and enter its host cell. However, T. gondii tachyzoites are known to display a strikingly low amount of actin filaments, which suggests that sequestration of actin monomers could play a key role in parasite actin dynamics. We isolated a 27-kDa tachyzoite protein on the basis of its ability to bind muscle G-actin and demonstrated that it interacts with parasite G-actin. Cloning and sequence analysis of the gene coding for this protein, which we named Toxofilin, showed that it is a novel actin-binding protein. In in vitro assays, Toxofilin not only bound to G-actin and inhibited actin polymerization as an actin-sequestering protein but also slowed down F-actin disassembly through a filament end capping activity. In addition, when green fluorescent protein-tagged Toxofilin was overexpressed in mammalian nonmuscle cells, the dynamics of actin stress fibers was drastically impaired, whereas green fluorescent protein-Toxofilin copurified with G-actin. Finally, in motile parasites, during gliding or host cell entry, Toxofilin was localized in the entire cytoplasm, including the rear end of the parasite, whereas in intracellular tachyzoites, especially before they exit from the parasitophorous vacuole of their host cell, Toxofilin was found to be restricted to the apical end.