Complex carbohydrates of the extracellular matrix structures, interactions and biological roles.
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Animals↗
Biomedical subjects
J D Aplin
Publications and source records attributed to J D Aplin.
A cell spreading factor, abundant in human placenta, contains fibronectin and fibrinogen.
A salt extract from human normal-term placental extracellular matrix contains material that rapidly adsorbs to glass or tissue-culture plastic and promotes the spreading of fibroblastic and epithelioid cell lines. The cellular response to placental spreading factor (PSF) is characterized and compared with the response to fibronectin. The active component is shown to contain antigenic determinants that cross-react with antibodies to fibronectin and fibrinogen. The activity is not retained on gelatin-Sepharose but another component of the extract (approximately 185 000 molecular weight) is retained. Partial purification is reported, and the possible involvement of PSF in trophoblastic invasion of the uterine wall is discussed.
Cell Adhesion↗
Protein-derivatised glass coverslips for the study of cell-to substratum adhesion.
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Animals↗
Reversible cross-linking of cellular components of adherent fibroblasts to fibronectin and lectin-coated substrata.
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Animals↗
Modification of fibroblast surface amines alters receptor-mediated cell spreading on protein-coated substrata but not adsorptive endocytosis.
Fluorescein isothiocyanate (FITC) and other anionic reagents specific for amine groups have previously been shown to inhibit the adhesion and spreading of cultured fibroblasts to fibronectin-coated surfaces (Butters, Devalia, Aplin & Hughes, 1980). Here it is demonstrated that a population of FITC-labelled cells can be separated using flow cytometry into fractions displaying greater and lesser adhesivity at lower and higher fluorescence, respectively, demonstrating that the inhibition is dose-dependent. Glass coverslips covalently derivatized with the lectins ricin and concanavalin A are used to show that the inhibition also occurs in lectinmediated cell adhesion as well as in adhesion to collagen coated with fibronectin and plastic coated with serum or antibody, suggesting that all of these responses share a common, FITC-sensitive component. Simple primary amine compounds inhibit adhesion to fibronectin, but specific inhibitors of transglutaminases do not affect the process. Transglutaminase activity of cell surfaces has been implicated in protein endocytosis and receptor recycling (Davies et al. 1980). FITC modification of cells appears to affect specifically adhesive interaction, since ricin cytotoxicity and infection of cells with influenza and Sendai viruses (phenomena thought to proceed by means of receptor-mediated endocytosis) are unaffected. Evidently, receptor-mediated cell attachment, spreading on protein-coated surfaces and protein endocytosis are functionally separate events requiring different cell-surface membrane components, even when the same protein (ricin) is used to trigger these 2 processes.
Adsorption↗
Cell adhesion on model substrata: threshold effects and receptor modulation.
Trypsinized BHK cells become attached to glass that has been derivatized with a variety of lectins with well-defined specificity for cell-surface carbohydrates. Provided a threshold concentration of glass-immobilized protein is present the cells undergo a transformation to a well-spread morphology. The matrix density of lectins (ricin and concanavalin A) required to trigger this morphological transformation is higher by 10 to 40-fold thant the value determined earlier (Hughes, Pena, Clark & Dourmashkin, 1979) for fibronectin. Cells resistant to the toxic lectin, ricin, and expressing 10% or less of ricin-binding carbohydrate groups at their cell surfaces require correspondingly greater matrix densities of ricin to promote active cell spreading. All cell lines spread equally well on concanavalin A-based matrices consistent with their similar binding properties. The quantitative interaction of complementary molecules on the cell surface and matrix, promoting cell adhesion, is demonstrated by these results and a model is proposed for the events leading to a well-spread cell morphology on a protein-coated substratum.
Animals↗
Preparation, properties, and applications of carbohydrate conjugates of proteins and lipids.
