模拟微重力效应通过微丝骨架调控成骨细胞BMP2-Smad信号

细胞微丝骨架在力信号传导和基因表达调控中起重要作用。为了研究微丝骨架在模拟微重力效应调控成骨细胞BMP2-Smad信号中的作用,作者通过构建反映Smad活性的报告基因载体转染MC3T3-E1细胞,并通过报告基因活性分析、Western blot等方法检测了微丝骨架解聚剂和回转模拟微重力效应对BMP2诱导Samd磷酸化、核质分布和转录活性的作用。结果显示,构建的报告基因载体在成骨细胞中正确表达并响应BMP2;破坏微丝骨架会抑制BMP2诱导的Smad1/5/8蛋白磷酸化、入核及转录活性;回转抑制Smad1/5/8磷酸化、入核及其转录活性,而微丝骨架稳定剂可对抗回转的抑制作用。因此,认为回转模拟微重力效应可通过解聚微丝骨架抑制BMP2-Smad信号传导。     

CX3CL1介导人脐静脉内皮细胞骨架改变的功能研究

该研究探讨了趋化因子CX3CL1对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)骨架的影响及其作用机制。CX3CL1刺激HUVECs后,采用免疫荧光染色技术检测细胞骨架蛋白纤维状肌动蛋白(F-actin)的分布和形态改变,采用Western blot技术检测胞质内F-actin和磷酸化促分裂原活化蛋白激酶(mitogen activated protein kinases,MAPKs)的三种亚型[p38、细胞外调节蛋白激酶1/2(extracellular regulated protein kinase 1/2,ERK1/2)和c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)]的表达水平。结果显示,10 nmol/L CX3CL1刺激HUVECs 30 min后,细胞的致密外周带逐渐被破坏,胞质内有应力纤维形成;120 min后,外周带消失,胞质内有大量的致密应力纤维形成;180 min后,胞质内应力纤维减少,少数细胞可见致密外周带。10 nmol/L CX3CL1刺激HUVECs30 min后,F-actin的表达水平逐渐升高,并于120 min后达峰值;10 nmol/L CX3CL1刺激HUVECs 1 min后,磷酸化p38、ERK1/2和JNK表达水平升高,5 min后三者的表达水平达峰值;5μg/m L抗CX3CR1抗体抑制10 nmol/L CX3CL1刺激HUVECs后,磷酸化p38、ERK1/2和JNK的表达水平降低;30μmol/L p38的特异性抑制剂SB203580和ERK1/2的特异性抑制剂PD98059抑制10 nmol/L CX3CL1刺激HUVECs后,胞质内应力纤维减少,应力纤维变短,F-actin的表达水平降低。以上研究结果表明,CX3CL1能通过p38和ERK1/2信号通路以时间依赖方式介导HUVECs细胞骨架的重构。     

脊髓水平细胞外信号调节激酶激活参与吗啡依赖的形成及戒断反应的表达

在大鼠吗啡依赖和戒断模型上,采用行为学、免疫组织化学和Western blot方法观察吗啡依赖及戒断大鼠脊髓神经元磷酸化细胞外信号调节激酶（phospho-extracellular signal-regulated kinase,pERK）表达的变化,及鞘内注射促分裂原活化蛋白激酶激酶（mitogen-activated protein kinase kinase,MEK）抑制剂U0126或ERK反义寡核苷酸对吗啡依赖大鼠纳洛酮催促戒断反应、触诱发痛及脊髓神经元pERK表达的影响,探讨脊髓水平pERK在介导吗啡依赖和戒断过程中的作用。结果显示：（1）在吗啡依赖形成过程中,大鼠脊髓胞浆与胞核非磷酸化ERK表达没有改变,但pERK表达逐渐增加,纳洛酮催促戒断后,仍有进一步增加的趋势,戒断1h后,其表达量明显下降,但仍高于对照组。（2）鞘内预先注射MEK抑制剂U0126或ERK反义寡核苷酸能明显抑制吗啡戒断反应和戒断引起的痛觉异常;与行为学结果一致,脊髓背角pERK阳性神经元表达与脊髓胞浆和胞核pERK表达也明显降低。上述结果提示,脊髓水平ERK激活和核转位参与吗啡依赖的形成及戒断反应的表达。     

