Integrins in point contacts mediate cell spreading: factors that regulate integrin accumulation in point contacts vs. focal contacts

We have studied the function and distribution of the alpha 1 beta 1, alpha 5 beta 1 and alpha 6 beta 1 heterodimers on type-1 astrocytes with antibodies specific for integrin subunits (alpha 1, alpha 5, alpha 6, and beta 1). The alpha 1 beta 1 heterodimer mediates adhesion to laminin and collagen, the alpha 5 beta 1 to fibronectin in an RGD- dependent manner. The alpha 5 beta 1 integrin is found in focal contacts in long-term cultures of well-spread astrocytes colocalizing with vinculin and the termini of actin stress fibers. alpha 1 beta 1 heterodimers can occasionally be found as small aggregates within focal contacts but they do not accumulate there. Instead, alpha 1 beta 1 integrins are found in punctate deposits called point contacts which are distributed over the upper and the lower cell surfaces whether laminin, collagen, fibronectin or polylysine is used as a substratum. Unlike focal contacts, point contacts contain clathrin but rarely codistribute with actin or vinculin. Two observations indicate that these point contacts are functional. First, mAb 3A3, directed against the rat alpha 1 subunit, inhibits the attachment of astrocytes to laminin and collagen. Second, during the spreading of astrocytes, a band of point contacts forms around the cell perimeter at a time when no focal contacts are visible. While alpha 1 beta 1 integrins are found only in point contacts in astrocytes, the alpha 6 beta 1 integrin, another laminin receptor, is localized within focal contacts. Moreover, alpha 1 beta 1 heterodimers accumulate in focal contacts in fibroblasts. Thus, the alpha subunit contributes, independent of its ligand, to functional integrin heterodimer accumulation in focal contacts or in point contacts. This accumulation varies among different cell types with apparently identical heterodimers as well as with the motile state (spreading vs. flattened) of the same cells.     

Structure and function of a vimentin-associated matrix adhesion in endothelial cells

The alpha4 laminin subunit is a component of endothelial cell basement membranes. An antibody (2A3) against the alpha4 laminin G domain stains focal contact-like structures in transformed and primary microvascular endothelial cells (TrHBMECs and HMVECs, respectively), provided the latter cells are activated with growth factors. The 2A3 antibody staining colocalizes with that generated by alphav and beta3 integrin antibodies and, consistent with this localization, TrHBMECs and HMVECs adhere to the alpha4 laminin subunit G domain in an alphavbeta3-integrin-dependent manner. The alphavbeta3 integrin/2A3 antibody positively stained focal contacts are recognized by vinculin antibodies as well as by antibodies against plectin. Unusually, vimentin intermediate filaments, in addition to microfilament bundles, interact with many of the alphavbeta3 integrin-positive focal contacts. We have investigated the function of alpha4-laminin and alphavbeta3-integrin, which are at the core of these focal contacts, in cultured endothelial cells. Antibodies against these proteins inhibit branching morphogenesis of TrHBMECs and HMVECs in vitro, as well as their ability to repopulate in vitro wounds. Thus, we have characterized an endothelial cell matrix adhesion, which shows complex cytoskeletal interactions and whose assembly is regulated by growth factors. Our data indicate that this adhesion structure may play a role in angiogenesis.     

Parathyroid hormone (1-34) regulates integrin expression in vivo in rat osteoblasts.

Intermittent administration of parathyroid hormone (PTH) activates new sites of bone formation by stimulating osteoblast differentiation and function resulting in an increase in bone mass. Because integrins have been shown to play a crucial role in osteoblast differentiation and bone formation, in the present study, we evaluated whether human PTH (1-34) upon administration to rats, influenced integrin expression in osteoblastic cells isolated from the metaphysis and the diaphysis of rat long bones. Initial immunohistochemical evaluation of bone sections demonstrated that the osteoblasts expressed at least alphav, alpha2, alpha3, and alpha5beta1 integrins. Immunocolocalization studies for integrins and vinculin established that alphav, alpha2, and alpha5beta1, but not alpha3 integrins were present in the focal adhesion sites of osteoblasts attached to FN coated surfaces. Osteoprogenitor cells isolated from metaphyseal (but not diaphyseal) marrow of rats injected with intermittent PTH (1-34) exhibited greater alphav and reduced alpha2 levels, with no apparent changes in alpha3, and alpha5beta1 integrin levels, as assessed by immunohistochemistry, Northern, and Western blot analyses. However, these changes were not observed on the same cells treated with PTH in vitro. These observations suggest that integrin modulation by PTH is likely to be indirect and that selective phenotypic expression of integrin subtypes is part of the cascade of events that lead to PTH (1-34) mediated osteoblast differentiation.     

