The production and localization of laminin in cultured vascular and corneal endothelial cells

The production and localization of laminin, as a function of cell density (sparse versus confluent cultures) and growth stage (actively growing versus resting cultures), has been compared on the cell surfaces of cultured vascular and corneal endothelial cells. Comparison of the abilities of the two types of cells to secrete laminin and fibronectin into their incubation medium reveals that vascular endothelial cells can secrete 20-fold as much laminin as can corneal endothelial cells. In contrast, both cell types produce comparable amounts of fibronectin. Furthermore, if one compares the secretion of laminin and fibronectin as a function of cell growth, it appears that the laminin released into the medium by either vascular or corneal endothelial cells, is a function of cell density and cell growth, since this release is most pronounced when the cells are sparse and actively growing, and decreases by 10- and 30-fold, respectively, when either vascular or corneal endothelial cell cultures become confluent. With regard to fibronectin secretion, no such variation can be seen with vascular endothelial cell cultures, regardless of whether they are sparse and actively growing or confluent and resting. Corneal endothelial cell cultures, demonstrated a twofold increase in fibronectin production when they were confluent and resting as compared to when they were sparse and actively growing. When the distribution of laminin versus fibronectin within the apical and basal cell surfaces of cultured corneal and vascular endothelial cells is compared, one can observe that unlike fibronectin, which in sparse and subconfluent cultures can be seen to be associated with both the apical cell surface. In confluent cultures, laminin can be found associated primarily with the extracellular matrix beneath the cell monolayer, where it codistributes with type IV collagen.     

Geometric control of switching between growth, apoptosis, and differentiation during angiogenesis using micropatterned substrates

Summary Past studies using micropatterned substrates coated with adhesive islands of extracellular matrix revealed that capillary endothelial cells can be geometrically switched between growth and apoptosis. Endothelial cells cultured on single islands larger than 1500 μm² spread and progressed through the cell cycle, whereas cells restricted to areas less than 500 μm² failed to extend and underwent apoptosis. The present study addressed whether island geometries that constrained cell spreading to intermediate degrees, neither supporting cell growth nor inducing apoptosis, cause cells to differentiate. Endothelial cells cultured on substrates micropatterned with 10-μm-wide lines of fibronectin formed extensive cell-cell contacts and spread to approximately 1000 μm². Within 72 h, cells shut off both growth and apoptosis programs and underwent differentiation, resulting in the formation of capillary tube-like structures containing a central lumen. Accumulation of extracellular matrix tendrils containing fibronectin and laminin beneath cells and reorganization of platelet endothelial cell adhesion molecule-positive cell-cell junctions along the lengths of the tubes preceded the formation of these structures. Cells cultured on wider (30-μm) lines also formed cell-cell contacts and aligned their actin cytoskeleton, but these cells spread to larger areas (2200 μm²), proliferated, and did not form tubes. Use of micropatterned substrates revealed that altering the geometry of cell spreading can switch endothelial cells among the three major genetic programs that govern angiogenesis—growth, apoptosis and differentiation. The system presented here provides a well-defined adhesive environment in which to further investigate the steps involved in angiogenesis.     

Expression and adhesive properties of basement membrane proteins in cerebral capillary endothelial cell cultures

Previous studies have indicated the importance of basement membrane components both for cellular differentiation in general and for the barrier properties of cerebral microvascular endothelial cells in particular. Therefore, we have examined the expression of basement membrane proteins in primary capillary endothelial cell cultures from adult porcine brain. By indirect immunofluorescence, we could detect type IV collagen, fibronectin, and laminin both in vivo (basal lamina of cerebral capillaries) and in vitro (primary culture of cerebral capillary endothelial cells). In culture, these proteins were secreted at the subcellular matrix. Moreover, the interaction between basement membrane constituents and cerebral capillary endothelial cells was studied in adhesion assays. Type IV collagen, fibronectin, and laminin proved to be good adhesive substrata for these cells. Although the number of adherent cells did not differ significantly between the individual proteins, spreading on fibronectin was more pronounced than on type IV collagen or laminin. Our results suggest that type IV collagen, fibronectin, and laminin are not only major components of the cerebral microvascular basal lamina, but also assemble into a protein network, which resembles basement membrane, in cerebral capillary endothelial cell cultures.     

