CELL SURFACE LOCALIZATION OF THE 60 kDa HEAT SHOCK CHAPERONIN PROTEIN (hsp60) IN MAMMALIAN CELLS

To investigate whether the 60-kDa heat shock chaperonin protein (hsp60) is present on the surface of mammalian cells, we used immunogold labeling of intact cells and backscattered electron imaging to image gold particles. Chinese hamster ovary cells and the human leukemic CD4-positive T-cell line CEM-SS on glass coverslips were labeled using affinity-purified monoclonal and polyclonal antibodies specific for hsp60 and 30 nm gold markers. Cells were imaged using the scanning mode of the conventional transmission electron microscope. Backscattered electron imaging provided definitive identification of the gold markers while secondary electron imaging gave information on surface architecture. Labeling intensity was 250–800 gold particles per cell in Chinese hamster ovary cells and 600–2000 in CEM-SS human lymphoblasts. The finding of hsp60 on the cell surface of mammalian cells may signify chaperone involvement in surface functions.     

Retrograde tracing of neural pathways with a protein gold complex. II. Electron microscopic demonstration of projections and collaterals

This paper describes a sensitive method for tracing neural connections at the electron microscopic (EM) level using a new compound produced through the coupling of colloidal gold particles to a wheat germ agglutinin horseradish peroxidase conjugate (the WGA*HRP-gold complex). Visualization of retrogradely labeled cells at the EM level was achieved either directly by gold particles scanning or after silver enhancement. By using different sizes of gold particles individually coupled to WGA*HRP and injected in different brain areas EM detection of multiple retrograde labeling was possible. Thus retrogradely labeled cells were first identified at the light microscopic level through HRP histochemistry with tetramethylbenzidine as a chromogen and then examined under the electron microscope after osmication and embedding. Gold particles were readily identified as electron dense, round dots in spherical grey vesicles. Identification of different sizes of gold particles often localized in the same vesicle established that the protein-gold complex can be used to study collateralisation of parental axons.     

Retrograde tracing of neural pathways with a protein gold complex

Summary This paper describes a sensitive method for tracing neural connections at the electron microscopic (EM) level using a new compound produced through the coupling of colloidal gold particles to a wheat germ agglutinin horseradish peroxidase conjugate (the WGA^*HRP-gold complex). Visualization of retrogradely labeled cells at the EM level was achieved either directly by gold particles scanning or after silver enhancement. By using different sizes of gold particles individually coupled to WGA^*HRP and injected in different brain areas EM detection of multiple retrograde labeling was possible. Thus retrogradely labeled cells were first identified at the light microscopic level through HRP histochemistry with tetramethylbenzidine as a chromogen and then examined under the electron microscope after osmication and embedding. Gold particles were readily identified as electron dense, round dots in spherical grey vesicles. Identification of different sizes of gold particles often localized in the same vesicle established that the protein-gold complex can be used to study collateralisation of parental axons.     

Three-dimensional locations of gold-labeled proteins in a whole mount eukaryotic cell obtained with 3nm precision using aberration-corrected scanning transmission electron microscopy

Three-dimensional (3D) maps of proteins within the context of whole cells are important for investigating cellular function. However, 3D reconstructions of whole cells are challenging to obtain using conventional transmission electron microscopy (TEM). We describe a methodology to determine the 3D locations of proteins labeled with gold nanoparticles on whole eukaryotic cells. The epidermal growth factor receptors on COS7 cells were labeled with gold nanoparticles, and critical-point dried whole-mount cell samples were prepared. 3D focal series were obtained with aberration-corrected scanning transmission electron microscopy (STEM), without tilting the specimen. The axial resolution was improved with deconvolution. The vertical locations of the nanoparticles in a whole-mount cell were determined with a precision of 3nm. From the analysis of the variation of the axial positions of the labels we concluded that the cellular surface was ruffled. To achieve sufficient stability of the sample under electron beam irradiation during the recording of the focal series, the sample was carbon coated. A quantitative method was developed to analyze the stability of the ultrastructure after electron beam irradiation using TEM. The results of this study demonstrate the feasibility of using aberration-corrected STEM to study the 3D nanoparticle distribution in whole cells.     

