Organization of the migrating chick epiblast edge: attachment sites, cytoskeleton, and early developmental changes

The radial expansion of the chick extraembryonic epiblast on the inner side of the vitelline membrane in yolk sac formation provides a useful system for study of adhesion and migration of an epithelial cell sheet. A band of specialized cells at the epiblast edge adheres by its dorsal side to the overlying vitelline membrane. The attached edge was examined by scanning electron microscopy. The attachment region (av 0.06 mm wide) extends from the advancing edge to a transitional ridge. The ridge appears to be an area of adhesion and de-adhesion. The attached surface is smooth with small surface projections and filopodia. These become more numerous and prominent with cold treatment. Epiblast cells display a filopodial/lamellipodial mode of migration in vivo and in vitro. The distribution of 4- to 7-nm microfilaments in edge cells is examined using transmission electron microscopy of whole cells. Decoration with heavy meromyosin shows that these components of the cytoskeleton contain actin. Treatment of intact blastoderms and dissociated edge cells with cytochalasin B and cold suggests that microfilaments rather than microtubules are primarily responsible for edge cell morphology. Early blastoderm cells which have not initiated migration respond to cytochalasin B, cold, and colcemid in the same way as migrating edge cells. This suggests that the differentiative change that produces the rapidly migrating edge cells does not involve a shift in the relative contribution of microtubules and microfilaments to the cytoskeleton.     

Functional domains of proteoliaisin, the adhesive protein that orchestrates fertilization envelope assembly

Ovoperoxidase, the enzyme that hardens the sea urchin fertilization envelope, is inserted into the assembling extracellular matrix through the action of an intermediary protein, proteoliaisin (PLN). The domain structure of PLN, a large, rod-shaped protein that binds to ovoperoxidase and the vitelline layer, was examined by limited proteolytic cleavage. Purified proteolytic fragments of PLN were tested for their ability to bind ovoperoxidase, inhibit the binding of 125I-PLN to the vitelline layer, or act as substrates for the hardening reaction. Based on these results, the vitelline layer-binding domain can be placed near the amino terminus, followed by the binding site for ovoperoxidase; the distal two-thirds of the protein contain sites for ovoperoxidase-catalyzed dityrosine formation. The pentapeptide GRGDS (but not RGD) inhibited PLN-vitelline layer binding half-maximally at 0.2 mM. Moreover, PLN promoted adhesion of bovine aortic endothelial cells to plastic dishes, a process inhibited by GRGDS. Thus PLN is a new member of the adhesive protein family, the function of which is to coordinate the morphogenesis of a specific, rapidly assembled extracellular matrix.     

Role of integrin receptors in manganese-dependent BHK cell spreading on albumin-coated substrata.

In manganese-containing medium, tissue cells can spread on albumin and other substrata typically nonadhesive for cells in calcium/magnesium-containing medium. To learn whether integrin receptors play a role in Mn-dependent adhesion, we tested the effects of RGD peptides and polyclonal anti-fibronectin receptor antibodies on BHK cell spreading on fibronectin and albumin-coated substrata. In Ca/Mg-containing medium on fibronectin substrata, the RGD-related peptides GRG-DSP and GRGDS but not RGDS inhibited cell spreading. In Mn-containing medium, spreading on albumin was inhibited by GRGDSP and GRGDS and also by RGDS. GRGESP, on the other hand, did not inhibit cell spreading under any condition tested. Antibodies directed against fibronectin receptors also inhibited Mn-dependent cell spreading on albumin substrata, but higher levels of antibody were required than were necessary to inhibit Ca/Mg-dependent spreading on fibronectin. On the basis of these results, we suggest that integrin receptors, but probably not fibronectin receptors, mediate Mn-dependent BHK cell spreading on albumin.     

