The response of chicken embryo dermal fibroblasts to cytochalasin B is altered by Rous sarcoma virus-induced cell transformation.

The drug cytochalasin B (CB), which disrupts the cellular microfilament network, allows the identification of as yet unclassified structural differences between normal and Rous sarcoma virus-transformed chicken embryo fibroblasts. When exposed to CB, normal chick fibroblasts attain an arborized or dendritic morphology. This results as the cytoplasm collapses upon the remaining structural and adhesive components of the cell. Rous sarcoma virus-transformed cells did not form or maintain these dendritic-like processes in the presence of CB and, as a result, rounded up but still remained attached to the substrate. With a temperature-sensitive mutant of Rous sarcoma virus, LA24A, it was possible to show that these effects are completely reversible and dependent on the expression of pp60src. The cytoskeleton in these CB-treated cells was examined by both immunofluorescence and electron microscopy. After exposure to CB, the microfilaments were found to be disrupted similarly throughout both the transformed and the nontransformed cells. In the nontransformed cells arborized by exposure to CB, the extended processes were found to contain intermediate filaments in an unusually high concentration and degree of organization. The distribution of these filaments in the central body of the arborized cells was random. This lower concentration and random distribution was similar to that seen throughout the transformed cells rounded up by exposure to CB. The failure of these transformed cells to arborize in CB indicates that the structural component(s) which is necessary for the formation or maintenance or both of the arborized state is altered by the expression of pp60src.     

Morphological evidence for cyclic AMP-induced reverse transformation in vole cells infected with avian sarcoma virus.

Normal fibroblasts of the vole displayed moderately spread or flattened, spindle-shaped, or polygonal morphologies and attached firmly to a substrate. Topographic features of these cells included sparse microvilli, ruffles, and filopodia. Microfilament bundles, intermediate filaments, and long microtubules generally parallel to each other, and the long axis of the cell or its extensions were present in the cytoplasm. Fibronectin was abundant, and fibronectin fibrils often formed junctions at the cell membrane with microfilament bundles. Transformation with avian sarcoma virus converted 90% of the cells to spheres 5 to 10 microns in diameter. In contrast to the normal vole cells, microfilament bundles were absent, microtubules were short and randomly arranged, and fibronectin was no longer visible. Exposure to dibutyryl cyclic AMP and testololactone caused a majority of the spherical cells to stretch and flatten, a process referred to as reverse transformation. Microtubules radiated out to the cell periphery and became parallel in cell extensions, while long microfilament bundles appeared in the cytoplasm. Parallel intermediate filaments were arranged throughout the cell. This ultrastructural analysis of reverse transformation in avian sarcoma virus-transformed vole cells detailed the status of the cytoskeletal system and showed agreement with earlier findings (Puck et al., J. Cell. Physiol. 107:399-412, 1981) using indirect immunofluorescence.     

Regulation of integrin-mediated adhesion at focal contacts by cyclic AMP

Cyclic AMP (cAMP) elevation causes diverse types of cultured cells to round partially and develop arborized cell processes. Renal glomerular mesangial cells are smooth, muscle-like cells and in culture contain abundant actin microfilament cables that insert into substratum focal contacts. cAMP elevation causes adhesion loss, microfilament cable fragmentation, and shape change in cultured mesangial cells. We investigated the roles of the classical vitronectin (αVβ3 integrin) and fibronectin (α5β1 integrin) receptors in these changes. Mesangial cells on vitronectin-rich substrata contained microfilament cables that terminated in focal contacts that stained with antibodies to vitronectin receptor. cAMP elevation caused loss of focal contact and associated vitronectin receptor. Both fibronectin and its receptor stained in a fibrillary pattern at the cell surface under control conditions but appeared aggregated along the cell processes after cAMP elevation. This suggested that cAMP elevation caused loss of adhesion mediated by vitronectin receptor but not by fibronectin receptor. We plated cells onto fibronectin-coated slides to test the effect of ligand immobilization on the cellular response to cAMP. On fibronectin-coated slides fibronectin receptor was observed in peripheral focal contacts where actin filaments terminated, as seen with vitronectin receptor on vitronectin-coated substrata, and in abundant linear arrays distributed along microfilaments as well. Substratum contacts mediated by fibronectin receptor along the length of actin filaments have been termed fibronexus contacts. After cAMP elevation, microfilaments fragmented and fibronectin receptor disappeared from peripheral focal contacts, but the more central contacts along residual microfilament fragments appeared intact. Also, substratum adhesion was maintained after cAMP elevation on fibronectin—but not on vitronectincoated surfaces. Although other types of extracellular matrix receptors may also be involved, our observations suggest that cAMP regulates adhesion at focal contacts but not at fibronexus-type extracellular matrix contacts. © 1993 Wiley-Liss, Inc.     

