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。     

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.     

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 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.     

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.     

The Molecular Evolution of Actin

We have investigated the molecular evolution of plant and nonplant actin genes comparing nucleotide and amino acid sequences of 20 actin genes. Nucleotide changes resulting in amino acid substitutions (replacement substitutions) ranged from 3-7% for all pairwise comparisons of animal actin genes with the following exceptions. Comparisons between higher animal muscle actin gene sequences and comparisons between higher animal cytoplasmic actin gene sequences indicated less than 3% divergence. Comparisons between plant and nonplant actin genes revealed, with two exceptions, 11-15% replacement substitution. In the analysis of plant actins, replacement substitution between soybean actin genes SAc1, SAc3, SAc4 and maize actin gene MAc1 ranged from 8-10%, whereas these members within the soybean actin gene family ranged from 6-9% replacement substitution. The rate of sequence divergence of plant actin sequences appears to be similar to that observed for animal actins. Furthermore, these and other data suggest that the plant actin gene family is ancient and that the families of soybean and maize actin genes have diverged from a single common ancestral plant actin gene that originated long before the divergence of monocots and dicots. The soybean actin multigene family encodes at least three classes of actin. These classes each contain a pair of actin genes that have been designated kappa (SAc1, SAc6), lambda (SAc2, SAc4) and mu (SAc3, SAc7). The three classes of soybean actin are more divergent in nucleotide sequence from one another than higher animal cytoplasmic actin is divergent from muscle actin. The location and distribution of amino acid changes were compared between actin proteins from all sources. A comparison of the hydropathy of all actin sequences, except from Oxytricha, indicated a strong similarity in hydropathic character between all plant and nonplant actins despite the greater number of replacement substitutions in plant actins. These protein sequence comparisons are discussed with respect to the demonstrated and implicated roles of actin in plants and animals, as well as the tissue-specific expression of actin.     

Slow Axonal Transport of Soluble Actin with Actin Depolymerizing Factor, Cofilin, and Profilin Suggests Actin Moves in an Unassembled Form

Abstract: We examined the axonal transport of actin and its monomer binding proteins, actin depolymerizing factor, cofilin, and profilin, in the chicken sciatic nerve following injection of [³⁵S]methionine into the lumbar spinal cord. At intervals up to 20 days after injection, nerves were cut into 1-cm segments and separated into Triton X-100-soluble and particulate fractions. Actin and its binding proteins were then isolated by affinity chromatography on DNase I-Sepharose and by one- and two-dimensional polyacrylamide gel electrophoresis. Fluorographic analysis showed that the specific activity of soluble actin was two to three times that of its particulate form and that soluble actin, cofilin, actin depolymerizing factor, and profilin were transported at similar rates in slow component b of axonal flow. Our data strongly support the view that the mobile form of actin in slow transport is soluble and that a substantial amount of this actin may travel as a complex with actin depolymerizing factor, cofilin, and profilin. Along labeled nerves the specific activity of the unphosphorylated form of actin depolymerizing factor, which binds actin, was not significantly different from that of its "inactive" phosphorylated form. This constancy in specific activity suggests that continuous inactivation and reactivation of actin depolymerizing factor occur during transport, which could contribute to the exchange of soluble actin with the filamentous actin pool.     

Actin in Mucor rouxii

Rhodamine-conjugated phalloidin was used to analyze the actin distribution during hyphal formation in Mucor rouxii. The occurrence of actin patches in the cortical region of the cells was seen in the initial stages of growth. A fungal 43 kDa protein was isolated by affinity chromatography on DNase I-sepharose. This peptide was identified on immunoblots when polyclonal antibodies against rabbit muscle actin were used as a probe. These results indicate: (1) that changes in actin localization accompany the hyphal development and (2) the fungal 43 kDa protein shares properties that are common to muscle actin.     

Actin in Mucor rouxii

Abstract Rhodamine-conjugated phalloidin was used to analyze the actin distribution during hyphal formation in Mucor rouxii . The occurrence of actin patches in the cortical region of the cells was seen in the initial stages of growth. A fungal 43 kDa protein was isolated by affinity chromatography on DNase I-sepharose. This peptide was identified on immunoblots when polyclonal antibodies against rabbit muscle actin were used as a probe. These results indicate: (1) that changes in actin localization accompany the hyphal development and (2) the fungal 43 kDa protein shares properties that are common to muscle actin.     

