"Thiocyanate gold": small (2-3 nm) colloidal gold for affinity cytochemical labeling in electron microscopy

Reduction of HAuCl4 by NaSCN or KSCN produces colloidal gold particles of 2.6 nm in diameter and homogeneous in size (coefficient of variation approximately 15%). The AuSCN sol forms protein-gold complexes. The amount of protein required to form an AuSCN-protein complex is best determined in the electron microscope, where serial dilutions of protein with gold sol are inspected for the presence of aggregates. By immuno-electron microscopy SCN-gold complexed to protein A is active and visible as is shown by revealing alpha-amylase in rat pancreatic acinar cells.     

Cellular localization of perforin 1 in murine cloned cytotoxic T lymphocytes

The localization of perforin 1 (P1) in cytotoxic cells was studied by immuno-electron microscopy by using a monospecific rabbit antiserum against highly purified mouse P1 and protein A gold as a second ligand. P1 was found in specific granules of cloned cytotoxic T lymphocytes (CTL). Within the granules, P1 antigen was localized in the fine granular matrix, whereas the vesicular compartment remained free of gold particles. The amount of P1 antigen detectable by immuno-electron microscopy varied between different CTL clones. CTL with NK-like activity had the highest level of P1 antigen. A cytotoxicity loss CTL mutant had no detectable P1 antigen, suggesting an important role of P1 during cell-mediated cytolysis. P1 antigen was undetectable also in bone marrow macrophages, indicating a different cytolytic mechanism of these cells.     

βAmyloid蛋白在恒河猴脑组织中的形态学特点

目的观察βamyloid蛋白在不同年龄的恒河猴脑中的表达及其组织学和细胞超微结构水平的分布特点。方法分别取脑组织额叶、海马、颞叶和顶叶做免疫组化,观察βamyloid蛋白在组织学上的分布特点及与年龄的关系;选用23岁恒河猴一只,灌注后取上述部位做免疫电镜,观察βamyloid蛋白在细胞超微结构水平的分布特点。结果免疫组化染色可观察到年轻猴的神经细胞和胶质细胞中有少量的Aβ40颗粒,以额叶和海马居多。在老年猴脑中Aβ40常聚集成团状斑块。年轻猴脑细胞内未见明显的颗粒状Aβ42,而老年猴的额叶有多量的Aβ42细胞外散在斑块,神经细胞和胶质细胞内也见有Aβ42颗粒状沉积。免疫电镜可观察到Aβ40的胶体金颗粒大部分存在于老年猴神经细胞细胞质中,在细胞间质和小胶质细胞中也可见少量的胶体金颗粒,多见于额叶;标记Aβ42的胶体金颗粒也是多见于神经细胞中,在小胶质细胞中少量存在,同时在颞叶的神经纤维束中也可见Aβ42标记的胶体金颗粒。结论Aβ42是恒河猴老年斑的主要成分,并在其形成过程中起到重要作用。     

Actin Is Localized in the Nucleolar Skeleton of Physarum polycephalum

Nucleoli were isolated from the interphase nuclei of Physarum polycephalum Schw. 'lhe nucleolar skeleton was obtained after DNA and most of the nucleolar proteins were extracted with DNase I 0.25 mol/L ( NH4)2SO4 and 2 mol/L NaC1. The nucleolar skeleton appeared as a fibrous network structure composed of fibres about 10 to 30 nm in diameter when observed under the electron microscope. SDS-PAGE analyses revealed about 20 polypeptides in the nucleolar skeleton, including a 43 kD pelypeptide which is equivalent to actin in molecular weight, lmmunofiuorescence observations upon slide preparations of the nueleolar skeleton labeled with anti-actin antibody showed that the nucleolar skeleton emanated bright fluorescence, indicating the existence of thc antigen, lmmunodotting assays further localized actin in the protein preparations of the nucleolar skeleton. Results of immuno-electron microscopy, with anti-actin antibody and protein A-gold as probes, indicated that gold particles were distributed all over the nucleolus of the interphase nucleus.     

