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C A Feltkamp

Publications and source records attributed to C A Feltkamp.

At least 19 recordsLinked to original sources
PubMed1997

v-src-induced cell shape changes in rat fibroblasts require new gene transcription and precede loss of focal adhesions.

The mechanism of v-src-induced morphological transformation is still obscure. We compared LA29 rat fibroblasts, which express a temperature-sensitive (ts) v-src mutant, with D1025 rat fibroblasts, transfected with a ts mutant of v-fps. Upon transformation, LA29 cells adopted an elongated shape with reduced focal adhesions and loss of actin stress fibers. In contrast, activation of v-fps in D1025 cells had little effect on morphology. In both cells, paxillin was strongly tyrosine phosphorylated upon activation of the kinases. This indicates that paxillin phosphorylation is not required, or not sufficient, for the v-src-induced disruption of focal adhesions. As previously described by others, v-src activated the ras-MAP kinase (MAPK) pathway, as indicated by tyrosine phosphorylation of the rasGAP-associated proteins p62 and p190 and MAPK phosphorylation. Since MAPK affects transcription, this suggested that novel gene transcription was required. This notion was confirmed using actinomycin D and cycloheximide, which did not impair activation of v-src kinase activity, but completely blocked v-src-induced morphological changes, as demonstrated using image analysis. Furthermore, we observed that v-src-induced changes in cell shape occurred before the reduction in number and size of focal adhesions. We conclude that v-src-induced transformation of rat fibroblasts depends on synthesis of a protein, which induces rapid changes in cell shape that precede the loss of focal adhesions.

Actin Cytoskeleton↗
PubMed1997

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.

3T3 Cells↗
PubMed1995

Functional expression of adhesion receptors and costimulatory molecules by fresh and immortalized B-cell non-Hodgkin's lymphoma cells.

Peripheral blood lymphocytes of a patient with follicular B-cell non-Hodgkin's lymphoma (B-NHL) were immortalized in vitro by Epstein-Barr virus (EBV). Eight cell lines were obtained (termed BNS1, BNS2-1 through BNS2-7), which showed a pattern of idiotypic (id) Ig surface expression and Ig JH and kappa gene rearrangement, identical to that of the parent cells (termed NS), confirming their neoplastic origin. Induction of allogeneic T-cell proliferation by NS cells was mediated by HLA-DR, leukocyte function-associated antigen-1 (LFA-1), LFA-3, B7-1/CD80, and CTLA4 and resulted in the upregulation (LFA-3, intercellular adhesion molecule-1 [ICAM-1], CD40) and induction (B7-1/CD80, B7-2/CD86, L16/activated LFA-1) of accessory molecules on NS cells. In turn, responder T lymphocytes were induced to express B7-1/CD80, B7-2/CD86, CD40 ligand (CD40L), ICAM-1, L16/activated LFA-1, and HLA-DR, reflecting bidirectional signaling between T lymphocytes and B-NHL cells. Preactivation of NS cells by EBV transformation or CD40 engagement resulted in enhanced expression of accessory molecules and abolished the requirement for accessory cells during allostimulation. These resting and activated clonal B cells will be useful in further dissecting the requirements for B-NHL costimulation.

Antigens, CD↗
PubMed1994

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.

Animals↗
PubMed1994

LFA-1 integrin redistribution during T-cell hybridoma invasion of hepatocyte cultures and manganese-induced adhesion to ICAM-1.

We have reported previously that the integrin LFA-1 is essential for metastasis of T-cell hybridomas to the liver. We show here that hepatocytes isolated from normal non-inflamed rat liver express intercellular adhesion molecule-1 (ICAM-1) at the dorsal surface and more prominently at the lateral and substratum-adherent surfaces. Anti-rat ICAM-1 mAb inhibited adhesion of TAM8C4 T-cell hybridoma cells to hepatocytes. Invasion between hepatocytes was not affected, but this is probably due to lack of penetration of the mAb between the hepatocytes. In all hepatocyte-adherent TAM8C4 cells, LFA-1 was concentrated at the adhesion site. Redistribution of ICAM-1 to the interacting hepatocyte membrane was also seen, but only for part of the adherent TAM8C4 cells. LFA-1 was highly concentrated on pseudopods of invading TAM8C4 cells inserted between hepatocytes, and on the upper surface of invaded TAM8C4 cells located under the hepatocytes. ICAM-1 was concentrated in the hepatocyte membrane overlying TAM8C4 cells located underneath the monolayer. These results suggests that ICAM-1 is of major importance for liver invasion by these lymphoma cells. For optimal adhesion to ICAM-1, LFA-1 on T-cell hybridomas requires activation, which apparently occurs upon contact with cell layers that are invaded (G. La Rivière et al., J. Cell Sci. 107, 551-559, 1994). LFA-1 can be activated artificially by Mn2+. To study LFA-1 redistribution upon ICAM-1 interaction with higher resolution, we performed immuno-EM on cells before and after Mn(2+)-induced adhesion and spreading on immobilized ICAM-1. By immune fluorescence, LFA-1 was observed to redistribute to the ICAM-1-adherent surface, and to be concentrated in lamellipodia of spreading TAM8C4 cells. By immuno-EM, LFA-1 was localized in microclusters of approximately 10 gold particles. This was seen in cells fixed in suspension, and the size of these clusters did not change upon adhesion to ICAM-1. LFA-1 was present at high density in thin filopodia, but again in microclusters of similar size. Comparable results were obtained with a cytotoxic T-cell clone. We conclude that Mn(2+)-induced activation of LFA-1 is not associated with the formation or enlargement of LFA-1 clusters.

