α-Actinin arcs in megakaryocyte spreading

Cultured megakaryocytes, isolated from guinea pig bone marrow, were treated with buffer or adenosine diphosphate (ADP) (10 μM) on plain or coated glass surfaces. Control cells were rounded and non-adherent. The nucleotide induced the cells to spread to several times the initial diameter, and to become flattened and markedly adherent to the substratum. ‘Cytoskeletons’ were examined by transmission electron microscopy (TEM). Those from unspread cells contained only rare microfilaments and no filament bundles; those from spread cells contained large numbers of microfilaments in nets and many filament bundles, which were largely oriented circumferentially. Interference reflection microscopy demonstrated that the spread cells were attached to the substratum in arc-shaped regions, which corresponded to arcs containing alpha-actinin as seen by specific immunofluorescence of the same cells. However, other arcs of α-actinin staining did not correspond to arcs of tight attachment. We conclude that fibrous arcs, which appear to assemble as part of the spreading process, play a role in what are probably transient surface attachment sites. A survey of observations of spreading in other cell types suggests that similar arcs are more widespread than has been realized.     

Microfilament-associated proteins in tissue culture cells viewed by stereo immunofluorescence microscopy.

Stereo immunofluorescence microscopy avoids the problem of juxtaposition of structures often encountered in normal fluorescence microscopy. The procedure has been used in conjunction with antibodies against microfilament associated proteins to reveal the arrangement of microfilaments in a rat mammary cell line both in the fully spread state and in cells during the process of spreading on the substratum. use of antibodies to myosin, tropomyosin, alpha-actinin and filamin emphasizes that at early times during the spreading process these proteins are abundantly present underneath the upper plasma membrane, suggesting that the cortical layer present underneath this membrane may be contractile. In addition the results emphasize that even in well spread cells microfilament bundles are expressed both above and below the nucleus, in agreement with the assumption that microfilaments may form a supporting layer underneath the plasma membrane.     

Fibroblast adhesion to RGDS shows novel features compared with fibronectin

As previously shown by others, the fibroblast attachment and spreading activity of fibronectin is mimicked by a short peptide (RGDS or longer) from the cell binding domain. Normal rat kidney fibroblasts showed similar attachment kinetics on either peptide GRGDSC or bovine plasma fibronectin and binding to either substratum was inhibited by peptide alone. We now demonstrate, however, considerable differences in biological activity between peptide and fibronectin. In particular, cells developed novel adhesion structures on peptide-coated substrata. Interference reflection microscopy showed a predominance of small round dark grey/black patches of adherent membrane ("spots") with relatively few focal adhesions, which occurred only at the outermost cell margins in contrast to their distribution in cells spread on fibronectin. The spots were resistant to detergent extraction and stained less strongly or not at all for vinculin. Electron microscopy in vertical thin section showed that the ventral surface of the cell was characterized by "point-contacts", corresponding in size to the spot structures seen by interference reflection microscopy, and which were only occasionally associated with microfilaments. Cells also required a higher substratum loading of peptide than fibronectin to promote spreading and proceeded to spread less rapidly and to a lesser extent, developing very few and extremely fine actin cables.     

Cell-substratum interaction of cultured avian osteoclasts is mediated by specific adhesion structures

The cell-substratum interaction was studied in cultures of osteoclasts isolated from the medullary bone of laying hens kept on low calcium diet. In fully spread osteoclasts, cell-substratum adhesion mostly occurred within a continuous paramarginal area that corresponded also to the location of a thick network of intermediate filaments of the vimentin type. In this area, regular rows of short protrusions contacting the substratum and often forming a cup-shaped adhesion area were observed in the electron microscope. These short protrusions showed a core of F-actin-containing material presumably organized as a network of microfilaments and surrounded by a rosette-like structure in which vinculin and alpha-actinin were found by immunofluorescence microscopy. Rosettes were superposable to dark circles in interference- reflection microscopy and thus represented circular forms of close cell- substratum contact. The core of ventral protrusions also contained, beside F-actin, fimbrin and alpha-actinin. Villin was absent. This form of cell-substratum contact occurring at the tip of a short ventral protrusion differed from other forms of cell-substratum contact and represented an osteoclast-specific adhesion device that might also be present in in vivo osteoclasts as well as in other normal and transformed cell types.     

