Actomyosin contractility controls cell surface area of oligodendrocytes

Background  

To form myelin oligodendrocytes expand and wrap their plasma membrane multiple times around an axon. How is this expansion controlled?     

Cleavage of a 135 kD cell surface glycoprotein correlates with loss of fibroblast adhesion to fibronectin

We have previously described a group of three plasma membrane glycoproteins that are recognized by an adhesion-disrupting antiserum and that are involved in fibronectin-mediated BHK cell adhesion. A peculiar property of these molecules is their resistance to tryptic digestion. We have now extended this study in the attempt to identify the active component within this group of molecules. SR/BALB mouse fibroblasts, used in this work, expose at their surface only two trypsin-resistant glycoproteins, gp1 (150 K) and gp2 (135 K), that are recognized by the adhesion-disrupting anti-BHK serum. Controlled proteolysis of the cell surface in the presence of a reducing agent results in the loss of cell adhesion to fibronectin-coated substratum. gp2 is selectively cleaved under these conditions. Moreover, cells treated with trypsin and reducing agent can no longer adsorb the adhesion-relevant antibodies from the anti-BHK serum. These data indicate that gp2 plays a critical role in the adhesion of SR/BALB fibroblasts to fibronectin-coated substratum, and that disulfide bonds are important in the conformation and function of this molecule.     

Actomyosin organisation for adhesion, spreading, growth and movement in chick fibroblasts.

Examination of the actomyosin structures and their relation to adhesion, movement and growth in the first fibroblasts migrating from chick heart explants shows striking differences with fibroblasts adapted to grow in culture. The latter have focal adhesions which seem to immobilize them for anchorage-dependent growth, rather than facilitate their movement. The fibroblasts specialized for movement from the explants, though equally well spread, make contact with substratum through extensive areas of relatively unspecialized membrane, have less well developed stress fibres and a low growth rate.     

Structural and functional features of a cell surface phosphoglycoprotein associated with tumorigenic phenotype in human fibroblast x HeLa cell hybrids

Monoclonal antibodies have been raised against a dimeric cell surface antigen (p75/150) which is specifically associated with the tumorigenic phenotype in human fibroblast X HeLa hybrids. During biosynthesis, a precursor molecule (p70/140), was associated with microsomal membranes in vivo but possessed no detectable cytoplasmic domains. At this stage, each p70 monomer contained 3 "high-mannose" type N-linked glycans which were subsequently processed into endoglycosidase H-insensitive complex oligosaccharides on the mature cell surface forms. Cleavage of this cell surface form with endoglycosidase F yielded non-N-glycosylated polypeptides of Mr = 60,000/120,000. All the monoclonal antibodies identified similar non-N-glycosylated polypeptides in cells grown in the presence of tunicamycin. p75/150 could be weakly labeled with [3H]palmitic or myristic acid. In vivo, p75/150 was found to be phosphorylated on serine residues. Immunoprecipitates of p75/150 from HeLa or tumorigenic hybrid cell lysates exhibited protein kinase activity in vitro, which phosphorylated p75/150 itself, also on serine residues. We were unable to detect this kinase activity in normal fibroblasts and in the nontumorigenic hybrid cells. Furthermore, we were unable to detect p75/150 or its precursors by either cell surface labeling, metabolic labeling, or Western blotting in nontumorigenic cell hybrids; p75/150 thus represents a tumor-specific marker in this system. Tryptic peptides of highly purified p75/150 have been generated, but their amino acid sequences did not reveal any significant homology with previously described proteins.     

Fibronectin glycosylation modulates fibroblast adhesion and spreading

The role of the carbohydrate residues of fibronectin concerning the specificities of that glycoprotein to interact with fibroblastic cell surfaces, gelatin, and heparin was examined. Tunicamycin was used to produce carbohydrate-depleted fibronectin; it was synthesized by cultured fibroblasts. Unglycosylated and glycosylated fibronectins were analyzed for their ability to bind gelatin and heparin, using affinity columns. Fibronectin-coated surfaces were used to quantitatively measure cell adhesion and spreading. The results showed that the lack of carbohydrates significantly increased the interaction of the protein with gelatin and markedly enhanced its ability to promote adhesion and spreading of fibroblasts. In contrast, the binding of fibronectin to heparin was not influenced by glycosylation. The composite data indicate that the Asn-linked oligosaccharides of fibronectin act as modulators of biological functions of the glycoprotein.     