A multiple-staining ultrastructural procedure simultaneously detecting three membrane antigens on suspended cells by monoclonal antibodies and preembedding immunogold labelling

Summary A triple ultrastructural immunogold staining method for the simultaneous demonstration of three surface antigens of peripheral blood mononuclear cells at the electron microscope level is described. A six-step pre-embedding immunoelectron microscopy procedure was developed, using commercially available reagents. The CD11b antigen was first detected, through a two-step (indirect) method with 40 nm-sized gold particles; after a blocking step, the HLA-DR surface antigen was subsequently detected, through a two-step (biotin-streptavidin) method with 20 nm-sized gold particles; the CD4 antigen was finally detected, through a one-step (direct) method, using 5 nm-sized gold particles. Electron microscopic examination revealed firstly the presence of a triple-labelled cell subpopulation, which showed gold granules of the three sizes simultaneously decorating the cell membrane. Thus, the cells of such a subset simultaneously expressed the three antigens investigated. In contrast, either gold particles of only one size or no gold particles were observed on the cell surface of other subpopulations. This technique is a model demonstrating the importance of varying the size of particles in pre-embedding gold immunoelectron microscopy for a better analysis of the expression of surface antigens in isolated cells.     

Nanoscale Imaging of Whole Cells Using a Liquid Enclosure and a Scanning Transmission Electron Microscope

Nanoscale imaging techniques are needed to investigate cellular function at the level of individual proteins and to study the interaction of nanomaterials with biological systems. We imaged whole fixed cells in liquid state with a scanning transmission electron microscope (STEM) using a micrometer-sized liquid enclosure with electron transparent windows providing a wet specimen environment. Wet-STEM images were obtained of fixed E. coli bacteria labeled with gold nanoparticles attached to surface membrane proteins. Mammalian cells (COS7) were incubated with gold-tagged epidermal growth factor and fixed. STEM imaging of these cells resulted in a resolution of 3 nm for the gold nanoparticles. The wet-STEM method has several advantages over conventional imaging techniques. Most important is the capability to image whole fixed cells in a wet environment with nanometer resolution, which can be used, e.g., to map individual protein distributions in/on whole cells. The sample preparation is compatible with that used for fluorescent microscopy on fixed cells for experiments involving nanoparticles. Thirdly, the system is rather simple and involves only minimal new equipment in an electron microscopy (EM) laboratory.     

Immuno-gold localization of IAA in leaf cells of Prunus persica at different stages of development

Subcellular localization of indole-3-acetic acid (IAA) in leaf cells of peach ( Prunus persica [L.] Batsch 'Hakuho') was investigated using imuno-gold electron microscopy. The distribution pattern of the gold particles, which detected IAA, changed as cells matured. The most prominent feature was the accumulation of the gold label in the chloroplasts and mitochondria of the parenchyma cells of opened leaves. Throughout the development, the cytosol, nuclei, and cell wall were labelled, although the level was low and no significant changes occurred. The density of colloidal gold at each stage of leaf development was well correlated with the analytical data obtained by high-performance liquid chromatography (HPLC).     

rRNA sequence-based scanning electron microscopic detection of bacteria

A new scanning electron microscopic method was developed for gaining both phylogenetic and morphological information about target microbes using in situ hybridization with rRNA-targeted oligonucleotide probes (SEM-ISH). Target cells were hybridized with oligonucleotide probes after gold labeling. Gold enhancement was used for amplification of probe signals from hybridized cells. The hybridized cells released a strong backscatter electron signal due to accumulation of gold atoms inside cells. SEM-ISH was applied to analyze bacterial community composition in freshwater samples, and bacterial cell counts determined by SEM-ISH with rRNA-targeted probes for major phyla within the domain Bacteria were highly correlated to those by fluorescent in situ hybridization (FISH). The bacterial composition on surface of river sediment particles before and after cell dispersion treatment by sonication was successfully revealed by SEM-ISH. Direct enumeration of bacterial cells on the surface of sonicated sediment particles by SEM-ISH demonstrated that members of Cytophaga-Flavobacterium existed tightly on the surface of particles. SEM-ISH allows defining the number and distribution of phylogenetically defined cells adherent to material surfaces, which is difficult in FISH, and it gives new insight into electron microscopic studies of microorganisms in their natural environment.     

