cofilin蛋白的调节机制对神经系统的影响

正细胞骨架中的肌动蛋白参与了一系列的重要生理活动,包括肌肉收缩、胞质分裂、神经纤维再生与退行性改变等。这些生理过程的实现除了需要肌动蛋白参与,还需要一些能够起到调节肌动蛋白聚合和解聚作用的蛋白。根据最近的研究表明,分子量为15~20 kD a的肌动蛋白解聚因子/丝切蛋白分子家族(ADF/cofilin family)如肌动蛋白解聚因子(actin depolymerizing factor,ADF)、丝切蛋白(cofilin)、抑制蛋白(profilin)、载肌动蛋白(actophorin)等能够在一定条件下起到     

肌动蛋白和Tau蛋白荧光蛋白融合载体的构建、表达及其细胞内定位

背景：肌动蛋白和Tau蛋白分别是微丝和微管的重要组成成分,肌动蛋白和Tau蛋白的分布能否作为微丝和微管的观察指标。 目的：构建肌动蛋白和Tau蛋白的红色和绿色荧光蛋白融合表达载体并转染真核细胞,观察其在细胞内的表达和定位情况。 设计、时间及地点：单一样本观察实验,于2008-03/08在南方医科大学病理生理学教研室和广东省蛋白质组学重点实验室共同完成。 材料：克隆在pcDNA3载体上的肌动蛋白和Tau蛋白真核表达载体pcDNA3-actin和pcDNA3-Tau,以及红色荧光蛋白表达载体pmCherry-C2和绿色荧光蛋白表达载体pEGFP-C2、大肠杆菌株DH5α和小鼠NIH3T3细胞系由南方医科大学病理生理学教研室保存。 方法：将克隆在pcDNA3上的肌动蛋白和Tau蛋白亚克隆到红色荧光蛋白载体pmCherry-C2和绿色荧光蛋白载体pEGFP-C2上,然后转染NIH3T3细胞,利用荧光显微镜观察重组载体的表达和细胞内定位。 主要观察指标：NIH3T3细胞转染24 h后,利用Leica荧光显微镜观察上述重组载体在细胞中的表达和定位情况。 结果：重组质粒经酶切、PCR和测序鉴定正确无误,并在NIH3T3细胞中可获得高量表达。融合蛋白发出的红色和绿色荧光均表明,肌动蛋白在细胞质中呈短丝状弥散分布,Tau蛋白以细胞核为中心呈放射状排列。 结论：成功构建了肌动蛋白和Tau蛋白的不同荧光蛋白融合表达载体,并在真核细胞中得到有效表达,这为研究细胞骨架在信号分子移位中的作用提供了一个重要的工具。     

X-连锁凋亡蛋白通路调控及在脑缺血中的表达

X连锁凋亡蛋白(XIAP)是凋亡抑制蛋白家族(IAPs)中抗凋亡作用最强的成员,通过抑制始动或效应半胱天冬酶(Caspases)活性发挥抗凋亡作用。但受到第二个线粒体源性Caspase激活剂/低等电点的IAP直接结合蛋白(Smac/DIA-BLO)、XIAP相关因子1(XAF1)和需求高温的丝胺酸蛋白酶A2(HtrA2)等相关因子的负性调控。脑缺血后XIAP通路基因表达增高,参与了脑缺血后细胞凋亡的动态发展。     

肌动蛋白相关蛋白(ARP2/3)在神经树突棘可塑性中的研究进展

研究突触后结构——树突棘改变，是对学习记忆的重要的热点。F-actin经调节可以结合和／或解聚的动态特点是树突棘运动、生长和塑性的基础。在分枝状微丝形成过程中，肌动蛋白相关蛋白e／3（Actin-related proteine／3，ARP2／3）复合体起着重要的作用。细胞内的多种核化促进因子可以调节ARP2／3的活性。Arp2／3复合体结构、功能及调节的研究对于阐明微丝骨架形成机制，特别是树突棘的可塑性有重要意义。     