As a result of the growing awareness of the involvement of the oligosaccharide moieties of glycoproteins and glycolipids in cell surface recognition and binding phenomena, a wide variety of methods have been developed, many quite recently, for preparing glycoconjugates. The chemical methods used for the attachment of sugars and certain hydrophilic polymers (e.g., polyethylene glycol) are discussed, as are the effects of such modifications on various properties of the protein (immune response, thermal stability and resistance to proteolysis, clearance, and specific binding to cell surface receptors). Enzymatic approaches to glycoconjugate preparation are also considered, and several examples are given of the preparation of model glycolipids, useful in studying cell surface phenomena. In a final section, three areas are considered in which rapid advances seem likely to occur: improved methods for the preparation of glycoconjugates; direct modification of cell surface glycoconjugates; and modification for the purpose of studying location and environment of membrane glycoconjugates.
Animals↗
Cell surface molecules involved in fibronectin-mediated adhesion. A study using specific antisera.
Attachment and spreading of baby hamster kidney (BHK) fibroblasts to fibronectin-coated surfaces can be inhibited by antibodies, and the (Fab) 2 fragments, prepared against the cells. The antibodies reacted specifically with ricin-binding glycoproteins of the cell surface, the major components having molecular weights of 130-140 K. The antibodies reacted also with mouse fibroblasts (L-cells) and Chinese hamster ovary (CHO)-cells and inhibited their fibronectin-mediated adhesion to inert surfaces. Antisera raised against material isolated from the underside of BHK cells spread out on fibronectin-coated substrata using a cleavable, photolabile heterobifunctional cross-linking reagent (Aplin et al. Exptl. Cell Res. (1981) 134, 488-494) also affected the interactions of cells with fibronectin-coated surfaces. The antibodies stained the periphery of BHK cells and L-cells in a discontinuous periodic manner and immunoprecipitated as a major component a low molecular weight (approximately 47 K) glycoprotein from cell extracts. These results indicate that different specific antisera modify cell-substratum adhesion, probably by interacting with different cell surface components. The properties of multiple factors involved in the attachment and spreading of cells to suitably adhesive substrata are discussed.
Animals↗
Polypeptide heterogeneity of hamster and calf fibronectins.
The adhesive glycoprotein fibronectin has been isolated from fresh hamster plasma by affinity chromatography on gelatin coupled to Sepharose beads by the method of Engvall & Ruoslahti [Int. J. Cancer (1979) 20, 1-5]. Polyacrylamide-gel electrophoresis of material heated in sodium dodecyl sulphate and 2-mercaptoethanol shows two prominent polypeptide subunits of approx. mol.wts. 215 000 and 200 000, with variable amounts of lower-molecular-weight fragments. The unexpected polypeptide heterogeneity of different preparations of hamster fibronectins and bovine serum fibronectin is shown to be partly an artefact and is generated during isolation and storage of purified fibronectin. Treatment of each hamster fibronectin subunit or a smaller fragment of approx. mol.wt. 140 000 with thermolysin or trypsin after radioiodination produces similar patterns of tyrpsine-containing peptides, indicating similar primary amino-acid sequences. Antibodies raised against the major subunits of hamster plasma fibronectin were coupled to Sepharose beads and used in conjunction with gelatin affinity chromatography to isolate fibronectins extracted with urea from baby-hamster kidney (BHK) cells and present in the long-term culture medium of these cells. The cell and medium fibronectins are similar to hamster plasma fibronectin in amino-acid and carbohydrate composition and also produce very similar peptide 'maps'. We conclude that the various forms of hamster fibronectins are structurally analogous in agreement with indistinguishable biological properties in mediating the substance adhesion of BKH cells [Pena & Hughes (1978) Cell Biol. Int. Rep. 3, 339-344].
Amino Acids↗
Sepharose 4B as a matrix for affinity chromatography. A spin-labelling investigation using nitroxides as model ligands.
Nitroxide spin labels were attached to CNBr-activated Sepharose 4B directly and through oligoglycines and oo-amino-carboxylic acids of varying length. The homogeneity of the carbohydrate environments of directly attached labels was investigated by measuring dipolar interactions between nitroxides as a function of solvation and of spin dilution with a diamagnetic analogue, as well as by electron exchange between the nitroxides and paramagnetic metal ions in solution. Only the exchange experiment revealed any inhomogeneity, suggesting that a small proportion of sites may be less accessible than the majority. The distances between sites were sufficiently small to allow, in principle, multiple-site interactions between quite small proteins in solution and immobilized ligands. Reorientation of the label at the matrix, characterized by the correlation time t, became more rapid with increasing spacer length n. For n > 12, the decrease in t was less pronounced. The two types of spacer behaved similarly. Thus an ideal spacer length for affinity separations is 12 atoms; this is in good agreement with data from a variety of affinity separations. The results of electron spin resonance studies of the effect of non-aqueous solvent on directly and indirectly labelled Sepharose 4B were used to suggest reasons why enzymes immobilized on Sepharose may be stabilized to denaturing solvents.