Scanning electron microscopy of chick epiblast expansion on the vitelline membrane. Cell-substrate interactions

Cell-substrate interactions have been studied by examining migrating edge cells of the expanding chick extraembryonic epiblast on their normal substrate and in culture. Scanning electron microscopy shows that the outer face of the vitelline membrane is a random meshwork of fibrils (80 nm diam). The inner face, which is the normal substrate of epiblast expansion, is composed of a random branched system of fibers (400 nm diam) overlain by a network of fibrils (40 nm diam). The epiblast edge in situ has radially oriented filopodia (20 μm long, 200 nm diam.), frequently extending from broad lamellipodia. Blastoderms cultured on the inner face of unincubated vitelline membrane expand at a normal rate but display ruffles as well as filopodia and lamellipodia. When the blastoderm is cultured on the outer membrane face there is no expansion, but cells leave the edge and migrate across the membrane. In these cultures, ruffles are observed on the ventral epiblast face. Absence of the mass of yolk in culture appears to permit or provoke the observed ruffling. Comparison of dissociated epiblast edge cells and skin epithelial cells, cultured on glass and on the vitelline membrane inner face, indicates that epiblast cells remain flattened and display characteristic filopodia on both substrates, whereas skin cells display ruffles on the vitelline membrane but are flattened on glass. The mode of migration of epiblast edge cells seems to be more dependent on intrinsic factors than that of skin cells.     

Monoclonal antibody characterization of two distant sites required for function of the central cell-binding domain of fibronectin in cell adhesion, cell migration, and matrix assembly

Site-directed mutagenesis studies have suggested that additional peptide information in the central cell-binding domain of fibronectin besides the minimal Arg-Gly-Asp (RGD) sequence is required for its full adhesive activity. The nature of this second, synergistic site was analyzed further by protein chemical and immunological approaches using biological assays for adhesion, migration, and matrix assembly. Fragments derived from the cell-binding domain were coupled covalently to plates, and their specific molar activities in mediating BHK cell spreading were compared with that of intact fibronectin. A 37-kD fragment purified from chymotryptic digests of human plasma fibronectin had essentially the same specific molar activity as intact fibronectin. In contrast, other fragments such as an 11.5-kD fragment lacking NH2-terminal sequences of the 37-kD fragment had only poor spreading activity on a molar basis. Furthermore, in competitive inhibition assays of fibronectin-mediated cell spreading, the 37-kD fragment was approximately 325-fold more active than the GRGDS synthetic peptide on a molar basis. mAbs were produced using the 37-kD protein as an immunogen and their epitopes were characterized. Two separate mAbs, one binding close to the RGD site and the other to a site approximately 15 kD distant from the RGD site, individually inhibited BHK cell spreading on fibronectin by greater than 90%. In contrast, an antibody that bound between these two sites had minimal inhibitory activity. The antibodies found to be inhibitory in cell spreading assays for BHK cells also inhibited both fibronectin-mediated cell spreading and migration of human HT-1080 cells, functions which were also dependent on function of the alpha 5 beta 1 integrin (fibronectin receptor). Assembly of endogenously synthesized fibronectin into an extracellular matrix was not significantly inhibited by most of the anti-37-kD mAbs, but was strongly inhibited only by the antibodies binding close to the RGD site or the putative synergy site. These results indicate that a second site distant from the RGD site on fibronectin is crucial for its full biological activity in diverse functions dependent on the alpha 5 beta 1 fibronectin receptor. This site is mapped by mAbs closer to the RGD site than previously expected.     

Integrin-like Protein Is Involved in the Osmotic Stress-induced Abscisic Acid Biosynthesis in Arabidopsis thaliana

We studied the perception of plant cells to osmotic stress that leads to the accumulation of abscisic acid （ABA） in stressed Arabidopsis thaliana L. cells. A significant difference was found between protoplasts and cells in terms of their responses to osmotic stress and ABA biosynthesis, implying that cell wall and/or cell wall-plasma membrane interaction are essential in identifying osmotic stress. Western blotting and immunofluorescence localization experiments, using polyclonal antibody against human integrin β1, revealed the existence of a protein similar to the integrin protein of animals in the suspension-cultured cells located in the plasma membrane fraction. Treatment with a synthetic pentapeptide, Gly-Arg-Gly-Asp-Ser （GRGDS）, which contains an RGD domain and interacts specifically with integrin protein and thus blocks the cell wall-plasma membrane interaction, significantly inhibited osmotic stress-induced ABA biosynthesis in cells, but not in protoplasts. These results demonstrate that cell wall and/or cell wall-plasma membrane interaction mediated by integrin-Iike proteins played important roles in osmotic stress-induced ABA biosynthesis in Arabidopsis thaliana.     

Polarity, protrusion-retraction dynamics and their interplay during keratinocyte cell migration.