Actin, alpha-actinin, and tropomyosin interaction in the structural organization of actin filaments in nonmuscle cells

During the spreading of a population of rat embryo cells, approximately 40% of the cells develop a strikingly regular network which precedes the formation of the straight actin filament bundles seen in the fully spread out cells. Immunofluorescence studies with antibodies specific for the skeletal muscle structural proteins actin, alpha-actinin, and tropomyosin indicate that this network is composed of foci containing actin and alpha-actinin, connected by tropomyosin-associated actin filaments. Actin filaments, having both tropomyosin and alpha-actinin associated with them, are also seen to extend from the vertices of this network to the edges of the cell. These results demonstrate a specific interaction of alpha-actinin and tropomyosin with actin filaments during the assembly and organization of the actin filament bundles of tissue culture cells. The three-dimensional network they form may be regarded as the structural precursor and the vertices of this network as the organization centers of the ultimately formed actin filament bundles of the fully spread out cells.     

Cytoarchitecture of Kirsten sarcoma virus-transformed rat kidney fibroblasts: butyrate-induced reorganization within the actin microfilament network

Murine sarcoma virus-transformed rat fibroblasts (KNRK cells) undergo marked cytoarchitectural reorganization during in vitro exposure to sodium-n-butyrate (NaB) resulting in restoration of (1) a more typical fibroblastoid morphology, (2) proper cell-to-cell orientation, and (3) substratum adherence. Augmented cell spreading, involving greater than 90% of the population, was a function of culture density and time of exposure to NaB (2 mM final concentration). Induced cell spreading reflected a 2.5- to 3.0-fold increase in both total cellular actin content and deposition of actin into the detergent-resistant cytoskeleton. Cytoskeletal actin deposition in response to NaB was accompanied by the formation of occasionally dense, parallel alignments of F-actin-containing microfilaments and by a dramatic increase in the size and incidence of actin-enriched membrane ruffles. Long-term NaB-treated cells exhibited parallel orientations of microfilaments similar to those found in untransformed fibroblasts. Increased cytoskeletal actin occurred within 24 hr of NaB exposure, correlating with the initial reorganization of actin-containing microfilaments detected microscopically, and reflected concomitant 3-fold increases in cellular alpha-actinin and fibronectin content. In contrast, the amount of vimentin, tropomyosin, and tubulin in NaB-treated cells was significantly decreased. NaB-induced morphologic restructuring of sarcoma virus-transformed fibroblasts, thus, impacts on all three basic cytoskeletal systems. Selective increases, however, were evident in particular cytoskeletal proteins (actin, alpha-actinin, fibronectin) implicated in microfilament networking and cell spreading.     

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.     

Initial adhesion of human fibroblasts in serum-free medium: possible role of secreted fibronectin.

