Cell adhesion strengthening: contributions of adhesive area, integrin binding, and focal adhesion assembly

Mechanical interactions between a cell and its environment regulate migration, contractility, gene expression, and cell fate. We integrated micropatterned substrates to engineer adhesive area and a hydrodynamic assay to analyze fibroblast adhesion strengthening on fibronectin. Independently of cell spreading, integrin binding and focal adhesion assembly resulted in rapid sevenfold increases in adhesion strength to steady-state levels. Adhesive area strongly modulated adhesion strength, integrin binding, and vinculin and talin recruitment, exhibiting linear increases for small areas. However, above a threshold area, adhesion strength and focal adhesion assembly reached a saturation limit, whereas integrin binding transitioned from a uniform distribution to discrete complexes. Adhesion strength exhibited exponential increases with bound integrin numbers as well as vinculin and talin recruitment, and the relationship between adhesion strength and these biochemical events was accurately described by a simple mechanical model. Furthermore, adhesion strength was regulated by the position of an adhesive patch, comprised of bound integrins and cytoskeletal elements, which generated a constant 200-nN adhesive force. Unexpectedly, focal adhesion assembly, in particular vinculin recruitment, contributed only 30% of the adhesion strength. This work elucidates the roles of adhesive complex size and position in the generation of cell-extracellular matrix forces.     

Localization of a cryptic binding site for tenascin on fibronectin

Fibronectin and tenascin are large extracellular matrix proteins that interact with each other and with integrin receptors to regulate cell growth and movement. They are both modular proteins composed of independently folded domains (modules) that are arranged in linear fashion. Fibronectin is a covalent dimer and tenascin is a hexamer. The site on tenascin to which fibronectin binds has been localized to type III modules 3-5. In this study we use surface plasmon resonance to examine the interaction between various fragments of fibronectin and tenascin to further characterize and localize the binding sites. We found that tenascin fragments that contain type III modules 3-5 bind primarily to the N-terminal 29-kDa hep-1/fib-1 domain, which contains the first five type I modules of fibronectin. The dissociation constant, K(d), is approximately 1 microm. The binding site on fibronectin appears to be cryptic in the whole molecule in solution but is exposed on the proteolytic fragments and probably when fibronectin is in the extended conformation.     

Latent TGF-beta binding protein LTBP-2 decreases fibroblast adhesion to fibronectin

We have analyzed the effects of latent TGF-beta binding protein 2 (LTBP-2) and its fragments on lung fibroblast adhesion. Quantitative cell adhesion assays indicated that fibroblasts do not adhere to full-length LTBP-2. Interestingly, LTBP-2 had dominant disrupting effects on the morphology of fibroblasts adhering to fibronectin (FN). Fibroblasts plated on LTBP-2 and FN substratum exhibited less adherent morphology and displayed clearly decreased actin stress fibers than cells plated on FN. These cells formed, instead, extensive membrane ruffles. LTBP-2 had no effects on cells adhering to collagen type I. Fibroblasts adhered weakly to the NH2-terminal fragment of LTBP-2. Unlike FN, this fragment did not augment actin stress fiber formation. Interestingly, the adhesion-mediating and cytoskeleton-disrupting effects were localized to the same NH2-terminal proline-rich region of LTBP-2. LTBP-2 and its antiadhesive fragment bound to FN in vitro, and the antiadhesive fragment associated with the extracellular matrix FN fibrils. These observations reveal a potentially important role for LTBP-2 as an antiadhesive matrix component.     

