Localization of cell surface glycoproteins in membrane domains associated with the underlying filament network.

To visualize the localization of cell surface constituents in relation to the plasma membrane-associated filament network, we developed a method based on a combination of immunogold labeling and dry-cleaving. For labeling we used trinitrophenyl-derivatized ligand, anti-TNP antibodies, and protein A-coated colloidal gold. Dry-cleaving (Mesland, D. A. M., H. Spiele, and E. Roos, 1981, Exp. Cell Res., 132: 169-184) involves cleavage of lightly fixed critical point-dried cells by means of adhesive tape. Since cells cleave close to the cell surface, the remaining layer is thin enough to be examined in transmission electron microscopy. Using this method, we studied concanavalin A-binding constituents on the medium-facing surface of H35 hepatoma cells. The distribution of the gold particles, which was partly dispersed and partly patchy, coincided strikingly with membrane-associated filaments, and label was virtually absent from areas overlying openings in the filament network. In stereo pairs we observed the label to be localized to areas of somewhat enhanced electron density at the plane of the membrane. These areas were interconnected in a pattern congruent with the filament network. Preliminary observations on wheat germ agglutinin receptors on the hepatoma cells as well as concanavalin A receptors on isolated hepatocytes yielded comparable results. It thus appears that surface glycoproteins, although seemingly randomly distributed as observed in thin sections, may actually be localized to particular membrane domains associated with underlying filaments.

Regulated membrane localization of Tiam1, mediated by the NH2-terminal pleckstrin homology domain, is required for Rac-dependent membrane ruffling and C-Jun NH2-terminal kinase activation.

Rho-like GTPases, including Cdc42, Rac, and Rho, regulate signaling pathways that control actin cytoskeletal structures and transcriptional activation. The Tiam1 gene encodes an activator of Rac1, and similarly to constitutively activated (V12)Rac1, overexpression of Tiam1 in fibroblasts induces the formation of membrane ruffles. Tiam1 contains a Dbl homology (DH) domain and adjacent pleckstrin homology (PH) domain, hallmarks for activators of Rho-like GTPases. Unique for Tiam1 are an additional PH domain and a Discs-large homology region in the NH2-terminal part of the protein. Here we show that both in fibroblasts and COS cells, membrane localization of Tiam1 is required for the induction of membrane ruffling. A detailed mutational analysis, in combination with confocal laser scanning microscopy and immunoelectron microscopy, demonstrates that the NH2-terminal PH domain of Tiam1, but not the DH-adjacent PH domain, is essential for membrane association. This NH2-terminal PH domain of Tiam1 can be functionally replaced by the myristoylated membrane localization domain of c-Src, indicating that the primary function of this PH domain is to localize the protein at the membrane. After serum starvation, both membrane association of Tiam1 and ruffling can be induced by serum, suggesting that receptor stimulation induces membrane translocation of Tiam1. Similar to V12Rac1, Tiam1 stimulates the activity of the c-Jun NH2-terminal kinase (JNK). This Rac-dependent stimulation of JNK also requires membrane association of Tiam1. We conclude that the regulated membrane localization of Tiam1 through its NH2-terminal PH domain determines the activation of distinct Rac-mediated signaling pathways.

Quantitation of virus-induced (MLr) and normal (Thy.1.2) cell surface antigens in isolated plasma membranes and the extracellular ascites fluid of mouse leukemia cells.

Plasma membranes were isolated by two methods from mouse leukemia cells containing mammary tumor virus-induced (MLr) and normal (Thy.1.2) antigens on their surfaces. A number of chemical components, enzymic activities, and the antigenic contents were determined in subcellular fractions and found to be specifically concentrated in the plasma membrane fractions. The major part of the cellular MLr, in contrast to Thy.1.2, was present in the 105,000 X gmax supernatant of the cell homogenate. This and other results indicate an easy release of the antigen from the plasma membrane. A considerable amount of MLr was also present in the ascites fluid, partly free and partly bound, supposedly in an immune complex that allowed the isolation of three components of similar molecular weights as mammary tumor virus components. The extracellular presence of MLr may illustrate that, by shedding of antigen, the tumor may protect itself against the immunological defense of the host.

