Actin-binding proteins from Drosophila embryos: a complex network of interacting proteins detected by F-actin affinity chromatography

By using F-actin affinity chromatography columns to select proteins solely by their ability to bind to actin filaments, we have identified and partially purified greater than 40 proteins from early Drosophila embryos. These proteins represent approximately 0.5% of the total protein present in soluble cell extracts, and 2 mg are obtained by chromatography of an extract from 10 g of embryos. As judged by immunofluorescence of fixed embryos, 90% of the proteins that we have detected in F-actin column eluates are actin-associated in vivo (12 of 13 proteins tested). The distributions of antigens observed suggest that groups of these proteins cooperate in generating unique actin structures at different places in the cell. These structures change as cells progress through the cell cycle and as they undergo the specializations that accompany development. The variety of different spatial localizations that we have observed in a small subset of the total actin-binding proteins suggests that the actin cytoskeleton is a very complex network of interacting proteins.     

The COOH terminus of the c-Abl tyrosine kinase contains distinct F- and G-actin binding domains with bundling activity [published erratum appears in J Cell Biol 1994 Mar;124(5):865]

The myristoylated form of c-Abl protein, as well as the P210bcr/abl protein, have been shown by indirect immunofluorescence to associate with F-actin stress fibers in fibroblasts. Analysis of deletion mutants of c-Abl stably expressed in fibroblasts maps the domain responsible for this interaction to the extreme COOH-terminus of Abl. This domain mediates the association of a heterologous protein with F-actin filaments after microinjection into NIH 3T3 cells, and directly binds to F-actin in a cosedimentation assay. Microinjection and cosedimentation assays localize the actin-binding domain to a 58 amino acid region, including a charged motif at the extreme COOH-terminus that is important for efficient binding. F-actin binding by Abl is calcium independent, and Abl competes with gelsolin for binding to F- actin. In addition to the F-actin binding domain, the COOH-terminus of Abl contains a proline-rich region that mediates binding and sequestration of G-actin, and the Abl F- and G-actin binding domains cooperate to bundle F-actin filaments in vitro. The COOH terminus of Abl thus confers several novel localizing functions upon the protein, including actin binding, nuclear localization, and DNA binding. Abl may modify and receive signals from the F-actin cytoskeleton in vivo, and is an ideal candidate to mediate signal transduction from the cell surface and cytoskeleton to the nucleus.     

Evolutionary conservation of actin-binding proteins in Trypanosoma cruzi and unusual subcellular localization of the actin homologue

The actin cytoskeleton controls pivotal cellular processes such as motility and cytokinesis, as well as cell-cell and cell-substrate interactions. Assembly and spatial organization of actin filaments are dynamic events regulated by a large repertoire of actin-binding proteins. This report presents the first detailed characterization of the Trypanosoma cruzi actin (TcActin). Protein sequence analysis and homology modelling revealed that the overall structure of T. cruzi actin is conserved and that the majority of amino-acid changes are concentrated on the monomer surface. Immunofluorescence assays using specific polyclonal antibody against TcActin revealed numerous rounded and punctated structures spread all over the parasitic body. No pattern differences could be found between epimastigotes and trypomastigotes or amastigotes. Moreover, in detergent extracts, TcActin was localized only in the soluble fraction, indicating its presence in the G-actin form or in short filaments dissociated from the microtubule cytoskeleton. The trypanosomatid genome was prospected to identify actin-binding and actin-related conserved proteins. The main proteins responsible for actin nucleation and treadmilling in higher eukaryotes are conserved in T. cruzi.     