Contact relationships between chick embryo cells growing in monolayer culture after infection with Rous sarcoma virus

An electron microscopic examination was made of cell contacts and associated microfilament arrays in subconfluent cultures of chick embryo fibroblasts (CEF) and chick embryo retinal pigmented epithelium cells (RPE) transformed by strains of Rous sarcoma virus (RSV) imparting a rounded (Morph r) or fusiform (Morph f) transformed morphology. A few cell substrate contact specializations were found in Morph r-transformed CEF and RPE cells. These resembled cell/substrate plaques of uninfected fibroblasts, but lacked associated microfilament tracts. In contrast Morph f-transformed CEF and RPE resembled untransformed fibroblasts having well developed cell/substrate and cell/cell contact specializations with extensive associated microfilament arrays. Morph r- and Morph f-transformed RPE cells had lost the junctional complex typical of untransformed RPE cultures and additionally no melanosomes were found. SEM and TEM demonstrated differences in adhesive properties of CEF and RPE cell surfaces, few virions adhering to the free cell surface of RPE cells but being found in clumps and singly on CEF cells.     

Unphosphorylated gelsolin is localized in regions of cell-substratum contact or attachment in Rous sarcoma virus-transformed rat cells

Regions associated with cell-substratum contact or attachment in Rous sarcoma virus (RSV)-transformed rat fibroblasts (RR1022 cells) were identified by reflection-interference microscopy. Electron microscopy of such regions revealed the presence of discrete membrane-associated structures composed of a paracrystalline lattice of hexagons and pentagons to which actin filaments appear to be attached. Staining of actin by biotin-labeled heavy meromyosin showed that transformed cells, unlike normal fibroblasts, lack prominent actin fibers, and that, instead, much of the fluorescence is concentrated in loci corresponding to locations of transient association between the cell and the substratum. In stationary cells, such loci were found in rosette formation, predominantly in the region beneath the nucleus. In cells engaged in active movement, such as during migration into a wound, the actin-containing spots were concentrated in the region of the leading edge. A similar pattern of staining was observed with antibody to gelsolin, a 91,000-dalton Ca2+-dependent actin filament-shortening protein. Since the action of gelsolin on actin is reversible and dependent on physiologically relevant changes in calcium concentration, the localization of gelsolin, together with actin-bundling proteins such as alpha-actinin, in the regions containing many small microfilament bundles on the ventral side of cytoplasm suggests that gelsolin may be a component of the mechanism for the disassembly and assembly of actin during the dissolution and reformation of structures for cell-substratum contact during cell locomotion. Regulation of gelsolin activity was not dependent on protein phosphorylation, as shown by lack of 32P-incorporation into gelsolin in either transformed or normal fibroblasts.     

