Cytochemical study of concanavalin A binding sites and their mobility in normal, cystic fibrosis, and SV40 transformed human fibroblasts in vitro

Concanavalin A (Con A) binding sites and their mobility were studied by peroxidase (Po) and ferritin labeling techniques in normal and SV40 transformed human fibroblasts. Binding sites were visualized either as osmium black of 3'3-diaminobenzidine (DAB) reactions or as ferritin particles. DAB reaction products were localized at the external surface of the plasma membrane and in some multivesicular bodies of fixed cells. The labeling was continuous in normal and SV40 transformed human fibroblasts. When living cells were treated with Con A-Po at 4 degrees C and incubated at 37 degrees C, both normal and transformed cells showed remarkable changes. The foci of membrane indentations (caps or patches) are formed on the cell surface. Many labeled internalized vacuoles and vesicles appeared within the cytoplasm and in close proximity to the Golgi region of all cell types. The cellular changes occurred more quickly in transformed cells than in normal cells. It is concluded that normal cells do cap under certain conditions and that the plasma membranes of transformed cells are more fluid than those of normal cells.     

Acylated simian virus 40 large T-antigen: a new subclass associated with a detergent-resistant lamina of the plasma membrane

We have analyzed the plasma membrane association of the SV40 large tumor antigen (large T) in SV40-transformed BALB/c mouse tumor cells (mKSA). Isolated plasma membranes were subfractionated: treatment with the non-ionic detergent Nonidet P40 (NP40) resulted in a NP40-resistant plasma membrane lamina, which could be further extracted with the zwitterionic detergent Empigen BB. Analysis of the different plasma membrane fractions revealed that only about one third of large T associated with isolated plasma membranes could be solubilized with NP40. The residual plasma membrane-associated large T was tightly bound to the NP40-resistant lamina of the plasma membrane from which it was released by treatment with the zwitterionic detergent Empigen BB. Further evidence for a specific interaction of a distinct subclass of large T with the plasma membrane was provided by showing that only T associated with the NP40-resistant lamina of the plasma membrane contained covalently bound fatty acid. Neither nuclear large T nor large T in the NP40-soluble plasma membrane fraction could be labeled with [3H]palmitic acid. Our results indicate that an acylated subclass of large T interacts specifically with a structure of the plasma membrane, suggesting that it might be involved in a membrane-dependent biological function.     

Ultrastructural localization of concanavalin A receptors in the plasma membrane: association with underlying actin filaments

Whole-mount cell preparations of cultured rat 3Y1 cells were examined by stereo electron microscopy to identify the ultrastructural localization of concanavalin A (Con A) receptors in the plasma membrane, and to clarify the relationship between Con A receptors and cytoskeletal components. Well spread monolayer cells were extracted with saponin, briefly fixed, and then partially broken open with shearing force to facilitate the introduction of antibodies for identification of actin filaments. Stereo electron microscopy of such treated cells revealed a 3-dimensional image of filamentous structures such as fine filaments, microtubules (MT) and endoplasmic reticulum (ER) in the flattened areas of each cell. Just beneath the plasma membrane were meshworks of actin-containing fine filaments, as identified by an immunogold staining method. Microtubules and ER were observed to be either directly or indirectly associated with this meshwork. The broken open part of each cell exhibited a meshwork of filaments which were associated with the cytoplasmic surface of the plasma membrane. Some of the filaments were connected to the plasma membrane either by their ends or by their lateral surfaces. The localization of Con A receptors was examined by binding colloidal gold-labelled Con A to the surface of fixed, saponin-extracted cells. Virtually all gold particles bound externally at the same membrane sites where intracellular actin filaments attached internally. The observations strongly suggest that the distribution of Con A receptors was regulated by the underlying meshwork of actin filaments.     

Interaction of endocytotic vacuoles with the inner nuclear membrane in simian virus 40 entry into CV-1 cell nucleus.

