Altered vitronectin receptor (alphav integrin) function in fibroblasts adhering on hydrophobic glass.

Function of integrins is crucial for adhesion, movement, proliferation, and survival of cells. In a recent study we found impaired fibronectin receptor function on hydrophobic substrata (G. Altankov et al. J Biomater Sci Polym Edn 1997;8:712-740). Here, we have studied the distribution and function of the vitronectin receptor (alphav integrin) in fibroblasts adhering on hydrophilic glass and hydrophobic octadecyl glass (ODS). The morphology of fibroblasts and the organization of actin cytoskeleton were studied and found to be altered on ODS, where the cells did not spread and possessed condensed actin. Pretreatment of the surfaces with serum or pure vitronectin improved cell morphology on both substrata, resulting in the development of longitudinal actin stress fibers. It was found with biotinylated vitronectin that comparable quantities of vitronectin were adsorbed from single vitronectin solutions or serum on glass and on hydrophobic ODS. The organization of the vitronectin receptors on the ventral cell surface was investigated in permeabilized cells showing normal focal adhesions in fibroblasts plated on glass but none of these structures on ODS. The distribution of alphav integrin on the dorsal cell surface was studied on nonpermeabilized living cells after antibody tagging. While fibroblasts adhering on plain or serum-treated glass developed a linear organization of alphav integrin, cells on plain and serum-treated ODS were not able to reorganize the vitronectin receptor. Studies on signal transduction with antiphosphotyrosine antibodies revealed co-localization of alphav integrin and phosphotyrosine in focal adhesions on glass and serum-treated glass. However, signaling was almost absent on plain ODS and weak on serum-treated ODS. It was concluded that alterations in vitronectin receptor function on the ventral cell surface caused by the hydrophobic material surface inhibit signal transfer and subsequent intracellular events that are important for the organization and function of integrins.     

The dependence of fibrillar adhesions in human fibroblasts on substratum chemistry

Little is known about the influence of substratum properties and composition on the ability of cells to translocate alpha5beta1 integrins and to form fibrillar adhesion. We have examined the impact of self-assembled monolayers (SAMs) bearing different functional end groups (amines (NH2) or carboxylic acids (COOH)) on the presence of fibrillar adhesions in human fibroblasts attached to fibronectin-coated SAMs. Most of the fibroblasts incubated in serum-free medium for 2 h on COOH showed segregation of focal contact components (alphav integrin subunits, phosphotyrosine proteins) and fibrillar adhesions (alpha5 integrin subunits, tensin) while the majority of cells plated on NH(2) did not. Analysis of fibronectin fibril formation confirmed also that human fibroblasts plated on COOH formed matrix fibrils significantly better. The surface-associated alpha5 to alphav integrin ratio was smaller in cells on COOH than on NH2 due to a decreased alpha5 integrin binding. In addition, human fibroblasts migrated more readily on COOH in comparison to NH2 which points to a different binding strength of integrins to the substratum. Overall, the results indicate that the molecular composition of substrata has a strong influence on FN matrix formation by promoting or inhibiting segregation of focal and fibrillar adhesions.     

Influence of substratum surface chemistry/energy and topography on the human fetal osteoblastic cell line hFOB 1.19: Phenotypic and genotypic responses observed in vitro

Time-dependent phenotypic response of a model osteoblast cell line (hFOB 1.19, ATCC, and CRL-11372) to substrata with varying surface chemistry and topography is reviewed within the context of extant cell-adhesion theory. Cell-attachment and proliferation kinetics are compared using morphology as a leading indicator of cell phenotype. Expression of (alpha2, alpha3, alpha4, alpha5, alphav, beta1, and beta3) integrins, vinculin, as well as secretion of osteopontin (OP) and type I collagen (Col I) supplement this visual assessment of hFOB growth. It is concluded that significant cell-adhesion events-contact, attachment, spreading, and proliferation-are similar on all surfaces, independent of substratum surface chemistry/energy. However, this sequence of events is significantly delayed and attenuated on hydrophobic (poorly water-wettable) surfaces exhibiting characteristically low-attachment efficiency and long induction periods before cells engage in an exponential-growth phase. Results suggest that a 'time-cell-substratum-compatibility-superposition principle' is at work wherein similar bioadhesive outcomes can be ultimately achieved on all surface types with varying hydrophilicity, but the time required to arrive at this outcome increases with decreasing cell-substratum-compatibility. Genomic and proteomic tools offer unprecedented opportunity to directly measure changes in the cellular machinery that lead to observed cell responses to different materials. But for the purpose of measuring structure-property relationships that can guide biomaterial development, genomic/proteomic tools should be applied early in the adhesion/spreading process before cells have an opportunity to significantly remodel the cell-substratum interface, effectively erasing cause and effect relationships between cell-substratum-compatibility and substratum properties. IMPACT STATEMENT: This review quantifies relationships among cell phenotype, substratum surface chemistry/energy, topography, and cell-substratum contact time for the model osteoblast cell line hFOB 1.19, revealing that genomic/proteomic tools are most useful in the pursuit of understanding cell adhesion if applied early in the adhesion/spreading process.     