“Thiocyanate gold”: small (2–3 nm) colloidal gold for affinity cytochemical labeling in electron microscopy

Summary Reduction of HAuCl₄ by NaSCN or KSCN produces colloidal gold particles of 2.6 nm in diameter and homogeneous in size (coefficient of variation 15%). The Au_SCN sol forms protein-gold complexes. The amount of protein required to form an Au_SCN-protein complex is best determined in the electron microscope, where serial dilutions of protein with gold sol are inspected for the presence of aggregates.By immuno-electron microscopy SCN-gold complexed to protein A is active and visible as is shown by revealing -amylase in rat pancreatic acinar cells.     

Localization of ribosomal protein L27 at the peptidyl transferase centre of the 50 S subunit, as determined by immuno-electron microscopy

Summary Protein L27 has been localized on the ribosomal surface by immuno-electron microscopy by using antibodies specific for Escherichia coli L27, and by reconstituting 50 S subunits from an E. coli mutant, which lacks protein L27, with the homologous protein from Bacillus subtilis and using antibodies specific for the B. subtilis protein. With both approaches, protein L27 has been located at the base of the central protuberance at the interface side of the 50 S particle and thus in proximity to the peptidyl transferase centre. The immuno-electron microscopic data also suggest that the interface region of the 50 S particle is not as flat as most of the proposed three-dimensional models suggest, but instead there is a significant depression.     

Three-dimensional location of ribosomal protein BL2 from Bacillus stearothermophilus, a key component of the peptidyl transferase center

Protein BL2 from Bacillus stearothermophilus has been localized by immunoelectron microscopy on the interface side of the 50 S subunit, beneath the angle formed between the central protuberance and the L1 protuberance. The immuno-electron microscopic data suggest that the interface region of the 50 S particle is not as flat as most of the proposed three-dimensional models suggest, but instead there is a significant concavity. Since several studies demonstrated that BL2 is implicated in peptidyl transferase activity or at least located close to the peptidyl transferase center, the location of protein BL2 also provides information as to the location of this important functional domain.     

Properties of chicken cardiac dystrophin.

We investigated the presence of dystrophin by immunoblot and immunofluorescence analyses, negative staining, rotatory shadowing and immunogold electron microscopy in chicken cardiac muscle. Saponin was found to be better than Triton X-100 for providing a new 'dystrophin-enriched' solution for use in biochemical studies of the molecule. By Western blot analysis, only a 400-kDa band was revealed with polyclonal antibodies directed against a central region (residues 1178-1723) of the dystrophin molecule and no cross-reactions with other proteins or degraded products were observed. Specific cleavage of the dystrophin molecule showed that the central rod-shaped domain corresponded to a resistant 'core'. This structure might rigidify the protein. By immunofluorescence, dystrophin was localized at the periphery of cardiac ventricular cells. The molecule was examined by electron microscopy and found to have variable lengths (140-160 nm for the monomeric from and about 260 +/- 10 nm or more for oligomeric forms). These oligomeric structures are considered to be associated molecules which are only partially overlapped lengthwise. The precise distribution of dystrophin within the cardiac muscle was determined by visualisation of gold particles in immuno-electron microscopy. Gold particles were found on the sarcolemma with no evidence of any association with cytoplasmic structures. The present data provide further details on the cardiac dystrophin molecule and suggest that its capacity of self-association may elasticize the dystrophin dimer.     