Chromatography, Affinity↗
Inhibition of fibronectin-mediated adhesion of hamster fibroblasts to substratum: effects of tunicamycin and some cell surface modifying reagents.
Using baby hamster kidney (BHK) fibroblasts we have studied the effect of tunicamycin, a specific inhibitor of protein glycosylation, on the ability of trypsinized cells to attach and spread onto fibronectin. Tunicamycin inhibited mannose incorporation into total acid-precipitable glycoproteins by at least 95% while glucosamine and leucine incorporation were less or hardly inhibited. Hydrolysis and analysis of [3H]glucosamine-labelled glycoproteins showed that radioactivity incorporated into cells exposed to tunicamycin was present predominantly as galactosamine, presumably present in O-glycosidically linked glycan chains whose assembly is insensitive to the drug. Treated cells exhibit reduced amounts of surface-associated fibronectin and adhere relatively poorly to plastic or collagen surfaces pre-coated with plasma or BHK cell-derived fibronectins at the minimum concentrations required to induce nearly quantitative attachment and spreading of untreated cells. Drug-treated cells do adhere and spread into a bipolar configuration on surfaces saturated with fibronectin. Cells treated with tunicamycin and then grown in the absence of the drug revert to a more normal behaviour, indicating that under certain conditions the effects of the drug are reversible. Fibronectin-mediated spreading of trypsinized BHK cells is also inhibited by pre-treatment of cells with several non-penetrating reagents reactive with cell surface amino groups, namely pyridoxal phosphate, trinitrobenzene sulphonate and fluorescein 5-isothiocyanate. Analysis of surface substitution indicates a strong correlation between the extent of amino group substitution and inability of treated cells to interact with a fibronectin lattice. While the extent of attachment under these conditions is normal, cells pretreated with a specific non-penetrating thiol reagent, p-chloromercuribenzenesulphonate fail to attach to fibronectin-coated culture dishes in a dose-dependent fashion, indicating that a biochemical distinction can be made between the processes of attachment and spreading. We conclude that both N-glycosidically linked carbohydrate moieties of BHK cell surface glycoproteins and primary amine groups present in surface proteins or lipid head groups play a role in interactions of cells with fibronectin, leading to the formation and maintenance of a stable well-spread morphology. Both N-linked glycans and surface sulphydryl groups appear to be required for an attachment process which precedes spreading.
Amines↗
Spin-labelling of sialic acid in soluble and cell-surface glycoproteins.
A new, mild method is described for spin-labelling sialic acid residues in situ. The procedure involves the formation of C-1 sialamides and has been applied to a serum glycoprotein, a mucin, tissue sections from human colon, and erythrocyte membrane components. The selectivity of the method and its possible applicability to other types of labelling are discussed.
Colon↗
Expression of integrin alpha 6 beta 4 in human trophoblast and its loss from extravillous cells.
