GFP-tagged proteins visualized by freeze-fracture immuno-electron microscopy: a new tool in cellular and molecular medicine

GFP-tagging is widely used as a molecular tool to localize and visualize the trafficking of proteins in cells but interpretation is frequently limited by the low resolution afforded by fluorescence light microscopy. Although complementary thin-section immunogold electron microscopic techniques go some way in aiding interpretation, major limitations, such as relatively poor structural preservation of membrane systems, low labelling efficiency and the two-dimensional nature of the images, remain. Here we demonstrate that the electron microscopic technique freeze-fracture replica immunogold labelling overcomes these disadvantages and can be used to define, at high resolution, the precise location of GFP-tagged proteins in specific membrane systems and organelles of the cell. Moreover, this technique provides information on the location of the protein within the phospholipid bilayer, potentially providing insight into mis-orientation of tagged proteins compared to their untagged counterparts. Complementary application of the freeze-fracture replica immunogold labelling technique alongside conventional fluorescence microscopy is seen as a novel and valuable approach to verification, clarification and extension of the data obtained using fluorescent-tagged proteins. The application of this approach is illustrated by new findings on PAT-family proteins tagged with GFP transfected into fibroblasts from patients with Niemann-Pick type C disease.     

Plant SILAC: Stable-Isotope Labelling with Amino Acids of Arabidopsis Seedlings for Quantitative Proteomics

Stable Isotope Labelling by Amino acids in Cell culture (SILAC) is a powerful technique for comparative quantitative proteomics, which has recently been applied to a number of different eukaryotic organisms. Inefficient incorporation of labelled amino acids in cell cultures of Arabidopsis thaliana has led to very limited use of SILAC in plant systems. We present a method allowing, for the first time, efficient labelling with stable isotope-containing arginine and lysine of whole Arabidopsis seedlings. To illustrate the utility of this method, we have combined the high labelling efficiency (>95%) with quantitative proteomics analyses of seedlings exposed to increased salt concentration. In plants treated for 7 days with 80 mM NaCl, a relatively mild salt stress, 215 proteins were identified whose expression levels changed significantly compared to untreated seedling controls. The 92 up-regulated proteins included proteins involved in abiotic stress responses and photosynthesis, while the 123 down-regulated proteins were enriched in proteins involved in reduction of oxidative stress and other stress responses, respectively. Efficient labelling of whole Arabidopsis seedlings by this modified SILAC method opens new opportunities to exploit the genetic resources of Arabidopsis and analyse the impact of mutations on quantitative protein dynamics in vivo.     

Micromere-specific cell surface proteins of 16-cell stage sea urchin embryos

Evidence is presented of cell-type specificity of surface proteins from the 16-cell stage sea urchin embryo. The protein composition of the micromere cell surface has been examined by 125I labelling of intact cells followed by SDS-PAGE. In Arbacia punctulata, four high molecular weight (HMW) proteins are detected on the surface of isolated micromeres--but not on mesomere-macromere fractions. In Strongylocentrotus droebachiensis, a micromere-specific protein of 133 K molecular weight (MW) was identified. This 133 K protein binds to wheat germ agglutinin (WGA) but not to concanavalin A (conA). Lectin binding was studied using a new technique. The procedure involves the separation, by SDS-PAGE, of iodinated cell-surface proteins followed by their electrophoretic transfer to lectin-coated nitrocellulose membranes. Using this procedure, cell-type-specific surface proteins which are also lectin-binding-specific, were detected.     

Differentially isotope-coded N-terminal protein sulphonation: combining protein identification and quantification

