Use of biocarrier beads and flow cytometry for single-cell studies of fibronectin gene regulation in dibutyrl cyclic AMP reverse transformed CHO-K1 cells

The protease sensitivity of a number of cell surface or cytoskeletal components and the relationship of these to the substratum in attached cells has prevented the quantitative measurement of their expression by flow cytometry. Using traditional cell sorting techniques, cells must be treated with a protease to detach them from a substrate in order to produce a single-cell suspension. Unfortunately, proteolytic treatment alters or destroys a number of cellular proteins. Fibronectin either on the cell surface or as part of the substratum laid down by the cell is particularly sensitive to proteases, preventing its quantitative study by flow cytometry. To circumvent these problems and produce a single cell suspension necessary for flow cytometric analysis, CHO-K1, a Chinese hamster ovary cell line, were grown in suspension on specially-treated 25 μm biocarrier beads. The CHO-K1 cell line is composed of transformed epithelial-like cells that have lost the fibronectin deposit around their cell membranes. To restore the typical fibroblastic deposit of fibronectin, the cells attached to beads were induced by dibutyrl cAMP to undergo a reverse transformation reaction to restore fibroblastic morphology and the typical fibroblastic deposite of fibronectin on the cell surface and substratum. The cells attached to beads were then immunofluorescently labeled for the protease-sensitive, extracellular matrix component, fibronectin, and examined on a flow cytometer. Cell surface fibronectin heterogeneity was then examined on a cell-by-cell basis as a function of cell cycle using Hoechst 33342 dye that binds to AT base pairs of cellular DNA. Dual laser measurement and multiparameter list mode data analysis were used to study the relationship between cell surface fibronectin of biocarrier bead attached cells and cell cycle.     

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.     

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.     

Fucan effect on CHO cell proliferation and migration

Fucan is a term used to denominate sulfated l-fucose rich polysaccharides. Here, a heterofucan, named fucan B, was extracted from the Spatoglossum schröederi seaweed. This 21.5 kDa galactofucan inhibited CHO-K1 proliferation and migration when fibronectin was the substrate. Fucan B derivatives revealed that such effects depend on their degree of sulfation. Fucan B did not induce cell death, but promoted G₁ cell cycle arrest. Western blotting and flow cytometry analysis suggest that fucan B binds to fibronectin and activates integrin, mainly integrin α5β1, which induces FAK/RAS/MEK/ERK activation. FAK activation inhibits CHO-K1 migration on fibronectin and ERK blocks cell cycle progression. This study indicates that fucan B could be applied in developing new antitumor drugs.     

Contact formation by fibroblasts adhering to heparan sulfate-binding substrata (fibronectin or platelet factor 4)

The process of cell-substratum adhesion of BALB/c 3T3 fibroblasts on fibronectin (FN)-coated substrata was compared with that of cells adhering to substrata coated with the heparan sulfate (HS)-binding protein, platelet factor four (PF4). FN has binding domains for HS and an unidentified cell surface receptor, whereas PF4 binds to only HS on the surface of the cell. The attachment and early spreading sequences of cells on either substratum were similar as shown by scanning electron microscopy (SEM). Within 2 h of spreading, cells on FN developed typical fibroblastic morphologies, whereas those on PF4 lacked polygonal orientations and formed numerous broadly spread lamellae. Interference reflection microscopic analysis indicated that PF4-adherent cells formed only close adhesive contacts, whereas FN-adherent cells formed both close contacts and tight focal contacts. Cells on either substratum responded to Ca2+ chelation with EGTA by rounding up, but remained adherent to the substratum by relatively EGTA-resistant regions of the cell's undersurface, demonstrating that cell surface HS by binding to an appropriate substratum is capable of initiating a Ca2+-dependent spreading response. The EGTA-resistant substratum-attached material on PF4 was morphologically similar to that on FN, the latter of which was derived from both tight focal contacts and discrete specializations within certain close contacts. These studies show that heparan sulfate proteoglycans on the surface of these cells can participate in the formation of close contact adhesions by binding to an appropriate substratum and suggest that sub-specializations within close contact adhesions may evolve into tight focal contacts by the participation of an unidentified cell surface receptor which binds specifically to fibronectin but not to PF4. In addition, the functional role of FN in tight focal contact formation is demonstrated.     

