Biomedical subjects

B Geiger

Publications and source records attributed to B Geiger.

At least 55 records · Page 3Linked to original sources

PubMed1987

Formation of heterotypic adherens-type junctions between L-CAM-containing liver cells and A-CAM-containing lens cells.

Cultured cells from either chicken lens or liver plated on solid substrates form flat epithelial sheets with adherens-type junctions between them. In lens cells these junctions contain A-CAM, while the same type of intercellular junctions in liver cells contain another cell adhesion molecule, L-CAM. Coculturing of lens and liver cells in the same dish resulted in the formation of mixed (heterotypic) adherens junctions. Double immunofluorescent labeling for both A-CAM and L-CAM indicated that the mixed junctions contained both molecules, each of which was present on one of the two partner cells. Moreover, the formation of the heterotypic junctions could be effectively inhibited by both anti-A-CAM and anti-L-CAM antibodies. It has thus been proposed that A-CAM and L-CAM share significant functional homology and may be involved in heterophilic interactions leading to the establishment of molecularly and cellularly asymmetrical adherens-type junctions.

Animals↗

PubMed1987

Contact-dependent regulation of vinculin expression in cultured fibroblasts: a study with vinculin-specific cDNA probes.

Vinculin specific cDNA clones were isolated from chicken embryo fibroblast (CEF) cDNA library in lambda gt11. The clones, ranging in size from 2.8 to 5.0 kb, were initially selected by rabbit antibodies to vinculin. Their identity was further confirmed by their specific reactivities with a battery of different vinculin-specific monoclonal antibodies. Southern blot analysis of restriction enzyme digested chicken spleen DNA suggested that all the isolated cDNA clones correspond to the same gene(s). Northern blot hybridization revealed that the vinculin-specific cDNA clones react with a single 6.5 kb mRNA in total cellular RNA preparations of CEF, whole chicken embryos and chicken gizzard smooth muscle. Moreover, fractionation of CEF poly(A)+ RNA by sucrose gradient centrifugation followed by translation in cell free system indicated that the mRNA coding for vinculin has a size of about 6.0-7.0 kb. The identity of these clones was finally confirmed by selection hybridization assay. The isolated vinculin-specific cDNA probes were subsequently used in order to study the effect of substrate adhesiveness on the expression of vinculin. We show here that cells cultured on highly adhesive substrate, such as endothelial extracellular matrix (ECM), form large vinculin-rich focal contacts, while cells grown on poorly adhesive substrate poly(2-hydroxyethyl methacrylate) [poly(HEMA)] contain only small distorted vinculin spots. These morphological differences were accompanied by over 5-fold reduction in vinculin synthesis in cells growing on poly(HEMA), compared to those cultured on the ECM and over 7.5-fold decrease in the levels of vinculin-specific mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Cytokeratin polypeptides expression in different epithelial elements of human salivary glands.

Immunofluorescent labeling of human salivary glands was carried out with a battery of monoclonal antibodies reactive with specific cytokeratin polypeptides. All the epithelial elements of the glands were positively labelled by a broad-spectrum cytokeratin antibody (KG 8.13) and by antibody Ks 18.18, which reacts with cytokeratin No. 18 exclusively. Labelling of frozen sections with antibody KM 4.62, which is reactive with the 40 Kd (No. 19) cytokeratin, was confined to the ductal system and apparently absent from the acini. Antibody KA-1, reactive with polypeptides 4, 5 and 6 stained both the myoepithelial cells and the basal cells of the large ducts. Antibody KS 8.58, however, reacted with the basal cells exclusively. It is thus proposed that the combined use of the various monoclonal antibodies may provide a most useful probe in studies on epithelial cell diversity in normal salivary glands as well as in pathological disorders of that gland.

Child↗

PubMed1987

The relationship between intermediate filaments and microfilaments before and during the formation of desmosomes and adherens-type junctions in mouse epidermal keratinocytes.