Disintegration of adhesion plaques in chicken embryo fibroblasts upon Rous sarcoma virus-induced transformation: different dissociation rates for talin and vinculin

The localization of talin and vinculin in chicken embryo fibroblasts (CEF) during transformation was studied by immunoelectron microscopy. CEF cells were infected with a temperature-sensitive mutant of Rous sarcoma virus. After 16 h at 42 degrees C, transformation was induced by incubation at 37 degrees C for different intervals up to 3 h. Cells were cleaved by "wet cleaving" as reported previously by us (R. Brands and C.A. Feltkamp, 1988, Exp. Cell Res. 176, 309) and labeled with affinity-purified polyclonal antibodies to talin or vinculin, or monoclonal anti-vinculin. We observed a rapid reduction of vinculin in adhesion plaques within 15 min and a much slower dissociation of talin. This was found using single-labeling procedures and also within the same cell using double labeling. Seemingly intact microfilament bundles were observed associated with adhesion plaques that contained relatively little vinculin. These observations show that an early event in src-induced transformation is the release of vinculin from adhesion plaques. Furthermore, since adhesion plaques with attached filament bundles can exist at least transiently with very little or no vinculin present, it seems likely that vinculin is not, or not the only protein, linking actin filaments to adhesion plaques.     

Normal and transformed fibroblast spreading: Role of microfilament polymerization and actin-myosin contractility

The polymerization of microfilaments and their subsequent rearrangements under the control of actin-myosin interactions are two major processes that underlie the morphogenetic reactions of cells. We studied their role in the spreading of normal and transformed REF52tetRas fibroblasts with adjustable ras-oncogene expression. Treatment with inhibitors of cell contractility (Y27632 or blebbistatin) led to the disappearance of actin bundles and focal adhesions; however, pseudopodial activity in both normal and transformed cells remained high. Under these conditions, spreading was more accelerated in normal cells then in ras-transformed cells. In normal cells treated with low concentrations of latrunculin A actin polymerization was suppressed, stress fibers and focal adhesions were preserved, but lamellipodial activity was lost and spreading was dramatically inhibited. In transformed fibroblasts treated with low doses of latrunculin, actin bundles and focal adhesions almost disappeared, but pseudopodial activity was apparent and spreading was less suppressed. Therefore, the most significant process in the regulation of cell spreading and polarization is the microfilament polymerization at the leading edge. ras-Transformed cells are less sensitive to inhibitors that affecting the cytoskeletal structure than nontransformed cells. Possible mechanisms that underlie the difference are discussed.     

The cytoskeletal protein vinculin is acylated by myristic acid

In non-muscle cells the mechanism by which microfilament bundles interact with the plasma membrane is unclear. Vinculin, a 130 kDa protein found in adhesion plaques, has been postulated to have a role as a membrane anchor for microfilaments and we have investigated the biochemistry of this molecule in more detail. We report that a fraction of vinculin in chick embryo fibroblasts is acylated by myristic acid. This modification was present in both membrane-bound, cytoskeletal and cytosolic vinculin and thus did not determine preferential subcellular localisation. Myristic acid was also present in vinculin from cells transformed by Rous sarcoma virus.     

Relationships between fibronectin (LETS protein) and actin.

Double label immunofluorescence was used to study the distribution of fibronectin (LETS protein), actin and intermediate filaments in cultured cells. No relationship was observed between fibronectin and intermediated filaments, but fibronectin and actin showed coincident staining in a large proportion of cells during spreading or when fully spread. The distributions of actin and fibronectin staining during the course of cell spreading progressed through a series of patterns. Certain actin patterns correlated with certain fibronectin patterns. When fibrillar patterns developed, there was correspondence between the two fibrillar arrays in 80--100% of the cells. These results suggest a transmembrane relationship between microfilament bundles and fibronectin. We propose that fibronectin may participate in the formation of attachment plaques and discuss the interrelationship between plaques, microfilament bundles and fibronectin in cell-substratum and cell-cell contacts.     

A new protein of adhesion plaques and ruffling membranes

A protein with a molecular weight on SDS polyacrylamide gels of 215,000 (referred to here as 215K) was purified from chicken gizzard smooth muscle. Antibodies against this protein localized it in fibroblasts to adhesion plaques (focal contacts), to regions underlying cell surface fibronectin, and to ruffling membranes. In the first two distributions it was similar to vinculin in cellular location, and this was confirmed by double-label immunofluorescence microscopy, but the concentration of 215K in membrane ruffles distinguished it from vinculin. There was no cross-reaction of the antibody against 215K with vinculin, and immunoprecipitation and antibody staining of SDS gels of whole cells revealed a single cross-reactive component with a molecular weight of 215,000. Immunoprecipitation from cultures labeled with [32P]phosphate revealed 215K to be a phosphoprotein. Transformation of rat or chicken fibroblasts by Rous sarcoma virus resulted in a reorganization of 215K, in some cases into complex intracellular structures. The localization of 215K where microfilament bundles terminate as well as in close relation to cell surface fibronectin and in membrane ruffles suggests that the protein has some function in the organization of actin filaments at or close to regions of actin-membrane attachment.     

