荧光蛋白标签对gD囊膜蛋白在BHK-21细胞亚细胞定位的影响

【目的】探究荧光蛋白标签对马疱疹病毒I型(Equine herpes virus type 1,EHV-1)gD囊膜蛋白亚细胞定位的影响。【方法】以EHV-1基因组为模板利用PCR扩增gD全基因,分别克隆至pAcGFP1-C1和p Ds Red2-N1质粒,构建p Ac-GFP-gD(GFP-gD)和p Ds-gD-Red(gD-Red)重组质粒;将GFP基因插入gD基因信号肽序列之后并克隆至PVAX-1质粒,构建PVAX-S-GFP-gD’(S-GFP-gD’)重组质粒;将Flag标签序列与gD囊膜蛋白N端序列融合后并克隆至p VAX-1表达载体,构建p VAX-Flag-gD(Flag-gD)重组质粒。将4种不同重组真核表达质粒分别转染BHK-21细胞,通过激光共聚焦显微镜对不同融合蛋白gD进行亚细胞定位。【结果】成功构建4种不同的融合蛋白gD真核表达载体;在BHK-21细胞单独表达时,不同融合蛋白gD绝大部分都定位于高尔基体,极少量定位于细胞核内。【结论】不同插入位点的荧光蛋白标签对gD囊膜蛋白亚细胞定位无明显影响,这对今后研究其它蛋白亚细胞定位提供参考。     

Kinase activity and subcellular distribution of a chimeric green fluorescent protein-tagged Janus kinase 2

Summary Janus kinase 2 (JAK2) is an essential intracellular signal transducer for numerous cytokines and hormones. To examine how JAK2 structural modifications can affect cellular physiology, we created expression vectors for chimeric proteins containing an enhanced green fluorescent protein (EGFP) fused to rat JAK2 (EGFP/rJAK2), and a kinase-inactive variant, EGFP/rJAK2(K882E). The properties of EGFP/rJAK2 were examined following transient transfection of COS-7 cells. EGFP/rJAK2 was expressed throughout the cell, and was found in subcellular membrane, cytosolic and nuclear fractions. Interestingly, EGFP/rJAK2 phosphorylated other proteins in situ without additional cytokine stimulation. Furthermore, despite a much higher level of tyrosine phosphorylation arising from in situ autophosphorylation, the in vitro radiolabelling autokinase activity of EGFP/rJAK2 was significantly less than that of the endogenous JAK2. These results reveal a technical limitation of the application of the “conventional” in vitro radiolabelling autokinase assay to hyperphosphorylated forms of the enzyme and illustrate the potential weaknesses in individual assays commonly used to determine JAK2’s enzymatic activity and subcellular distribution. We also suggest that the EGFP/rJAK2 model can be very useful in studying JAK2-related cancers, because its ubiquitous distribution and abnormal constitutive hyperphosphorylation may distinguish it from the cytokine-regulated, membrane-proximal form of JAK2 associated with normal physiology.     

Three-color intranuclear staining for measuring mitosis and apoptosis in cells transfected with a GFP-tagged histone

Abstract

Green fluorescent protein (GFP) has become a powerful tool for monitoring the expression of transfected genes by flow cytometry including GFP-tagged histones for tracking chromatin and elucidating histone function. We describe here a method for simultaneous detection of three nucleus-localized signals: a GFP-tagged histone, DNA content and detection of phosphorylated histone H3, which labels mitotic cells. We also demonstrate another application of this method for simultaneous detection of a GFP-tagged histone, DNA content, and cleaved caspase-3.     

Arabidopsis thaliana subcellular responses to compatible Erysiphe cichoracearum infections

Subcellular events of Erysiphe cichoracearum infections of epidermal cells were visualized in living tissues of Arabidopsis plants carrying various green fluorescent protein (GFP)-tagged organelles via laser scanning confocal microscopy. Early in the infection sequence, cytoplasm and organelles moved towards penetration sites and accumulated near penetration pegs. Peroxisomes appeared to accumulate preferentially relative to the cytoplasm at penetration sites. Another early event, which preceded haustorium formation, was the aggregation of some GFP-tagged plasma membrane marker proteins into rings around penetration sites, which extended across cell-wall boundaries into neighboring cells. This feature localized to sites where papillae were deposited. The extrahaustorial membrane (EHM) encases the fungal feeding structure, the haustorium, separating it from the host cytoplasm. Eight plasma membrane markers were excluded from the EHM and remained in a collar-like formation around the haustorial neck. These observations support the suggestions that the EHM is a unique, specialized membrane and is different from the plasma membrane. Our results suggested two possibilities for the origin of the EHM: invagination of the plasma membrane coupled with membrane differentiation; or de novo synthesis of the EHM by targeted vesicle trafficking.     