Actin, keratin, vinculin and desmoplakin organization were studied in primary mouse keratinocytes before and during Ca2+-induced cell contact formation. Double-label fluorescence shows that in cells cultured in low Ca2+ medium, keratin-containing intermediate filament bundles (IFB) and desmoplakin-containing spots are both concentrated towards the cell center in a region bounded by a series of concentric microfilament bundles (MFB). Within 5-30 min after raising Ca2+ levels, a discontinuous actin/vinculin-rich, submembranous zone of fluorescence appears at cell-cell interfaces. This zone is usually associated with short, perpendicular MFB, which become wider and longer with time. Later, IFB and the desmoplakin spots are seen aligned along the perpendicular MFB as they become redistributed to cell-cell interfaces where desmosomes form. Ultrastructural analysis confirms that before the Ca2+ switch, IFB and desmosomal components are found predominantly within the perimeter defined by the outermost of the concentric MFB. Individual IF often splay out, becoming interwoven into these MFB in the region of cell-substrate contact. In the first 30 min after the Ca2+ switch, areas of submembranous dense material (identified as adherens junctions), which are associated with the perpendicular MFB, can be seen at newly formed cell-cell contact sites. By 1-2 h, IFB-desmosomal component complexes are aligned with the perpendicular MFB as the complexes become redistributed to cell-cell interfaces. Cytochalasin D treatment causes the redistribution of actin into numerous patches; keratin-containing IFB undergo a concomitant redistribution, forming foci that coincide with the actin-containing aggregates. These results are consistent with an IF-MF association before and during desmosome formation in the primary mouse epidermal keratinocyte culture system, and with the temporal and spatial coordination of desmosome and adherens junction formation.

Actin Cytoskeleton↗

PubMed1987

Modulation of intermediate-filament expression in developing cochlear epithelium.

The present study was designed to characterize the expression and distribution of intermediate filaments (IFs) in the diverse cellular elements of inner-ear epithelium in guinea pig and man. Using immunofluorescence microscopy with a battery of IF-specific monoclonal antibodies, we show that the epithelium of the otocyst expresses cytokeratin (CK) polypeptides typical of simple epithelia. Cells in the early otic ganglion were also positively labelled for cytokeratins, suggesting that they are of otocystic epithelial origin. Cytokeratin distribution was largely homogeneous in the early cochlear duct as the epithelium differentiated, differences in the distribution of cytokeratin between the various cell types became detectable. Characteristically, cochlear hair cells became devoid of cytokeratin labelling, and remained unlabelled with antibodies specific for all other IF classes. The neural tissue of the inner ear was also devoid of cytokeratins and was typically positive for neurofilaments. Vimentin IFs were abundant in the mesenchymal tissues around the membranous labyrinth. Desmin and glial fibrillary acidic protein were not detectable in the cochlea. The apparent absence of all IFs from the cochlear hair cells in both guinea pig and man, as revealed by immunofluorescence and electron microscopy, and the possible significance of their absence for cochlear physiology, are discussed.

Animals↗

PubMed1987

Cytokeratin polypeptide expression during the histogenesis of guinea pig submandibular salivary gland.

The present study was directed towards the characterization of cell-specific histogenetic markers for the various epithelial elements of the adult and the developing guinea pig submandibular salivary gland. We have employed immunofluorescent labelling using three cytokeratin monoclonal antibodies, for which the polypeptide specificities towards guinea pig cytokeratins were determined. All the epithelial elements of the adult gland were positively labelled with two monoclonal antibodies, namely KG 8.13 ('broad spectrum' anti-cytokeratin) and antibody Ks B.18 (reactive with a simple cytokeratin-specific polypeptide of 49 X 10(3) Mr). Antibody KS 8.58 (reactive with a guinea pig cytokeratin polypeptide of 50 X 10(3) Mr) labelled the basal cells of the large ducts, as well as the myoepithelium. During development of the gland, the submandibular anlage and its primary and secondary branches with their terminal buds, were uniformly labelled with the three antibodies; however, the cytokeratin polypeptides reactive with antibody KS 8.58, which were apparently expressed in all cells of the developing ducts, gradually disappear from most of the ductal cells, starting at about 6 weeks of gestation, and remain only in the basal or reserve cells of the large ducts and the myoepithelium. These observations support the notion that the basal cells retain at least some of the properties of the embryonic glandular epithelium and could be considered as pluripotent reserve cells which may function as progenitors for other epithelial elements in the salivary glands epithelia.

