PCDH17 restricts dendritic spine morphogenesis by regulating ROCK2-dependent control of the actin cytoskeleton,modulating emotional behavior

Proper regulation of synapse formation and elimination is critical for establishing mature neuronal circuits and maintaining brain function.Synaptic abnormalities,such as defects in the density and morphology of postsynaptic dendritic spines,underlie the pathology of various neuropsychiatric disorders.Protocadherin 17(PCDH17)is associated with major mood disorders,including bipolar disorder and depression.However,the molecular mechanisms by which PCDH17 regulates spine number,morphology,and behavior remain elusive.In this study,we found that PCDH17 functions at postsynaptic sites,restricting the number and size of dendritic spines in excitatory neurons.Selective overexpression of PCDH17 in the ventral hippocampal CA1 results in spine loss and anxiety-and depression-like behaviors in mice.Mechanistically,PCDH17 interacts with actin-relevant proteins and regulates actin filament(F-actin)organization.Specifically,PCDH17 binds to ROCK2,increasing its expression and subsequently enhancing the activity of downstream targets such as LIMK1 and the phosphorylation of cofilin serine-3(Ser3).Inhibition of ROCK2 activity with belumosudil(KD025)ameliorates the defective F-actin organization and spine structure induced by PCDH17 overexpression,suggesting that ROCK2 mediates the effects of PCDH17 on F-actin content and spine development.Hence,these findings reveal a novel mechanism by which PCDH17 regulates synapse development and behavior,providing pathological insights into the neurobiological basis of mood disorders.

Scinderin promotes glioma cell migration and invasion via remodeling actin cytoskeleton

BACKGROUND Glioma is one of the most common intracranial tumors,characterized by invasive growth and poor prognosis.Actin cytoskeletal rearrangement is an essential event of tumor cell migration.The actin dynamics-related protein scinderin(SCIN)has been reported to be closely related to tumor cell migration and invasion in several cancers.AIM To investigate the role and mechanism of SCIN in glioma.METHODS The expression and clinical significance of SCIN in glioma were analyzed based on public databases.SCIN expression was examined using real-time quantitative polymerase chain reaction and Western blotting.Gene silencing was performed using short hairpin RNA transfection.Cell viability,migration,and invasion were assessed using cell counting kit 8 assay,wound healing,and Matrigel invasion assays,respectively.F-actin cytoskeleton organization was assessed using F-actin staining.RESULTS SCIN expression was significantly elevated in glioma,and high levels of SCIN were associated with advanced tumor grade and wild-type isocitrate dehydrogenase.Furthermore,SCIN-deficient cells exhibited decreased proliferation,migration,and invasion in U87 and U251 cells.Moreover,knockdown of SCIN inhibited the RhoA/focal adhesion kinase(FAK)signaling to promote F-actin depolymerization in U87 and U251 cells.CONCLUSION SCIN modulates the actin cytoskeleton via activating RhoA/FAK signaling,thereby promoting the migration and invasion of glioma cells.This study identified the cancer-promoting effect of SCIN and provided a potential therapeutic target for the treatment of glioma.

Dynamics of perinuclear actin ring regulating nuclear morphology

Cells are capable of sensing and responding to the extracellular mechanical microenvironment via the actin skeleton.In vivo,tissues are frequently subject to mechanical forces,such as the rapid and significant shear flow encountered by vascular endothelial cells.However,the investigations about the transient response of intracellular actin networks under these intense external mechanical forces,their intrinsic mechanisms,and potential implications are very limited.Here,we observe that when cells are subject to the shear flow,an actin ring structure could be rapidly assembled at the periphery of the nucleus.To gain insights into the mechanism underlying this perinuclear actin ring assembly,we develop a computational model of actin dynamics.We demonstrate that this perinuclear actin ring assembly is triggered by the depolymerization of cortical actin,Arp2/3-dependent actin filament polymerization,and myosin-mediated actin network contraction.Furthermore,we discover that the compressive stress generated by the perinuclear actin ring could lead to a reduction in the nuclear spreading area,an increase in the nuclear height,and a decrease in the nuclear volume.The present model thus explains the mechanism of the perinuclear actin ring assembly under external mechanical forces and suggests that the spontaneous contraction of this actin structure can significantly impact nuclear morphology.

