肌动蛋白解聚因子/cofilin功能的研究进展

肌动蛋白解聚因子（actin depolymerizing factor,ADF）/cofilin家族是一类肌动蛋白结合蛋白,它们通过切断肌动蛋白纤丝并结合到肌动蛋白单体上,在重塑肌动蛋白骨架中发挥重要作用。就ADF/cofilin家族的结构特点、调控肌动蛋白动力学的机制及其功能的最新研究进展做一简要综述,并指出了目前在ADF/cofilin功能研究方面的不足和尚需解决的问题。     

肌动蛋白解聚因-子/丝切蛋白:肌动蛋白重塑蛋白质家系

肌动蛋白解聚因子／丝切蛋白(actin depolymerizing factor／cofilin,ADF／cofilin)是肌动蛋白结合蛋白(actin—binding protein)家系。迄今为止,可以在所有的真核细胞中检测到ADF／cofilin。它们调节(纤)丝状肌动蛋白细胞骨架(F—actin eytoskeleton),影响细胞的各种生理功能。不同的生物有不同的ADF／cofilin,但其功能基本相似。ADF／cofilin可以使(纤)丝状肌动蛋白(F—actin)解聚合,而且这种解聚合活性是可逆的,ADF／cofilin切割F-actin并且能提高球状肌动蛋白(G—actin)离开纤维突出端(pointedend)的能力,其作用受很多因素调控。     

ADF／cofilin分子家族的研究进展

细胞骨架中的肌动蛋白参与了一系列重要生理活动,如肌肉收缩、胞质环流、细胞运动、胞质分裂等。这些过程的发生除了需要肌动蛋白以外,还需要一些与之结合的调节蛋白参与,现在已经发现了100多种肌动蛋白结合蛋白,其中有一类分子量为15—20KD的蛋白,如肌动蛋白解聚因子(actin depolymerizing factor,ADF)、cofilin、profilin、actophorin、depactin、de-strin、UNC-60)等,在一定条件下可以使肌动蛋白微丝解聚,统称为ADF/cofilin分子家族。     

李斯特菌诱导宿主细胞骨架重排与磷脂酶D

无论是免疫细胞对病原体的主动吞噬,还是病原体诱导非吞噬细胞的被动吞噬,均是不同细胞膜受体介导的细胞肌动蛋白骨架重排过程,受到单体G蛋白和肌动蛋白骨架相关蛋白的精密调控。细胞内重要信号蛋白,磷脂酰胆碱专一性磷脂酶D(PLD)的活性变化与细胞肌动蛋白骨架重排密切相关,其参与调节了由抗体受体(FcγR)及补体受体(CR3)介导的免疫细胞的主动吞噬,而细胞肌动蛋白骨架解聚蛋白cofilin被磷酸化后可与PLD结合并激活PLD,进而调节肌动蛋白骨架重排。另一方面,cofilin磷酸化状态严格调控李斯特菌感染细胞过程中的肌动蛋白骨架重排。因此,阐明PLD是否在李斯特菌感染细胞过程中被激活并参与调节肌动蛋白骨架重排,将有助于揭示PLD激活对感染发生的调控作用,对透彻理解细菌感染宿主细胞的分子机制具有重要意义。     

宿主细胞丝切蛋白(cofilin)与病原微生物感染

摘要:病原微生物侵入宿主细胞是其引发有效感染的必要环节,该过程依赖于宿主细胞内肌动蛋白骨架的重排。丝切蛋白(cofilin)是细胞内一种重要的肌动蛋白解聚因子,参与多种病毒、细菌及真菌的感染过程。病原微生物感染可诱导宿主细胞肌动蛋白发生两相变化,同时伴随cofilin的磷酸化水平改变。通过突变、抑制或过表达改变cofilin 的活性均能有效的抑制病原微生物的感染。本文将对宿主细胞cofilin在病原微生物感染过程中的具体变化及可能的调控机制进行综述。     

