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B Geiger

Publications and source records attributed to B Geiger.

At least 37 records · Page 2Linked to original sources
PubMed1991

Expression of a novel cadherin (EP-cadherin) in unfertilized eggs and early Xenopus embryos.

Two distinct cadherin cDNA clones of Xenopus laevis were isolated from a stage 17 embryo cDNA library. Analysis of the complete deduced amino acid sequences indicated that one of these molecules is closely homologous to chicken and mouse N-cadherin, while the other displays comparable homology to both E- and P-cadherins and was thus denoted EP-cadherin. This molecule has an apparent relative molecular mass of 125 x 10(3) (compared to approx. 138 x 10(3) or approx. 140 x 10(3) of E-cadherin and N-cadherins, respectively). Northern and Western blot analyses indicated that N-cadherin is first expressed at the neurula stage while EP-cadherin is the only cadherin detected in unfertilized eggs and cleavage stage embryos. Immunolabeling of Xenopus eggs with antibodies prepared against a fusion protein, containing a segment of EP-cadherin, indicated that the protein is highly enriched at the periphery of the animal hemisphere. EP-cadherin was also found in A6 epithelial cells derived from Xenopus kidneys, and was apparently localized in the intercellular adherens junctions.

Amino Acid Sequence↗
PubMed1991

EP-cadherin in muscles and epithelia of Xenopus laevis embryos.

EP-cadherin is a novel Xenopus Ca+2-dependent adhesion molecule, which shares comparable homology with mouse E- and P-cadherins (Ginsberg, De Simone and Geiger; 1991, Development 111, 315-325). We report here the patterns of expression of this molecule in Xenopus laevis embryos at different developmental stages ranging from cleavage to postmetamorphic. EP-cadherin is already expressed in the oocyte and egg and can then be detected in close association with the membrane of all blastomeres up to late blastula stages. Starting at late gastrula stages, the level of EP-cadherin expression increases sharply in non-neural ectodermal cells, in the somites and in the notochord; it persists in endodermal cells and decreases rapidly in all migratory cells. During neurulation the level of EP-cadherin expression declines gradually in the nervous system and is undetectable here throughout later development except in the optic nerve and in the neural part of the olfactory organ. This pattern continues during later development so that in the tailbud stage and up to metamorphosis the most prominent staining is detected in the epidermis and skeletal muscle. After metamorphosis, the molecule gradually disappears from the muscle tissue and the major site of expression remains the skin. EP-cadherin is invariably present in close association with the cell membrane. In the muscle it is associated with the sarcolemma at regions of myoblast-myoblast or myotube-myotube contact. In epidermal cells, EP-cadherin is usually coexpressed with E-cadherin. Yet, while E-cadherin staining is always restricted to the basolateral aspects of the cells, EP-cadherin is often distributed throughout the plasmalemma including the apical surface.

Animals↗
PubMed1991

Study of vitrified, unstained frozen tissue sections by cryoimmunoelectron microscopy.

We describe the development and application of a novel approach to high-resolution ultrastructural analysis of cells and tissues. It is based on the preparation of ultrathin frozen sections of fixed tissues, rinsing of the sections, followed by their embedding on the grid in a layer of vitrified ice, and direct observation with a cryoelectron microscope. Examination of smooth muscle, kidney and heart tissues showed that although no heavy metal staining was used, high-contrast images are obtained. Fine details of cytoplasmic filaments and organelles, membranes and membrane-associated structures, as well as connective-tissue elements are all visible. The new method is suitable for immunolabeling, including high resolution localization of specific molecules within the cytoplasm.

Animals↗
PubMed1990

Immunocytochemical characterization of lung tumors in fine-needle aspiration. The use of cytokeratin monoclonal antibodies for the differential diagnosis of squamous cell carcinoma and adenocarcinoma.