Analysis and Review of Downregulated Actin Cytoskeletal Proteins in Non-Small Cell Lung Cancer

Actin, a highly conserved protein, plays a dominant role in Non-small cell lung cancer (NSCLC). Late diagnosis and the aggressive nature of NSCLC pose a significant threat. Studying the clinic pathological properties of NSCLC proteins is a potential alternative for developing treatment strategies. Towards this, 35 downregulated actin cytoskeletal proteins on NSCLC prognosis and treatment were studied by examining their protein-protein interactions, gene ontology enrichment terms, and signaling pathways. Using PubMed, various proteins in NSCLC were identified. The protein-protein interactions and functional associations of these proteins were examined using the STRING database. The focal adhesion signaling pathway was selected from all available KEGG and Wiki pathways because of its role in regulating gene expression, facilitating cell movement and reproduction, and significantly impacting NSCLC. The protein-protein interaction network of the 35 downregulated actin cytoskeleton proteins revealed that ACTG1, ACTR2, ACTR3, ANXA2, ARPC4, FLNA, TLN1, CALD1, MYL6, MYH9, MYH10, TPM1, TPM3, TPM4, PFN1, IQGAP1, MSN, and ZXY exhibited the highest number of interactions. Whereas HSPB1, CTNNA1, KRT17, KRT7, FLNB, SEPT2, and TUBA1B displayed medium interactions, while UTRN, TUBA1B, and DUSP23 had relatively fewer interactions. It was discovered that focal adhesions are critical in connecting membrane receptors with the actin cytoskeleton. In addition, protein kinases, phosphatases, and adapter proteins were identified as key signaling molecules in this process, greatly influencing cell shape, motility, and gene expression. Our analysis shows that the focal adhesion pathway plays a crucial role in NSCLC and is essential for developing effective treatment strategies and improving patient outcomes.

Cloning and characterization of an actin gene of Chlamys farreri and the phylogenetic analysis of mollusk actins

An actin gene (CfACT1) was cloned by using RT-PCR, 3’and 5’RACE from hemocytes of the sea scallop Chlamys farreri. The full length of the transcript is 1 535 bp, which contains a long 3’ un-translated region of 436bp and 59bp of a 5’ un-translated sequence. The open reading frame encodes a polypeptide of 376 amino acids. Sequence comparisons indicated that CfACT1 is more closely related to vertebrate cytoplasmic actins than muscle types. Phylogenetic analysis showed that molluscan actins could be generally divided into two categories: muscle and cytoplasmic, although both are similar to vertebrate cytoplasmic actins. It was also inferred that different isotypes existed in muscle or cytoplasma in mollusks. The genomic sequence of CfACT1 was cloned and sequenced. Only one intron was detected: it was located between codons 42 and 43 and different from vertebrate actin genes.

Regulation of actin cytoskeleton via photolithographic micropatterning

Actin cytoskeleton plays crucial roles in various cellular functions.Extracellular matrix(ECM)can modulate cell morphology by remodeling the internal cytoskeleton.To define how geometry of ECM regulates the organization of actin cytoskeleton,we plated individual NIH 3T3 cells on micropatterned substrates with distinct shapes and sizes.It was found that the stress fibers could form along the nonadhesive edges of T-shaped pattern,but were absent from the opening edge of V-shaped pattern,indicating that the organization of actin cytoskeleton was dependent on the mechanical environment.Furthermore,a secondary actin ring was observed on 50μm circular pattern while did not appear on 30μm and 40μm pattern,showing a size-dependent organization of actin cytoskeleton.Finally,osteoblasts,MDCK and A549 cells exhibited distinct organization of actin cytoskeleton on T-shaped pattern,suggesting a cell-type specificity in arrangement of actin cytoskeleton.Together,our findings brought novel insight into the organization of actin cytoskeleton on micropatterned environments.

Inhibiting phosphatase and actin regulator 1 expression is neuroprotective in the context of traumatic brain injury

Studies have found that the phosphatase actin regulatory factor 1 expression can be related to stroke,but it remains unclear whether changes in phosphatase actin regulatory factor 1 expression also play a role in traumatic brain injury.In this study we found that,in a mouse model of traumatic brain injury induced by controlled cortical impact,phosphatase actin regulatory factor 1 expression is increased in endothelial cells,neurons,astrocytes,and microglia.When we overexpressed phosphatase actin regulatory factor 1 by injection an adeno-associated virus vector into the contused area in the traumatic brain injury mice,the water content of the brain tissue increased.However,when phosphatase actin regulatory factor 1 was knocked down,the water content decreased.We also found that inhibiting phosphatase actin regulatory factor 1 expression regulated the nuclear factor kappa B signaling pathway,decreased blood-brain barrier permeability,reduced aquaporin 4 and intercellular adhesion molecule 1 expression,inhibited neuroinflammation,and neuronal apoptosis,thereby improving neurological function.The findings from this study indicate that phosphatase actin regulatory factor 1 may be a potential therapeutic target for traumatic brain injury.