Cofilin蛋白功能及活性调节

Cofilin是普遍存在于真核细胞的一种肌动蛋白结合蛋白. Cofilin的基本功能是在细胞内结合和解聚F肌动蛋白（F-actin）,其活性是通过磷酸化、去磷酸化、磷酸肌醇、pH改变等进行调节.cofilin介导了细胞内的信号途径,从而调节肌动蛋白骨架的重组,对肌肉形态发育起到重要作用.目前,在各种有机体中发现了许多特征性的cofilin蛋白同系物.其中鼠和人有2种cofilin蛋白：cofilin-1（non-muscle cofilin）和cofilin-2（muscle cofilin）.本文根据目前对cofilin的研究结果,从cofilin蛋白结构、功能、活性调节、在肌肉发育中的作用及相关疾病等方面进行阐述.     

凝溶胶蛋白基因的生物学功能及其应用研究进展

凝溶胶蛋白(gelsolin,GSN)是Gelsolin/Villin超家族的核心成员,是一种多功能的钙依赖性肌动蛋白结合蛋白,在细胞中Ca^2+和PIP2等多因素的调控下,对细胞凋亡、吞噬功能、肌动蛋白微丝切割、细胞信号转导等方面起着重要的作用。近年来,凝溶胶蛋白还被频繁用于相关疾病的预防、诊断与治疗,但其在调控细胞凋亡、炎症等病理生理中的作用机制还存在些许争议。本研究综述了凝溶胶蛋白的结构特点、生物学功能以及对疾病的诊断和治疗,旨在了解凝溶胶蛋白在生物医学及动物科学等领域的应用以及未来凝溶胶蛋白的发展前景。     

Cofilin在疾病中的研究进展

Cofilin是一种肌动蛋白结合蛋白,属于肌动蛋白解聚因子家族成员,普遍存在于真核细胞。它与肌动蛋白微丝(F-actin)结合,调节F-actin的解聚和重构。Cofilin具有多种生物学功能,如参与肌动蛋白骨架重组、核转运、胞质分裂以及心血管生成等。在应激条件下,cofilin可通过相应作用通路进行调节,改变细胞的功能。Cofilin在多种疾病如肿瘤、神经系统性疾病、血管性疾病、感染性疾病等的发生与发展中都起到了非常重要的作用。     

肌动蛋白解聚因子在动物生殖和胚胎发育中的作用

肌动蛋白解聚因子家族Cofilin/ADF(AC蛋白家族)属于肌动蛋白结合蛋白,是微丝骨架的一个重要调节者。AC蛋白家族能够截断微丝,促进肌动蛋白单体的解离和循环以及微丝解聚,调控微丝骨架的重建,进而影响与微丝骨架相关的一些生理功能如细胞增殖、迁移、凋亡及胚胎发育等。本文将着重介绍AC蛋白家族在动物生殖诸如精子发生、卵巢发育、卵子发生、卵裂,以及胚胎发育等过程中的调节与功能。     

成蛋白 (Formin): 植物细胞形态与发育的重要调控者

成蛋白(Formin)广泛存在于真菌、植物和动物等真核生物中,它们在调控肌动蛋白的聚合、协调肌动蛋白与微管之间的协同作用、决定细胞生长和形态等过程中发挥着决定性作用。一般认为,与真菌、动物不同,植物成蛋白家族结构在进化中因基因进化事件形成了植物特有的两类成蛋白家族,其中Ⅱ类成蛋白主要负责细胞极性生长,而Ⅰ类成蛋白可能调控细胞的膨胀。近年来,随着相关领域研究的深入,植物两类成蛋白在细胞内行使的功能也逐渐被阐明,而最近的研究结果也表明简单地根据蛋白结构对成蛋白的功能进行分类是不恰当的。据此,文中重点归纳了成蛋白结构域的组成与其对应功能,总结了成蛋白在代表性植物中的作用机理和最近研究进展,并就目前植物成蛋白研究中尚未解决的问题和尚未探索的领域进行了分析,最后对未来的植物成蛋白的研究方向提出了相关建议。     

Microscopic evidence that actin-interacting protein 1 actively disassembles actin-depolymerizing factor/Cofilin-bound actin filaments