Regulation of Actin Dynamics in Pollen Tubes: Control of Actin Polymer Level

Actin cytoskeleton undergoes rapid reorganization in response to internal and external cues. How the dynamics of actin cytoskeleton are regulated, and how its dynamics relate to its function are fundamental questions in plant cell biology. The pollen tube is a well characterized actin-based cell morphogenesis in plants. One of the striking features of actin cytoskeleton characterized in the pollen tube is its surprisingly low level of actin polymer. This special phenomenon might relate to the function of actin cytoskeleton in pollen tubes. Understanding the molecular mechanism underlying this special phenomenon requires careful analysis of actin-binding proteins that modulate actin dynamics directly. Recent biochemical and biophysical analyses of several highly conserved plant actin-binding proteins reveal unusual and unexpected properties, which emphasizes the importance of carefully analyzing their action mechanism and cellular activity. In this review, we highlight an actin monomer sequestering protein, a barbed end capping protein and an F-actin severing and dynamizing protein in plant. We propose that these proteins function in harmony to regulate actin dynamics and maintain the low level of actin polymer in pollen tubes.     

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.     

Cofilin Changes the Twist of F-Actin: Implications for Actin Filament Dynamics and Cellular Function

Cofilin is an actin depolymerizing protein found widely distributed in animals and plants. We have used electron cryomicroscopy and helical reconstruction to identify its binding site on actin filaments. Cofilin binds filamentous (F)-actin cooperatively by bridging two longitudinally associated actin subunits. The binding site is centered axially at subdomain 2 of the lower actin subunit and radially at the cleft between subdomains 1 and 3 of the upper actin subunit. Our work has revealed a totally unexpected (and unique) property of cofilin, namely, its ability to change filament twist. As a consequence of this change in twist, filaments decorated with cofilin have much shorter ‘actin crossovers' (~75% of those normally observed in F-actin structures). Although their binding sites are distinct, cofilin and phalloidin do not bind simultaneously to F-actin. This is the first demonstration of a protein that excludes another actin-binding molecule by changing filament twist. Alteration of F-actin structure by cofilin/ADF appears to be a novel mechanism through which the actin cytoskeleton may be regulated or remodeled.     

Role of actin cytoskeleton in dendritic spine morphogenesis

Dendritic spines are the postsynaptic receptive regions of most excitatory synapses, and their morphological plasticity play a pivotal role in higher brain functions, such as learning and memory. The dynamics of spine morphology is due to the actin cytoskeleton concentrated highly in spines. Filopodia, which are thin and headless protrusions, are thought to be precursors of dendritic spines. Drebrin, a spine-resident side-binding protein of filamentous actin (F-actin), is responsible for recruiting F-actin and PSD-95 into filopodia, and is suggested to govern spine morphogenesis. Interestingly, some recent studies on neurological disorders accompanied by cognitive deficits suggested that the loss of drebrin from dendritic spines is a common pathognomonic feature of synaptic dysfunction. In this review, to understand the importance of actin-binding proteins in spine morphogenesis, we first outline the well-established knowledge pertaining to the actin cytoskeleton in non-neuronal cells, such as the mechanism of regulation by small GTPases, the equilibrium between globular actin (G-actin) and F-actin, and the distinct roles of various actin-binding proteins. Then, we review the dynamic changes in the localization of drebrin during synaptogenesis and in response to glutamate receptor activation. Because side-binding proteins are located upstream of the regulatory pathway for actin organization via other actin-binding proteins, we discuss the significance of drebrin in the regulatory mechanism of spine morphology through the reorganization of the actin cytoskeleton. In addition, we discuss the possible involvement of an actin-myosin interaction in the morphological plasticity of spines.     

hhLIM is a novel F-actin binding protein involved in actin cytoskeleton remodeling