Mechanism for transition from initial to stable cell-cell adhesion: kinetic analysis of E-cadherin-mediated adhesion using a quantitative adhesion assay

A centrifugal force-based adhesion assay has been used to quantitatively examine the kinetics of formation of cell-cell contacts mediated specifically by expression of E-cadherin under the control of a glucocorticoid-inducible promoter in mouse fibroblasts. Analysis of cells expressing maximal or minimal levels of E-cadherin showed that the strength of E-cadherin-mediated adhesion developed in a single exponential step over a short time (half-maximal adhesion, 13-17 min). At 37 degrees C, adhesion strength increased rapidly in the first 20 min without an apparent lag phase. After 90 min, adhesion strength reached a plateau. Differences in final strengths of adhesion were commensurate with the level of E-cadherin expression. Strengthening of adhesion was temperature dependent. At 19 degrees C, strengthening of adhesion was delayed and subsequently developed with a slower rate compared to adhesion at 37 degrees C. At 4 degrees C, adhesion was completely inhibited. Strengthening of adhesion was absolutely dependent on a functional actin cytoskeleton since adhesion did not develop when cells were treated with cytochalasin D. Together, our current and previous (McNeill et al., 1993.J. Cell Biol. 120:1217-1226) studies indicate that the rate of initial strengthening of E-cadherin- mediated adhesion is neither dependent on the amount of E-cadherin expressed nor on long-range protein diffusion in the membrane to the adhesion site. However, initial strengthening of adhesion is dependent on temperature-sensitive cellular activities that may locally couple clusters of E-cadherin to the actin cytoskeleton.     

Kinase-dependent adhesion to fibronectin: regulation by calreticulin

We studied the phosphorylation (activation status) of c-Src and CaMKII in MEFs either wild type for calreticulin, calreticulin-null, or rescued with full-length calreticulin. We found that calreticulin-null cells were poorly spread on the substratum and formed few, if any, focal contacts. Fibronectin expression and deposition were lower in calreticulin-null MEFs compared to calreticulin-expressing cells, which also exhibited increased c-Src and CaMKII phosphorylation (activity). Plating MEFs on preformed fibronectin rescued the poor adhesive phenotype of calreticulin-null cells, and caused a decrease in c-Src Y418 phosphorylation (activity). c-Src inhibition caused the calreticulin-null MEFs to become well spread on the substratum and to make many prominent focal contacts. Calmodulin and CaMKII inhibition caused similar results, along with a notable increase in paxillin phosphorylation (activation). To test if the calcium storage function of calreticulin was responsible for these effects, we manipulated intracellular [Ca(2+)]. Lowering [Ca(2+)](ER) caused an increase in c-Src phosphorylation and a decrease in fibronectin abundance. Conversely, increasing [Ca(2+)] caused opposite effects. These results suggest that calreticulin regulates both the c-Src and calmodulin/CaMKII pathways, enabling cells to be better spread on the substratum by allowing greater fibronectin deposition and increased focal contact formation.     

Involvement of gangliosides and glycoprotein fibronectin receptors in cellular adhesion to fibronectin

We have used a rat neural cell line, B65, to investigate the relative contributions of gangliosides and glycoprotein receptors in adhesion to fibronectin. Monoclonal antibodies against two neuroectoderm-associated gangliosides, D1.1 and GD3, inhibit the rate of B65 attachment to fibronectin, suggesting that these gangliosides are involved in the adhesion process. Adhesion to fibronectin is not affected by a third monoclonal antibody against a separate, unidentified cell-surface component of B65 cells. Furthermore, B65 cells lacking D1.1 adhere to fibronectin at a slower rate than B65 cells that express D1.1. The involvement of glycoprotein receptors in adhesion is demonstrated by the ability of antibodies against human fibronectin receptor to inhibit B65 attachment to fibronectin. In addition, adhesion is blocked by a hexapeptide containing the Arg-Gly-Asp fibronectin sequence which is necessary for binding to the receptor. Trypsin treatment of B65 cells in the absence of divalent cations results in proteolysis of the fibronectin receptor with an accompanying loss of ability of the cells to attach to fibronectin. D1.1 and GD3 expression is not affected by this trypsinization, indicating that the gangliosides alone are incapable of mediating attachment. The glycoprotein receptors must be primarily responsible for adhesion to fibronectin with the gangliosides playing a secondary role as enhancers or modulators.     