The transfer of endocytosed simian virus 40 (SV40) to the nuclear position was investigated ultrastructurally using cationized ferritin (CF), ferritin labelled concanavalin A (Fer-Con A) and Con A as cell membrane markers. In the cells incubated with these markers and SV40 at 4 degrees C, and then chased for 2 h at 37 degrees C in serum-free medium, ferritin particles representing CF and/or Fer-Con A binding sites were found in vacuoles with SV40. The membrane of some vacuoles seemed to be in contact with the outer nuclear membrane. Several ferritin particles were located in the perinuclear cisterna and within the nucleoplasm, but not within the nuclear pores. In addition, there were vacuoles with ferritin particles and SV40 near the nuclear membrane, which looked like a single diaphragm with heterochromatins inside it. The outer nuclear and vacuole membranes were often obscure in the areas where the vacuole was very close to the diaphragm. In the case of cells incubated with CF, SV40 and Con A at 4 degrees C, chased for 2 h at 37 degrees C, and then reacted with horseradish peroxidase (HRP), HRP activity showing Con A-binding sites was also observed along the nuclear side of the inner nuclear membrane as well as in the perinuclear cisterna along the outer membrane. These results confirm that SV40-induced endocytotic vacuoles fuse with the outer nuclear membrane, and further indicate that some endocytotic vacuoles may well interact directly with the diaphragm, suggesting another path for migration of SV40 into CV-1 cell nuclei besides the path going through the process of fusion of the vacuole membrane with the outer nuclear membrane.     

Surface morphology and agglutinability with concanavalin A in normal and transformed murine fibroblasts

The surface morphology of attached and suspended normal and transformed fibroblasts has been studied with the scanning electron microscope. Normal murine fibroblasts (3T3) grow in vitro with widely extended leading lamellae. During most parts of the cell cycle the surfaces of these cells are practically free of microvilli. When the cells round up for mitosis, their cell surfaces become adorned with many microvilli. In contrast, simian virus 40-transformed fibroblasts (SV3T3) grow more compact, and their cell surfaces remain smooth throughout the life cycle. When confluent 3T3 and SV3T3 cells are suspended with ethylenediaminetetraacetic acid (EDTA) for agglutination assays, similar differences in surface morphology are found: 3T3 cells always bear many microvilli, whereas most SV3T3 cells are essentially free of microvilli. The addition of concanavalin A (Con A) does not influence the surface morphology of the suspended cells. The morphological differences described here may be important for the agglutination process of the normal and transformed 3T3 cells, because they affect the real cell surface area and thus the density of Con A-binding sites.     

Simian virus 40 T-antigen-related cell surface antigen: serological demonstration on simian virus 40-transformed monolayer cells in situ.

Simian virus 40 (SV40)-transformed monolayer cells were analyzed in situ by indirect immunofluorescence microscopy for the postulated cell surface location of SV40 T-antigen-related molecules. With antisera prepared against purified, sodium dodecyl sulfate-denatured SV40 T-antigen, positive surface staining was obtained when the cells had been treated with formaldehyde before immunofluorescence analysis. In contrast, living SV40-transformed cells analyzed in monolayer were surface fluorescence negative. The fixation procedure developed in this study combined with a double staining immunofluorescence technique allowed the simultaneous analysis of the same cells for the expression of both SV40 T-antigen-related surface antigen and nuclear T-antigen. The localization of SV40 T-antigen-related surface antigen on the outer surface of the plasma membrane of formaldehyde-fixed SV40-transformed cells was demonstrated directly by the protein A-mediated binding of Staphylococcus aureus bacteria on formaldehyde-fixed SV40-transformed cells precoated with antiserum against sodium dodecyl sulfate-denatured T-antigen. Both cell surface staining and S. aureus binding were found to be highly specific for SV40 T-antigen-related binding sites. These results indicate that T-antigen-related molecules in a cryptic form are located on the surface of SV40-transformed monolayer cells and can be detected in situ after modification of the cell surface architecture.     

Cytolocalization artifacts with immunofluorescent probes

Formaldehyde fixation, nonionic detergent extraction, and ligand binding are commonly used in conjunction with immunofluorescence microscopy to visualize antigens and lectin-reactive molecules in cytoskeletal preparations. These procedures have the potential to produce serious artifacts in cytolocalization studies, as is shown in the present investigation of wheat-germ agglutinin (WGA) binding and localization in BeWo choriocarcinoma cells. Formaldehyde fixation of intact cells reduced the binding of 125I-labeled WGA by 30% and altered the pattern of staining with fluorescein isothyocyanate (FITC)-WGA. Except for perinuclear sites which were brightly stained, the binding of FITC-WGA to other cell surface regions was significantly decreased. Nonionic detergent extractions had two different effects on lectin binding activity depending on whether or not the cells had been pretreated with lectin. In lectin-pretreated cells, 50% of bound lectin was solubilized by detergent but all of the surface binding sites appeared to be retained in active form in the detergent-insoluble residue. Nuclear-cytoskeletal monolayers prepared from cells that were not lectin pretreated lost considerable binding activity, however. These results suggest that a number of erroneous conclusions can be drawn from studies on cytoskeletal associations with membrane components using immunofluorescence microscopy on fixed and detergent-extracted cells.     