Animals↗

PubMed1987

Molecular interactions in adherens-type contacts.

Adherens junctions are members of a molecularly and structurally heterogeneous family of cell contacts sharing a common association with the microfilament system. Various topics related to the biogenesis of these cellular contacts and the molecular interactions involved in their formation are discussed. The role of vinculin, a cytoplasmic 'plaque' component present in all adherens junctions tested to date and its possible interactions with the other junctional domains have been investigated by both biochemical analyses and studies of molecular dynamics in microinjected living cells. The importance of A-CAM, which apparently functions as a 'junctional receptor' is emphasized and its roles in junction formation in cell cultures and in developing embryos are discussed. In addition, its relationship to other Ca2+-dependent cell adhesion molecules (in particular L-CAM) is considered. The evidence indicating that the level of expression of vinculin-specific mRNA is affected by culture conditions and may be markedly modulated by changes in the adhesiveness of the substratum on which the cells grow is reviewed.

Animals↗

PubMed1987

Fibrosarcoma of the urinary bladder. Study of a case showing extensive chondroid differentiation.

A case of primary fibrosarcoma of the urinary bladder showing extensive chondroid differentiation was studied by light microscopy and immunofluorescent microscopy using tissue-specific antibodies against intermediate filaments. The tumor cells were uniformly and positively labeled with vimentin antibodies and were negative for desmin and keratin, thus confirming the nonmuscle mesenchymal origin of the neoplasm. The value of intermediate filament typing in the differential diagnosis of spindle cell tumors of the urinary bladder is discussed, and a review of the literature on the subject is presented. It is postulated that the retained capacity for continued differentiation displayed by this tumor may account for the relatively better prognosis observed for this patient.

Aged↗

PubMed1986

Sclerosing hemangioma of the lung. Immunohistochemical demonstration of mesenchymal origin using antibodies to tissue-specific intermediate filaments.

A case of pulmonary sclerosing hemangioma of the lung was studied by light microscopy and indirect immunofluorescence using tissue-specific antibodies against intermediate filament subunits. All the tumor cells stained positively and exclusively with antivimentin antibodies thus indicating their mesenchymal origin. In addition, positive staining with cytokeratin antibodies was observed in cells lining cystic spaces and elongated slit-like spaces were occasionally encountered throughout the tumor, disclosing residual epithelial elements. Using brightfield microscopy, the keratin-positive areas were identified as distorted alveolar spaces lined by hyperplastic respiratory epithelium entrapped within the tumor. It is proposed that these entrapped epithelial elements may account for the conflicting results obtained by different investigators in previous attempts to determine the histogenesis of this tumor.

Antibodies↗

PubMed1986

Antigenic interrelationship between the 40-kilodalton cytokeratin polypeptide and desmoplakins.

We describe here antigenic cross-reactivity between the human 40-kilodalton cytokeratin polypeptide [Moll et al] and components of bovine desmosomal plaque, namely desmoplakins I and II. This relationship was revealed by an antibody (KM 4.62), raised against cytoskeletal preparation of cultured human breast adenocarcinoma cells (MCF-7) and selected by immunoblotting and immunofluorescent labeling. In cultured human cells that contain the 40-kD cytokeratin, antibody KM 4.62 labeled arrays of filaments throughout the cytoplasm. This antibody labeled the basal layer of nonkeratinizing squamous epithelia as well as various simple (normal and malignant) epithelia and epithelial elements of the thymus. In liver tissue, labeling was obtained only in bile ducts and canaliculi but not in the hepatocytes. In bovine cells and tissues, on the other hand, immunofluorescent labeling with antibody KM 4.62 was strictly confined to desmosomes. This was verified by double immunolabeling with both antibody KM 4.62 and specific cytokeratin or desmosomal antibodies. Immunoblotting analysis indicated that the former antibody reacts specifically with the high molecular weight components of the bovine desmosomal plaque, namely desmoplakins I and II. These immunochemical results suggest that bovine desmoplakins share same structural relationship with the human acidic, 40-kD cytokeratin.

