Cell adhesion: old and new questions

Metazoans clearly need cell adhesion to hold themselves together, but adhesion does much more than that. Adhesion receptors make transmembrane connections, linking extracellular matrix and adjacent cells to the intracellular cytoskeleton, and they also serve as signal transducers. In this article, I briefly summarize our present understanding of the molecular basis and biological consequences of cell adhesion and discuss how our current knowledge sheds light on questions of specificity of cell adhesion. I offer some thoughts and speculations about the evolution of cell-adhesion molecules and processes, consider their inter-relationships with other forms of cell–cell communication and discuss unresolved questions ripe for investigation as we enter the postgenomic era.     

Amphibian Pleurodeles waltl Fibronectin: cDNA Cloning and Developmental Expression of Spliced Variants

Partial cDNA clones encoding approximately the carboxy terminal half of Pleurodeles fibronectin (FN) were isolated. They account for 4.7 Kbp of the 3' region of the FN mRNA. The cDNA nucleotide sequence comprises all three alternatively spliced segments designated EIIIA, EIIIB and V-segment, respectively. All three segments are included in FN mRNA synthesized during early embryogenesis whereas, from the tailbud stage onward the V-region was partially excluded. The isolation of Pleurodeles cDNA clones including the three different spliced EIIIA, EIIIB and V segment raises new possibilities for the study of the precise role of specific regions of FN in early amphibian development.     

Density- and serum-dependent regulation of the Reck tumor suppressor in mouse embryo fibroblasts

Reck is a membrane-anchored glycoprotein identified as a transformation suppressor. Accumulating evidence indicates that Reck negatively regulates a wide spectrum of matrix metalloproteinases and is commonly down-regulated in a variety of malignant solid tumors. Physiological cues that regulate Reck expression, however, remained unknown. In this study, we found that Reck expression was up-regulated at high cell density, low serum, or after treatment with some kinase inhibitors, such as PP2 (Src inhibitor), LY294002 (PI3-kinase inhibitor), and PF573228 (FAK inhibitor), in mouse embryo fibroblasts. Curve fitting indicated that the levels of Reck protein and Reck mRNA are quadratic in the cell density. Other factors, including serum, extracellular matrix components (type I collagen and fibronectin), the kinase inhibitors, and some of their oncogenic targets (v-Src and PIK3CA mutants), modify the shape of the quadratic curve. Comparison of these modifications implicated Src in Reck down-regulation under sparse conditions, PI3-kinase in serum-induced Reck down-regulation, and FAK in Reck down-regulation at high cell density. Fibronectin and type I collagen down-regulated Reck, supporting the role of integrin-FAK signaling in Reck down-regulation at high cell density. Our study has revealed multiple signaling pathways impinging on Reck in cultured mouse embryo fibroblasts and sets a foundation for future studies to find effective Reck inducers of potential value in cancer therapy.     

Synthesis and distribution of laminin-related polypeptides in early amphibian embryos

Summary Western blotting experiments carried out with several heterospecific antibodies against mouse-derived laminin allowed the identification of four laminin-related polypeptides in early Pleurodeles waltlii embryos. Synthesis of all four polypeptides was detected from the early blastula stage to late gastrula stage. Immunofluorescent staining with anti-laminin and anti-fibronectin antibodies provided evidence for a close association of these laminin-related polypeptides with the fibronectin fibrillar network.     

A novel method for purification of plasma fibronectin

Plasma fibronectin was purified from a gelatin-affinity chromatography column by elution with glucose. This procedure was effective only if the gelatin was particulate when it was attached to the Sepharose 4B. Glucose could not elute fibronectin from the gelatin if the gelatin was melted before it was attached to the Sepharose 4B. This new purification technique has the advantage of using very mild conditions for the isolation of plasma fibronectin.     