Variations in distribution of con A receptor sites and anionic groups during red blood cell differentiation in the rat

The distribution of receptors for concanavalin A (Con A) and anionic groups on plasma membranes of developing blood cells was investigated in the rat. Glutaraldehyde-fixed bone marrow and circulating blood cells were exposed to ferritin-conjugated Con A or positively chaged ferric oxide (CI) and processed for electro n microscopy. The frequency of Con A and CI binding sites varied during different erythroid developmental stages and amont different leukoid cell types. There was a constant inverse relationship between Con A and CI binding sites. Among leukoid cells, Con A binding was high on plasma cells and macrophages, lower on neutrophils and lymphocytes, and still lower on eosinophils and basophils; CI binding was highest in the latter and lowest in plasma cells and macrophages. Among erythroid cells, there was a progressive increase in Con A and a decrease in CI binding after successive divisions of erythroblasts, and a progressive decrease in Con A and an increase in CI binding upon maturation of the orthochromatic erythroblast to the reticulocyte. The most pronounced modification in distribution of these sites occurred during nuclear expulsion: that protion of the plasma membrane surrounding the extruded nucleus was heavily labeled by Con A (up to four times that of the orthochromatic erythroblast) whereas the reticulocyte had considerably fewer sites. The situation was reversed with CI. The results suggest that the concentration of nonsialated glycoproteins (to which Con A binds) varies inversely to that of sialoproteins (to which CI binds) in the membrane of the differentiating erythroid cell. The remarkable changes observed at the time of nuclear extrusion suggest that there is either local modification of glycosylated groups with removal of sialyl residues from the membrane surrounding the extruded nucleus of selective segregation of membrane glycoproteins leading to a high concentration of sialoproteins (glycophroin) in the membrane of the mature erythrocyte.     

Effects of plant lectins on the adhesive properties of baby hamster kidney cells

We studied the effects of different lectins on the adhesive properties of baby hamster kidney (BHK) cells. The purpose of these studies was to learn more about the cell surface receptors involved in cell adhesion. Three adhesive phenomena were analyzed: 1) the adhesion of BHK cells to lectin-coated substrata; 2) the effects of lectins on the adhesion of cells to substrata coated by plasma fibronectin (pFN); and 3) the effects of lectins on the binding of pFN-coated beads to cells. Initial experiments with fluorescein-conjugated lectins indicated that concanavalin A (Con A), ricinus communis agglutinin I (RCA I), and wheat germ agglutinin (WGA) bound to BHK cells but peanut agglutinin (PNA), soybean agglutinin (SBA), and ulex europaeus agglutinin I (UEA I) dod not bind. All three of the lectins which bound to the cells promoted cell spreading on lectin substrata, and the morphology of the spread cells was similar to that observed with cells spread on pFN substrata. Protease treatment of the cells, however, was found to inhibit cell spreading on pFN substrata or WGA substrata more than on Con A substrata or RCA I substrata. In the experiment of cells with Con A or WGA inhibited cell spreading on pFN substrata, but RCA I treatment had no effect. Finally, treatment of cells with WGA inhibited binding to cells of pFN beads, but neither Con A nor RCA I affected this interaction. These results indicate that the lectins modify cellular adhesion in different ways, probably by interacting with different surface receptors. The possibility that the pFN receptor is a WGA receptor is discussed.     

Differences in the redistribution of concanavalin A and wheat germ agglutinin binding sites on mouse neuroblastoma cells