The role of surface wettability on hepatocyte adhesive interactions and function

In this paper the effect of surface wettability on hepatocyte morphology and function was studied, using clean and octadecylsylane (ODS)-coated glass as a model for hydrophilic and hydrophobic surfaces, respectively. C3A cells - a hepatoblastoma cell line, and freshly obtained porcine hepatocytes were cultured for a short-time period of up to 4 days on the above substrata. Hepatocyte adhesive interactions were characterized monitoring the initial cell attachment, the overall cell morphology, the formation of focal adhesions, and actin filaments. Since hepatocytes showed a clear tendency for homotypic adhesion on ODS, specific E-cadherin staining was used to visualize the intercellular contacts by immunofluorescence microscopy. Additionally, functional assays were carried out to monitor proliferation, metabolic activity, and albumin synthesis of C3A cells. It could be shown that both C3A cells and normal porcine hepatocytes spread better on hydrophilic glass; spreading being accompanied by the development of pronounced actin stress fibers and focal adhesion contacts. In contrast, on hydrophobic substrata predominant cell-cell interactions took place which led to intense E-cadherin staining in the intercellular contacts of porcine hepatocytes but not in C3A cells. On the other hand, metabolic activity and growth of C3A cells were reduced on hydrophobic ODS, but albumin synthesis was similar on both surfaces. It was concluded that the wettability of materials has a strong influence on the attachment and morphology of hepatocytes while the influence of surface properties on the functional activity of hepatocytes still remains to be elucidated.     

The role of surface wettability on hepatocyte adhesive interactions and function

In this paper the effect of surface wettability on hepatocyte morphology and function was studied, using clean and octadecylsylane (ODS)-coated glass as a model for hydrophilic and hydrophobic surfaces, respectively. C3A cells--a hepatoblastoma cell line, and freshly obtained porcine hepatocytes were cultured for a short-time period of up to 4 days on the above substrata. Hepatocyte adhesive interactions were characterized monitoring the initial cell attachment, the overall cell morphology, the formation of focal adhesions, and actin filaments. Since hepatocytes showed a clear tendency for homotypic adhesion on ODS, specific E-cadherin staining was used to visualize the intercellular contacts by immunofluorescence microscopy. Additionally, functional assays were carried out to monitor proliferation, metabolic activity, and albumin synthesis of C3A cells. It could be shown that both C3A cells and normal porcine hepatocytes spread better on hydrophilic glass; spreading being accompanied by the development of pronounced actin stress fibers and focal adhesion contacts. In contrast, on hydrophobic substrata predominant cell-cell interactions took place which led to intense E-cadherin staining in the intercellular contacts of porcine hepatocytes but not in C3A cells. On the other hand, metabolic activity and growth of C3A cells were reduced on hydrophobic ODS, but albumin synthesis was similar on both surfaces. It was concluded that the wettability of materials has a strong influence on the attachment and morphology of hepatocytes while the influence of surface properties on the functional activity of hepatocytes still remains to be elucidated.     

Integrin pattern and effect on contraction in cultured testicular peritubular myoid cells

PROBLEM: Neither the integrin pattern nor the biological functions of integrins have been extensively documented in human cultured testicular peritubular myoid cells (TPMC). The integrin pattern and the presence of some proteins of the immunoglobulin superfamily on human TPMC as well as the role of integrins in TPMC contraction were examined. METHOD OF STUDY: Integrin expression was evaluated by immunofluorescence and FACS analysis. To assess the role of integrin in TPMC contraction, human and rat cells were added to a collagen gel system and exposed to contractile stimuli. RESULTS: The immunofluorescence and cytofluorimetric analysis showed that human cultured TPMC express alpha1, alpha2, alpha3, alpha4, alpha5, alpha6, alphav, beta1, beta3, and beta4 integrin subunits, and significant amounts of intercellular adhesion molecule-1 (ICAM-1), whereas they do not present alpha4, beta2, beta7 subunits, nor intercellular adhesion molecule-2 (ICAM-2) and neural cell adhesion molecule (NCAM). The preincubation of human cells with an anti-beta1 mAb and of rat cells with a polyclonal anti-beta1 antibody inhibited TPMC contraction induced by different contractile stimuli. CONCLUSION: Our investigation documented a broad integrin pattern on human cultured TPMC as well as a role for integrins in human and rat TPMC contraction.     

