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.     

Contact formation by fibroblasts adhering to heparan sulfate-binding substrata (fibronectin or platelet factor 4)

The process of cell-substratum adhesion of BALB/c 3T3 fibroblasts on fibronectin (FN)-coated substrata was compared with that of cells adhering to substrata coated with the heparan sulfate (HS)-binding protein, platelet factor four (PF4). FN has binding domains for HS and an unidentified cell surface receptor, whereas PF4 binds to only HS on the surface of the cell. The attachment and early spreading sequences of cells on either substratum were similar as shown by scanning electron microscopy (SEM). Within 2 h of spreading, cells on FN developed typical fibroblastic morphologies, whereas those on PF4 lacked polygonal orientations and formed numerous broadly spread lamellae. Interference reflection microscopic analysis indicated that PF4-adherent cells formed only close adhesive contacts, whereas FN-adherent cells formed both close contacts and tight focal contacts. Cells on either substratum responded to Ca2+ chelation with EGTA by rounding up, but remained adherent to the substratum by relatively EGTA-resistant regions of the cell's undersurface, demonstrating that cell surface HS by binding to an appropriate substratum is capable of initiating a Ca2+-dependent spreading response. The EGTA-resistant substratum-attached material on PF4 was morphologically similar to that on FN, the latter of which was derived from both tight focal contacts and discrete specializations within certain close contacts. These studies show that heparan sulfate proteoglycans on the surface of these cells can participate in the formation of close contact adhesions by binding to an appropriate substratum and suggest that sub-specializations within close contact adhesions may evolve into tight focal contacts by the participation of an unidentified cell surface receptor which binds specifically to fibronectin but not to PF4. In addition, the functional role of FN in tight focal contact formation is demonstrated.     

Microvilli and blebs as sources of reserve surface membrane during cell spreading

The increase in surface area that occurs as cells spread from the rounded to the flattened state has been examined in synchronized BHK 21 cells in the scanning electron microscope. Rounded cells, whether in mitosis or dissociated and freshly seeded in culture, are covered with a mixture of folds, blebs, and microvilli. As cells spread, these protuberances disappear, first in the flattening marginal region and progressively submarginally until the entire cell surface is virtually smooth. The estimated surface area of rounded post-mitotic daughter cells, taking microvilli into account, is close to that of fully spread cells 4 h after mitosis. Likewise, rounded early mitotic mother cells, which are also covered with microvilli, have approximately the same surface as fully spread cells just prior to mitosis. These findings suggest that cells possess a membrane reserve in their microvilli and other protuberances which can be utilized for spreading and initiating cell locomotion.     

Effects of a tumour promoter, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), on expression of differentiated phenotype in the chick retinal pigmented epithelial cells and on their interactions with the native basement membrane and with artificial substrata

Chick retinal pigmented epithelial (RPE) cells grown in vitro on basement membrane matrices from the Engelbreth-Holm-Swarm tumour (BM-matrigel) do not spread, and they maintain their differentiated phenotype, most notably the heavy pigmentation. Maintenance of the differentiated phenotype by RPE cells on BM-matrigel is promoted not only by the biochemical composition of the gel but also by its mechanical properties, i.e., its low rigidity prevents cell spreading. In this report, RPE cells on BM-matrigel were treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to promote the transformed phenotype and diminish cell traction. In contrast to most cell types TPA treatment induced RPE cells to increase their spread area. TPA promoted RPE cell spreading on BM-matrigel and changed the spatial organization of actin and actin-associated proteins in the cytoskeleton-ECM linkage complexes, uncoupling actin from its extracellular counterpart. TPA did not affect other components of the cytoskeleton in RPE cells. TPA also affected labile adhesions i.e., focal contacts and adherens junctions in statu nascendi, but preformed, stable adherens junctions were resistant to TPA. TPA enhanced proliferation, blocked melanogenesis and thus inhibited differentiation of RPE cells grown on either artificial substrata or their natural basement membrane.     

Substratum attachment of embryonic mesoderm cells in culture

Summary The cell-substratum adhesive characteristics of cultured chick embryo primary mesoderm cells have been examined by inteference reflection microscopy and transmission electron microscoy 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 substrate, focal contacts (10 to 15-nm gap) were rare and close contacts (about 30-nm gap) were pedominant. By contrast, when the cells were immobile, after 5 d in cultue, 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. This work was supported by grants from the Medical Research Council of Canada and the Alberta Heritage Foundation for Medical Research.     

