Concanavalin A surface receptors and cytoplasmic actin in cell adhesion.

A double immunofluorescence staining technique to locate concanavalin A (Con A) surface receptors and cytoplasmic actin in the same cell was applied to monolayer cultures of rat foetal fibroblasts during cell detachment induced by trypsin and during cell attachment to glass substratum. Con A receptors were demonstrated by fluorescein-isothiocyanate-labelled Con A (FITC-Con A) and actin by specific anti-actin antibody (AAA) traced with rhodamine-labelled goat anti-human globulin (R-AHG). Untreated, control cells had an elongated shape, Con A receptors restricted to cell margins and prominent actin filaments. After 2 min treatment with 0.001% trypsin the cells became angular with Con A receptors in clusters and actin in a diffuse or aggreagate staining pattern. Progressive cell rounding followed and this was accompanied by the development of long, thin, arborized cell processes, studded with Con A receptors and containing fine actin filaments. Complete cell rounding preceded cell detachment. The sites of detached cells were marked by fine aggregates containing Con A receptors and actin. In cells attaching to a glass substratum, actin was present in a diffusely stained or aggregate pattern in round cells, in filaments restricted to cell margins in partially spread cells and in numerous filaments in fully spread cells. Con A receptors were present in clusters in round cells, in clusters or caps in partially spread cells and in cell margins in fully spread cells. Binding of FITC-Con A to partially spread cells resulted in dissolution of the few, newly formed, actin filaments. We believe our observations are consistent with the idea that actin filaments, formed during cell attachment, contribute towards the maintenance of cell adhesion by helping in the preservation of cell shape and by anchorage of Con A receptors at points of cell attachment to the substratum.     

The contributions of microtubules and F-actin to the in vitro migratory mechanisms of Hydra nematocytes as determined by drug interference experiments.

In order to investigate the contributions of microtubules and of F-actin to the in vitro migration mechanisms of Hydra nematocytes we have studied the effects of agents directed against cytoskeletal structures. Disassembly of microtubules by treatment with the drug nocodazole in moving nematocytes resulted in the loss of all locomotory activity within 20 min after the onset of treatment and in the detachment from the substratum after about 30 min. Depolymerization of microtubules by exposure to low temperatures had the same effect but was reversible in this case. Locomoting cells treated with cytochalasin D, which disrupts the actin filaments, stopped movement 2 min after drug administration and detached from the substratum after 15 min. The pattern of F-actin, alpha-tubulin, and tyrosinated tubulin in drug- or cold-treated cells was determined by immunocytochemical techniques and confocal laser scanning microscopy. These patterns and the reactions of the cells to the various drug treatments suggest that both actin filaments and microtubules play a crucial role in nematocyte locomotion. Analysis of the cytoskeletal pattern in drug-treated cells shows that the microtubules which are involved in locomotion are mostly tyrosinated. Furthermore it is suggested that microtubules and actin filaments interact with each other during the locomotion of nematocytes.     

Morphology and microfilament organization in human blood lymphocytes : Effects of substratum and mitogen exposure

During culture in serum-containing medium normal human blood lymphocytes, depleted of phagocytic and adherent cells, do not attach to adhesive surfaces. Concanavalin A (ConA) or phytohemagglutinin (PHA) in appropriate concentrations mediate adhesion of these lymphocytes to tissue culture plastic or glass. This process consists of two phases.

1. 1. The mitogen-mediated contact with a surface induces an almost instantaneous alteration of cell shape and a simultaneous redistribution of actin in the majority of the cells.

2. 2. The initial morphological changes are accompanied by an accumulation of actin-containing material in prominent peripheral cytoplasmic outgrowths formed by the spread cells. The contact-induced spreading and rearrangement of actin are inhibited by cytochalasin B (CB) but not by colchicine or vinblastine. The distribution of detectable actin in spread lymphocytes is similar to the distribution of footprints of actin after detachment of spread cells suggesting that actin is involved in the attachment of lymphocytes to substratum. In contrast to lymphocytes on glass or tissue culture plastic which show morphological changes and redistribution of actin cells cultured with ConA on non-adhesive surfaces of bacterial plastic or poly-2-hydroxy-methacrylate do not exhibit any morphological alterations and no rearrangement of actin.

The present approach enables visualization of cytoskeletal structures in lymphocytes to an extent which is not possible using conventional methods with the cells in suspension. The results indicate that contact is a regulator of lymphocyte shape and that actin-containing structures mediate and maintain contact-induced changes of lymphocyte morphology.     