Adhesion to extracellular materials by neural crest cells at the stage of initial migration

Summary Trunk-level neural anlagen bearing neural crest cells at the stage of initiation of migration were isolated from chick embryos and explanted in serum-free medium onto glass substrates which had previously been treated with extracellular materials. After 0.5–2 h incubation, the expiants were dislodged with a stream of culture medium and the substrate examined for adherent crest cells. Crest cells adhered to collagen gels, and adhered to and spread on adsorbed fibronectin; antiserum to fibronectin prevented adhesion to fibronectin but not to collagen gels. Air-dried collagen gels and collagen solutions were less adhesive, the adhesivity declining with longer drying time and lower collagen concentration. Crest cells adhered poorly to dried gelatin and not at all to adsorbed collagen. Fibronectin increased the adhesion to dried collagen and gelatin. Pretreatment of collagen gels with hyaluronate retarded adhesion. Hyaluronate pretreatment also retarded adhesion to adsorbed fibronectin but only when adsorbed collagen was also present. Pretreatment of collagen gels with the proteoglycan monomer from bovine nasal cartilage had no effect of the adhesion of crest cells, but the proteoglycan almost completely inhibited adhesion to adsorbed fibronectin, but only when absorbed collagen was also present. The results are discussed in terms of the control of migration of neural crest cells by extracellular materials.     

Type-I collagen production by human odontoblast-like cells in expiants cultured on cyanoacrylate films

Summary Odontoblast-like cells derived from human tooth pulps were maintained in expiant culture and grown either on glass coverslips only (used as control) or on glass coverslips coated with cyanoacrylate films. Ultrastructural and cyto-morphometric evidence showed that cells exposed to cyanoacrylate, in contrast to controls, display a significant decrease of rough endoplasmic reticulum and mitochondria. In addition, immunofluorescent staining and radioimmunoassays for type-I collagen suggested disturbances in production for the exposed cells. The use of anti-fibronectin antibodies with electron-microscopic immunoperoxidase-labelling demonstrated that the adherence of cells to cyanoacrylate can involve both adhesion plaques and fibronectin. These results therefore suggest that there were no apparent differences in the adhesion interaction of cells between glass and cyanoacrylate substrates.     

Staphylococcus aureus PSMα3 Cross-α Fibril Polymorphism and Determinants of Cytotoxicity

1. Download : Download high-res image (281KB)
2. Download : Download full-size image

     

Probing the role of aromatic residues in the self-assembly of Aβ(16–22) in fluorinated alcohols and their aqueous mixtures

The Aβ(16–22) sequence KLVFFAE spans the hydrophobic core of the Aβ peptide and plays an important role in its self-assembly. Apart from forming amyloid fibrils, Aβ(16–22) can self-associate into highly ordered nanotubes and ribbon-like structures depending on the composition of solvent used for dissolution. The Aβ(16–22) sequence which has FF at the 19th and 20th positions would be a good model to investigate peptide self-assembly in the context of aromatic interactions. In this study, self-assembly of Aβ(16–22) and its aromatic analogs obtained by replacement of F19, F20 or both by Y or W was examined after dissolution in fluorinated alcohols and their aqueous mixtures in solvent cluster forming conditions. The results indicate that the presence of aromatic residues Y and W and their position in the sequence plays an important role in self-assembly. We observe the formation of amyloid fibrils and other self-assembled structures such as spheres, rings and beads. Our results indicate that 20% HFIP is more favourable for amyloid fibril formation as compared to 20% TFE, when F is replaced with Y or W. The dissolution of peptides in DMSO followed by evaporation of solvent and dissolution in water appears to greatly influence peptide conformation, morphology and cross-β content of self-assembled structures. Our study shows that positioning of aromatic residues F, Y and W have an important role in directing self-assembly of the peptides.     

Novel fluorescent trimethine cyanine dye 7519 for amyloid fibril inhibition assay

Abstract

Fluorescence spectroscopy was used to study the ability of dye 7519 to follow the transition of monomeric insulin into fibrils and applicability of the dye to the insulin aggregation inhibition assay. The commercially available classic amyloid stain, thioflavin T, was used as the reference dye. For selecting potential inhibitors, the QSAR approach was applied. Dye 7519 appeared to be suitable for monitoring insulin aggregation into fibrils in vitro. The properties of the dye allowed us to test it as a potential probe in the screening assay of potential inhibitors of insulin fibrillization. One hundred forty-four flavonoids were tested as potential inhibitors of amyloid fibril formation using the quantitative structure activity relationship approach. Among them, 10 candidates with high indexes of inhibition were selected for tests in vitro using dye 7519 and the reference amyloid dye thioflavine T. Using dye 7519 fluorescence, we found that two compounds had inhibitory effects on insulin amyloid formation. These results agree with inhibition data using the thioflavine T assay. Our studies demonstrated that the fluorescent cyanine dye 7519 is a sensitive probe for quantitative detection of insulin amyloid formation and can be applied to screen agents capable of affecting aggregation of amyloid proteins.