Inhibition of fibronectin binding to platelets by proteolytic fragments and synthetic peptides which support fibroblast adhesion

To define regions within fibronectin (Fn) recognized by platelet binding sites, inhibition of Fn binding by an Fn fragment and synthetic peptides has been analyzed. A highly purified 120-kDa chymotryptic fragment, which has cell attachment activity but did not bind to insolubilized heparin or gelatin, inhibited Fn binding to platelets with an ID50 approximately 3 microM. Previous work indicates that fibroblasts attach to an 11.5-kDa subfragment of this 120-kDa fragment, and that one of four 30-residue synthetic peptides containing sequences from this region supports cell attachment. Only the peptide containing the COOH terminus of the 11.5-kDa fragment inhibited Fn binding to platelets, with an ID50 approximately 10 microM and is the peptide which supports fibroblast attachment. Of the smaller peptides studied from this sequence, all peptides containing the Arg-Gly-Asp-Ser sequence, including the tetrapeptide itself, were active in inhibiting Fn binding to platelets (ID50 values approximately 10-20 microM). The same peptides support fibroblast attachment. Those which lacked this sequence including Gly-Asp-Ser-Pro and Thr-Gly-Arg-Gly (immediately adjacent tetrapeptides) lacked both activities. Further evidence for specificity of inhibition was provided by structurally modified peptides in which substitution of a Glu for Asp abolished inhibitory activity and substitution of Lys for Arg or Ala for Gly reduced activity 6- and 8-fold, respectively. In addition, Arg-Gly-Asp-Ser-containing peptides inhibited the rate and extent of thrombin-induced platelet aggregation. These data suggest that the Arg-Gly-Asp-Ser tetrapeptide contains a recognition specificity involved in the binding of Fn to platelets and that platelets share features of this recognition specificity with fibroblasts.     

Concurrent modulation of cell surface fibronectin and adhesion to fibronectin in hepatoma cells

Rat hepatoma cells grown in vitro were poorly adhesive to plastic surfaces coated with fibronectin and lacked cell surface fibronectin matrix. They synthesized soluble fibronectin into the medium. The cell surface fibronectin matrix and the ability to attach to fibronectin-coated surface were restored in the 7777 cells upon passage as a tumor in rats and by coculturing these cells with normal liver-derived cells in vitro. Fibronectin matrix and the ability of cells to attach to fibronectin were thus modulated in a coordinated fashion, suggesting that the formation of a cell surface fibronectin matrix is dependent on the cell surface property that enables cells to interact with fibronectin.     

Localization and chemical synthesis of fibronectin peptides with melanoma adhesion and heparin binding activities

Tumor cell adhesion to the extracellular matrix is an important consideration in tumor metastasis. Recent results show that multiple adhesion-promoting domains for melanoma cells can be purified from proteolytic digests of fibronectin [McCarthy, J. B., Hagen, S. T., & Furcht, L. T. (1986) J. Cell Biol. 102, 179-188]. Monoclonal antibodies were generated against a tryptic/catheptic 33K heparin binding fragment of fibronectin derived from the carboxyl terminal of the A chain. This region contains a tumor cell adhesion-promoting domain(s). The amino-terminal sequence was determined for this fragment, as well as a tryptic 31K fragment which is located to the carboxyl-terminal side of the 33K heparin binding fragment in A chains of fibronectin. The partial sequence data demonstrate that arginyl-glycyl-aspartyl-serine (RGDS) or the related arginyl-glutamyl-aspartyl-valine (REDV) is not present in the 33K heparin binding fragment, confirming earlier results which demonstrated that cells adhere to this fragment by an RGDS-independent mechanism. Two monoclonal antibodies, termed AHB-1 and AHB-2, recognized epitopes common to heparin binding fragments derived from the carboxyl terminus of both the A and B chains of fibronectin. Monoclonal antibody AHB-2 inhibited melanoma adhesion to the 33K heparin binding fragment of fibronectin in a concentration-dependent manner, whereas monoclonal antibody AHB-1 had no effect on adhesion to this fragment. Neither monoclonal antibody inhibited adhesion to intact fibronectin. However, monoclonal AHB-2 potentiated the inhibitory effect of suboptimal levels of exogenous RGDS on cell adhesion to intact fibronectin. AHB-2 recognized an epitope common to both the A- and B-chain carboxyl-terminal heparin binding region of fibronectin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heparan sulfate proteoglycans in the substratum adhesion sites of human neuroblastoma cells: modulation of affinity binding to fibronectin

