隧道纳米管：神经系统中的新型细胞间交流结构

隧道纳米管(tunneling nanotubes,TNTs)是基于细胞骨架尤其是纤维状肌动蛋白形成的细胞间管道样结构,其功能主要是介导广泛的细胞间物质交换,包括各种信号分子、RNA、蛋白质、细胞器甚至病原体,在生理和病理过程中都发挥重要作用.各种细胞类型中均发现有TNTs的形成,尤其在神经元细胞和神经胶质细胞中得到广泛关注.神经元细胞间或神经元细胞与星形胶质细胞间形成的TNTs,能够介导电耦合,还参与神经退行性疾病相关致病蛋白质的转移和/或传播,进而在神经系统发育和疾病进展中发挥作用.本文简要总结了在神经系统细胞间形成TNTs的研究进展,包括调节其形成的分子机制、功能和在神经系统疾病治疗中的潜在优势.     

糖基转移酶在神经系统中的作用

神经系统的发育及其正常功能的维持受到精确的控制,其调控异常导致的神经系统疾病成为危害健康的重要因素。研究神经系统的发育及其疾病发生的分子机制是生命科学的热点。糖基转移酶是一组催化糖链合成及糖链与蛋白质或者脂质形成复合物的酶类。糖基转移酶可以调节神经细胞表面多种蛋白质及脂质的糖基化,参与神经系统的发生及多种疾病发病过程的调控。对糖基转移酶在神经系统发育和疾病中的作用做一综述。     

5-羟甲基胞嘧啶及其TET氧合酶在神经系统发育和相关疾病中的研究进展

神经系统的正常发育是多种因素相互协调作用的结果,一旦特定因素失衡将引起相关疾病的发生。近年来不断有研究发现,DNA去甲基化过程的一类中间产物5-羟甲基胞嘧啶(5-hydroxymethylcytosine, 5hmC)作为一种新的表观遗传标记,在神经系统中高水平分布,并参与认知、记忆等重要的神经功能。5hmC的形成由氧合酶家族分子(ten-eleven translocation protein, TET)催化,在多种神经系统相关疾病中,5hmC水平和TETs分子的表达都发生改变,提示TET-5hmC表观遗传机制在复杂的神经系统发生发展过程中发挥了重要的调控作用。此外,作为基因表达调控的DNA标记物,5hmC的基因定位与基因表达水平的关系也是重要的研究方向。本文就近年来5hmC和TET家族蛋白分子在神经系统发育和相关疾病方面的重要研究发现进行了综述总结,希望为相关领域研究人员深入开展研究提供重要的思路,并为相关疾病设计治疗策略提供理论支持。     

核纤层蛋白B1的功能及其在神经系统疾病和肿瘤中的研究新进展

核纤层蛋白B1 (Lamin B1)是核纤层蛋白家族重要成员之一,其主要功能在于维持细胞核骨架完整性,并通过影响染色体分布、基因表达及DNA损伤修复等参与细胞的增殖和衰老。其表达异常与多种疾病有关,如神经系统疾病(神经管畸形,ADLD)及肿瘤(胰腺癌)等,是潜在的药物靶点和肿瘤标志物。对Lamin B1功能的深入研究,将有助于对相关神经系统疾病和肿瘤发生发展的分子机制的了解并为治疗靶点研究提供新方向。     

2D-DIGE技术在蛋白质组学中的应用

双向荧光差异凝胶电泳(2D-D IGE)作为一种新型的蛋白质组分析技术,已经被广泛应用于动物、植物、微生物以及人类差异蛋白的研究。在动物医学方面,采用DIGE技术,通过对不同类型,不同个体的细胞、组织、或经过不同处理和不同生长条件下蛋白质表达差异分析,在研究疾病的分子机理、分子诊断、药物作用机理、毒理学等方面都有广泛的应用。在植物学方面,该技术可以用来分离和分析植物亚细胞结构蛋白质组以及在生物和非生物胁迫下,植物细胞中蛋白质表达的差异性,从而建立差异蛋白相互作用网络图,从而为研究伤害机制提供一定的依据。     