Regulation of fibroblast adhesion by polyreactive antibodies

The effect of polyreactive antibodies on fibroblast adhesion was studied. Dose-dependent decrease of adhesion was found at the stage of adhesive complex formation. A possible mechanism of this effect was determined as a steric influence on integrin agregation and clastering.     

Actomyosin interactions with insulin-storage granules in vitro.

Interactions between actomyosin and insulin storage granules isolated from rat islets of Langerhans have been examined in a simple system in vitro, which allows comparison of the sedimentation of the granules in the presence of absence of actomyosin in various conditions. Actomyosin altered granule-sedimentation rates in a manner consistent with the binding of the granules of actomyosin filaments. This interaction was enhanced by addition of ATP (1.5 mM) but unaltered by addition of CaCl2, by calmodulin or by calmodulin in the presence of 10 microM-CaCl2. Addition of EGTA (0.1 mM), cyclic AMP (10 microM) of cytochalasin B (10 microgram/ml) were also without effects in these conditions. Pre-incubation of granules with phospholipase c did not affect granule-actomyosin interaction. Ultrastructural studies showed close contacts between the membranes of the granules and actomyosin filaments. The results indicate the possibility that actomyosin might provide the motile force for granule translocation during the insulin secretory process.     

An adhesion molecule in free-living Dictyostelium amoebae with integrin beta features

The study of free-living amoebae has proven valuable to explain the molecular mechanisms controlling phagocytosis, cell adhesion and motility. In this study, we identified a new adhesion molecule in Dictyostelium amoebae. The SibA (Similar to Integrin Beta) protein is a type I transmembrane protein, and its cytosolic, transmembrane and extracellular domains contain features also found in integrin beta chains. In addition, the conserved cytosolic domain of SibA interacts with talin, a well-characterized partner of mammalian integrins. Finally, genetic inactivation of SIBA affects adhesion to phagocytic particles, as well as cell adhesion and spreading on its substrate. It does not visibly alter the organization of the actin cytoskeleton, cellular migration or multicellular development. Our results indicate that the SibA protein is a Dictyostelium cell adhesion molecule presenting structural and functional similarities to metazoan integrin beta chains. This study sheds light on the molecular mechanisms controlling cell adhesion and their establishment during evolution.     

Cell surface adhesion receptors

Several new structural motifs found in cell surface adhesion receptors have been described in the past few years. Also, several two-domain structures of extracellular portions of cell surface proteins have been reported. Structural models for complexes between receptors and counter-receptors have been proposed. The first reports on carbohydrate conformation in intact glycoprotein domains have recently appeared. These new data are presented within a more general review of the field of cell adhesion receptors.     

N-WASP plays a critical role in fibroblast adhesion and spreading

N-WASP (Neural Wiskott Aldrich Syndrome Protein) regulates actin polymerization by activating the Arp2/3 complex and promotes the formation of actin-rich structures such as filopodia. Such actin-rich structures play critical roles in cell adhesion and cell motility. Analysis of the adhesion properties of N-WASP^+/+ and N-WASP^−/− mouse embryonic fibroblasts to extracellular matrix proteins revealed that N-WASP is critical for cell adhesion to fibronectin. There was no significant difference in the localization of paxillin in the two cell lines, however the vinculin patches in WASP^+/+ cells were thicker and more prominent than those in N-WASP^−/− cells. The β1 integrins in N-WASP^+/+ cells were found in large clusters, while β1 integrins were more dispersed in N-WASP^−/− cells. The N-WASP^−/− cells migrated more rapidly than N-WASP^+/+ cells in a scratch migration assay. Thus, our data suggest that N-WASP deficiency leads to reduced adhesion to fibronectin and increased cell motility.     