Actin cytoskeleton dynamics and the cell division cycle

The network of actin filaments is one of the crucial cytoskeletal structures contributing to the morphological framework of a cell and which participates in the dynamic regulation of cellular functions. In adherent cell types, cells adhere to the substratum during interphase and spread to assume their characteristic shape supported by the actin cytoskeleton. This actin cytoskeleton is reorganized during mitosis to form rounded cells with increased cortical rigidity. The actin cytoskeleton is re-established after mitosis, allowing cells to regain their extended shape and attachment to the substratum. The modulation of such drastic changes in cell shape in coordination with cell cycle progression suggests a tight regulatory interaction between cytoskeleton signalling, cell–cell/cell–matrix adhesions and mitotic events. Here, we review the contribution of the actin cytoskeleton to cell cycle progression with an emphasis on the effectors responsible for the regulation of the actin cytoskeleton and integration of their activities with the cell cycle machinery.     

Proteoglycan synthesis by fibroblasts from different regions of bovine tendon cultured in alginate beads

The ability of cell shape to modulate proteoglycan synthesis in tendon fibroblasts was investigated by placing freshly isolated tendon fibroblasts and chondrocytes into primary culture either as adherent cells on a polystyrene substratum or as rounded cells in alginate beads. Chondrocytes and cells from the compressed region of adult tendon synthesized predominantly large proteoglycan when maintained either as dense monolayers, where actin stress fibers in the cytoskeleton were prominent, or in alginate beads, where actin fibers could not be detected. After three rounds of proliferation as elongated adherent cells the synthesis of large proteoglycan was greatly reduced, i.e. the chondrocytic cells underwent 'dedifferentiation'. Cells from the tensional region of adult tendon synthesized predominantly small proteoglycan when in primary culture as a monolayer, after proliferation on a flat substratum, or as round cells in alginate beads. Fibroblasts from the tensional region of newborn tendon showed no tendency toward increased synthesis of large proteoglycan when maintained as round cells in alginate beads for 7 weeks. In tendon there appears to be a mechanically induced developmental transition from fibroblastic to chondrocytic cells. However, neither the change to a rounded cell shape nor the lack of organized cytoskeletal actin fibers was sufficient to induce chondrocyte-like proteoglycan synthesis in differentiated tendon fibroblasts in culture.     

Focal Adhesion and Stress Fiber Formation Is Regulated by Tyrosine Phosphatase Activity

Tyrosine phosphorylation of cytoskeletal proteins plays an important role in the regulation of focal adhesions and stress fiber organization. In the present study we examined the role of tyrosine phosphatases in this process using p125FAK and paxillin as substrates. We show that tyrosine phosphatase activity in Swiss 3T3 cells was markedly increased when actin stress fibers were disassembled by cell detachment from the substratum, by serum starvation, or by cytochalasin D treatment. This activity was blocked by phenylarsine oxide, an inhibitor of a specific class of tyrosine phosphatases characterized by two vicinal thiol groups in the active site. Phenylarsine oxide treatment of serum-starved cells induced increased tyrosine phosphorylation of p125FAK and paxillin in a dose-dependent manner and induced assembly of focal adhesions and actin stress fibers, showing that inhibition of one or more phenylarsine oxide-sensitive tyrosine phosphatases is a sufficient stimulus for triggering focal adhesion and actin stress fiber formation in adherent cells.     

Integrin and cytoskeleton behaviour in human neuroblastoma cells during hyperthermia-related apoptosis

Hyperthermia induces several cellular responses leading to morphological changes, cell detachment and death. Loss of integrins from the cell surface after acute heat-treatment may block several physiological signalling pathways, but whether the assembly network between integrin and cytoskeletal actin is perturbed during hyperthermic treatment is unknown. In this study we tested this hypothesis by evaluating cell morphology, protein cytoskeletal profile and integrin CD11a content in both adherent and floating SK-N-MC human neuroblastoma cells. Morphological and cytometric analyses confirmed that hyperthermia is an effective apoptotic trigger, revealing the typical chromatin margination, cell shape changes and 7-AAD incorporation. After hyperthermia, cytoskeletal proteins showed an increase of high-molecular-weight aggregates and a significant decrease of both actin and CD11a content with respect to control cells. The integrin CD11a and membrane-bound actin alterations found in detached floating neuroblastoma cells recovered after heat-shock may cause the cytoskeletal abnormalities related to the observed surface cell rounding/blebbing and anoikis, early events of hyperthermia-induced programmed cell death.     