单细胞转录组测序在少突胶质谱系细胞异质性与神经系统疾病中的应用

少突胶质细胞是中枢神经系统中形成髓鞘的高度特化的胶质细胞，由少突胶质前体细胞分化而来。长期以来，围绕少突胶质谱系细胞开展的研究主要集中在少突胶质细胞发育、髓鞘形成以及少突胶质谱系细胞在神经系统疾病中的作用等。新兴的单细胞转录组测序技术可以在转录组层面鉴定出特定类型细胞，为少突胶质谱系细胞的研究提供助力。本综述主要关注常见单细胞测序技术的发展以及它们在少突胶质细胞功能异质性和神经系统疾病研究中的应用，并对已取得的成果进行总结阐述，为单细胞测序技术在中枢神经系统疾病中少突胶质谱系细胞相关研究的应用和开发提供思路和参考。     

象甲科线粒体基因组特征及系统发育分析

昆虫线粒体基因组广泛应用于系统发育关系的重新建立、分子进化、谱系地理学及物种诊断等领域。为揭示象甲科昆虫线粒体全基因组序列的主要结构特征,探究其系统发育相关信息,为进化遗传学研究和分子标记选取等提供参考依据,本研究利用比较基因组学和生物信息学方法,对NCBI上已公布的35种象甲科物种线粒体全基因组序列进行了分析。结果显示:(1)象甲科tRNA基因存在排序及数目异常情况,不同物种中蛋白质编码基因和2种rRNAs排列相同,线粒体全基因组具有明显AT偏向;(2)COX1、ATP6、ND5、ND4、ND4L和ND1基因除标准三联密码子外,还存在特殊的起始密码子AAT、TTG和GTG;(3)13种蛋白质编码基因的进化速率顺序为COX3ATP8ND2ND5ND1ND4ND6ND4LND3ATP6CytbCOX1COX2;(4)13个蛋白编码基因和rRNAs基因中,ND5、rrnL、ND4和ND2基因变异位点数较高,可作为备选的分子标记;(5)各亚科的系统发育关系可能为(((小蠹亚科Scolytinae+长小蠹亚科Platypodinae)+(隐喙象亚科Cryptorhynchinae+魔喙象亚科Molytinae+象虫亚科Curculioninae)+((孢喙象亚科Cyclominae+粗喙象亚科Entiminae)+(隐颏象亚科Dryophthorinae+长小蠹亚科))),为象甲科的系统发育分析有提供参考。     

两种实验技术在蛋白质-蛋白质相互作用检测中的应用

蛋白质是生命活动的主要承担者。蛋白质种类繁多,结构多样,具有十分广泛的生物学功能,可作为载体蛋白、酶蛋白和信号肽等参与调控细胞内的各种代谢活动。生物体内蛋白质与其他分子的相互作用,尤其是蛋白质-蛋白质之间的相互作用,是蛋白质行使这些重要生物学功能的基础。通过研究可以相互作用的蛋白质形成的各种复合体,对揭示蛋白质的功能,更清楚地阐明细胞生长、发育、分化和凋亡的生命活动规律,为重大疾病的预防、治疗和新药开发提供了理论基础。目前科研工作者在基于分子生物学、生物化学、微生物学和生物物理学的基础上已经发展出了许多蛋白质相互作用的研究技术,本文着重对现有研究方法中的生物膜干涉技术和微量热泳动技术进行介绍和综述。     

液-液相分离在细胞信号调控过程中的作用机制

近年来,液-液相分离(简称相分离)因其独特的功能与组织性,在细胞生物学研究中发展迅速。细胞内部分蛋白质及核酸(多为RNA)等生物大分子通过由多种弱多价相互作用及构象熵共同介导的相变(phase transition)形成无膜细胞器(membraneless organelles, MLOs)。这些无膜结构具有明显的流体性质,包括其圆形外表、可浸润、滴落和彼此融合,并具有动态的内部成分交换。在体内形成的无膜细胞器,广泛参与到包括细胞膜信号传导、膜结合蛋白质组装、染色质重塑、RNA代谢、突触传递、活跃转录中心形成、有丝分裂结构形成,以及蛋白质病理性转变等多种重要的细胞内信号调控过程。本文从相分离的研究背景,相分离发生的分子机制,正常相分离过程参与的多种细胞生理活动,异常相分离与神经性疾病及癌症发生的关系等方面,阐述了生物大分子的相分离在细胞信号调控过程中的普遍性及重要作用,并对研究相分离的实验技术和常用的相分离数据库进行了介绍。生物大分子相分离行为的发现,为重新理解众多结构及细胞生物学现象提供了全新的角度,生物大分子相分离可能作为一种新的生物学过程,帮助重新认识众多信号通路的调控方式,也有望为相关疾病的治疗提供新的方向。     

