Cell surface heparan sulfate mediates some adhesive responses to glycosaminoglycan-binding matrices, including fibronectin

Proteins with affinities for specific glycosaminoglycans (GAC's) were used as probes for testing the potential of cell surface GAG's to mediate cell adhesive responses to extracellular matrices (ECM). Plasma fibronectin (FN) and proteins that bind hyaluronate (cartilage proteo-glycan core and link proteins) or heparan sulfate (platelet factor 4 [PF4]) were adsorbed to inert substrata to evaluate attachment and spreading of several 3T3 cell lines. Cells failed to attach to hyaluronate-binding substrata. The rates of attachment on PF4 were identical to those on FN; however, PF4 stimulated formation of broad convex lamellae but not tapered cell processes fibers during the spreading response. PF4-mediated responses were blocked by treating the PF4-adsorbed substratum with heparin (but not chondroitin sulfate), or alternatively the cells with Flavobacter heparinum heparinase (but not chondroitinase ABC). Heparinase treatment did not inhibit cell attachment to FN but did inhibit spreading. Cells spread on PF4 or FN contained similar Ca2+-independent cell-substratum adhesions, as revealed by EGTA-mediated retraction of their substratum-bound processes. Microtubular networks reorganized in cells on PF4 but failed to extend into the broadly spread lamellae, where fine microfilament bundles had developed. Stress fibers, common on FN, failed to develop on PF4. These experiments indicate that (a) heparan sulfate proteoglycans are critical mediators of cell adhesion and heparan sulfate-dependent adhesion via PF4 is comparable in some, but not all, ways to FN-mediated adhesion, (b) the uncharacterized and heparan sulfate-independent "cell surface" receptor for FN permits some but not all aspects of adhesion, and (c) physiologically compatible and complete adhesion of fibroblasts requires binding of extracellular matrix FN to both the unidentified "cell surface" receptor and heparan sulfate proteoglycans.     

Substratum contacts and cytoskeletal reorganization of BALB/c 3T3 cells on a cell-binding fragment and heparin-binding fragments of plasma fibronectin

BALB/c 3T3 cells make both close contacts and tight-focal contacts (with associated microfilament stress fibers) on plasma fibronectin (pFN)-coated substrata. To resolve the importance of the heparan sulfate-binding or cell-binding activities of the pFN molecule in these adhesive responses, a cell-binding fragment (120K) (CBF) free of any heparan sulfate-binding activity was prepared from human pFN by chymotrypic digestion and isolated as described by Pierschbacher et al. (Cell 26 (1981) 259). These adhesive responses to CBF were also compared to those of the model heparan sulfate-binding protein, platelet factor-4 (PF4), or heparin-binding fragments (HBF) of pFN. On intact pFN, greater than 70% of the cells formed tight-focal contacts and associated stress fibers by 4 h, the latter staining with NBD-phallacidin. In contrast, cells spread differently on CBF and failed to form tight-focal contacts; staining with NBD-phallacidin was localized to spiky projections at the cell margin with no detectable stress fiber formation. On PF4 or HBF, cells failed to form tight-focal contacts but did spread well and formed long microfilament bundles in peripheral lamellae. Spreading on CBF, HBF, or PF4 was paralleled by formation of close contacts. Spreading and to some extent attachment of cells on CBF was inhibited with a small peptide containing the Arg-Gly-Asp-Ser sequence; responses on HBF were unaffected by this peptide. When mixtures of CBF and PF4 were tested, cells still failed to form tight-focal contacts and stress fibers. These results demonstrate that the binding of CBF to its probable receptor under conditions routinely used to assay spreading activity results in an incomplete adhesive response compared with intact pFN. While this partial response may result from quantitative differences in the density of active cell-binding domains on the substratum, the pattern of microfilament reorganization produced by the binding of PF4 to cell surface heparan sulfate proteoglycans suggests that the ability of pFN to promote formation of tight-focal contacts and stress fibers may reside in the coordinate interaction of two or more binding activities in the intact molecule.     

