前纤维蛋白影响花粉肌动蛋白体外聚合分析

利用超速离心沉淀及紫外分光光度测定等技术 ,研究了不同比例的玉米 (ZeamaysL .)花粉内源前纤维蛋白对玉米 (ZeamaysL .)花粉肌动蛋白 (前纤维蛋白与肌动蛋白摩尔数比分别为 2∶1,1.5∶1,1∶1,0 .5∶1,0 .1∶1)聚合与解聚的影响。初步实验结果显示 ,前纤维蛋白在各种比例下均可与Mg_ATP_肌动蛋白结合并抑制肌动蛋白的聚合作用。这种抑制作用随着前纤维蛋白比例的增加而增大。其解离常数值 (Kd)为 (1.30± 0 .33) μmol/L。在本实验条件下尚未见到有前纤维蛋白促进植物肌动蛋白聚合的作用 ,表明玉米花粉前纤维蛋白具有螯合G_肌动蛋白的作用。     

ATP和钾离子对肌动蛋白与前纤维蛋白结合的影响及其在植物肌动蛋白纯化中的应用

根据ATP和钾离子影响肌动蛋白聚合及肌动蛋白及肌动蛋白与前纤维蛋白结合的实验结果,通过对以往报道的利用多聚脯氨酸-琼脂糖亲和柱层析法纯化植物肌动蛋白过程中ATP及钾离子浓度的改变,不需要加源前纤维蛋白。可得到较大量高纯度具活性的花粉肌动蛋白。SDS凝胶电泳、免疫印迹、电镜负染及紫外检测表明,所得肌动蛋白在纯度、聚合特性等方面均与原方法所得结果相同,肌动蛋白产率为原方法的73.5%,而整个肌动蛋白提纯过程所需时间缩短了1/3,节省了纯化重组前纤维蛋白所需的费用,消除了外源前纤维蛋白的使用对一些实验室的限制,而且同时得到了大量纯化的花粉前纤维蛋白。     

前纤维蛋白影响花粉肌动蛋白聚合分析

利用超速离心沂淀及紫外分光光度测定等技术,研究了不同比例的玉米（Ｚｅａ ｎａｙｓ Ｌ．）花粉内泊前纤维蛋白对玉米（Ｚｅａ ｍａｙｓ Ｌ．）花烩肌动蛋白（前纤维蛋白与肌动蛋白摩尔数比分别为２：１,１．５：１,１：１,０．５：１,０．１：１）聚合与解聚的影响。初步实验结果显示,前纤维蛋白在各种比例下均可与Ｍｇ＝ＡＴＰ肌动蛋白结合并抑制肌动蛋白的聚实验条件下尚水见玻有前纤维蛋白促进植物肌动蛋白聚合的作用     

植物细胞中的前纤维蛋白

肌动蛋白组成的微丝骨架是真核细胞中的重要结构,在体内处于高度动态变化之中,受多种肌动蛋白结合蛋白（actin-binding proteins）的调节。前纤维蛋白（profilin）是一种单体肌动蛋白结合蛋白,存在于所有的真核细胞中,在植物细胞中也得到较多的研究。前纤维蛋白除可以结合单体肌动蛋白之外,还可以与磷脂酰肌醇及富含多聚脯氨酸的蛋白质等多种分子结合,在细胞信号转导中行使着重要的功能。本文结合本实验室的研究结果,概述了前纤维蛋白的最新研究进展。     

粗糙脉孢菌前纤维蛋白Y86和R88位点突变抑制菌丝生长

微丝骨架在真菌的生长发育过程中发挥着重要的作用,而动力学特性是其实现功能的关键．前纤维蛋白（profilin）是肌动蛋白动态组装的主要调控因子,对其功能研究有助于阐明微丝骨架在真菌生长发育中的机制．本文以丝状真菌模式生物粗糙脉孢菌(Neurospora crassa)为材料,利用定点突变技术和同源重组技术,分别将前纤维蛋白上肌动蛋白结合位点86位酪氨酸（Y86）和88位精氨酸（R88）进行了单突变和双突变,获得了Y86R、R88E和Y86RR86E前纤维蛋白点突变株．进一步利用平板培养和竞争性生长管培养对点突变株的表型进行分析后发现,与野生型相比,3个前纤维蛋白点突变株的菌丝生长均明显减慢．这些结果表明,前纤维蛋白与肌动蛋白的相互作用对于N. crassa的生长和发育至关重要．     