Intracellular Localization of Phospholipase D in Leaves and Seedling Tissues of Castor Bean

The intracellular distribution of phospholipase D (PLD; EC 3.1.4.4) in castor bean (Ricinus communis L.) tissues was investigated by subcellular fractionation and by immuno-electron microscopy. Centrifugal fractionation revealed that most PLD in young leaves was soluble, whereas in mature leaves a majority of PLD was associated with microsomal membranes. Further separation of microsomal membranes by a two-phase partitioning system indicated that PLD was associated with both plasma and intracellular membranes. Sucrose gradient separation of intracellular membranes showed PLD present in the endoplasmic reticulum, a submicrosomal band, and in soluble fractions but not in mitochondria and glyoxysomes of postgermination endosperm. Immunocytochemical studies found high gold labeling in vacuoles in young leaves, suggesting that the high level of soluble PLD in young leaves is due to release of PLD from vacuoles during tissue disruption. In addition to the labeling in vacuoles, gold particles were also found in the cytoplasmic matrices and plasma membrane in leaves and in 2-d postgermination seedlings. Collectively, these results show that PLD in castor bean leaf and seedling tissues is localized in the vacuole and is associated with the endoplasmic reticulum and plasma membrane and that the relative distribution between the soluble and membrane compartments changes during castor bean leaf development.     

Evidence for mitochondrial localization of P5, a member of the protein disulphide isomerase family

This report demonstrates for the first time that P5, a member of the protein disulphide isomerase (PDI) family, is present in the mitochondria. Various organelles were screened for proteins bearing the CGHC motif using an affinity column conjugated with the phage antibody 5E, which cross-reacts with PDI family proteins. P5 was found in bovine liver mitochondrial extract and identified by Western blot analysis using anti-P5 antibody and by mass spectrometric analysis. Results of cell fractionation, proteinase sensitivity experiments and immuno-electron microscopy supported the mitochondrial localization of P5 and also indicated the presence of ERp57, another PDI family protein, in mitochondria. Our findings will be useful for the elucidation of the translocation mechanism of PDI family proteins and their roles in mitochondria.     

Identification and characterization of a novel peritrophic matrix protein, Ae-Aper50, and the microvillar membrane protein, AEG12, from the mosquito, Aedes aegypti

Immuno-screening of an adult Aedes aegypti midgut cDNA expression library with anti-peritrophic matrix antibodies identified cDNAs encoding a novel peritrophic matrix protein, termed Ae. aegypti Adult Peritrophin 50 (Ae-Aper50), and the epithelial cell-surface membrane protein, AEG12. Both genes are expressed exclusively in the midguts of adult female mosquitoes and their expression is strongly induced by blood feeding. Ae-Aper50 has a predicted secretory signal peptide and five chitin-binding domains with intervening mucin-like domains. Localization of Ae-Aper50 to the peritrophic matrix was demonstrated by immuno-electron microscopy. Recombinant Ae-Aper50 expressed in baculovirus-infected insect cells binds chitin in vitro. Site-directed mutagenesis was used to study the role that cysteine residues from a single chitin-binding domain play in the binding to a chitin substrate. Most of the cysteine residues proved to be critical for binding. AEG12 has a putative secretory signal peptide at the amino-terminus and a putative glycosyl-phosphatidylinositol (GPI) anchor signal at its carboxyl-terminus and the protein was localized by immuno-electron microscopy to the midgut epithelial cell microvilli.     

FluoroNanogold: an important probe for correlative microscopy

Correlative microscopy is a powerful imaging approach that refers to observing the same exact structures within a specimen by two or more imaging modalities. In biological samples, this typically means examining the same sub-cellular feature with different imaging methods. Correlative microscopy is not restricted to the domains of fluorescence microscopy and electron microscopy; however, currently, most correlative microscopy studies combine these two methods, and in this review, we will focus on the use of fluorescence and electron microscopy. Successful correlative fluorescence and electron microscopy requires probes, or reporter systems, from which useful information can be obtained with each of the imaging modalities employed. The bi-functional immunolabeling reagent, FluoroNanogold, is one such probe that provides robust signals in both fluorescence and electron microscopy. It consists of a gold cluster compound that is visualized by electron microscopy and a covalently attached fluorophore that is visualized by fluorescence microscopy. FluoroNanogold has been an extremely useful labeling reagent in correlative microscopy studies. In this report, we present an overview of research using this unique probe.     