Close and focal contact adhesions of fibroblasts to a fibronectin-containing matrix

The fibroblast cell line Balb/c 3T3 makes both close and tight-focal adhesive contacts with the plasma fibronectin (pFn)-coated tissue culture substratum. Detachment of these cells mediated by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid leaves tight-focal contacts and a subset of close contacts as substratum-attached material (SAM). The enrichment of heparan sulfate (HS) proteoglycan (HS-PG) in SAM under specific attachment conditions, as well as the recent demonstration of HS binding to pFn or cellular Fn, has evolved a series of experiments with the selective HS-binding protein platelet factor-4 to analyze the requirement of the HS-binding activity of pFn in the formation of these two types of adhesive contacts. In addition, the cell-binding domain (CBD) of pFn, which recognizes an unidentified cell surface receptor, has been isolated free of HS-binding domains after proteolytic cleavage of pFn. These functional studies indicate that the binding of pFn on the substratum to cell surface HS-PG is necessary and sufficient to generate close contacts with transmembrane signaling of this proteoglycan to reorganize lengthy microfilament bundles in the cytoplasm. Cells spread incompletely on CBD alone, form only close contacts, and reorganize highly concentrated actin filaments only in their spiky projections. Furthermore, the formation of tight-focal contacts with associated stress fibers appears to require both reactions of pFn, binding to cell surface HS-PG and to its unidentified receptor. The importance of HS-PG in these functional studies has led to its better biochemical characterization in SAM. Initially formed close contacts of these cells contain principally a large HS-PG that aggregates into high-molecular-weight complexes by some unknown mechanism. With time, there are two different mechanisms of catabolism of HS-PG, one of which includes the liberation of single-chain HS. The importance of these changes in the HS-PG in SAM are now being analyzed with regard to the formation and disappearance of close and tight-focal adhesions.     

Dissecting focal adhesions in cells differentially expressing calreticulin: a microscopy study

Background information. Our previous studies have shown that calreticulin, a Ca²⁺‐binding chaperone located in the endoplasmic reticulum, affects cell—substratum adhesions via the induction of vinculin and N‐cadherin. Cells overexpressing calreticulin contain more vinculin than low expressers and make abundant contacts with the substratum. However, cells that express low levels of calreticulin exhibit a weak adhesive phenotype and make few, if any, focal adhesions. To date, the identity of the types of focal adhesions made by calreticulin overexpressing and low expressing cells has not been dissected. Results. The results of the present study show that calreticulin affects fibronectin matrix assembly in L fibroblast cell lines that differentially express the protein, and that these cells also differ profoundly in focal adhesion formation. Although the calreticulin overexpressing cells generate numerous interference‐reflection‐microscopy‐dark, vinculin‐ and paxillin‐containing classical focal contacts, as well as some fibrillar adhesions, the cells expressing low levels of calreticulin generate only a few weak focal adhesions. The fibronectin receptor was found to be clustered in calreticulin overexpressing cells, but diffusely distributed over the cell surface in low expressing cells. Plating L fibroblasts on fibronectin‐coated substrata induced extensive spreading in all cell lines tested. However, although calreticulin overexpressing cells were induced to form classical vinculin‐rich focal contacts, the low calreticulin expressing cells overcame their weak adhesive phenotype by induction of many tensin‐rich fibrillar adhesions, thus compensating for the low level of vinculin in these cells. Conclusions. We propose that calreticulin affects fibronectin production and, thereby, assembly, and it indirectly influences the formation and/or stability of focal contacts and fibrillar adhesions, both of which are instrumental in matrix assembly and remodelling.     