Human heart LIM protein (hhLIM) is a newly cloned protein. In vitro analyses showed that green fluorescent protein (GFP)-tagged hhLIM protein accumulated in the cytoplasm of C2C12 cells and colocalized with F-actin, indicating that hhLIM is an actin-binding protein in C2C12 cells. Overexpression of hhLIM-GFP in C2C12 cells significantly stabilized actin filaments and delayed depolymerization of the actin cytoskeleton induced by cytochalasin B treatment. Expression of hhLIM-GFP in C2C12 cells also induced significant changes in the organization of the actin cytoskeleton, specifically, fewer and thicker actin bundles than in control cells, suggesting that hhLIM functions as an actin-bundling protein. This hypothesis was confirmed using low-speed co-sedimentation assays and direct observation of F-actin bundles that formed in vitro in the presence of hhLIM. hhLIM has two LIM domains. To identify the essential regions and sites for association, a series of truncated mutants was constructed which showed that LIM domain 2 has the same activity as full-length hhLIM. To further characterize the binding sites, the LIM domain was functionally destructed by replacing cysteine with serine in domain 2, and results showed that the second LIM domain plays a central role in bundling of F-actin. Taken together, these data identify hhLIM as an actin-binding protein that increases actin cytoskeleton stability by promoting bundling of actin filaments.     

Stable interaction between G-actin and neurofilament light subunit in dopaminergic neurons

Excessive accumulation of neurofilaments in the cell bodies and proximal axons of motor neurons is a major pathological hallmark of motor neuron diseases. In this communication we provide evidence that the neurofilament light subunit (68 kDa) and G-actin are capable of forming a stable interaction. Cytochalasin B, a cytoskeleton disrupting agent that interrupts actin-based microfilaments, caused aggregation of neurofilaments in cultured mesencephalic dopaminergic neurons, suggesting a possible interaction between neurofilaments and actin; which was tested further by using crosslinking reaction and affinity chromatography techniques. In the cross-linking experiment, G-actin interacted with individual neurofilament subunits and covalently cross-linked disuccinimidyl suberate, a homobifunctional cross-linking reagent. Furthermore, G-actin was extensively cross-linked to the light neurofilament subunit with this reagent. The other two neurofilament subunits showed no cross-linking to G-actin. Moreover, neurofilament subunits were retained on a G-actin coupled affinity column and were eluted from this column by increasing salt concentration. All three neurofilament subunits became bound to the G-actin affinity column. However, a portion of the 160 and 200 kDa neurofilament subunits did not bind to the column, and the remainder of these two subunits eluted prior to the 68 kDa subunit, suggesting that the light subunit exhibited the highest affinity for G-actin. Moreover, neurofilaments demonstrated little or no binding to F-actin coupled affinity columns. The phosphorylation of neurofilament proteins with protein kinase C reduced its cross-linking to G-actin. The results of these studies are interpreted to suggest that the interaction between neurofilaments and actin, regulated by neurofilament phosphorylation, may play a role in maintaining the structure and hence the function of dopaminergic neurons in culture.     

The Actin Cytoskeleton and Signaling Network during Pollen Tube Tip Growth

The organization and dynamics of the actin cytoskeleton play key roles in many aspects of plant cell development. The actin cytoskeleton responds to internal developmental cues and en-vironmental signals and is involved in cell division, subcellular organelle movement, cell polarity and polar cell growth. The tip-growing pollen tubes provide an ideal model system to investigate fundamental mechanisms of underlying polarized cell growth. In this system, most signaling cascades required for tip growth, such as Ca~(2+)-, small GTPases- and lipid-mediated signaling have been found to be involved in transmitting signals to a large group of actin-binding proteins. These actin-binding proteins subsequently regulate the structure of the actin network, as well as the rapid turnover of actin filaments (F-actin), thereby eventually controlling tip growth. The actin cytoskeleton acts as an integrator in which multiple signaling pathways converge, providing a general growth and regulatory mechanism that applies not only for tip growth but also for polarized diffuse growth in plants.     

