Publisher Summary This chapter describes the study of the intracellular location of proteins by immunocytochemical procedures. Although the methods given in the chapter are generally applicable, they are documented for tubulin containing structures and antibodies to tubulin. Further examples of the usefulness of these methods in studying cell structure can be found in several recent reviews. Immunofluorescence microscopy is a very sensitive method, theoretically requiring only one antigenic site on the structural protein. It offers several unique advantages for the study of cytoskeletal structures. First, only the protein of choice is visualized by use of its specific antibody. Second, if the protein forms part of a supramolecular structure, the organization of that structure throughout the cell can be demonstrated. Third, numerous cells can be observed simultaneously, and thus the unique features of the organization under different experimental conditions can be observed. Thus, immunofluorescence microscopy can bridge the gap between light microscopy and electron microscopy.

Immunofluorescence and immunocytochemical procedures with affinity purified antibodies: tubulin-containing structures.

Microtubule organization and nucleation were studied during in vitro human myogenesis by immunocytology that used monoclonal and polyclonal antitubulin antibodies and a rabbit nonimmune serum that reacts with human centrosomes. In myoblasts, we observed a classical microtubule network centered on juxtanuclear centrosomes. Myotubes possessed numerous microtubules organized in parallel without any apparent nucleation centers. Centrosomes in these cells were not associated one to each nucleus but were often clustered in the vicinity of nuclei groups. They were significantly smaller than those of the mononucleated cells. The periphery of each nucleus in myotubes was labeled with the serum that labels centrosomes suggesting a profound reorganization of microtubule-nucleating material. Regrowth experiments after Nocodazole treatment established that microtubules were growing from the periphery of the nuclei. The redistribution of nucleating material was shown to take place early after myoblast fusion. Such a phenomenon appears to be specific to myogenic differentiation in that artificially induced polykaryons behaved differently: the centrosomes aggregated to form only one or a few giant nucleating centers and the nuclei did not participate directly in the nucleation of microtubules. The significance of these results is discussed in relation to the possible role of the centrosome in establishing cell polarity.

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Fate of microtubule-organizing centers during myogenesis in vitro.

Publisher Summary Living organisms abound in a large variety of proteins that possess the ability to specifically bind different compounds. Most important among these are the enzymes that combine specifically with the substrates and inhibitors, and antibodies that bind antigens. A third class of proteins with specific combining sites, found mainly in plants, has recently moved to the forefront of biologic research; these are lectins. The main characteristics of lectins are their ability to bind sugars, to agglutinate cells, and to stimulate lymphocytes. In all their activities, lectins exhibit varying degrees of specificity. Most lectins interact preferentially with a single sugar structure. For some lectins, the specificity is broader and includes a number of closely related sugars; whereas certain lectins interact only with complex carbohydrate structures. Through their sugar-combining sites, lectins interact specifically with polysaccharides and glycoproteins to form precipitates. This reaction is similar to the precipitin reaction between antibody and antigen, in that it is specific, exhibits concentration dependence on lectin and polysaccharide, and might be inhibited specifically by low molecular weight “haptens”—compounds identical with or derived from the sugar(s) for which, the lectin is specific. Therefore, lectins are similar to antibodies in many respects.

Lectins: Their Chemistry and Application to Immunology

The cytoskeleton in cancer cells.

The localization of pp60src within adhesion structures of epithelioid rat kidney cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus was compared to the organization of actin, alpha-actinin, vinculin (a 130,000-dalton protein), tubulin, and the 58,000-dalton intermediate filament protein. The adhesion structures included both adhesion plaques and previously uncharacterized adhesive regions formed at cell-cell junctions. We have termed these latter structures "adhesion junctions." Both adhesion plaques and adhesion junctions were identified by interference-reflection microscopy and compared to the location of pp60src and the various cytoskeletal proteins by double fluorescence. The results demonstrated that the src gene product was found within both adhesion plaques and the adhesion junctions. In addition, actin, alpha-actinin, and vinculin were also localized within the same pp60src-containing adhesion structures. In contrast, tubulin and the 58,000-dalton intermediate filament protein were not associated with either adhesion plaques or adhesion junctions. Both adhesion plaques and adhesion junctions were isolated as substratum-bound structures and characterized by scanning electron microscopy. Immunofluorescence revealed that pp60src, actin, alpha-actinin, and vinculin were organized within specific regions of the adhesion junctions. Heavy accumulations of actin and alpha-actinin were found on both sides of the junctions with a narrow gap of unstained material at the midline, whereas pp60src stain was more intense in this central region. Antibody to vinculin stained double narrow lines defining the periphery of the junctional complexes but was excluded from the intervening region. In addition, the distribution of vinculin relative to pp60src within adhesion plaques suggested an inverse relationship between the presence of these two proteins. Overall, these results establish a close link between the src gene product and components of the cytoskeleton and implicate the adhesion plaques and adhesion junctions in the mechanism of Rous sarcoma virus-induced transformation.