Bacterial adhesion to hydroxyl- and methyl-terminated alkanethiol self-assembled monolayers.

The attachment of bacteria to solid surfaces is influenced by substratum chemistry, but to determine the mechanistic basis of this relationship, homogeneous, well-defined substrata are required. Self-assembled monolayers (SAMs) were constructed from alkanethiols to produce a range of substrata with different exposed functional groups, i.e., methyl and hydroxyl groups and a series of mixtures of the two. Percentages of hydroxyl groups in the SAMs and substratum wettability were measured by X-ray photoelectron spectroscopy and contact angles of water and hexadecane, respectively. SAMs exhibited various substratum compositions and wettabilities, ranging from hydrophilic, hydroxyl-terminated monolayers to hydrophobic, methyl-terminated monolayers. The kinetics of attachment of an estuarine bacterium to these surfaces in a laminar flow chamber were measured over periods of 120 min. The initial rate of net adhesion, the number of cells attached after 120 min, the percentage of attached cells that adsorbed or desorbed between successive measurements, and the residence times of attached cells were quantified by phase-contrast microscopy and digital image processing. The greatest numbers of attached cells occurred on hydrophobic surfaces, because (i) the initial rates of adhesion and the mean numbers of cells that attached after 120 min increased with the methyl content of the SAM and the contact angle of water and (ii) the percentage of cells that desorbed between successive measurements (ca. 2 min) decreased with increasing substratum hydrophobicity. With all surfaces, 60 to 80% of the cells that desorbed during the 120-min exposure period had residence times of less than 10 min, suggesting that establishment of firm adhesion occurred quickly on all of the test surfaces.     

Regulation of cell morphology and cytochrome P450 expression in human hepatocytes by extracellular matrix and cell-cell interactions

The influence of extracellular matrix conditions and plating density on cell cytoarchitecture and the constitutive and chemically induced expression of cytochrome P450 3A4 (CYP3A4) was examined in primary cultures of human hepatocytes. Constitutive and drug-induced microsomal CYP3A4 expression occurred equally well in human hepatocyte cultures maintained on either a complex or simple substratum (Matrigel vs collagen, type I), or in a sandwich configuration (i.e., between two layers of extracellular matrix), despite the markedly different morphological properties exhibited by each condition. However, a density-dependent decrease in both the constitutive and induced levels of CYP3A4 was observed in hepatocytes maintained on a simple collagen substratum as plating density was reduced from 100% to 25%. Marked alterations in cell shape and cytoarchitecture were noted concomitant with decreases in the expression and localization of intercellular gap junctions and E-cadherin-mediated cell adhesions. In addition, the intracellular distribution of microtubules and microfilaments was altered substantially and the expression of immunoreactive actin and beta-tubulin increased as cell density was decreased. These effects were reversed to some extent by overlaying monolayers with extracellular matrix or by co-culturing with another cell type. Efforts to maintain normal cell shape and cytoskeletal distribution in hepatocytes at low cell density with a Matrigel substratum failed to restore normal basal levels of CYP3A4 expression or responsiveness to rifampicin (RIF). Likewise, E-cadherin and Cx-32 expression was again reduced, even though the distribution and expression of cytoskeletal elements returned to normal levels. These results suggest that cell-cell contacts, but not the extracellular matrix configuration or composition, play a critical role in determining normal responsiveness to chemical modulators in human hepatocytes.     

Characterization of the intermediate filament proteins of Murine Mammary Gland epithelial cells : Response to collagen substratum

The insoluble cytoskeletal material remaining after detergent lysis of 'Normal' Murine Mammary Gland (NMuMG) cells, growing on plastic or collagen gel substrata, was analyzed by two-dimensional gel electrophoresis. The identity of the cytoskeletal elements was determined by their solubility properties, electrophoretic separation pattern, and immunoreactivity using monoclonal antibodies against intermediate filament proteins (AIF), keratins (AE1 and AE3) and actin. The electrophoretic pattern of the cytoskeletal elements from the NMuMG cell strain was found to be very similar to that of primary mouse mammary epithelial cells. Both NMuMG and primary mammary epithelial cells when grown on collagen exhibited an increased expression of a 49 kD protein with a pI of 5.6, that appeared to be a cytokeratin. Many of the cytoskeletal proteins remained tightly attached to the collagen gel substratum after cell lysis. These results demonstrate that the NMuMG cell strain has retained a stable expression of cytokeratins that remains responsive to the presence of extracellular matrix material.     