Animals↗

PubMed1986

Interaction of fibronectin-coated beads with attached and spread fibroblasts. Binding, phagocytosis, and cytoskeletal reorganization.

After 15 min incubations, binding of 0.8-, 6-, and 16-microns fibronectin-coated latex beads occurred primarily at the margins of chick embryo fibroblasts that previously were attached and spread on fibronectin-coated glass coverslips. Extensive phagocytosis of the smallest beads and some phagocytosis of the larger beads occurred within 2 h. Following binding of the 16-micron beads, there were no changes in overall cell shape or in the distribution of several cytoskeletal proteins. There was, however, a local accumulation of actin and alpha-actinin patches adjacent to the sites where the beads were bound. The formation of alpha-actinin patches could be detected with 6- or 16-microns beads shortly after initial bead binding to the cells, but a similar reorganization of alpha-actinin in response to the binding of 0.8-micron beads was not detected. The patches of alpha-actinin appeared to be associated with membrane ruffles, since such structures were observed by scanning electron microscopy (SEM) to be sites of cell interaction with 6- but not 0.8-micron beads. Also, two other cytoskeletal proteins normally absent from membrane ruffles, tropomyosin and vinculin, were not detected at the sites of cell-bead interaction. No reorganization of vinculin at the cell-bead interaction sites was observed even when the 16-microns beads remained bound at the cell surfaces for up to 6 h. Nevertheless, prominent vinculin plaques were observed at the marginal attachment sites on the ventral cell surfaces. Consequently, formation of mature focal adhesions may be restricted to linear regions of cell-substratum interaction.

Actinin↗

PubMed1986

Changes in membrane-microfilament interaction in intercellular adherens junctions upon removal of extracellular Ca2+ ions.

EGTA-induced depletion of Ca2+ ions from the culture medium of Madin-Darby bovine kidney epithelial cells results in rapid splitting of adherens-type junctions and the detachment of the vinculin- and actin-containing filament bundle from the cytoplasmic faces of the plasma membrane of the zonula adhaerens. This process was monitored by phase-contrast microscopy, combined with electron microscopy and immunofluorescent localization of the two proteins. It is shown that shortly after extracellular free Ca2+ concentration is lowered to the micromolar range, the actin-containing, junction-associated belt of microfilaments, together with the vinculin-rich junctional plaque material, is irreversibly detached as one structural unit from the plasma membrane, contracts, and is displaced towards the perinuclear cytoplasm where it gradually disintegrates. Other actin- and vinculin-containing structures present in the same cells, notably the focal contacts at the substratum, are not similarly affected by the Ca2+ depletion and retain both the adhesion to the external surface and the association with the plaque and microfilament components. Electron microscopic examination has shown that the membrane domain of the zonulae adhaerentes, unlike that of desmosomes, is not endocytosed after Ca2+ removal and that the displaced actin- and vinculin-containing plaque and filament belt are not associated with a particular membrane. It is further shown that upon restoration of normal Ca2+ levels in the culture medium, new intercellular contacts are established gradually by accretion of both vinculin and actin into new belt-like plaque- and microfilament-containing structures.

Actin Cytoskeleton↗

PubMed1986

A-CAM: a 135-kD receptor of intercellular adherens junctions. I. Immunoelectron microscopic localization and biochemical studies.