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.     

Expression of the differentiated phenotype by epithelial cells in vitro is regulated by both biochemistry and mechanics of the substratum

In this paper I sought to determine how the expression of differentiated traits of chick retinal pigmented epithelial (RPE) cells in vitro can be modulated by varying both the biochemical and the spatial complexity, and the mechanical properties, of the growth substratum. I have used glass derivatized with proteins of a basement membrane extract (nondeformable, two-dimensional substratum) and gels of reconstituted basement membrane extract (viscoelastic, three-dimensional substratum). These two biochemically similar substrata were compared to an inert substratum (untreated glass) and to the native basement membrane of the RPE, i.e., Bruch's Membrane. With immunofluorescence microscopy, I have shown that RPE cells, given space, will spread on their native basement membrane and form stress fibres and focal contacts, analogous to the stress fibres and integrin-, talin-, and vinculin-containing focal contacts of the cells grown on glass. Therefore, the stress fibres and focal contacts present in cultured cells are not artifacts of growth in vitro, but are a natural cellular response to the nondeformability of commonly used tissue culture substrata. The proteins of the basement membrane promote expression of some of the differentiated traits by RPE cells in vitro: however, the fully differentiated phenotype is expressed by RPE cells only when their spreading is prevented by low resilience of a substratum. Basement membrane gels generally are not resilient enough to support RPE cell spreading; however, the cells spread and form stress fibres, and integrin-, talin-, and vinculin-containing focal contacts when they are presented with areas of the gel which locally acquired higher resilience. The extent of cell spreading is determined by the deformability of substratum, hence elastic forces operating within the substratum determine the maximal cell traction allowable and, indirectly, the cytoarchitecture. Therefore, in addition to biochemical composition, the mechanical properties of substrata play important role in regulation of expression of the differentiated phenotype of cells in vitro and, possibly, in vivo.     

The LD4 motif of paxillin regulates cell spreading and motility through an interaction with paxillin kinase linker (PKL).

The small GTPases of the Rho family are intimately involved in integrin-mediated changes in the actin cytoskeleton that accompany cell spreading and motility. The exact means by which the Rho family members elicit these changes is unclear. Here, we demonstrate that the interaction of paxillin via its LD4 motif with the putative ARF-GAP paxillin kinase linker (PKL) (Turner et al., 1999), is critically involved in the regulation of Rac-dependent changes in the actin cytoskeleton that accompany cell spreading and motility. Overexpression of a paxillin LD4 deletion mutant (paxillinDeltaLD4) in CHO.K1 fibroblasts caused the generation of multiple broad lamellipodia. These morphological changes were accompanied by an increase in cell protrusiveness and random motility, which correlated with prolonged activation of Rac. In contrast, directional motility was inhibited. These alterations in morphology and motility were dependent on a paxillin-PKL interaction. In cells overexpressing paxillinDeltaLD4 mutants, PKL localization to focal contacts was disrupted, whereas that of focal adhesion kinase (FAK) and vinculin was not. In addition, FAK activity during spreading was not compromised by deletion of the paxillin LD4 motif. Furthermore, overexpression of PKL mutants lacking the paxillin-binding site (PKLDeltaPBS2) induced phenotypic changes reminiscent of paxillinDeltaLD4 mutant cells. These data suggest that the paxillin association with PKL is essential for normal integrin-mediated cell spreading, and locomotion and that this interaction is necessary for the regulation of Rac activity during these events.     