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.     

Concomitant loss of cell surface fibronectin and laminin from transformed rat kidney cells

Both fibronectin and laminin were found by immunofluorescence as a matrix at the surface of normal rat kidney cells. These matrices were absent from the surface of virally transformed rat kidney cells. Soluble fibronectin and laminin were detected in the culture media of the transformed as well as the normal cells. Culture supernates of the transformed cells contained even more fibronectin than the supernates of the transformed cells contained even more fibronectin than the supernates of the normal cells while laminin was present in similar amounts in both culture media. This shows that the loss of fibronectin and laminin from the surface of the transformed cells is caused by failure of the cells to deposit these proteins into an insoluble matrix and not caused by inadequate production. Fibronectins isolated from culture media of the normal and transformed cells were similar in SDS polyacrylamide gel electrophresis. Laminin isolated from culture media by affinity chromatography on heparin-Sepharose followed by immunoprecipitation was composed of three main polypeptides, one with a molecular weight of 400,000 and two with a molecular weight close to 200,000 in both cell types. Fibronectins from both cell types were equally active in promoting cell attachment. Rat fibronectin from transformed cells, like normal cells, when applied to culture dishes coated with fibronectin, readily attached and spread on the substratum, requiring approximately the same amount of fibronectin as the normal cells. On the basis of these results it seem that the failure of the transformed cells to incorporate fibronectin into an insoluble cell surface matix is not a consequence of a demonstrable change in the functional characteristics of the fibronectin molecule or in the ability of the cells to interact with fibronectin. It may depend on as yet unidentified interactions of the cell surface. Similar interactions may be needed for the deposition of laminin into the matrix, because laminin was also absent from the surface of transformed cells, despite its being synthesized by these cells.     

Stages in specialization of fibroblast adhesion and deposition of extracellular matrix

Fibroblast cells seeded on a serum glycoprotein shown previously to mediate a spread shape without focal adhesions or microfilament bundles (Stage 1 spread) are now shown to have substratum contacts in which coated pits are abundant and associated with small globular deposits of glycocalyx bridging to substratum and staining for fibronectin and acidic glycoconjugates. After stimulation with serum or fibronectin to form focal adhesions and microfilament bundles (Stage 2 spread), clathrin-based structures remain at the cell underside but no longer in conspicuously higher concentration than on the dorsal surface; extracellular material at adhesions is now as regular strands which stain for acidic glycoconjugates but (as reported earlier by Chen and Singer) not always for fibronectin. During these stages of adhesion, striking changes are seen in the cellular display of fibronectin monitored by immunofluorescence. In rounded cells this is granular and cytoplasmic, concentrated around the submembranous cortex; on spreading to Stage 1, it remains granular and intracellular but is now oriented strongly towards the lower cell surface; only in Stage 2 does externalisation proceed to deposit fibrillar fibronectin on the substratum. While cytoplasmic orientation of matrix precursors can be determined by cell contact, organised externalisation is therefore coupled to fully developed adhesion status.     

Fluorescent gangliosides as probes for the retention and organization of fibronectin by ganglioside-deficient mouse cells

Ganglioside-deficient transformed mouse fibroblasts (NCTC 2071A cells), which grow in serum-free medium, synthesize fibronectin but do not retain it on the cell surface. When fluorescent derivatives of gangliosides, containing either rhodamine or Lucifer yellow CH attached to the sialic acid residues, were added to the culture medium, the cells incorporated the derivatives and their surfaces became highly fluorescent. When the cells were stained with anti-fibronectin antibodies and a fluorescent second antibody, fibrillar strands of fibronectin were observed to be attached to the cell surface, with partial coincidence of the patterns of direct ganglioside fluorescence and indirect fibronectin immunofluorescence at the cell surface. When the cells were exposed to bacterial neuraminidase during the time of ganglioside insertion, similar patterns of fluorescence were observed. Because the fluorescent gangliosides are resistant to the enzyme, these results suggest that neuraminidase-sensitive endogenous glycoconjugates were not involved in the ganglioside-mediated retention and organization of endogenous fibronectin. After cells were exposed to exogenous chicken fibronectin, most of the fibronectin was attached to the substratum and only a few fibrils were attached to the cells. When exogenous gangliosides were included in the incubation, there was a striking increase in cell-associated exogenous fibronectin, which was highly organized into a fibrillar network. Conversely, cells incubated for 18 h with exogenous unmodified gangliosides exhibited a highly organized network of endogenously derived fibronectin. Upon further incubation of the cells for 2 h with fluorescent gangliosides, there was considerable co-distribution of the fluorescent gangliosides with the fibronectin network as revealed by immunofluorescence. Our results support the concept that gangliosides can mediate the attachment of fibronectin to the cell surface and its organization into a fibrillar network.     