Concanavalin A (Con A), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA) bound with either ¹²⁵I, fluorescent dyes, or fluorescent polymeric microspheres were used to quantitate and visualize the distribution of lectin binding sites on mouse neuroblastoma cells. As viewed by fluorescent light and scanning electron microscopy, over 10⁷ binding sites for Con A, WGA, and RCA appeared to be distributed randomly over the surface of differentiated and undifferentiated cells. An energy-dependent redistribution of labeled sites into a central spot occurred when the cells were labeled with a saturating dose of fluorescent lectin and maintained at 37°C for 60 min. Reversible labeling using appropriate saccharide inhibitors indicated that the labeled sites had undergone endocytosis by the cell. A difference in the mode of redistribution of WGA or RCA and Con A binding sites was observed in double labeling experiments. When less than 10% of the WGA or RCA lectin binding sites were labeled, only these labeled sites appeared to be removed from the cell surface. In contrast, when less than 10% of the Con A sites were labeled, both labeled and unlabeled Con A binding sites were removed from the cell surface. Cytochalasin B uncoupled the coordinate redistribution of labeled and unlabeled Con A sites, suggesting the involvement of microfilaments. Finally, double labeling experiments employing fluorescein-tagged Con A and rhodamine-tagged WGA indicate that most Con A and WGA binding sites reside on different membrane components and redistribute independenty of each other.     

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.     

EFFECTS OF COLCHICINE, CYTOCHALASIN B, AND 2-DEOXYGLUCOSE ON THE TOPOGRAPHICAL ORGANIZATION OF SURFACE-BOUND CONCANAVALIN A IN NORMAL AND TRANSFORMED FIBROBLASTS

The distribution of surface-bound concanavalin A on the membranes of 3T3, and simian virus 40-transformed 3T3 cultured mouse fibroblasts was examined using a shadow-cast replica technique with a hemocyanin marker. When cells were prefixed in paraformaldehyde, the binding site distribution was always random on both cell types. On the other hand, labeling of transformed cells with concanavalin A (Con A) and hemocyanin at 37°C resulted in the organization of Con A binding sites (CABS) into clusters (primary organization) which were not present on the pseudopodia and other peripheral areas of the membrane (secondary organization). Treatment of transformed cells with colchicine, cytochalasin B, or 2-deoxyglucose did not alter the inherent random distribution of binding sites as determined by fixation before labeling. However, these drugs produced marked changes in the secondary (but not the primary) organization of CABS on transformed cells labeled at 37°C. Colchicine treatment resulted in the formation of a caplike aggregation of binding site clusters near the center of the cell, whereas cytochalasin B and 2-deoxyglucose led to the formation of patches of CABS over the entire membrane, eliminating the inward displacement of patches observed on untreated cells. The distribution of bound Con A on normal cells (3T3) at 37°C was always random, in both control and drug-treated preparations. Pretreatment of cells with Con A enhanced the effect of colchicine on cell morphology, but inhibited the morphological effects of cytochalasin B. The mechanisms that determine receptor movement and disposition are discussed.     

Receptor mobility and the binding of cells to lectin-coated fibers

The ability of cells to bind to nylon fibers coated with lectin molecules interspaced with varying numbers of albumin molecules has been analyzed. The cells used were lymphoma cells, normal lymphocytes, myeloid leukemia cells, and normal and transformed fibroblasts, and the fibers were coated with different densities of concanavalin A or the lectins from soybean or wheat germ. Cells fixed with glutaraldehyde did not bind to lectin-coated fibers. The number of cells bound to fibers could be increased by increasing the density of lectin molecules on the fiber, the density of specific receptors on the cell, or the mobility of the receptors. It is suggested that binding of cells to fibers involves alignment and binding of specific cell surface receptors with lectin molecules immobilized on the fibers, and that this alignment requires short-range rapid lateral mobility (RLM) of the receptors. The titration of cell binding to fibers coated with different densities of lectin and albumin has been used to measure the relative RLM of unoccupied cell surface receptors for the lectin. The results indicate a relationship of RLM to lectin-induced cell-to-cell binding. The RLM or receptors for concanavalin A (Con A) was generally found to be higher than that of receptors for the lectins from wheat germ or soybean. Receptor RLM could be decreased by use of metabolic inhibitors or by lowering the temperature. Receptors for Con A had a lower RLM on normal fibroblasts than on SV40-transformed fibroblasts, and trypsinization of normal fibroblasts increased Con A receptor RLM. Normal lymphocytes, lymphoma cells, and lines of myeloid leukemia cells that can be induced to differentiate had a high receptor RLM, whereas lines of myeloid leukemia cells that could not be induced to differentiate had a low receptor RLM. These results suggest that the RLM of Con A receptors is related to the transformation of fibroblasts and the ability of myeloid leukemia cells to undergo differentiation     