Association of intermediate filaments with vinculin-containing adhesion plaques of fibroblasts

Double immunofluorescence staining of quail embryo fibroblasts with rabbit antibody to vinculin and mouse monoclonal antibody to vimentin revealed a coincidence between fluorescence patterns for cell-substrate focal contacts and intermediate filaments. Most of the vinculin-containing adhesion plaques coincided with the ends of vimentin-positive fibrils. This association was further corroborated by immunoelection microscopic observations of the cytoskeletons of quail and mouse fibroblasts using a platinum replica technique. The intermediate filaments were identified either by direct treatment with antivimentin IgM or by an indirect immunogold staining method. Colcemid treatment of the cells caused a collapse of intermediate filaments and destroyed their association with focal contacts. During the early stages of the colcemid-induced collapse of the intermediate filaments, single vimentin fibrils appeared to retain their association with focal contacts. The possible role of the intermediate filaments in the formation and maintenance of focal contacts is discussed.     

Focal contacts: transmembrane links between the extracellular matrix and the cytoskeleton

The sites of tightest adhesion that form between cells and substrate surfaces in tissue culture are termed focal contacts. The external faces of focal contacts include specific receptors, belonging to the integrin family of proteins, for fibronectin and vitronectin, two common components of extracellular matrices. On the internal (cytoplasmic) side of focal contacts, several proteins, including talin and vinculin, mediate interactions with the actin filament bundles of the cytoskeleton. The changes that occur in focal contacts as a result of viral transformation are discussed.     

Effects of hyperthermia on the cytoskeleton and focal adhesion proteins in a human thyroid carcinoma cell line.

Hyperthermia is reported to act as a sensitizer to chemotherapeutic drugs in the treatment of cancer. Thyroid follicular carcinoma were used to elucidate the effects of hyperthermic treatment (41-43 degrees C) on cell morphology, cytoskeleton, and the focal adhesion complex. The critical temperature that resulted in inhibition of cell proliferation as the cell number in the same area did not increase over a 23 h time course and irreversible changes in cell morphology was 42-43 degrees C. An immunofluorescence study on heat-treated cells (43 degrees C, 1-5 h) demonstrated that depolymerization of actin filaments, intermediate filaments, and microtubules accounted for the rounding-up of cells and detachment from the substratum. Characteristic staining patterns for integrin alphav, focal adhesion kinase, and vinculin were noted in untreated cells, but the immunoreactive intensities for these proteins became weaker with time of heat treatment. Anti-phosphotyrosine staining revealed less immunoreactivity in the focal adhesions in treated cells compared with control cells. The disappearance of integrin alphav from the cell surface may result in inhibition of integrin-mediated activation of focal adhesion kinase, which results in dephosphorylation of focal adhesion components and its disassembly. These results indicate that hyperthermia induces disruption of integrin-mediated actin cytoskeleton assembly and, possibly, of other integrin-mediated signaling pathways.     

Overexpression of vinculin suppresses cell motility in BALB/c 3T3 cells.

The content of vinculin, a cytoplasmic protein found in focal contacts and cell-cell junctions, was increased in BALB/c 3T3 cells by gene transfection. The vinculin expressed from the full length chicken cDNA, incorporated into focal contacts and its pattern was identical to that of the endogenous protein. Cells stably expressing vinculin by 20% over the endogenous level had altered locomotory properties. In these cells, the ability to migrate into a wound formed in a confluent monolayer and the locomotion of individual cells were drastically reduced. The results provide direct evidence that cell locomotion can be regulated by modulating vinculin expression.     

Del1 mediates VSMC adhesion, migration, and proliferation through interaction with integrin alpha(v)beta(3)

Del1 is a matrix protein transiently expressed by embryonic endothelial cells. It was recently demonstrated that vascular endothelial cells adhere and interact with Del1 through alpha(v)beta(3)- integrins, providing an autocrine angiogenic signaling pathway in this cell type. To determine whether Del1 might signal to other cell types in the vessel wall in a paracrine fashion, studies were conducted with vascular smooth muscle cells (VSMC). Del1 promoted adhesion and migration of VSMC in a dose-dependent fashion. These functions were mediated through alpha(v)beta(3)-integrins, as the vitronectin receptor inhibitory peptide containing penacillamine (PCN) arginine-glycine-aspartic acid (PCN-RGD) and an antibody specific for the alpha(v)beta(3)-integrin specifically blocked both adhesion and migration. Adhesion of VSMC to Del1 was associated with organization of actin filaments and formation of focal contacts enriched in vinculin and alpha(v)beta(3). Furthermore, Del1 supported VSMC proliferation at least in part by inhibiting these cells from undergoing apoptosis. These data, in conjunction with evidence that Del1 expression is reactivated in vascular injury, suggest that Del1 may have a paracrine role in vessel wall development and remodeling.     