脑红蛋白的神经保护作用及可能机制

脑红蛋白是血红素蛋白家族的新成员,与氧具有高度亲和力,主要表达于代谢活跃、耗氧剧烈的脊椎动物神经细胞。在缺血缺氧、氧化应激、毒物损伤等广泛的病理状态下,脑红蛋白作为一种内源性神经保护因子,通过调节线粒体功能、协助氧的转运、清除自由基、抑制凋亡、与细胞色素C等蛋白相互作用等多种途径增强组织对缺血/缺氧性损伤的耐受,发挥了神经保护作用,是一种备受期待的神经元损伤修复介质,为脑缺血缺氧性损伤的研究提供了新思路。     

法舒地尔对戊四氮点燃大鼠海马丝切蛋白及苔藓纤维出芽的影响

目的 探讨法舒地尔对戊四氮(PTZ)点燃大鼠海马组织中丝切蛋白(cofilin,非磷酸化形式)表达与苔藓纤维出芽程度关系的影响.方法 210只SD雄性大鼠分成戊四氮组、法舒地尔干预组和生理盐水对照组,采用PTZ慢性点燃癫癎模型,应用SABC法检测cofilin表达,用Timm染色检测苔鲜纤维出芽情况.结果 PTZ组大鼠点燃率、病死率与法舒地尔组比较差异无统计学意义.PTZ组和法舒地尔组CA3区苔藓纤维出芽评分差异无统计学意义,与对照组相比差异均有统计学意义(P＜0.05).PTZ组和法舒地尔组海马非磷酸化cofilin表达差异无统计学意义.结论 丝切蛋白可能通过苔藓纤维出芽与癫癎的发生相关.     

下调磷酸甘油酸激酶 1 对胶质瘤 U373 细胞的放疗增敏作用及其机制的研究

目的研究下调磷酸甘油酸激酶1(PGK1)对U373细胞的放疗增敏作用;探讨PGK1参与的放疗增敏的可能机制。方法建立放射抵抗U373(RR-U373)细胞;采用Lipofectamine~(TM) 2000将shRNA-PGK1转染至U373细胞;对照组为0 Gy,处理组为5 Gy放射治疗。应用MTT法、划痕实验、Matrigel侵袭实验分别检测放疗前后各组细胞的增殖能力、迁移能力和侵袭能力; Western-Blot法检测各组细胞PGK1、CIN(chronophin)和丝切蛋白1(cofilin 1,CFL1)蛋白表达量。结果在U373细胞和RR-U373细胞中,相对于对照组,下调PGK1的细胞在放疗前后的增殖、迁移及侵袭能力均减弱;下调PGK1表达后,CIN、CFL1蛋白表达水平降低。结论下调U373细胞中PGK1的表达对胶质瘤U373细胞有放疗增敏作用。PGK1可能通过调节CIN、CFL1蛋白表达影响胶质瘤细胞对放射的敏感性。     

14—3—3蛋白在细胞凋亡中的作用

14—3—3蛋白是存在于所有真核细胞中的蛋白家族分子，参与蛋白激酶信号通路。通过磷酸化丝氨酸或磷酸化苏氨酸的方式，14—3—3蛋白与磷酸化依赖的蛋白间相互作用，调控细胞周期的进展，启动并维持了DNA损害的枪金点，激活促分裂素原活化蛋白激酶（mitogen—activated protein kinases，MAPK），调节生存信号及细胞骨架，对细胞凋亡发挥重要作用。本文对近年来关于14—3—3蛋白与细胞凋亡的研究进展作一综述。     

β-catenin蛋白在肝细胞癌中表达的意义

β-连环蛋白是一种重要的胞内蛋白,它具有双重功能,一是介导细胞粘附的E钙粘连素的重要结构分子,其异常改变在肿瘤浸润、转移过程中发挥重要作用;二是WNT信号传导途径的关键信号分子,降解障碍致使胞质内游离的β-连环蛋白积累,并与T细胞因子/淋巴细胞增强因子(TCF/LEF)结合进入细胞核,激活下游靶基因cmyc转录,引起细胞增殖和分化失控,导致肿瘤发生.     