Substratum attachment of embryonic mesoderm cells in culture

The cell-substratum adhesive characteristics of cultured chick embryo primary mesoderm cells have been examined by interference reflection microscopy and transmission electron microscopy under various conditions. Correlations were drawn between the type of adhesion and the degree of motility shown by the cells. During the rapid spreading and motility of cells cultured on fibronectin-containing substrata, focal contacts (10 to 15-nm gap) were rare and close contacts (about 30-nm gap) were predominant. By contrast, when the cells were immobile, after 5 d in culture, extensive focal contacts were present, together with stress fibers. The results indicate that tight cell-substratum contact is incompatible with rapid cell motility and that fibronectin acts by inducing the formation of close contacts rather than focal contacts.     

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.     

Cell adhesion and migration in the early vertebrate embryo: location and possible role of the putative fibronectin receptor complex

Using a combined in vivo and in vitro approach, we have analyzed the immunofluorescent localization and function of a 140,000-mol-wt glycoprotein complex implicated in cell adhesion to fibronectin (FN), with particular emphasis on neural crest cell adhesion and migration. This putative fibronectin receptor complex (FN-receptor) was detectable in almost all tissues derived from each of the three primary germ layers. It was present in both mesenchymal and epithelial cells, and was particularly enriched at sites close to concentrations of FN, e.g., at the basal surfaces of epithelial cells. It was also present on neural crest cells. The distribution and function of this putative receptor was then analyzed on individual cells in vitro. It was diffusely organized on highly locomotory neural crest cells and somitic fibroblasts. Both motile cell types also displayed relatively low numbers of focal contacts and microfilament bundles and limited amounts of localized vinculin, alpha-actinin, and endogenous FN. In contrast, the FN-receptor in stationary embryonic cells, i.e., somitic cells after long-term culture or ectodermal cells, existed in characteristic linear patterns generally co-distributed with alpha-actinin and fibers of endogenous FN. Anti-FN-receptor antibodies inhibited the adhesion to FN of motile embryonic cells, but not of stationary fibroblasts. However, these same antibodies adsorbed to substrata readily mediated adhesion and spreading of cells, but were much less effective for cell migration. Our results demonstrate a widespread occurrence in vivo of the putative FN-receptor, with high concentrations near FN. Embryonic cell migration was associated with a diffuse organization of this putative receptor on the cell surface in presumably labile adhesions, whereas stationary cells were anchored to the substratum at specific sites linked to the cytoskeleton near local concentrations of FN- receptor.     

3D coupling of fibronectin fibril arrangement with topology of ventral plasma membrane

Interaction of integrins with extracellular matrices is essential for cell adhesion to substrata. Ventral surfaces of fibroblasts adhering to flat substrata are not flat but have uneven 3D topology. However, spatial relationship between the topology of the ventral cell surface and arrangement of extracellular matrix fibrils remains unclear. Here, we report a novel and simple method based on total internal reflection fluorescence microscopy to quantify the distance between the ventral plasma membrane and the glass substratum. We observe that the distance varies from < 25 nm at focal adhesions to 40-50 nm at close contacts and > 80 nm in other regions. Furthermore, by applying this novel method, we show that fibronectin fibrils are also separated from the substratum in regions where the ventral cell surface-substratum distance is > 80 nm. Our results reveal that fibronectin fibrils are not simply adsorbed to the glass substratum but follow the ventral cell surface topology.     

