Scanning electron microscopy of cell surface antigens labeled with colloidal gold

A method is described and discussed that permits the specific labeling of the surface of prefixed cells with the colloidal gold marker viewed with the scanning electron microscope. Its value depends exclusively on the use of backscattered electron imaging. Its advantages include the possibility of preserving the surface features of the labeled cells, the ease with which specificity can be established, the possibility of making total counts of the labeled surface antigenic sites, and the possibility of achieving distinct labeling for two different antigens expressed on the surface of the same cell.     

Rapid redistribution of myosin II in livingDictyostelium amoebae,as revealed by fluorescent probes introduced by electroporation

Summary Fluorescently labeled myosin II fromDictyostelium and fluorescently labeled antibody Fab fragments against myosin II fromDictyostellium were introduced into livingDictyostelium amoebae by electroporation. Fluorescent labeling of myosin II impairs neither actin-activated ATPase activity nor the ability to form filaments in vitro. Fluorescently labeled Fab also did not interfere with the functions of myosin II in vitro. After electroporation, introduced fluorescently labeled myosin II was distributed diffusely in the endoplasm but some of it accumulated at the tail cortical region of migrating cells. During the course of observations, intense fluorescence due to myosin II disappeared and then it appeared again instantaneously in the cortical regions during amoeboid movement. Fluorescently labeled Fab, after electroporation, bound to endogenous myosin II in amoebae and the dynamic changes in its distribution were similar to those of fluorescently labeled myosin II. The fluorescence due to myosin II also underwent dynamic redistribution during the division of cells and chemotactic stimulation. The introduction of labeled Fab and labeled myosin II did not impair the motility ofDictyostelium. During changes in direction associated with cell locomotion, myosin II accumulated at the original front region of the cell and, thereafter, the accumulation was observed at the new tail region of the cell. These results are consistent with the hypothesis that myosin II has two possible roles for cell locomotion. One is that myosin II accumulates at tail regions to produce the power required for contraction. The other is that it hinders the extension of pseudopods in directions other than the frontal direction.     

Three-dimensional immunogold labeling of nuclear matrix proteins in permeabilized cells.

A preembedment labeling procedure is described for the three-dimensional (3D) labeling of nuclear matrix proteins in permeabilized cells. The procedure is based on the use of ultra-small (1 nm) gold particles as a marker system. This marker penetrates the nucleus more efficiently than the conventionally used 5-10 nm colloidal gold probes. Dehydration is performed by freeze-substitution to preserve the ultrastructure of the cell as optimally as possible. During freeze-substitution the samples are stained by uranyl ions to stain the cellular material throughout the resin section. The 3D gold-labeled and uranyl-stained specimen is embedded in Epon resin and semi-thin (0.2-0.5 microns) sections are made for stereo electron microscopy. The applicability of this method is illustrated by the localization of nuclear matrix-associated nuclear bodies in permeabilized interphase and mitotic HeLa cells.     

A 1.4-nm gold cluster covalently attached to antibodies improves immunolabeling.

A large gold cluster (Au1.4nm) was covalently coupled to IgG and Fab' fragments. Its gold core is 1.4 nm in diameter and the Fab'-Au1.4nm immunoconjugate is the smallest gold immunoprobe that can be seen directly in the conventional electron microscope. It is useful in high-resolution immunolabeling, providing a resolution of 7.0 nm. The cluster's visibility can be enhanced with silver development for use in EM or light microscopy for histological purposes, or to detect less than or equal to 0.2 pg of antigen in immunoblots. By using a gold compound with covalent attachment, a number of advantages over colloidal gold probes are realized, including better resolution, stability, uniformity, sensitivity, and complete absence of aggregation; its small size should also improve penetration and more quantitative labeling of antigenic sites.     