Most proteomic labelling technologies intend to improve protein quantification and/or facilitate (de novo) peptide sequencing. We present here a novel stable-isotope labelling method to simultaneously identify and quantify protein components in complex mixtures by specifically derivatizing the N-terminus of proteins with 4-sulphophenyl isothiocyanate (SPITC). Our approach combines protein identification with quantification through differential isotope-coded labelling at the protein N-terminus prior to digestion. The isotope spacing of 6 Da (unlabelled vs. six-fold 13C-labelled tag) between derivatized peptide pairs enables the detection on different MS platforms (MALDI and ESI). Optimisation of the reaction conditions using SPITC was performed on three model proteins. Improved detection of the N-terminally derivatized peptide compared to the native analogue was observed in negative-ion MALDI-MS. Simpler fragmentation patterns compared to native peptides facilitated protein identification. The 13C-labelled SPITC resulted in convenient peptide pair spacing without isotopic overlap and hence facilitated relative quantification by MALDI-TOF/TOF and LC-ESI-MS/MS. The combination of facilitated identification and quantification achieved by differentially isotope-coded N-terminal protein tagging with light/heavy SPITC represents, to our knowledge, a new approach to quantitative proteomics.     

Protein turnover: measurement of proteome dynamics by whole animal metabolic labelling with stable isotope labelled amino acids

The measurement of protein turnover in tissues of intact animals is obtained by whole animal dynamic labelling studies, requiring dietary administration of precursor label. It is difficult to obtain full labelling of precursor amino acids in the diet and if partial labelling is used, calculation of the rate of turnover of each protein requires knowledge of the precursor relative isotope abundance (RIA). We describe an approach to dynamic labelling of proteins in the mouse with a commercial diet supplemented with a pure, deuterated essential amino acid. The pattern of isotopomer labelling can be used to recover the precursor RIA, and sampling of urinary secreted proteins can monitor the development of liver precursor RIA non-invasively. Time-series analysis of the labelling trajectories for individual proteins allows accurate determination of the first order rate constant for degradation. The acquisition of this parameter over multiple proteins permits turnover profiling of cellular proteins and comparisons of different tissues. The median rate of degradation of muscle protein is considerably lower than liver or kidney, with heart occupying an intermediate position.     

Selective proton labelling of amino acids in deuterated bovine calbindin D9K. A way to simplify 1H-NMR spectra

A technique for proton labelling of selected amino acids in deuterated calbindin D9K, heterologously expressed in E. coli, was developed in order to simplify and obtain higher resolution in 1H-NMR spectra. The spectra from two double-labelling experiments, Val plus Ser and Val plus Leu, when compared to the uniformly protonated protein showed a dramatically simpler pattern with low background signals and gave considerably sharper resonances due to reduced relaxation rates in the deuterated proteins. The selective proton labelling technique will enable detailed and rapid analysis of interesting domains of proteins and will also make the analysis of larger proteins feasible.     

Adding value to fusion proteins through covalent labelling

Recombinant proteins are often expressed with an additional polypeptide the role of which is to aid purification, detection or functional studies. Recently, the role of these tags has been extended to mediate the labelling of the protein of interest with chemically diverse compounds. These approaches are of importance for protein science as they allow proteins to be equipped with properties that cannot be genetically encoded and also enable the use of a single fusion protein for several different applications. Several new approaches have been developed for the covalent labelling of fusion proteins both in living cells and in vitro.     

Ricin and alpha-sarcin alter the conformation of 60S ribosomal subunits at neighboring but different sites

The effects of ricin and alpha-sarcin separately or in combination on the conformation of rat liver ribosomes were investigated by measuring the relative accessibility of individual ribosomal proteins to N-ethylmaleimide after 80S ribosomes were treated with these toxins. By using a double-labelling technique in which ribosomes were incubated with the toxins and then treated with 3H-labelled or 14C-labelled N-ethylmaleimide, it was found that labelling of protein L14 was specifically reduced by treatment with ricin, and that of proteins L3 and L4 by treatment with alpha-sarcin, suggesting that the toxins alter the conformation of ribosomes in the vicinity of these proteins. When ribosomes were treated with both ricin and alpha-sarcin, the extent of labelling of protein L3 was reduced compared to that observed after treatment with alpha-sarcin alone. These results are discussed in relation to previous observations showing that these three proteins are neighbours in the 60S ribosomal subunit and probably play important roles in protein biosynthesis, and in the actions of ricin and alpha-sarcin on 28S rRNA.     

Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis

Differential protein expression analysis based on modification of selected amino acids with labelling reagents has become the major method of choice for quantitative proteomics. One such methodology, two-dimensional difference gel electrophoresis (2-D DIGE), uses a matched set of fluorescent N-hydroxysuccinimidyl (NHS) ester cyanine dyes to label lysine residues in different samples which can be run simultaneously on the same gels. Here we report the use of iodoacetylated cyanine (ICy) dyes (for labelling of cysteine thiols, for 2-D DIGE-based redox proteomics. Characterisation of ICy dye labelling in relation to its stoichiometry, sensitivity and specificity is described, as well as comparison of ICy dye with NHS-Cy dye labelling and several protein staining methods. We have optimised conditions for labelling of nonreduced, denatured samples and report increased sensitivity for a subset of thiol-containing proteins, allowing accurate monitoring of redox-dependent thiol modifications and expression changes. Cysteine labelling was then combined with lysine labelling in a multiplex 2-D DIGE proteomic study of redox-dependent and ErbB2-dependent changes in epithelial cells exposed to oxidative stress. This study identifies differentially modified proteins involved in cellular redox regulation, protein folding, proliferative suppression, glycolysis and cytoskeletal organisation, revealing the complexity of the response to oxidative stress and the impact that overexpression of ErbB2 has on this response.     

Mapping the proteome of thylakoid membranes by de novo sequencing of intermembrane peptide domains

The proteome of a membrane compartment has been investigated by de novo sequence analysis after tryptic in gel digestion. Protein complexes and corresponding protein subunits were separated by a 2-D Blue Native (BN)/SDS-PAGE system. The transmembrane proteins of thylakoid membranes from a higher plant (Hordeum vulgare L.) were identified by the primary sequence of hydrophilic intermembrane peptide domains using nano ESI-MS/MS-analysis. Peptide analysis revealed that lysine residues of membrane proteins are primarily situated in the intermembrane domains. We concluded that esterification of lysine residues with fluorescent dyes may open the opportunity to label membrane proteins still localized in native protein complexes within the membrane phase. We demonstrate that covalent labelling of membrane proteins with the fluorescent dye Cy3 allows high sensitive visualization of protein complexes after 2-D BN/SDS-PAGE. We show that pre-electrophoretic labelling of protein subunits supplements detection of proteins by post-electrophoretic staining with silver and CBB and assists in completing the identification of the membrane proteome.     

Localisation of the major high-molecular-weight protein on microtubules in vitro and in cultured cells

An antiserum has been produced which is specific for the major high-molecular-weight protein (HMW) associated with pig brain microtubules assembled in vitro. The HMW protein can be localised on the surface of brain microtubules assembled in vitro as a corase helix by using a peroxidase labelling technique. In cultured ovarian granulosa cells, by using indirect immunofluorescence, the HMW is present on cold- and colchicine-sensitive structures which have an intracellular distribution similar to microtubules. It seems likely, therefore, that the brain protein is representative of a class of proteins associated with non-neuronal cytoplasmic microtubules.     

Use of surface enhanced infrared absorption spectroscopy (SEIRA) to probe the functionality of a protein monolayer

We present a novel infrared method to investigate the functionality of a protein monolayer tethered to a metal substrate. The approach employs Surface Enhanced Infrared Absorption Spectroscopy (SEIRAS), which renders high surface sensitivity by enhancing the signal of the adsorbed protein by up to approximately 2 orders of magnitude. We demonstrate that the electrochemically induced absorption changes of a cytochrome c monolayer can be observed with excellent signal-to-noise ratio when the protein is adhered to a modified gold surface. To probe membrane proteins, a concept is introduced for the oriented incorporation into solid supported lipid bilayers. Recombinant cytochrome c oxidase solubilized in detergent is immobilized on a chemically modified gold surface via the affinity of its histidine (His)-tag to a nickel-chelating nitro-triacetic acid (NTA) surface. The protein monolayer is reconstituted into the lipid environment by detergent removal. Changing the orientation of the protein with respect to the metal surface is achieved by inserting the His-tag on either side of the membrane protein surface. Orientational control is mandatory for experiments in which electrons are injected from the electrode into the protein. The presented methodology opens new avenues to study the mechanism of the biomedically relevant class of electron and voltage-gated proteins on the atomic level.     