The integrin complex alpha v beta 3 participates in the adhesion of microvascular endothelial cells to fibronectin.

Fibronectin is a major adhesive glycoprotein of the vascular basement membrane. Since fibronectin is also found in the interstitium, it may be important not only for attachment but also for endothelial cell migration during neovascularization. We have analyzed how human dermal microvascular endothelial cells use their diverse set of integrin receptors to interact with this ligand. Immunofluorescent staining with specific antibodies identified both beta 1 and beta 3 integrin receptor complexes in focal adhesion plaques on cells adhering to immobilized fibronectin. Adhesion assays with blocking monoclonal antibodies implicated both beta 1 and beta 3 complexes, specifically alpha 5 beta 1 and alpha v beta 3, in the initial adhesion of cells to fibronectin. Finally, ligand affinity chromatography of extracts of surface radiolabeled cells established that both alpha 5 beta 1 and alpha v beta 3 could bind to the 110-kDa cell-binding fragment of fibronectin. An additional receptor complex composed of an alpha v subunit and a beta 5-like subunit was also detected. These results provide evidence that microvascular endothelial cells use multiple integrin receptors, from several beta families, to attach to fibronectin surfaces.     

Developmental Regulation of Focal Contact Protein Expression in Human Melanocytes

Focal contacts are transmembrane links between the extracellular matrix and the actin cytoskeleton that play a critical role in directed cell migration, adhesion, and normal growth. Several different component proteins of the focal contact show develop-mentally dependent changes in expression, suggesting that this is an important mechanism by which focal contact formation is controlled during embryogenesis. In this report we examine the expression of focal contact-associated proteins in human fetal and neonatal melanocytes using Western blotting. We show that expression of paxillin, a 69-kDa vinculin binding protein, is fourfold higher in neonatal melanocytes than in fetal melanocytes. Further, we show that talin, a high molecular weight structural protein that links integrins to the actin cytoskeleton, is proteolytically cleaved in fetal, but not in neonatal melanocytes. Immunofluorescence microscopy of cells grown on fibronectin confirmed the presence of paxillin, talin, and vinculin at the ends of actin stress fibers at presumptive focal contacts in melanocytes. Adhesion experiments to extracellular matrix ligands revealed significant differences in adhesion of fetal and neonatal melanocytes to fibronectin. The developmentally specific changes in focal contact protein expression observed suggest that this may be an important mechanism by which focal contact assembly is controlled in human melanocytes during development.     

Shark cartilage extract interferes with cell adhesion and induces reorganization of focal adhesions in cultured endothelial cells

In this study, we examined the effects of shark cartilage extract on the attachment and spreading properties and the focal adhesion structure of cultured bovine pulmonary artery endothelial cells. Treatment with cartilage extract resulted in cell detachment from the substratum. Immunofluorescence staining of those treated cells that remained attached showed that, instead of being present in both central and peripheral focal adhesions as in control cells, both integrin alpha(v)beta(3) and vinculin were found only in peripheral focal adhesion and thinner actin filament bundles were seen. In addition to causing cell detachment, cartilage extract partially inhibited the initial adherence of the cells to the substratum in a dose-dependent manner. Integrin alpha(v)beta(3) and vinculin staining of these cells also showed a peripheral focal adhesion distribution pattern. Vitronectin induced cell spreading in the absence of serum, but was blocked by simultaneous incubation with cartilage extract, which was shown to inhibit both integrin alpha(v)beta(3) and vinculin recruitment to focal adhesion and the formation of stress fibers. Dot binding assays showed that these inhibitory effects on cell attachment and spreading were not due to direct binding of cartilage extract components to integrin alpha(v)beta(3) or vitronectin. Shark cartilage chondroitin sulfate had no inhibitory effect on either cell attachment or spreading of endothelial cells. These results show that the inhibitory effects of cartilage extract on cell attachment and spreading are mediated by modification of the organization of focal adhesion proteins.     