Characterization of a novel portal protein from deep-sea thermophilic bacteriophage GVE2

Portal proteins, located asymmetrically at one of the twelve vertices of the capsid, play very important roles in viral DNA packaging. Compared with the well-studied portal proteins of bacteriophages infecting mesophilic bacteria, portal proteins of thermophilic bacteriophages from deep sea have not been characterized. In this investigation, a novel portal protein was identified from a deep-sea thermophilic bacteriophage GVE2 for the first time. The GVE2 portal protein (designated as VP411 protein) shared low similarity to known portal proteins from other species, but they showed high similarities in the predicted secondary structures, suggesting that they had the same function in viral DNA packaging. The Northern blot and Western bolt results demonstrated that the vp411 gene was expressed in the late stage of GVE2 infection, implying that it might be a viral late gene. As revealed by immuno-electron microscopy, the gold particles were observed in the junction between the phage head and the phage tail when the anti-VP411 IgG was used as the primary antibody, indicating that it had the location in the virion expected of a portal protein.     

HIV-1 egress is gated through late endosomal membranes

HIV-1 buds from the surface of activated T lymphocytes. In macrophages, however, newly formed HIV-1 particles amass in the lumen of an intracellular compartment. Here, we demonstrate by live-cell imaging techniques, by immunocytochemistry and by immuno-electron microscopy that HIV-1 structural proteins, particularly the internal structural protein Gag, accumulate at membranes of the late endocytic compartment in a variety of cell types and not just in monocyte/macrophage-derived cells. Recent biochemical and genetic studies have implicated components of the mammalian vacuolar protein sorting pathway in retroviral budding. Together with those observations, our study suggests that HIV-1 morphogenesis is thoroughly rooted in the endosomal system.     

Serotonin organelles of rabbit platelets contain synaptophysin

Synaptophysin, an integral membrane protein of synaptic vesicles in nerve terminals and a class of small translucent vesicles in neuroendocrine cells, was detected in intact rabbit platelets by immunoblotting, immunofluorescence staining and immuno-electron microscopy. In a highly purified preparation of serotonin organelles isolated from rabbit platelets, synaptophysin was enriched approximately 10-15-fold over platelet homogenate. About 80% of total platelet synaptophysin was present in this purified fraction. The apparent molecular mass (approximately 38 kDa) and the extent of glycosylation of platelet-derived synaptophysin was more similar to the neuronal than to the neuroendocrine form of the protein. Immunofluorescence microscopy revealed that synaptophysin was compartmentalized in intact rabbit platelets and immuno-electron microscopy of subcellular fractions showed that it was localized exclusively to the membrane surface of serotonin organelles. No synaptophysin-like immunoreactivity was detected in platelets from other species such as human, guinea pig and rat. Another integral membrane protein of synaptic vesicles, p65, and a family of synaptic vesicle-associated phosphoproteins, the synapsins, were not detected in platelets of any species tested. These results provide evidence that serotonin organelles from rabbit platelets share a subset of protein components with synaptic vesicles from neurons. Synaptophysin in serotonin organelles from rabbit platelets, as suggested for small synaptic vesicles in neurons, might play a role in the formation of protein channels for the exocytotic release of serotonin.     

A new method of preparing gold probes for multiple-labeling cytochemistry

A new method is described for preparing colloidal gold particles in any size between approximately 3 and 17 nm for electron microscopy. The gold particles are homogeneous in size (homodisperse). When bound to various proteins (e.g. IgG and protein A), the complexes were stable for long periods and suitable for affinity cytochemistry. We demonstrated the usefulness of the new gold probes bound to protein A for multiple labeling in a current immunocytochemical study on receptor mediated transport of IgA in human duodenal crypt cells. Other gold-protein complexes were useful for studying macromolecular arrangements at high resolution.     

Characterization of a monoclonal antibody specific to rainbow trout thrombocytes.