In the current study, immunocytochemical typing of intermediate filaments was used for a differential diagnosis of human lung tumors from transthoracic fine-needle aspiration biopsies (TFNAB). The authors have compared the cytologic diagnosis of 53 lung cancer cases with the immunofluorescence patterns obtained using a panel of monoclonal antibodies, five of which (KG 8.13, KM 4.62, Ks B.17, KS 8.12, KK 8.60) react with specific cytokeratin polypeptides and one with vimentin (VIM 13.2). Only in six of 23 samples cytologically diagnosed as squamous cell carcinoma did the immunocytochemical typing of cytokeratins (ICTC) confirm the cytologic diagnosis. In seven cases some of the tumor cells stained positively with antibody Ks B.17 specific for simple epithelial keratin (No: 18), suggesting the presence of some cells of glandular origin. In ten additional cases the ICTC was in conflict with the cytologic diagnosis of squamous cell carcinoma (i.e., antibodies Ks 8.12 and KK 8.60 were negative, and antibody Ks B.17, positive) supporting a diagnosis of adenocarcinoma. In 14 of 18 cases cytologically diagnosed as adenocarcinoma, the ICTC confirmed the diagnosis whereas in four cases additional presence of some squamous cells was noticed. The ICTC labeling of cases cytologically diagnosed as undifferentiated and large cell carcinomas was similar to that of the group of adenocarcinomas. Thus, the application of cytokeratin typing for TFNAB samples seems to provide a vital complementation to routine cytologic study, especially for cases cytologically diagnosed as squamous carcinoma.

Adenocarcinoma↗
PubMed1990

Cleavage of A-CAM by endogenous proteinases in cultured lens cells and in developing chick embryos.

We describe two truncated forms of A-CAM (N-cadherin) and present evidence suggesting that both forms are proteolytically derived from the intact A-CAM molecule. The first is a membrane-bound fragment of A-CAM displaying an apparent molecular weight of 78 kDa. This polypeptide, containing the C-terminal portion of the protein, may be generated in cultured chicken lens cells, either by a short treatment with trypsin-EGTA, or by endogenous proteinase(s) during incubation in low Ca2+ medium. Immunofluorescent labeling of normal and EGTA-treated cells indicated that the 78-kDa fragment is uniformly distributed over the cell surface. Moreover, staining of developing chick embryos with pairs of antibodies which distinguish the 78-kDa fragment from intact A-CAM indicated that, at early stages of sclerotome dissociation in developing somites, a truncated derivative of the molecule is generated. The second truncated form of A-CAM is a 97-kDa polypeptide which is constitutively released by cultured lens cells into the culture medium in the presence of normal medium. We present evidence that the 97-kDa molecule is proteolytically derived from A-CAM by the action of an endogenous proteinase. We discuss possible mechanisms leading to the formation of these two truncated derivatives and their possible involvement in the physiological modulation of A-CAM-mediated interactions.

Amino Acid Sequence↗
PubMed1990

Broad spectrum pan-cadherin antibodies, reactive with the C-terminal 24 amino acid residues of N-cadherin.

We describe here the preparation and application of antibodies directed against a synthetic, 24 amino acid long, peptide corresponding to the conserved cytoplasmic C terminus of N-cadherin. We demonstrate here that the antibodies to the synthetic peptide react extensively with all known members of the cadherin family and, in addition, recognize novel cadherins in a variety of cells and tissues, suggesting that these antibodies indeed exhibit pan-cadherin reactivity. By Western blot screening of chicken tissues at least 4 different immunoreactive bands were resolved, commonly disclosing 2-3 distinct bands within the same tissue. The pan-cadherin antibodies also displayed a broad interspecies cross reactivity, recognizing cadherins in man, bovine, canine, avian, amphibian and teleost cells. This property renders these antibodies excellent reagents for the cloning and identification of novel cadherins. Immunocytochemical labelling with the pan-cadherin antibodies, at the light- and electron-microscope levels, revealed an extensive reactivity with intercellular adherens junctions in cardiac muscle and in various epithelia. We thus propose that the pan-cadherin antibodies may be used as ubiquitous cadherin probes and serve as markers for adherens junctions.

Amino Acid Sequence↗
PubMed1989

Primary sequence and domain structure of chicken vinculin.

We have determined the complete sequence of chick vinculin from two overlapping cDNA clones. The vinculin mRNA consists of 262 bp of 5' untranslated sequence, an open reading frame of 3195 bp (excluding the initiation codon) and a long 3' untranslated sequence (greater than 2 kb). Chick vinculin contains 1066 amino acid residues, and has a deduced molecular mass of 116,933 Da. Analysis of the domain structure of vinculin shows that the molecule can be cleaved by V8 proteinase into a 90 kDa globular head and a 32 kDa tail region, the latter of which could further be cleaved into a 27 kDa polypeptide. The 90 kDa globular head contains the N-terminus of vinculin, three 112-residue repeats (residues 259-589), and extends to approximately residue 850. Gel overlay experiments show that it also contains a binding site for the cytoskeletal protein talin. The talin-binding domain was further localized to the N-terminal 398 amino acid residues of the protein by expression in vitro of this region from a vinculin cDNA cloned into the Bluescript SK+ vector. The head and tail domains are apparently separated by a proline-rich region that contains V8-proteinase-cleavage sites and a candidate tyrosine (822)-phosphorylation site. Secondary-structure prediction suggests that the head and tail domains contain alpha-helical regions separated by short stretches of turn/coil. Comparison of the chick with a partial human sequence reveals that vinculin is a highly conserved protein. In chickens Southern-blot analysis is consistent with a single vinculin gene, and it is therefore likely that vinculin, and its higher-molecular-mass isoform termed metavinculin, arise through alternative splicing.