Immunoelectron microscopic localization of idiotypes and allotypes on immunoglobulin molecules

We have developed a novel approach to the analysis of antigenic (allotypic and idiotypic) determinants on intact immunoglobulin molecules. Immune complexes composed of IgG in combination with anti-idiotype or anti-allotype antibody were "visualized" by transmission electron microscopy. Individual Fab fragments of anti-idiotype or anti-allotype antibody, when bound to the IgG, altered the "Y" configuration in a reproducible and interpretable manner. Anti-idiotype antibody (either as Fab or IgG) bound to the terminus of the presumed V region of the IgG molecule, thus extending the apparent length of the Fab arms. Analysis of a rabbit VH framework allotype (a1) revealed that the determinant(s) is (are) located on the lateral portion of the V region of IgG. Binding of the anti-a1 Fab fragments was always at approximately right angles to the axis of the Fab arms of IgG. Fab antibody to the rabbit kappa light chain (b4) allotype bound to the lateral portion of the terminal half of the IgG Fab arms. This technique should be of value in localizing less well defined immunoglobulin determinants.     

Filter screening of antibody Fab fragments secreted from individual bacterial colonies: specific detection of antigen binding with a two-membrane system

Recently antibody fragments have been expressed in a functional form from bacteria. We have devised a simple method to detect the binding of antigen to antibody Fab fragments secreted by bacterial colonies. Bacteria harboring plasmid vectors that direct the secretion of Fab fragments into the bacterial periplasm are grown on one membrane. The secreted fragments are allowed to diffuse to a second "capture" membrane coated with anti-globulin, and are probed with antigen. Using enzyme or colloidal gold conjugates, the binding of antigen is detected on the second membrane as a colored spot. The colonies can be regrown on the first membrane, and the antigen binding signal on the second membrane is free of noise contributed by bacterial debris.     

Use of antibodies specific to defined regions of scorpion alpha-toxin to study its interaction with its receptor site on the sodium channel

Five antibody populations selected by immunoaffinity chromatography for their specificity toward various regions of toxin II of the scorpion Androctonus australis Hector were used to probe the interaction of this protein with its receptor site on the sodium channel. These studies indicate that two antigenic sites, one located around the disulfide bridge 12-63 and one encompassing residues 50-59, are involved in the molecular mechanisms of toxicity neutralization. Fab fragments specific to the region around disulfide bridge 12-63 inhibit binding of the 125I-labeled toxin to its receptor site. Also, these two antigenic regions are inaccessible to their antibodies when the toxin is bound to its receptor site. In contrast, the two other antigenic sites encompassing the only alpha-helix region (residues 23-32) and a beta-turn structure (residues 32-35) are accessible to their respective antibodies when the toxin is bound to its receptor. Together, these data support the recent proposal that a region made of residues that are conserved in the scorpion toxin family is involved in the binding of the toxin to the receptor.     

Preembedding colloidal gold immunostaining of hypothalamic neurons: light and electron microscopic localization of beta-endorphin-immunoreactive perikarya

A preembedding immunogold staining (IGS) procedure was developed to identify beta-endorphin/adrenocorticotropic hormone immunoreactive neurons at the light and electron microscopic levels. Colchicine-treated rats were perfused with Nakane's periodate-lysine-paraformaldehyde fixative. Vibratome sections were incubated in primary antisera followed by goat anti-rabbit immunoglobulin G coupled to 16 nm colloidal gold, and, in some cases, rabbit immunoglobulin G coupled to gold. The appearance to pink to light red perikarya, corresponding to colloidal gold deposition at antigenic sites, was monitored under the light microscope. Positive cell bodies in the arcuate region sometimes extended lateral to the nucleus. Only proximal portions of neuronal processes were stained. At the ultrastructural level, colloidal gold labeled the periphery of 90-110 nm dense neurosecretory granules in the perikaryal cytoplasm and a few proximal axons. Clusters of gold particles, appearing free in the neuroplasm, actually labeled secretory granules in adjacent thin sections. Granules associated with the Golgi apparatus were not stained. Colloidal gold labeling of mature beta-endorphin granules, but not progranules, in rat hypothalamic neurons was confirmed using the peroxidase-antiperoxidase technique. The results correlate well with data on the intracellular processing of pro-opiomelanocortin in pituitary cells and prepropressophysin in the paraventricular nucleus. These data demonstrate the first application of the preembedding colloidal gold staining method for the identification of intracellular antigens within the central nervous system. The IGS method provides a definitive marker for single or double labeling of nervous tissue at both the light and electron microscopic levels.     