Actin-depolymerizing factor (ADF)/cofilin and gelsolin are the two major factors to enhance actin filament disassembly. Actin-interacting protein 1 (AIP1) enhances fragmentation of ADF/cofilin-bound filaments and caps the barbed ends. However, the mechanism by which AIP1 disassembles ADF/cofilin-bound filaments is not clearly understood. Here, we directly observed the effects of these proteins on filamentous actin by fluorescence microscopy and gained novel insight into the function of ADF/cofilin and AIP1. ADF/cofilin severed filaments and AIP1 strongly enhanced disassembly at nanomolar concentrations. However, gelsolin, gelsolin-actin complex, or cytochalasin D did not enhance disassembly by ADF/cofilin, suggesting that the strong activity of AIP1 cannot be explained by simple barbed end capping. Barbed end capping by ADF/cofilin and AIP1 was weak and allowed filament elongation, whereas gelsolin or gelsolin-actin complex strongly capped and inhibited elongation. These results suggest that AIP has an active role in filament severing or depolymerization and that ADF/cofilin and AIP1 are distinct from gelsolin in modulating filament elongation.     

ADF/cofilin proteins translocate to mitochondria during apoptosis but are not generally required for cell death signaling

Non-muscle cofilin (n-cofilin) is a member of the ADF/cofilin family of actin depolymerizing proteins. Recent studies reported a mitochondrial translocation of n-cofilin during apoptosis. As these studies also revealed impaired cytochrome c release and a block in apoptosis upon small interfering RNA-mediated n-cofilin knockdown, n-cofilin was postulated to be essential for apoptosis induction. To elucidate the general importance of ADF/cofilin activity for apoptosis, we exposed mouse embryonic fibroblasts deficient for n-cofilin, ADF (actin depolymerizing factor), or all ADF/cofilin isoforms to well-characterized apoptosis inducers. Cytochrome c release, caspase-3 activation, and apoptotic chromatin condensation were unchanged in all mutant fibroblasts. Thus, we conclude that ADF/cofilin activity is not generally required for induction or progression of apoptosis in mammalian cells. Interestingly, mitochondrial association of ADF and n-cofilin during apoptosis was preceded by, and dependent on, actin that translocated by a yet unknown mechanism to mitochondria during cell death.     

Involvement of slingshot in the Rho-mediated dephosphorylation of ADF/cofilin during Xenopus cleavage

ADF/cofilin is a key regulator for actin dynamics during cytokinesis. Its activity is suppressed by phosphorylation and reactivated by dephosphorylation. Little is known, however, about regulatory mechanisms of ADF/cofilin function during formation of contractile ring actin filaments. Using Xenopus cycling extracts, we found that ADF/cofilin was dephosphorylated at prophase and telophase. In addition, constitutively active Rho GTPase induced dephosphorylation of ADF/cofilin in the egg extracts. This dephosphorylation was inhibited by Na(3)VO (4) but not by other conventional phosphatase-inhibitors. We cloned a Xenopus homologue of Slingshot phosphatase (XSSH), originally identified in Drosophila and human as an ADF/cofilin phosphatase, and raised antibody specific for the catalytic domain of XSSH. This inhibitory antibody significantly suppressed the Rho-induced dephosphorylation of ADF/cofilin in extracts, suggesting that the dephosphorylation at telophase is dependent on XSSH. XSSH bound to actin filaments with a dissociation constant of 0.4 microM, and the ADF/cofilin phosphatase activity was increased in the presence of F-actin. When latrunculin A, a G-actin-sequestering drug, was added to extracts, both Rho-induced actin polymerization and dephosphorylation of ADF/cofilin were markedly inhibited. Jasplakinolide, an actin-stabilizing drug, alone induced actin polymerization in the extracts and lead to dephosphorylation of ADF/cofilin. These results suggest that Rho-induced dephosphorylation of ADF/cofilin is dependent on the XSSH activation that is caused by increase in the amount of F-actin induced by Rho signaling. XSSH colocalized with both actin filaments and ADF/cofilin in the actin patches formed on the surface of the early cleavage furrow. Injection of inhibitory antibody blocked cleavage of blastomeres. Thus, XSSH may reorganize actin filaments through dephosphorylation and reactivation of ADF/cofilin at early stage of contractile ring formation.     