Gab2信号转导通路与阿尔茨海默病(英文)

作为一类骨架/接头蛋白,生长因子受体结合蛋白2(Grb2)的相关结合蛋白家族(Grb2-associated binder protein)参与了细胞内多种信号转导通路,是酪氨酸激酶(PTKs)激活的下游信号转导通路的关键分子。Gab2是这个家族的重要成员,可被PTKs磷酸化激活,进而招募多种信号分子(如p85、SHP2和Crk),在细胞增殖、存活、分化和凋亡等过程中发挥重要作用。最近研究表明,Gab2基因多态性可增加阿尔茨海默病(Alzheimer’s disease,AD)的患病风险,从而与AD的发病密切相关。本文拟对Gab2的结构特点、相关信号转导通路及其在AD发生过程中可能扮演的角色作一系统综述。     

Spatiotemporal expression of testicular protein kinase 1 after rat sciatic nerve injury

Testicular protein kinase 1 (TESK1), a serine/threonine kinase, has been found expressing in various tissues and cell lines. Previous reports have shown that TESK1 plays an important role in regulating actin reorganization of spreading cell on fibronectin via phosphorylating cofilin. Because of the importance of actin reorganization in radial sorting and remyelination of peripheral nerve regeneration, we investigated the spatiotemporal expression of TESK1 in a rat sciatic nerve crush model. We observed that sciatic nerve crush resulted in a significant upregulation of TESK1 from 5 days to 2 weeks and subsequent return to the control level at 4 weeks. At its peak expression, TESK1 expressed mainly in both Schwann cells (SCs) and macrophages of the distal sciatic nerve segment, but had few colocalization in axons. In addition, upregulation of TESK1 was approximately in parallel with Oct-6, and numerous SCs expressing TESK1 were Oct-6 positive. Experiments with Schwann cell primary cultures revealed that TESK1 accumulated at F-actin-rich lamellipodia of the cell periphery when SCs were plated on fibronectin, whereas it was distributed in the cytoplasm in the case of non-stimulated cells. Thus, these findings suggest that TESK1 plays important roles in promyelinating SCs, potentially through subcellular localization change and participation in integrin-mediated actin reorganization.     

Activation of ezrin/radixin/moesin mediates attractive growth cone guidance through regulation of growth cone actin and adhesion receptors

The development of a functioning neural network relies on responses of axonal growth cones to molecular guidance cues that are encountered en route to their target tissue. Nerve growth factor (NGF) and neurotrophin-3 serve as attractive cues for chick embryo sensory growth cones in vitro and in vivo, but little is known about the actin-binding proteins necessary to mediate this response. The evolutionarily conserved ezrin/radixin/moesin (ERM) family of proteins can tether actin filaments to the cell membrane when phosphorylated at a conserved threonine residue. Here we show that acute neurotrophin stimulation rapidly increases active phospho-ERM levels in chick sensory neuron growth cone filopodia, coincident with an increase in filopodial L1 and β-integrin. Disrupting ERM function with a dominant-negative construct (DN-ERM) results in smaller and less motile growth cones with disorganized actin filaments. Previously, we found that NGF treatment increases actin-depolymerizing factor (ADF)/cofilin activity and growth cone F-actin (Marsick et al., 2010). Here, we show this F-actin increase, as well as attractive turning to NGF, is blocked when ERM function is disrupted despite normal activation of ADF/cofilin. We further show that DN-ERM expression disrupts leading edge localization of active ADF/cofilin and free F-actin barbed ends. Moreover, filopodial phospho-ERM levels are increased by incorporation of active ADF/cofilin and reduced by knockdown of L1CAM.Together, these data suggest that ERM proteins organize actin filaments in sensory neuron growth cones and are crucial for neurotrophin-induced remodeling of F-actin and redistribution of adhesion receptors.     