Animals↗
PubMed1991

Characterization of a human plasmacytoma line.

The TH line was established by bringing tumour cells from a multiple myeloma patient into suspension culture and subsequently cloning them by limiting dilution. The cultured cells show marked heterogeneity; there are ultrastructural differences between small and large TH cells, particularly with respect to the rough endoplasmatic reticulum (RER). Karyotyping revealed chromosome numbers in the triploid range, with many structural abnormalities, at the 14q32 region among others. A t(14;18) could not be demonstrated. TH was shown to have germline and a rearranged allele for kappa light chain, and only a single rearranged gene for heavy chain immunoglobulin. TH expressed PCA-1, CD9, CD28 and CD38 antigens, HLA class II, RER and kappa light chain, but few or no other antigens associated with the B-cell lineage. Light chain kappa and trace amounts of IgG3 were found intracellularly as well as in culture supernatant. The addition of IL-6 to cultures of TH increased proliferation, as well as the secretion of kappa light chain and the membrane expression of CD28 and CD38 antigens. Because TH has relatively few B cell markers on its membrane, it may be useful for the induction of monoclonal antibodies specific for human plasma cells. It also provides a model for the demonstration that IL-6 can act as a paracrine growth and differentiation factor for cells of myelomal origin.

Adult↗
PubMed1991

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.

Animals↗
PubMed1990

Disintegration of adhesion plaques in chicken embryo fibroblasts upon Rous sarcoma virus-induced transformation: different dissociation rates for talin and vinculin.

The localization of talin and vinculin in chicken embryo fibroblasts (CEF) during transformation was studied by immunoelectron microscopy. CEF cells were infected with a temperature-sensitive mutant of Rous sarcoma virus. After 16 h at 42 degrees C, transformation was induced by incubation at 37 degrees C for different intervals up to 3 h. Cells were cleaved by "wet cleaving" as reported previously by us (R. Brands and C.A. Feltkamp, 1988, Exp. Cell Res. 176, 309) and labeled with affinity-purified polyclonal antibodies to talin or vinculin, or monoclonal anti-vinculin. We observed a rapid reduction of vinculin in adhesion plaques within 15 min and a much slower dissociation of talin. This was found using single-labeling procedures and also within the same cell using double labeling. Seemingly intact microfilament bundles were observed associated with adhesion plaques that contained relatively little vinculin. These observations show that an early event in src-induced transformation is the release of vinculin from adhesion plaques. Furthermore, since adhesion plaques with attached filament bundles can exist at least transiently with very little or no vinculin present, it seems likely that vinculin is not, or not the only protein, linking actin filaments to adhesion plaques.

Animals↗
PubMed1988

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.

Animals↗
PubMed1987

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.

Animals↗
PubMed1985

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.

Actin Cytoskeleton↗
PubMed1984

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.

Adult↗
PubMed1984

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.

Antigens, Surface↗
PubMed1983

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.

Adult↗
PubMed1983

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.

Acetone↗
PubMed1982

T cell leukemia presenting as chronic polyarthritis.

T cell leukemia was detected in a woman who suffered from chronic polyarthritis. The peripheral blood leukocytes were increased in number and consisted of lymphocytes, 95% of which could be identified as T lymphocytes. T cell infiltration was found in the bone marrow, the synovial fluid, and tissue, and in nodules macroscopically resembling rheumatoid skin lesions. Further investigation of these cells by enzyme chemistry, immunohistochemistry, electron microscopy, and cytochemistry revealed that they had irregularly indented nuclei, no alpha-naphthyl acetate esterase activity, and only faint granular acid-phosphatase activity. The cells were negative for Ia-like antigen and surface immunoglobulin. Analysis of the cell surface glycopeptides showed the presence of abnormally enlarged carbohydrate structures. These data suggest that these leukemic T cells are a malignant equivalent of immature T cells.

Adult↗