Electron microscopic immunocytochemistry. Silver enhancement of colloidal gold marker allows double labeling with the same primary antibody

Electron microscopic sections, immunocytochemically labeled with colloidal gold, can be prepared for double labeling by applying the "EM-silver enhancement" procedure. This method, a photographic, so-called physical, development, increases the size of the gold marker to a predeterminable value and thereby inactivates the anti-species antibody present on the gold grain, thus allowing the labeling of a second antigen with antibody raised in the same species.     

Interrelationship of concanavalin-A-binding and antigenic sites on the acetylcholine receptor from Torpedo marmorata

A preparation of purified 125I-labelled acetylcholine receptor was shown to bind to concanavalin A and to be totally bound by rabbit antiserum to Torpedo acetylcholine receptor. Pre-incubation of the receptor with F(ab')2 and Fab fragments from antibodies against Torpedo acetylcholine receptor, or with corresponding fragments from control immunoglobulin G showed that subsequent binding of the receptor to concanavalin A was specifically inhibited to a maximum of approximately 25% by the immune fragments. Treatment of acetylcholine receptor with periodate or with glycosidases apparently destroyed or removed carbohydrate residues without affecting the antigenicity of the receptor as assessed by radioimmunoassay. These results suggest that although there is a steric interrelatonship between the antigenic and concanavalin-A-binding sites of the receptor the latter sites do not contain its major antigenic determinants.     

Preferential association of glycoproteins to the euchromatin regions of cross-fractured nuclei is revealed by fracture-label

We used fracture-label to establish ultrastructural localization of glycoproteins in cross-fractured nuclei of duodenal columnar and exocrine pancreatic cells. Mannose residues were detected in cell nuclei by labeling freeze-fractured tissues with concanavalin A-horseradish peroxidase X colloidal gold (Con A-HRP X CG) or direct concanavalin A X colloidal gold (Con A X CG); fucose residues were detected with Ulex Europaeus I X colloidal gold (UEA I X CG) markers. Areas of the three main intranuclear compartments (euchromatin, heterochromatin, and nucleolus) exposed by freeze-fracture were determined by automated image analysis. Colloidal gold particles bound to each nuclear subcompartment were counted and the results expressed in number of colloidal gold particles per square micrometer +/- SEM. Duodenal and pancreatic tissues fractured and labeled with Con A-HRP X CG complex or direct Con A X CG conjugates showed that the vast majority of Con A binding sites was confined to euchromatin regions with only sparse labeling of the heterochromatin and nucleolus. UEA I labeling of duodenal columnar cells showed that colloidal gold particles were almost exclusively confined to cross-fractured areas where euchromatin is exposed. Trypsinization of the fractured tissues before labeling with Con A and UEA I abolished 95-100% of the original label. Our results show that, within the nucleoplasm, mannose and fucose are residues of glycoproteins preferentially located within the regions of euchromatin.     

Localization of cell surface sites involved in fibronectin fibrillogenesis

Fibronectin binding sites on cultured human fibroblasts were localized by high voltage electron microscopy using either 5- or 18-nm colloidal gold beads (Au5 or Au18) bound to intact fibronectin, the 70-kD amino- terminal fragment of fibronectin that blocks incorporation of exogenous fibronectin into extracellular matrix, or 160-180-kD fragments of fibronectin with cell adhesion and heparin-binding activities. Binding sites for Au18-fibronectin on the cell surface were localized to specific regions along the edge of the fibroblast and on retraction fibers. Au18-fibronectin complexes at these sites were initially localized in clusters that co-aligned with intracellular microfilament bundles. With longer incubations, Au18-fibronectin complexes were arranged into long fibrillar networks on the cell surface and in the extracellular space. The appearance of Au18-fibronectin in these fibrillar networks and disappearance of clusters of Au18-fibronectin suggest that Au18-fibronectin complexes are arranged into matrix at specific regions of the cell surface. Au18-70-kD fragment complexes initially had a similar distribution to Au18-fibronectin complexes. With longer incubations, Au18-70-kD fragment complexes were found in long linear arrangements on the cell surface. Double labeling experiments using Au18-70-kD fragment and Au5-160-180-kD fragments showed that the 70-kD fragment and the 160-180-kD fragments bind to different regions of the cell.     