Keratinocyte migration on a two-dimensional substrate can be split into four distinct phases: cell extension, attachment, contraction, and detachment. It is preceded by polarization of the cell which leads to a functional asymmetry observable by the formation of a leading lamella. In this work variation of fibronectin coating concentrations and competitive inhibition with RGD peptides are used to investigate the dependency of polarization, migration, lamella dynamics, and ruffling on substrate adhesiveness. Looking at migrating human epidermal keratinocytes with a well-defined polarity we find that a fibronectin-coating concentration of 10 microg/cm(2) stimulates migration and ruffling speed twofold, whereas protrusion speed increases only by 20% (compared to 2.5 microg/cm(2) fibronectin). Nonpolar cells show a constant migration and ruffling speed independent of the amount of fibronectin. In contrast protrusion speeds of polar and nonpolar cells are equal. Treatment of cells on 10 microg/cm(2) fibronectin with 1 mg/ml GRGDS reduces the characteristic migration, protrusion, and ruffling speed of polar cells which corresponds to lowering the effective coating concentration to under 5 microg/cm(2). The probability of being polarized (quantified by a polarity index) increases with increasing fibronectin concentration. However, addition of soluble RGD on 10 microg/cm(2) fibronectin does not simply reduce the polarity index like one would expect from the corresponding changes in the other motility parameters, but it remains unchanged.     

A peptide containing the cell adhesion sequence RGD can mediate degranulation and cell adhesion of crayfish granular haemocytes in vitro

The synthetic peptide Gly-Arg-Gly-Asp-Ser (GRGDS) (which corresponds to a fragment of fibronectin and contains its cell adhesion sequence RGD) caused degranulation and spreading of monolayers of isolated granular haemocytes of the crayfish Pacifastacus leniusculus in vitro. When coated on glass coverslips, this RGD-containing peptide could mediate cell attachment of granular cells in vitro. A control peptide, Gly-Arg-Gly-Glu-Ser (GRGES), did not have these activities.Thus, GRGDS imitates the biological activities in vitro of the cell adhesion factor recently purified from crayfish haemocytes. This suggests that the sequence Arg-Gly-Asp (RGD), which is responsible for the cell adhesion activities of a number of vertebrate proteins, may also be involved in degranulation and cell adhesion of arthropod haemocytes.This is the first report describing direct activities by an RGD-containing peptide towards invertebrate cells in vitro, and the first indication of the presence of an RGD-recognizing receptor on invertebrate haemocytes.     

Cell surface proteoglycan binds mouse mammary epithelial cells to fibronectin and behaves as a receptor for interstitial matrix

The proteoglycan (PG) on the surface of NMuMG mouse mammary epithelial cells consists of at least two functional domains, a membrane- intercalated domain which anchors the PG to the plasma membrane, and a trypsin-releasable ectodomain which bears both heparan and chondroitin sulfate chains. The ectodomain binds cells to collagen types I, III, and V, but not IV, and has been proposed to be a matrix receptor. Because heparin binds to the adhesive glycoproteins fibronectin, an interstitial matrix component, and laminin, a basal lamina component, we asked whether the cell surface PG also binds these molecules. Cells harvested with either trypsin or EDTA bound to fibronectin; binding of trypsin-released cells was inhibited by the peptide GRGDS but not by heparin, whereas binding of EDTA-released cells was inhibited only by a combination of GRDS and heparin, suggesting two distinct cell binding mechanisms. In the presence of GRGDS, the EDTA-released cells bound to fibronectin via the cell surface PG. Binding via the cell surface PG was to the COOH-terminal heparin binding domain of fibronectin. In contrast with the binding to fibronectin, EDTA-released cells did not bind to laminin under identical assay conditions. Liposomes containing the isolated intact cell surface PG mimic the binding of whole cells. These results indicate that the mammary epithelial cells have at least two distinct cell surface receptors for fibronectin: a trypsin- resistant molecule that binds cells to the sequence RGD and a trypsin- labile, heparan sulfate-rich PG that binds cells to the COOH-terminal heparin binding domain. Because the cell surface PG binds cells to the interstitial collagens (types I, III, and V) and to fibronectin, but not to basal lamina collagen (type IV) or laminin, we conclude that the cell surface PG is a receptor on epithelial cells specific for interstitial matrix components.     