An RGD spacing of 440 nm is sufficient for integrin alpha V beta 3- mediated fibroblast spreading and 140 nm for focal contact and stress fiber formation

The synthetic peptide Gly-Arg-Gly-Asp-Tyr (GRGDY), which contains the RGD sequence of several adhesion molecules, was covalently grafted to the surface of otherwise poorly adhesive glass substrates and was used to determine the minimal number of ligand-receptor interactions required for complete spreading of human foreskin fibroblasts. Well-defined adhesion substrates were prepared with GRGDY between 10(-3) fmol/cm2 and 10(4) fmol/cm2. As the adhesion ligand surface concentration was varied, several distinct morphologies of adherent cells were observed and categorized. The population of fully spread cells at 4 h reached a maximum at 1 fmol/cm2, with no further increases up to 10(4) fmol/cm2. Although maximal cell spreading was obtained at 1 fmol/cm2, focal contacts and stress fibers failed to form at RGD surface concentrations below 10 fmol/cm2. The minimal peptide spacings obtained in this work correspond to 440 nm for spreading and 140 nm for focal contact formation, and are much larger than those reported in previous studies with adsorbed adhesion proteins, adsorbed RGD-albumin conjugates, or peptide-grafted polyacrylamide gels. Vitronectin receptor antiserum specific for integrin alpha V beta 3 blocked cell adhesion and spreading on substrates containing 100 fmol/cm2 of surface-bound GRGDY, while fibronectin receptor antiserum specific for alpha 5 beta 1 did not. Furthermore, alpha V beta 3 was observed to cluster into focal contacts in spread cells, but alpha 5 beta 1 did not. It was thus concluded that a peptide-to-peptide spacing of 440 nm was required for alpha V beta 3-mediated cellular spreading, while 140 nm was required for alpha V beta 3-mediated focal contact formation and normal stress fiber organization in human foreskin fibroblasts; these spacings represent much fewer ligands than were previously thought to be required.     

NBT-II cell locomotion is modulated by restricting the size of focal contacts and is improved through EGF and ROCK signaling

Focal contacts, large macromolecular complexes that link the extracellular matrix and the internal cell cytoskeleton, are thought to govern cell locomotion. However, the maturation process through which focal contacts control the cellular migratory machinery by changes in size and molecular composition remain unclear. Here, we fabricated cell growth substrates that contained linear ECM strips of micron- or submicron-width in order to limit the enlargement of focal contacts. We found that NBT-II cells plated on the submicron substrate possessed smaller focal complexes that exhibited a highly dynamic turnover. These cells possessed various leading edges at multiple sites of the cell periphery, which prevented the cell from advancing. In contrast, cells grown on the micron-width substrate possessed large and stable focal adhesions. Most of these cells were elongated bipolar cells that were tethered at both ends and were immobile. Further, EGF and ROCK signaling pathways can modulate the cellular migratory responses according to the substrate guidance. On the submicron-width substrate, EGF treatment increased the focal contact size and the contractile force, causing these cells to develop one leading edge and migrate along the submicron-sized ECM paths. In contrast, inhibition of ROCK signaling decreased the focal contact size for cells plated on the micron substrate. These cells became less tethered and were able to migrate along or even across the micron-sized ECM paths. Our results indicate that formation and maturation of focal contacts is controlled by both ECM cues and intracellular signaling and they play a central role in directed cell motion.     

Immunocytochemical localization of polyamines during attachment and spreading of retinal pigment epithelial and intestinal epithelial cells

In order to form and maintain a protective barrier for photoreceptors, the retinal pigment epithelium relies on integrin signaling and related pathways to form adhesion complexes, undergo cell spreading, and establish a confluent cellular monolayer. Polyamines are multifunctional polycations that are essential for cell attachment and spreading, although their exact mechanisms of action are as yet unclear. We report new immunocytochemical evidence suggesting that in the cells of retinal pigment epithelium and also the intestinal epithelium, polyamines are present in a population of intracellular vesicles that appear transiently during initial stages of cell spreading. In newly attached cells with minimal spreading, the vesicles are seen near the nucleus, whereas in more highly spread cells, the vesicles are localized to the plasma membrane, near, but not precisely co-localized with an enzyme marker for adhesion complexes, focal adhesion kinase. We also observe pronounced nuclear staining in newly attached cells that have not spread, whereas this staining is decreased in cells that have spread. Nuclear staining has been previously reported in other cell types and has been attributed to DNA binding of polyamines, which is known to stabilize chromatin structure. We hypothesize that the appearance of polyamine vesicles near focal adhesions of cells undergoing attachment and spreading may reflect the mechanism by which polyamine pools are targeted to appropriate interaction sites necessary for the assembly of adhesion complexes. Alternatively, the vesicles could represent the mechanism by which polyamines are removed from the nucleus and possibly released from the cell.     