Immunoelectron microscopic double labeling of alkaline phosphatase and penicillinase with colloidal gold in frozen thin sections of Bacillus licheniformis 749/C.

The subcellular distribution of alkaline phosphatase and penicillinase was determined by double labeling frozen thin sections of Bacillus licheniformis 749/C with colloidal gold-immunoglobulin G (IgG). Antipenicillinase and anti-alkaline phosphatase antibodies were used to prepare complexes with 5- and 15-nm colloidal gold particles, respectively. The character of the labeling of membrane-bound alkaline phosphatase and penicillinase was different: the immunolabels for alkaline phosphatase (15-nm particles) were bound to a few sites at the inner surface of the plasma membrane, and the gold particles formed clusters of various sizes at the binding sites; the immunolabels for penicillinase (5-nm particles), on the other hand, were bound to the plasma membrane in a dispersed and random fashion. In the cytoplasm, immunolabels for both proteins were distributed randomly, and the character of their binding was similar. The labeling was specific: pretreating the frozen thin sections with different concentrations of anti-alkaline phosphatase or penicillinase blocked the binding of the immunolabel prepared with the same antibody. Binding could be fully blocked by pretreatment with 800 micrograms of either antibody per ml.     

Organization of glycosphingolipids in phosphatidylcholine bilayers: use of antibody molecules and Fab fragments as morphologic markers

The techniques of ultrafast freezing and freeze-etch electron microscopy have been successfully employed to visualize IgG molecules and Fab fragments specifically bound to the neutral glycosphingolipids Forssman and asialo-GM1 incorporated into phosphatidylcholine liposomes. Monovalent Fab is the superior marker because of its small size and because it does not cause liposomal aggregation with concomitant glycolipid reorganization. Analysis of Fab labeling of liposomes containing these neutral glycosphingolipids leads to the conclusion that the Forssman glycosphingolipid is dispersed in clusters of not more than several molecules when present at low mole fraction in fluid-phase 1-palmitoyl-2-oleoylphosphatidylcholine liposomes. In contrast to this, asialo-GM1 under the same conditions is present in clusters of about 15 molecules in this phospholipid matrix.     

Vitronectin at sites of cell-substrate contact in cultures of rat myotubes

Affinity-purified antibodies to the serum glycoprotein, vitronectin, were used to study sites of cell-substrate contact in cultures of rat myotubes and fibroblasts. Cells were removed from the substrate by treatment with saponin, leaving fragments of plasma membrane attached to the glass coverslip. When stained for vitronectin by indirect immunofluorescence, large areas of the substrate were brightly labeled. The focal contacts of fibroblasts and the broad adhesion plaques of myotubes appeared black, however, indicating that the antibodies had failed to react with those areas. Contact sites within the adhesion plaque remained unlabeled after saponin-treated samples were extracted with Triton X-100, or after intact cultures were sheared with a stream of fixative. These procedures expose extracellular macromolecules at the cell-substrate interface, which can then be labeled with concanavalin A. In contrast, when samples were sheared and then sonicated to remove all the cellular material from the coverslip, the entire substrate labeled extensively and almost uniformly with anti-vitronectin. Extracellular molecules associated with substrate contacts were also studied after freeze-fracture, using a technique we term "post-release fracture labeling." Platinum replicas of the external membrane were removed from the glass with hydrofluoric acid to expose the extracellular material. Anti-vitronectin, bound to the replicas and visualized by a second antibody conjugated to colloidal gold, labeled the broad areas of close myotube-substrate attachment and the nearby glass equally well. Our results are consistent with the hypothesis that vitronectin is present at all sites of cell-substrate contact, but that its antigenic sites are obscured by material deposited by both myotube and fibroblast cells.     

Following single antibody binding to purple membranes in real time

Antibody binding to surface antigens in membranes is the primary event in the specific immune defence of vertebrates. Here we used force microscopy to study the dynamics of antibody recognition of mutant purple membranes from Halobacterium salinarum containing a genetically appended anti-Sendai recognition epitope. Ligation of individual anti-Sendai antibodies to their antigenic epitopes was observed over time. Their increase in number within a small selected area revealed an apparent kinetic on-rate. The membrane-bound antibodies showed many different conformations that ranged from globular to V- and Y-like shapes. The maximum distance of two Fab fragments of the same antibody was observed to be approximately 18 nm, indicating an overall strong intrinsic flexibility of the antibody hinge region. Fab fragments of bound anti-Sendai antibodies were allocated to antigenic sites of the purple membrane, allowing the identification and localization of individual recognition epitopes on the surface of purple membranes.     