The use of Rous sarcoma virus transformation mutants with differing tyrosine kinase activities to study the relationships between vinculin phosphorylation, pp60v-src location and adhesion plaque integrity

Tyrosine-specific phosphorylation of cellular proteins has been implicated in the neoplastic transformation of cells by Rous sarcoma virus (RSV). One of the putative substrates for the src gene product (pp60v-src) of RSV is the cytoskeletal protein vinculin, giving rise to the hypothesis that tyrosine-specific phosphorylation of vinculin disrupts adhesion plaque integrity, leading to the characteristic rounded morphology of RSV-transformed cells. We have investigated this hypothesis by analysing the properties of fibroblasts transformed by conditional and non-conditional mutants of RSV which confer different morphologies on infected cells, with respect to formation of microfilament bundles, formation of vinculin-containing adhesion plaques, the deposition of a fibronectin-containing extracellular matrix, the localization of pp60v-src and the tyrosine-specific phosphorylation of vinculin. Cells transformed by the temperature-sensitive (ts) RSV mutant LA32 cultured at 41 degrees C were morphologically normal, and contained prominent microfilament bundles and well-developed adhesion plaques. However, these cells had a fully active pp60v-src kinase, had pp60v-src concentrated in their adhesion plaques and contained vinculin which was heavily phosphorylated on tyrosine residues. Cells transformed by a recovered avian sarcoma virus, rASV 2234.3 exhibited a markedly fusiform morphology with pp60v-src concentrated in well-developed adhesion plaques and an elevation of the phosphotyrosine content of vinculin. Cells transformed by LA32 at restrictive temperature comprise morphologically normal cells, indistinguishable from untransformed CEF, yet which contain tyrosine-phosphorylated vinculin and suggest that neither tyrosine-specific phosphorylation of vinculin nor pp60v-src concentration in adhesion plaques is sufficient for the rounded morphology of RSV-transformed cells.     

Serum-free growth of normal and transformed fibroblasts in milk: differential requirements for fibronectin

Bovine milk may be used as a supplement for the serum-free growth of certain fibroblastic cells in culture. The growth properties of three representative cell types in milk-supplemented medium were examined; fibroblastic cell strains, fibroblastic cell lines, and transformed fibroblasts. Transformed fibroblasts, which included RNA and DNA tumor virus-transformed cells and carcinogen-transformed cells, grew in milk. Instead of growing attached to the culture dishes, as they normally do in serum, transformed fibroblasts grew in milk as large clusters in suspension. In contrast, nontransformed fibroblastic cell strains and cell lines did not grow in milk-supplemented medium. Fibroblasts transformed by a temperature-sensitive transformation mutant of Rous sarcoma virus were temperature-sensitive for growth in milk. The failure of cells to adhere to the substratum in milk-supplemented medium suggested that milk might be deficient in attachment factors for fibroblasts. When the attachment of fibroblastic cells in milk- supplemented medium was facilitated by pretreating culture dishes with fibronectin, (a) transformed cells grew attached rather than in suspension, (b) normal cell lines attached and grew to confluence, and (c) normal cell strains adhered and survived but did not exhibit appreciable cell proliferation.     