Amino Acid Sequence↗
PubMed1989

Identification of two distinct functional domains on vinculin involved in its association with focal contacts.

We report here on the identification of two distinct functional domains on chicken vinculin molecule, which can, independently, mediate its interaction with focal contacts in living cells. These findings were obtained by immunofluorescent labeling of COS cells transfected with a series of chicken vinculin-specific cDNA constructs derived from clones cVin1 and cVin5 (Bendori, R., D. Salomon, and B. Geiger. 1987. EMBO [Eur. Mol. Biol. Organ.] J. 6:2897-2905). These included a chimeric construct consisting of 5' sequences of cVin1 attached to the complementary 3' region of cVin5, as well as several constructs of either cVin1 or cVin5 from which 3' or 5' sequences were deleted. We show here that the products of both cVin1 and cVin5, and of the cVin1/cVin5 chimera, readily associated with focal contacts in transfected COS cells. Furthermore, 78 and 45 kD NH2-terminal fragments encoded by a deleted cVin1 and the 78-kD COOH-terminal portion of vinculin encoded by cVin5 were capable of binding specifically to focal contact areas. In contrast 3'-deletion mutants prepared from clone cVin5 and a 5'-deletion mutant of cVin1, lacking both NH2- and COOH-terminal sequences, failed to associate with focal contacts in transfected cells. The loss of binding was accompanied by an overall disarray of the microfilament system. These results, together with previous in vitro binding studies, suggest that vinculin contains at least two independent sites for binding to focal contacts; the NH2-terminal domain may contain the talin binding site while the COOH-terminal domain may mediate vinculin-vinculin interaction. Moreover, the disruptive effect of the double-deleted molecule (lacking the two focal-contact binding sites) on the organization of actin suggests that a distinct region involved in the binding of vinculin to the microfilament system is present in the NH2-terminal 45-kD region of the molecule.

Animals↗
PubMed1989

Cingulin: characterization and localization.

Cingulin, a protein component associated with the tight junctions of chicken intestinal epithelium, has been purified to homogeneity by a new procedure and characterized. Purified cingulin is a heat-stable elongated dimer, composed of two polypeptides of Mr 108,000 (cingulin-108), with a Stokes' radius of approximately 15 nm, and a molecular length of 130 nm +/- 32 nm. Monoclonal antibodies were used to determine the tissue distribution and subcellular localization of cingulin in a variety of avian tissues and cultured cells. Indirect immunofluorescence analysis of semi-thin frozen sections demonstrated that cingulin is localized in the junctional complex of various polarized epithelia and in the endothelium, whereas it is essentially absent from mesenchymal and myogenic cells. In permeabilized and fixed cultured chick embryo kidney cells, the antibodies stained solely the regions of contacts between the epithelial cells. Double immunofluorescent labeling of these cells with anti-cingulin and anti-vinculin antibodies showed that cingulin is localized close to the vinculin-rich cytoskeletal belt associated with adherens junctions, but is absent from focal contacts and stress fibers. In cultured kidney cells, actin was detected mainly in stress fibers and in the peripheral junctional regions, where it showed a distribution similar to that of cingulin, suggesting that actin filaments may be part of the submembrane cytoskeleton at the level of the tight junction. Indirect immunoelectron microscopic labeling of ultrathin frozen sections of chicken intestine showed that cingulin is localized along the endofacial surfaces of the tight junction (zonula occludens), and is apparently excluded from the more basal zonula adhaerens, and from the desmosomes.

Animals↗
PubMed1988

Characterization of an inhibitor of actin polymerization in vinculin-rich fraction of turkey gizzard smooth muscle.