Serum-dependent "cannibalism" and autodestruction in cultures of human small cell carcinoma of the lung.

In cell lines of human small cell carcinoma of the lung (SCCL) and in all subclones of one of the cell lines, cells were observed which completely interiorized other cells, leading to death of the interiorized cells and sometimes to complete autodestruction of the cultures. This phenomenon, which we have called "cannibalism," is also observed in fresh tumor biopsies from SCCL patients. Cannibalistic cells appeared to be of SCCL origin. "Cannibalism" is never observed in serum-free cultures but can be reinduced by serum exposure. It is likely that "cannibalism" may contribute to the frequent failure to establish SCCL cell lines in serum-containing medium. The potential to induce autodestruction of tumor cells in SCCL patients by as yet unknown serum factor(s) may be of therapeutic value.

Lymphoid proliferations in the orbit: malignant or benign?

Clinical, pathological, and immunological analysis of 20 patients with ocular adnexal lymphoid disease has demonstrated several parameters which are useful for distinguishing malignant from benign lesions. Patients in the fourth or fifth decade of life presenting with an acute history of pain, oedema, epiphora, double vision, and ptosis, with a mass localised in the lacrimal gland area, are more likely to have a pseudolymphoma or a chronic inflammatory lesion than a true non-Hodgkin lymphoma (NHL). It is not possible to obtain a definite diagnosis without surgical intervention, because only three out of nine patients with orbital NHL had evidence of a monoclonal B cell population in peripheral blood on admission to the Orbital Centre. Furthermore it was confirmed that the identification of the various orbital lymphoid infiltrates becomes more distinct when immunological techniques are added to the clinical and histopathological methods of investigation. Multidisciplinary cooperation leads to further improvement of diagnosis and treatment of ocular adnexal lymphoproliferative disease.

Sarcoma arising from interdigitating cells. Cytology and cytochemistry.

The cytologic features of a highly malignant sarcomatous tumor in a 37-year-old male, arising from interdigitating cells and localized in the mediastinum, lymph nodes and skin, are described. Cytologically this sarcoma was characterized by large cells with ill-defined, faintly basophilic cytoplasm, monocytoid or multilobulated nuclei and a reticular chromatin structure; very prominent nucleoli were seen in some of the cells. Some of the tumor cells were spindle shaped. The ultrastructurally characteristic invaginations of the cell membrane were not obvious in the cytologic smear, although the nuclear membrane showed deep, narrow, channel-like indentations. The specific ultrastructural, immunologic and cytochemical characteristics of the interdigitating cells were recognized in the tumor cells. Adenosine triphosphatase was present in the tumor cells in large amounts, while acid phosphatase, acid alpha-naphthyl acetate esterase and other enzymes were absent. The described tumor must be considered another tumor of the mononuclear phagocyte system; the proposed name is "interdigitating-cell sarcoma."

Wet cleaving of cells: a method to introduce macromolecules into the cytoplasm. Application for immunolocalization of cytosol-exposed antigens.

A new method, called "wet cleaving," has been introduced to allow direct exposure of cytoplasm to externally supplied macromolecules by mechanical rupture of the plasma membrane. Monolayers of adherent cells or poly-L-lysine-attached suspension cells are overlayed with nitrocellulose sheets. By subsequent removal of the sheets, cells are cleaved, thereby exposing the cytoplasm. The method allows bulk quantities of cells to be cleaved in an efficient manner. Cleavage, although imposing some mechanical stress on the cells, leaves most if not all organelles morphologically intact, as shown by electron microscopy. Mechanically ruptured cells are well suited for use in immunocytochemical studies, as is demonstrated with the immunofluorescence localization of vinculin in chicken embryo fibroblasts.

Organization of talin and vinculin in adhesion plaques of wet-cleaved chicken embryo fibroblasts.