Transformation of chick embryo fibroblasts by rous sarcoma virus does not inhibit the assembly of fibronectin into reduction-sensitive dimer and high molecular weight complex

Chick embryo fibroblasts (CEFs) transformed by Rous sarcoma virus synthesize reduced amounts of fibronectin and also shed this protein into the medium more rapidly than do uninfected cells. We wanted to know whether or not the increased fibronectin shedding rate observed in RSV-transformed CEFs could be explained by an inability of fibronectin to form dimers and/or HMW complex. Our studies on normal and RSV-transformed fibroblasts labelled either metabolically or by lactoperoxidase-catalysed iodination indicate that RSV-induced transformation does not alter the subunit structure of either cell-bound or shed fibronectin, nor does it appear to alter the kinetics of conversion of dimeric fibronectin into HMW complex. We conclude that transformation of CEFs by Rous sarcoma virus does not prevent the assembly of fibronectin into dimeric and HMW complex forms and we offer alternative hypotheses for the more rapid shedding of fibronectin protein by these cells.     

Formation of bundles of microfilaments during spreading of fibroblasts on the substrate

Electron microscopy was used to study the sites of formation of bundles of parallel microfilaments in the early stages of spreading of normal mouse embryo fibroblasts on the substrate. Bundles of microfilaments were not found in suspended cells. Contact of the surface of spherical cells with the substrate was not sufficient for the formation of bundles: these bundles were not seen near the under surface of cells that were already attached to the substrate but had not yet developed cytoplasmic outgrowths at their periphery. Peripheral cytoplasmic outgrowths (microspikes and lamellar processes) attached to the substrate were found to be the only sites of localization of the first bundles of microfilaments seen in the spreading cells. It is suggested that surface and/or cytoplasm of the newly-formed peripheral cytoplasmic outgrowth may have some special properties necessary for the initiation of the development of microfilament bundles.     

Trifluoperazine inhibits spreading and migration of cells in culture

Trifluoperazine (TFP) blocks spreading and migration of cultured mammalian cells. These are calcium-dependent and microfilament-mediated processes. Calmodulin, a regulator of many calcium-dependent processes in cells, is selectively inhibited by TFP. Cell spreading on a plastic- or collagen-coated substratum was reversibly inhibited by 10 μM TFP. The drug blocks cell spreading even in the presence of 1 mM cAMP. TFP is as effective as cytochalasin B (CB), an inhibitor of microfilament function, in blocking cell spreading. All cell lines tested, whether “normal” or virally transformed, failed to spread in TFP. The drug, at a concentration sufficient to inhibit spreading, does not interfere with the initial attachment of a cell to a plastic surface. Cells plated in the presence of 10 μM TFP attach at a rate and to an extent equal to untreated controls. TFP added to already spread cells results in a reversible cell rounding. Detection of fibronectin by indirect immunofluorescence suggests TFP-induced cell rounding is not due to shedding of fibronectin from the cell surface. TFP reversibly blocks cell migration into a wound edge almost as effectively as CB. We suggest that TFP interferes with these microfilament-mediated functions by direct action on the microfilaments or indirect action by inactivating calmodulin.     

Temperature-dependent and transformation-associated changes in polyamine metabolism in normal and Rous sarcoma virus-infected chick embryo fibroblasts

Tertiary cultures of chick embryo fibroblasts infected and transformed by the wild-type Rous sarcoma virus, when actively growing at 35 degrees C, had higher putrescine levels than the respective uninfected cells. Transformed cells also had much higher specific activity of ornithine decarboxylase (EC 4.1.1.17) than the normal fibroblasts. At 41 degrees C the difference in putrescine levels between the normal and the transformed cells was less marked, and both cell types showed a relative accumulation of spermine. Cultures infected with the NY68 mutant virus, which is temperature-sensitive for transformation, showed at 41 degrees C normal cell morphology and intermediate polyamine patterns, while at 35 degrees C a transformed phenotype was found in both aspects. In shift-down experiments a change towards the permissive temperature pattern of polyamine metabolism was evident within 2-3 h. Difluoromethylornithine, a specific and irreversible inhibitor of ornithine decarboxylase efficiently reduced the enzyme activity as well as the levels of both putrescine and spermidine in all culture types and temperatures. Incubation of Rous sarcoma virus-transformed cells with 3 mM difluoromethylornithine for 36 h did not affect the maintenance of the transformed state. Likewise, when NY68-infected cultures were exposed to difluoromethylornithine at 41 degrees C for 12 h and then shifted down to 35 degrees C, the appearance of the transformed morphology took place concomitantly with that of the control cultures without respective changes in the polyamine levels. This suggests that the transformation-associated pattern of polyamines in chick embryo fibroblasts is not a prerequisite for morphological transformation of these cells.     