大鼠侧脑室注射瘦素对卵巢摘除给予雌激素子宫腔液2-DE图谱分析

瘦素(leptin)通过动物下丘脑-垂体轴对其生殖活动予以调节,但瘦素通过下丘脑对子宫调节作用仍不清楚.本研究在大鼠侧脑室微量注射瘦素6 h后,采用双向凝胶电泳(2-DE)结合基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)技术分离、鉴定了卵巢摘除给予雌激素的子宫腔液蛋白质表达谱.我们获得了分辨率较高、重复性较好的2-DE图谱|质谱分析结合SwissProt蛋白数据库检索,共鉴定了24个差异表达的蛋白质与生理盐水注射（对照）比较,侧脑室微量注射瘦素引起22个蛋白质分子显著上调,2个显著下调.重要的是,在上调的蛋白质分子中包括转铁蛋白、基质金属蛋白酶7、膜联蛋白A1、补体B因子、磷酸甘油酸酯变位酶1、烯醇酶、冠蛋白-1A等15个蛋白质分子,下调的2个蛋白质分子是补体C3、溶解物载体家族4成员3.这些蛋白质分子涉及细胞分化、细胞迁移、细胞凋亡,以及炎症反应等功能.实验结果提示,瘦素可能通过下丘脑-垂体轴参与免疫调节、雌性生殖及炎症反应等,从而对子宫发挥调节作用.本研究将为加深对瘦素调节子宫作用的认识,并为深入研究女性生殖系统疾病提供新的启示.     

An improved bimolecular fluorescence complementation tool based on superfolder green fluorescent protein

Bimolecular fluorescence complementation (BiFC) has been widely used in the analysis of protein-protein interactions (PPIs) in recent years. There are many notable advantages of BiFC such as convenience and direct visualization of PPI in cells. However, BiFC has one common limitation: the separated non-fluorescent fragments can be spontaneously self-assembled into an intact protein, which leads to false-positive results. In this study, a pair of complementary fragments (sfGFPN and sfGFPC) was constructed by splitting superfolder GFP (sfGFP) between the 214 and 215 amino acid residue, and sfGFPC was mutated by site-directed gene mutagenesis to decrease the signal of negative control. Our results showed that mutations in sfGFPC (sfGFPC(m12)) can effectively decrease the signal of negative control. Thus, we provide an improved BiFC tool for the analysis of PPI. Further, since the self-assembly problem is a common shortcoming for application of BiFC, our research provides a feasible strategy for other BiFC candidate proteins with the same problem.     

New Gateway-compatible vectors for a high-throughput protein–protein interaction analysis by a bimolecular fluorescence complementation (BiFC) assay in plants and their application to a plant clathrin structure analysis

Protein–protein interactions (PPI) play key roles in various biological processes. The bimolecular fluorescence complementation (BiFC) assay is an excellent tool for routine PPI analyses in living cells. We developed new Gateway vectors for a high-throughput BiFC analysis of plants, adopting a monomeric Venus split just after the tenth β-strand, and analyzed the interaction between Arabidopsis thaliana coated vesicle coatmers, the clathrin heavy chain (CHC), and the clathrin light chain (CLC). In competitive BiFC tests, CLC interacted with CHC through a coiled-coil motif in the middle section of CLC. R1340, R1448, and K1512 in CHC and W94 in CLC are potentially key amino acids underlying the inter-chain interaction, consistent with analyses based on homology modeling. Our Gateway BiFC system, the V10-BiFC system, provides a useful tool for a PPI analysis in living plant cells. The CLC–CHC interaction identified may facilitate clathrin triskelion assembly needed for cage formation.     