Non-RGD domains of osteopontin promote cell adhesion without involving αv integrins

Osteopontin (OPN) is an integrin-binding secreted protein that contains an Arg-Gly-Asp (RGD) amino acid sequence and binds to various cell types via RGD-mediated interaction with the αvβ3 integrin. We have identified a cell line whose binding to OPN does not require RGD or αv interactions. We compared the ability of two murine cell lines, L929 fibroblastic cells and B16-BL6 melanoma cells, to interact with OPN (from human milk, and recombinant human and mouse OPN) as well as recombinant OPN prepared to include either the N-terminal or C-terminal halves but lacking the RGD sequence. Both cell lines adhered to GRGDS peptides coupled to BSA, and these interactions were inhibited by addition of GRGDS (but not GRGES) peptides or a monoclonal antibody specific to the αv integrin subunit. Adhesion of L929 cells to OPN was also dependent on the RGD sequence and the αv integrin subunit. However, the binding of B16-BL6 cells was not inhibited by either GRGDS peptides or the anti-αv antibody. B16-BL6 (but not L929) cells were also able to adhere to and spread on both N-terminal and C-terminal OPN proteins that lack the RGD sequence, and these interactions were not inhibited by either GRGDS peptides or anti-αv antibody. Together these results indicate that B16-BL6 cells can adhere to OPN by interactions that are independent of either the RGD sequence or the αv integrin subunit, and suggest that some cells can interact with additional, non-RGD binding sites in OPN. © 1996 Wiley-Liss, Inc.     

The synergy peptide PHSRN and the adhesion peptide RGD mediate cell adhesion through a common mechanism

This work reports on the role of the synergy peptide PHSRN in mediating the adhesion of cells. The attachment of baby hamster kidney cells and 3T3 Swiss fibroblasts to model substrates presenting either GRGDS or PHSRN was evaluated using self-assembled monolayers of alkanethiolates on gold presenting the peptide ligands mixed with tri(ethylene glycol) groups. These substrates permit rigorous control over the structures and densities of peptide ligands and at the same time prevent nonspecific interactions with adherent cells. Both cell types attached efficiently to monolayers presenting either the RGD or the PHSRN peptide but not to monolayers presenting scrambled peptide GRDGS or HRPSN. Cell attachment was comparable on substrates presenting either peptide ligand but less efficient than on substrates presenting the protein fibronectin. The degree of cell spreading, however, was substantially higher on substrates presenting RGD relative to PHSRN. Staining of 3T3 fibroblasts with anti-vinculin and phalloidin revealed clear cytoskeletal filaments and focal adhesions for cells attached by way of either RGD or PHSRN. Inhibition experiments showed that the attachment of 3T3 fibroblasts to monolayers presenting RGD could be inhibited completely by a soluble RGD peptide and partially by a soluble PHSRN peptide. IMR 90 fibroblast attachment to monolayers presenting PHSRN could be inhibited with anti-integrin alpha(5) or anti-integrin beta(1) antibody. This work demonstrates unambiguously that PHSRN alone can support the attachment of cells and that the RGD and PHSRN bind competitively to the integrin receptors.     

Intracellular localization of integrin-like protein and its roles in osmotic stress-induced abscisic acid biosynthesis in Zea mays

Summary. Plants have evolved many mechanisms to cope with adverse environmental stresses. Abscisic acid (ABA) accumulates significantly in plant cells in response to drought conditions, and this is believed to be a major mechanism through which plants enhance drought tolerance. In this study, we explore the possible mechanisms of osmotic stress perception by plant cells and the consequent induction of ABA biosynthesis. Immunoblotting and immunofluorescence localization experiments, using a polyclonal antibody against human integrin β1, revealed the presence of a protein in Zea mays roots that is similar to the integrin proteins of animals and mainly localized in the plasma membrane. Treatment with GRGDS, a synthetic pentapeptide containing an RGD domain, which interacted specifically with the integrin protein and thus blocked the cell wall–plasma membrane interaction, significantly inhibited osmotic stress-induced ABA biosynthesis in cells, and the GRGDS analog which does not contain the RGD domain had no effect. Our results show that a strong interaction exists between the cell wall and plasma membrane and that this interaction is largely mediated by integrin-like proteins. They also imply that the cell wall and/or cell wall–plasma membrane interaction plays important roles in the perception of osmotic stress. Accordingly, we conclude that the cell wall and/or cell wall–plasma membrane interaction mediated by the integrin-like protein plays important roles in osmotic stress-induced ABA biosynthesis in Zea mays. Correspondence: J. S. Liang, College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225009, People’s Republic of China.     