Vaccinia virus utilizes microtubules for movement to the cell surface

Vaccinia virus (VV) egress has been studied using confocal, video, and electron microscopy. Previously, intracellular-enveloped virus (IEV) particles were proposed to induce the polymerization of actin tails, which propel IEV particles to the cell surface. However, data presented support an alternative model in which microtubules transport virions to the cell surface and actin tails form beneath cell-associated enveloped virus (CEV) particles at the cell surface. Thus, VV is unique in using both microtubules and actin filaments for egress. The following data support this proposal. (a) Microscopy detected actin tails at the surface but not the center of cells. (b) VV mutants lacking the A33R, A34R, or A36R proteins are unable to induce actin tail formation but produce CEV and extracellular-enveloped virus. (c) CEV formation is inhibited by nocodazole but not cytochalasin D or 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine (PP1). (d) IEV particles tagged with the enhanced green fluorescent protein fused to the VV B5R protein moved inside cells at 60 microm/min. This movement was stop-start, was along defined pathways, and was inhibited reversibly by nocodazole. This velocity was 20-fold greater than VV movement on actin tails and consonant with the rate of movement of organelles along microtubules.     

Construction and characterization of an enhanced GFP-tagged anti-BAFF scFv antibody

Elevated levels of B-cell-activating factor of the TNF family (BAFF) have been implicated in the pathogenesis of autoimmune diseases in human. In this study, an anti-BAFF single-chain antibody fragment (scFv) was genetically linked to the C terminus of the enhanced green fluorescent protein (EGFP) to generate an EGFP/scFv fusion protein. The EGFP/scFv fusion protein had an apparent molecular weight of 52 kDa and was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis. After being purified by immobilized metal affinity chromatography, the fusion protein exhibited similar fluorescence spectra with native EGFP. Enzyme-linked immunosorbent assay and fluorescence-activated cell sorting showed the EGFP/scFv could bind to human soluble BAFF and BAFF positive cell lines in vitro. The binding of EGFP/scFv can also be visualized under laser scanning confocal microscopy. Furthermore, the results of the competition assay indicated its antigen binding specificity. Therefore, the fusion protein EGFP/scFv has several characteristics including high sensitivity, stability and convenience for manipulation, and can be a powerful tool for the study of the underlying pathology of BAFF relevant to autoimmune diseases.     

盐穗木功能未知蛋白 (HcUKPP)的亚细胞定位

藜科的极端盐生植物盐穗木（Halostachys caspica）的高盐胁迫抑制差减文库中有39%的功能未知蛋白（proteins with obscure features, POFs）,利用亚细胞定位分析可以初步判断其可能的功能.将盐穗木的1个POF-cDNA序列HcUKPP的编码区构建至pCAMBIA1301-GFP植物表达载体上,冻融法将重组质粒pCAMBIA1301-HcUKPP-GFP转化农杆菌EH105A,利用花序浸染法将基因导入拟南芥,经潮霉素筛选获得T1代阳性幼苗.通过激光扫描共聚焦显微镜观察转基因拟南芥植株的根部细胞. 结果显示,表达GFP蛋白的对照转基因植株中,绿色荧光在细胞核、细胞膜以及细胞质中均能检测到,而表达HcUKPP-GFP融合蛋白的转基因植株中,绿色荧光只在细胞质膜上表达,说明HcUKPP蛋白为细胞质膜相关蛋白.本研究为深入探讨盐穗木未知蛋白的功能奠定了基础.     