Expression of desmin, laminin and fibronectin during in situ differentiation (decidualization) of rat uterine stromal cells

Immunocytochemical analysis of frozen rat uterine sections containing decidual tissue, formed in response to normal or artificial stimulation of uteri sensitized by endogenous or exogenous hormonal regimens, demonstrated an elevated expression of the intermediate filament protein desmin in decidual cells. Changes in the expression of extracellular matrix (ECM) components were coordinated with the elevated expression of desmin as stromal cells underwent decidualization. In parallel with the pattern of regional decidualization, as determined by elevated desmin expression, laminin accumulated in ECM of decidual cells while an apparent decrease in fibronectin was associated with altered organization at the decidual cell surface. The in situ observations confirm previous results, which indicated that the expression of desmin in decidual cells formed in vivo or in vitro is a valid marker of their differentiation, and resolve questions unanswered in the previous study: (a) desmin (and laminin) appear to be constitutively expressed in non-decidualized stroma at barely detectable levels, (b) desmin is a valid marker of stromal cell differentiation because it is expressed similarly in decidual cells, irrespective of varying experimental protocols for uterine sensitization and stimulation, and (c) desmin expression follows the same regional progression described for the process of decidualization in morphological and histochemical studies.     

Syndecans in cell adhesion and differentiation

Syndecans are transmembrane proteoglycans expressed on adherent cells. They are a family of four proteins, which participate in cell-matrix adhesion, the regulation of growth factors (FGFs, VEGF, HGF) binding and signaling. The extracellular domain of syndecans contains heparan sulfate and chondroitin sulfate glycosaminoglycan chains. Syndecans have transmembrane region and a short cytoplasmic domain. The cytoplasmic domain attaches activated protein kinase Calpha, phosphatidyl-inositol-4,5-bisphosphate, syntenin, beta-catenin and many others molecules. Syndecans bind numerous ligands, which are present in extracellular matrix: growth factors, enzymes, extracellular matrix molecules (fibronectin, laminin). They form connections with actin cytoskeleton. The changes in syndecan expression influence on cell adhesion and migration, structure of focal contacts and cytoskeleton. Syndecans participate in cell differentiation and tissue regeneration.     

Substratum effects on cell dispersal, morphology, and differentiation in cultures of avian neural crest cells

Adhesive extracellular matrix (ECM) molecules appear to play roles in the migration of neural crest cells, and may also provide cues for differentiation of these cells into a variety of phenotypes. We are studying the influences of specific ECM components on crest differentiation at the levels of both individual cells and cell populations. We report here that the glycoproteins fibronectin and laminin differentially affect melanogenesis in cultures of avian neural crest-derived cells. Clusters of neural crest cells were allowed to form on explanted neural tubes for 24 and 48 hr, and then subcultured on uncoated glass coverslips or coverslips coated with fibronectin or laminin. The morphology of cells varied on the three substrata, as did patterns of cell dispersal. Crest cells dispersed most rapidly and extensively on fibronectin. In contrast, cells on laminin dispersed initially, but then assumed a stellate morphology and rapidly formed small aggregates. Cell dispersal was minimal on glass substrata, resulting in a uniformly dense distribution. These patterns of dispersal were similar in subcultures of both 24- and 48-hr clusters, although dispersal of cells from older clusters was less extensive. The rate and extent of melanogenesis correlated with patterns of cell dispersal. Cell from 24-hr clusters underwent melanogenesis significantly more slowly on fibronectin than on the other two substrata. Pigment cells began to differentiate by 2 days of subculture in the cell aggregates on laminin and in the dense centers of cultures on untreated glass. By 5 days, there was significantly more melanogenesis in cultures on laminin and glass than on fibronectin substrata. Melanogenesis in cultures of 48-hr clusters was more rapid and extensive on control (glass) substrata than on fibronectin or laminin, correlating with reduced cell dispersal. We conclude that fibronectin and laminin, which are found along neural crest migratory pathways in vivo, can affect melanogenesis in vitro by regulating patterns of cell dispersal.     

