Amantadine treatment of cells infected with H7 strains of influenza A viruses causes an M2 protein-mediated conversion of hemagglutinin (HA) from its native to its low pH conformation. Immunofluorescence and electron microscopic observations showed that the structural alteration and hence drug action occur shortly after HA exits from the Golgi complex during its passage through the strans Golgi region. Using the DAMP/anti-DNP pH probe it is evident that virus infection causes increased acidity of the trans Golgi region and that vesicles containing low pH HA in amantadine-treated virus-infected cells are particularly acidic. These results indicate therefore that the alteration in HA is the direct consequence of exposure to an adverse low pH and provide further support for the conclusion that the M2 protein, the target of amantadine action, is involved in regulating vesicular pH, a function important for the correct maturation of the HA glycoprotein.

A novel procedure for reversing cell monolayers is described. Cells are embedded in liquid gelatin containing ethylenediaminetetraacetic acid, cooled down to solidify gelatin, and then reversed. The main advantage of this technique is that cells are fixed after reversing so that the extracellular matrix does not obscure the cell surface. No substantial migration of receptors is likely to have taken place judging from the concentration of fibronectin receptor in typical focal or extracellular matrix contacts.

Cell/substratum adhesions have been studied in rat fibroblasts transformed by a ts-mutant of Rous sarcoma virus (LA-29) using light and electron microscopy and a variety of preparative methods including immunolabeling. Cells were studied both during the process of transformation, i.e., shifting from 39 degrees to 35 degrees C, and in a fully transformed state (passaged at 35 degrees C continuously). The typical focal contacts observed at 39 degrees C (restrictive temperature) were replaced by "point-contacts" (100-200 per cell) which were classified by immunolabeling as podosome-like adhesions containing actin, beta 1 integrin subunit, vinculin, talin, alpha-actinin, and small membrane patches containing clathrin and integrin. Tyrosine-phosphorylated proteins and pp60src were found in association with groups of small particles on the protoplasmic surface of ventral membranes by gold immunolabeling. Both types of point-contacts were visualized by electron microscopy of ultrathin sections and shadowed replicas and characterized by gold immunolabeling wherever possible. The overall composition of podosome-like adhesions is similar to focal contacts but there are differences in the three-dimensional organization of the microfilaments and in the topography of vinculin which is associated more with actin filaments than with the plasma membrane. The presence of talin and extracellular matrix receptor in podosomes together with the adhesive properties of these actin-containing structures argues against the hypothesis that pp60src affects the interaction of actin with the plasma membrane by phosphorylating the fibronectin receptor and/or other associated proteins.

The mechanism by which Rous sarcoma virus (RSV) induces a reorganization of actin and its associated proteins and a reduction in microfilament bundles is at present poorly understood. To examine the relationship between the organization of the microfilament system and the polymerization state of actin after transformation, we have investigated these changes in a Rat-1 cell line transformed by LA29, a temperature-sensitive (ts) mutant of RSV. Parallel immunofluorescence and biochemical analysis demonstrated that LA29 pp60v-src was ts for tyrosine kinase activity and cytoskeletal association. Changes in the distribution and organization of actin, alpha-actinin and vinculin were dependent on the association of a kinase-active pp60v-src molecule with the detergent-insoluble cytoskeleton. Whilst there was a transformation-dependent loss of microfilament bundles, biochemical quantitation demonstrated that the polymerization state of the actin in both detergent-soluble and insoluble fractions of these cells grown at temperatures either permissive or restrictive for transformation was quantitatively unchanged. These results indicate that the loss of microfilament bundles after transformation is not due to a net depolymerization of filamentous actin but rather to a reorganization of polymeric actin from microfilament bundles and stress fibers to other polymeric forms within the cell. The polymeric nature of the actin in these cells was confirmed by electron microscopy of cytoskeletons and substrate-adherent membranes.