The interaction of fibronectin fragments with fibroblastic cells

We have examined the interaction of the purified cell-binding domain of fibronectin with fibroblastic baby hamster kidney cells. When the cell-binding region of fibronectin is part of a large 75,000-dalton fragment, the direct binding of the tritium-labeled fragment to cells in suspension can be observed. There is a single class of 10(5) sites/cell with an apparent dissociation constant of 4 X 10(-7) M. When the cell-binding region is part of a smaller 11,500-dalton fragment, an interaction with cells can only be observed indirectly via inhibition assays. The apparent affinity of this fragment for the cell surface fibronectin receptor is low. This 11,500-dalton fragment competitively inhibits both the direct binding of soluble [3H]fibronectin to cells in suspension and the spreading of cells on fibronectin-coated substrates, suggesting that the fragment binds to the same receptor site as intact fibronectin. Possible models describing the mechanism of the interaction of fibronectin with its receptor are proposed.     

Cellular growth control: properties of a unique adhering derivative of L5178Y cells

A new cell line derived from L5178Y murine lymphoma cells with unique properties is described. The cells, L5178Y Adh, grow simultaneously in suspension culture and attached to a substratum but not in contact with each other. Limitation of growth of the cells in either the suspended state or attached to substratum is not linked to a limiting surface or cell density in a manner analogous to “contact inhibition.” The adhering and non-adhering cells grow with different doubling times. Cell size analysis indicates that initial gravity settling of the inoculum determines which cells attach. Contact of these cells' external surface with substratum may then initiate events which ensure attachment of adhering populations.     

Association between fibronectin receptor and the substratum: Spare receptors for cell adhesion

We have investigated the association of the recently described 140-kDa cell membrane receptor for fibronectin with the cytoskeleton or with substratum-bound fibronectin. Using a monospecific polyclonal antibody to the 140-kDa receptor, we have demonstrated that most of the receptor molecules are soluble in nonionic detergent either in suspension culture CHO cells or in CHO cells attached to and spread on a fibronectin-coated substratum. This may suggest that putative linkages of the receptor either to fibronectin or to detergent-insoluble cytoskeletal components are labile to nonionic detergent and thus are rather weak. Alternatively, it may mean that only a small fraction of the cell's receptors are needed to mediate adhesion. In order to explore this latter concept, we have coated substrata with various concentrations of PB1, a monoclonal antibody with a high affinity for fibronectin receptor. We demonstrate that coating the substratum with increasing concentrations of PB1 results in increasing amounts of 140-kDa receptor becoming bound to the substratum in detergent-insoluble form. However, the amount of receptor bound does not necessarily correlate with the degree of cell adhesion and spreading. Thus, coating the substratum with 5 μg/ml of PB1 results in essentially complete attachment and spreading of CHO cells, but only a small fraction of the 140-kDa receptor becomes substratum bound. Coating with 50 μg/ml of PB1 produces no further increase in cell adhesion and spreading, but results in the detergent-stable association of a large fraction of the total receptor pool with the substratum. These observations suggest the possibility of there being “spare” receptors for cell adhesion processes.     