Actin mRNA Levels and Actin Synthesis During the Encystation of Entamoeba invadens

ABSTRACT. Parasitic amebas propagate among hosts through cysts, the resistant forms in their life cycle. In spite of their key role in infection, little is known about the encystation process and the mechanisms involved in reaching this stage. Two features drastically affected by encystation are motility and cell shape, both of which are determined by the cytoskeleton, composed mainly of actin in these organisms. Therefore, we studied the occurrence and relative levels of actin and actin synthesis during encystation of Entamoeba invadens. Using a cDNA actin probe obtained from a library of E. histolytica and a monoclonal antibody against actin, we found that, while the total actin levels sharply decrease as encystation proceeds, the levels of actin mRNA are reduced only in mature cysts. Moreover, actin synthesis does not take place in precysts and the later stages of cyst formation. In contrast, the levels of other proteins remain stable in trophozoites, precysts and cysts, and stage specific peptides are actively synthesized in precysts. The results indicate that encystation is accompanied by a preferential down-regulation of actin synthesis and a decrease in actin levels. The reorganization of the cytoskeleton occurring as trophozoites transform into round, quiescent cells, could be a regulatory factor in the observed changes.     

LL-37 Induces Polymerization and Bundling of Actin and Affects Actin Structure

Actin exists as a monomer (G-actin) which can be polymerized to filaments) F-actin) that under the influence of actin-binding proteins and polycations bundle and contribute to the formation of the cytoskeleton. Bundled actin from lysed cells increases the viscosity of sputum in lungs of cystic fibrosis patients. The human host defense peptide LL-37 was previously shown to induce actin bundling and was thus hypothesized to contribute to the pathogenicity of this disease. In this work, interactions between actin and the cationic LL-37 were studied by optical, proteolytic and surface plasmon resonance methods and compared to those obtained with scrambled LL-37 and with the cationic protein lysozyme. We show that LL-37 binds strongly to CaATP-G-actin while scrambled LL-37 does not. While LL-37, at superstoichiometric LL-37/actin concentrations polymerizes MgATP-G-actin, at lower non-polymerizing concentrations LL-37 inhibits actin polymerization by MgCl₂ or NaCl. LL-37 bundles Mg-F-actin filaments both at low and physiological ionic strength when in equimolar or higher concentrations than those of actin. The LL-37 induced bundles are significantly less sensitive to increase in ionic strength than those induced by scrambled LL-37 and lysozyme. LL-37 in concentrations lower than those needed for actin polymerization or bundling, accelerates cleavage of both monomer and polymer actin by subtilisin. Our results indicate that the LL-37-actin interaction is partially electrostatic and partially hydrophobic and that a specific actin binding sequence in the peptide is responsible for the hydrophobic interaction. LL-37-induced bundles, which may contribute to the accumulation of sputum in cystic fibrosis, are dissociated very efficiently by DNase-1 and also by cofilin.     

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.     

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.     

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.     

MARCKS-Related Protein Binds to Actin without Significantly Affecting Actin Polymerization or Network Structure

Actinis a 42-kDa protein which, due to its ability to polymerize into filaments (F-actin), is one of the major constituents of the cytoskeleton. It has been proposed that MARCKS (an acronym for myristoylated alanine-rich C kinase substrate) proteins play an important role in regulating the structure and mechanical properties of the actin cytoskeleton by cross-linking actin filaments. We have recently reported that peptides corresponding to the effector domain of MARCKS proteins promote actin polymerization and cause massive bundling of actin filaments. We now investigate the effect of MARCKS-related protein, a 20-kDa member of the MARCKS family, on both filament structure and the kinetics of actin polymerization in vitro. Our experiments document that MRP binds to F-actin with micromolar affinity and that the myristoyl chain at the N-terminus of MRP is not required for this interaction. In marked contrast to the effector peptide, binding of MRP is not accompanied by an acceleration of actin polymerization kinetics, and we also could not reliably observe an actin cross-linking activity of MRP.     