Villous trophoblast from first trimester and term placenta expresses the integrin subunits alpha 6 and beta 4, as monitored by immunohistochemistry. The subunits, which co-distribute in all locations, are present at the basal cell surface where cyto- and syncytiotrophoblast make contact with the villous basement membrane and, in addition, in areas of contact between cytotrophoblast or cyto- and syncytiotrophoblast, but both are absent from the apical syncytial surface. This distribution, which together with other evidence suggests that the two subunits are associated in trophoblast as the heterodimer alpha 6 beta 4, differs from other epithelia in which alpha 6 beta 4 is predominantly basally disposed. Anti-cytokeratin antibodies were used to identify cytotrophoblast columns at the tips of anchoring villi and the migratory interstitial extravillous cytotrophoblast population in the placental bed. Cells in the columns showed a level of expression that diminished with distance from the villous basement membrane, and the alpha 6 and beta 4 subunits were not detected in interstitial cytotrophoblast. In trophoblast of the chorion laeve, both the subunits were expressed in all layers from the pseudobasement membrane to the edge of the parietal decidua, but not in the decidua itself. Integrin alpha 6 beta 4 distribution was also examined in primary cultures of term trophoblast and in the choriocarcinoma cell line BeWo. The alpha 6 and beta 4 subunits were present at or near cell contact areas, where basement membrane components including laminin were also detected. The results suggest that integrin alpha 6 beta 4 plays a role in anchoring trophoblast to basement membrane and its loss may play a role in enabling cells to migrate into the maternal tissue compartment.
Antigens, Surface↗
An immunofluorescence study of extracellular matrix associated with cytotrophoblast of the chorion laeve.
Using immunofluorescence we have studied the distribution within and beneath the cytotrophoblast of chorion laeve of five extracellular matrix components: types I, III and IV collagen, fibronectin and laminin. Fibronectin, laminin and types I and IV collagen are located in a network of extracellular matrix which encapsulates cells of the cytotrophoblast multilayer. The results suggest that cytotrophoblast in all layers is active in matrix biosynthesis. The pseudo-basement membrane, which links this intercellular matrix to the underlying reticular layer, contains rich deposits of fibronectin and type III collagen along with more restricted foci of laminin and type IV collagen. The reticular layer contains fibronectin, type III collagen and small amounts of type I collagen, but no laminin or type IV collagen. Type III collagen is also found in the maternal decidua. Extracellular matrix markers may be useful in defining trophoblast differentiation pathways. Possible reasons are discussed for the unusual architecture of extracellular matrix of cytotrophoblast of the chorion laeve. Cytoplasmic actin occurs in cells throughout the chorion laeve, while cytokeratin appears only in cytotrophoblast. Plasminogen also occurs throughout cytotrophoblast in pericellular locations.
Actins↗
Importance of vitamin C in maintenance of the normal amnion: an experimental study.
Collagenous matrix in amnion accounts for most of the dry weight of the tissue and provides its mechanical strength and resistance to rupture. Cell and organ culture techniques have been utilized to study the influence of vitamin C upon the synthesis and deposition of extracellular matrix by cells of normal amnion at term. The cultures have been examined using light and electron microscopy and metabolic labelling. These studies show that both epithelial cells and fibroblasts of the deeper stromal layer are active in the production of fibrillar matrix at this time. Matrix deposition by epithelial cells in culture increases several-fold when the vitamin C concentration in the supernatant medium is increased from zero to 50 micrograms/ml, and, at the latter concentration, a continuous anastomosing fibrillar collagenous meshwork appears beneath the cells. This study thus provides new evidence that an adequate dietary supply of vitamin C is probably needed to maintain the strength of the chorioamnion.
Amnion↗
The cell biology of human implantation.
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Cell Adhesion Molecules↗
Trophoblast invasion of spiral arteries: a novel in vitro model.
Extravillous trophoblasts invade the uterine wall (interstitial invasion) and the spiral arteries (endovascular invasion), replacing the cells of the vessel wall and creating a high-flow low-resistance vessel. We have developed a novel model to allow the interactions between the invading trophoblast cells and the cells of the spiral artery to be directly examined. Unmodified (non-placental bed) spiral arteries were obtained from uterine biopsies at caesarean section. Fluorescently labelled trophoblasts were seeded on top of artery segments embedded in fibrin gels (to study interstitial invasion) or perfused into the lumen of arteries mounted on a pressure myograph (to study endovascular invasion). Trophoblasts were incubated with the vessels for 3-5 days prior to cryo-sectioning. Both interstitial and endovascular interactions/invasion could clearly be detected and a comparison of the extravillous trophoblast cell line, SGHPL-4 and primary first trimester cytotrophoblasts showed both to be invasive in this model. This novel method will prove useful in an area where in vitro studies have been hampered by the lack of suitable models directly examining cellular interactions during invasion.
Adult↗