Evidence for a Conformational Change in Actin Induced by Fimbrin (N375) Binding

Fimbrin belongs to a superfamily of actin cross-linking proteins that share a conserved 27-kD actin-binding domain. This domain contains a tandem duplication of a sequence that is homologous to calponin. Calponin homology (CH) domains not only cross-link actin filaments into bundles and networks, but they also bind intermediate filaments and some signal transduction proteins to the actin cytoskeleton. This fundamental role of CH domains as a widely used actin-binding domain underlines the necessity to understand their structural interaction with actin. Using electron cryomicroscopy, we have determined the three-dimensional structure of F-actin and F-actin decorated with the NH₂-terminal CH domains of fimbrin (N375). In a difference map between actin filaments and N375-decorated actin, one end of N375 is bound to a concave surface formed between actin subdomains 1 and 2 on two neighboring actin monomers. In addition, a fit of the atomic model for the actin filament to the maps reveals the actin residues that line, the binding surface. The binding of N375 changes actin, which we interpret as a movement of subdomain 1 away from the bound N375. This change in actin structure may affect its affinity for other actin-binding proteins and may be part of the regulation of the cytoskeleton itself. Difference maps between actin and actin decorated with other proteins provides a way to look for novel structural changes in actin.     

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.     

Interaction of the low-molecular-weight GTP-binding protein rap2 with the platelet cytoskeleton is mediated by direct binding to the actin filaments

The interaction of the low-molecular-weight GTP-binding protein rap2 with the cytoskeleton from thrombin-aggregated platelets was investigated by inducing depolymerization of the actin filaments, followed by in vitro-promoted repolymerization. We found that the association of rap2 with the cytoskeleton was spontaneously restored after one cycle of actin depolymerization and repolymerization. Exogenous rap2, but not unrelated proteins, added to depolymerized actin and solubilized actin-binding proteins, was also specifically incorporated into the in vitro reconstituted cytoskeleton. The incorporation of exogenous rap2 was also observed when the cytoskeleton from resting or thrombin-activated platelets was subjected to actin depolymerization-repolymerization. Moreover, such interaction occurred equally well when exogenous rap2 was loaded with either GDP or GTPgammaS. We also found that polyhistidine-tagged rap2 immobilized on Ni(2+)-Sepharose and loaded with either GDP or GTPgammaS, could specifically bind to cytoskeletal actin. Moreover, when purified monomeric actin was induced to polymerize in vitro in the presence of rap2, the small G-protein specifically associated with the actin filaments. Finally, rap2 loaded with either GDP or GTPgammaS was able to bind to purified F-actin immobilized on a plastic surface. These results demonstrate that rap2 interacts with the platelet cytoskeleton by direct binding to the actin filaments and that this interaction is not regulated by the activation state of the protein.     

Spectrin and protein 4.1 as an actin filament capping complex

Spectrin and protein 4.1, when added to G- or F-actin, cause the formation of short filaments, as judged by the appearance of powerful nucleating activity for G-actin polymerisation. F-Actin filaments are rapidly fragmented under physiological solvent conditions. The effect of cytochalasin E on the polymerisation reaction and the extent of reduction in the critical monomer concentration of actin when spectrin and 4.1 are added suggest that these proteins form a capping system for the more slowly growing, or 'pointed' ends of actin filaments. The interaction is not affected by calcium or by 4.9, the remaining constituent of the purified red cell membrane cytoskeleton.     

Regulation of actin cytoskeleton dynamics in cells

The dynamic remolding of the actin cytoskeleton is a critical part of most cellular activities, and malfunction of cytoskeletal proteins results in various human diseases. The transition between two forms of actin, monomeric or G-actin and filamentous or F-actin, is tightly regulated in time and space by a large number of signaling, scaffolding and actin-binding proteins (ABPs). New ABPs are constantly being discovered in the post-genomic era. Most of these proteins are modular, integrating actin binding, protein-protein interaction, membrane-binding, and signaling domains. In response to extracellular signals, often mediated by Rho family GTPases, ABPs control different steps of actin cytoskeleton assembly, including filament nucleation, elongation, severing, capping, and depolymerization. This review summarizes structure-function relationships among ABPs in the regulation of actin cytoskeleton assembly.     