Bioprocess-inspired Actin Biomineralized Hematite Mesocrystals for Energy Storage

Biomineralization is a biological process of synthesizing inorganic minerals within organisms.It has been found that intracellular proteins are involved in the room temperature synthesis process of anatase Ti O2in living mussels.Here,we used intracellular actin to synthesize hematite by biomineralization.Biomineralized hematite has a nano spindle structure with a particle size of approximately 150 nm.The microstructure indicates that the prepared hematite is a mesocrystals composed of ordered arrangement and assembly of primary nanoparticles.In addition,hematite mesocrystals exhibit good lithium storage performance as electrode materials for lithium batteries.The discharge specific capacity of the battery remained at 560.7 m Ah·g^(-1)after 130 cycles at a current density of 200 m A·g^(-1).This work expands the synthesis methods of hematite by biomineralization,and provides a new strategy for preparing inorganic materials by intracellular proteins.

Holistic Approach in the Treatment of Actinic Keratosis: Benefits and Disadvantages of 5-Fluorouracil, Imiquimod, Diclofenac and Curaderm

Background Actinic keratosis is the most prevalent premalignant skin disorder in the white population. Current guidelines provide no clear recommendations about preferred treatments. Methods The parameters;effectiveness, treatment duration, recurrence, side effects and cost of treatment were investigated for three frequently used topical therapies which were then compared with a most recent developed topical therapy. Published clinical data obtained from the literature was used to compare these parameters for 5-fluorouracil, imiquimod and diclofenac and relate them with the newly developed Curaderm. Results A wide variation in the concentrations of the active anti-keratotic ingredients, application frequency, duration of treatment, recurrence rates and cost of treatment exist between the different topical therapies. The efficacy rates and side effects were less variable. Overall, Curaderm is the most suitable treatment for actinic keratosis. Clinical evidence is presented illustrating the effects of Curaderm on field-directed treatments and solitary treatments of actinic keratoses. Conclusions Current medical guidelines do not provide clear recommendations on which treatment approach for actinic keratosis is preferred. Direct head-to-head comparison between treatments with emphasis on efficacy, safety, treatment duration, compliance, convenience, cosmetic outcome, patient acceptance and cost should be available to the patient, the practising physician, healthcare system and should assist in therapeutic treatment guidelines and policymaking. Given the very favourable profiles of these parameters with Curaderm when compared with other home-based treatments, it should be considered that Curaderm is first-in-line.

拟南芥、水稻和杨树ACTIN家族全基因组分析

鉴定了覆盖拟南芥、水稻和杨树3种模式植物全基因组的20个拟南芥、18个水稻、22个杨树ACTIN蛋白基因,对其染色体定位、基因结构、基因复制等进行了综合分析.并在系统进化分析基础上,将ACTIN基因家族分为12个亚家族,有助于揭示植物ACTIN基因家族的进化历史,为后续ACTIN基因家族的功能提供线索,对研究植物ACTIN基因家族功能和进化上的多样性提供理论基础.

紧密连接蛋白ZO-1、occludin和actin参与缺氧缺血诱导的血脑屏障通透性增加

目的探讨血脑屏障紧密连接(blood-brain barrier-tight junction,BBB-TJ)蛋白ZO-1、occludin和ac-tin在缺氧缺血诱导的血脑屏障(blood-brain barrier,BBB)通透性增加中的变化及其机制。方法利用人脐静脉内皮细胞系ECV304与星形胶质细胞(astrocytes,AS)共培养建立体外BBB模型,模型随机分为正常对照组和缺氧缺血两组。透射电镜观察两组间BBB-TJ的变化,直接免疫荧光观察细胞骨架蛋白actin分布的改变。γ计数仪检测大分子物质125I-牛血清白蛋白(125I-BSA)通透曲线观察BBB通透性的改变,Western blot检测细胞骨架蛋白actin,胞浆附着蛋白ZO-1,跨膜蛋白occludin的表达量的改变。结果透射电镜观察培养第10天的体外BBB模型,可见内皮细胞连接紧密,细胞间形成光滑、连续、较高密度的紧密连接。缺氧缺血后5 h,内皮细胞间连接开放,形成裂隙。直接免疫荧光下检测可见周边Actin丝带模糊,部分断裂,形成细胞间裂隙。缺氧缺血组125I-BSA的通透量增加,与对照组比较差异有统计学意义(P<0.01)。同时ZO-1的表达量显著减少,而occludin和actin的表达量无明显改变。结论缺氧缺血诱导occludin的位置分布改变和ZO-1的表达量减少进而促使actin蛋白发生重排,是导致缺氧缺血后BBB通透性增加的可能机制之一。