Stochastic severing of actin filaments by actin depolymerizing factor/cofilin controls the emergence of a steady dynamical regime

Actin dynamics (i.e., polymerization/depolymerization) powers a large number of cellular processes. However, a great deal remains to be learned to explain the rapid actin filament turnover observed in vivo. Here, we developed a minimal kinetic model that describes key details of actin filament dynamics in the presence of actin depolymerizing factor (ADF)/cofilin. We limited the molecular mechanism to 1), the spontaneous growth of filaments by polymerization of actin monomers, 2), the ageing of actin subunits in filaments, 3), the cooperative binding of ADF/cofilin to actin filament subunits, and 4), filament severing by ADF/cofilin. First, from numerical simulations and mathematical analysis, we found that the average filament length, 〈L〉, is controlled by the concentration of actin monomers (power law: 5/6) and ADF/cofilin (power law: −2/3). We also showed that the average subunit residence time inside the filament, 〈T〉, depends on the actin monomer (power law: −1/6) and ADF/cofilin (power law: −2/3) concentrations. In addition, filament length fluctuations are ∼20% of the average filament length. Moreover, ADF/cofilin fragmentation while modulating filament length keeps filaments in a high molar ratio of ATP- or ADP-P_i versus ADP-bound subunits. This latter property has a protective effect against a too high severing activity of ADF/cofilin. We propose that the activity of ADF/cofilin in vivo is under the control of an affinity gradient that builds up dynamically along growing actin filaments. Our analysis shows that ADF/cofilin regulation maintains actin filaments in a highly dynamical state compatible with the cytoskeleton dynamics observed in vivo.     

Efficient Salmonella entry requires activity cycles of host ADF and cofilin

Entry of Salmonella into mammalian cells is strictly dependent on the reorganization of actin cytoskeleton induced by a panel of Salmonella type III secreted proteins. Although several factors have been identified to be responsible for inducing the actin polymerization and stability, little is known about how the actin depolymerization contributes to Salmonella-induced actin rearrangements. We report here that activity cycles of host actin depolymerizing factor (ADF and cofilin) are modulated by Salmonella during bacterial entry. Efficient Salmonella internalization involves an initial dephosphorylation of ADF and cofilin followed by phosphorylation, suggesting that ADF and cofilin activities are increased briefly. Expression of a kinase dead form of an ADF/cofilin kinase (LIM kinase 1) or a catalytically inactive ADF/cofilin phosphatase (Slingshot), but not constitutively active LIM kinase 1 or wild-type Slingshot, resulted in decreased invasion. These data suggest that ADF/cofilin activities play a key role in the actin polymerization/depolymerization process induced by Salmonella. The activation of ADF/cofilin is brief and has to be reversed to facilitate efficient bacterial entry. Surprisingly, co-expression of constitutive active ADF and cofilin prevented efficient Salmonella entry, whereas expression of either one alone had no effect. We propose that ADF and cofilin actin-dynamizing activities and their activity cycling via phosphorylation are required for efficient Salmonella internalization.     

Structure of the actin-depolymerizing factor homology domain in complex with actin

Actin dynamics provide the driving force for many cellular processes including motility and endocytosis. Among the central cytoskeletal regulators are actin-depolymerizing factor (ADF)/cofilin, which depolymerizes actin filaments, and twinfilin, which sequesters actin monomers and caps filament barbed ends. Both interact with actin through an ADF homology (ADF-H) domain, which is also found in several other actin-binding proteins. However, in the absence of an atomic structure for the ADF-H domain in complex with actin, the mechanism by which these proteins interact with actin has remained unknown. Here, we present the crystal structure of twinfilin's C-terminal ADF-H domain in complex with an actin monomer. This domain binds between actin subdomains 1 and 3 through an interface that is conserved among ADF-H domain proteins. Based on this structure, we suggest a mechanism by which ADF/cofilin and twinfilin inhibit nucleotide exchange of actin monomers and present a model for how ADF/cofilin induces filament depolymerization by weakening intrafilament interactions.     