Regulation of spine morphology and synaptic function by LIMK and the actin cytoskeleton

Filamentous actin (F-actin) is highly enriched in the dendritic spine, a specialized postsynaptic structure on which the great majority of the excitatory synapses are formed in the mammalian central nervous system (CNS). The protein kinases of the Lim-kinase (LIMK) family are potent regulators of actin dynamics in many cell types and they are abundantly expressed in the CNS, including the hippocampus. Using a combination of genetic manipulations and electrophysiological recordings in mice, we have demonstrated that LIMK-1 signaling is important in vivo in the regulation of the actin cytoskeleton, spine morphology, and synaptic function, including hippocampal long-term potentiation (LTP), a prominent form of long lasting synaptic plasticity thought to be critical to memory formation. Our results provide strong genetic evidence that LIMK and its substrate ADF/cofilin are involved in spine morphology and synaptic properties and are consistent with the notion that the Rho family small GTPases and the actin cytoskeleton are critical to spine structure and synaptic regulation.     

Developmental expression of the actin depolymerizing factor ADF in the mouse inner ear and spiral ganglia

Hair cells, the inner ear's sensory cells, are characterized by tens to hundreds of actin‐rich stereocilia that form the hair bundle apparatus necessary for mechanoelectrical transduction. Both the number and length of actin filaments are precisely regulated in stereocilia. Proper cochlear and vestibular function also depends on actin filaments in nonsensory supporting cells. The formation of actin filaments is a dynamic, treadmill‐like process in which actin‐binding proteins play crucial roles. However, little is known about the presence and function of actin binding molecules in the inner ear, which set up, and maintain, actin‐rich structures and regulate actin turnover. Here we examined the expression and subcellular location of the actin filament depolymerizing factor (ADF) in the cochlea and vestibular organs. By means of immunocytochemistry and confocal microscopy, we analyzed whole‐mount preparations and cross‐sections in fetal and postnatal mice (E15–P26). We found a transient ADF expression in immature hair cells of the organ of Corti, the utricle, and the saccule. Interestingly, the stereocilia were not labeled. By P26, ADF expression was restricted to supporting cells. In addition, we localized ADF in presynaptic terminals of medio‐olivocochlear projections after hearing onset. A small population of spiral ganglion neurons strongly expressed ADF. Based on their relative number, peripheral location within the ganglion, smaller soma size, and coexpression of neurofilament 200, we identified these cells as Type II spiral ganglion neurons. The developmentally regulated ADF expression suggests a temporally restricted function in the stereocilia and, thus, a hitherto undescribed role of ADF. J. Comp. Neurol. 518:1724–1741, 2010. © 2009 Wiley‐Liss, Inc.     

Pyridoxal‐5′‐phosphate phosphatase/chronophin inhibits long‐term potentiation induction in the rat dentate gyrus

Pyridoxal‐5′‐phosphate (PLP)‐phosphatase/chronophin (PLPP/CIN) directly dephosphorylates actin‐depolymerizing factor (ADF)/cofilin as well as PLP. Although PLPP/CIN plays a role in the regulation of F‐actin and vitamin B₆ metabolism, there is no direct evidence to support a correlation between PLPP/CIN and F‐actin polymerization during long‐term potentiation (LTP) induction. In this study, we investigated whether the expression of PLPP/CIN is altered following LTP induction, and whether Tat‐PLPP/CIN transduction affects LTP induction in the rat dentate gyrus (DG). PLPP/CIN immunoreactivity was markedly decreased in dentate granule cells after the induction of LTP. Tat‐PLPP/CIN transduction (20 and 200 μg/kg) decreased the efficiency of high frequency stimulus‐induced potentiation of populations spike amplitude as compared to saline or Tat‐protein‐treated animals. The PLPP/CIN protein level showed an inverse correlation with phosphorylated ADF/cofilin levels and F‐actin content. These findings suggest that PLPP/CIN‐mediated actin dynamics may play an important role in the changes of morphological properties (dendritic spine reorganization) of the hippocampus in LTP. © 2009 Wiley‐Liss, Inc.     