Mechanism of concanavalin A-induced anchorage of the major cell surface glycoproteins to the submembrane cytoskeleton in 13762 ascites mammary adenocarcinoma cells

Concanavalin A (Con A)-induced anchorage of the major cell surface sialoglycoprotein component complex (ASGP-1/ASGP-2) was studied in 13762 rat mammary adenocarcinoma sublines with mobile (MAT-B1 subline) and immobile (MAT-C1 subline) cell surface Con A receptors. Treatment of cells, isolated microvilli, or microvillar membranes with Con A resulted in marked retention of ASGP-1 and ASGP-2, a Con A-binding protein, in cytoskeletal residues of both sublines obtained by extraction with Triton X-100 in PBS. When Con A-treated microvillar membranes were extracted with a buffer containing Triton X-100, the sialoglycoprotein complex was found associated in the residues with a transmembrane complex composed of actin, a 58,000-dalton polypeptide, and a cytoskeleton-associated glycoprotein (CAG), also a Con A-binding protein, in MAT-C1 membranes, and of actin and CAG in MAT-B1 membranes. Untreated membrane Triton residues retained very little ASGP-1/ASGP-2 complex. Association of the sialoglycomembrane complex and the transmembrane complex was also demonstrated in Con A-treated, but not untreated, microvilli by their comigration on CsCl gradients. Association of both complexes with the cytoskeleton of microvilli was shown by sucrose density gradient centrifugation. A fraction of the polymerized actin comigrated with the transmembrane complex alone in the absence of Con A and with both the transmembrane complex and the sialoglycoprotein complex in the presence of Con A. From these results we propose that anchorage of the sialoglycoprotein complex to the cytoskeleton on Con A treatment occurs by cross-linking ASGP-2, the major cell surface Con A-binding component, to CAG of the transmembrane complex, which is natively linked to the cytoskeleton via its actin component. Since Con A-induced anchorage occurs in sublines with mobile and immobile receptors, the anchorage process cannot be responsible for the differences in receptor mobility between the sublines.     

THE ROLE OF F-ACTIN DURING FERTILIZATION IN THE RED ALGA AGLAOTHAMNION OOSUMIENSE (RHODOPHYTA)

The actin cytoskeletons in spermatia and trichogynes of Aglaothamnion oosumiense Itono were studied using fluorescein isothiocyanate (FITC) conjugated phalloidin and the cytoskeletal inhibitors, potassium iodide (KI), cytochalasin-B, and latrunculin-A. Microfilaments were localized to the distal ends of elongated spermatia and trichogynes and were more prominent in the trichogyne before spermatium binding. The actin cytoskeleton in spermatia and trichogynes was disrupted by treatment with 0.6 M KI, 100 μM cytochalasin-B, or 10 μM latrunculin-A. The actin cytoskeleton in trichogynes recovered within 24 h of removal from the inhibitor, but no recovery was observed in spermatia. Spermatial nuclei entered mitosis as soon as spermatia attached to the trichogyne. The greatest percentage (50%– 60%) of spermatia having completed mitosis was obtained at 60 min after spermatial binding to trichogynes. During mitosis, actin accumulated in the center of the spermatium, thereby separating the two daughter nuclei. Cytoskeletal inhibitors did not affect initial binding of spermatia to trichogynes but did block subsequent stages of fertilization, including spermatial mitosis and gamete fusion. The accumulation of cellulose or β-linked polysaccharide on the spermatial surface was also blocked by treatment with actin inhibitors. Exposure of the trichogyne to actin inhibitors after gamete fusion caused spermatial nuclei in trichogynes to stop moving and to condense. These results suggest that the microfilaments involved in nuclear division, cellulose deposition into the spermatial wall, gamete fusion, and migration of spermatial nuclei in trichogynes during fertilization in Aglaothamnion oosumiense.     

Cytoskeleton in human mammary carcinoma cells forming three-dimensional cellular structures within collagen gels

Human mammary carcinoma cell line MCF-7 cells grown on type I collagen gels floating in a medium occasionally invaginated into the gels as a cell mass and formed cylindrical or domed structures within it. The 0.05% Triton-insoluble cytoskeleton of such cellular structures sedimented as a white flocculent layer at the boundary between 60 and 70% sucrose layers by ultracentrifugation, and consisted of 4 basal components: 54-kD (beta-tubulin), 45-kD, 42-kD (actin), and 39-kD polypeptides. By contrast, the isolated cytoskeleton of MCF-7 cells grown as monolayers on plastic substratum formed a finer cytoskeletal network with a smaller buoyant density and consisted of two distinct polypeptides with apparent molecular sizes of 80-kD and 65-kD in addition to the 4 basal components found in the morphologically developing cells. The present results indicate that the cytoskeleton of MCF-7 cells forming the three-dimensional cellular structures within collagen gels is lacking in these two polypeptides, and that it has a coarser cytoskeletal network with a greater buoyant density than that of the monolayered cells on plastic.     