弱极低频磁场对Actin骨架组装效率的频率窗口效应初探

前期研究发现,50 Hz弱磁场辐照能明显降低细胞的微丝含量和组装效率,对actin骨架形态也有明显影响.电磁生物学效应是否与辐照场频率相关,一直受到研究者的关注.单体球状肌动蛋白(G-actin)是带电结构,电磁场频率会影响其振荡频率并对微丝聚合效率产生影响.本文从细胞骨架形态和蛋白质两层次,采用免疫荧光技术考察0.4 m T,在35~140 Hz范围内5个频率的极低频磁场(ELF-MF)对FL细胞中纤维状肌动蛋白(F-actin)含量的影响,并采用荧光共振能量转移技术(FRET)验证效应最明显的频率对离体G-actin组装效率的干扰程度.结果显示,相比假辐照组,细胞中F-actin含量在50 Hz辐照组下降了(34.66±3.14)%,110 Hz次之,而另外3组(35、70和140 Hz)无显著性差异.同时利用FRET方法验证,在50 Hz磁场辐照下,离体环境中G-actin组装成F-actin的效率较假辐照组、35和70 Hz组显著降低.经初步分析,G-actin在弱ELF-MF中受到以洛伦兹力和感生电场力的合力为主的相关电磁力干扰,致使组装效率下降,且由于工频磁场周期与微丝组装周期的特殊相干性,在50 Hz频率附近可能存在一个外磁场干扰actin骨架组装的频率窗口.     

回转模拟失重对心肌成纤维细胞Ⅰ型胶原代谢的影响

为探讨模拟失重对心肌成纤维细胞Ⅰ型胶原代谢的影响 ,本研究采用回转器模拟失重效应 ,通过免疫细胞化学和反转录聚合酶链式反应 (RT PCR)研究了回转模拟失重对原代培养的新生大鼠心肌成纤维细胞Ⅰ型胶原蛋白及mRNA表达 ,以及胶原降解抑制物———金属蛋白酶组织抑制因子 (Tissueinhibitorofmetallopro teinase ,TIMP)mRNA表达的影响。免疫细胞化学染色显示回转组Ⅰ型胶原蛋白沉积增加 ;RT PCR分析显示Ⅰ型胶原α1链 (TypeⅠcollagenα1chain ,ColⅠA1)mRNA表达没有明显变化 ,TIMP 1、TIMP 2及TIMP 3的mRNA表达均增强。提示在回转模拟失重条件下 ,心肌成纤维细胞Ⅰ型胶原蛋白沉积增加 ,作为胶原降解抑制物的TIMP可能是造成胶原沉积的原因     

重组SM22αC端肽段促进细胞骨架重构

目的:探讨SM22αC端功能域肽段与细胞骨架F-actin聚合的关系,明确SM22α在血管平滑肌细胞(VSMC)骨架重构中的作用。方法:构建GST-SM22αC端功能域融合蛋白原核表达质粒pGEX3X-SM22α,诱导E coli高效表达可溶性GST-SM22α融合蛋白,制备抗SM22α抗体,VSMC蛋白分步提取及Western blot检测F-actin/G-actin中SM22α的含量变化,GST-pull down分析和免疫共沉淀检测SM22α与actin的相互作用,细胞免疫双荧光染色观察SM22α和actin在VSMC中的定位关系。结果:所构建的pGEX3X-SM22α原核表达质粒,在0.5mmol/LIPTG,30℃诱导6h条件下,表达可溶性GST-SM22α融合蛋白的水平最高,用纯化的融合蛋白免疫新西兰白兔获得的抗血清效价为1∶16。免疫双荧光染色和蛋白分步提取分析结果表明,在VSMC再分析过程中,SM22α与F-actin共定位,GSTpull down分析和免疫共沉淀结果均显示,SM22α通过C端功能域与F-actin相互作用而参与细胞骨架的重构;但是,SM22α与G-actin的结合能力较弱。结论:本研究重组得到的SM22αC端功能域具有与F-actin结合的活性,SM22α通过该区域与actin相互作用而参与细胞骨架重构。     