Dynamics of epidermal growth factor receptor internalization studied by Nanovid light microscopy and electron microscopy in combination with immunogold labeling

Individual gold particles with a diameter of approximately 10 to 40 nm can be visualized using video-enhanced contrast microscopy (Nanovid) (De Brabander et al., Cell Motil. Cytoskel. 6, 105-113 (1986)). This technique allows a study of the dynamic properties of receptors and ligands in living cells at high resolution. We have studied epidermal growth factor (EGF) receptor internalization in human epidermoid carcinoma A431 cells, using a monoclonal anti-EGF-receptor antibody conjugated to 20-nm gold particles, referred to as 2E9-gold. Exposure of A431 cells to 2E9-gold at 37 degrees C resulted in binding of the complex at the cell surface. Most of the gold particles exhibit a Brownian type of movement, while a minority appeared immobile. Binding of the 2E9-gold complex is followed by internalization, as judged from Nanovid light microscopy studies in combination with electron microscopic observations. The internalized gold particles clearly cluster into large aggregates, most likely multivesicular bodies. Individual gold particles as well as aggregates are characterized by a saltatory movement, by which the gold particles eventually move from the cell periphery towards the cell center. Addition of EGF results in an increased rate of internalization of 2E9-gold, while Na-azide and nocodazole completely immobilize the intracellular gold particles, as has been demonstrated previously for the transferrin receptor.     

Quantitative immunogold localization of Na, K-ATPase along rat nephron.

Ultrastructural localization of Na, K-ATPase alpha-subunit along rat nephron segments was investigated quantitatively by immunogold electron microscopy on LR-White ultrathin sections using affinity-purified antibody against alpha-subunit of the enzyme. Ultrathin sections were incubated with the antibody at a saturation level and the number of gold particles bound per micron of the plasma membrane (particle density) of the tubular epithelial cells from the proximal tubule to the collecting duct was determined. In all the tubular epithelial cells, gold particles were located exclusively on the basolateral surface, and no significant binding of gold particles to the apical surface was observed. Distribution of gold particles on the basolateral membranes was quite heterogeneous; lateral membranes and infolded basal membranes were highly labeled, whereas the basal membranes which are in direct contact with the basal lamina were scarcely labeled. The average particle density on the basal surface was highest in the distal straight tubule cells (11.4 units), very high in the distal convoluted tubule cells (9.8 units), intermediate in the proximal tubule cells (3.3 units), in the connecting tubule cells (4.3 units), and in the principal cells of the collecting duct (5.6-3.8 units), low in the thin limb of Henle's loop (1.0 unit), and at the control level in the intercalated cells in the connecting and collecting duct. The relative number of gold particles/mm nephron segment and the relative number of gold particles in the various nephron segments were calculated using quantitative morphological data. The estimated distribution profile of the former was in good agreement with the Na, K-ATPase activity profile in rat nephron, which was determined biochemically with a microenzymatic method.     

Observation of fibronectin distribution on the cell undersurface using immunogold scanning electron microscopy.

Immunogold staining followed by observation with scanning electron microscopy (SEM) has been quite effective in showing the distribution of proteins on dorsal cell surfaces. However, observation of proteins on the ventral cell surface using SEM has not been developed to the same extent. In this study, human gingival fibroblasts cultured on titanium-coated wafers were embedded in resin. After fracturing the wafers off the embedded cells, the undersurface of the cell was exposed by argon gas glow discharge etching. After 15 min of glow discharge etching, the resin covering the cell undersurface was completely removed. The distribution of fibronectin (FN) on the cell undersurface was demonstrated using an anti-FN antibody and colloidal gold (30 nm) conjugated with IgG. The undersurface was then coated with carbon or gold-palladium and observed by SEM. Using backscattered electron detection, gold beads could be identified in high contrast. On cells cultured for 5 hr, gold beads were distributed randomly on the entire cell undersurface. However, a line of gold beads was sometimes observed close to the edge of the cell. These results indicated that this immunogold/SEM etching method provides a powerful means for studying cell adhesion molecules on the cell undersurface. (J Histochem Cytochem 47:1487-1493, 1999)     

Localization of a phytohormone using immunocytochemistry

The localization of cytokinins in corn root tips was investigated using antibodies or antibody fragments directed against dihydrozeatin riboside and labeled with rhodamine or colloidal gold. Roots were sectioned at -30 degrees to -40 degrees for immunofluorescence or freeze-substituted in ethanol or acetone and embedded in plastic for electron microscopy. Meristematic cells surrounding the quiescent center as well as root cap cells were specifically labeled using direct immunofluorescence techniques, whereas cells of the quiescent center did not bind label. Tissue sections treated with colloidal gold-labeled antibody fragments had gold particles widely distributed in the cytoplasm. The results show that the quiescent center is not the major site of cytokinin localization in root tips.     