Association of fibronectin and vinculin with focal contacts and stress fibers in stationary hamster fibroblasts

We have recently observed a transmembrane association between extracellular fibronectin (FN) fibers and elongated focal patches or fibers of vinculin (VN) in G1-arrested stationary Nil 8 hamster fibroblasts, with double-label immunofluorescence microscopy (Singer and Paradiso, 1981, Cell. 24:481-492). We hypothesized that these FN-VN complexes might correspond to focal contacts, the membrane sites that are probably mainly responsible for attaching cells to their substrata, because vinculin is often localized in focal contacts. However, because fibronectin-vinculin associations may not be restricted to the substrate adhesive surface of the cell, it became necessary to determine whether some or all of the various kinds of FN-VN complexes which we described are in proximity to the substrate. Using interference reflection optics and double-label immunofluorescence microscopy for fibronectin and vinculin, many elongated (up to 38 micrometer) FN-VN associations were found to be strikingly coincident with focal contacts in the perinuclear area of extremely flattened arrested Nil 8 fibroblasts in 0.3% fetal bovine serum (FBS). In addition, the long FN-VN adhesion complexes were precisely aligned with the major phase-dense stress fibers observed at the ventral surfaces of these stationary cells with phase contrast microscopy. Fibronectin was neither associated with vinculin-containing focal contacts of Nil 8 cells cultured in medium with 5% FBS nor with vinculin-negative focal contacts located at the extreme edges of stationary cells arrested in 0.3 FBS. Our time-course experiments suggest that early FN-VN lacking- focal contacts, which form at the cellular margins, develop into mature substrate adhesion complexes containing both fibronectin and vinculin, localized in the major stress fibers at the centers of sessile fibroblasts.     

3-D surface charges modulate protrusive and contractile contacts of chondrosarcoma cells

Up to now, most of the studies addressing the critical roles played by protrusive and contractile cell-matrix contacts in cell adhesion, guidance, migration, matrix assembly, and activation of signaling molecules have been performed on two-dimensional surfaces. Here, we analysed the organization of chondrosarcoma cell contacts in a new three-dimensional environment made of titanium beads. Surface charges were modified by deposition of polyelectrolyte multilayer films built up by alternated polycations poly-(L-lysine) or poly(allylamine hydrochloride) and polyanions poly-(L-glutamic acid) or poly(sodium 4-styrenesulfonate). Negatively charged 3-D titanium surfaces amplified the occurrence and length of cell protrusions. These protrusions had pseudopod characteristics extended to 200 microm in length, growing off the substratum to distant beads. Pseudopod formation is inhibited by the exocytosis inhibitor concanamycin A and is triggered by a secreted factor. Chondrosarcoma cells adhering on uncoated or on negatively charged surfaces contained discrete focal spots of vinculin and actin cables. In cells plated onto these surfaces, phosphorylation of p44/42 MAPK/ERK was twofold increased. In contrast, no cytoskeletal vinculin and actin organization was observed when the surface was positively charged. These data suggest that chondrosarcoma cells adapt a more stable adhesion on uncoated or negatively charged surfaces. This point may be critical in tissue engineering strategies designed for cartilage repair.     

Lamellipodial motility in wounded endothelial cells exposed to physiologic flow is associated with different patterns of beta1-integrin and vinculin localization

Integrins- and cytoskeletal-associated focal adhesion proteins may participate in the process of endothelial wound closure, but their relationship in these wounds and in the presence of shear forces has not been defined. The goal in this study was to test the hypotheses that (1) modulation of beta(1)-integrin in human coronary artery endothelial cells (HCAEC) would alter endothelial wound closure under shear stress, and (2) beta(1)-integrin association with vinculin would be necessary for mediating this closure. HCAEC monolayers were pre-conditioned to attain alignment by shearing at 12 dynes/cm(2) for 18 h in a parallel-plate flow chamber. Subsequently, they were divided into three groups: (a) control, (b) treated with anti-beta(1)-integrin adhesion blocking antibody, or (c) treated with anti-beta(1)-integrin adhesion promoting antibody. Next, the monolayers were wounded with a metal spatula, and re-sheared at 20 dynes/cm(2) or left static. Time-lapse imaging and deconvolution microscopy were then performed for 3 h. Immunocytochemistry for beta(1)-integrin expression and vinculin was performed on all wounded monolayers. Under shear stress, vinculin localized to the ends of stress fibers, while beta(1)-integrin took on an intracellular macroaggregate appearance. Treatment with anti-beta(1)-integrin adhesion blocking antibody enhanced wound closure, left the vinculin staining at the lamellipodial tips unchanged, but was associated with beta(1)-integrin staining at the lateral cell edges. Treatment with the anti-beta(1)-integrin adhesion promoting antibody retarded wound closure, increased vinculin staining at cell-cell junctions, and was associated with a fibrillar pattern of beta(1)-integrin staining. Modulation of beta(1)-integrin and changes in beta(1)-integrin and vinculin localization may further our understanding of laminar shear stress-induced endothelial repair in the coronary circulation.     