Tolrestat对高糖所致肾小球系膜细胞肌动蛋白去组装的影响

研究了醛糖还原酶抑制剂Tolrestat对高浓度葡萄糖(HG)所致肾小球系膜细胞(MC)肌动蛋白(actin)组装的影响。结果证明,与正常浓度葡萄糖(NG)相比,在HG培养的MC,F-actin失去束状外观呈不规则网状,显示F-actin部分去组装;F-actin荧光强度降低,G-actin荧光强度升高和F-/G-actin荧光强度比值下降。Tolrestat加入培养后,明显防止HG引起的F-actin去组装及F-和G-actin荧光强度的变化。提示多元醇通路激活在HG引起的MCactin去组装改变中起一定作用。     

维生素E和伊那普利对高糖改变肾小球系膜细胞肌动蛋白组装的影响

研究了维生素E(VE)和伊那普利(EN)对高浓度葡萄糖(HG)所致肾小球系膜细胞(MC)肌动蛋白组装的影响。结果证明,MC在HG培养时,F-actin失去粗大束状外观呈不规则网状,显示F-actin部分去组装。与正常浓度葡萄糖(NG)培养的MC相比,HG引起F-actin荧光强度降低,G-actin荧光强度升高和F/G-actin荧光强度比值下降。VE和EN加入培养后,HG引起的F-actin部分去组装及F-和G-actin荧光强度的变化均恢复正常,提示,VE和EN可防止HG引起的MC actin去组装。     

失重/模拟失重条件下心肌萎缩的发生机制

心肌能够应对内外环境改变而发生重塑。失重/模拟失重等去负荷条件可导致心肌萎缩、心脏功能下降。从系统和细胞分子层面揭示失重/模拟失重造成心肌萎缩的机制对于航天飞行后心血管功能紊乱的对抗研究至关重要。失重/模拟失重导致机体血流动力负荷下降、代谢需求降低和神经内分泌变化;同时导致包括钙相关信号、NF-κB通路、ERK通路、泛素-蛋白酶体途径以及自噬等通路的改变,上述变化在心肌萎缩的发生发展过程中发挥着关键调控作用。本文从系统和细胞分子层面对失重/模拟失重引起心肌萎缩的发生机制进行综述。     

反义封闭NGAL基因表达对SHEEC食管癌细胞微丝骨架的影响

为了研究反义封闭NGAL基因表达对SHEEC食管癌细胞微丝骨架以及肿瘤细胞生物学行为的影响,以不同长度NGAL基因片段反义表达载体和硫代修饰反义寡核苷酸单链片段转染SHEEC食管癌细胞,通过G418筛选,建立一系列旨在封闭SHEEC食管癌细胞NGAL基因表达的亚细胞克隆.在细胞内F-肌动蛋白(F-actin)及DNA荧光双标记基础上,通过流式细胞术、激光共聚焦显微镜扫描术等技术手段检测封闭反义NGAL基因表达后, SHEEC食管癌细胞中F-actin和DNA含量、F-actin形态结构以及肿瘤细胞生物学行为的变化特征.结果显示,反义封闭NGAL基因表达后,SHEEC食管癌细胞F-actin的含量明显降低,与永生化食管上皮细胞SHEE相近,但细胞分裂增殖指数未见明显变化.表明反义封闭NGAL基因表达对SHEEC食管癌细胞的微丝骨架有明显影响,而对SHEEC食管癌细胞的分裂增殖影响不明显.激光共聚焦显微镜扫描观测显示,反义封闭NGAL基因表达可使SHEEC食管癌细胞F-actin分布均匀,F-actin小体减少,细胞间连接重新建立,结构较紧密,主要形态结构特征与SHEE细胞趋于一致.提示反义封闭NGAL基因表达可对SHEEC食管癌细胞的微丝骨架F-actin产生明显影响,推测癌细胞的微丝骨架F-actin可能是NGAL基因在SHEEC食管癌细胞中发挥功能的一种作用环节.     

Rac1/MAPK/ERK 通路介导脂多糖LPS 诱导的人内皮细胞 单层通透性增高机制研究

目的:探讨LPS诱导的人内皮细胞单层通透性改变的分子机制。方法:应用逆转录病毒为载体,感染并筛选稳定表达持续活化型Rac1和主导抑制型Rac1的人HUVEC细胞,应用LPS刺激并观察细胞骨架蛋白F-actin和HUVEC单层通透性的改变。同时应用Western blot方法检测LPS刺激前后细胞中MAPK/ERK信号通路的改变及加入PD98059阻断ERK表达后,细胞内F-actin的改变情况。结果:与正常HUVEC相比较,LPS刺激后,感染活化型Rac1和主导抑制型Rac1的HUVEC中F-actin重构并形成大量应力纤维,细胞单层通透性显著增加。而抑制型Rac1感染后的HUVEC中F-actin无重构现象,同时细胞单层通透性无明显增加。LPS刺激前后,各组细胞中ERK1/2总蛋白均无明显改变。LPS刺激后,感染活化型Rac1的HUVEC中,p-ERK增加。经PD98059阻断后,细胞内p-ERK表达下降同时伴随F-actin解聚发生。结论:LPS诱导的内皮细胞通透性增加是经过Rac1-MAPK/ERK通路介导的。     