植物的肌动蛋白结合蛋白

文章介绍植物细胞内几类肌动蛋白结合蛋白（如：前纤维蛋白、形成素、肌动蛋白相关蛋白2／3复合体、肌动蛋白解聚因子和成束蛋白）的结构、性质和功能的研究进展。     

微丝骨架的构成及其对花粉管极性生长的调控作用

微丝骨架是细胞骨架的重要组成部分,它由肌动蛋白和肌动蛋白结合蛋白组成,广泛存在于真核细胞中。近年来,大量研究表明植物花粉及花粉管中存在丰富的微丝骨架。目前,在微丝骨架作为信号转导途径的靶标参与对花粉管极性生长的调控、微丝骨架在花粉和花粉管中的分布及其在花粉管生长过程中与其他信号分子之间的相互作用等方面取得了一系列突破性进展。     

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。     

心肌肌钙蛋白Ⅰ基因突变与心肌病的研究进展

心肌肌钙蛋白I(cTnI)是心肌肌钙蛋白复合物的亚单位之一,与心肌肌钙蛋白T和C相互作用,与肌动蛋白-原肌球蛋白结合从而抑制肌动蛋白-肌球蛋白的收缩作用。在肥厚型、扩张型和限制型心肌病中发现30多种cTnI基因的突变,cTnI基因突变转基因小鼠也反映了心肌病的特征。本文总结了cTnI基因突变在心肌病发病机制中的研究情况。     

IRSp53和MIM同源结构域IMD相互作用蛋白质的鉴定

IRSp53(胰岛素受体酪氨酸激酶底物)和MIM(肿瘤转移消失蛋白)的同源结构域(IMD结构域)在IR-Sp53/MIM家族调控肌动蛋白动态变化的过程中起重要作用.IMD结构域具有使肌动蛋白纤维成束的活性,与小分子GTPase家族Racl也存在相互作用.然而,IMD结构域是否存在其它相互作用蛋白并不清楚.本研究利用GST pull down技术结合质谱分析从大鼠小脑中筛选IMD结构域的相互作用蛋白,得到了几个候选蛋白.其中,神经发育中下调蛋白NEDD9与IRSp53及MIM在小脑中存在类似的分布,体外实验进一步证明了两者之间的相互作用,暗示NEDD9是一种新的IMD结构域相互作用蛋白质.     

Effect of tropomyosin on formin-bound actin filaments

Formins are conservative proteins with important roles in the regulation of the microfilament system in eukaryotic cells. Previous studies showed that the binding of formins to actin made the structure of actin filaments more flexible. Here, the effects of tropomyosin on formin-induced changes in actin filaments were investigated using fluorescence spectroscopic methods. The temperature dependence of the Förster-type resonance energy transfer showed that the formin-induced increase of flexibility of actin filaments was diminished by the binding of tropomyosin to actin. Fluorescence anisotropy decay measurements also revealed that the structure of flexible formin-bound actin filaments was stabilized by the binding of tropomyosin. The stabilizing effect reached its maximum when all binding sites on actin were occupied by tropomyosin. The effect of tropomyosin on actin filaments was independent of ionic strength, but became stronger as the magnesium concentration increased. Based on these observations, we propose that in cells there is a molecular mechanism in which tropomyosin binding to actin plays an important role in forming mechanically stable actin filaments, even in the case of formin-induced rapid filament assembly.     

A new model of cooperative myosin-thin filament binding

Cooperative myosin binding to the thin filament is critical to regulation of cardiac and skeletal muscle contraction. This report delineates and fits to experimental data a new model of this process, in which specific tropomyosin-actin interactions are important, the tropomyosin-tropomyosin polymer is continuous rather than disjointed, and tropomyosin affects myosin-actin binding by shifting among three positions as in recent structural studies. A myosin- and tropomyosin-induced conformational change in actin is proposed, rationalizing the approximately 10,000-fold strengthening effect of myosin on tropomyosin-actin binding. Also, myosin S1 binding to regulated filaments containing mutant tropomyosins with internal deletions exhibited exaggerated cooperativity, implying an allosteric effect of tropomyosin on actin and allowing the effect's measurement. Comparisons among the mutants suggest the change in actin is promoted much more strongly by the middle of tropomyosin than by its ends. Regardless of calcium binding to troponin, this change in actin facilitates the shift in tropomyosin position to the actin inner domain, which is required for tight myosin-actin association. It also increases myosin-actin affinity 7-fold compared with the absence of troponin-tropomyosin. Finally, initiation of a shift in tropomyosin position is 100-fold more difficult than is its extension from one actin to the next, producing the myosin binding cooperativity that underlies cooperative activation of muscle contraction.     