A monoclonal antibody (MAb) specific for rainbow trout thrombocytes was produced and its reactivity was demonstrated by flow cytometry and immuno-electron microscopy. Flow cytometry analysis showed that this MAb (TTL-7D11) reacted positively with about 30% of the peripheral blood leucocytes (PBL) and about 1%, 2%, and 11% of the pronephros, mesonephros, and spleen cells, respectively. Electron microscopy using immunogold labeling demonstrated that this MAb reacted strongly with thrombocytes, where gold beads could be seen attached only to the membrane and canalicular system of these cells. Positive and negative leucocytes for this MAb were obtained by magnetic cell separation. In the positive fraction, 96% of the cells were thrombocytes, while in the negative fraction no more than 3% were, which clearly showed a high purity of the positive fraction. Aggregation studies showed that about 75% of the positive fraction cells aggregated after being mixed with U-46619 thromboxane-mimetic, whereas in the negative fraction only 10% of the cells did so. Thus, utilizing the TTL-7D11 we have succeeded in isolating a pure thrombocyte population, and this would facilitate further studies, particularly on their characteristics and function(s).     

Combined Use of Immunoferritin and Immunocolloidal Gold for the Simultaneous Demonstration of Two Antigens by Immuno-Electron Microscopy

In this report we describe a double-label bridging technique for the simultaneous visualization of two cell surface antigens by immuno-electron microscopy. Mouse IgG-labeled horse spleen ferritin and rabbit IgG-labeled gold sols served as the electron dense marker-conjugates. The technique was found to discriminate reproducibly two cell surface antigens simultaneously in two separate murine systems, one an in vivo cell line and the other an in vitro cell line. The technique described in this report differs from other double-labeling procedures in two notable ways: (1) the electron dense markers are easily distinguished from one another, and (2) the primary and link antibodies are not modified.     

Subcellular Localization and Differentiation-Induced Redistribution of the Protein Tyrosine Phosphatase PTP-BL in Neuroblastoma Cells

1.In cells of epithelial origin the protein tyrosine phosphatase PTP-BL is predominantly localized at the apical membrane of polarized cells. This large submembranous multidomain PTP is also expressed in cells of neuronal origin. We studied the localization of PTP-BL in mouse neuroblastoma cells utilizing EGFP-tagged versions of the protein. 2. In proliferating Neuro-2a cells, immunofluorescence and immuno-electron microscopy revealed a submembranous FERM domain-dependent localization at cell-cell boundaries for EGFP-PTP-BL. Additionally, significant amounts of EGFP-PTP-BL are located in the cytoplasm as well as in nuclei. Upon serum depletion-induced differentiation of Neuro-2a cells, a partial shift of EGFP-PTP-BL from a cortical localization to cytoskeleton-like F-actin-positive structures is observed. Parallel biochemical studies corroborate this finding and reveal a serum depletion-induced shift of EFGP-PTP-BL from a membrane(-associated) fraction to an NP40-soluble cytoskeletal fraction. 3. Different pools of PTP-BL-containing protein complexes can be discerned in neuronal cells, reflecting distinct molecular microenvironments in which PTP-BL may exert its function.     

Activated FcgammaRII and signalling molecules revealed in rafts by ultra-structural observations of plasma-membrane sheets

To reveal topography of FcgammaRII components of the receptor-signalling complex, large plasma-membrane sheets were obtained by cell cleavage and analysed by immuno-electron microscopy. Non-activated FcgammaRII was dispersed in the plane of the plasma membrane and only rarely was localized in the proximity of Lyn, an Src family tyrosine kinase, and CD55, a glycosylphosphatidylinositol-anchored protein. After FcgammaRII activation by cross-linking with antibodies, clusters of an electron-dense material acquiring about 86% of FcgammaRII and reaching up to 300 nm in diameter were formed within 5 min. These structures also accommodated about 85% of Lyn and 63% of CD55 labels that were located in close vicinity of gold particles attributed to the cross-linked FcgammaRII . The electron-dense structures were also abundant in tyrosine phosphorylated proteins. At their margins PIP2 was preferentially located. Based on a concentration of Lyn, CD55 and activated FcgammaRII , the electron-dense structures seem to reflect coalescent membrane rafts.