Vinculin Nucleates Actin Polymerization and Modifies Actin Filament Structure

Vinculin links integrins to the actin cytoskeleton by binding F-actin. Little is known with respect to how this interaction occurs or affects actin dynamics. Here we assess the consequence of the vinculin tail (VT) on actin dynamics by examining its binding to monomeric and filamentous yeast actins. VT causes pyrene-labeled G-actin to polymerize in low ionic strength buffer (G-buffer), conditions that normally do not promote actin polymerization. Analysis by electron microscopy shows that, under these conditions, the filaments form small bundles at low VT concentrations, which gradually increase in size until saturation occurs at a ratio of 2 VT:1 actin. Addition of VT to pyrene-labeled mutant yeast G-actin (S265C) produced a fluorescence excimer band, which requires a relatively normal filament geometry. In higher ionic strength polymerization-promoting F-buffer, substoichiometric amounts of VT accelerate the polymerization of pyrene-labeled WT actin. However, the amplitude of the pyrene fluorescence caused by actin polymerization is quenched as the VT concentration increases without an effect on net actin polymerization as determined by centrifugation assays. Finally, addition of VT to preformed pyrene-labeled S265C F-actin causes a concentration-dependent decrease in the maximum amplitude of the pyrene fluorescence band demonstrating the ability of VT to remodel the conformation of the actin filament. These observations support the idea that vinculin can link adhesion plaques to the cytoskeleton by initiating the formation of bundled actin filaments or by remodeling existing filaments.Cell migration is critical for embryonic development, adult homeostasis, inflammatory responses, and wound healing. To migrate, a cell must coordinate a number of different inputs into appropriate cellular responses. The cell must polarize in the direction of migration and extend lamellipodial and/or filopodial protrusions. Nascent adhesions that assemble within the branched actin network of the lamellipodium must link to the underlying actin cytoskeleton. This process allows for the maturation of adhesions to structures that anchor the protrusion. These adhesions also provide the traction forces necessary to pull the cell body forward and break older adhesions at the cell rear. Perturbation of any of these events affects a cell''s migratory ability. For example, nascent adhesions that do not form linkages to the actin cytoskeleton cannot effectively anchor the protrusion to the substratum. The result is an extension that folds back upon itself, forming a membrane ruffle that cannot provide the traction forces necessary for migration.How adhesions establish links to the underlying actin cytoskeleton has been an area of intense investigation. Integrin-containing structures are active areas of actin polymerization suggesting that adhesion plaques can initiate actin filament formation (reviewed in Refs. 1–3). Focal complexes are small integrin clusters that are found exclusively at the tips of lamellipodia and filopodia. Formation of these structures is closely coupled with actin assembly in protruding regions of cells. Accumulating evidence indicates that adhesion complex components recruit the Arp2/3 complex, a potent nucleator of actin polymerization. Our work (4) and that of others (5–7) demonstrates that the Arp2/3 complex is recruited to focal complexes or transient adhesion structures reminiscent of focal complexes by binding vinculin. FAK has also been implicated in linking focal complexes to the actin cytoskeleton by virtue of its ability to recruit and activate the Arp2/3 complex (8). Furthermore, efficient focal complex assembly requires the actin-binding protein, cortactin, which could affect adhesion assembly by interacting with the Arp2/3 complex (9). Hence, many of the known mechanisms for initiating filament formation involve recruitment of the Arp2/3 complex, which initiates the formation of branched actin filaments (55). It is surprising then that the earliest detectable forms of actin-associated adhesions are interconnected by short actin bundles, not branched filaments (10). These observations suggest that our current understanding for how nascent adhesions initiate filament formation is incomplete.The earliest detectable actin-associated adhesions are “dots or doublets of dots” and are highly enriched in integrins, paxillin, and vinculin (10), suggesting that one of these molecules has the capability to initiate actin filament formation from such a plaque. Vinculin has long been implicated in linking adhesion plaques to the actin cytoskeleton by virtue of the ability of its tail to bind (11) and bundle F-actin (12). The interaction of vinculin with actin has been extensively studied from the perspective of vinculin (11, 13–23). Studies of recombinant proteins identified two regions of the vinculin tail (VT)² that bind F-actin independently (21, 17), but mapping these sites onto the VT crystal structure reveals that these peptides do not correspond to distinct sites (25). Upon binding actin, vinculin undergoes a conformational change that promotes dimerization suggesting that vinculin self-association may be important for its bundling activities (15).Less is known with respect to the effect of vinculin on actin filament formation and structure. This lack of knowledge stems from the fact that many of the early studies showed vinculin to have no effect on actin dynamics (26–28). However, these experiments were performed using chicken gizzard vinculin, which exists almost exclusively in a conformation where the actin binding sites are inaccessible, or from preparations that contain contaminants that produce false negatives (29). More recently, recombinant VT proteins were shown to cross-link and bundle actin (23). However, the interaction of vinculin with G-actin and the effect of vinculin on actin filament dynamics have not been explored. In this study, we have assessed the interaction of vinculin with pyrene-labeled wild-type and mutant yeast actins. We show that the VT can promote the formation of an actin nucleus from which filaments arise and alter the assembly and structure of actin filaments. These findings provide novel insights into how adhesion plaques may be linked to the actin cytoskeleton.     

Actin Cytoskeleton: A Team Effort during Actin Assembly

Two recent studies highlight how tandems of previously described actin nucleators collaborate to produce new actin filaments. One key player in these collaborations is formin, which appears to function as a modulator of filament elongation.     