Metabolic changes in fibroblast adhesion sites induced by growth factors

The mechanisms involved in the association between membrane movements and the initiation of DNA synthesis were approached measuring the synthesis of membrane-associated substances after stimulation of resting phase cultures. The inducement of the division cycle by fresh serum, FGF and a growth factor produced during RSV infection, caused a decreased incorporation of glucosamine and SO4 into focal adhesion sites. The decreased incorporation of glucosamine occurred during the G1 period, remained at a plateau during S and increased back to initial levels when DNA synthesis declined; it was inversely related to the size of the fraction of cells that entered the division cycle.     

Fibroblast adhesion to RGDS shows novel features compared with fibronectin

As previously shown by others, the fibroblast attachment and spreading activity of fibronectin is mimicked by a short peptide (RGDS or longer) from the cell binding domain. Normal rat kidney fibroblasts showed similar attachment kinetics on either peptide GRGDSC or bovine plasma fibronectin and binding to either substratum was inhibited by peptide alone. We now demonstrate, however, considerable differences in biological activity between peptide and fibronectin. In particular, cells developed novel adhesion structures on peptide-coated substrata. Interference reflection microscopy showed a predominance of small round dark grey/black patches of adherent membrane ("spots") with relatively few focal adhesions, which occurred only at the outermost cell margins in contrast to their distribution in cells spread on fibronectin. The spots were resistant to detergent extraction and stained less strongly or not at all for vinculin. Electron microscopy in vertical thin section showed that the ventral surface of the cell was characterized by "point-contacts", corresponding in size to the spot structures seen by interference reflection microscopy, and which were only occasionally associated with microfilaments. Cells also required a higher substratum loading of peptide than fibronectin to promote spreading and proceeded to spread less rapidly and to a lesser extent, developing very few and extremely fine actin cables.     

Strengthening relationships: amyloids create adhesion nanodomains in yeasts

Budding yeasts adhere to biotic or abiotic surfaces and aggregate to form biofilms, using wall-anchored glycoprotein adhesins. The process is paradoxical: adhesins often show weak binding to specific ligands, yet mediate remarkably strong adherence. Single-molecule atomic force microscopy (AFM), genomics, biochemistry and cell biology have recently explained the puzzle, with Candida albicans Als adhesins as the paradigm. The strength of adhesion results partly from force-activated amyloid-like clustering of hundreds of adhesin molecules to form arrays of ordered multimeric binding sites. The various protein domains of eukaryotic adhesins cooperate to facilitate this fascinating new mechanism of activation.     

Influence of decorin on fibroblast adhesion to fibronectin.

Decorin is a ubiquitous small dermatan sulfate proteoglycan carrying a single glycosaminoglycan chain. It is known for its ability to bind, via its core protein, to interstitial collagens. Decorin was purified from the secretions of cultured human skin fibroblasts under non-denaturing conditions. The intact proteoglycan and its glycosaminoglycan-free core protein were tested for their interference with fibroblast adhesion to a fibronectin substrate. Concentrations of 40 nmoles or more of hexuronic acid/ml of decorin or equivalent amounts of core protein inhibited cell adhesion. Inhibition was caused by an interaction of core protein with fibronectin and not by masking of the fibronectin receptor. When cell-binding fragments of fibronectin were used as substrates, a similar inhibition of cell adhesion by decorin core protein was found, and in vitro assays demonstrated an interaction of core protein with the cell-binding domain of fibronectin. Decorin core protein also inhibited the low degree of cell adhesion to heparin-binding fragments on the N-terminus and near the C-terminus of the fibronectin molecules.     