The removal of extracellular fibronectin from areas of cell-substrate contact

Immunofluorescent labeling for fibronectin was largely excluded from sites of closest contact between spreading chicken gizzard fibroblasts and the substratum. This was observed by double immunofluorescent labeling of fixed cells for fibronectin and vinculin, a smooth muscle intracellular protein that is specifically associated with focal adhesion plaques, in conjunction with interference-reflection microscopy. When the cells were plated on a fibronectin-coated substratum they adhered to its surface and rapidly spread on it. The immunofluorescent labeling for fibronectin in those cultures (after fixation and triton permeabilization) was usually absent from the newly formed, vinculin-containing focal adhesion plaques. We have found, however, that the accessibility to the cell-substrate gap at the focal adhesion plaques is limited and therefore a more direct approach was adopted. We have found that cells spreading on a substrate coated with rhodamine-labeled fibronectin progressively removed the underlying protein from the substrate. The removal of fibronectin involved at least two distinct mechanisms. Part of the substrate-associated fibronectin was removed from small areas and displaced toward the cell center. The arrowhead-shaped areas from which fibronectin was removed often coincided with vinculin-rich focal contacts. We observed, however, many areas where focal contacts were found over unperturbed fibronectin carpet, as well as fibronectin-free areas with no overlapping focal contacts. The possibilities that fibronectin is actively displaced from areas of cell-substrate contact, that the focal adhesion plaques are transiently associated with these areas and their implications on the dynamics of cell spreading and locomotion are discussed. The second route of fibronectin removal from the substrate was endocytosis. The rhodamine-labeled fibronectin was found in the cells in a partial or transient association with clathrin-containing structures.     

Adhesion of glycosaminoglycan-deficient chinese hamster ovary cell mutants to fibronectin substrata

We have examined the role of cell surface glycosaminoglycans in fibronectin-mediated cell adhesion by analyzing the adhesive properties of Chinese hamster ovary cell mutants deficient in glycosaminoglycans. The results of our study suggest that the absence of glycosaminoglycans does not affect the initial attachment and subsequent spreading of these cells on substrata composed of intact fibronectin or a fibronectin fragment containing the primary cell-binding domain. However, in contrast to wild-type cells, the glycosaminoglycan- deficient cells did not attach to substrate composed of a heparin- binding fibronectin fragment. Furthermore, the wild-type but not the glycosaminoglycan-deficient cells formed F-actin-containing stress fibers and focal adhesions on substrata composed of intact fibronectin. We propose, therefore, that cell surface proteoglycan(s) participate in the transmembrane linking of intracellular cytoskeletal components to extracellular matrix components which occurs in focal adhesions.     

Fibrinogen induces adhesion, spreading, and microfilament organization of human endothelial cells in vitro

Human umbilical vein endothelial cells (ECs) have been shown to attach to a substratum of fibrinogen (fg). Later, ECs undergo spreading, organization of thick microfilament bundles of the stress fiber type, and formation of focal contacts (adhesion plaques) that correspond to accumulation of vinculin at the cytoplasmic aspect of the ventral membrane. The rate of attachment to fg and the type of spreading is virtually identical to that obtained on substrata coated with fibronectin (FN). Antibodies to fg, but not to FN, prevent EC adhesion to fg; conversely, antibodies to FN, but not to fg, prevent adhesion of ECs to a FN-coated substratum. The removal of residual FN contamination from fg preparations by means of DEAE-cellulose chromatography does not result in any difference in EC adhesion on fg. Moreover, pretreatment of cells with inhibitors of synthesis and release of proteins does not impair their adhesion capacity on an fg-coated substratum. In contrast, human arterial smooth muscle cells do not adhere and spread on fg substrata but do so on FN. The synthetic peptides (Gly-Arg-Gly-Asp[GRGD] and Gly-Arg-Gly-Asp-Ser-Pro[GRGDSP]) containing the tripeptide Arg-Gly-Asp (RGD), originally found to be responsible for the cell binding activity of FN, have been found to inhibit EC spreading and the redistribution of their cytoskeleton, including the formation of stress fibers and the localization of vinculin either on fg or on FN. Conversely, the synthetic peptide Arg-Gly-Gly (RGG) was completely uneffective in inhibiting the adhesion and the sequence of events leading to spreading and cytoskeletal organization. These results indicate that ECs, but not smooth muscle cells, specifically adhere and spread on an fg substratum and this occurs by recognition mechanisms similar to those reported for FN.     