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Organization of pp60src and selected cytoskeletal proteins within adhesion plaques and junctions of Rous sarcoma virus-transformed rat cells.

Sera of normal nonimmunized rabbits, pigs, calves, and humans contain tubulin-reactive antibodies Usually, low amounts of antibodies against tubulin of the IgG class (25-4 mg/100 ml of serum from nonimmunized animals) were isolated Anti-tubulin antibodies were also produced by injecting pig tubulin in complete Freund's adjuvant into rabbits Slightly higher amounts of anti-tubulin antibody were isolated from sera of immunized rabbits (7 mg/100 ml of serum) The cytoplasmic network of microtubules of Tcc 36 mouse cells in culture was not clearly stained by natural anti-tubulin antibodies, but dense staining of the centrosphere was observed In contrast, induced anti-tubulin antibodies densely stained cytoplasmic microtubular networks Vinblastine-induced tubulin paracrystals were equally stained by natural and induced anti-tubulin antibodies

Antibodies to tubulin in normal nonimmunized animals.

The mode of binding of 125I-labelled concanavalin A and succinyl-concanavalin A to rat thymocytes at 4 degrees C was investigated. Simultaneously, the free binding sites of the cell-bound lectin molecules were quantified by horseradish peroxidase binding. Concanavalin A showed cooperative binding while succinyl-concanavalin A did not. The number of molecules of concanavalin A bound to the cell surface when it was saturated was twice the number of molecules of succinyl-concanavalin A. We interpret these results as showing that the binding of native concanavalin A to thymocytes at 4 degrees C brings about a cooperative modification of the membrane which leads to appearance of new receptors. Divalent succinyl-concanavalin A has no such effect. Horseradish peroxidase binding to cell-bound lectin was shown to be related to the immobilization of membrane receptors; the more they are immobilized, the more receptor-associated lectin can bind horseradish peroxidase. This allowed us to establish that post-binding events, which we called micro-redistribution, occurred at 4 degrees C when either concanavalin A or succinyl-concanavalin A binds to cells. A cooperative restriction of the micromobility of cell receptors is produced by increasing concentrations of concanavalin A. Succinyl-concanavalin A does not restrict cell receptor mobility at any concentration tested. The results are discussed in terms of cell stimulation and cell agglutination.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1976.tb10389.x

Cooperative Binding of Concanavalin A to Thymocytes at 4°C and Micro‐redistribution of Concanavalin A Receptors

This paper reports on the preparation of immunsera against tubulin and actin, and the purification of anti-tubulin and anti-actin antibodies on immunoadsorbent columns. These purified antibodies were used in an indirect immunoperoxidase assay to visualize microtubules and microfilaments in various cell lines. The specificity of antibodies and the methods of cell fixation required are discussed, as well as some aspects of microtubule and microfilament organization, as visualized by this technique.

Detection of tubulin and actin in various cell lines by an immunoperoxidase technique.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2873%2980841-5

The use of concanavalin A in the study of the dynamics of lymphocyte membrane glycans.

Wheat germ agglutinin, which has been found unable to stimulate rat thymocytes, is shown to strongly inhibit DNA synthesis of normal as well as concanavalin A-stimulated cells.

E Karsenti

Papers

Antibodies to tubulin in normal nonimmunized animals.

Cooperative Binding of Concanavalin A to Thymocytes at 4°C and Micro‐redistribution of Concanavalin A Receptors

Detection of tubulin and actin in various cell lines by an immunoperoxidase technique.

The use of concanavalin A in the study of the dynamics of lymphocyte membrane glycans.

Stimulation and inhibition of DNA synthesis in rat thymocytes: action of concanavalin A and wheat germ agglutinin.