Selective eosinophil adhesion to fibroblast via IFN-gamma-induced galectin-9

Among galectin family members, galectin-9 was first described as a potent eosinophil chemoattractant derived from Ag-stimulated T cells. In the present study a role of galectin-9 in the interaction between eosinophils and fibroblasts was investigated using a human lung fibroblast cell line, HFL-1. RT-PCR, real-time PCR, and Western blot analyses revealed that both galectin-9 mRNA and protein in HFL-1 cells were up-regulated by IFN-gamma stimulation. On the one hand, IL-4, known as a Th2 cytokine, did not affect the galectin-9 expression in HFL-1 cells. We further confirmed that IFN-gamma up-regulated the expression of galectin-9 in primary human dermal fibroblasts. Flow cytometric analysis revealed that IFN-gamma up-regulated surface galectin-9 expression on HFL-1 cells. Stimulation of HFL-1 cells with IFN-gamma up-regulated adhesion of eosinophils, but not neutrophils, to HFL-1 cells. This adherence of eosinophils to HFL-1 cells was inhibited by both lactose and anti-galectin-9 Ab. These findings demonstrate that IFN-gamma-induced galectin-9 expression in fibroblasts mediates eosinophil adhesion to the cells, suggesting a crucial role of galectin-9 in IFN-gamma-stimulated fibroblasts as a physiological modulator at the inflammatory sites.     

Stages in specialization of fibroblast adhesion and deposition of extracellular matrix

Fibroblast cells seeded on a serum glycoprotein shown previously to mediate a spread shape without focal adhesions or microfilament bundles (Stage 1 spread) are now shown to have substratum contacts in which coated pits are abundant and associated with small globular deposits of glycocalyx bridging to substratum and staining for fibronectin and acidic glycoconjugates. After stimulation with serum or fibronectin to form focal adhesions and microfilament bundles (Stage 2 spread), clathrin-based structures remain at the cell underside but no longer in conspicuously higher concentration than on the dorsal surface; extracellular material at adhesions is now as regular strands which stain for acidic glycoconjugates but (as reported earlier by Chen and Singer) not always for fibronectin. During these stages of adhesion, striking changes are seen in the cellular display of fibronectin monitored by immunofluorescence. In rounded cells this is granular and cytoplasmic, concentrated around the submembranous cortex; on spreading to Stage 1, it remains granular and intracellular but is now oriented strongly towards the lower cell surface; only in Stage 2 does externalisation proceed to deposit fibrillar fibronectin on the substratum. While cytoplasmic orientation of matrix precursors can be determined by cell contact, organised externalisation is therefore coupled to fully developed adhesion status.     

Transformation-induced alterations in fibroblast adhesion: masking by trypsin treatment.

As assessed by initial rates of cell aggregation, adhesivity of EDTA-suspended cells is increased by SV 40 transformation of mouse 3T3 and human WI 38 fibroblasts. Preparation of cell suspensions by trypsin treatment decreases the aggregation rate and abolishes the difference between transformed and untransformed cells. These findings suggest that increased cellular adhesivity accompanies the surface alterations induced by viral transformation and that trypsinization masks these changes. Thus, use of freshly trypsinized cells in quantitative assessments of cellular adhesivity may yield results which do not reflect the actual cell properties.     

Cell surface carbohydrates in adhesion and migration

Cell surface sugar chains extend away from the cell membraneand offer a first line of contact with approaching cells andsubstrates. These sugars are candidates for mediating cell-celladhesion and migration. Here, I review experiments that implicatecarbohydrate-containing molecules in cell-cell adhesion of ascitestumor and embryonic cells and that correlate the mobility ofcarbohydrate-containing receptor sites in the membrane withcellular migratory activity. The experiments show that L-glutamineis required to form complex carbohydrates implicated in mediatingintercellular adhesion and that a controlling factor in determiningcellular adhesiveness may be the specific activity of intracellularglutamme synthetase. Molecules that promote ascites tumor celladhesion have been isolated. These molecules are large, appearto contain terminal D-galactose residues that bind to cell surfacereceptor sites and consist of more than one component on DEAEcellulose. Studies with sea urchin embryonic cells, utilizingplant lectins that bind to surface carbohydrates, indicate thatcell surface sugar-containing receptor sites change during development.In addition only the micromeres, that become actively migratory,possess mobile cell surface lectin receptor sites. Other seaurchin embryonic cell populations (mesonieres and macromeres)do not exhibit lectin receptor site mobility. Cell surface sugar-containingreceptor sites potentially mediate adhesion and migration inembryos and tumors.