Interactions of tensin with actin and identification of its three distinct actin-binding domains

Tensin, a 200-kD phosphoprotein of focal contacts, contains sequence homologies to Src (SH2 domain), and several actin-binding proteins. These features suggest that tensin may link the cell membrane to the cytoskeleton and respond directly to tyrosine kinase signalling pathways. Here we identify three distinct actin-binding domains within tensin. Recombinant tensin purified after overexpression by a baculovirus system binds to actin filaments with Kd = 0.1 microM, cross- links actin filaments at a molar ratio of 1:10 (tensin/actin), and retards actin assembly by barbed end capping with Kd = 20 nM. Tensin fragments were constructed and expressed as fusion proteins to map domains having these activities. Three regions from tensin interact with actin: two regions composed of amino acids 1 to 263 and 263 to 463, cosediment with F-actin but do not alter the kinetics of actin assembly; a region composed of amino acids 888-989, with sequence homology to insertin, retards actin polymerization. A claw-shaped tensin dimer would have six potential actin-binding sites and could embrace the ends of two actin filaments at focal contacts.     

A review of actin binding proteins: new perspectives

Actin binding proteins (ABPs) have been considered components of the cytoskeleton, which gives structure and allows mobility of the cell. The complex dynamic properties of the actin cytoskeleton are regulated at multiple levels by a variety of proteins that control actin polymerization, severing of actin filaments and cross-linking of actin filaments into networks, which may be used by molecular motors. Proteins that cross-link F-actin are important for the maintenance of the viscoelastic properties of the cytoplasm and for the integrity of plasma membrane-associated macromolecules. Most of these F-actin cross-linking proteins have an actin-binding domain homologous to calponin. In addition, some of them have been considered scaffolds. Through the years, several research groups have found different proteins that interact with ABPs; however, the effect of these interactions on ABPs remains mostly unknown. In addition to organize the cytoskeletal structure, recent data indicate that ABPs can also migrate to the nucleus. This fact is in agreement and could be relevant to the recently found role that actin might play in nuclear function. Recent data and analysis of published results have also indicated that scaffold proteins like filamin A (FLNa) may be processed by proteolysis and that the degradation products generated by this reaction may play a role as signaling molecules, integrating nuclear and cytosolic pathways. Some of the relevant information in this area is reviewed here.     

植物微丝骨架动态变化的调节

由球形肌动蛋白聚合而成的微丝骨架,又称肌动蛋白纤维,它在细胞运动、细胞形态建成以及物质运输等诸多生命活动中发挥重要作用。细胞内微丝的解聚和聚合动态特性是微丝骨架行使功能的重要基础,并受到如微丝结合蛋白、金属离子、小G蛋白等各种因素的严格控制。植物细胞微丝骨架的研究虽然晚于动物细胞,但也取得了飞速发展。本文对植物细胞内微丝骨架动态变化的作用机制及一些主要调节因子的最新研究进展做一介绍。     

The CH-domain of calponin does not determine the modes of calponin binding to F-actin

Many actin-binding proteins have been observed to have a modular architecture. One of the most abundant modules is the calponin-homology (CH) domain, found as tandem repeats in proteins that cross-link actin filaments (such as fimbrin, spectrin and alpha-actinin) or link the actin cytoskeleton to intermediate filaments (such as plectin). In proteins such as the eponymous calponin, IQGAP1, and Scp1, a single CH-domain exists, but there has been some controversy over whether this domain binds to actin filaments. A previous three-dimensional reconstruction of the calponin-F-actin complex has led to the conclusion that the visualized portion of calponin bound to actin belongs to its amino-terminal homology (CH) domain. We show, using a calponin fragment lacking the CH-domain, that this domain is not bound to F-actin, and cannot be positioning calponin on F-actin as hypothesized. Further, using classification methods, we show a multiplicity in cooperative modes of binding of calponin to F-actin, similar to what has been observed for other actin-binding proteins such as tropomyosin and cofilin. Our results suggest that the form and function of the structurally conserved CH-domain found in many other actin-binding proteins have diverged. This has broad implications for inferring function from the presence of structurally conserved domains.