A quantitative analysis of G-actin binding proteins and the G-actin pool in developing chick brain

The large G-actin pool in individual actively motile cells has been shown to be maintained primarily by the actin sequestering protein thymosin beta four (Tbeta4). It is not clear whether Tbeta4 or an isoform also plays a primary role in neural tissue containing highly motile axonal growth cones. To address this question we have made a definitive analysis of the relative contributions of all the known G-actin sequestering proteins: Tbeta4, Tbeta10, profilin, and phosphorylated (inactive) and unphosphorylated (potentially active) forms of both ADF and cofilin, in relation to the G-actin pool in developing chick brain at embryonic days 13 and 17. From our measurements we estimate the intracellular concentration of G-actin as 30-37 microM and of Tbeta4 as 50-60 microM in an 'average' brain cell in embryonic chick brain. No other beta thymosin isoforms were detected in these brain extracts. The ratio of soluble, unphosphorylated ADF to Tbeta4 is only 1:7 at 13 embryonic days, but increases to 1:4 at 17 days. Profilin and cofilin concentrations are an order of magnitude lower than Tbeta4. Combining the contributions of Tbeta4, unphosphorylated ADF and unphosphorylated cofilin, we estimate a mean G-actin critical concentration of approximately 0.45 microM and approximately 0.2 microM, respectively, in day 13 and day 17 embryonic brain extracts, suggesting a significant developmental decrease. We conclude that (a) Tbeta4 is the major actin sequestering protein in embryonic chick brain and the only beta thymosin isoform present; (b) ADF may play a significant developmental role, as its concentration changes significantly with age; (c) the known G-actin binding proteins can adequately account for the G-actin pool in embryonic chick brain.     

Pyridoxal‐5′‐phosphate phosphatase/chronophin induces astroglial apoptosis via actin‐depolymerizing factor/cofilin system in the rat brain following status epilepticus

Actin‐depolymerizing factor (ADF)/cofilin is a small cytoskeletal protein that is a stimulus‐responsive mediator of actin dynamics. ADF/cofilin also translocates into mitochondria and nuclei in response to apoptotic stimuli for cytochrome c release. These ADF/cofilin translocations are negatively regulated by phosphorylation. Recently, it has been reported that pyridoxal‐5′‐phosphate (PLP) phosphatase/chronophin (PLPP/CIN) regulates phosphorylation of ADF/cofilin levels. Therefore, we investigated whether PLPP/CIN contributes to apoptosis‐like events via modulation of ADF/cofilin phosphorylation following status epilepticus (SE). In the present study, apoptosis‐like astroglial damages were detected in the dentate gyrus after SE. Upregulation of ADF/cofilin and PLPP/CIN expression in the cytoplasm and nucleus were accompanied by apoptosis‐like events. PLPP/CIN level showed a direct proportionality to nuclear translocation of ADF/cofilin. Moreover, nuclear accumulation of apoptosis‐inducing factor was simultaneously observed with that of ADF/cofilin. Tat‐PLPP/CIN pretreatment accelerated astroglial apoptosis‐like degeneration following SE, although Tat‐PLPP/CIN transduction alone could not induce apoptosis or necrosis in astrocytes. Therefore, our findings suggest that nuclear accumulation of ADF/cofilin itself may not induce apoptogenic events, but may play a synergic role in apoptosis‐like astroglial loss following SE. © 2010 Wiley‐Liss, Inc.     

Actin-depolymerizing factor (ADF) in the cerebellum of the developing rat: a quantitative and immunocytochemical study.