Scatter factor affects major changes in the cytoskeletal organization of epithelial cells.

The effects of scatter factor on the cytoskeleton of MDCK and PtK2 cells are described. During the first 6 h after the addition of scatter factor, MDCK cells were found to increase their projected areas twofold, as well as the number and size of their F-actin stress fibers. In contrast PtK2 cells showed no change in their projected areas or in their stress fiber content. However, when both MDCK and PtK2 cells began to separate and scatter after approximately 6 h, the size and number of stress fibers was found to decrease considerably. Unscattered PtK2 cells and cells treated with scatter factor which had yet to scatter showed focal contacts present over the whole ventral surface, as judged by staining for both vinculin and talin. After treated cells separated, both vinculin and talin staining were mainly present in focal contacts on the ventral surfaces of the cell bodies and the distal ends of the processes. However, the cell processes showed few focal contacts along their lengths. The distribution of microtubules and vimentin and keratin intermediate filaments also did not change significantly until scattering had occurred. After cell separation, the processes were always packed with microtubules which were often, but not always, rich in detyrosinated alpha-tubulin and often, but not always, packed with intermediate filaments. All these changes in cytoskeletal organization are consistent with the adoption of a much more motile phenotype. The changes found are compared with those brought about by transformation.     

Human microvascular endothelial cells express integrin-related complexes that mediate adhesion to the extracellular matrix

Microvascular endothelial cells (MEC) must use a set of surface receptors to adhere not only to the vascular basement membrane but, during angiogenic stimulation, to the interstitium. We examined how cultured MEC isolated from human foreskin interact with their subendothelial matrix. MEC were able to attach to diverse extracellular matrix proteins, including fibronectin (Fn), vitronectin (Vn), laminin (Ln), type I and IV collagen, as well as to fibrinogen and gelatin. Adhesion to Fn, but not to laminin or collagens, was specifically blocked in the presence of Arg-Gly-Asp (RGD)-containing peptides. When surface radioiodinated MEC were solubilized and subjected to affinity chromatography on Fn-Sepharose columns, two polypeptides of 150 and 125 kD, corresponding to the integrin heterodimer alpha 5 beta 1, were identified. MEC also express a complex of 150 (alpha) and 95 kD (beta 3) that is related to the Vn receptor. Immunofluorescent staining of MEC cultures with antibodies to the integrin beta 1 subunit demonstrated receptors on the basolateral surface at focal adhesion plaques that co-localized with vinculin and with Fn-positive matrix fibers. Occasionally, antibodies to the Vn receptor stained the vinculin-positive focal adhesion plaques that frequently co-localized with the beta 1 complex. However, in cultures of MEC that were attached to substrates coated with alternating strips of Fn and Vn, the beta 1 complex was preferentially localized to the Fn substrate, while the Vn receptor was concentrated on the Vn substrate. The results indicate that MEC express at least two different heterodimer adhesion receptors that belong to the integrin super-family and appear to have distinct ligand specificities: the Fn receptor and the Vn receptor. These receptors mediate cell adhesion to the extracellular matrix and presumably have an important role in hemostasis and neovascularization.     

Calreticulin affects focal contact-dependent but not close contact-dependent cell-substratum adhesion.

We used two cell lines expressing fast (RPEfast) and slow (RPEslow) attachment kinetics to investigate mechanisms of cell-substratum adhesion. We show that the abundance of a cytoskeletal protein, vinculin, is dramatically decreased in RPEfast cells. This coincides with the diminished expression level of an endoplasmic reticulum chaperone, calreticulin. Both protein and mRNA levels for calreticulin and vinculin were decreased in RPEfast cells. After RPEfast cells were transfected with cDNA encoding calreticulin, both the expression of endoplasmic reticulum-resident calreticulin and cytoplasmic vinculin increased. The abundance of other adhesion-related proteins was not affected. RPEfast cells underexpressing calreticulin displayed a dramatic increase in the abundance of total cellular phosphotyrosine suggesting that the effects of calreticulin on cell adhesiveness may involve modulation of the activities of protein tyrosine kinases or phosphatases which may affect the stability of focal contacts. The calreticulin and vinculin underexpressing RPEfast cells lacked extensive focal contacts and adhered weakly but attached fast to the substratum. In contrast, the RPEslow cells that expressed calreticulin and vinculin abundantly developed numerous and prominent focal contacts slowly, but adhered strongly. Thus, while the calreticulin overexpressing RPEslow cells "grip" the substratum with focal contacts, calreticulin underexpressing RPEfast cells use close contacts to "stick" to it.     