Dual cytoplasmic and nuclear distribution of the novel arsenite-stimulated human ATPase (hASNA-I)

The arsenite-stimulated human ATPase (hASNA-I) protein is a distinct human ATPase whose cDNA was cloned by sequence homology to the Escherichia coli ATPase arsA. Its subcellular localization in human malignant melanoma T289 cells was examined to gain insight into the role of hASNA-I in the physiology of human cells. Immunocytochemical staining using the specific anti-hASNA-I monoclonal antibody 5G8 showed a cytoplasmic, perinuclear, and nucleolar distribution. Subcellular fractionation indicated that the cytoplasmic hASNA-I was soluble and that the perinuclear distribution was due to association with the nuclear membrane rather than with the endoplasmic reticulum. Its presence in the nucleolus was confirmed by showing colocalization with an antibody of known nucleolar specificity. Further immunocytochemical analysis showed that the hASNA-I at the nuclear membrane was associated with invaginations into the nucleus in interphase cells. These results indicate that hASNA-I is a paralogue of the bacterial ArsA protein and suggest that it plays a role in the nucleocytoplasmic transport of a nucleolar component. J. Cell. Biochem. 71:1–10, 1998. © 1998 Wiley-Liss, Inc.     

低温胁迫对两种圆柏属植物亚细胞抗氧化酶活性的影响

以祁连圆柏和圆柏幼苗为材料,研究不同处理时间下低温胁迫对圆柏属植物叶片亚细胞抗氧化酶活性的影响,探讨其在圆柏属植物叶片中的亚细胞定位。结果表明：低温胁迫下,丙二醛(MDA)含量和抗氧化酶活性随时间变化均呈先升后降趋势,祁连圆柏中抗氧化酶的种类比圆柏的多且活性强,而 MDA 含量低于圆柏,表明祁连圆柏在低温胁迫下具有更广泛的适应性。此外,两种圆柏植物叶片超氧化物歧化酶(SOD)和抗坏血酸过氧化物酶(APX)定位为叶绿体＞细胞溶质＞线粒体,过氧化氢酶(CAT)定位为线粒体＞叶绿体＞细胞溶质,谷胱甘肽还原酶(GR)定位为线粒体＞细胞溶质＞叶绿体,祁连圆柏过氧化物酶(POD)定位为细胞溶质＞叶绿体＞线粒体,圆柏POD定位为细胞溶质＞线粒体＞叶绿体,且抗氧化酶SOD、APX和 GR在亚细胞中分布差异达到极显著,这说明抗氧化酶在其中一种亚细胞中发挥主要作用,为克隆亚细胞组分中的抗氧化酶基因提供了理论依据。     

A bicistronic, Ubiquitin-10 promoter-based vector cassette for transient transformation and functional analysis of membrane transport demonstrates the utility of quantitative voltage clamp studies on intact Arabidopsis root epidermis

To date the use of fluorescent reporter constructs in analysing membrane transport has been limited primarily to cell lines expressing stably either the tagged transporter protein(s) or markers to identify lineages of interest. Strategies for transient expression have yet to be exploited in transport analysis, despite their wide application in cellular imaging studies. Here we describe a Gateway-compatible, bicistronic vector, incorporating the constitutive Ubiqutin-10 gene promoter of Arabidopsis that gives prolonged expression after transient transformation and enables fluorescence marking of cells without a fusion construct. We show that Arabidopsis root epidermal cells are readily transformed by co-cultivation with Agrobacterium and are tractable for quantitative electrophysiological analysis. As a proof of principle, we transiently transformed Arabidopsis with the bicistronic vector carrying GFP as the fluorescent marker and, separately, the integral plasma membrane protein SYP121 essential for the inward K+ channel current. We demonstrate that transient expression of SYP121 in syp121 mutant plants is sufficient to rescue the K+ current in vivo. The combination of transient expression and use of the bicistronic vector promises significant advantages for studies of membrane transport and nutrient acquisition in roots.     