We report here on the purification and characterization of a new 25-kDa inhibitor of actin polymerization from turkey gizzard smooth muscle. The protein was purified by chromatography on DEAE-cellulose and hydroxyapatite, as well as by affinity chromatography on an immobilized-antibody column. The purified polypeptide reduced the low-shear viscosity of actin, apparently due to its inhibitory effect on actin polymerization. We demonstrate that this protein is largely responsible for the apparent inhibitory activity previously reported to be associated with smooth muscle vinculin preparations. Three independent monoclonal antibodies prepared against the 25-kDa inhibitor of actin polymerization can effectively adsorb the inhibiting activity of actin polymerization from the crude vinculin preparation or inhibit it. We also show here that the 25-kDa inhibitor of actin polymerization tends to undergo dimerization when maintained in non-reducing buffers, concomitant with the loss of its inhibitory activity. Immunohistochemical labeling of frozen sections, as well as immunoblotting analyzes, indicated that the 25-kDa inhibitor of actin polymerization is particularly enriched in smooth muscle cells and that its distribution is apparently homogenous throughout the cytoplasm showing no apparent enrichment in the vinculin-rich dense plaques located along the endofacial surface of the plasma membrane.

Actins↗
PubMed1988

Cingulin, a new peripheral component of tight junctions.

The tight junction (Zonula occludens), a belt-like region of contact between cells of polarized epithelia, serves as a selective barrier to small molecules and as a total barrier to large molecules, and is involved in the separation between lumenal and basolateral compartments of the epithelium. In the electron microscope, tight junctions show focal regions of apparent fusion between the adjoining cell membranes, and freeze-fractured membranes display an elaborate network of branching and anastomosing strands. Very little is known about the molecular composition and architecture of tight junctions. The first specific zonula occludens-associated protein, designated ZO-1, has recently been identified in mammalian epithelial and endothelial cells. Here we describe the identification and purification of a new component of this junctional complex in avian brush-border cells, which we name cingulin. Cingulin is an acidic, heat-stable protein, with a highly elongated shape. Immunofluorescence and immunoelectron microscopy of brush-border cells with anti-cingulin antibodies show that cingulin is localized in the apical zone of the terminal web, at the endofacial surfaces of the zonula occludens.

Amino Acids↗
PubMed1988

The modulation of cell adhesion molecule expression and intercellular junction formation in the developing avian inner ear.

The cells that constitute the membranous labyrinth in the vertebrate inner ear are all derived from a single embryonic source, namely, the otocyst. The mature inner ear epithelia contain different regions with highly differentiated cells, displaying a highly specialized cytoarchitecture. The present study was designed to determine the presence of adherens-type intercellular junctions in this tissue and study the expression of cell adhesion molecules (CAMs) associated with these junctions, namely, A-CAM and L-CAM, in the developing avian inner ear epithelia. The results presented here show that throughout the early otocyst, A-CAM is coexpressed with L-CAM. The formation of asymmetries between sensory and nonsensory areas in the epithelium is accompanied by the modulation of CAMs expression and the assembly of intercellular junctional complexes. A-CAM and L-CAM display reciprocal expression patterns, the former being expressed mostly in the mosaic sensory epithelium, while L-CAM becomes conspicuous in the nonsensory areas but its expression in the sensory region is markedly reduced. Adherens-type junctions and numerous desmosomes are found in the junctional complexes of early otocyst cells. The former persist to maturity of the various inner ear epithelia, whereas desmosomes disappear from junctional complexes of hair cells but remain in the intercellular junctional complexes of all other cell types in the membranous labyrinth. Thus, adherens type intercellular junctions comprise the only defined cytoskeleton-bound junction in mature hair cells. A-CAM-positive cells are also found in the region of the acoustic ganglion in early developmental stages but not in the mature neural elements.

Actins↗
PubMed1988

Selective expression of cytokeratin polypeptides in various epithelia of human Brenner tumor.

A human ovarian Brenner tumor presenting a wide spectrum of benign and malignant histologic features was studied for its patterns of intermediate filament expression. All epithelial elements of the tumor, regardless of their morphologic type, contained cytokeratins as their only intermediate filament component. Differences were detected, however, between tumor nests that displayed transitional epithelium and those with squamoid features. These differences were manifested by the presence of cytokeratin 18, in the former type only, and by the abundance of cytokeratins 10/11 in the latter. We also detected mixed epithelial nests in which both features were present, suggesting that the transitional epithelium transforms in polar fashion into squamous epithelium. Examination of cytokeratin patterns found in urothelium and in the surface epithelium of the ovary pointed to certain differences from the Brenner tumor epithelia. The significance of these latter findings with regard to cellular transformation and histogenesis of the Brenner tumor are discussed.

Antibodies, Monoclonal↗
PubMed1988

Contact formation during fibroblast locomotion: involvement of membrane ruffles and microtubules.