Imaging remodeling of the actin cytoskeleton in vascular smooth muscle cells after mechanosensitive arteriolar constriction

Experiments were performed to determine whether remodeling of the actin cytoskeleton contributes to arteriolar constriction. Mouse tail arterioles were mounted on cannulae in a myograph and superfused with buffer solution. The alpha1-adrenergic agonist phenylephrine (0.1-1 micromol/l) caused constriction that was unaffected by cytochalasin D (300 nmol/l) or latrunculin A (100 nmol/l), inhibitors of actin polymerization. In contrast, each compound abolished the mechanosensitive constriction (myogenic response) evoked by elevation in transmural pressure (PTM; 10-60 or 90 mmHg). Arterioles were fixed, permeabilized, and stained with Alexa-568 phalloidin and Alexa-488 DNAse I to visualize F-actin and G-actin, respectively, using a Zeiss 510 laser scanning microscope. Elevation in PTM, but not phenylephrine (1 micromol/l), significantly increased the intensity of F-actin and significantly decreased the intensity of G-actin staining in arteriolar vascular smooth muscle cells (VSMCs). The increase in F-actin staining caused by an elevation in PTM was inhibited by cytochalasin D. In VSMCs at 10 mmHg, prominent F-actin staining was restricted to the cell periphery, whereas after elevation in PTM, transcytoplasmic F-actin fibers were localized through the cell interior, running parallel to the long axis of the cells. Phenylephrine (1 micromol/l) did not alter the architecture of the actin cytoskeleton. In contrast to VSMCs, the actin cytoskeleton of endothelial or adventitial cells was not altered by an elevation in PTM. Therefore, the actin cytoskeleton of VSMCs undergoes dramatic alteration after elevation in PTM of arterioles and plays a selective and essential role in mechanosensitive myogenic constriction.     

Conformation changes of actin during formation of filaments and paracrystals and upon interaction with DNase I, cytochalasin B, and phalloidin

Spin labels attached to rabbit muscle actin became more immobilized upon conversion of actin from the G state to the F state with 50 mM KCl. Titration of G-actin with MgCl2 produced F-actin-like EPR spectra between 2 and 5 mM-actin filaments by electron microscopy. Higher concentrations of MgCl2 produced bundles of actin and eventually paracrystals, accompanied by further immobilization of spin labels. The effects of MgCl2 and KCl were competitive: addition of MgCl2 to 50 mM could convert F-actin (50 mM KCl) to paracrystalline (P) actin; the reverse titration (0 to 200 mM KCl in the presence of 20 mM MgCl2) was less complete. Addition of DNase I to G- or F-actin gave the expected amorphous electron micrographic pattern, and the actin was not sedimentable at (400,000 x g x h). EPR showed that the actin was in the G conformation. Addition of DNase I to paracrystalline actin gave the F conformation (EPR) but the actin was "G" by electron microscopy. Phalloidin converted G-actin to F-actin, had no effect on F-actin, and converted P-actin to the F state by electron microscopy but maintained the P conformation by EPR. Cytochalasin B produced no effects observable by EPR or centrifugation but "untwisted" paracrystals into nets. Since actin retained its P conformation by EPR in two states which were morphologically not P, we conclude that the P state is a distinct conformation of the actin molecule and that actin filaments aggregate to form bundles (and eventually paracrystals) when actin monomers are able to enter the P conformation.     