Cooperativity between Sertoli cells and testicular peritubular cells in the production and deposition of extracellular matrix components

We examined the synthesis and deposition of extracellular matrix (ECM) components in cultures of Sertoli cells and testicular peritubular cells maintained alone or in contact with each other. Levels of soluble ECM components produced by populations of isolated Sertoli cells and testicular peritubular cells were determined quantitatively by competitive enzyme-linked immunoabsorbent assays, using antibodies shown to react specifically with Type I collagen, Type IV collagen, laminin, or fibronectin. Peritubular cells in monoculture released into the medium fibronectin (432 to 560 ng/microgram cell DNA per 48 h), Type I collagen (223 to 276 ng/microgram cell DNA per 48 h), and Type IV collagen (350 to 436 ng/microgram cell DNA per 48 h) during the initial six days of culture in serum-free medium. In contrast, Sertoli cells in monoculture released into the medium Type IV collagen (322 to 419 ng/microgram cell DNA per 48 h) but did not form detectable amounts of Type I collagen or fibronectin during the initial six days of culture. Neither cell type produced detectable quantities of soluble laminin. Immunocytochemical localization investigations demonstrated that peritubular cells in monoculture were positive for fibronectin, Type I collagen, and Type IV collagen but negative for laminin. In all monocultures most of the ECM components were intracellular, with scant deposition as extracellular fibrils. Sertoli cells were positive immunocytochemically for Type IV collagen and laminin but negative for fibronectin and Type I collagen. Co-cultures of peritubular cells and Sertoli cells resulted in interactions that quantitatively altered levels of soluble ECM components present in the medium. This was correlated with an increased deposition of ECM components in extracellular fibrils. The data correlated with an increased deposition of ECM components in extracellular fibrils. The data presented here we interpret to indicate that the two cell types in co-culture act cooperatively in the formation and deposition of ECM components. Results are discussed with respect to the nature of interactions between mesenchymal peritubular cell precursors and adjacent epithelial Sertoli cell precursors in the formation of the basal lamina of the seminiferous tubule.     

The impact of cell-cell contact, e-cadherin and EGF receptor on the cellular radiosensitivity of a431 cancer cells

Cell-cell contact is thought to be critically involved in mechanisms leading to radioresistance. Here, we assessed the influence of confluent compared to subconfluent cell culture conditions and the radiosensitizing ability of E-cadherin inhibition alone or in combination with C225-mediated EGFR inhibition in human squamous cell carcinoma cells. Our results show higher radioresistance under subconfluent growth conditions than under confluency. Delayed plating only resulted in higher radiation survival in confluently growing cells. While E-cadherin depletion significantly reduced basal clonogenic survival, increased the rate of apoptosis, and diminished cell adhesion, the cellular radiosensitivity remained unchanged under both subconfluent and confluent conditions. C225 decreased basal cell survival but failed to modify radiation survival. Additional treatment of E-cadherin knockdown cell cultures with C225 did not further reduce basal cell survival or lead to radiosensitization. Interestingly, E-cadherin silencing in 3D cell cultures did not alter radiosensitivity. Our data indicate that cell-cell contact and E-cadherin are not prominently involved in the regulation of radioresistance of human squamous cell carcinoma cells. In addition, no regulatory interaction between E-cadherin and EGFR in the radiation response was observed.     

Antagonistic effects of laminin and fibronectin on the expression of the myogenic phenotype

Skeletal myoblasts from fetal muscle respond adversely to fibronectin and laminin substrata: when primary mouse skeletal myoblasts are plated onto laminin, more myosin and desmin-positive myoblasts (myo+ cells) develop than on plates coated with fibronectin or collagen. In clonal cultures virtually all cells differentiate into postmitotic, fusion-capable myo + myoblasts on laminin after 3 days. In contrast, on fibronectin, the majority of the cells becomes myosin- and desmin-negative, partially due to proliferation of undifferentiated myoblast precursor cells, partially due to dedifferentiation or modulation of myoblasts into fibroblast-like myo- cells. Loss of the myogenic phenotype on fibronectin was also observed in cloned mouse myoblasts and in cultures of a differentiating mouse satellite cell line, MM14Dy, confirming that the appearance of desmin-negative cells is a result of myoblast modulation and not due simply to overgrowth by muscle fibroblasts. In the light of other effects of laminin on myoblasts, such as the stimulation of migration, differentiation and proliferation, our findings are consistent with the notion that laminin and fibronectin may be counteracting factors in the control of muscle differentiation.     