Polarity, Protrusion–Retraction Dynamics and Their Interplay during Keratinocyte Cell Migration

Keratinocyte migration on a two-dimensional substrate can be split into four distinct phases: cell extension, attachment, contraction, and detachment. It is preceded by polarization of the cell which leads to a functional asymmetry observable by the formation of a leading lamella. In this work variation of fibronectin coating concentrations and competitive inhibition with RGD peptides are used to investigate the dependency of polarization, migration, lamella dynamics, and ruffling on substrate adhesiveness. Looking at migrating human epidermal keratinocytes with a well-defined polarity we find that a fibronectin-coating concentration of 10 μg/cm² stimulates migration and ruffling speed twofold, whereas protrusion speed increases only by 20% (compared to 2.5 μg/cm² fibronectin). Nonpolar cells show a constant migration and ruffling speed independent of the amount of fibronectin. In contrast protrusion speeds of polar and nonpolar cells are equal. Treatment of cells on 10 μg/cm² fibronectin with 1 mg/ml GRGDS reduces the characteristic migration, protrusion, and ruffling speed of polar cells which corresponds to lowering the effective coating concentration to under 5 μg/cm². The probability of being polarized (quantified by a polarity index) increases with increasing fibronectin concentration. However, addition of soluble RGD on 10 μg/cm² fibronectin does not simply reduce the polarity index like one would expect from the corresponding changes in the other motility parameters, but it remains unchanged.     

Inhibition of in vitro tumor cell invasion by Arg-Gly-Asp-containing synthetic peptides [published erratum appears in J Cell Biol 1989 Jun;108(6):following 2546]

The interaction of cells with extracellular matrix components such as fibronectin, vitronectin, and type I collagen has been shown to be mediated through a family of cell-surface receptors that specifically recognize an arginine-glycine-aspartic acid (RGD) amino acid sequence within each protein. Synthetic peptides containing the RGD sequence can inhibit these receptor-ligand interactions. Here, we use novel RGD-containing synthetic peptides with different inhibition properties to investigate the role of the various RGD receptors in tumor cell invasion. The RGD-containing peptides used include peptides that inhibit the attachment of cells to fibronectin and vitronectin, a peptide that inhibits attachment to fibronectin but not to vitronectin, a cyclic peptide with the opposite specificity, and a peptide, GRGDTP, that inhibits attachment to type I collagen in addition to inhibiting attachment to fibronectin and vitronectin. The penetration of two human melanoma cell lines and a glioblastoma cell line through the human amniotic basement membrane and its underlying stroma was inhibited by all of the RGD-containing peptides except for the one that inhibits only the vitronectin attachment. Various control peptides lacking RGD showed essentially no inhibition. This inhibitory effect on cell invasion was dose-dependent and nontoxic. A hexapeptide, GRGDTP, that inhibits the attachment of cells to type I collagen in addition to inhibiting fibronectin- and vitronectin-mediated attachment was more inhibitory than those RGD peptides that inhibit only fibronectin and vitronectin attachment. Analysis of the location of these cells that were prevented from invading indicated that they attached to the amniotic basement membrane but did not proceed further into the tissue. These results suggest that interactions between RGD-containing extracellular matrix adhesion proteins and cells are necessary for cell invasion through tissues and that fibronectin and type I collagen are important for this process.     

Site-directed mutagenesis of the arginine-glycine-aspartic acid sequence in osteopontin destroys cell adhesion and migration functions

Osteopontin (OPN) is a secreted calcium-binding phosphoprotein produced in a variety of normal and pathological contexts, including tissue mineralization and cancer. OPN contains a conserved RGD (arg-gly-asp) amino acid sequence that has been implicated in binding of OPN to cell surface integrins. To determine whether the RGD sequence in OPN is required for adhesive and chemotactic functions, we have introduced two site-directed mutations in the RGD site of the mouse OPN cDNA, in which the RGD sequence was either deleted or mutated to RGE (arg-gly-glu). In order to test the effect of these mutations on OPN function, we expressed control and mutated mouse OPN in E. coli as recombinant glutathione-S-transferase (GST)-OPN fusion proteins. Control mouse GST-OPN was functional in cell adhesion assays, supporting attachment and spreading of mouse (malignant PAP2 ras-transformed NIH 3T3, and, to a lesser extent, normal NIH 3T3 fibroblasts) and human (MDA-MB-435 breast cancer, and normal gingival fibroblast) cells. In contrast, neither of the RGD-mutated OPN proteins (“delRGD” or “RGE”) supported adhesion of any of the cell lines, even when used at high concentrations or for long assay times. GRGDS (gly-arg-gly-asp-ser) peptides inhibited cell adhesion to intact GST-OPN, as well as to fibronectin and vitronectin. In chemotaxis assays, GST-OPN promoted directed cell migration of both malignant (PAP2, MDA-MB-435) and normal (gingival fibroblast, and NIH 3T3) cells, while RGD-mutated OPN proteins did not. Together these results suggest that the conserved RGD sequence in OPN is required for the majority of the protein's cell attachment and migration-stimulating functions.     