Amount and surface structure of albumin adsorbed to solid substrata with different wettabilities in a parallel plate flow cell

In this article we studied the adsorption of serum albumin to substrata with a broad range of wettabilities from solutions with protein concentrations between 0.03 and 3.00 mg.mL-1 in a parallel-plate flow cell. Wall shear rates were varied between 20 and 2000 s-1. The amount of albumin adsorbed in a stationary state was always highest on PTFE, the most hydrophobic material employed and decreased with increasing wettability of the substrata. Increasing stationary amounts of adsorbed albumin were observed with increasing wall shear rates at the lowest protein concentration. Inverse observations were made at the highest protein concentration. Transmission electron micrographs of replicas from the albumin-coated substrata showed that proteins were mostly adsorbed in islandlike structures on the hydrophobic substrata. The tendency to form islandlike structures was shear rate- and concentration-dependent and disappeared gradually going to more hydrophilic substrata. On glass, the most hydrophilic material employed, a homogeneous, well distributed, fine knotted, reticulated structure was found. In conclusion, this study demonstrates that both the amount of adsorbed albumin as well as the surface structure of the adsorbed proteins are regulated by the substratum wettability. This observation may well account for the fact that substratum properties can be transferred by an adsorbed protein film to the interface with adhering cells or microorganisms.     

The regulation of integrin-mediated osteoblast focal adhesion and focal adhesion kinase expression by nanoscale topography

An important consideration in developing physical biomimetic cell-stimulating cues is that the in vivo extracellular milieu includes nanoscale topographic interfaces. We investigated nanoscale topography regulation of cell functions using human fetal osteoblastic (hFOB) cell culture on poly(l-lactic acid) and polystyrene (50/50 w/w) demixed nanoscale pit textures (14, 29, and 45nm deep pits). Secondary ion mass spectroscopy revealed that these nanotopographic surfaces had similar surface chemistries to that of pure PLLA because of PLLA component surface segregation during spin casting. We observed that 14 and 29nm deep pit surfaces increased hFOB cell attachment, spreading, selective integrin subunit expression (e.g., alphav relative to alpha5, beta1, or beta3), focal adhesive paxillin protein synthesis and paxillin colocalization with cytoskeletal actin stress fibers, and focal adhesion kinase (FAK) and phosphorylated FAK (pY397) expression to a greater degree than did 45nm deep pits or flat PLLA surfaces. Considering the important role of integrin-mediated focal adhesion and intracellular signaling in anchorage-dependent cell function, our results suggest a mechanism by which nanostructured physical signals regulate cell function. Modulation of integrin-mediated focal adhesion and related cell signaling by altering nanoscale substrate topography will have powerful applications in biomaterials science and tissue engineering.     

Surface energy effects on osteoblast spatial growth and mineralization

While short-term surface energy effects on cell adhesion are relatively well known, little is revealed as regards its later stage effects on cell behavior. We examined surface energy effects on osteoblastic cell growth and mineralization by using human fetal osteoblastic (hFOB) cells cultured on plasma-treated quartz (contact angle, theta=0 degrees) and octadecyltrichlorosilane (OTS)-treated quartz (theta=113 degrees). hFOB cells formed a homogeneous cell layer on plasma-treated quartz, while those cultured on OTS-treated quartz produced randomly distributed clump-like structures that were filled with cells (confirmed by confocal microscopy). Mineral deposition by hFOB cells was spatially homogeneous when cultured on hydrophilic surfaces. Furthermore, cells on hydrophilic surfaces exhibited increased mineralized area as well as enhanced mineral-to-matrix ratio (assessed by Fourier transform infrared spectroscopy), relative to cells on hydrophobic surfaces. Experiments using other types of osteoblast-like cells (MC3T3-E1, MG63, and SAOS-2) revealed more or less similar effects in spatial growth morphology. It was concluded that hydrophilic surfaces induce homogeneous spatial osteoblastic cell growth and mineral deposition and enhance the quantity (e.g., area) and quality (e.g., mineral-to-matrix ratio) of mineralization relative to hydrophobic surfaces. Our data suggest that surface energy effects on osteoblastic cell differentiation, especially mineralization, may be correlated with surface energy dependent changes in spatial cell growth.     