We have correlated the motility of the leading edge of fibroblasts, monitored by phase-contrast cinematography, with the relative distributions of several cytoskeletal elements (vinculin, tubulin, and actin) as well as with the contact patterns determined by interference reflection microscopy. This analysis has revealed the involvement of both ruffles and microspikes, as well as microtubules in the initiation of focal contact formation. Nascent vinculin sites within the leading edge or at its base, taken as primordial cell-substrate contacts, were invariably colocalized with sites that showed a history of transient, prolonged, or cyclic ruffling activity. Extended microspike structures, often preceded the formation of ruffles. Immunofluorescent labeling indicated that some of these primordial contacts were in close apposition to the ends of microtubules that penetrated into the leading edge. By fluorescence and electron microscopy short bundles of actin filaments found at the base of the leading edge were identified as presumptive, primordial contacts. It is concluded that ruffles and microspikes, either independently or in combination, initiate and mark the sites for future contact. Plaque proteins then accumulate (within 10-30 s) at the contract site and, beneath ruffles, induce localized bundling of actin filaments. We propose that all primordial contacts support traction for leading edge protrusion but that only some persist long enough to nucleate stress fiber assembly. Microtubules are postulated as the elements that select, stabilize, and potentiate the formation of these latter, long-lived contacts.

Actins↗
PubMed1988

Subtyping of epithelial cells of normal and metaplastic human uterine cervix, using polypeptide-specific cytokeratin antibodies.

The aim of the present study was to explore the histogenesis of metaplastic cells in the human uterine cervix. In a previous study we demonstrated that squamous cervical metaplasia expresses a unique set of cytokeratin polypeptides different from that expressed by the various normal epithelial elements of both the exo- and endocervix. It was thus proposed that the formation of squamous metaplasia represented a new route of differentiation. In the present study we further investigated this aspect by expanding the battery of monoclonal antibodies directed against specific cytokeratin epitopes used for immunohistochemical labelling. The antibodies used were: KS-1 A3, which specifically stains cytokeratin polypeptide no. 13; antibody KS-2.1, which is an anti-cytokeratin reacting with pseudostratified transitional and some simple epithelia; and antibody KS-B17.2 reacting with cytokeratin polypeptide no. 18. Examination of the staining patterns obtained with these antibodies revealed specific staining of ciliated cells with antibody KS-2.1 and of endocervical reserve cells with antibody KS-1A3. In 6 out of 19 cases tested reserve cells were also stained with antibody KS-2.1. These results enabled us to distinguish between at least four types of cells residing within the simple epithelium of the endocervix, namely columnar nonciliated cells, ciliated cells, and two subpopulations of reserve cells. Since metaplasia was positively stained by antibodies KS-1A3 and KS-2.1, we propose that the endocervical reserve cells that express cytokeratin polypeptide no. 13 are most probably the cells from which endocervical metaplasia is derived.

Antibodies, Monoclonal↗
PubMed1988

Spatial and temporal distribution of the adherens-junction-associated adhesion molecule A-CAM during avian embryogenesis.

A-CAM (adherens-junction-specific cell adhesion molecule) is a calcium-dependent adhesion molecule which is associated with intercellular adherens junctions in various tissues (Volk & Geiger, 1986, J. Cell Biol. 103, 1441-1450 and 1451-1464). In the present report, we have investigated the distribution of A-CAM during avian morphogenesis by immunofluorescence microscopy and immunoblotting. A-CAM appeared at the onset of gastrulation on developing mesodermal and endodermal cells and was then expressed on tissues derived from the three primary germ layers. During embryonic life, A-CAM was constitutively expressed in a number of tissues including the central and peripheral nervous system, myocardium, muscles, notochord, skin and lens whereas it was found transiently in many tissues ranging from the nephritic tubules and the endoderm of visceral arches to ectodermal placodes. In the adult, in addition to the nervous system, A-CAM was restricted to the skin, lens, heart and testis, and exhibited an apparent molecular weight higher than the one found in the embryo. The prevalence and cell-surface modulation of A-CAM could frequently be correlated with morphogenetic events such as mesenchyme condensation into epithelia or cell clusters (e.g. formation of the somitic epithelium, kidney tubules and peripheral ganglia), dissociation of epithelia (e.g. dissociation of the somitic epithelium and segregation of neural crest from the neural tube), separation of cell populations (e.g. fibroblasts and myotubes in the heart) and reorganizations of epithelia (e.g. neurulation). In addition, using electron microscopy, the expression of A-CAM on the surface of aggregating and separating cells could be correlated with the formation and disappearance of adherens junctions. This precisely scheduled control of A-CAM correlated with early morphogenetic events during embryogenesis suggests that this CAM could play a crucial role in these processes.

Animals↗