The recently described adherens junction-specific 135-kD protein (Volk, T., and B. Geiger, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 3:2249-2260) was localized along cardiac muscle intercalated discs by immunogold labeling of ultrathin frozen sections. Analysis of this labeling indicated that the 135-kD protein, adherens junction-specific cell adhesion molecule (A-CAM), is tightly associated with the plasma membrane unlike vinculin labeling, which was present along the membrane-bound plaques of the fascia adherens. In cultured chick lens cells, A-CAM was associated with Ca2+-dependent junctions that were cleaved upon a decrease of extracellular Ca2+ concentrations to less than or equal to 0.5 mM. In the chelator-separated junction, A-CAM became exposed to exogenously added antibodies or to proteolytic enzymes. Upon addition of trypsin to EGTA-treated cells, A-CAM was cleaved into three major cell-bound antigenic peptides with apparent molecular masses of 78, 60, and 46 kD, suggesting that the extracellular domain of A-CAM has a size greater than or equal to kD. Incubation of electrophoretic gels with 125I-concanavalin A (Con A) indicated that one of the major Con A-binding proteins in chicken lens membranes is a integral of 135-kD glycoprotein that was partially purified on Con A-Sepharose column and identified as A-CAM by immunoblotting. Detergent partitioning assay using Triton X-114 biphasic system was carried out to determine whether A-CAM displays properties of an integral membrane protein. This assay indicated that the intact A-CAM molecule was recovered in the buffer phase but its cell-associated tryptic peptides, which presumably lost a great part of the A-CAM extracellular extension, readily partitioned into the detergent phase. The results obtained in this and in the following paper (Volk, T., and B. Geiger, 1986, J. Cell Biol., 103:1451-1464) strongly suggest that A-CAM is a Ca2+-dependent adherens junction-specific membrane glycoprotein that is involved in intercellular adhesion in these sites.

Animals↗

PubMed1986

A-CAM: a 135-kD receptor of intercellular adherens junctions. II. Antibody-mediated modulation of junction formation.

Intercellular adherens junctions between cultured lens epithelial cells are highly Ca2+-dependent and are readily dissociated upon chelation of extracellular Ca2+ ions. Addition of Ca2+ to EGTA-treated cells results in the recovery of cell-cell junctions including the reorganization of adherens junction-specific cell adhesion molecule (A-CAM), vinculin, and actin (Volk, T., and B. Geiger, 1986, J. Cell Biol., 103:000-000). Incubation of cells during the recovery phase with Fab' fragments of anti-A-CAM specifically inhibited the re-formation of cell-cell adherens junctions. This inhibition was accompanied by remarkable changes in microfilament organization manifested by an apparent deterioration of stress fibers and the appearance of fragmented actin bundles throughout the cytoplasm. Incubation of EGTA-dissociated cells with intact divalent anti-A-CAM antibodies in normal medium had no apparent inhibitory effect on junction formation and did not affect the assembly of actin microfilament bundles. Moreover, adherens junctions formed in the presence of the divalent antibodies became essentially Ca2+-independent, suggesting that cell-cell adhesion between them was primarily mediated by the antibodies. These studies suggest that A-CAM participates in intercellular adhesion in adherens-type junctions and point to its involvement in microfilament bundle assembly.

Actin Cytoskeleton↗

PubMed1986

Monoclonal antibodies to various acidic (type I) cytokeratins of stratified epithelia. Selective markers for stratification and squamous cell carcinomas.

We determined the reactivity of two monoclonal antibodies to cytokeratins that are typically expressed in certain stratified epithelia and several human squamous cell carcinomas using immunoblotting techniques and immunofluorescence microscopy. Antibody KS 8.12 reacted specifically with cytokeratin polypeptides nos. 13 and 16, and stained noncornified squamous epithelia in a rather uniform way. The examination of diverse human carcinomas showed all squamous cell carcinomas to be positively stained with this antibody, whereas all adenocarcinomas were negative. Another antibody, KK 8.60, reacted with polypeptides nos. 10 and 11, and uniformly stained the suprabasal layers of the epidermis. In several noncornified squamous epithelia (e.g., tongue, exocervix), in thymus reticulum epithelial cells, and in moderately and well differentiated squamous cell carcinomas this antibody exhibited a nonuniform labeling pattern that allowed the detection of individual cytokeratin-10/11-positive cells scattered throughout the tissue. It is concluded that antibodies KS 8.12 and KK 8.60 represent specific molecular probes for the definition of certain stages of squamous differentiation in normal development as well as in pathological processes such as squamous metaplasia and carcinogenesis. We propose the use of these antibodies in the differential diagnosis of carcinomas and their metastases.