Polymeric membranes for hybrid liver support devices: The effect of membrane surface wettability on hepatocyte viability and functions

Extracorporeal therapies based on membrane hybrid liver support devices using primary hepatocytes are an interesting approach to the treatment of acute hepatic failure. In such devices, semipermeable polymeric membranes are effectively used as immunoselective barriers between a patient's blood and the xenocytes in order to prevent the immune rejection of the graft. The membranes may act also as the substratum for cell adhesion, thus favouring the viability and functions of anchorage-dependent cells such as the hepatocytes. Membrane cytocompatibility is expected to depend on the surface properties of the polymer, such as its morphology and its physico-chemical properties. In this paper, we report our investigation on the effect of the surface wettability of membranes on hepatocyte viability and functions. Polypropylene microporous membranes were modified to increase their surface wettability and were used as substrata for rat hepatocyte adhesion culture. Isolated hepatocytes were also cultured on collagen as a reference substratum. Hepatocyte viability generally improved as the cells were cultured on more wettable membranes. In agreement with the viability data, the increasing wettability of the membrane surface also improved some metabolic functions.     

Short-term effects of adhesion peptides on the responses of preosteoblasts to pBMP-9

Adhesion peptides are currently used to enhance the interactions of osteoblasts with biomaterials. However, little is known about the effects of adhesion peptides on cell responses to growth factors, especially the bone morphogenetic proteins (BMPs). We used adhesion peptides Ac-CGGNGERPRGDTYRAY-NH(2) (pRGD), derived from bone sialoprotein, and Ac-CGGDGEA-NH(2) (pDGEA), derived from collagen, which interact with alpha(v)beta(3) and alpha(2)beta(1) integrins, respectively. We analyzed the effects of pRGD- and pDGEA-coated polystyrene (PS) on the responses of murine MC3T3-E1 preosteoblasts to a peptide derived from human BMP-9 (pBMP-9) in serum-free medium. After 1h, pRGD favoured interactions with alpha(v) while pDGEA bound beta(1) integrin subunits. Adding pBMP-9 (400 ng/mL) increased the amount of alpha(v) integrin subunits in cell membranes on pRGD-coated PS, but had no effect on beta(1) integrin subunits. Only on this substratum, collagen type I mRNA was enhanced and the addition of pBMP-9 promoted the early cell differentiation, increasing their alkaline phosphatase (ALP) activity within 24 h. These cells also organized beta(1) integrin subunits at their focal adhesion points. Inhibiting alpha(2)beta(1) integrins by pDGEA pre-treatment decreased this ALP activity. It is therefore important to understand the impact of adhesion peptides on the early cell responses to growth factors in order to improve biomimetic materials.     

Arrangement of type IV collagen and laminin on substrates with controlled density of -OH groups

Collagen IV (Col IV) and laminin (Lam) are the main structural components of the basement membrane where they form two overlapping polymeric networks. We studied the adsorption pattern of these proteins on five model surfaces with tailored density of -OH groups obtained by copolymerization of different ratios ethyl acrylate (EA) and hydroxyl EA (HEA): X(OH)=0, X(OH)=0.3, X(OH)=0.5, X(OH)=0.7, and X(OH)=1 (where X refers the ratio of HEA). Atomic force microscopy revealed substratum-specific adsorption patterns of Col IV and Lam, ranging from single molecules deposition on more hydrophilic substrata to the formation of complex networks on hydrophobic ones. Human umbilical endothelial cells were used to study the biological performance of adsorbed proteins, following the overall cell morphology, the quantities for cell adhesion and spreading, and the development of focal adhesion complexes and actin cytoskeleton. Surprisingly, two optima in the cellular interaction were observed-one on the most hydrophilic X(OH)=1 and other on the relatively hydrophobic X(OH)=0.3 substrate-valid for both Col IV and Lam. When the proteins were adsorbed consecutively, a hydrophobic shift to X(OH)=0 substratum was obtained. Collectively, these data suggest that varying with the density of -OH groups one can tailor the conformation and the functional activity of adsorbed basement membrane proteins.     