An improved bimolecular fluorescence complementation assay with a high signal-to-noise ratio

Protein-protein interactions (PPIs) play crucial roles in various biological processes. Among biochemical, genetic, and imaging approaches that have been used for the study of PPIs, visualization of PPIs in living cells is the key to understanding their cellular functions. The bimolecular fluorescence complementation (BiFC) assay represents one of these imaging tools for direct visualization of PPIs in living cells. The BiFC assay is based on the structural complementation of two nonfluorescent N- and C-terminal fragments of a fluorescent protein when they are fused to a pair of interacting proteins. Although over 10 different fluorescent proteins have been used for BiFC assays, the two nonfluorescent fragments from all of these fluorescent proteins can spontaneously self-assemble, which contributes to background fluorescence and decreases the signal-to-noise (S/N) ratio in the BiFC assay. Here we report the identification of a mutation, I152L, that can specifically reduce self-assembly and decrease background fluorescence in a Venus-based BiFC system. This mutation allows a 4-fold increase in the S/N ratio of the BiFC assay in living cells. This improved Venus-based BiFC system will facilitate PPI studies in various biological research fields.     

Monitoring G protein-coupled receptor activation using an adenovirus-based β-arrestin bimolecular fluorescence complementation assay

G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors and are involved in a variety of pathological conditions including cancer and cardiovascular, metabolic, neurological, and autoimmune diseases. GPCRs are being intensively investigated as targets for therapeutic intervention, and the β-arrestin recruitment assay has become a popular tool for analyzing GPCR activation. Here, we report a high-throughput method for cloning GPCR cDNAs into adenoviral bimolecular fluorescence complementation (BiFC) vectors and performing the β-arrestin BiFC assay in cells transduced with recombinant adenoviruses. An analysis of the activation of somatostatin receptor 2 (SSTR2) with the adenovirus-based β-arrestin BiFC assay showed that the assay is suitable for quantifying SSTR2 activation in response to specific agonists or antagonists. Furthermore, the adenovirus-based β-arrestin BiFC assay was able to detect the activation of a broad range of GPCRs. Collectively, our data indicate that the adenovirus-based β-arrestin BiFC assay can serve as a simple and universal platform for studying GPCR activation and thus will be useful for high-throughput screening of drugs that target GPCRs.     

In vivo visualization of actin dynamics and actin interactions by BiFC

The method of bimolecular fluorescence complementation (BiFC) enables selective visualization of protein interactions. While BiFC complex formation under in vitro conditions is considered to be essentially irreversible, there are hints that under in vivo conditions BiFC complex formation can be reversible. In the present study we used the BiFC method to visualize in vivo actin cytoskeleton dynamics. We demonstrate that in living cells formation of actin/actin BiFC complexes is reversible. Furthermore, we show heterologous binding between actin and protein kinase C delta (PKCdelta). Treatment with phorbol esters caused translocation of actin/PKCdelta complexes from the cytosol to the plasma membrane independent of an intact actin cytoskeleton. Our experiments demonstrate that the BiFC method might be a useful tool to investigate participation of the actin cytoskeleton in regulation of cell function.     

双分子荧光互补技术

双分子荧光互补（bimolecular fluorescence complementation, BiFC）是近年发展起来的用于体内或体外检测蛋白质相互作用的一项新技术.该技术是将荧光蛋白在合适的位点切开形成不发荧光的2个片段,这2个片段借助融合于其上的目标蛋白的相互作用,彼此靠近,重新形成能具有活性的荧光蛋白.BiFC方法简单直观,既可以检测蛋白之间的相互作用,也可以定位相互作用蛋白质的位点.多色BiFC系统共用或与荧光共振能量转移（FRET）技术联用,还可以检测细胞内多个蛋白质的相互作用.     

A novel far-red bimolecular fluorescence complementation system that allows for efficient visualization of protein interactions under physiological conditions

Fluorescent protein (FP) has enabled the analysis of biomolecular interactions in living cells, and bimolecular fluorescence complementation (BiFC) represents one of the newly developed imaging technologies to directly visualize protein–protein interactions in living cells. Although 10 different FPs that cover a broad range of spectra have been demonstrated to support BiFC, only Cerulean (cyan FP variant), Citrine and Venus (yellow FP variants)-based BiFC systems can be used under 37 °C physiological temperature. The sensitivity of two mRFP-based red BiFC systems to higher temperatures (i.e., 37 °C) limits their applications in most mammalian cell-based studies. Here we report that mLumin, a newly isolated far-red fluorescent protein variant of mKate with an emission maximum of 621 nm, enables BiFC analysis of protein–protein interactions at 37 °C in living mammalian cells. Furthermore, the combination of mLumin with Cerulean- and Venus-based BiFC systems allows for simultaneous visualization of three pairs of protein–protein interactions in the same cell. The mLumin-based BiFC system will facilitate simultaneous visualization of multiple protein–protein interactions in living cells and offer the potential to visualize protein–protein interactions in living animals.     