Objective evaluation of differences in protein subcellular distribution

Research on the cell and molecular biology of proteins involved in membrane traffic often requires determination of the effects of various experimental conditions on the subcellular distributions of those proteins. This is most often accomplished by acquiring fluorescence microscope images and visually comparing these images. While this approach is quite suitable for detecting major changes in distributions, it is not sensitive to small changes and does not permit a quantitative and objective analysis. We therefore describe the application of pattern analysis methods to the comparison of sets of fluorescence microscope images. This approach provides a high throughput and reproducible technique to determine whether image distributions differ within a specified statistical confidence, and is shown to resolve image sets indistinguishable by visual inspection.     

Expression,subcellular localization and protein‑protein interaction of four isoforms of EcR/USP in the common cutworm

Ecdysone receptor (EcR) and ultraspiracle (USP) form heterodimers to mediate ecdysteroid signaling during molting and metamorphosis. Various EcR/USP heterodimers have been reported. However, it is unclear what kind of EcR/USP combination is adopted by lepidopteran insects during the larval?pupal metamorphosis and whether the EcR/USP heterodimer varies among different tissues. To address these questions, two isoforms of each EcR and USP were cloned from the common cutworm, their messenger RNA expression patterns were examined by real‐time quantitative polymerase chain reaction in different tissues during the larval–pupal metamorphosis and in the midgut in response to hormonal induction. Furthermore, their subcellular localization and protein?protein interaction were explored by transient expression and far‐western blotting, respectively. All the four genes were significantly up‐regulated in prepuae and/or pupae. The expression profiles of EcRB1 and USP1 were nearly identical to each other in the epidermis, fat body and midgut, and a similar situation also applied to EcRA and USP2. The three genes responded to 20‐hydroxyecdysone (20E) induction except for USP2, and USP1 could be up‐regulated by both 20E and juvenile hormone. The four proteins mainly localized in the nucleus and the nuclear localization was promoted by 20E. The protein?protein interaction between each EcR and USP was found in vitro. These results suggest that two types of EcR/USP heterodimer (EcRA/USP2 and EcRB1/USP1) may exist simultaneously in the common cutworm, and the latter should play more important roles during the larval?pupal metamorphosis. In addition, the types of EcR/USP heterodimer do not vary in the tissues which undergo histolysis and regeneration during metamorphosis.     

Time-lapse,single cell based confocal imaging analysis of caspase activation and phosphatidylserine flipping during cellular apoptosis

Abstract

Apoptosis is an important phenomenon for investigating the efficacy of anti-cancer drug candidates. The conventional assays for cellular apoptosis, such as enzyme-linked immunosorbent assay, absorbance monitoring for the activity of caspase, and flow cytometric assay, have focused only on biochemical events. We investigated the staurosporine (STS)-induced apoptosis of the murine macrophage RAW-264.7 cell using a cell based bioimaging technique. Using time-lapse confocal microscopy, we monitored caspase-3 activation during apoptosis by imaging the translocation of green fluorescent protein from the cytosol to the nuclei. Five hours after 1 μM STS treatment, caspase-3 was observed to be activated and membrane blebbing was observed simultaneously. Also, the loss of phosphatidylserine (PS) asymmetry in the phospholipid bilayer of plasma membrane during early apoptosis was monitored by imaging annexin-V labeled with fluorescein isocyanate binding to the externalized PS at various concentrations of STS. Moreover, disintegration of the plasma membrane during late apoptosis was confirmed using a nuclear dye, propidium iodide. The single cell based bioimaging data agreed well with those of the biochemical assays for caspase activation and morphological observation for membrane integrity.     

Plant cell biology in the new millennium: new tools and new insights

The highly regulated structural components of the plant cell form the basis of its function. It is becoming increasingly recognized that cellular components are ordered into regulatory units ranging from the multienzyme complexes that allow metabolic channeling during primary metabolism to the "transducon" complexes of signal transduction elements that allow for the highly efficient transfer of information within the cell. Against this structural background the highly dynamic processes regulating cell function are played out. Recent technological advances in three areas have driven our understanding of the complexities of the structural and functional dynamics of the plant cell. First, microscope and digital camera technology has seen not only improvements in the resolution of the optics and sensitivity of detectors, but also the development of novel microscopy applications such as confocal and multiphoton microscopy. These technologies are allowing cell biologists to image the dynamics of living cells with unparalleled three-dimensional resolution. The second advance has been in the availability of increasingly powerful and affordable computers. The computer control/analysis required for many of the new microscopy techniques was simply unavailable until recently. Third, there have been dramatic advances in the available probes to use with these new microscopy approaches. Thus the plant cell biologist now has available a vast array of fluorescent probes that will report cell parameters as diverse as the pH of the cytosol, the oxygen level in a tissue, or the dynamics of the cytoskeleton. The combination of these new approaches has led to an increasingly detailed picture of how plant cells regulate their activities.     