Evidence for a Role of the Epithelial Glycoprotein 40 (Ep-CAM) in Epithelial Cell-Cell Adhesion

Recently we have demonstrated that a 40kD human epithelium-specific glycoprotein exhibits the features of a homophilic cell-cell adhesion molecule, when expressed in transfected murine cells. We suggested the name Ep-CAM for this molecule (Litvinov et al., J. Cell Biol., 125: 437-446). Here we investigate the possible biological function of Ep-CAM in its natural environment—cells of epithelial origin. Immunolocalization of Ep-CAM in tissues and in cultures of epithelial/carcinoma cells showed that the majority of the Ep-CAM molecules are localized at cell-cell boundaries, predominantly along the whole lateral domain of polarized cells. In vitro, on single cells in suspension, the Ep-CAM molecules are present on the entire cell surface, and when the single cells grow attached, Ep-CAM is present at their pseudo-apical domain. During formation of intercellular contacts by such single cells, the majority of the Ep-CAM molecules are redistributed from the pseudoapical to the lateral domain of the cell membrane. Attachment of cells to the substrate does not cause redistribution of the molecules to the site of substrate attachment irrespective of the adhesive substrate (fibronectin, collagens, laminin, EHS-matrigel were tested). The monoclonal antibody 323/A3, reactive with the extracellular domain of the Ep-CAM molecule, has a strong negative effect on the aggregating behaviour of COV362 ovarian carcinoma cells and RC-6 immortalized mammary epithelial cells. The mAb affected cell aggregation in both cell lines in the presence of Ca⁺⁺, but with RC-6 cells the effect was more pronounced in low-calcium medium. The effects of the 323/A3 mAb on the already established intercellular contacts was not significant. The data presented demonstrate that the Ep-CAM molecules are functionally active in the epithelial and carcinoma cells tested, are capable of mediating Ca¹⁺-independent intercellular adhesions, and are not likely to be involved in cell-substrate adhesion.     

Real-time quantitative polymerase chain reaction and flow cytometric analyses of cell adhesion molecules expressed in human cell–multilayered periosteal sheets in vitro

Background aimsCultured human periosteal sheets more effectively function as an osteogenic grafting material at implantation sites than do dispersed periosteal cells. Because adherent cell growth and differentiation are regulated by cell-cell and cell–extracellular matrix contacts, we hypothesized that this advantage is a result of the unique cell adhesion pattern formed by their multiple cell layers and abundant extracellular matrix. To test this hypothesis, we prepared three distinct forms of periosteal cell cultures: three-dimensional cell-multilayered periosteal sheets, two-dimensional dispersed cell cultures, and three-dimensional hybrid mock-ups of cells dispersed onto collagen sponges.MethodsPeriosteal cells were obtained from human alveolar bone. Cell adhesion and extracellular matrix molecules were quantitatively determined at the messenger RNA and protein levels by means of real-time quantitative polymerase chain reaction and flow cytometry, respectively.ResultsReal-time quantitative polymerase chain reaction analysis demonstrated that regardless of culture media α1 integrin, vascular cell adhesion molecule-1, fibronectin and collagen type 1 were substantially upregulated, whereas CD44 was strongly downregulated in periosteal sheets compared with dispersed cell monolayers. With increased thickness, stem cell medium upregulated several integrins (β1, α1 and α4), CD146, vascular cell adhesion molecule-1, fibronectin and collagen type 1 in the periosteal sheets. Flow cytometric analysis revealed that the active configuration of β1 integrin was substantially downregulated in the stem cell medium–expanded cell cultures. The cell adhesion pattern found in the mock-up cultures was almost identical to that of genuine periosteal sheets.ConclusionsIntegrin α1β1 and CD44 function as the main cell adhesion molecule in highly cell-multilayered periosteal sheets and dispersed cells, respectively. This difference may account for the more potent osteogenic activity shown by the thicker periosteal sheets.     