The role of endogenous heparan sulfate proteoglycan in adhesion and neurite outgrowth from dorsal root ganglia

Some phases of dorsal root ganglion (DRG) substratum attachment and growth cone morphology are mediated through endogenous cell surface heparan sulfate proteoglycan. The adhesive behavior of intact embryonic chicken DRG (spinal sensory ganglia) is examined on substrata coated with fibronectin, fibronectin treated with antibody to the cell-binding site (anti-CBS), and the heparan sulfate-binding protein platelet factor four. DRG attach to fibronectin, anti-CBS-treated fibronectin, and platelet factor four. The ganglia extend an extensive halo of unfasciculated neurites on fibronectin and produce fasciculated neurite outgrowth on platelet factor four and anti-CBS antibody-treated FN. Treatment with heparinase, but not chondroitinase, abolishes adhesion to fibronectin and platelet factor four. Growth cones of DRG on fibronectin have well-spread lamellae and microspikes. On platelet factor four, and anti-CBS-treated FN, growth cones exhibit microspikes only. Isolated Schwann cells adhere equally well to fibronectin and platelet factor four, spreading more rapidly on fibronectin. Isolated DRG neurons adhere equally well on both substrata, but only 10% of the neurons extend long neurites on platelet factor four. The majority of the isolated neurons on platelet factor four exhibit persistent microspike production resembling that of the early stages of normal neurite extension. Endogenous heparan sulfate proteoglycan supports the adhesion of whole DRG, isolated DRG neurons, and Schwann cells, as well as extensive microspike activity by DRG neurons, one important part of growth cone activity.     

Proteolytic activity of specialized surface protrusions formed at rosette contact sites of transformed cells

Surface protrusions at the leading edge of a moving cell that make contact with the surrounding extracellular matrix (ECM) are its main motor for locomotion and invasion. Chicken embryonic fibroblasts transformed by Rous sarcoma virus (RSV-CEF) form specialized membrane rosette-shaped contact sites on planar substrata as shown by interference reflection microscopy (IRM). Such activity is lacking in normal cells. These rosette contacts are more labile than other adhesion sites, such as focal and close contacts. Ultrastructural studies demonstrate that rosettes are sites at which membrane protrusions from the ventral cell surface contact the substratum. These protrusions are filled with meshworks of microfilaments and contain the pp60src oncogene product, actin, vinculin, and alpha-actinin. However, unlike focal contacts, at the rosettes these proteins interact to extend a highly motile membrane. Rosettes have the biological activity of degrading ECM components, as demonstrated by (1) local degradation of fibronectin substrata at sites of rosette contacts, but not focal and close contacts; (2) localization of putative antiprotease antibody at sites of rosette contacts, but not at focal an close contacts; and (3) local disruption of fibronectin matrix at sites of protrusive activity seen by transmission electron microscopy (TEM). In addition, formation of the rosette contact is insensitive to the ionophore monensin, and to inhibitors of proteolytic enzymes, while local fibronectin degradation at rosette contacts is inhibited by inhibitors of metalloproteases, 1,10-phenanthroline and NP-20. I consider these membrane protrusions of the rosette contacts in RSV-transformed cells specialized structural entities--invadopodia--that are involved in the local degradation of the ECM.     

3D Coupling of Fibronectin Fibril Arrangement with Topology of Ventral Plasma Membrane

Abstract

Interaction of integrins with extracellular matrices is essential for cell adhesion to substrata. Ventral surfaces of fibroblasts adhering to flat substrata are not flat but have uneven 3D topology. However, spatial relationship between the topology of the ventral cell surface and arrangement of extracellular matrix fibrils remains unclear. Here, we report a novel and simple method based on total internal reflection fluorescence microscopy to quantify the distance between the ventral plasma membrane and the glass substratum. We observe that the distance varies from <?25 nm at focal adhesions to 40–50 nm at close contacts and >?80 nm in other regions. Furthermore, by applying this novel method, we show that fibronectin fibrils are also separated from the substratum in regions where the ventral cell surface-substratum distance is >?80 nm. Our results reveal that fibronectin fibrils are not simply adsorbed to the glass substratum but follow the ventral cell surface topology.     