Dynamic cytoskeleton-integrin associations induced by cell binding to immobilized fibronectin

We have examined the early events of cellular attachment and spreading (10-30 min) by allowing chick embryonic fibroblasts transformed by Rous sarcoma virus to interact with fibronectin immobilized on matrix beads. The binding activity of cells to fibronectin beads was sensitive to both the mAb JG22E and the GRGDS peptide, which inhibit the interaction between integrin and fibronectin. The precise distribution of cytoskeleton components and integrin was determined by immunocytochemistry of frozen thin sections. In suspended cells, the distribution of talin was diffuse in the cytoplasm and integrin was localized at the cell surface. Within 10 min after binding of cells and fibronectin beads at 22 degrees C or 37 degrees C, integrin and talin aggregated at the membrane adjacent to the site of bead attachment. In addition, an internal pool of integrin-positive vesicles accumulated. The mAb ES238 directed against the extracellular domain of the avian beta 1 integrin subunit, when coupled to beads, also induced the aggregation of talin at the membrane, whereas ES186 directed against the intracellular domain of the beta 1 integrin subunit did not. Cells attached and spread on Con A beads, but neither integrin nor talin aggregated at the membrane. After 30 min, when many of the cells were at a more advanced stage of spreading around beads or phagocytosing beads, alpha-actinin and actin, but not vinculin, form distinctive aggregates at sites along membranes associated with either fibronectin or Con A beads. Normal cells also rapidly formed aggregates of integrin and talin after binding to immobilized fibronectin in a manner that was similar to the transformed cells, suggesting that the aggregation process is not dependent upon activity of the pp60v-src tyrosine kinase. Thus, the binding of cells to immobilized fibronectin caused integrin-talin coaggregation at the sites of membrane-ECM contact, which can initiate the cytoskeletal events necessary for cell adhesion and spreading.     

Triflavin, an Arg-Gly-Asp-containing peptide, inhibits human cervical carcinoma (HeLa) cell-substratum adhesion through an RGD-dependent mechanism

Triflavin, a 7.5-kDa cysteine-rich polypeptide purified from Trimeresurus flavoviridis snake venom, belongs to a family of RGD-containing peptides, termed disintegrins, that have been isolated from the venoms of various vipers and shown to be potent inhibitors of platelet aggregation. The interaction of tumor cells with extracellular matrices such as fibronectin, vitronectin, and collagen has been shown to be mediated through a family of cell surface receptors that specifically recognize an arginine-glycine-aspartic acid (RGD) sequence within each adhesive protein. In this study, we show that triflavin dose-dependently inhibited adhesion of human cervical carcinoma (HeLa) cells to extracellular matrices (ECMs; i.e., fibronectin, fibrinogen, and vitronectin). On the other hand, triflavin exerted a limited inhibitory effect on cell adhesion to laminin and collagen (type I and IV). On a molar basis, triflavin is approximately 800 times more potent than Gly-Arg-Gly-Asp-Ser (GRGDS) at inhibiting cell adhesion. When immobilized on plate, triflavin significantly promoted HeLa cell adhesion, and this attachment was inhibited by GRGDS. Furthermore, FITC-conjugated triflavin bound to cells in a saturable manner and its binding was inhibited by GRGDS. In addition, triflavin did not affect [³H]thymidine uptake of HeLa cells during a 3-day incubation. These results suggest that triflavin probably binds to integrin receptors expressed on HeLa cell surface via its RGD sequence within its molecule, thereby inhibiting the adhesion of extracellular matrices to HeLa cells.     