转基因在苹果蠹蛾不育昆虫释放技术中的应用

使用不育昆虫释放技术是一项新兴的苹果蠹蛾Cydia pomonella(L.)防治方法,使用转基因得到不育雄蛾具有比传统辐射方法更多的优点。转基因技术通过使用基于piggyBac等转座子的质粒载体,并插入显性条件致死基因以培育遗传性别品系,同时插入荧光蛋白等标记基因来显示转基因的效果;在人工控制的发育条件,建立成熟的稳定苹果蠹蛾品系,用以最终的田间释放以达到防治的目的。     

Self-assembly of proteins and their nucleic acids

We have developed an artificial protein scaffold, herewith called a protein vector, which allows linking of an in-vitro synthesised protein to the nucleic acid which encodes it through the process of self-assembly. This protein vector enables the direct physical linkage between a functional protein and its genetic code. The principle is demonstrated using a streptavidin-based protein vector (SAPV) as both a nucleic acid binding pocket and a protein display system. We have shown that functional proteins or protein domains can be produced in vitro and physically linked to their DNA in a single enzymatic reaction. Such self-assembled protein-DNA complexes can be used for protein cloning, the cloning of protein affinity reagents or for the production of proteins which self-assemble on a variety of solid supports. Self-assembly can be utilised for making libraries of protein-DNA complexes or for labelling the protein part of such a complex to a high specific activity by labelling the nucleic acid associated with the protein. In summary, self-assembly offers an opportunity to quickly generate cheap protein affinity reagents, which can also be efficiently labelled, for use in traditional affinity assays or for protein arrays instead of conventional antibodies.     

Comparative proteomic analysis using samples obtained with laser microdissection and saturation dye labelling

Comparative proteomic methods are rapidly being applied to many different biological systems including complex tissues. One pitfall of these methods is that in some cases, such as oncology and neuroscience, tissue complexity requires isolation of specific cell types and sample is limited. Laser microdissection (LMD) is commonly used for obtaining such samples for proteomic studies. We have combined LMD with sensitive thiol-reactive saturation dye labelling of protein samples and 2-D DIGE to identify protein changes in a test system, the isolated CA1 pyramidal neurone layer of a transgenic (Tg) rat carrying a human amyloid precursor protein transgene. Saturation dye labelling proved to be extremely sensitive with a spot map of over 5,000 proteins being readily produced from 5 mug total protein, with over 100 proteins being significantly altered at p < 0.0005. Of the proteins identified, all showed coherent changes associated with transgene expression. It was, however, difficult to identify significantly different proteins using PMF and MALDI-TOF on gels containing less than 500 mug total protein. The use of saturation dye labelling of limiting samples will therefore require the use of highly sensitive MS techniques to identify the significantly altered proteins isolated using methods such as LMD.     

Increased degradation rates of protein synthesized in hepatoma cells in the presence of amino acid analogues.

1. Reuber H35 hepatoma cells incorporate the arginine analogue canavanine into cell protein when arginine is omitted from the incubation medium. 2. By labelling arginine-containing proteins with (14-C)leucine and then canavanine-containing proteins with (3-H)leucine in the same cells, it is possible to measure the degradation of both types of protein during a subsequent 'chase' period. With this technique it has been shown that canavanine-containing proteins are degraded at a rate severalfold greater than normal proteins. Comparable results were found when 6-fluorotryptophan was used as an analogue to tryptophan. 3. Control experiments in which the labelling order was reversed or where the animo acid and its analogue were incubated in separate cell cultures support the conclusion that abberrant proteins are rapidly degraded in vivo.     

A comparision of normal submaxillary gland proteins with proteins obtained from a transplantable murine salivary gland carcinoma (myoepithelioma).

A new transplantable murine salivary gland carcinoma (myoepithelioma) was further characterized by comparing total protein patterns with normal submaxillary gland proteins. Analysis by isoelectric focussing or by labelling studies with radioactive leucine showed considerable differences between tumour and normal proteins. In contrast, analysis of staining patterns after electrophoresis in sodium dodecyl sulphate containing gels revealed a striking similarity between normal proteins, tumour proteins and proteins obtained from metastatic growths. It thus appears that tumour proteins closely resemble the normal proteins from the tissue of origin with respect to molecular size as determined by electrophoretic mobility, while significant differences occur in charge and labelling kinetics.     