Regulation of integrin-mediated adhesion at focal contacts by cyclic AMP

Cyclic AMP (cAMP) elevation causes diverse types of cultured cells to round partially and develop arborized cell processes. Renal glomerular mesangial cells are smooth, muscle-like cells and in culture contain abundant actin microfilament cables that insert into substratum focal contacts. cAMP elevation causes adhesion loss, microfilament cable fragmentation, and shape change in cultured mesangial cells. We investigated the roles of the classical vitronectin (αVβ3 integrin) and fibronectin (α5β1 integrin) receptors in these changes. Mesangial cells on vitronectin-rich substrata contained microfilament cables that terminated in focal contacts that stained with antibodies to vitronectin receptor. cAMP elevation caused loss of focal contact and associated vitronectin receptor. Both fibronectin and its receptor stained in a fibrillary pattern at the cell surface under control conditions but appeared aggregated along the cell processes after cAMP elevation. This suggested that cAMP elevation caused loss of adhesion mediated by vitronectin receptor but not by fibronectin receptor. We plated cells onto fibronectin-coated slides to test the effect of ligand immobilization on the cellular response to cAMP. On fibronectin-coated slides fibronectin receptor was observed in peripheral focal contacts where actin filaments terminated, as seen with vitronectin receptor on vitronectin-coated substrata, and in abundant linear arrays distributed along microfilaments as well. Substratum contacts mediated by fibronectin receptor along the length of actin filaments have been termed fibronexus contacts. After cAMP elevation, microfilaments fragmented and fibronectin receptor disappeared from peripheral focal contacts, but the more central contacts along residual microfilament fragments appeared intact. Also, substratum adhesion was maintained after cAMP elevation on fibronectin—but not on vitronectincoated surfaces. Although other types of extracellular matrix receptors may also be involved, our observations suggest that cAMP regulates adhesion at focal contacts but not at fibronexus-type extracellular matrix contacts. © 1993 Wiley-Liss, Inc.     

Immunocytochemical localization of 140 kD cell adhesion molecules in cultured chicken fibroblasts, and in chicken smooth muscle and intestinal epithelial tissues

A monoclonal antibody (JG22 MAb) that was previously raised to a chick embryo myogenic cell preparation had been shown to produce rounding and other morphological changes in myogenic cells in culture, and, in some cases, their detachment from the substratum. In other studies it was shown that the epitope recognized by JG22 was associated with a set of 140 kD cell surface glycoproteins. It is shown that this antigen occurs in a wide variety of cell types; in cultured fibroblasts, it is distributed equally between the dorsal and ventral cell surfaces shortly after plating, but appears to become concentrated on the ventral surface as cell spreading proceeds; by immunoelectron microscopic labeling experiments, it is absent from the focal adhesion contact sites formed by fibroblasts with their substrata and with one another, but is present in clusters at the edge of focal adhesions, and within the close contact sites and extracellular matrix contact sites; in smooth muscle cells, it is absent from the membrane-associated dense plaques, but is located in clusters at adjacent membrane sites; in intestinal epithelium, it is present in clusters at the basolateral membranes, but not at the microvilli or within junctional complexes of the brush border of the cell layers. These and other results are consistent with the suggestion that the antigen recognized by JG22 MAb is important cell adhesion molecules, and performs a characteristic function in a variety of cell-cell contacts and cell adhesions.     