Interplay between Rac and Rho in the control of substrate contact dynamics.

BACKGROUND: Substrate anchorage and cell locomotion entail the initiation and development of different classes of contact sites, which are associated with the different compartments of the actin cytoskeleton. The Rho-family GTPases are implicated in the signalling pathways that dictate contact initiation, maturation and turnover, but their individual roles in these processes remain to be defined. RESULTS: We monitored the dynamics of peripheral, Rac-induced focal complexes in living cells in response to perturbations of Rac and Rho activity and myosin contractility. We show that focal complexes formed in response to Rac differentiated into focal contacts upon upregulation of Rho. Focal complexes were dissociated by inhibitors of myosin-II-dependent contractility but not by an inhibitor of Rho-kinase. The downregulation of Rac promoted the enlargement of focal contacts, whereas a block in the Rho pathway not only caused a dissolution of focal contacts but also stimulated membrane ruffling and formation of new focal complexes, which were associated with the advance of the cell front. CONCLUSIONS: Rac functions to signal the creation of new substrate contacts at the cell front, which are associated with the induction of ruffling lamellipodia, whereas Rho serves in the maturation of existing contacts, with both contact types requiring contractility for their formation. The transition from a focal complex to a focal contact is associated with a switch to Rho-kinase dependence. Rac and Rho also influence the development of focal contacts and focal complexes, respectively, through mutually antagonistic pathways.     

Force sensing and generation in cell phases: analyses of complex functions.

Cellular morphology is determined by motility, force sensing, and force generation that must be finely controlled in a dynamic fashion. Contractile and extensile functions are integrated with the overall cytoskeleton, including linkages from the cytoplasmic cytoskeleton to the extracellular matrix and other cells by force sensing. During development, as cells differentiate, variations in protein expression levels result in morphological changes. There are two major explanations for motile behavior: either cellular motility depends in a continuous fashion on cell composition or it exhibits phases wherein only a few protein modules are activated locally for a given time. Indeed, in support of the latter model, the quantification of cell spreading and other motile activities shows multiple distinct modes of behavior, which we term "phases" because there exist abrupt transitions between them. Cells in suspension have a basal level of motility that enables them to probe their immediate environment. After contacting a matrix-coated surface, they rapidly transition to an activated spreading phase. After the development of a significant contact area, the cells contract repeatedly to determine the rigidity of the substrate and then develop force on matrix contacts. When cells are fully spread, extension activity is significantly decreased and focal complexes start to assemble near the cell periphery. For each of these phases, there are significant differences in protein activities, which correspond to differences in function. Thus overall morphological change of a tissue is driven by chemical signals and force-dependent activation of one or more motile phases in limited cell regions for defined periods.     

Effects of surface-bound water and surface stereochemistry on cell adhesion to crystal surfaces

Crystals of calcium-(R,S)-tartrate trihydrate were used as adhesion substrates (for A6 epithelial cells), to study specific stages in cell adhesion. Events such as surface recognition, cell attachment, spreading, motility, cell-cell aggregation, and cell penetration into the crystal bulk are all shown to depend on the molecular structure of the various crystal faces. These crystals exhibit three chemically equivalent, yet structurally distinct, faces. On the {100}, a layered surface exposing bound water, the cells attach, are motile, and tend to form multicellular aggregates, but do not spread and do not form focal contacts. Following prolonged incubation, single cells attached to the {100} surface undergo apoptosis, while those interacting with other cells are rescued. Macroscopic spiral dislocations emerging on the {100} face of the crystal are highly adhesive for cells. Cells attached to these sites develop long protrusions that penetrate into the crystal. The {011} faces expose mainly hydroxyls attached to the chiral carbons. The cells interact extensively with these faces, are immobilized, do not spread, do not form focal contacts, and subsequently die. The faces belonging to the {0kl}? family are characterized by molecular and topographical steps. The cells attach to these faces, spread, and form focal contacts and stress fibers. Thus the molecular character of the crystal surfaces, including the presence of bound water, the exposure of determinants that promote rapid surface recognition, and the effective association with extracellular adhesive proteins, affect the patterns of cell adhesive behavior and fate.     