溴氰菊酯对鼠脑蛋白质磷酸化作用的影响

以小鼠大脑碎片与[γ-~(32)P]ATP一起保温,观察到溴氰菊酯对蛋白1—3磷酸化的刺激作用和对4、5磷酸化的抑制作用,表明溴氰菊酯对大脑蛋白质磷酸化产生了影响。从鼠脑分离了C、D、S三个组分,分别进行的蛋白质磷酸化试验结果表明,C、D组分可能是重要的磷酸化部位。 蛋白1、2、3的磷酸化明显地受到溴氰菊酯的刺激,这三个蛋白质可能是“蛋白Ⅲb”的几种形式。溴氰菊酯对“蛋白Ⅲb”磷酸化的刺激,可能会影响神经末梢的神经激素释放,从而影响到与其相关的某些神经功能。     

Effect of oxidative stress on protein thiols in the blue mussel Mytilus edulis: proteomic identification of target proteins

Protein thiols are targets of oxidative stress. Their modification was analysed in gill extracts of the mussel Mytilus edulis, exposed to menadione. Diagonal gel electrophoresis revealed two clusters of carbonylated proteins involved in interchain disulphide linkages. Immunoblotting identified these as being associated with protein disulphide isomerase (PDI) and actin and this was confirmed by immunoprecipitation. Protein free thiols (-SH) were identified in 2-DE separations by labelling with 5-iodoacetamidofluorescein (IAF). Cysteines involved in disulphide bridges were identified by blocking free -SH with N-ethylmaleimide, reducing disulphides with DTT and IAF labelling. Several protein spots containing free thiols disappeared on exposure to menadione. Conversely, new protein spots containing disulphides appeared in response to menadione which may be protective against oxidative stress. In-gel tryptic digestion followed by LC/MS-MS and database searching identified some of the free thiol targets: PDI; hsp gp96; calreticulin; heavy metal binding protein. Tubulin, PDI, enolase and gelsolin contained new disulphide bridges in response to menadione. Our findings indicate a protein level response to oxidative stress principally involving PDI, chaperone-like and cytoskeletal proteins. Since many environmental pollutants cause oxidative stress, studies on PDI and structural proteins may be particularly relevant to understanding toxicity in this popular sentinel species.     

A Transgenic Mouse Line Expressing the Red Fluorescent Protein tdTomato in GABAergic Neurons

GABAergic inhibitory neurons are a large population of neurons in the central nervous system (CNS) of mammals and crucially contribute to the function of the circuitry of the brain. To identify specific cell types and investigate their functions labelling of cell populations by transgenic expression of fluorescent proteins is a powerful approach. While a number of mouse lines expressing the green fluorescent protein (GFP) in different subpopulations of GABAergic cells are available, GFP expressing mouse lines are not suitable for either crossbreeding to other mouse lines expressing GFP in other cell types or for Ca²⁺-imaging using the superior green Ca²⁺-indicator dyes. Therefore, we have generated a novel transgenic mouse line expressing the red fluorescent protein tdTomato in GABAergic neurons using a bacterial artificial chromosome based strategy and inserting the tdTomato open reading frame at the start codon within exon 1 of the GAD2 gene encoding glutamic acid decarboxylase 65 (GAD65). TdTomato expression was observed in all expected brain regions; however, the fluorescence intensity was highest in the olfactory bulb and the striatum. Robust expression was also observed in cortical and hippocampal neurons, Purkinje cells in the cerebellum, amacrine cells in the retina as well as in cells migrating along the rostral migratory stream. In cortex, hippocampus, olfactory bulb and brainstem, 80% to 90% of neurons expressing endogenous GAD65 also expressed the fluorescent protein. Moreover, almost all tdTomato-expressing cells coexpressed GAD65, indicating that indeed only GABAergic neurons are labelled by tdTomato expression. This mouse line with its unique spectral properties for labelling GABAergic neurons will therefore be a valuable new tool for research addressing this fascinating cell type.