Neurite extension by neuroblastoma cells on substratum-bound fibronectin''s cell-binding fragment but not on the heparan sulfate-binding protein, platelet factor-4

Human and rat neuroblastoma cells extend neurites over plasma fibronectin (pFN)-coated substrata. For resolution of which fibronectin binding activities (the cell-binding domain (CBD), the heparan sulfate-binding domains, or a combination of the two) are responsible for neurite outgrowth, CBD was prepared free of heparan sulfate-binding activity as described by Pierschbacher et al. (Cell 26 (1981) 259-267). Neuroblastoma cells attached and extended neurites as stably and as effectively on CBD-coated substrata as on intact pFN, while cytoplasmic spreading was more extensive on pFN-coated substrata. The structures of growth cones on CBD or pFN were virtually identical. On substrata coated with the model heparan sulfate-binding protein, platelet factor 4 (PF4), cells attached and spread somewhat but never extended neurites. When cells were challenged with substrata coated with various ratios of CBD and PF4, PF4 was found to be an effective inhibitor of CBD-mediated neurite extension. Similarly, cells grown on substrata coated at different locations with CBD or PF4 in order to evaluate topographical dependence of growth cone formation extended neurites only onto the CBD-coated region or along the interface between these two proteins, but never onto the PF4 side of cells that bridged the interface. These studies indicate that (a) the CBD activity of pFN, and not its heparan sulfate-binding activity, is the critical determinant in neurite extension of these neural tumor cells from the central nervous system; (b) under some circumstances, heparan sulfate-binding activity can be antagonistic to neurite extension; (c) the chemical nature of the substratum controls the direction of neurite extension; (d) these neuroblastoma cells respond to these binding proteins very differently than fibroblasts or neurons from the peripheral nervous system.     

Adhesive responses of fibroblast and neuroblastoma cells to substrata coated with polyvalent or monoclonal antibody to fibronectin

Both polyvalent and hybridoma-produced antibodies to fibronectin (Fn) were used to ‘map’ the immunoaccessible subsets of cell surface fibronectin on virus-transformed murine fibroblast SVT2 and rat neuroblastoma B104 cells. As one approach to this end, attachment and spreading responses of cells were measured on tissue culture substrata coated with antibody or with plasma fibronectin to compare their adhesive responses. Both SVT2 and B104 cells adhere poorly to polyvalent anti-Fn-coated substrata over short time intervals, but within several hours changes occur which permit cells to attach and spread as well on anti-Fn as on Fn (post-adsorption of the anti-Fn with Fn also generates a maximal response). This adhesive response could be completely prevented by predigesting the cells with Flavobacterium heparanase, but not with chondroitinase ABC, indicating that the cell surface Fn responsible for antibody-mediated adhesion is associated with heparan sulfate proteoglycans on the cell surface. The compositions of the substratum-attached material (left bound after EGTA-mediated detachment of cells) from cells attaching to anti-Fn or Fn were analysed by SDS-PAGE and found to be identical within the same cell type for the two different substrata. Three hybridoma-produced antibodies, which recognize different determinants on Fn, generated different adhesive responses for SVT2 or B104 cells when adsorbed to the substratum. SVT2 cells adhered well to antibody no. 32-coated substrata but poorly to antibodies 92 or 136; on the other hand, B104 cells responded similarly to all three antibodies over short times of attachment but much better to no. 32 after a several hour incubation. These experiments indicate that (1) much of the cell surface fibronectin is complexed with heparan sulfate proteoglycan and is initially inaccessible to bind to polyvalent antibody on the substratum to promote adhesion; (2) the surface of neuroblastoma cells contains a fibronectin-like molecule which is important in their substratum adhesion; and (3) monoclonal antibodies are valuable tools in ‘mapping’ the orientation of cell surface molecules like fibronectin by measuring adhesive responses to antibody-coated substrata.     

Adhesion of cells to protein carpets: do cells' feet have to be black?

In most physiological situations, cell contact with a substratum is mediated by proteins of extracellular matrix. Therefore, an increasing number of cell-substratum adhesion studies employ substrata covered with one or more proteins of extracellular matrix. To visualize the most adhesive cell structures, focal contacts and focal adhesions, the interference reflection microscopy has been widely used. It has been generally accepted that these strongly adhesive structures can be seen as black streaks in interference reflection microscopy. Calculations are presented herein, which although simplified, suggest that when cells are plated on protein-covered substrata, their focal contacts may not always appear black in interference reflection microscopy.