Positional information by Rauber's sickle and a new look at the mechanisms of primitive streak initiation in avian blastoderms

The present experimental in vitro study suggests that a primitive streak (PS) in avian blastoderms is induced by diffusion of morphogenetic substances emanating from Rauber's sickle. Indeed, even without direct contact between a quail Rauber's sickle and the reacting upper layer (by interposition of a vitelline membrane), a PS can be induced in the isolated area centralis or antisickle region of unincubated chicken blastoderms. The so-formed PSs are localized below the vitelline membrane in the immediate neighborhood of the apposed Rauber's sickle material. This seems to indicate that Rauber's sickle organizes the formation of the avian PS according to the basic concept of "positional information." The morphogenetic substances seem to have an effect only on the formation of a PS. Each part of Rauber's sickle seems to have, point by point, the same thickening and PS-inducing effect on each corresponding part of the underlying upper layer (UL). By a mechanism of sliding over the basement membrane and fusion, this finally results in the formation of one single median PS. Our study shows that a PS can be induced in the total absence of hypoblast (sickle endoblast) or caudal marginal zone, by only the presence of Rauber's sickle material. In contrast, the differentiation of mesoblast into blood islands under the influence of Rauber's sickle and neural tissue development are impaired by the interposition of a vitelline membrane. The latter could be due to the absence of a normal interaction of Rauber's sickle-derived sickle endoblast with endophyll and/or upper layer and the absence of cranial migration of the mesoblast. Thus, earlier studies and the present study indicate the existence of a temporospatially bound cascade of gastrulation and neurulation phenomena and blood island formation in the avian blastoderm, starting from Rauber's sickle, the primary major organizer with inducing, inhibiting, and dominating potencies. The latter not only plays a role by secretion of signaling molecules, but also influences development by its cell lineages (junctional endoblast and sickle endoblast).     

VAMP3 regulates podosome organisation in macrophages and together with Stx4/SNAP23 mediates adhesion, cell spreading and persistent migration

The ability of cells to adhere, spread and migrate is essential to many physiological processes, particularly in the immune system where cells must traffic to sites of inflammation and injury. By altering the levels of individual components of the VAMP3/Stx4/SNAP23 complex we show here that this SNARE complex regulates efficient macrophage adhesion, spreading and migration on fibronectin. During cell spreading this complex mediates the polarised exocytosis of VAMP3-positive recycling endosome membrane into areas of membrane expansion, where VAMP3's surface partner Q-SNARE complex Stx4/SNAP23 was found to accumulate. Lowering the levels of VAMP3 in spreading cells resulted in a more rounded cell morphology and most cells were found to be devoid of the typical ring-like podosome superstructures seen normally in spreading cells. In migrating cells lowering VAMP3 levels disrupted the polarised localisation of podosome clusters. The reduced trafficking of recycling endosome membrane to sites of cell spreading and the disorganised podosome localisation in migrating macrophages greatly reduced their ability to persistently migrate on fibronectin. Thus, this important SNARE complex facilitates macrophage adhesion, spreading, and persistent macrophage migration on fibronectin through the delivery of VAMP3-positive membrane with its cargo to expand the plasma membrane and to participate in organising adhesive podosome structures.     

In vitro studies on the expansion of endothelial cell monolayers on components of the basement membrane

The purpose of the present study was to observe the expansion of a monolayer of endothelial cells over specific components of the basement membrane. This was performed in vitro in a monolayer expansion assay over 5 days. The control surface was uncoated glass in the form of coverslips. Test substances were coated at a concentration of 10 micrograms/ml. The highest expansion was obtained with a high molecular weight fragment mixture of collagen type IV (IV-F, consisting of 75, 120 and 140 KD fragments), followed by fibronectin. Collagens type I, III and IV tetramer gave similar results, less than fibronectin or collagen type IV-F, although all of the above basement membrane coatings promoted expansion significantly above that of the control (P less than 0.01). The poorest expansion was obtained with laminin, which was significantly less than the control. The pentapeptide GRGDS, related to the fibronectin cell binding region, gave expansion significantly below that of the intact fibronectin molecule, as did the intact collagen type IV molecule compared with type IV-F (P less than 0.025). This indicates that sequences of the fibronectin molecule other than the cell binding sequence may be involved in promoting endothelial cell expansion. In addition, the integrity of the collagen type IV molecule does not appear necessary for this effect. On the contrary, the higher movement on IV-F may represent an inherent repair mechanism in damaged endothelium. Autoradiographic studies show that endothelial cell proliferation at the expanding front is involved in the migration assay.