There is no single method which would provide an unambiguous image of all types of biological macromolecules. The choice of method depends largely on the size and properties of the macromolecule. Obviously small molecules are best visualized by negative staining, the problems appear with negative staining of larger structures. Here, the uncertainty about which part of the complex is actually stained (top or bottom) makes correct interpretation difficult. Shadowing techniques have the advantage of both visualizing the surface and also delineating the whole macromolecule, but suffer from lower resolution due to the graininess of the metal. However, they are superior to negative staining for the visualization of thin linear macromolecules. The next series of problems includes the interaction of macromolecules with supporting films, glass coverslips or mica, which can be hydrophobic, hydrophilic or charged and these properties can influence the orientation of the molecules. Surface tension forces during air-drying must also be considered. We have used a variety of preparative techniques in our studies of biological macromolecules: (a) negative staining; (b) air-drying from ethanol; (c) glycerol-spraying; (d) adsorption freeze-drying; (e) monolayer freeze-etching. These methods have been tested on small viruses, water soluble proteins (ribosomes, F-actin, microtubules) and transmembrane proteins requiring the presence of detergents (sarcoplasmic reticulum ATPase, fibronectin receptor). We find that freeze-drying is the most reliable and easy method for molecules that withstand distilled water; freeze-etching can be successfully applied to transmembrane proteins (even in the presence of detergents or salt); the glycerol-spray technique provides an excellent alternative to the cryotechniques in particular for studies of single linear molecules.

Over the past decade new methods have been developed to visualize both the external and the protoplasmic surfaces of cultured cells in the electron microscope. In this review the emphasis is on cell monolayers, though some of the techniques are also applicable to cells in suspension. There is no universal method which would satisfy all our requirements i.e. the preservation of native structure and antigenicity and the visualization of the whole cell surface at high resolution. While surface replicas of freeze-dried or critical point-dried cells are eminently suited for high resolution studies including gold immunolabelling, scanning electron microscopy provides a view of the whole cell and a large sample for 'statistical' evaluation. Whole mount preparations of cleaved cells prove useful in studies of plasma membrane associated structures such as the cytoskeleton. A series of new procedures have been developed for studies of cytoskeleton/membrane interactions, identification of intramembrane particles and their contacts with the glycocalyx, to mention some of the biological problems. Although the lysis-squirting technique appears most suitable for the visualization and immunolabelling of protoplasmic surfaces of ventral membranes, dry- or wet-cleaving represent a useful alternative for studies of the protoplasmic surfaces of dorsal membranes and of the ventral membrane associated cytoplasmic domains. An assessment of the methods is given though this should only serve as guidance and it is up to the experimentor to choose the most useful technique for the project under study. Briefly the aim of the project determines the choice of the method. A multi-methodical approach is recommended when one method does not provide satisfactory results.

ABSTRACT Rous sarcoma virus-transformed BHK (RSV/B4-BHK) cells develop peculiar dot-like adhesions, that have been named podosomes, which, in the presence of serum, aggregate into ring- or crescentshaped adhesion sites, the rosettes of podosomes. We have used the lysis-squirting technique and gold-immunolabelling to study the 3D- organisation of podosomes and the location of vinculin, gelsolin, phosphotyrosine-containing proteins and pp60src at an ultrastructural level. Podosomes appear to be conical bodies, 0 ·l-0 ·5 μm high, made by a dense aggregation of actin oligomers and several associated proteins, connected in the rosette by actin filaments. Gelsolin and some phosphotyrosine-containing proteins are found within the podosomes, often associated with the actin filaments, while vinculin is found predominantly at the podosome periphery, associated with microfilaments, and pp60arc is located on the adjacent plasma membrane.