We have studied the fine structure of adhesion plaques in chicken embryo fibroblasts (CEF) and visualized the localization of vinculin and talin using immunoelectron microscopy on CEF opened by 'wet-cleaving'. This procedure, performed with nitrocellulose on cells grown on electron microscope grids, cleaved the CEF close to the inner face of the ventral membrane or at a slightly higher level through the cytoplasm. In the resulting preparations, adhesion plaques were identified by their localization at the end of microfilament bundles and by their density of vinculin and talin. The plaques showed a substructure of moderately electron-dense parallel bands that were interconnected. Both the parallel bands as well as the interconnecting threads showed a high density of vinculin and talin labels, whereas neither the surrounding membrane cytoskeleton nor the overlaying bundled microfilaments were labeled. In stereomicrographs, we observed no difference between the distances from vinculin or talin label, respectively, to the plasma membrane. In early spreading cells, vinculin and talin were found to be deposited simultaneously in fine radiating streaks that covered rather large parts of the ventral membrane at areas of close contact with the substratum. These streaks, which were initially overlayed by an isotropic cytoskeletal network without filament bundles, were the apparent precursors of later formed adhesion plaques. These observations suggest that there are no separate layers of talin and vinculin, but rather that adhesion plaques consist of a dense network of talin and vinculin. The observations strongly support the model proposed by Bendori et al. (1989), J. Cell Biol. 108, 2383-2393, that was based on the location of vinculin- and talin-binding sites in the vinculin molecule.

Junction formation between cultured normal rat hepatocytes. An ultrastructural study on the presence of cholesterol and the structure of developing tight-junction strands.

In cultures of normal rat hepatocytes, isolated by collagenase perfusion, formation of junctions started 4-7 h after seeding. The junctional complexes were localized at two preferential sites of the contiguous membrane: apically and on both sides of open spaces with microvilli at some distance from the upper surface. The domains of early contact formation are characterized by a flattening of the membranes, a decrease in intercellular space, a depletion of intramembrane particles (IMPs) and a concentration of electron-dense and of fine fibrillar material in the cytoplasm immediately adjacent to the membrane. We were able to demonstrate the presence of cholesterol in these domains by formation of cholesterol-filipin complexes that deformed the membrane. However, membrane-deformation in these domains was often inhibited. This inhibition appeared to be due to the presence of pepsin-sensitive material, since full deformation was induced after mild proteolysis. The arrangement of IMPs into small gap junctions and short tight-junction strands is synchronous in the membranes of the neighbouring cells. The tight junctions are composed of two strands arranged in a slightly offset configuration. The possibility that the strands are lipidic in nature is not excluded.

Interaction of plasma membrane-associated filaments and H-2 histocompatibility antigens before and after induced patching and capping.

The interaction of H-2 antigens and plasma membrane-associated filaments was studied on dry-cleaved preparations of immunogold-labelled lymphoma cells. In prefixed cells, the plasma membrane-associated network was isotropic without any prevailing direction of the filaments, and the gold-labelled H-2 antigens were preferentially localized over or at a very short distance from membrane-associated filaments. Incubation of unfixed cells with anti-H-2 antibodies followed by fixation and incubation with anti-Ig, did not induce detectable redistribution of H-2 antigens or of the filament network. Notwithstanding this apparent absence of rearrangement of H-2 antigens and filaments, a detergent-resistant linkage to the cytoskeleton was induced. Before immune incubations, virtually all H-2 antigens were solubilized by extraction with Triton X-100, while after incubation with anti-H-2 antibodies about 50% of the H-2 antigens were linked to the Triton X-100-insoluble cytoskeleton. Sequential addition of anti-H-2 and anti-Ig antibodies to unfixed cells induced formation of patches and caps of H-2 antigens. Under these conditions, the majority of the H-2 antigens became linked to the detergent-resistant cytoskeleton. Redistribution into patches and caps was often accompanied by a local rearrangement of the isotropic network into bundles of parallel filaments immediately adjacent to the plasma membrane. Patches were seen to overly both isotropic networks and these parallel filaments. Large sheets of plasma membrane overlying parallel filaments were frequently devoid of gold-labelled H-2 antigens and coated pits, and thus most probably represented areas away from caps. This observation suggests that capping is accompanied by a rearrangement of filaments close to the membrane.

A malignant tumor arising from interdigitating cells; light microscopical, ultrastructural, immuno-and enzyme-histochemical characteristics.