Comparisons of Endothelial Cell G- and F-Actin Distribution in Situ and in Vitro

Numerous studies have described the F-actin cytoskeleton; however, little information relevant to C-actin is available. The actin pools of bovine aortic endothelial cells were examined using in situ and in vitro conditions and fluorescent probes for G-(deoxyribonuclease I.0.3 μM) or F-actin (phalloidin, 0.2 μM). Cells in situ displayed a diffuse G-actin distribution, while F-actin was concentrated in the cell periphery and in fine stress fibers that traversed some cells. Cells of subconfluent or just confluent cultures demonstrated intense fluorescence, with many F-actin stress fibers. Postconfluent cultures resembled the condition in situ; peripheral F-actin was prominent, traversing actin stress fibers were greatly reduced and fluorescent intensity was diminished. Postconfluency had little influence on G-actin. with only an enhancement in the intensity of G-actin punctate fluorescence. When post-confluent cultures were incubated with cytochalasin D (15 min; 10^--⁴ M), F-actin networks were disrupted and actin punctate and diffuse fluorescence increased. G-actin fluorescence was not altered by the incubation. Although its unstructured nature may account for the minor changes observed, the stability of the G-actin pool in the presence of notable F-actin modulations suggested that filamentous actin was the key constituent involved in these actin cytoskeletal alterations. A separate finding illustrated that the concomitant use of actin probes with image enhancement and fluorescent microscopy could reveal simultaneously the G- and F-actin pools within the same cell.     

Polymerization of G-actin by myosin subfragment 1

The polymerization of actin from rabbit skeletal muscle by myosin subfragment 1 (S-1) from the same source was studied in the depolymerizing G-actin buffer. The polymerization reactions were monitored in light-scattering experiments over a wide range of actin/S-1 molar rations. In contrast to the well resolved nucleation-elongation steps of actin assembly by KC1 and Mg2+, the association of actin in the presence of S-1 did not reveal any lag in the polymerization reaction. Light scattering titrations of actin with S-1 and vice versa showed saturation of the polymerization reaction at stoichiometric 1:1 ratios of actin to S-1. Ultracentrifugation experiments confirmed that only stoichiometric amounts of actin were incorporated into a 1:1 acto-S-1 polymer even at high actin/S-1 ratios. These polymers were indistinguishable from standard complexes of S-1 with F-actin as judged by electron microscopy, light scattering measurements, and fluorescence changes observed while using actin covalently labeled with N-(1-pyrenyl)iodoacetamide. F-actin obtained by polymerization of G-actin by S-1 could initiate rapid assembly of G-actin in the presence of 10 mM KC1 and 0.5 mM MgCl2 and showed normal activation of MgATPase hydrolysis by myosin.     

Inhibition of sliding movement of F-actin by crosslinking emphasizes the role of actin structure in the mechanism of motility

The effects of crosslinking of monomeric and polymeric actin with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), disuccinimidyl suberate (DSS) and glutaraldehyde on the interaction with heavy meromyosin (HMM) in solution and on the sliding movement on glass-attached HMM were examined. The Vmax values of actin-activated HMM ATPase decreased in the following order: intact actin = EDC F-actin greater than DSS actin greater than glutaraldehyde F-actin = glutaraldehyde G-actin greater than EDC G-actin. The affinity of actin for HMM in the presence of ATP decreased in the following order: DSS actin greater than glutaraldehyde F-actin = glutaraldehyde G-actin greater than intact actin greater than EDC F-actin greater than EDC G-actin. However, sliding movement was inhibited only in the case of glutaraldehyde-crosslinked F and G-actin and EDC-crosslinked G-actin. Interestingly, after copolymerization of "non-motile" glutaraldehyde or EDC-crosslinked monomers with "motile" monomers of intact actin sliding of the copolymers was observed and its rate was independent of the type of crosslinked monomer, i.e. of the manner of their interaction with HMM. These data strongly indicate that inhibition of the sliding of actin by crosslinking cannot be explained entirely by changes in the Vmax value or affinity for myosin heads. We conclude that movement is generated by interaction of myosin with segments of F-actin containing a number of intact monomers, and the mechanism of inhibition involves an effect of the crosslinkers on the structure of F-actin itself.     