Adenocarcinoma↗

PubMed1986

Cytokeratin expression in squamous metaplasia of the human uterine cervix.

The expression of cytokeratin polypeptides in squamous metaplasia of the human uterine cervix was investigated by immunocytochemical labeling with polypeptide-specific antibodies against cytokeratins. Immunofluorescence microscopic examination of cervical tissues using various monoclonal antibodies indicated that squamous cervical metaplasia expresses a unique set of cytokeratin polypeptides, this being distinctively different from that expressed by all of the normal epithelial elements of the exo- and endocervix. The development of metaplastic foci was accompanied by the expression of cytokeratin polypeptide no. 13, which is commonly detected in stratified epithelia, and by a reduction in the level of polypeptide no. 18, which is typical of simple epithelia. The 40-kilodalton cytokeratin (no. 19) described by Moll et al., which is abundant in the columnar and reserve cells of the endocervix, was found throughout the metaplastic lesions. Only in 'well-differentiated' metaplasias did we detect polarity of cytokeratin expression reminiscent of the staining patterns in the exocervix. This was manifested by the exclusive labeling of the basal cell layer(s) with antibodies KB 8.37 and KM 4.62, which stain the basal cells of the exocervix. Furthermore, a comparison of cervical metaplasia with squamous areas occurring within endometrial adenocarcinomas pointed to a close similarity in the cytokeratin expression of the two. We discuss the use of cytokeratins as specific markers of squamous differentiation, the relationships between squamous metaplasia and cervical neoplasia, and the involvement of reserve cells in the metaplastic process.

Adult↗

PubMed1986

Spatial and temporal relationships between vinculin and talin in the developing chicken gizzard smooth muscle.

The spatiotemporal relationships between vinculin and talin in developing chicken gizzard smooth muscle were investigated. Immunofluorescence and immunoelectron-microscopic labeling revealed that both proteins are associated with membrane-bound dense plaques in muscle cells; however, the most intense labeling for vinculin was located rather closer to the membrane than that for talin. The localization of vinculin and talin in embryonic chicken gizzards indicated that both are primarily cytoplasmic during the first 2 embryonic weeks. Only around days 16-18 does talin apparently become associated with the plasma membrane, this being concomitant with the appearance of distinct myofilament-bound dense plaques. Vinculin, on the other hand, remains primarily cytoplasmic and appears in the plaques only 1-3 days after hatching. It is thus proposed that the interactions of the dense plaque with myofilaments or with the membrane do not depend on the presence of vinculin in the plaque. Electrophoretic analyses indicated that, during development, there is no major change in the differential expression of specific vinculin isoforms. Quantitative immunoblotting analysis indicated that the vinculin content (relative to total extracted protein) is virtually constant during the last week of embryonic life. However, within 3 days of hatching, the vinculin concentration increases remarkably to over twice the embryonic level, and then slowly increases until it reaches the adult levels, which are three to four times higher than the embryonic level. The concentration of metavinculin (a 160-Kd vinculin-related protein) showed only a limited increase after hatching. We discuss the possible roles of vinculin and talin in the assembly of membrane-bound dense plaques during the different phases of smooth-muscle development.

Animals↗

PubMed1985

Spatial interrelationships between proteoglycans and extracellular matrix proteins in cell cultures.

Immunolabelling of cultured cells for chondroitin-sulfate proteoglycan (CSPG), in conjunction with antibodies to fibronectin, collagen and laminin, revealed the spatial interrelationships between the different matrix components. CSPG was organized in two major forms. Fibronectin-independent dotted patterns of CSPG were detected on the substrate and cell surfaces at early stages after plating. At later stages, however, significant overlapping was found between the two extracellular matrix components. Immunoelectron microscopic examination indicated that the CSPG was organized as granules of varying sizes which were associated with the cell surface, the substrate, or with the periphery of the fibronectin network.

Animals↗