The significance of alpha 5 beta 1 integrin dependent and independent actin cytoskelton organization in cell transformation and survival

Cell-matrix and cell-cell interactions are important physiological determinants of cell growth, survival and transformation. Cell adhesion to the extra cellular matrix (ECM) via integrins also crucially influences the organization of the cytoskeleton. It triggers a cascade of intracellular biochemical events, which regulate cell viability and growth. We have studied the relationship between cell attachment to the substratum and cytoskeletal organization and cell survival and transformation. Our results demonstrate that in the absence of attachment to the substratum, adhesion-dependent fibroblasts exhibit rapid loss of viability. However, a small percentage of cells survive even after remaining non-adherent for 16h. The adherent and non-adherent cells differ from one another both morphologically and physiologically. The latter show a loss of alpha5beta1 integrin expression on their surface and bind non-specifically to the substratum and ECM, thereby activating certain pathways more efficiently than adherent cells. We have also shown that non-adherent cells grow faster and have worse cytoskeletal organization after attachment to the substratum, and do not form focal adhesions or actin stress fibres. Hence, our data suggests that rat fibroblasts in prolonged suspension exhibit some properties that are comparable to cells undergoing transformation, by adapting integrin-dependent or independent signalling pathways for their survival.     

Three-dimensional study of the cytoskeleton in macrophages and multinucleate giant cells by quick-freezing and deep-etching method

The three-dimensional ultrastructure of multinucleate giant cells in subcutaneous granulomas was compared with those of peritoneal macrophages using a quick-freezing and deep-etching method. Subcutaneous granulomas were induced by implanting plastic coverslips in the dorsal subcutaneous tissue of rats. The quick-freezing and deep-etching replicas were prepared from the cells attached to the coverslips. Dense networks of actin filaments were distributed along all peripheral aspects (beneath the plasma membrane, and on free and coverslip-attached surfaces) of the multinucleate giant cells. On the coverslip-attached surface, numerous clathrin-coated pits and vesicles occurred between the actin filaments. In these cells, intermediate filaments, but not actin filaments, were the predominant cytoskeletal components in perinuclear regions and were attached to the cell nucleus, mitochondria and other vesicular cell organelles. A similar distribution of cytoskeletal components was observed in the mononuclear macrophages of the granulomas and the peritoneal macrophages. These results show that the cytoskeletal organization varies in different regions of the cytoplasm of multinucleate giant cells, while the characteristic cytoskeletal arrangement, resembling that of mononuclear macrophages, is maintained.     

Adhesion of culture cells to their substratum

The attachment of cells to culture dishes has been investigated by replica techniques and scanning electron microscopy. Cells were removed from their substratum either in a stream of medium or by micromanipulation. In the most usual case of interphase cells one finds many small points of attachment uniformly distributed over the whole cell underside. Attachment sites are also found under the lamellipodia and at the trailing edge of the cells. A large portion of the cell underside is sometimes left behind. This is probably because the sole plate is reinforced by cytoskeletal elements, and does not necessarily indicate the presence of large adhesion points. The distal ends of the retraction fibers formed as the cells round-up in trypsin, or in the cold, represent the attachment points of the cell to the substratum. Agents which tend to stabilize microtubules greatly slow cell detachment by proteolytic agents. The primary effect of trypsin is not on the glue which holds the cells to the substratum but rather on the cell shape itself, affecting the rigidity of cytoskeletal elements.     

Three-dimensional study of the cytoskeleton in macrophages and multinucleate giant cells by quick-freezing and deep-etching method.