香蕉果实SMART cDNA文库的构建及利用PCR方法筛选香蕉Actin2基因

利用SMART(switching mechanismat5’end of RNA transcript)技术,提取果实少量总RNA,经15-25轮LD-PCR扩增获得全长ds-cDNA,构建了海南主栽的食用香蕉巴西蕉(Musa AAA Group Cavendish)果实的cDNA文库。所构建的文库容量为5×106Pfuml-1,重组率93%。利用此cDNA文库,采用96孔板PCR法筛选香蕉Actin2基因,测序结果显示,序列全长1723bp,编码区长1134bp,编码378个氨基酸,与蝴蝶兰Actin2基因序列同源率达83%,已递交GenBank,接受号692696。

多浆旱生植物霸王Actin基因片段的克隆及序列分析

根据其他植物Actin基因的保守序列设计一对简并性引物,以霸王叶片总RNA为模板,采用RT-PCR的方法扩增出Actin基因片段并克隆到PUCm-T载体。阳性克隆经PCR鉴定后进行测序,序列分析结果表明:该片段长598bp,编码198个氨基酸;所得序列与GenBank中注册的Actin基因序列的同源性均在82%以上,与其他肌动蛋白的氨基酸序列的同源性达91%以上。

盐生植物碱蓬Actin基因片段的克隆及序列分析

目的:克隆盐生植物碱蓬(Suaeda glauca)Actin基因片段,为研究其它基因在碱蓬的表达和调控提供内参基因。方法:根据已知植物Actin基因的保守序列设计一对简并性引物,采用RT-PCR的方法扩增Actin基因片段,使用分子生物学软件进行序列分析。结果:获得一段大小为598bp的基因片段,编码198个氨基酸;该序列与其它Actin基因核苷酸序列的同源性均在80%以上,与氨基酸序列的同源性达93%以上。结论:克隆的基因为Actin基因片段,将其命名为SgACT,并登录在GenBank,登录号为EU429457。

香樟Actin基因的克隆及表达分析

Actin作为一个看家基因,经常在实时定量PCR中被用作内参对不同样品中的m RNA进行定量,因此在基因表达分析中扮演重要的角色。利用同源克隆的方法,根据Gen Bank中已经公布的其他植物的Actin基因的保守序列设计简并引物,采用RT-PCR(Reverse Transcription Polymerase Chain Reaction)技术从香樟中获得了4个c DNA片段。分子生物学分析软件分析结果显示,4个片段大小为均为998 bp,编码332个氨基酸;同源性分析表明,所获得的片段属于Actin亚家族,分别命名为Cc ACTa、Cc ACTb、Cc ACTc和Cc ACTd并提交Gen Bank(登录号:KM086736、KM086737、KM086738和KM086739)。实时定量PCR结果显示,Cc ACTc在根、茎、叶及低温处理的叶片中表达量相对稳定,可以作为候选内参基因。

内参基因β-actin质粒标准品的构建

为构建检测内参基因β-actin mRNA表达水平差异的标准品,以成人外周血细胞的总RNA为模板、Random 6 mers为引物反转录合成cDNA,用该cDNA为模板PCR扩增人β-actin基因相应的cDNA片段,构建PMD-18-β-actin重组质粒,鉴定测序后,用荧光定量PCR制作标准曲线。结果外周血提取的总RNA完整性良好,构建的β-actin质粒经PCR扩增后得到一186bp的清晰条带,测序结果与目的片段完全一致,且质粒的原始浓度为2.39×1013 copies/mL,倍比稀释至2.0×104copies/mL均能得到良好的标准曲线(R2=1),提示构建β-actin基因荧光定量PCR标准质粒成功。

中国卤虫actin基因的实时荧光定量PCR方法的建立和优化

根据GenBank中卤虫actin基因的保守区域序列设计并合成一对引物,采用SYBR GreenⅠ作为染料建立了实时荧光定量PCR(real-time fluorescence quantitative PCR)方法.以Ct值为纵坐标,以稀释倍数的对数为横坐标,建立标准曲线,其相关系数达到了0.999.溶解曲线分析显示产物为单一的特异峰,Tm为83.6℃.结果表明:本实验建立的中国卤虫actin基因的实时荧光定量PCR法扩增效率高、特异性强、线性范围广、检测周期短,为actin基因作为内参基因进行卤虫早期胚胎发育基因表达的定量分析奠定了基础.