A tumor in a 37 years old male is described in which the tumor cells appeared to be derived from interdigitating cells normally found in the T-cell area of lymph nodes. The patient presented with superior vena caval obstruction due to a mediastinal mass, followed by lymph node enlargement and skin lesions leading to death within 4 months. The tumor cells lacked immune markers for lymphocytic cells. They showed Ia-like antigens and high adenosine triphosphatase activity, while acid phosphatase and alpha-naphthyl acetate esterase activity was absent. Their fine morphology was strikingly similar to that of interdigitating cells. A combination of these data led us to the conclusion that this tumor represents a specific subtype of the tumors derived from the mononuclear phagocyte system, namely a sarcoma of interdigitating cells.

Immuno-EM localization of the beta 1 integrin subunit in wet-cleaved fibronectin-adherent fibroblasts.

Using immuno-EM, we have studied the distribution of the beta 1 integrin subunit in chicken embryo fibroblasts allowed to adhere and spread for 3 hours on a fibronectin-coated surface in serum-free medium. The cells were wet-cleaved, which removed most of the cell body, yielding ventral plasma membranes with little, and sometimes virtually no, associated cytoskeleton. The beta 1 integrin subunit was detected with antibodies against the cytoplasmic domain. In immune fluorescence, it colocalized with adhesion plaques, in a punctate staining pattern, and often seemed to be at the periphery of the plaque. By immuno-EM, beta 1 was in fact found in discrete clusters, not throughout the plaque. In deep-cleaved cells from which virtually all cytoskeleton was removed, clusters could often be seen to be located on fibronectin fibrils. Furthermore, beta 1 was present in clusters at the cell margins, and isolated or in small groups at the very edge of the cell. When fibronectin synthesis, and consequently fibril formation, was inhibited by cycloheximide, large adhesion plaque-like structures were formed at the cell margin. This phenotype was reversed by addition of soluble fibronectin, which was incorporated into fibrils. As in normal plaques, talin and vinculin were present, the plasma membrane was very close (10-20 nm) to the substratum and the fibronectin layer underneath was removed. These plaques did contain beta 1 integrins but they were not in clusters. These observations indicate that the talin-vinculin network of an adhesion plaque is normally anchored to the substratum at discrete beta 1 integrin clusters that may be located on fibronectin fibrils, and that elsewhere the plaque is not necessarily attached to the substratum by interaction of integrins with matrix proteins. In the absence of fibronectin fibrils, adhesion plaque-like structures can be formed, but these are aberrant in size, location and fine structure.

Rigid plasma-membrane-derived vesicles, enriched in tumour-associated surface antigens (MLr), occurring in the ascites fluid of a murine leukaemia (GRSL).

Extracellular membraneous vesicles of GRSL leukaemia cells were isolated from the ascites fluid bathing the cells in vivo, and from cell washes. Mammary tumour virus-induced antigens (MLr) expressed on the surface of the cells are enriched on these vesicles as compared to plasma membranes isolated from the cell homogenate. The lipid fluidity of the vesicles is much smaller than that of the plasma membranes, and the content of the pertinent lipid parameters, cholesterol and sphingomyelin, are accordingly greatly increased. The extracellular vesicles which are also enriched in sialic acid and 5'-nucleotidase are apparently derived from the plasma membrane, probably at least partly by exfoliation of selected parts or domains of the surface of living cells. An analogy between this shedding of vesicles, the formation of endocytotic vesicles and the budding of viruses is noted; all these processes select or assemble rigid lipid domains of the cell membrane. The possible participation of surface microvilli and sub-lethal autolysis in the process of shedding is discussed.

Sural nerve immune deposits in polyneuropathy as a remote effect of malignancy.

Sural nerves from six patients with polyneuropathy as a remote accompaniment of malignancy were studied by light microscopy, immunofluorescence, electron microscopy, and immuno-electron microscopy. Sural nerves also were studied in twelve control patients with malignant diseases without peripheral neuropathy and in fifteen with neuropathy associated with nonmalignant conditions. Only nerves from the first group of six patients showed immune deposits of immunoglobulin M, C3, and C1q as granular or confluent layers in the inner laminae of the perineurium as well as in and around endoneural capillaries. The findings are similar to those in published studies of Waldenström's macroglobulinemia, chronic relapsing polyneuropathy, and neuropathy of monoclonal gammopathy and extend the list of conditions to include several forms of carcinoma and Hodgkin's disease.