Fusion of SV40-induced endocytotic vacuoles with the nuclear membrane

The interaction between simian virus 40(SV40)-induced endocytotic vacuoles and the nuclear membrane was investigated using cationized ferritin (CF) and concanavalin A (Con A) as cell membrane markers. These markers bound to the cell surfaces of CV-1 cells together with SV40 at 4 degrees C. Following incubation of these modified cells at 37 degrees C in serum-free medium, the cell membranes showed many invaginations. After incubation for 60 min at 37 degrees C in the same medium, many various-sized vacuoles were present that contained membrane-bound CF, Con A and SV40. After 2 h of incubation at 37 degrees C, Con A was present in some areas of the perinuclear cisterna along the nuclear membrane. The control experiment, however, showed no localization of Con A-binding on the nuclear membrane. These results provide evidence that SV40-induced endocytotic vacuoles migrate toward the nucleus and fuse with its membrane.     

Rescue of infectious virus from permissive monkey cells containing simian virus 40 DNA fragments.

Permissive TC7 cells were separately transfected with simian virus 40 (SV40) EcoRI/Hap II A (74% genome) DNA fragments and EcoRI/Hap II B (26% genome) DNA fragments in the presence of DEAE-dextran. Fusion of the progeny of recipient cells receiving the A fragment, TC7 (SV40/74) cells, with TC7 (SV40/26) cells, which had received the B fragment, resulted in SV40 rescue. TC7 (SV40/74 + 26) cells, which had simultaneously received both complementary subgenomes, either spontaneously produced SV40 upon subculture or yielded virus upon treatment with iododeoxyuridine. In addition, fusion of rat cells containing the EcoRI/Hap II A fragment with TC7 (SV40/26) cells resulted in SV40 rescue. Cytopathology, V-antigen production, neutralization, and electron microscopy were parameters used to verify that the rescued virus was SV40. No infectious virus was produced when the combinations of cells fused did not total a complete SV40 genome equivalent.     

Electron microscopy of SV40 DNA cross-linked by anti-Z DNA IgG.

Electron microscopy has revealed the specific binding of bivalent anti-Z DNA immunoglobulin G (IgG) to different sites on supercoiled Form I SV40 DNA. The anti-Z IgG links together left-handed regions located within individual or on multiple SV40 DNA molecules at the superhelix density obtained upon extraction. Velocity sedimentation, electrophoresis, and electron microscopy all show that two or more Z DNA sites in the SV40 genome can be intermolecularly cross-linked with bivalent IgG into high mol. wt. complexes. The formation and stability of the intermolecular antibody-DNA complexes are dependent on DNA superhelix density, as judged by three criteria: (1) relaxed circular (Form II) DNA does not react; (2) release of torsional stress by intercalation of 0.25 microM ethidium bromide removes the antibody; and (3) linearization with specific restriction endonucleases reverses antibody binding and DNA cross-linking. Non-immune IgG does not bind to negatively supercoiled SV40 Form I DNA, nor are complexes observed in the presence of competitive synthetic polynucleotides constitutively in the left-handed Z conformation; B DNA has no effect. Using various restriction endonucleases, three major sites of anti-Z IgG binding have been mapped by electron microscopy to the 300-bp region containing nucleotide sequences controlling SV40 gene expression. A limited number of minor sites may also exist (at the extracted superhelix density).     

Microtubule regulation of cell surface receptor topography during granulosa cell differentiation

Abstract. A possible role for cytoplasmic microtubules in modulating lectin binding site topography has been examined during the hormone-directed differentiation of rat ovarian granulosa cells in vitro. Indirect immunofluorescence staining with anti-tubulin antibodies indicates that undifferentiated cultured granulosa cells contain a network of microtubules which radiate from the cell center to the cell periphery. Cultures induced to differentiate by a three day treatment with 1 μg/ml prolactin exhibit a marginal distribution of microtubules and a centrally-located primary cilium. Prolactin enhances the incidence of granulosa cells containing a primary colium from 9% in undifferentiated cultures to 53% in hormone-treated cultures. The pattern of lectin binding site redistribution induced by Concanavalin A (Con A) is also modified by prolactin treatment. In contrast to undifferentiated cells, which randomly endocytose fluorescein Con A, granulosa cells exposed to prolactin respond to fluorescein Con A by forming central surface caps to a greater extent (75%) than undifferentiated controls (25%). Double label fluorescence microscopy and transmission electron microscopy on Con A labeled cells show that caps form at central cell surface sites which contain the primary cilium. Disruption of cytoplasmic microtubules by colchicine, in undifferentiated granulosa cells, results in the formation of cell surface caps upon Con A addition. These data suggest that cytoplasmic microtubules modulate the topography of lectin bindings sites which is subject to hormonal control during the in vitro differentiation of ovarian granulosa cells.     