Interactions between the budding yeast IQGAP homologue Iqg1p and its targets revealed by a split-EGFP bimolecular fluorescence complementation assay

A split-EGFP based bimolecular fluorescence complementation (BiFC) assay has been used to detect interactions between the Saccharomyces cerevisiae cytoskeletal scaffolding protein Iqg1p and three targets: myosin essential light chain (Mlc1p), calmodulin (Cmd1p) and the small GTPase Cdc42p. The format of the BiFC assay used ensures that the proteins are expressed at wild type levels thereby avoiding artefacts due to overexpression. This is the first direct in vivo detection of these interactions; in each case, the complex is localised to discrete regions of the yeast cytoplasm. The labelling with EGFP fragments results in changes in growth kinetics, cell size and budding frequency. This is partly due to the reassembled EGFP locking the complexes into essentially permanent interactions. The consequences of this for Iqg1p interactions and BiFC assays in general are discussed.     

基于关联基因本体论注释的蛋白质相互作用预测

细胞中的生理活动主要是通过蛋白质 - 蛋白质之间的相互作用来调控完成 . 详尽细致的蛋白质 - 蛋白质相互作用网络的解析对于理解细胞中复杂的调控、代谢和信号通路有重要的意义 . 近年来,关于新的蛋白质 - 蛋白质相互作用预测领域进展快速,这里,利用贝叶斯算法结合关联的 GO (Gene Ontology) ,来预测蛋白质的相互作用 . 利用非冗余的蛋白质相互作用数据来观察 GO 对的特性,得到 GO 关联的概率 . 通过阳性的和阴性的标准对照数据证实这个新方法可以很好地区别这两类不同的数据,显示出较好的灵敏度和非常低的假阳性预测率 . 通过与已知的高通量的实验数据比较,这个方法具有灵敏度高、速度快的优点 . 而且,运用这个新方法可以提供一些新的关于细胞内蛋白质之间相互作用的信息,为进一步的实验提供理论依据 .     

Bimolecular Fluorescence Complementation Analysis of Inducible Protein Interactions: Effects of Factors Affecting Protein Folding on Fluorescent Protein Fragment Association

Bimolecular fluorescence complementation (BiFC) analysis enables visualization of the subcellular locations of protein interactions in living cells. Using fragments of different fluorescent proteins, we investigated the temporal resolution and the quantitative accuracy of BiFC analysis. We determined the kinetics of BiFC complex formation in response to the rapamycin-inducible interaction between the FK506 binding protein (FKBP) and the FKBP-rapamycin binding domain (FRB). Fragments of yellow fluorescent protein fused to FKBP and FRB produced detectable BiFC complex fluorescence 10 min after the addition of rapamycin and a 10-fold increase in the mean fluorescence intensity in 8 h. The N-terminal fragment of the Venus fluorescent protein fused to FKBP produced constitutive BiFC complexes with several C-terminal fragments fused to FRB. A chimeric N-terminal fragment containing residues from Venus and yellow fluorescent protein produced either constitutive or inducible BiFC complexes depending on the temperature at which the cells were cultured. The concentrations of inducers required for half-maximal induction of BiFC complex formation by all fluorescent protein fragments tested were consistent with the affinities of the inducers for unmodified FKBP and FRB. Treatment with the FK506 inhibitor of FKBP-FRB interaction prevented the formation of BiFC complexes by FKBP and FRB fusions, but did not disrupt existing BiFC complexes. Proteins synthesized before the addition of rapamycin formed BiFC complexes with the same efficiency as did newly synthesized proteins. Inhibitors of protein synthesis attenuated BiFC complex formation independent of their effects on fusion protein synthesis. The kinetics at which they inhibited BiFC complex formation suggests that they prevented association of the fluorescent protein fragments, but not the slow maturation of BiFC complex fluorescence. Agents that induce the unfolded protein response also reduced formation of BiFC complexes. The effects of these agents were suppressed by cellular adaptation to protein folding stress. In summary, BiFC analysis enables detection of protein interactions within minutes after complex formation in living cells, but does not allow detection of complex dissociation. Conditional BiFC complex formation depends on the folding efficiencies of fluorescent protein fragments and can be affected by the cellular protein folding environment.