The Effects of ADF/Cofilin and Profilin on the Conformation of the ATP-Binding Cleft of Monomeric Actin

Actin depolymerizing factor (ADF)/cofilin and profilin are small actin-binding proteins, which have central roles in cytoskeletal dynamics in all eukaryotes. When bound to an actin monomer, ADF/cofilins inhibit the nucleotide exchange, whereas most profilins accelerate the nucleotide exchange on actin monomers. In this study the effects of ADF/cofilin and profilin on the accessibility of the actin monomer''s ATP-binding pocket was investigated by a fluorescence spectroscopic method. The fluorescence of the actin bound ɛ-ATP was quenched with a neutral quencher (acrylamide) in steady-state and time dependent experiments, and the data were analyzed with a complex form of the Stern-Volmer equation. The experiments revealed that in the presence of ADF/cofilin the accessibility of the bound ɛ-ATP decreased, indicating a closed and more compact ATP-binding pocket induced by the binding of ADF/cofilin. In the presence of profilin the accessibility of the bound ɛ-ATP increased, indicating a more open and approachable protein matrix around the ATP-binding pocket. The results of the fluorescence quenching experiments support a structural mechanism regarding the regulation of the nucleotide exchange on actin monomers by ADF/cofilin and profilin.     

AIP1/WDR1 supports mitotic cell rounding

The actin cytoskeleton plays a fundamental role in configuring cell shapes and movements. Actin interacting protein 1 (AIP1)/tryptophan-aspartate-repeat protein 1 (WDR1) induces actin severing and disassembly cooperating with ADF/cofilin. We found that mitotic cell flattening but not rounding was manifested by suppression of AIP1/WDR1 in cells. This mitotic cell flattening was not due to any changes in phosphorylation and distribution of cofilin in cells. We carried out a direct observation of actin filament severing/disassembly assay and found that phosphorylated cofilin still somewhat severs/disassembles actin filaments and that AIP1/WDR1 effaces this in vitro. We suggest that the phosphorylation of ADF/cofilin will be insufficient to completely inhibit actin turnover during mitosis, and that AIP1/WDR1 could abort the severing/disassembly activity somewhat still carried out due to phosphorylated ADF/cofilin. This mechanism could be required to induce cell morphologic changes, especially mitotic cell rounding.     

Actin depolymerization factor/cofilin activation regulates actin polymerization and tension development in canine tracheal smooth muscle

The contractile activation of airway smooth muscle tissues stimulates actin polymerization, and the inhibition of actin polymerization inhibits tension development. Actin-depolymerizing factor (ADF) and cofilin are members of a family of actin-binding proteins that mediate the severing of F-actin when activated by dephosphorylation at serine 3. The role of ADF/cofilin activation in the regulation of actin dynamics and tension development during the contractile activation of smooth muscle was evaluated in intact canine tracheal smooth muscle tissues. Two-dimensional gel electrophoresis revealed that ADF and cofilin exist in similar proportions in the muscle tissues, and that approximately 40% of the total ADF/cofilin in unstimulated tissues is phosphorylated. Phospho-ADF/cofilin decreased concurrently with tension development in response to stimulation with acetylcholine (ACh) or potassium depolarization indicating the activation of ADF/cofilin. Expression of an inactive phospho-cofilin mimetic (cofilin S3E) but not wild type cofilin in the smooth muscle tissues inhibited endogenous ADF/cofilin dephosphorylation and ACh-induced actin polymerization. Expression of cofilin S3E in the tissues depressed tension development in response to ACh, but it did not affect myosin light chain phosphorylation. The ACh-induced dephosphorylation of ADF/cofilin required the Ca2+-dependent activation of calcineurin (PP2B). The results indicate that the activation of ADF/cofilin is regulated by contractile stimulation in tracheal smooth muscle and that cofilin activation is required for actin polymerization and tension development in response to contractile stimulation.