Construction of bifunctional fusion proteins consisting of duck BAFF and EGFP

We constructed fusion proteins consisting of fluorescence-enhanced green fluorescent protein (EGFP) and soluble domain of duck B-cell-activating factor of the TNF family (dsBAFF). The soluble EGFP/dsBAFF was efficiently expressed in Escherichia coli BL 21 (DE3) and was purified in milligram amounts using metal chellate affinity chromatography. The fusion protein exhibited similar fluorescence spectra with free EGFP and promoted the survival of duck bursal B cells in vitro as well as dsBAFF. EGFP/dsBAFF has shown specific binding to duck BAFF receptors positive-cells and the stained cells could be analyzed with flow cytometry. Thus, the fusion protein represents a readily obtainable source of biologically active dsBAFF that may prove useful in further studies on duck BAFF and its receptors.     

Characterization of stably transfected fusion protein GFP-estrogen receptor-alpha in MCF-7 human breast cancer cells

Tagging hormone receptors with the green fluorescent protein (GFP) has increased our knowledge of ligand dependent sub-cellular trafficking of hormone receptors. However, the effect of the tagged hormone receptor expression on the corresponding wild type hormone receptor and endogenous gene expression has not been investigated. In this study, we constructed a MCF-7 cell line stably expressing GFP-tagged human estrogen receptor-alpha (ER) under control of the tetracycline-on system to determine the effect of GFP-ER expression on cell proliferation and expression of endogenous ER and hormone-responsive genes. Further, the inducible system was applied to determine the ligand dependent turnover rates of GFP-ER protein and mRNA. Our results demonstrate that GFP-ER expression did not affect cell cycling. Independent of ligand, GFP-ER markedly reduced the level of endogenous ER mRNA and protein, suggesting that ER negatively autoregulates its expression. Cisplatin cross-linking studies showed that GFP-ER is associated with nuclear DNA in situ, suggesting that GFP-ER is partially replacing ER at estrogen response elements. Furthermore, GFP-ER expression did not affect the estradiol induced temporal expression of hormone responsive genes c-myc and pS2.     

Phosphorylation of the N-terminal domain regulates subcellular localization and DNA binding properties of the peptidyl-prolyl cis/trans isomerase hPar14

Human parvulin 14 (hPar14) is a folding helper enzyme belonging to the parvulin family of peptidyl-prolyl cis/trans isomerases (PPIases). This enzyme is thought to play a role in cell-cycle and chromatin remodeling. Although hPar14 was nuclearly localized and bound to double-stranded DNA, the molecular basis of the subcellular localization and the functional regulation remained unknown.Here we show that subcellular localization and DNA-binding ability of hPar14 is regulated by posttranslational modification of its N-terminal domain. As proved by MALDI-TOF mass spectrometry and MS/MS fragmentation, hPar14 is phosphorylated at Ser19 in vitro and in vivo. In human HeLa cells the protein is most likely modified by casein kinase 2 (CK2). Phosphorylation of hPar14 is inhibited both in vitro and in vivo by 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB), a specific inhibitor of CK2 activity. Mutation of Ser19 to Ala abolishes phosphorylation and alters the subcellular localization of hPar14 from predominantly nuclear to significantly cytoplasmic. Immunostaining shows that a Glu19 mutant of hPar14, which mimics the phosphorylated state of Ser19, is localized around the nuclear envelope, but does not penetrate into the nucleoplasm. In contrast to wild-type hPar14, the in vitro DNA-binding affinity of the Glu19 mutant is strongly reduced, suggesting that only the dephosphorylated protein is the active DNA-binding form of hPar14 in the nucleus.     