Microtubule regulation of cell surface receptor topography during granulosa cell differentiation

A possible role for cytoplasmic microtubules in modulating lectin binding site topography has been examined during the hormone-directed differentiation of rat ovarian granulosa cells in vitro. Indirect immunofluorescence staining with anti-tubulin antibodies indicates that undifferentiated cultured granulosa cells contain a network of microtubules which radiate from the cell center to the cell periphery. Cultures induced to differentiate by a three day treatment with 1 microgram/ml prolactin exhibit a marginal distribution of microtubules and a centrally-located primary cilium. Prolactin enhances the incidence of granulosa cells containing a primary cilium from 9% in undifferentiated cultures to 53% in hormone-treated cultures. The pattern of lectin binding site redistribution induced by Concanavalin A (Con A) is also modified by prolactin treatment. In contrast to undifferentiated cells, which randomly endocytose fluorescein Con A, granulosa cells exposed to prolactin respond to fluorescein Con A by forming central surface caps to a greater extent (75%) than undifferentiated controls (25%). Double label fluorescence microscopy and transmission electron microscopy on Con A labeled cells show that caps form at central cell surface sites which contain the primary cilium. Disruption of cytoplasmic microtubules by colchicine, in undifferentiated granulosa cells, results in the formation of cell surface caps upon Con A addition. These data suggest that cytoplasmic microtubules modulate the topography of lectin bindings sites which is subject to hormonal control during the in vitro differentiation of ovarian granulosa cells.     

Dynamic cytoskeleton-integrin associations induced by cell binding to immobilized fibronectin

We have examined the early events of cellular attachment and spreading (10-30 min) by allowing chick embryonic fibroblasts transformed by Rous sarcoma virus to interact with fibronectin immobilized on matrix beads. The binding activity of cells to fibronectin beads was sensitive to both the mAb JG22E and the GRGDS peptide, which inhibit the interaction between integrin and fibronectin. The precise distribution of cytoskeleton components and integrin was determined by immunocytochemistry of frozen thin sections. In suspended cells, the distribution of talin was diffuse in the cytoplasm and integrin was localized at the cell surface. Within 10 min after binding of cells and fibronectin beads at 22 degrees C or 37 degrees C, integrin and talin aggregated at the membrane adjacent to the site of bead attachment. In addition, an internal pool of integrin-positive vesicles accumulated. The mAb ES238 directed against the extracellular domain of the avian beta 1 integrin subunit, when coupled to beads, also induced the aggregation of talin at the membrane, whereas ES186 directed against the intracellular domain of the beta 1 integrin subunit did not. Cells attached and spread on Con A beads, but neither integrin nor talin aggregated at the membrane. After 30 min, when many of the cells were at a more advanced stage of spreading around beads or phagocytosing beads, alpha-actinin and actin, but not vinculin, form distinctive aggregates at sites along membranes associated with either fibronectin or Con A beads. Normal cells also rapidly formed aggregates of integrin and talin after binding to immobilized fibronectin in a manner that was similar to the transformed cells, suggesting that the aggregation process is not dependent upon activity of the pp60v-src tyrosine kinase. Thus, the binding of cells to immobilized fibronectin caused integrin-talin coaggregation at the sites of membrane-ECM contact, which can initiate the cytoskeletal events necessary for cell adhesion and spreading.     

The solubility characteristics of SV 40 tumor antigen

The solubility characteristics of SV40 tumor (T) antigen were investigated. Cell nuclei were isolated from cover glass cultures of SV 40-infected or transformed cells by detergent treatment. When treated in a buffered isotonic sucrose solution, the isolated nuclei contained typical SV40 T antigen by immunofluorescence. If the detergent was dissolved in an isotonic electrolyte solution containing 0.15 M monovalent cations (K+), the T antigen was extracted from the nuclei. Complement fixation tests demonstrated that the various solutions had no deleterious effects on the antigenicity of T antigen. Under identical conditions, the SV 40 capsid (V) antigen was not extracted from infected cell nuclei. These solubility characteristics place SV40 T antigen in the class of “loosely bound” nuclear proteins which includes non-histone proteins that appear to have cell regulatory functions. This supports the suggestion that the biological function of T antigen may be regulatory in nature.