Actin lessons from pathogens

Salmonella typhimurium secretes proteins that co-opt the host actin cytoskeleton to induce membrane ruffling, leading to the uptake of the bacterium. New information about the biochemical activities of the Salmonella protein SipA suggests that this protein might inhibit host cell actin dynamics by competing with ADF/cofilin and gelsolin, two key proteins that promote the turnover of actin filaments.     

Actin from Saccharomyces cerevisiae.

Inhibition of DNase I activity has been used as an assay to purify actin from Saccharomyces cerevisiae (yeast actin). The final fraction, obtained after a 300-fold purification, is approximately 97% pure as judged by sodium dodecyl sulfate-gel electrophoresis. Like rabbit skeletal muscle actin, yeast actin has a molecular weight of about 43,000, forms 7-nm-diameter filaments when polymerization is induced by KCl or Mg2+, and can be decorated with a proteolytic fragment of muscle myosin (heavy meromyosin). Although heavy meromyosin ATPase activity is stimulated by rabbit muscle and yeast actins to approximately the same Vmax (2 mmol of Pi per min per mumol of heavy meromyosin), half-maximal activation (Kapp) is obtained with 14 micro M muscle actin, but requires approximately 135 micro M yeast actin. This difference suggests a low affinity of yeast actin for muscle myosin. Yeast and muscle filamentous actin respond similarly to cytochalasin and phalloidin, although the drugs have no effect on S. cerevisiae cell growth.     

Polymerization of Actin from Maize Pollen

Here we describe the in vitro polymerization of actin from maize (Zea mays) pollen. The purified actin from maize pollen reported in our previous paper (X. Liu, L.F. Yen [1992] Plant Physiol 99: 1151-1155) is biologically active. In the presence of ATP, KCl, and MgCl2 the purified pollen actin polymerized into filaments. During polymerization the spectra of absorbance at 232 nm increased gradually. Polymerization of pollen actin was evidently accompanied by an increase in viscosity of the pollen actin solution. Also, the specific viscosity of pollen F-actin increased in a concentration-dependent manner. The ultraviolet difference spectrum of pollen actin is very similar to that of rabbit muscle actin. The activity of myosin ATPase from rabbit muscle was activated 7-fold by the polymerized pollen actin (F-actin). The actin filaments were visualized under the electron microscope as doubly wound strands of 7 nm diameter. If cytochalasin B was added before staining, no actin filaments were observed. When actin filaments were treated with rabbit heavy meromyosin, the actin filaments were decorated with an arrowhead structure. These results imply that there is much similarity between pollen and muscle actin.     

Human Muscle LIM Protein Dimerizes along the Actin Cytoskeleton and Cross-Links Actin Filaments

The muscle LIM protein (MLP) is a nucleocytoplasmic shuttling protein playing important roles in the regulation of myocyte remodeling and adaptation to hypertrophic stimuli. Missense mutations in human MLP or its ablation in transgenic mice promotes cardiomyopathy and heart failure. The exact function(s) of MLP in the cytoplasmic compartment and the underlying molecular mechanisms remain largely unknown. Here, we provide evidence that MLP autonomously binds to, stabilizes, and bundles actin filaments (AFs) independently of calcium and pH. Using total internal reflection fluorescence microscopy, we have shown how MLP cross-links actin filaments into both unipolar and mixed-polarity bundles. Quantitative analysis of the actin cytoskeleton configuration confirmed that MLP substantially promotes actin bundling in live myoblasts. In addition, bimolecular fluorescence complementation (BiFC) assays revealed MLP self-association. Remarkably, BiFC complexes mostly localize along actin filament-rich structures, such as stress fibers and sarcomeres, supporting a functional link between MLP self-association and actin cross-linking. Finally, we have demonstrated that MLP self-associates through its N-terminal LIM domain, whereas it binds to AFs through its C-terminal LIM domain. Together our data support that MLP contributes to the maintenance of cardiomyocyte cytoarchitecture by a mechanism involving its self-association and actin filament cross-linking.