核定位信号分析示踪载体——pGST-EGFP的构建及功能鉴定

目的 构建谷胱甘肽转硫酶（GST）与EGFP相融合的新型蛋白质示踪载体--pGST-EGFP,以用于蛋白质细胞亚定位信号序列的深入分析.方法 以质粒pEGFP-N1为骨架,融合从pGEX-2TK载体中扩增的GST编码序列,构建成pGST-EGFP融合表达质粒;再插入人工合成的已知核定位蛋白SV40的核定位序列（NLS）,构建成pGST-EGFP-SV40 NLS作为阳性对照;另外,构建小分子量蛋白TNNI2在pGST-EGFP的融合表达质粒.将对照pEGFP-N1和各重组质粒分别用脂质体介导,瞬时转染HeLa细胞,荧光显微镜下观察蛋白的核定位情况.结果 单独表达的EGFP呈全细胞分布,而GST-EGFP融合蛋白只存在于细胞浆;SV40 NLS能将GST-EGFP融合蛋白带进细胞核.虽然TNNI2-EGFP融合蛋白的细胞亚定位呈现核内丰度更高的特点,但TNNI2-GST-EGFP融合蛋白仅限定于胞浆分布,提示TNNI2不能主动定位到细胞核中.结论 成功构建了蛋白质细胞亚定位示踪载体--pGST-EGFP.作为核定位信号分析系统,其对小分子蛋白细胞亚定位的示踪效果优于传统的pEGFP载体,更适用于科研工作中小分子量蛋白质核定位信号序列的研究.     

Expression and subcellular targeting of canine parvovirus capsid proteins in baculovirus-transduced NLFK cells

A mammalian baculovirus delivery system was developed to study targeting in Norden Laboratories feline kidney (NLFK) cells of the capsid proteins of canine parvovirus (CPV), VP1 and VP2, or corresponding counterparts fused to EGFP. VP1 and VP2, when expressed alone, both had equal nuclear and cytoplasmic distribution. However, assembled form of VP2 had a predominantly cytoplasmic localization. When VP1 and VP2 were simultaneously present in cells, their nuclear localization increased. Thus, confocal immunofluorescence analysis of cells transduced with the different baculovirus constructs or combinations thereof in the absence or presence of infecting CPV revealed that the VP1 protein is a prerequisite for efficient targeting of VP2 to the nucleus. The baculovirus vectors were functional and the genes of interest efficiently introduced to this CPV susceptible mammalian cell line. Thus, we show evidence that the system could be utilized to study targeting of the CPV capsid proteins.     

Identification of regions of leukotriene C4 synthase which direct the enzyme to its nuclear envelope localization

Leukotrienes (LTs) are fatty acid derivatives formed by oxygenation of arachidonic acid via the 5-lipoxygenase (5-LO) pathway. Upon activation of inflammatory cells 5-LO is translocated to the nuclear envelope (NE) where it converts arachidonic acid to the unstable epoxide LTA4. LTA4 is further converted to LTC4 by conjugation with glutathione, a reaction catalyzed by the integral membrane protein LTC4 synthase (LTC4S), which is localized on the NE and endoplasmic reticulum (ER). We now report the mapping of regions of LTC4S that are important for its subcellular localization. Multiple constructs encoding fusion proteins of green fluorescent protein (GFP) as the N-terminal part and various truncated variants of human LTC4S as C-terminal part were prepared and transfected into HEK 293/T or COS-7 cells. Constructs encoding hydrophobic region 1 of LTC4S (amino acids 6-27) did not give distinct membrane localized fluorescence. In contrast hydrophobic region 2 (amino acids 60-89) gave a localization pattern similar to that of full length LTC4S. Hydrophobic region 3 (amino acids 114-135) directed GFP to a localization indistinguishable from that of full length LTC4S. A minimal directing sequence, amino acids 117-132, was identified by further truncation. The involvement of the hydrophobic regions in the homo-oligomerization of LTC4S was investigated using bioluminescence resonance energy transfer (BRET) analysis in living cells. BRET data showed that hydrophobic regions 1 and 3 each allowed oligomerization to occur. These regions most likely form transmembrane helices, suggesting that homo-oligomerization of LTC4S is due to helix-helix interactions in the membrane.