Immunomorphological research on the formation of the extracellular matrix in epithelial cell cultures

Formation of extracellular matrix structures in cultures of rat liver epithelial nontransformed cell line IAR2 was studied with antisera to fibronectin, laminin and type IV collagen by immunofluorescence and immunoelectron microscopy of platinum replicas. Fibronectin formed peripheral spots of variable size some of which outlined free cell edges, as well as fibrils located towards the center of single cells or of cellular islands. Similarly distributed structures were seen in isolated matrices. Codistribution of fibronectin and actin was observed only for the peripheral line of fibronectin spots and marginal circular actin bundle. Basement membrane components. laminin and type IV collagen, formed mainly spots of variable size predominantly beneath the cell or each cell in an island. Occasional fibrils were seen also. Essentially the same results were obtained by immunofluorescence and immunogold electron microscopy. Cytochalasin D treated cells displayed spots of both fibronectin and laminin. The relevance of previously postulated receptor-mediated assembly of extracellular matrix structures to the epithelial cells is discussed.     

Development of the electromotor system of Torpedo marmorata: Distribution of extracellular matrix and cytoskeletal components during acetylcholine receptor focalization

Summary A combination of direct fluorescence and indirect immunofluorescence microscopy has been used to compare the distribution of the acetylcholine receptor with the distribution of major cytoskeletal and extracellular matrix components during electrocyte differentiation in the electric organs of Torpedo marmorata. Laminin, fibronectin and extracellular matrix proteoglycan are always more extensively distributed around the differentiating cell than the acetylcholine receptor-rich patch that forms on the ventral surface of the cell. The distribution of acetylcholinesterase within the ventral surface of the differentiating electrocyte closely resembles the distribution of the acetylcholine receptor. Areas of apparently high acetylcholine receptor density within the ventrally forming acetylcholine receptor-rich patch are always areas of apparently high extracellular matrix proteoglycan density but are not always areas of high laminin or fibronectin density. Desmin levels appear to increase at the onset of differentiation and desmin initially accumulates in the ventral pole of each myotube as it begins to form an electrocyte. During differentiation F-actin-positive filament bundles are observed that extend from the nuclei down to the ventrally forming acetylcholine receptorrich patch. Most filament bundles terminate in the acetylcholine receptor-rich region of the cell membrane. Electronmicroscopic autoradiography suggests that the filament bundles attach to the membrane at sites where small acetylcholine receptor clusters are found. The results of this study suggest that, out of the four extracellular matrix components studied, only the distribution of acetylcholinesterase (which may be both matrix- and membrane-bound at this stage) closely parallels that of the acetylcholine receptor, and that F-actin filament bundles terminate in a region of the cell that is becoming an area of high acetylcholine receptor density.Abbreviations ACHR nicotinic acetylcholine receptor - ACHE acetylcholinesterase - BSA bovine serum albumin - EMPG extracellular matrix proteoglycan fraction - FITC fluorescein isothiocyanate - FN fibronectin - LN laminin - TBS Tris-HCl-buffered saline - SDS PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis     

Colocalization of laminin and fibronectin in bovinelens epithelial cells in vitro

Summary The distribution and organization of the extracellular matrix (ECM) proteins laminin, fibronectin, entactin, and type IV collagen were investigated in primary colonies and secondary cultures of bovine lens epithelial cells using species-specific antisera and indirect immunofluorescence microscopy. Primary cell colonies fixed in formaldehyde and permeabilized with Triton X-100 displayed diffuse clonies. In contrast, thick bundles of laminin and fibronectin were located on the basal cellsurfaces and in between cells in the densely packed center of the colonies, and as “adhesive plaques” and fine extracellular matrix cords in the sparsely populated (migratory) outer edge of the colonies. The distribution of ECM proteins observed in secondary lens epithelial cell cultures was similar to that observed at the periphery of the primary colony. Extraction of the secondary cell cultures with sodium deoxycholate confirmed that laminin and fibronectin were deposited on the basal cell surface. Indeed, the patterns of laminin and fibronectin deposition suggested that these proteins codistribute. These results establish that lens epithelial cells in culture can be used as a model system to study the synthesis and extracellular deposition of the basement membrane proteins, laminin and fibronectin. Supported by Public Health Service grant EY05570 from the National Eye Institute Bethesda, MD.     