13种植物actin基因的密码子使用特性分析

【目的】分析13种植物actin基因的密码子组成、密码子偏性及聚类关系,了解其密码子使用模式及影响密码子使用的因素,为深入研究分子进化及物种进化提供参考。【方法】运用Codon W 1.4.4软件分析13种植物的肌动蛋白基因（actin）密码子组成及使用参数,并对影响密码子偏性的因素进行研究。应用MEGA 4.1对13条基因的CDS序列进行聚类分析,采用SPSS 20.0进行密码子偏性的聚类分析。【结果】双子叶植物中actin基因的GC含量为45.0%～51.2%、GC3s含量为36.3%～53.8%,单子叶植物中GC含量为53.0%～58.3%、GC3s含量为60.9%～75.8%;actin基因在单子叶植物中偏爱G/C结尾的密码子,双子叶植物中偏爱A/T结尾的密码子。GC和GC3s与有效密码子数（ENC）呈极显著负相关（P〈0.01）,相关系数均为-0.906。ENC绘图分析结果表明,actin基因密码子偏性同时受突变和选择压力影响,单子叶植物受选择压力影响的程度大于双子叶植物。基于actin基因密码子偏性的聚类将单子叶植物高粱、玉米聚为一类,水稻、大麦、竹聚为一类,8种双子叶植物聚为一类。【结论】actin基因密码子偏性与碱基组成密切相关,其密码子偏性在单、双子叶植物间存在差异,依据密码子偏性的聚类能在一定程度上反映物种间的亲缘关系。

葡萄Actin基因家族的鉴定及进化和表达分析

采用生物信息学方法从葡萄(Vitis vinifera Linn.)全基因组中鉴定Actin基因家族,并对各基因的染色体定位和结构特征,编码蛋白质的理化性质、亚细胞定位、二级结构、三级结构和系统进化,以及不同组织的基因表达进行研究。结果表明:葡萄Actin基因家族16个基因分布在12条染色体上。16个基因的结构特征及其编码蛋白质的理化性质差异较大。16个基因的长度及其内含子总长度的变化范围较大,编码序列(CDS)和外显子总长度的变化范围较小。除登录号GSVIVG01008254001和GSVIVG01014035001的基因外,其他14个基因的GC含量均低于其CDS的GC含量。除登录号GSVIVG01008254001的基因外,其他15个基因编码的蛋白质的理论相对分子质量为12 534.54~82 612.33,理论等电点为p I 4.92~p I 9.13。16个基因编码蛋白质的消光系数为14 105~73 645,脂肪族氨基酸指数为65.54~92.06,其中9个为稳定蛋白,7个为不稳定蛋白。除登录号GSVIVG01014035001的基因外,其他15个基因编码的蛋白质均为亲水性蛋白。登录号GSVIVG01016517001的基因编码的蛋白质定位于细胞质和细胞核,其他15个基因编码的蛋白质定位于细胞质。二级结构和三级结构显示:葡萄Actin基因家族16个基因编码的蛋白质均由α螺旋、无规则卷曲和延伸链构成,且总体以无规则卷曲为主。系统进化分析和不同组织的基因表达分析结果显示:与拟南芥[Arabidopsis thaliana(Linn.)Heynh.]相似,葡萄Actin基因家族16个基因编码的蛋白质分为3个亚家族,ClassⅡ亚家族(营养型)包括登录号GSVIVG01003099001和GSVIVG01026580001的基因编码的蛋白质,这2个基因在所有组织中的表达均较高;ClassⅢ亚家族(生殖型)包括登录号GSVIVG01033494001、GSVIVG01024980001和GSVIVG01016550001的基因编码的蛋白质,这3个基因在花粉、雄蕊和花中的表达均较高;ClassⅠ亚家族包括其他11个基因编码的蛋白质,这11个基因在各组织中的表达总体上较低。研究结果显示:葡萄Actin基因家族的表达具有组织特异性。

海州香薷Actin基因片段克隆及表达分析

通过克隆海州香薷Actin基因片段并分析其组织表达,为研究海州香薷重金属抗性相关基因的表达调控奠定基础。根据Gen Bank中其他植物Actin基因保守序列设计兼并引物,以海州香薷根总RNA为模板,利用RT-PCR技术分离得到Actin基因片段。序列分析结果表明,海州香薷Actin基因片段长576 bp,编码192个氨基酸,与其他植物同源基因的氨基酸序列相似性为84%-97%,所克隆的序列为Actin基因的同源片段,将其命名为Eh ACT,在Gen Bank中提交序列,获得登录号AGT37260。半定量RT-PCR分析结果表明,Eh ACT在海州香薷的根、茎和叶中表达相对稳定,初步表明其可作为研究海州香薷基因表达的内参基因。