Structural and functional aspects of filamins

Filamins are a family of high molecular mass cytoskeletal proteins that organize filamentous actin in networks and stress fibers. Over the past few years it has become clear that filamins anchor various transmembrane proteins to the actin cytoskeleton and provide a scaffold for a wide range of cytoplasmic signaling proteins. The recent cloning of three human filamins and studies on filamin orthologues from chicken and Drosophila revealed unexpected complexity of the filamin family, the biological implications of which have just started to be addressed. Expression of dysfunctional filamin-A leads to the genetic disorder of ventricular heterotopia and gives reason to expect that abnormalities in the other isogenes may also be connected with human disease. In this review aspects of filamin structure, its splice variants, binding partners and biological function will be discussed.     

A genetic dissection of Aip1p's interactions leads to a model for Aip1p-cofilin cooperative activities

Actin interacting protein 1 (Aip1p) and cofilin cooperate to disassemble actin filaments in vitro and are thought to promote rapid turnover of actin networks in vivo. The precise method by which Aip1p participates in these activities has not been defined, although severing and barbed-end capping of actin filaments have been proposed. To better describe the mechanisms and biological consequences of Aip1p activities, we undertook an extensive mutagenesis of AIP1 aimed at disrupting and mapping Aip1p interactions. Site-directed mutagenesis suggested that Aip1p has two actin binding sites, the primary actin binding site lies on the edge of its N-terminal beta-propeller and a secondary actin binding site lies in a comparable location on its C-terminal beta-propeller. Random mutagenesis followed by screening for separation of function mutants led to the identification of several mutants specifically defective for interacting with cofilin but still able to interact with actin. These mutants suggested that cofilin binds across the cleft between the two propeller domains, leaving the actin binding sites exposed and flanking the cofilin binding site. Biochemical, genetic, and cell biological analyses confirmed that the actin binding- and cofilin binding-specific mutants are functionally defective, whereas the genetic analyses further suggested a role for Aip1p in an early, internalization step of endocytosis. A complementary, unbiased molecular modeling approach was used to derive putative structures for the Aip1p-cofilin complex, the most stable of which is completely consistent with the mutagenesis data. We theorize that Aip1p-severing activity may involve simultaneous binding to two actin subunits with cofilin wedged between the two actin binding sites of the N- and C-terminal propeller domains.     

Cortactin phosphorylation as a switch for actin cytoskeletal network and cell dynamics control

Cortactin is an important molecular scaffold for actin assembly and organization. Novel mechanistic functions of cortactin have emerged with more interacting partners identified, revealing its multifaceted roles in regulating actin cytoskeletal networks that are necessary for endocytosis, cell migration and invasion, adhesion, synaptic organization and cell morphogenesis. These processes are mediated by its multi-domains binding to F-actin and Arp2/3 complex and various SH3 targets. Furthermore, its role in actin remodeling is subjected to regulation by tyrosine and serine/threonine kinases. Elucidating the mechanisms underlying cortactin phosphorylation and its functional consequences would provide new insights to various aspects of cell dynamics control.     

Utrophin binds laterally along actin filaments and can couple costameric actin with sarcolemma when overexpressed in dystrophin-deficient muscle

Dystrophin is widely thought to mechanically link the cortical cytoskeleton with the muscle sarcolemma. Although the dystrophin homolog utrophin can functionally compensate for dystrophin in mice, recent studies question whether utrophin can bind laterally along actin filaments and anchor filaments to the sarcolemma. Herein, we have expressed full-length recombinant utrophin and show that the purified protein is fully soluble with a native molecular weight and molecular dimensions indicative of monomers. We demonstrate that like dystrophin, utrophin can form an extensive lateral association with actin filaments and protect actin filaments from depolymerization in vitro. However, utrophin binds laterally along actin filaments through contribution of acidic spectrin-like repeats rather than the cluster of basic repeats used by dystrophin. We also show that the defective linkage between costameric actin filaments and the sarcolemma in dystrophin-deficient mdx muscle is rescued by overexpression of utrophin. Our results demonstrate that utrophin and dystrophin are functionally interchangeable actin binding proteins, but that the molecular epitopes important for filament binding differ between the two proteins. More generally, our results raise the possibility that spectrin-like repeats may enable some members of the plakin family of cytolinkers to laterally bind and stabilize actin filaments.     