The fibronectin-binding integrins alpha5beta1 and alphavbeta3 differentially modulate RhoA-GTP loading,organization of cell matrix adhesions,and fibronectin fibrillogenesis

We have studied the formation of different types of cell matrix adhesions in cells that bind to fibronectin via either alpha5beta1 or alphavbeta3. In both cases, cell adhesion to fibronectin leads to a rapid decrease in RhoA activity. However, alpha5beta1 but not alphavbeta3 supports high levels of RhoA activity at later stages of cell spreading, which are associated with a translocation of focal contacts to peripheral cell protrusions, recruitment of tensin into fibrillar adhesions, and fibronectin fibrillogenesis. Expression of an activated mutant of RhoA stimulates alphavbeta3-mediated fibrillogenesis. Despite the fact that alpha5beta1-mediated adhesion to the central cell-binding domain of fibronectin supports activation of RhoA, other regions of fibronectin are required for the development of alpha5beta1-mediated but not alphavbeta3-mediated focal contacts. Using chimeras of beta1 and beta3 subunits, we find that the extracellular domain of beta1 controls RhoA activity. By expressing both beta1 and beta3 at high levels, we show that beta1-mediated control of the levels of beta3 is important for the distribution of focal contacts. Our findings demonstrate that the pattern of fibronectin receptors expressed on a cell dictates the ability of fibronectin to stimulate RhoA-mediated organization of cell matrix adhesions.     

Calreticulin affects fibronectin-based cell-substratum adhesion via the regulation of c-Src activity

Calreticulin is an endoplasmic reticulum Ca2+-storage protein, which influences gene expression and cell adhesion. In this study, we show that calreticulin induces fibronectin gene expression and matrix deposition, leading to differences in cell spreading and focal adhesion formation in cells differentially expressing calreticulin. We further show that these effects of calreticulin occur via a c-Src-regulated pathway and that c-Src activity is inversely related to calreticulin abundance. Since c-Src is an important regulator of focal contact turnover, we investigated the effect of c-Src inhibition on cells differentially expressing calreticulin. Inhibition of c-Src rescued the poorly adhesive phenotype of the calreticulin-underexpressing cells in that they became well spread, commenced formation of numerous focal contacts, and deposited a rich fibronectin matrix. Importantly, we show that c-Src activity is dependent on releasable Ca2+ from the endoplasmic reticulum, thus implicating Ca2+-sensitive pathways that are affected by calreticulin in cell-substratum adhesion. We propose that calreticulin affects fibronectin synthesis and matrix assembly via the regulation of fibronectin gene expression. In parallel, calcium-dependent effects of calreticulin on c-Src activity influence the formation and/or stability of focal contacts, which are instrumental in matrix assembly and remodeling.     

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.     

The p16(INK4a) tumour suppressor protein inhibits alphavbeta3 integrin-mediated cell spreading on vitronectin by blocking PKC-dependent localization of alphavbeta3 to focal contacts

Expression of full-length p16(INK4a) blocks alphavbeta3 integrin-dependent cell spreading on vitronectin but not collagen IV. Similarly, G1-associated cell cycle kinases (CDK) inhibitory (CKI) synthetic peptides derived from p16(INK4a), p18(INK4c) and p21(Cip1/Waf1), which can be delivered directly into cells from the tissue culture medium, do not affect non-alphavbeta3-dependent spreading on collagen IV, laminin and fibronectin at concentrations that inhibit cell cycle progression in late G1. The alphavbeta3 heterodimer remains intact after CKI peptide treatment but is immediately dissociated from the focal adhesion contacts. Treatment with phorbol 12-myristate 13-acetate (PMA) allows alphavbeta3 to locate to the focal adhesion contacts and the cells to spread on vitronectin in the presence of CKI peptides. The cdk6 protein is found to suppress p16(INK4a)-mediated inhibition of spreading and is also shown to localize to the ruffling edge of spreading cells, indicating a function for cdk6 in controlling matrix-dependent cell spreading. These results demonstrate a novel G1 CDK-associated integrin regulatory pathway that acts upstream of alphavbeta3-dependent activation of PKC as well as a novel function for the p16(INK4a) tumour suppressor protein in regulating matrix-dependent cell migration.     

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.     

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.