Distribution in clusters of complement receptor type one (CR1) on human erythrocytes

The distribution of CR1 on human E was studied using label-fracture and thin section electron microscopy. CR1 was found to be organized in clusters on unfixed cells and on cells that had been prefixed with paraformaldehyde or glutaraldehyde before labeling. The number of clusters/E ranged from 8 to 20 as estimated from the examination of freeze-fracture replicas of labeled cells. Clusters contained an average of 30 to 75 gold particles on cells from two donors which expressed 462 and 586 CR1 Ag sites/cell, as determined by flow cytometry. In thin section electron micrographs, gold complexes were seen surrounding an electron-dense material protruding from the membrane which represents compact aggregates of CR1. The maximal distance between gold particles and the membrane was 100 nm, which corresponds to the estimated length of the major allotypic form of CR1, as calculated from the primary DNA sequence of the molecule. The distribution in clusters of CR1 on the E membrane may provide the basis for an enhanced affinity of C3b-CR1 interactions on the plasma membrane of the cells and may explain the preferential binding of C3b-bearing immune complexes to E in vivo.     

Colloidal gold, a useful marker for transmission and scanning electron microscopy.

Electron dense markers of a size suitable for transmission electron microscopy and scanning electron microscopy have been prepared with gold granules labeled with a monolayer of specific macromolecules. The optimum conditions for preparing the markers have been ascertained. The method is simple, rapid and seems to be general since gold granules have been labeled with polysaccharides and proteins. As homogeneous populations of gold granules having different sizes can be prepared, the method is also suitable for double marking experiments. The gold technique is illustrated by the localization of polysaccharides and glycoproteins on yeast cell walls and erythrocyte membranes by transmission electron microscopy and on yeast cells and intact erythrocytes by scanning electron microscopy. Good spatial resolution of the marker was achieved in all cases. The method is also suitable for marking thin sections. Spectrophotometric measurements were used to determine the number of gold granules adsorbed per cell.     

Immunoelectron microscopic demonstration of pancreatic polypeptide in midgut epithelium of hematophagous dipterans

Midguts of mosquitoes, Aedes aegypti and Anopheles stephensi, and of the tsetse fly, Glossina morsitans morsitans, as well as guinea pig pancreas, were prepared for electron microscopy by using low-temperature embedding in Lowicryl K4M. Rabbit antiserum to bovine pancreatic polypeptide (PP) crossreacted with secretory granules of pancreatic PP-producing cells and of the clear cells in mosquito gut. Rabbit antiserum to human somatostatin crossreacted with the control tissue, guinea pig pancreas D-cells, but not with the mosquito clear cells. None of the antisera used showed a distinct reaction with the endocrine-like cells of tsetse fly midgut. Positive reactions were revealed by gold as electron-dense marker. The gold particles were coated with protein A-gold or goat antibodies to rabbit immunoglobulin.     

rRNA Sequence-Based Scanning Electron Microscopic Detection of Bacteria

A new scanning electron microscopic method was developed for gaining both phylogenetic and morphological information about target microbes using in situ hybridization with rRNA-targeted oligonucleotide probes (SEM-ISH). Target cells were hybridized with oligonucleotide probes after gold labeling. Gold enhancement was used for amplification of probe signals from hybridized cells. The hybridized cells released a strong backscatter electron signal due to accumulation of gold atoms inside cells. SEM-ISH was applied to analyze bacterial community composition in freshwater samples, and bacterial cell counts determined by SEM-ISH with rRNA-targeted probes for major phyla within the domain Bacteria were highly correlated to those by fluorescent in situ hybridization (FISH). The bacterial composition on surface of river sediment particles before and after cell dispersion treatment by sonication was successfully revealed by SEM-ISH. Direct enumeration of bacterial cells on the surface of sonicated sediment particles by SEM-ISH demonstrated that members of Cytophaga-Flavobacterium existed tightly on the surface of particles. SEM-ISH allows defining the number and distribution of phylogenetically defined cells adherent to material surfaces, which is difficult in FISH, and it gives new insight into electron microscopic studies of microorganisms in their natural environment.     