Experiments were carried out to test the hypothesis that the initial attachment and spreading of human fibroblasts in serum-free medium occurs to cell fibronectin which has been secretd spread on tissue culture substrata in serum-free medium in 60 min. When potential protein adsorption sites on the substratum were covered with bovine serum albumin before initial human fibroblasts attachment, their subsequent attachment to the substratum was prevented. When substratum adsorption sites were covered immediately after initial attachment, subsequent cell spreading was prevented. The distribution of fibronectin on human fibroblast surfaces during initial attachment and spreading was studied by indirect immunofluorescence analysis using a monospecific anti-cold-insoluble globulin antiserum. The initial appearance (10 min) of fibronectin was in spots over the entire cell surface. Concomitant with human fibroblast spreading, the random distribution of sites disappeared, and most fibronectin was subsequently observed in spots at the cell substratum interface (60 min). A fibrillar pattern of fibronectin appeared later (2-8 hr). The sites beneath the cells could be visualized as footprints on the substratum following treatment of the attached human fibroblasts with 0.1 M NaOH. A second fluorescence pattern of fibronectin secreted on the substratum was characterized by a diffuse halo around the cells and a very faint, diffuse staining elsewhere on the substratum. Another cell type (baby hamster kideny cells) was used to assay biologically for the presence or absence of the factor secreted by human fibroblasts on the substratum. Human fibroblasts were found to secrete an adhesion factor for baby hamster kidney cells into the substratum in a time- and temperature-dependent fashion, and immunological studies indicated that the factor secreted by human fibroblasts was cross-reactive with cold-in-soluble globulin, the plasma form of fibronectin. The conditioning factor secreted by the human fibroblasts was also found to be an attachment and spreading factor for human fibroblasts in experiments measuring human fibroblast adhesion to fibronectin footprints of human fibroblasts. Substratum-adsorbed cold-insoluble globulin was also found to be an attachment and spreading factor for human fibroblasts. Based upon the timing of appearance of conditioning factors on the substratum and the immunofluorescence patterns, it seems that the diffusely organized fibronectin on the substratum constitutes the sites to which cell attachment occurs. The bright spots of fibronectin that appear beneath the cells may represent fibronectin reorganization during cell spreading.     

Surface movements during the spreading of blood platelets

When human blood platelets spread on a substratum they increase their surface area as much as 4-fold. We investigated the mechanism of spreading by light microscopy and by scanning and transmission electron microscopy. Contact of a platelet with a glass surface induces formation of thin extensions which spread out over the substratum. These extensions resemble the actin-containing microspikes and lammelipodia of tissue cells in culture and appear to be drawn from the peripheral cortical layer associated with the plasma membrane. If platelets are initially labeled on their external surface with cationic ferritin or lentil-conjugated gold particles and then allowed to spread, the labels are retained in the central region, or granulomere. Proteins released by the spreading platelet--fibronectin and fibrinogen--also remain in this central unspread region. Peripheral regions of spread platelet surface (hyalomere) were unlabeled following the above procedures but could be labeled with cationic ferritin or lentil-conjugated gold provided these were applied after spreading was completed. These markers are cleared with time from the periphery, moving centripetally to accumulate at the granulomere. We suggest, on the basis of these observations, that platelets spread onto a substratum by a closely similar mechanism to that used by cells such as fibroblasts. In both cases the spreading involves the peripheral actin cortex and is accompanied by a continual centripetal movement of surface components--a "membrane flow"--which continues even after spreading is completed.     

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.     

HeLa cell adhesion to microcarriers in high shear conditions: evidence for membrane receptors for collagen but not laminin or fibronectin

HeLa-S3 cells were analyzed for their ability to attach and spread on cell culture microcarriers that were made either positively or negatively charged with polymeric plastics or were coated with BSA, gelatin, fibronectin or laminin. The cells stuck to all microcarriers under low shear, i.e. low stirring conditions with similar rates of attachment. Except in the case of gelatin microcarriers where cells fully spread, cells did not or only partially spread on the others. Under high shear, cells attached with the following rates: positive = negative = gelatin = BSA greater than laminin greater than fibronectin. Cells detached from all but the gelatin and BSA coated beads. However, the cells did not fully spread on BSA beads. The observation that cells not only attached but also spread on gelatin beads indicated that gelatin could be a specific substratum adhesion protein while the other surfaces were 'non-specific'. It should be noted that neither antibodies to laminin nor fibronectin interfered with attachment to gelatin. Protein synthesis inhibitors reduced the attachment and spreading on gelatin beads under high but not low shear conditions. With low shear, attachment and spreading appeared normal. We concluded that the density of the cell surface attachment proteins was reduced by the protein synthesis inhibitors and there were not enough present to facilitate attachment under high shear. The results also indicated that protein synthesis was not essential for cell spreading. Proteolysis of the cell surface with low concentrations of trypsin abolished the attachment of cells to gelatin-coated beads. The reappearance of attachment ability took several hours and was inhibited by actinomycin-D.     