Tissue culture substratum adhesion sites from EGTA-detached Platt human neuroblastoma cells were extracted with a buffer containing ocytlglucoside, NaCl, guanidine hydrochloride, and a variety of protease inhibitors, an extraction which resulted in quantitative solubilization of the 35SO4 = -radiolabeled proteoglycans and 3H-leucine-radiolabeled proteins. Of the sulfate-radiolabeled material, the vast majority was heparan sulfate proteoglycan (Kav = 0.15 on Sepharose C14B columns) and the remainder was chondroitin sulfate chains (no single chains of heparan sulfate were observed). This extract was then fractionated on DEAE-Sephadex columns under two different buffer elution conditions. Under DEAE-I conditions in low ionic strength acetate buffer, two major peaks of 35SO4 = -radiolabeled material (A,B) and a minor peak (C) could be resolved in the NaCl gradient; however, three-fourths of the material required 4 M guanidine hydrochloride to elute it from the column (peak D). Under DEAE-II conditions in acetate buffer supplemented with 8 M urea, the vast majority of the proteoglycan material could be eluted in the NaCl gradient as peak AB. Peak D material was shown to contain aggregated proteoglycan, along with nonproteoglycan protein, which high concentrations of urea or guanidine could dissociate, but not nonionic or zwitterionic detergents. Three different affinity chromatography systems were used to further characterize these components. Approximately 60% of peak A heparan sulfate proteoglycan from DEAE-I binds to the hydrophobic matrix, octyl-Sepharose, while 80% of the proteoglycan in DEAE-I peak D binds to this hydrophobic column. A sizable fraction of peak A proteoglycan fails to bind to plasma fibronectin but does bind to platelet factor-4 affinity columns. In contrast, peak AB proteoglycan from DEAE-II columns yields a much higher proportion of molecules which do bind to fibronectin. To examine the basis for these differences in affinity binding, nonproteoglycan protein from these adhesion sites was mixed with peak AB proteoglycan prior to affinity chromatography; proteoglycan binding to fibronectin decreased markedly while binding to platelet factor-4 was unaffected. This modulating activity involves the binding of nonproteoglycan protein in adhesion site extracts to both fibronectin on the column, as well as to heparan sulfate proteoglycan itself, and it could not be mimicked by a number of known proteins in adhesion site extracts or several other proteins. These results demonstrate selectivity and specificity in this modulation and indicate that a previously unidentified protein(s) is responsible.(ABSTRACT TRUNCATED AT 400 WORDS)     

A neuroectoderm-associated ganglioside participates in fibronectin receptor-mediated adhesion of germinal cells to fibronectin

Previous studies with a rat neural cell line have shown that the D1.1 ganglioside, an O-acetylated derivative of GD3, is involved in cellular adhesion to fibronectin. In vivo, D1.1 is present on germinal cells of the developing rat central nervous system, but not on postmitotic cells that migrate away from the germinal zones. These observations suggest that D1.1 could participate in adhesive interactions with germinal zones and that the loss of D1.1 could be involved in the decision to being migration. In support of this hypothesis, immunofluorescence histochemistry shows that both fibronectin and fibronectin receptor are colocalized with D1.1 in the ventricular zones of the embryonic rat brain and in the external granule cell layer of the postnatal cerebellum. Dishes coated with monoclonal antibody against D1.1 were used to isolate D1.1-positive germinal cells from Embryonic Day 14 cerebrum and from Postnatal Day 6 cerebellum. These cells are able to adhere to fibronectin-coated dishes by a mechanism that is inhibitable by a synthetic hexapeptide containing the arg-gly-asp cell recognition sequence of fibronectin. Adhesion is also partially inhibited by antibody against fibronectin receptor and is slowed by anti-D1.1 antibody, implicating both the receptor and the ganglioside in the adhesion process. During 3 days in culture these D1.1-positive, fibronectin receptor-positive cells exhibit a neuronal phenotype, as judged by morphology and staining with tetanus toxin. This further confirms the neuroepithelial origin of the cells. The cells do not synthesize detectable amounts of fibronectin, thus leaving unidentified the source of the fibronectin seen in the germinal zones in tissue sections. Immunoprecipitation experiments show that the fibronectin receptors present on these cells are heterodimers. Under nonreducing conditions, the immunoprecipitates contain an alpha-subunit of 150-160 kDa and a beta-subunit of 115-125 kDa.     