The three-dimensional ultrastructure of multinucleate giant cells in subcutaneous granulomas was compared with those of peritoneal macrophages using a quick-freezing and deep-etching method. Subcutaneous granulomas were induced by implanting plastic coverslips in the dorsal subcutaneous tissue of rats. The quick-freezing and deep-etching replicas were prepared from the cells attached to the coverslips. Dense networks of actin filaments were distributed along all peripheral aspects (beneath the plasma membrane, and on free and coverslip-attached surfaces) of the multinucleate giant cells. On the coverslip-attached surface, numerous clathrin-coated pits and vesicles occurred between the actin filaments. In these cells, intermediate filaments, but not actin filaments, were the predominant cytoskeletal components in perinuclear regions and were attached to the cell nucleus, mitochondria and other vesicular cell organelles. A similar distribution of cytoskeletal components was observed in the mononuclear macrophages of the granulomas and the peritoneal macrophages. These results show that the cytoskeletal organization varies in different regions of the cytoplasm of multinucleate giant cells, while the characteristic cytoskeletal arrangement, resembling that of mononuclear macrophages, is maintained.     

Location of actin, myosin, and microtubular structures during directed locomotion of Dictyostelium amebae

During their life cycle, amebae of the cellular slime mould Dictyostelium discoideum aggregate to form multicellular structures in which differentiation takes place. Aggregation depends upon the release of chemotactic signals of 3',5'-cAMP from aggregation centers. In response to the signals, aggregating amebae elongate, actively more toward the attractive source, and may be easily identified from the other cells because of their polarized appearance. To examine the role of cytoskeletal components during ameboid locomotion, immunofluorescence microscopy with antibodies to actin, myosin, and to a microtubule-associated component was used. In addition, rhodamine-labeled phallotoxin was employed. Actin and myosin display a rather uniform distribution in rounded unstretched cells. In polarized locomoting cells, actin fluorescence (due to both labeled phallotoxin and specific antibody) is prevalently concentrated in the anterior pseudopod while myosin fluorescence appears to be excluded from the pseudopod. Similarly, microtubules in locomoting cells are excluded from the leading pseudopod. The cell nucleus is attached to the microtubule network by way of a nucleus-associated organelle serving as a microtubule-organizing center and seems to be maintained in a rather fixed position by the microtubules. These findings, together with available morphological and biochemical evidences, are consistent with a mechanism in which polymerized actin is moved into the pseudopod through its interaction with myosin at the base of the pseudopod. Microtubules, apparently, do not actively participate in movement but seem to behave as anchorage structures for the nucleus and possibly other cytoplasmic organelles.     

Action of adenosine triphosphate on human skin and gingival fibroblasts. Protective action of fibronectin

If cultured in media supplemented with adenosine triphosphate (ATP), EDTA, trypsin, thrombin, or incubated at 0-15 degrees C, human skin fibroblasts (HSF) and human gingival fibroblasts (HGF) change from long attached elliptical to round floating cell cultures. Also, if treated with ATP, EDTA, trypsin, thrombin, or incubated at 0-15 degrees C, the attached HFS or HGF monolayers detach from plastic substratum and form floating round cells that progressively aggregate together and die. The described experiments examined the role of cellular and extracellular ATP on HSF and HGF attachment. These two types of fibroblasts differed in their cellular ATP levels and their response to metabolic inhibitors. ATP causes destruction of microtubules as monitored by colcemid uptake and cellular detachment. Fibronectin protects both HSF and HGF from the effects of extracellular ATP.     

Tumor promoter and fibronectin induce actin stress fibers and focal adhesion sites in spreading human erythroleukemia (HEL) cells

The effects of plasma fibronectin (pFn) and the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) on adhesion and cytoskeletal organization of human erythroleukemia (HEL) cells were studied. HEL cells, that normally grow in suspension, attached rapidly on pFn-coated growth substratum and some cells showed spreading. Upon exposure to TPA most of the cells adhered and showed some degree of spreading also when plated on plastic. The spread cells showed mostly peripheral accumulations of F-actin in addition to actin fibers seen in some of the cells. When the cells were plated in the presence of TPA on pFn or on pFn-fragments, containing the cell binding site, all the cells adhered rapidly, spread extensively, organized prominent F-actin stress fibers and typical ventral plaques of vinculin and alpha-actinin. Both proteins were revealed also in the suspended cells by Western blot analysis. When plated on substratum coated with other pFn-fragments or laminin, the HEL cells did not adhere or spread. Both adhesion on pFn as well as formation of stress fibers in the presence of TPA could be prevented by the synthetic peptide Arg-Gly-Asp-Ser (RGDS). HEL cells were also able to organize typical ventral fibrillar arrays of Fn. Immunostaining and metabolic labeling experiments showed that the cells did not contain or synthesize Fn, indicating that the plaques were formed from exogenous pFn by the cells. The results suggest that Fn and TPA synergistically induce the organization of the actomyosin system in HEL cells by promoting the formation of prominent actin stress fibers and focal adhesion sites.