Development of the cytodetachment technique to quantify mechanical adhesiveness of the single cell

Adhesion of cells to biomaterials or to components of the extracellular matrix is fundamental in many tissue engineering and biotechnological processes, as well as in normal development and tissue maintenance. Many cells on adhesive molecules will spread and form an organized actin cytoskeleton and complex transmembrane signaling regions called focal adhesions. Focal adhesions appear to function as both signaling and stabilizing components of normal adherent cell activity. To better understand adhesion formations between cells and their underlying substrata, we have designed, developed, and utilized a novel 'cytodetachment' methodology to quantify the force required to displace attached cells. We allowed bovine articular chondrocytes to attach and spread on a substratum of either fibronectin, bovine serum albumin, or standard microscope glass. The cytodetacher was then employed to displace the cells from the substratum. Our results demonstrate that a significantly greater force is required to detach cells from fibronectin versus the two other substrata, suggesting that a cell's actin cytoskeleton and perhaps focal adhesions contribute significantly to its mechanical adhesiveness. The cytodetacher allows us to directly measure the force required for cell detachment from a substratum and to indirectly determine the ability of different substrata to support cell adhesion.     

Analogies in the two-dimensional spatial arrangement of adsorbed proteins and adhering bacteria: bovine serum albumin and Streptococcus sanguis 12

Neither proteins nor bacteria adsorb or adhere homogeneously to a substratum surface. The final two-dimensional spatial arrangement depends on a complicated interplay between protein-protein (bacterium-bacterium) and protein (bacterium)-substratum interactions and the prevailing hydrodynamic conditions. In this paper, results are presented of two separate experiments in which bovine serum albumin (BSA) was adsorbed to, or Streptococcus sanguis 12 was deposited on substrata with different wettabilities in a search for analogies in the two-dimensional spatial arrangement of the adsorbed proteins and adhering bacteria. The spatial arrangement of adsorbed BSA, visualized by transmission electron microscopy on replicas of the surface, was island-like on substrata with a low wettability and well distributed on substrata with a high wettability. The spatial arrangement of adhering S. sanguis 12 was observed directly by light microscopy during the experiment and showed a relatively large collection of near-neighbour sites on the low wettability substrata compared with the high wettability substrata. Thus, it seems that the occurrence of island-like structures in protein adsorption is concurrent with a large collection of near-neighbour sites in bacterial adhesion. As a possible explanation for the above analogy, it is suggested that proteins or bacteria are insufficiently immobilized on low wettability substrata owing to weak interaction forces, and they can move over the substratum surface to yield the two-dimensional spatial arrangements observed.     

Analogies in the two-dimensional spatial arrangement of adsorbed proteins and adhering bacteria: bovine serum albumin and Streptococcus sanguis 12.

Neither proteins nor bacteria adsorb or adhere homogeneously to a substratum surface. The final two-dimensional spatial arrangement depends on a complicated interplay between protein-protein (bacterium-bacterium) and protein (bacterium)-substratum interactions and the prevailing hydrodynamic conditions. In this paper, results are presented of two separate experiments in which bovine serum albumin (BSA) was adsorbed to, or Streptococcus sanguis 12 was deposited on substrata with different wettabilities in a search for analogies in the two-dimensional spatial arrangement of the absorbed proteins and adhering bacteria. The spatial arrangement of adsorbed BSA, visualized by transmission electron microscopy on replicas of the surface, was island-like on substrata with a low wettability and well distributed on substrata with a high wettability. The spatial arrangement of adhering S. sanguis 12 was observed directly by light microscopy during the experiment and showed a relatively large collection of near-neighbour sites on the low wettability substrata compared with the high wettability substrata. Thus, it seems that the occurrence of island-like structures in protein adsorption is concurrent with a large collection of near-neighbour sites in bacterial adhesion. As a possible explanation for the above analogy, it is suggested that proteins or bacteria are insufficiently immobilized on low wettability substrata owing to weak interaction forces, and they can move over the substratum surface to yield the two-dimensional spatial arrangements observed.     