Rapid Nucleotide Exchange Renders Asp-11 Mutant Actins Resistant to Depolymerizing Activity of Cofilin,Leading to Dominant Toxicity in Vivo

Conserved Asp-11 of actin is a part of the nucleotide binding pocket, and its mutation to Gln is dominant lethal in yeast, whereas the mutation to Asn in human α-actin dominantly causes congenital myopathy. To elucidate the molecular mechanism of those dominant negative effects, we prepared Dictyostelium versions of D11N and D11Q mutant actins and characterized them in vitro. D11N and D11Q actins underwent salt-dependent reversible polymerization, although the resultant polymerization products contained small anomalous structures in addition to filaments of normal appearance. Both monomeric and polymeric D11Q actin released bound nucleotides more rapidly than the wild type, and intriguingly, both monomeric and polymeric D11Q actins hardly bound cofilin. The deficiency in cofilin binding can be explained by rapid exchange of bound nucleotide with ATP in solution, because cofilin does not bind ATP-bound actin. Copolymers of D11Q and wild type actins bound cofilin, but cofilin-induced depolymerization of the copolymers was slower than that of wild type filaments, which may presumably be the primary reason why this mutant actin is dominantly toxic in vivo. Purified D11N actin was unstable, which made its quantitative biochemical characterization difficult. However, monomeric D11N actin released nucleotides even faster than D11Q, and we speculate that D11N actin also exerts its toxic effects in vivo through a defective interaction with cofilin. We have recently found that two other dominant negative actin mutants are also defective in cofilin binding, and we propose that the defective cofilin binder is a major class of dominant negative actin mutants.     

Role of Drosophila gene dunc-115 in nervous system

Axonal guidance signals are transduced through growth cone surface receptors to the interior leading to changes of actin dynamics and actin binding proteins, which are critical in determining the outcome of actin cytoskeleton reorganization. We report here the characterization of the Drosophila actin binding protein abLIM/Unc-115 homolog Dunc-115 and its role in the nervous system. Three Dunc-115 isoforms are identified as Dunc-115L, M and S, respectively. While Dunc-115L is a canonical homolog of Unc-115 with four LIM domains and one villin headpiece domain, Dunc-115M and S are novel isoforms without counterparts in other species. Our molecular modeling shows Dunc-115L is likely to bind to actin. Mutant analysis reveals that Dunc-115 is involved in axonal projection in both the visual and central nervous system.     

Troponin Revealed: Uncovering the Structure of the Thin Filament On-Off Switch in Striated Muscle

Recently, our understanding of the structural basis of troponin-tropomyosin’s Ca²⁺-triggered regulation of striated muscle contraction has advanced greatly, particularly via cryo-electron microscopy data. Compelling atomic models of troponin-tropomyosin-actin were published for both apo- and Ca²⁺-saturated states of the cardiac thin filament. Subsequent electron microscopy and computational analyses have supported and further elaborated the findings. Per cryo-electron microscopy, each troponin is highly extended and contacts both tropomyosin strands, which lie on opposite sides of the actin filament. In the apo-state characteristic of relaxed muscle, troponin and tropomyosin hinder strong myosin-actin binding in several different ways, apparently barricading the actin more substantially than does tropomyosin alone. The troponin core domain, the C-terminal third of TnI, and tropomyosin under the influence of a 64-residue helix of TnT located at the overlap of adjacent tropomyosins are all in positions that would hinder strong myosin binding to actin. In the Ca²⁺-saturated state, the TnI C-terminus dissociates from actin and binds in part to TnC; the core domain pivots significantly; the N-lobe of TnC binds specifically to actin and tropomyosin; and tropomyosin rotates partially away from myosin’s binding site on actin. At the overlap domain, Ca²⁺ causes much less tropomyosin movement, so a more inhibitory orientation persists. In the myosin-saturated state of the thin filament, there is a large additional shift in tropomyosin, with molecular interactions now identified between tropomyosin and both actin and myosin. A new era has arrived for investigation of the thin filament and for functional understandings that increasingly accommodate the recent structural results.     

Inhibition of interdomain motion in g-actin by the natural product latrunculin: a molecular dynamics study

As part of the cytoskeleton, actin is essential for the morphology, motility, and division of eukaryotic cells. Recent X-ray fiber diffraction studies have shown that the conformation of monomeric actin is flattened upon incorporation into the filament by a relative rotation of its two major domains. The antiproliferative activity of latrunculin, a macrolide toxin produced by sponges, seems to be related to its binding to monomeric actin and inhibition of polymerization. Yet, the mechanism of inhibition is not known in detail. Here, multiple explicit water molecular dynamics simulations show that latrunculin binding hinders the conformational transition related to actin polymerization. In particular, the presence of latrunculin at the interface of the two major domains of monomeric actin reduces the correlated displacement of Domain 2 with respect to Domain 1. Moreover, higher rotational flexibility between the two major domains is observed in the absence of ATP as compared to ATP-bound actin, offering a possible explanation as to why actin polymerizes more favorably in the absence of nucleotides.