A specific antiserum against actin-depolymerizing factor (ADF) was used in a quantitative and immunocytochemical study of ADF in the cerebellum of developing rats. The Triton-soluble ADF concentration remained stable throughout development. Light and electron microscopic immunocytochemistry showed that ADF was not detected in all cerebellar cells. ADF immunoreactivity was found in Purkinje cells, but not in granule cells. It was found in the Bergmann astrocytes and the astrocytes of the white matter, but not in the oligodendrocytes. The cell bodies and dendrites of Purkinje cells were immunoreactive for ADF but the axons were not. In contrast, the other axons of the white matter (mossy and climbing fibres) were labeled. Thus, ADF was not restricted to either the dendritic or axonal compartments. However, dendritic spines and postsynaptic densities were immunoreactive, whereas presynaptic varicosities were unlabeled. The immunoreactivities for ADF and actin were compared. ADF staining was uniformly distributed throughout the entire dendritic arborization of the Purkinje cell, while filamentous actin is highly concentrated in the dendritic spines, indicating that ADF activity might vary according to its cellular localization.     

WDR1 colocalizes with ADF and actin in the normal and noise-damaged chick cochlea

Auditory hair cells of birds, unlike hair cells in the mammalian organ of Corti, can regenerate following sound-induced loss. We have identified several genes that are upregulated following such an insult. One gene, WDR1, encodes the vertebrate homologue of actin-interacting protein 1, which interacts with actin depolymerization factor (ADF) to enhance the rate of actin filament cleavage. We examined WDR1 expression in the developing, mature, and noise-damaged chick cochlea by in situ hybridization and immunocytochemistry. In the mature cochlea, WDR1 mRNA was detected in hair cells, homogene cells, and cuboidal cells, all of which contain high levels of F-actin. In the developing inner ear, WDR1 mRNA was detected in homogene cells and cuboidal cells by embryonic day 7, in the undifferentiated sensory epithelium by day 9, and in hair cells at embryonic day 16. We also demonstrated colocalization of WDR1, ADF, and F-actin in all three cell types in the normal and noise-damaged cochlea. Immediately after acoustic overstimulation, WDR1 mRNA was seen in supporting cells. These cells contribute to the structural integrity of the basilar papilla, the maintenance of the ionic barrier at the reticular lamina, and the generation of new hair cells. These results indicate that one of the immediate responses of the supporting cell after noise exposure is to induce WDR1 gene expression and thus to increase the rate of actin filament turnover. These results suggest that WDR1 may play a role either in restoring cytoskeletal integrity in supporting cells or in a cell signaling pathway required for regeneration.     

Intracellular LINGO-1 negatively regulates Trk neurotrophin receptor signaling

Neurotrophins, essential regulators of many aspects of neuronal differentiation and function, signal via four receptors, p75, TrkA, TrkB and TrkC. The three Trk paralogs are members of the LIG superfamily of membrane proteins, which share extracellular domains consisting of leucine-rich repeat and C2 Ig domains. Another LIG protein, LINGO-1 has been reported to bind and influence signaling of p75 as well as TrkA, TrkB and TrkC. Here we examine the manner in which LINGO-1 influences the function of TrkA, TrkB and TrkC. We report that Trk activation promotes Trk association with LINGO-1, and that this association promotes Trk degradation by a lysosomal mechanism. This mechanism resembles the mechanism by which another LIG protein, LRIG1, promotes lysosomal degradation of receptor tyrosine kinases such as the EGF receptor. We present evidence indicating that the Trk/LINGO-1 interaction occurs, in part, within recycling endosomes. We show that a mutant form of LINGO-1, with much of the extracellular domain deleted, has the capacity to enhance TrkA signaling in PC12 cells, possibly by acting as an inhibitor of Trk down-regulation by full length LINGO-1. We propose that LINGO-1 functions as a negative feedback regulator of signaling by cognate receptor tyrosine kinases including TrkA, TrkB and TrkC.