Endothelial cell motility, integrin receptor clustering, and microfilament organization are inhibited by agents that increase intracellular cAMP

Adhesion of human umbilical vein endothelial cells (ECs) to various extracellular matrix proteins is mostly mediated by receptors of the integrin family. The interaction of ECs with extracellular matrix proteins is accompanied by cell spreading, cytoskeletal organization, and clustering of the specific integrin receptors in complex supramolecular structures known as adhesion plaques or focal contacts. Little is known on the functional role of focal contacts in EC adhesion and motility and on the possibility to modulate their organization. In this article we report that an increase in intracellular cAMP levels severely impaired focal contact formation. This process did not affect cell attachment, but increased cell adhesion and strongly inhibited cell motility. ECs were treated with the cAMP-increasing agents forskolin and 2-chloro-adenosine or with the cAMP analogue 8-bromo-cAMP. When treated cells were seeded on purified vitronectin, fibrinogen, or fibronectin little modification in the number of attached cell was observed. In contrast ECs showed impaired organization of microfilaments and poorly developed clusters of beta 3- and beta 1-integrin receptors. On a vitronectin substrate, vinculin followed the distribution of beta 3-receptors. It was typically enriched at the focal contacts in control cells but was fragmented in small dots at the cell periphery in treated cells, as were bundles of actin stress fibers. Similarly, when forskolin was added to ECs spread on vitronectin or on fibrinogen, there was a progressive but reversible disruption of actin microfilaments and diffusion of beta 3 receptors. This was accompanied by a tighter adhesion of the cells to substrata. Migration of ECs in response to different matrix proteins was severely inhibited by cAMP-increasing agents. These data indicate that EC adhesion can occur very efficiently in the absence of fully developed beta 3- or beta 1-integrin receptor-containing focal contacts but suggest that the capacity to normally assemble focal contacts and cytoskeletal proteins is required for full cell spreading and migration.     

Pharmacological inhibition of Hsp90 as a novel antitumor strategy to target cytoarchitecture through extracellular matrix signaling

Pharmacological inhibition of Hsp90 in tumor cells induces anticancer effects through the destabilization of several oncogenic signaling molecules. Although there were reports that Hsp90 inhibition compromises cellular integrity, how this affects the cell adhesion through extracellular matrix (ECM) and integrin signaling is not known. Using human neuroblastoma (IMR-32), cervical (HeLa) and breast (MCF-7) cancer cells, and mouse embryonic carcinoma (PCC-4) cells, and using different substratum, glass, plastic, fibronectin, and matrigel, we demonstrate 17AAG induced alterations in integrin cross-linking with the actin cytoskeleton. The 17AAG treatment of cells resulted in decreased mRNA levels and confined surface expression of three major beta1 family of integrins namely α2, α3, and α5 in IMR-32, HeLa and PCC-4 cells, but showed induced mRNA levels and surface expression in MCF-7 cells. Loss of surface expression of integrins correlated with inhibition of focal adhesion kinase (FAK) and mitogen regulated kinase (ERK1/2) activities, in contrast, induced integrin expression in MCF-7 correlated with activation of these kinases. Prolonged treatment but not the pretreatment (2 h) with 17AAG resulted in destabilized actin cytoskeleton, delayed wound repair, and limited colony forming ability of tumor cells on soft agar. Conclusively, we show that Hsp90 inhibition targets cell adhesion, which may relate to the inhibition of integrin signaling and inhibition of integrin-cytoskeleton crosslinking.     

Laminin receptors in the integrin family.

Integrins are membrane receptors, consisting of an alpha and a beta subunit, which are involved in cell adhesion. Their extracellular domain is able to bind to ligands such as laminin which occurs in basement membranes of various kinds of cells. Most of these integrins, with their intracellular domains, interact with the actin-containing cytoskeleton, via linking proteins such as vinculin and talin, while one of them interacts with the keratin filaments, via an as yet unknown linking molecule(s). Among more than eighteen integrins which have been identified to date, integrins alpha 3 beta 1 and alpha 6 beta 1 have been characterized as laminin receptors. They recognize the laminin long arm E8 fragment obtained after elastase digestion of the molecule. The binding requires the presence of divalent cations which bind to specific sites on the integrin alpha subunit. The affinities of the alpha 3 beta 1 and alpha 6 beta 1 integrins for murine and human laminin are different, which is probably depended on the existence of different isoforms of laminin. When cells have adhered to laminin, the alpha 6 beta 1 integrin localizes in focal contacts in which actin microfilaments are anchored to the plasma membrane. Whether another integrin, the alpha 6 beta 4 complex, of epidermal cells is also a laminin receptor has not yet been confirmed. The alpha 6 beta 4 integrin localizes in hemidesmosomes which are attachment structures to the substratum where intermediate (keratin) filaments are anchored.(ABSTRACT TRUNCATED AT 250 WORDS)