Cell-cell and cell-substrate adhesion in cultured Drosophila imaginal disc cells

Summary Drosophila imaginal disc cell lines were used to investigate various aspects of cellular adhesion in vitro. The distribution of PS integrins and their involvement in cell-cell and cell-substrate adhesion were assessed with the monoclonal antibody aBG-1 against the βPS subunit, and both forms of adhesion were found to be impeded by the presence of the antibody. Adhesion to a number of extracellular matrix components was investigated, and the cells were found to adhere to human fibronectin. This adhesion was inhibited by aBG-1. The adhesion molecule fasciclin III was also found in these cells. Given that the cells are competent to perform cell-cell and cell-substrate adhesion, it was thought that apical basal polarity might be restored when other suitable conditions were provided, i.e., an artificial basement layer with feeder cells to provide nutrients basally to the cells, and some features of apical-basal morphology were seen in cells cultured under these conditions.     

Effects of cell density and extracellular matrix on the lateral diffusion of major histocompatibility antigens in cultured fibroblasts

We have studied the effect of cell density on the lateral diffusion of major histocompatibility (MHC) antigens in the plasma membranes of fibroblasts using fluorescence recovery after photobleaching. The percent recovery of fluorescence was decreased in fibroblasts grown in confluent cultures. While recovery of fluorescence was measurable in greater than 90% of the cells from sparse cultures, measurable recovery was detected in only 60-80% of the cells from dense cultures; no mobile antigens were detectable in 20-40% of cells examined. The diffusion coefficient on human skin fibroblast cells that did show recovery was the same for cells grown in sparse or dense conditions. In WI-38, VA-2, and c1 1d cultures the diffusion coefficients of mobile antigens were smaller in cells from dense cultures. Changes in lateral diffusion occurred with increased cell-cell contact and with age of cell culture but were not observed in growth-arrested cells or in sparse cells cultured in medium conditioned by confluent cells. Decreased mobile fractions of MHC antigens were observed when cells were plated on extracellular matrix materials derived from confluent cultures. Treatment of the extracellular matrix materials with a combination of proteolytic enzymes or by enzymes that degrade proteoglycans abolished this effect. Matrices produced by cells from other cell lines were less effective in inducing changes in mobile fractions and purified matrix components alone did not induce changes in lateral diffusion. Finally, there were no differences in the proportion of MHC antigens that were resistant to Triton X-100 extraction in sparse and dense cells. These results suggest that cell-cell interactions mediated through extracellular matrix materials can influence the lateral diffusion of at least part of the population of MHC antigens.     

Extracellular matrix components synthesized by human amniotic epithelial cells in culture

Epithelial cells from human post-partal amniotic membrane in primary culture secreted two major matrix proteins, fibronectin and procollagen type III, and small amounts of laminin and basement membrane collagens (types IV and AB). Identified in the culture medium by immunoprecipitation, these components were located by immunofluorescence to a pericellular matrix beneath the cell monolayer. Deposition of fibronectin, laminin and procollagen type III occurred under freshly seeded spreading cells. In the matrix of confluent cultures, fibronectin and procollagen type III had a moss-like distribution. Matrix laminin had predominantly a punctate pattern and was sometimes superimposed on the fibronectin-procollagen type III matrix. In the human amniotic membrane in vivo, laminin, type IV collagen and fibronectin were located to a narrow basement membrane directly beneath the epithelial cells. Fibronectin and procollagen type III were detected in the underlying thick acellular compact layer. Fibronectin secreted by amniotic epithelial cells is a disulfide-bonded dimer of slightly higher apparent molecular weight (240 kilodaltons) than fibronectins isolated from human plasma or fibroblast cultures. Laminin was detected in small amounts in the culture medium. Laminin antibodies precipitated a polypeptide of about 400 kilodaltons, and two polypeptides with slightly faster mobility in electrophoresis under reducing conditions than fibronectin. Procollagen type III was by far the major collagenous protein whereas little or no production of procollagen type I could be observed. Basement membrane collagens were identified as minor components in the medium by immunoprecipitation (type IV) or chemical methods (αA and αB chains).     