Actin Filament Length Tunes Elasticity of Flexibly Cross-Linked Actin Networks

Networks of the cytoskeletal biopolymer actin cross-linked by the compliant protein filamin form soft gels that stiffen dramatically under shear stress. We demonstrate that the elasticity of these networks shows a strong dependence on the mean length of the actin polymers, unlike networks with small, rigid cross-links. This behavior is in agreement with a model of rigid filaments connected by multiple flexible linkers.     

Preparation of Actin and Fluorescent Actin Analogs from Plant Cells

The plant actin cytoskeleton provides a dynamic cytoplasmic framework for many fundamental cellular processes like cytoplasmic streaming,cytokinesis and morphogenesis.Understanding the actin organization and structure in plants requires the generation of new probes for measuring actin dynamics in living cells. Fluorescent analog cytochemistry presents an unrivaled opportunity to probe the actin cytoskeleton in living cells. Such method using in the study of plant actin cytoskeleton has not been reported. By using this method, based on the affinity chromatography of profilin with PLP-Sepharose (PLP: poly-L-proline) for actin purification, the author obtained 6 mg of > 98% in purity, polymerizable actin from 10 g of maize (Zea mays L. ) pollen, and this actin was successfully labeled with Oregon Green 488 carboxylic acid. From 10 g of maize pollen, 1.2 mg with 60 % dye/protein ratio, polymerizable, fluorescent actin analog was obtained. The study yields an effective method for purifying plant actin and preparing fluorescent analog, which may provide facilities for the study of actin dynamics in plant ceils.     

Actin in oogenesis

Summary

The laser-scanning confocal microscope employed in conjunction with various specific agents and antibodies conjugated to fluorescent dyes reveals details of the actin scaffolding of developing oocytes and the nuclei of attendant cells. The employment of DNase I followed by anti-DNase I antibody has been particularly useful in revealing otherwise cryptic actin-containing structures. The cortical cytoskeleton of developing moth eggs was found to bind both poly (A)+RNA and RNA Pol II. Exposure to cytochalasin D disrupted the actin of the cortex, and at the same time caused redistribution of the proteins and RNA associated with the cytoskeleton. Cytochalasin also had dramatic effects on the structure of nuclei of nurse and follicle cells. Taken in context of the actin network in nuclei uncovered by DNase-anti-DNase treatment, these results suggest that actin plays a major structural and perhaps functional role in insect nuclei.     

肌动蛋白结合蛋白

肌动蛋白结合蛋白是一类调节肌动蛋白聚合、成束或交联的蛋白质,迄今已经发现160多种。通过与肌动蛋白相互作用,直接或间接参与肌动蛋白纤丝的聚合及解聚、纤丝成束与交联,从而介导细胞形态的维持、细胞运动等众多生物学功能。     

Actin on trafficking

During an infection, neutrophils are the first immune cells to arrive armed to clear the invading pathogen. In order to do so, neutrophils need to transmigrate from the peripheral blood through the endothelial layer toward the site of inflammation. This process is in most cases dependent on integrins, adhesion molecules present on all immune cells. These molecules are functionally regulated by “inside-out” signaling, where stimulus-induced signaling pathways act on the intracellular integrin tail to regulate the activity of the receptor on the outside. Both a change in conformation (affinity) and clustering (avidity/valency) of the receptors occurs and many factors have been linked to regulation of integrins on neutrophils. Control of integrin conformation and clustering is of pivotal importance for proper cell adhesion, migration, and bacterial clearance. Recently, gelsolin was found to be involved in β₁-integrin affinity regulation and cell adhesion. Here, I summarize the role of neutrophil integrin regulation in the essential steps to reach the site of inflammation and clearance of bacterial pathogens.     

Actin binding proteins

In order to metastasize away from the primary tumor site and migrate into adjacent tissues, cancer cells will stimulate cellular motility through the regulation of their cytoskeletal structures. Through the coordinated polymerization of actin filaments, these cells will control the geometry of distinct structures, namely lamella, lamellipodia and filopodia, as well as the more recently characterized invadopodia. Because actin binding proteins play fundamental functions in regulating the dynamics of actin polymerization, they have been at the forefront of cancer research. This review focuses on a subset of actin binding proteins involved in the regulation of these cellular structures and protrusions, and presents some general principles summarizing how these proteins may remodel the structure of actin. The main body of this review aims to provide new insights into how the expression of these actin binding proteins is regulated during carcinogenesis and highlights new mechanisms that may be initiated by the metastatic cells to induce aberrant expression of such proteins.     

Actin in Xenopus Development: Indirect Immunofluorescence Study of Actin Localization

Actin was studied in Xenopus unfertilized eggs and early developmental stages. Immunochemical proof is given of structural differences between Xenopus laevis muscle actin and nonmuscle cell actin. Actin localization and changes of actin aggregation during Xenopus development were observed using indirect immunofluorescence. We have also tried to explain the presence of an actin shell around the yolk platelets that appeared in our experiments.     

玉米花粉肌动蛋白与兔骨骼肌肌动蛋白的比较研究

本文对玉米花粉肌动蛋白和兔骨骼肌肌动蛋白进行了比较研究。玉米花粉肌动蛋白与兔骨骼肌肌动蛋白具有相同的分子量(42KD)。玉米花粉肌动蛋白可与兔抗鸡胃肌动蛋白抗血清产生免疫沉淀反应。玉米花粉肌动蛋白与兔骨骼肌肌动蛋白的氨基酸组成以及胰蛋白酶水解所得到的肽谱都相似。它们的羧基未端氨基酸顺序完全一致,其顺序都是Lys.Cys.Phe(COOH)。它们的圆二色谱基本相同,由圆二色谱计算得到的二级结构数据也相近。以上的结果表明了玉米花粉肌动蛋白与兔骨骼肌肌动蛋白的相似性。