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.     

Unique morphology of HeLa cell attachment, spreading and detachment from microcarrier beads covalently coated with a specific and non-specific substratum

A SEM and TEM evaluation of adhesion of HeLa-S3 cells to suspensions of culture microcarriers coated with various substrata revealed two unique cell morphologies. One is similar to that for cells attaching to culture dishes and the other one only appeared with microcarriers stirred under high shear conditions. The usual appearance of a spreading cell is to change from a sphere to the shape of a 'fried egg'. This proceeded in HeLa cells by a radial extension of the filopodia in between which the cytoplasm subsequently filled. Fluorescent antibody staining of actin suggested that more actin was present at the periphery of the spreading edges of the cell than inwards. The above morphology was characteristic of HeLa cell attachment to gelatin-coated microcarriers. However, the morphology of the attachment to microcarriers coated with non-biological substances such as negatively charged sulfonate groups or positively charged polyethyleneimine or even with the attachment protein laminin was quite different. Here the cells attached and began to spread as with gelatin-microcarriers, however, the spreading was not radial but occurred from one or two major regions of the cell periphery. The cell then appeared to constrict with the formation of a substratum attached pedestal upon which the cell body was perched. With time the cell pinched-off from pedestal. Evidence indicated that the pedestal was quite fragile. Furthermore, fluorescent antiactin staining indicated that the initial spreading region contained abundant actin which was depleted upon pedestal formation and detachment. The above in addition to previous kinetic measurements provided the information to classify cell substrate attachment materials into two distinct types. One is specific substrata which promote normal attachment and spreading and appear to interact with specific cell surface proteins. The other is non-specific substrata which in high shear conditions induces pedestal formation followed by pinching-off of the cells. Had previous attachment assays been done under high shear as done with the microcarriers and HeLa cells it is likely that substrata classified as specific might be reclassified into non-specific.     

The serum dependence of baby hamster kidney cell attachment to a substratum

Normal attachment and spreading of baby hamster kidney cells onto a non-living substratum requires the presence of a specific serum component adsorbed to the substratum surface and Ca²⁺ ions in the medium. In the absence of the adsorbed serum factor or Ca²⁺ ions cells attach but do not spread. Thus, although the initial rate of BHK cell attachment is faster in serum-free medium than serum-containing medium, no cell spreading occurs in serum-free medium. Adsorption of serum onto the substratum results in a lag phase in the time course of cell attachment which can be eliminated by blocking the negatively charged groups of the serum components adsorbed to the substratum surface; blocking positively charged groups or free sulfhydryl groups of the adsorbed serum components is without effect. The requirement for serum components can be substituted for by adsorbing molecules such as concanavalin A or polycationic ferritin to the substratum surface; however, only ConA results in normal morphology of cell spreading. The data are discussed in terms of a non-electrostatic direct cell-substratum binding model of cell attachment.     

Induction of cell spreading by substratum-adsorbed ligands directed against the cell surface

Studies were carried out to compare the spreading of baby hamster kidney (BHK) cells, which occurs by an interaction between the cells and a specific serum glycoprotein (ASF) adsorbed onto the substratum surface, with the spreading of BHK cells that occurs by an interaction between the cells and substrata coated with ligands directed at various cell surface determinants. The ligands tested were polycationic ferritin, concanavalin A (ConA) and antibody directed against BHK plasma membranes. Cell spreading onto ASF and ligand-coated substrata were similar even though different cell surface components were apparently involved. The similarities were:

1. 1. The shape of the spread cells.

2. 2. The inhibition of cell spreading by conditions that interfere with metabolic activity, block free sulfhydryl groups, or interfere with microtubules and microfilaments.

3. 3. The similar reorganization of certain cell surface antigenic determinants during cell spreading onto any of the substrata.

The results indicate that cell spreading is a general cellular response to specific cell-substratum interactions but does not depend upon binding between a unique cell surface receptor and the substratum.     