The role of integrins alpha 2 beta 1 and alpha 3 beta 1 in cell-cell and cell-substrate adhesion of human epidermal cells

We have examined cultures of neonatal human foreskin keratinocytes (HFKs) to determine the ligands and functions of integrins alpha 2 beta 1, and alpha 3 beta 1 in normal epidermal stratification and adhesion to the basement membrane zone (BMZ) in skin. We used three assay systems, HFK adhesion to purified extracellular matrix (ECM) ligands and endogenous secreted ECM, localization of integrins in focal adhesions (FAs), and inhibition of HFK adhesion with mAbs to conclude: (a) A new anti-alpha 3 beta 1 mAb, P1F2, localized alpha 3 beta 1 in FAs on purified laminin greater than fibronectin/collagen, indicating that laminin was the best exogeneous ligand for alpha 3 beta 1. However, in long term culture, alpha 3 beta 1 preferentially codistributed in and around FAs with secreted laminin-containing ECM, in preference to exogenous laminin. Anti-alpha 3 beta 1, mAb P1B5, detached prolonged cultures of HFKs from culture plates or from partially purified HFK ECM indicating that interaction of alpha 3 beta 1 with the secreted laminin-containing ECM was primarily responsible for HFK adhesion in long term culture. (b) In FA assays, alpha 2 beta 1 localized in FAs conincident with initial HFK adhesion to exogenous collagen, but not laminin or fibronectin. However, in inhibition assays, anti-alpha 2 beta 1 inhibited initial HFK adhesion to both laminin and collagen. Thus, alpha 2 beta 1 contributes to initial HFK adhesion to laminin but alpha 3 beta 1 is primarily responsible for long-term HFK adhesion to secreted laminin-containing ECM. (c) Serum or Ca2(+)-induced aggregation of HFKs resulted in relocation of alpha 2 beta 1 and alpha 3 beta 1 from FAs to cell-cell contacts. Further, cell-cell adhesion was inhibited by anti-alpha 3 beta 1 (P1B5) and a new anti-beta 1 mAb (P4C10). Thus, interaction of alpha 3 beta 1 with either ECM or membrane coreceptors at cell-cell contacts may facilitate Ca2(+)-induced HFK aggregation. (d) It is suggested that interaction of alpha 3 beta 1 with a secreted, laminin-containing ECM in cultured HFKs, duplicates the role of alpha 3 beta 1 in basal cell adhesion to the BMZ in skin. Further, relocation of alpha 2 beta 1 and alpha 3 beta 1 to cell-cell contacts may result in detachment of cells from the BMZ and increased cell-cell adhesion in the suprabasal cells contributing to stratification of the skin.     

N-cadherin cell-cell adhesion complexes are regulated by fibronectin matrix assembly

Fibronectin is a principal component of the extracellular matrix. Soluble fibronectin molecules are assembled into the extracellular matrix as insoluble, fibrillar strands via a cell-dependent process. In turn, the interaction of cells with the extracellular matrix form of fibronectin stimulates cell functions critical for tissue repair. Cross-talk between cell-cell and cell-extracellular matrix adhesion complexes is essential for the organization of cells into complex, functional tissue during embryonic development and tissue remodeling. Here, we demonstrate that fibronectin matrix assembly affects the organization, composition, and function of N-cadherin-based adherens junctions. Using fibronectin-null mouse embryonic myofibroblasts, we identified a novel quaternary complex composed of N-cadherin, β-catenin, tensin, and actin that exists in the absence of a fibronectin matrix. In the absence of fibronectin, homophilic N-cadherin ligation recruited both tensin and α5β1 integrins into nascent cell-cell adhesions. Initiation of fibronectin matrix assembly disrupted the association of tensin and actin with N-cadherin, released α5β1 integrins and tensin from cell-cell contacts, stimulated N-cadherin reorganization into thin cellular protrusions, and decreased N-cadherin adhesion. Fibronectin matrix assembly has been shown to recruit α5β1 integrins and tensin into fibrillar adhesions. Taken together, these studies suggest that tensin serves as a common cytoskeletal link for integrin- and cadherin-based adhesions and that the translocation of α5β1 integrins from cell-cell contacts into fibrillar adhesions during fibronectin matrix assembly is a novel mechanism by which cell-cell and cell-matrix adhesions are coordinated.