Effect of Hypothyroidism on Different Forms of Actin in Rat Cerebral Neuronal Cultures Studied by an Improved DNase I Inhibition Assay

An improved DNase I inhibition assay for the filamentous actin (F-actin) and monomeric actin (G-actin) in brain cells has been developed. Unlike other methods, the cell lysis conditions and postlysis treatments, established by us, inhibited the temporal inactivation of actin in the cell lysate and maintained a stable F-actin/G-actin ratio for at least 4-5 h after lysis. The new procedure allowed separate quantitation of the noncytoskeletal F-actin in the Triton-soluble fraction (12,000 g, 10 min supernatant) that did not readily sediment with the Triton-insoluble cytoskeletal F-actin (12,000 g, 10 min pellet). We have applied this modified assay system to study the effect of hypothyroidism on different forms of actin using primary cultures of neurons derived from cerebra of neonatal normal and hypothyroid rats. Our results showed a 20% increase in the Triton-insoluble cytoskeletal F-actin in cultures from hypothyroid brain relative to normal controls. In the Triton-soluble fraction, containing the G-actin and the noncytoskeletal F-actin, cultures from hypothyroid brain showed a 15% increase in G-actin, whereas the F-actin remained unaltered. The 10% increase in total actin observed in this fraction from hypothyroid brain could be totally accounted for by the enhancement of G-actin. The mean F-actin/G-actin ratio in this fraction was about 30% higher in the cultures from normal brain compared to that of the hypothyroid system, which indicates that hypothyroidism tends to decrease the proportion of noncytoskeletal F-actin relative to G-actin.     

Structural effects of cofilin on longitudinal contacts in F-actin

Structural effects of yeast cofilin on skeletal muscle and yeast actin were examined in solution. Cofilin binding to native actin was non-cooperative and saturated at a 1:1 molar ratio, with K(d)相似文献   

Influence of botulinum C2 toxin on F-actin and N-formyl peptide receptor dynamics in human neutrophils

Stimulation of human neutrophils with the chemotactic N-formyl peptide causes production of oxygen radicals and conversion of monomeric actin (G-actin) to polymeric actin (F-actin). The effects of the binary botulinum C2 toxin on the amount of F-actin and on neutrophil cell responses were studied. Two different methods for analyzing the actin response were used in formyl peptide-stimulated cells: staining of F-actin with rhodamine-phalloidin and a transient right angle light scatter. Preincubation of neutrophils with 400 ng/ml component I and 1,600 ng/ml component II of botulinum C2 toxin for 30 min almost completely inhibited the formyl peptide-stimulated polymerization of G-actin and at the same time decreased the amount of F-actin in unstimulated neutrophils by an average of approximately 30%. Botulinum C2 toxin preincubation for 60 min destroyed approximately 75% of the F-actin in unstimulated neutrophils. Right angle light scatter analysis showed that control neutrophils exhibited the transient response characteristic of actin polymerization; however, after botulinum C2 toxin treatment, degranulation was detected. Single components of the binary botulinum C2 toxin were without effect on the actin polymerization response. Fluorescence flow cytometry and fluorospectrometric binding studies showed little alteration in N-formyl peptide binding or dissociation dynamics in the toxin-treated cells. However, endocytosis of the fluorescent N-formyl peptide ligand-receptor complex was slower but still possible in degranulating neutrophils treated with botulinum C2 toxin for 60 min. The half-time of endocytosis, estimated from initial rates, was 4 and 8 min in control and botulinum C2 toxin-treated neutrophils, respectively.     

Mechanism of regulation of actin polymerization by Physarum profilin

Physarum profilin reduces the rates of nucleation and elongation of F- actin and also reduces the extent of polymerization of actin at the steady state in a concentration-dependent fashion. The apparent critical concentration for polymerization of actin is increased by the addition of profilin. These results can be explained by the idea that Physarum profilin forms a 1:1 complex with G-actin and decreases the concentration of actin available for polymerization. The dissociation constant for binding of profilin to G-actin is estimated from the kinetics of polymerization of G-actin and elongation of F-actin nuclei and from the increase of apparent critical concentration in the presence of profilin. The dissociation constants for binding of Physarum profilin to Physarum and muscle actins under physiological ionic conditions are in the ranges of 1.4-3.7 microM and 11.3-28.5 microM, respectively. When profilin is added to an F-actin solution, profilin binds to G-actin which co-exists with F-actin, and then G- actin is dissociated from F-actin to compensate for the decrease of the concentration of free G-actin and to keep it constant at the critical concentration. At the steady state, free G-actin of the critical concentration is in equilibrium not only with F-actin but also with profilin-G-actin complex. The stoichiometry of 1:1 for the formation of complex between profilin and G-actin is directly shown by means of chemical cross-linking.