Highly‐purified human fibronectin receptor (a heterodimer of two distinct subunits, alpha and beta) was studied using electron microscopy and a variety of preparative procedures. It was found that the receptor consists of a globular head approximately 80 by 120 A and two tails about 20 A thick and 180‐200 A long. The whole complex is approximately 280 A long. At low concentrations of detergent the receptor forms doublets, triplets or rosettes associated with the tails which possess the transmembrane portion of the molecule. Computer‐assisted structure prediction using the published amino acid sequence of both subunits showed differences in the secondary structure of the tails, the alpha‐tail being rich in beta‐strands, the beta‐tail having five cysteine‐rich repeats analogous to the EGF‐like repeats of laminin. Estimates of the length of the tails from the predicted structure conformed well with the dimensions obtained from electron micrographs.

Adenoviruses are non-enveloped isometric particles with an icosahedral surface shell (capsid) and a DNA-containing core. They replicate and assemble in nuclei of infected cells and cause typical cytopathic effects. Many subgenera agglutinate red blood cells of various animal species. Adenoviruses are mild pathogens whose host ranges are usually confined to single species of animals or birds. Some human strains can transform cells in tissue culture and many virus species are oncogenic in experimental animals. This chapter discusses the structural, chemical, and physical characteristics of human adenovirus. The chapter also discusses the two “structural complexes” of adenovirion: the capsid (outer icosahedral shell) and the core (internal body comprising the nucleocapsid and the core shell). The complex structure of the adenovirion is maintained by the energy of the bonds between structural elements (macromolecules) and structural complexes.

This chapter discusses methods for studies of virus particles and virus-infected cells. Negative staining is one of the most useful methods for the visualization of virus particles and is frequently used in both research and clinical diagnosis. The most useful “negative stains” are those solutions that do not tend to bind with biological structures or interact to cause disruption. The chapter discusses unidirectional shadow-casting with chromium, which is one of the first methods used to increase contrast of viruses in the electron microscope. Low-temperature techniques in virus research are described. The chapter concludes with a discussion on the tactics of structural analysis of viruses. The aim of structural analysis of viruses is to determine the shape, size, surface structure and internal organization of the virion.

Image analysis of freeze-etch replicas of cylindrical aberrant forms of FV3 provided evidence for three morphological subunits protruding from the six-coordinated capsomers. Negatively stained capsomers displayed both triangular and hexagonal profiles which suggests that their innermost portion is pseudohexagonal. Images from underfocused micrographs of capsomers are indicative of a central channel. The trimeric nature of the capsomer has been established by electrophoresis in the presence of Triton X-100, which showed that the molecular weight of the nondissociated capsomer is about 140,000 whereas that of the polypeptide itself is 48,000. This trimeric association does not occur via disulfide bonds, and inside the capsomers there are no free amino groups accessible to the usual bifunctional reagents. Thus, the chemical nature of the interpolypeptide bonds inside the trimers is still unknown. We have previously estimated the triangulation number (T) of FV3 to be 147 or 133 (Darcy-Tripier et al., 1984). The present study, using optical diffraction of the facets of FV3, allowed a better determination of the angle of skewness and is in favor of T = 133 (h = 9, k = 4, 18 degrees).

The effect of specific DNA binding of the cAMP · cAMP receptor protein complex to two DNA fragments (301 and 2685 base-pairs in length) containing the lac operon has been investigated by electron microscopy. It is shown that specific DNA binding of the cAMP · cAMP receptor protein complex induces a kink of 30 to 45 ° in the DNA with the apex of the kink located at the site of protein attachment. These findings lend direct visual support for the kinking hypothesis based on the observation of anomalous electrophoretic mobility of DNA fragments containing specifically bound cAMP receptor protein.

The mass of adenovirus type 5 was determined by means of computer-assisted scanning transmission electron microscopy (STEM). Arithmetic mean of 157 ± 10 (SD) × 106 daltons and mode between 160 and 170 × 106 daltons compare favourably with previously reported data. The advantages of the STEM-procedure over the physical and chemical techniques are: low amounts of purified virus particles are needed; visual control of the physical state of virus particles; no need to know the chemical composition or protein concentration of the virus sample.