Electron microscopic study of the interaction between neoplastic fibroblasts and the substrate in tissue culture]

Electron microscope study of neoplastic L fibroblasts was carried out in 15, 30, 60, and 90 min after their attachment to solid substratum. Comparative analysis of neoplastic and normal fibroblasts at the same stages was carried out. Spreading rate of L fibroblasts proved slower than that of normal fibroblasts. Primary reaction of neoplastic cells was disturbed on the contact with substratum: it was morphologically manifested in changing of structure of the cell lower surface. Bundles of microfilaments were absent from the neoplastic fibroblasts' cytoplasm. The above changes, apparently, may entail the lesser degree of spreading and the weaker attachement of neoplastic fibroblasts to the substratum.     

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.     

Cytoplasmic elongation and rupture in megakaryoblastic leukemia cells via activation of adhesion and motility by staurosporine on fibronectin-bound substratum

Human megakaryoblastic leukemia Meg-01 cells were attached to fibronectin (FN)-coated substratum, on which remarkable spreading and cytoplasmic elongation was induced by treatment with a protein kinase inhibitor, staurosporine (stp). This effect was inhibited by RGDS and was also not seen on FN-lacking substratum. The extended cytoplasm had swollen terminals and nodes, which contained GpIIb and beta-thromboglobulin, occasionally included alpha granules, and tended to form particles (2-5 microm) after rupture of the narrowed cytoplasm. Among other protein kinase modulators tested, only K252a promoted the elongation, while calphostin, herbimycin, TPA, and calyculin suppressed it. The cells began to migrate soon after addition of stp, with attachment to the substratum held at some sites during the migration. This tethered movement seemed to cause the cytoplasmic elongation and the rupture into particles. The elongation was retarded by pretreating the cells with cytochalasin A and Clostridium C3 toxin but not with demecolcine. Actin microfilaments in the stp-treated Meg-01 cells accumulated in the filopodia and periphery of the extended cytoplasm, in which vinculin was colocalized as adhesion plaques. The microtubules were longitudinally oriented through the cytoplasmic extension and showed no ring profile in the nodes and particles. Thus, stp in the presence of FN appears to stimulate reorganization of actin-based cytoskeleton and formation of focal contacts in Meg-01 cells. This leads to the activation of cell adhesion and motility, and then cytoplasmic elongation and rupture into particles.     

Transfection of chicken skeletal muscle alpha-actinin cDNA into nonmuscle and myogenic cells: dimerization is not essential for alpha-actinin to bind to microfilaments.

alpha-Actinins from striated muscle, smooth muscle, and nonmuscle cells are distinctive in their primary structure and Ca2+ sensitivity for the binding to F-actin. We isolated alpha-actinin cDNA clones from a cDNA library constructed from poly(A)+ RNA of embryonic chicken skeletal muscle. The amino acid sequence deduced from the nucleotide sequence of these cDNAs was identical to that of adult chicken skeletal muscle alpha-actinin. To examine whether the differences in the structure and Ca2+ sensitivity of alpha-actinin molecules from various tissues are responsible for their tissue-specific localization, the cDNA cloned into a mammarian expression vector was transfected into cell lines of mouse fibroblasts and skeletal muscle myoblasts. Immunofluorescence microscopy located the exogenous alpha-actinin by use of an antibody specific for skeletal muscle alpha-actinin. When the protein was expressed at moderate levels, it coexisted with endogenous alpha-actinin in microfilament bundles in the fibroblasts or myoblasts and in Z-bands of sarcomeres in the myotubes. These results indicate that Ca2+ sensitivity or insensitivity of the molecules does not determine the tissue-specific localization. In the cells expressing high levels of the exogenous protein, however, the protein was diffusely present and few microfilament bundles were found. Transfection with cDNAs deleted in their 3' portions showed that the expressed truncated proteins, which contained the actin-binding domain but lacked the domain responsible for dimerization, were able to localize, though less efficiently in microfilament bundles. Thus, dimer formation is not essential for alpha-actinin molecules to bind to microfilaments.