Neutralisation of TGF beta or binding of VLA-4 to fibronectin prevents rat tendon adhesion following transection

Following tendon injury, severe loss of function often occurs either as a result of obliteration of the synovial canal with fibrous scar tissue or from rupture of the repaired tendon. The role of cell engineering in tendon repair is to promote strong and rapid healing of tendon whilst at the same time facilitating rapid reconstitution of the synovial canal. Modification of the immediate inflammatory response around healing tendon has been found to be of value. Experimentally this has been achieved by neutralisation of transforming growth factor-beta over the first 3 days following injury, or by blockade of inflammatory cell binding to the CS-1 locus on fibronectin with an anti-VLA-4 antibody, or with the synthetic VLA-4 inhibitor, CS-1 peptide, in a rat model of tendon transection. It is concluded from this pilot study that the treatments described hold promise in improving outcomes of the common clinical problem of tendon injury in man.     

The effect of surface charge property on Escherichia coli initial adhesion and subsequent biofilm formation

Polyethylene (PE) sheets were modified by radiation-induced graft polymerization (RIGP) of an epoxy-group containing monomer glycidyl methacrylate (GMA). The epoxy group of GMA was opened by introducing sodium sulfite (SS) and diethylamine (DEA) as representatives of negatively and positively charged functional groups, respectively. These modified surfaces by RIGP, termed GMA, SS, and DEA sheets, were investigated to elucidate their effects on initial adhesion and subsequent biofilm formation of Escherichia coli. Initial adhesion test revealed that E. coli density and viability were governed by sheet surface electrostatic property: E. coli cell density on the DEA sheet was 23 times higher than that on the SS sheet after 8 h incubation. The viability of E. coli cells dramatically decreased after contact with the DEA sheet, but remained high on the SS sheet. E. coli biofilm structure on the DEA sheet was dense, homogeneous, and uniform, with biomass higher than that of the GMA and SS sheets by factors of 14.0 and 37.5, respectively. On the contrary, biofilm structure on the SS sheet was sparse, heterogeneous, and mushroom-shaped. More than 40% of E. coli biofilm on the DEA sheet was retained under a high liquid shear force condition (5,000 s(-1)), whereas 97% and 100% of biofilms on the GMA and SS sheets were sloughed, indicating that E. coli biofilm robustness depends on surface charge property of the substratum. This suggests that substratum surface fabrication by RIGP may enhance or suppress biofilm formation, a finding with potentially important practical implications.     