Serological Relationships of Salmonella A Bacteriophages Isolated from Salmonellae in Different Kauffmann- White Groups

Six different Salmonella group A phages from salmonellae in Kauffmann- White groups B, C(1), C(2), and D were examined serologically. Those phages which were specific for a particular somatic antigen were found to be serologically very similar. Antiserum against a phage with one specificity was able to neutralize a different phage with the same specificity but unable to neutralize, in the normal way, a phage with a different specificity. Phages mixed with heterologous phage antiserum responded with an "inhibition response" in which there appeared to be a neutralization of the phage infectivity for the first 10 min, followed by a reversal of the neutralization until, by 20 or 25 min, there was no apparent neutralization. This response was interpreted to indicate that the adsorption antigens, probably situated on the tail fibers, were different for phages with different specificities but sufficiently similar so that heterologous antibodies could react with the antigens; but the antigen-antibody complex was quickly disassociated, resulting in a modification of the antibody molecules but no change in the specificity sites of the antigen. A subgrouping of the Salmonella A phages based on their antigenic specificity is suggested.     

T-even-type bacteriophages use an adhesin for recognition of cellular receptors

Protein 38 of the Escherichia coli phage T4 is thought to be required catalytically for the assembly of the long tail fibers of this phage. It is shown that this protein of phage T2 and the T-even-type phage K3 and Ox2 act differently. It was found that NH2-terminal fragments of the protein, expressed from cloned fragments of gene 38 of phage K3, bind to gene 38 amber mutants of phage T2. Such phage or T2 gene 38 amber mutants, grown on a non-permissive host, possess a complete set of six tail fibers but are non-infectious. Both types of non-infectious phage could be repaired by incubation with an extract of cells harboring a cloned gene 38 of a host range mutant of phage K3, K3hx. The repaired phages had the host range of K3hx and not of T2. Immuno-electron microscopy showed that protein 38 is located at the free ends of the long tail fibers of phages T2, K3 and Ox2. The protein serves the recognition of the cellular receptor, i.e. it acts as an adhesin.     

Evaluation of immunoelectron microscopic techniques in the study of basement membrane antigens

 Recent technical advances in immunoelectron microscopy (IEM), including methods of pre- and postembedding IEM and cryoultramicrotomy, have helped to elucidate the precise ultrastructural localization of various basement membrane-related molecules. Our objective was to evaluate the advantages and disadvantages of several different techniques for studying the ultrastructural organization of basement membrane components. We found that, while ”on-surface” immunolabeling of postembedding IEM and cryoultramicrotomy with anti-type IV collagen or anti-laminin-5 antibody clearly demonstrated dense labeling on the lamina densa, preembedding IEM with a 1-nm ultra-small gold probe showed labeling only on the epidermal and/or dermal surfaces of the lamina densa, with no specific gold particles being seen within the lamina densa itself. These results indicate that even ultra-small colloidal gold-labeled antibody fails to penetrate the lamina densa in preembedding IEM. However, labeling with a GB3 monoclonal antibody against laminin-5 was demonstrable with preembedding IEM and cryoultramicrotomy, but not with post-embedding IEM, probably due to a loss of antigenicity. These results confirm the advantages and limitations of these techniques of IEM and emphasize the importance of using different techniques of IEM in determining the precise ultrastructural distribution of basement membrane antigens. Accepted: 30 January 1998     

Phage-display libraries of murine and human antibody Fab fragments

We provide efficient and detailed procedures for construction, expression, and screening of comprehensive libraries of murine or human antibody Fab fragments displayed on the surface of filamentous phage. In addition, protocols for producing and using ultra-electrocompetent cells, for producing Fab phages from libraries, and for selecting antigen binders by panning are presented. The latter protocol includes a procedure for trypsin elution of bound phage.