alpha 2-macroglobulin adsorbed to colloidal gold: a new probe in the study of receptor-mediated endocytosis

alpha 2-Macroglobulin (alpha 2 M) was adsorbed to colloidal gold and used as a new tool in the study of receptor-mediated endocytosis. alpha 2 M-gold is easy to prepare and is clearly visualized at the electron microscope level. When cells were incubated with alpha 2 M-gold at 0 degrees C, gold was visualized both diffusely over the cell surface and concentrated in coated pits. After cells to which alpha 2 M-gold had been bound at 0 degrees C were warmed, the gold was rapidly internalized into uncoated vesicles, previously termed receptosomes. After 30 min of incubation or longer, gold was found in small lysosomes and, later, in large lysosomes and very small vesicles in the region of the Golgi complex. This pattern of localization is similar to that previously described, using peroxidase-labeled anti-alpha 2 M antibodies. By incubating cells with both alpha 2 M-gold and vesicular stomatitis virus (VSV), we studied the internalization of these two markers simultaneously. VSV and alpha 2 M-gold rapidly clustered in the same coated pits and were internalized in the same receptosomes. Proteins and hormones adsorbed to gold may be useful in the study of receptor-mediated endocytosis.     

Filopodia are enriched in a cell cohesion molecule of Mr 80,000 and participate in cell-cell contact formation in Dictyostelium discoideum

During the early phase of Dictyostelium discoideum development, cells undergo chemotactic migration to form tight aggregates. A developmentally regulated surface glycoprotein of Mr 80,000 (gp80) has been implicated in mediating the EDTA-resistant type of cell cohesion at this stage. We have used a monoclonal antibody directed against gp80 to study the topographical distribution of gp80 on the cell surface. Indirect immunofluorescence studies showed that gp80 was primarily localized on the cell surface, with a higher concentration at contact areas. Immunoelectron microscopy was carried out by indirect labeling using protein A-gold, and a nonrandom distribution of gp80 was revealed. In addition to contact regions, gold particles were found preferentially localized on filopodia. Quantitative analysis using transmission electron microscopy (TEM) showed that approximately 60% more gold particles were localized in contact regions in comparison with the noncontact regions, and the filopodial surfaces had a twofold higher gold density. Both TEM and scanning electron microscopy showed that contact areas were enriched in filopodial structures. Filopodia often appeared to adhere to either smooth surfaces or similar filopodial structures of an adjacent cell. These observations suggest that the formation of stable cell-cell contacts involves at least four sequential steps in which filopodia and gp80 probably play an important role in the initial stages of recognition and cohesion among cells.     

Construction and direct electrochemistry of orientation controlled laccase electrode

A laccase has multiple redox centres. Chemisorption of laccases on a gold electrode through a polypeptide tag introduced at the protein surface provides an isotropic orientation of laccases on the Au surface, which allows the orientation dependent study of the direct electrochemistry of laccase. In this paper, using genetic engineering technology, two forms of recombinant laccase which has Cys-6×His tag at the N or C terminus were generated. Via the Au-S linkage, the recombinant laccase was assembled orientationally on gold electrode. A direct electron transfer and a bioelectrocatalytic activity toward oxygen reduction were observed on the two orientation controlled laccase electrodes, but their electrochemical behaviors were found to be quite different. The orientation of laccase on the gold electrode affects both the electron transfer pathway and the electron transfer efficiency of O₂ reduction. The present study is helpful not only to the in-depth understanding of the direct electrochemistry of laccase, but also to the development of laccase-based biofuel cells.     

Demonstration and cytochemical analysis of anionic sites on ejaculated boar spermatozoa: a scanning electron microscopy study using cationised colloidal gold

The plasmalemma of spermatozoa bears negative charges as is the case for most mammalian cells. This has been concluded from the sperm cell's electrophoretic behaviour and from labelling experiments with various cationic probes followed by transmission electron microscopy of ultrathin sections. An overall view of the cell surface, however, is necessary in order to assess the distribution and density of the anionic sites adequately. We, therefore, used scanning electron microscopy in combination with cationised colloidal gold labelling to analyse the presence of anionic sites on ejaculated boar spermatozoa. Incubations were performed at pH 3.5, 2.5 and 1.0. Labelling was specific and bound gold particles were unequivocally identified using the backscattered electron signal. The chemical nature of the anionic sites involved was investigated by treating spermatozoa with pronase, phosphatase and neuraminidase as well as by methylation, acid hydrolysis and beta-elimination prior to cationised gold labelling. Our results suggest that besides phosphates, carboxyl groups are predominantly accountable for the binding of cationised colloidal gold. Presumptive macromolecules bearing these anionic sites are phospholipids and sialic acid residues. The combination of methods presented herewith should be of value in order to elucidate charge interactions which have been shown to play a role in cellular recognition events and adhesion.