Effect of hypoxia on cellular adhesion to vitronectin and fibronectin

Cellular invasion of extracellular matrix (ECM) occurs during normal and pathological settings. For cells to invade, they must adhere to the underlying substratum, break down barrier molecules, and detach from the substratum prior to migrating through the ECM. We previously demonstrated that incubation under reduced oxygen levels increases the in vitro invasiveness of trophoblast and breast carcinoma cells, an effect linked to elevated expression of the cell surface receptor for urokinase-type plasminogen activator (uPAR). This study examined the role of oxygen, integrins and the urokinase-type plasminogen activator (uPA) system on the adhesion of trophoblast and breast carcinoma cells to the ECM molecules vitronectin and fibronectin. Compared to exposure to 20 and 5% oxygen, exposure to 1% oxygen decreased adhesion of these cells to vitronectin and fibronectin, an effect that was reversible by re-exposure to 20% oxygen. Incubation in 1% oxygen also resulted in reduced expression of surface alpha(5) integrin. Furthermore, adhesion to vitronectin and fibronectin was reduced by compounds that interfere with integrin function, such as EDTA, anti-integrin antibodies, or by antibodies that interfere with the binding of pro-uPA to uPAR, soluble uPAR, soluble vitronectin, phosphatidylinositol-specific phospholipase C, as well as plasminogen activator inhibitor-1. These findings suggest an important role for oxygen in the regulation of cellular invasion, possibly in part through its effects on integrin and uPAR-mediated mechanisms of adhesion.     

Syndecan-4 binding to the high affinity heparin-binding domain of fibronectin drives focal adhesion formation in fibroblasts

Cell adhesion to extracellular matrix involves signaling mechanisms which control attachment, spreading and the formation of focal adhesions and stress fibers. Fibronectin can provide sufficient signals for all three processes, even when protein synthesis is prevented by cycloheximide. Primary fibroblasts attach and spread following integrin ligation, but do not form focal adhesions unless treated with a heparin-binding fragment of fibronectin (HepII), a peptide from this domain, or phorbol esters to activate protein kinase C. Syndecan-4 heparan sulfate proteoglycan is a transmembrane component present together with integrins in focal adhesions. Syndecan-4 binds and activates protein kinase Calpha, whose activity is needed for focal adhesion formation. We now report that the glycosaminoglycan chains of syndecan-4 bind recombinant HepII and it is incorporated into forming focal adhesions.     

The binding of calcium to human fibronectin

The binding of calcium to human plasma fibronectin has been measured by equilibrium dialysis at 25° in 0.1 M NaCl 50mM Tris HCL, pH 7.4. Curve fitting of the binding data indicates that fibronectin has two strong calcium binding sites per chain (Mr 220,000), KD = 1.3 mM and approximately 12 weak sites, KD = 2.3 mM. No significant displacement of bound calcium by magnesium was observed at magnesium concentrations up to 1 mM. Calcium binding to a pair of tryptic fragments of fibronectin (Mr ? 160,000 and 180,000) that bind to gelatin has also been investigated. These fragments have a single class of calcium binding sites, with 2.2 sites per chain, KD = 1.1 mM. Negligible calcium binding to tryptic fragments derived from other regions of the fibronectin molecule was observed.     

Cell adhesion promotes Ebola virus envelope glycoprotein-mediated binding and infection

Ebola virus infects a wide variety of adherent cell types, while nonadherent cells are found to be refractory. To explore this correlation, we compared the ability of pairs of related adherent and nonadherent cells to bind a recombinant Ebola virus receptor binding domain (EboV RBD) and to be infected with Ebola virus glycoprotein (GP)-pseudotyped particles. Both human 293F and THP-1 cells can be propagated as adherent or nonadherent cultures, and in both cases adherent cells were found to be significantly more susceptible to both EboV RBD binding and GP-pseudotyped virus infection than their nonadherent counterparts. Furthermore, with 293F cells the acquisition of EboV RBD binding paralleled cell spreading and did not require new mRNA or protein synthesis.     

The fibronectin binding proteins of Staphylococcus aureus are required for adhesion to and invasion of bovine mammary gland cells

We recently described adhesion to and invasion of bovine mammary gland cells by Staphylococcus aureus in vitro. Here, we show that the levels of adhesion and invasion are dependent on the bacterial growth phase and are controlled by the agr locus. Incubation of exponential growth phase cells of S. aureus with mammary gland cells resulted in bacterial cell clumping. Strains of S. aureus deficient in expression of the fibronectin binding proteins (FnBPA and FnBPB) failed to clump and their ability to adhere to and to invade the bovine mammary gland cells is strongly reduced. This indicates that the fibronectin binding proteins are essential for S. aureus adhesion to and invasion of bovine mammary gland cells.