Focal contacts of normal and RSV-transformed quail cells : Hypothesis of the transformation-induced deficient maturation of focal contacts

Morphology and distribution of cell-substrate contacts and their association with microfilament bundles in normal and RSV-transformed quail fibroblasts (16Q line) were studied by indirect immunofluorescence. The focal contacts were visualized by antibody exclusion method using monoclonal antibody to 80 kD serum protein adsorbed on the substratum. Embryo quail cells formed focal contacts of two morphological types: (1) small punctate; and (2) elongated large contacts. These two variants of contacts were designated respectively as dot and dash contacts. Both of focal contacts contained vinculin and alpha-actinin. Double immunofluorescence staining with polyclonal antibody to actin and monoclonal antibody to vinculin revealed that the dot contacts, in contrast to the dash ones, were not associated with microfilament bundles. The dot contacts were localized mostly near the active cell edges, while the dash contacts were found near the retracted cell edges and also under the central parts of the cell. We suppose that dot contacts represent initial structures which then can undergo maturation transforming them into dash contacts. RSV-transformed 16Q cells had predominantly the dot contacts which were not only located at the edges but also in the more central parts of the lamella. The dash contacts were present only in the minority of 16Q cells. RSV transformation is assumed to affect not the ability of cells to form initial dot contacts, but the maturation of dot contacts into dash contacts.     

Investigation of cell adhesion to structured surfaces using total internal reflection fluorescence and confocal laser scanning microscopy

Adhesion of adherent cells on structured surfaces is influenced by the surface pattern given. Here, we designed a structured gold relief surface based on cell adhesion patterns we had previously observed. We analysed the geometric parameters and the overall distribution of focal adhesion kinase in focal adhesions on unstructured glass surfaces using optical microscopy. The basic structural elements obtained from this analysis were arranged in regular clusters that resembled the shape of a polarised migratory cell. In time-lapse studies we observed that the cells adhere preferentially to the gold pads and adopt the shape of the clusters. Staining of the actin cytoskeleton revealed that the actin filaments are aligned to the gold pads of the elementary structure.     

Migratory phenotypes of HSC-3 squamous carcinoma cell line induced by EGF and PMA: relevance to migration of loosening of adhesion and vinculin-associated focal contacts with prominent filopodia

Cell migration is involved in carcinoma cell invasion and wound healing. We examined motogenic cytokines that potentiated migration of human HSC-3 carcinoma cells. To assess migratory activity, modified Boyden chambers were used. Among a variety of potential motogenic cytokines, epidermal growth factor (EGF) enhanced migration of HSC-3 cells both on collagen and fibronectin. Phorbol myristate acetate (PMA) also enhanced migration. Inhibitors of protein kinase C completely inhibited PMA-induced migration, but only partly inhibited EGF-induced migration. Protein kinase A was also involved in the EGF-induced signaling pathway for migration. Although the signaling pathways were independent, and the cell shape on collagen was different from that on fibronectin, migratory cells stimulated by EGF or PMA showed common morphology on different ligands. The cells were polygonal or round in shape and the loss of long cytoplasmic extensions was noted. Migratory HSC-3 cells stimulated by EGF or PMA became less adhesive to collagen and fibronectin. Since both EGF- and PMA-stimulated migration did not require de novo protein synthesis, the signaling pathways possibly lead to assembly and disassembly of an actin cytoskeleton. Immunofluorescence for vinculin was concentrated into focal contacts in EGF- and PMA-stimulated HSC-3 cells, whereas the fluorescence signal was hardly detected in non-stimulated cells. Talin and beta1 integrin were immunolocalized at focal contacts in non-stimulated cells, and it remained unchanged in stimulated cells. Numerous filopodia visualized with actin immunofluorescence were formed around stimulated HSC-3 cells, whereas filopodia were short and sparse around elongated cytoplasms in non-stimulated cells. Thus, shortening of cytoplasmic extensions with numerous filopodia, loosening of adhesion, and vinculin-associated focal contacts were regarded as migratory phenotypes.