Vinculin: a cytoskeletal target of the transforming protein of Rous sarcoma virus

Vinculin, a protein associated with the cytoplasmic face of the focal adhesion plaques which anchor actin-containing microfilaments to the plasma membrane and attach a cell to the substratum, contains 8-fold more phosphotyrosine in cells transformed by Rous sarcoma virus than in uninfected cells. Because the transforming protein of RSV, p60src, is a protein kinase that modifies cellular proteins through the phosphorylation of tyrosine and because phosphotyrosine is a very rare modified amino acid, this result is a very rare modified amino acid, this result suggests that vinculin is a primary substrate of p60src. Only trace amounts of phosphotyrosine were detected in myosin heavy chains, alpha-actinin, filamin, and the intermediate filament protein vimentin. The modification of vinculin by p60src may be responsible in part for the disruption of the microfilament organization and for the changes in cell shape and adhesiveness which accompany transformation by Rous sarcoma virus.     

Cytoskeletal dynamics in rabbit synovial fibroblasts: II. Reformation of stress fibers in cells rounded by treatment with collagenase-inducing agents

Modulation of the synthesis and secretion of extracellular matrix proteins and matrix-degrading metalloproteases by rabbit synovial fibroblasts is an important model system for studying the control of tissue-specific gene expression. Induction of collagenase expression is correlated with changes in cell shape and actin filament distribution, but the role of the cellular cytoskeleton in the sustained synthesis and secretion of metalloproteases has not been closely examined. When cells were allowed to respread after rounding by trypsin or cytochalasin, two known metalloprotease inducers, reformation of stress fibers was observed within 2 h in the presence of serum. In the absence of serum, trypsin-treated cells did not respread substantially, even after 24 h in culture. In contrast, cytochalasin-treated cells recovered almost as rapidly in the absence as in the presence of serum, showing reformation of well-formed microfilament bundles within 30 min of drug removal, especially at the spreading cell edges. High resolution electron-microscopic views of detergent-extracted cytoskeletons confirmed the rapid rebundling of peripheral microfilaments. Acrylamide-treated cells fell between these two extremes, spreading slowly in the absence of serum, but almost as rapidly as cytochalasin-treated cells in its presence. Reestablishment of normal intermediate filament distribution generally lagged slightly behind actin for all treatments, and intermediate filaments always appeared to spread back into the cellular cytoplasm within the confines of the reforming peripheral microfilament bundles. No obvious interaction between these two cytoskeletal elements was observed after any treatment, and no specific role for intermediate filaments in modulating gene expression in these cells is suggested by these results. The serum dependence displayed after trypsin or acrylamide treatment may be due to the disturbances in fibronectin synthesis observed in these cells and is consistent with evidence that both induction and sustained expression of matrix-degrading metalloprotease may involve signals transduced through plasma membrane matrix receptors (integrins).     

F-actin aggregates in transformed cells contain alpha-actinin and fimbrin but apparently lack tropomyosin

Transformation-specific F-actin structures are examined in tumor cells after in vitro tumor cell growth alone or on an untransformed cell monolayer. In transformed cells F-actin aggregates near the ventral plasma membrane in close substrate adhesion areas contain the cytoskeletal proteins alpha-actinin and fimbrin but, unlike microfilament bundles, are not labeled with antibody against tropomyosin. By electron microscopy the dense ventral aggregates in transformed cells resemble stress fiber termini found at the membrane in normal cells. These transformed-cell cytoskeletal structures are not limited solely to substrate adhesion areas; they are also expressed at cell-cell contacts about 48 h after transformed cells are plated on untransformed cells. These specialized F-actin aggregates appear to be implicated in the processes of penetration of these transformed cells between adjoining untransformed cells in vitro.