Flow cytometric evaluation of cell cycle characteristics during in vitro differentiation of chick embryo chondrocytes

The cell cycle kinetic characteristics of chick endochondral chondrocytes differentiating in vitro were studied by flow cytometry. In addition, the synthesis of type I and type X collagens of the same cells was evaluated by immunoprecipitation. Dedifferentiated cells, derived from chick embryo tibiae and grown attached to a substratum, were characterized by type I collagen synthesis, a high growth fraction (GF = 0.94), minimal cell loss factor (phi = 0.02), and a total cell cycle time of the proliferating cells of about 17 h (tG1 = 8 h, tS = 5 h, and tG2 + M = 4 h). Transfer of dedifferentiated cells to suspension culture on agarose-coated dishes induced differentiation to hypertrophic chondrocytes. These were characterized by type X collagen synthesis, a low growth fraction (GF = 0.52), maximal cell loss factor (phi = 1.0), and a total cell cycle time of the proliferating cells of about 73 h (tG1 = 53 h, tS = 12 h, and tG2 + M = 8 h). The transition from dedifferentiated chondrocytes to hypertrophic chondrocytes was accompanied by large increases of the duration of all the cell cycle phases and of the number of quiescent and degenerating cells. Associated with these alterations in cell cycle kinetics was a switch from type I to type X collagen synthesis. Further preliminary data suggest that the population of differentiating chondrocytes (a state between dedifferentiated and hypertrophic chondrocytes) comprises a heterogeneous population of fast and slow growing cells.     

Transfection of myeloid leukaemia cell lines is distinctively regulated by fibronectin substratum

Gene transfer into haematopoietic cells is a challenging approach. The extracellular matrix component fibronectin has been known to modulate the cell cycle dynamics, viability and differentiation of leukaemia cells. Thus, our aim was to investigate the influence of fibronectin substratum on the liposomal transfection of myeloid leukaemia cell lines. Liposomal transfection was performed with K562 and HL-60 as representative lines of transfection-competent and -incompetent myeloid leukaemia cells, respectively. Flow cytometry analyses were performed to determine transfection efficiency monitored by green fluorescent protein (GFP) expression and to assess cell viability and cell cycle status. Quantitation of GFP gene expression and DNA uptake was assayed by real time PCR. The current data showed that the adhesion to fibronectin deteriorated the transfection of K562 cells. In contrary, it enhanced the delivery of plasmid DNA into HL-60 cells. Correspondingly, the adhesion to fibronectin influenced the transfection efficiency mainly by modulating the intracellular presence of plasmid DNA. The cell cycle and viability which is regulated by fibronectin had a minor impact on the success of gene delivery. This phenomenon may be considered as an important factor which may modulate the potential gene transfer approaches for myeloid leukaemia.     

Quantification and Characterization of Phagocytosis in the Soil Amoeba Acanthamoeba castellanii by Flow Cytometry

Phagocytosis in the common grazing soil amoeba Acanthamoeba castellanii was characterized by flow cytometry. Uptake of fluorescently labelled latex microbeads by cells was quantified by appropriate setting of thresholds on light scatter channels and, subsequently, on fluorescence histograms. Confocal laser scanning microscopy was used to verify the effectiveness of sodium azide as a control for distinguishing between cell surface binding and internalization of beads. It was found that binding of beads at the cell surface was complete within 5 min and 80% of cells had beads associated with them after 10 min. However, the total number of phagocytosed beads continued to rise up to 2 h. The prolonged increase in numbers of beads phagocytosed was due to cell populations containing increasing numbers of beads peaking at increasing time intervals from the onset of phagocytosis. Fine adjustment of thresholds on light scatter channels was used to fractionate cells according to cell volume (cell cycle stage). Phagocytotic activity was approximately threefold higher in the largest (oldest) than in the smallest (newly divided) cells of A. castellanii and showed some evidence of periodicity. At no stage in the cell cycle did phagocytosis cease. Binding and phagocytosis of beads were also markedly influenced by culture age and rate of rotary agitation of cell suspensions. Saturation of phagocytosis (per cell) at increasing bead or decreasing cell concentrations occurred at bead/cell ratios exceeding 10:1. This was probably a result of a limitation of the vacuolar uptake system of A. castellanii, as no saturation of bead binding was evident. The advantages of flow cytometry for characterization of phagocytosis at the single-cell level in heterogeneous protozoal populations and the significance of the present results are discussed.     

Capture of rare cells in suspension with antibody-coated polystyrene beads

A method for the separation of one cell type present in small number from a predominant mixture of cell types using macroscopic polystyrene beads is demonstrated. An antibody specific to murine leukocytes (CD45) was adsorbed to the surface of the beads. Beads and murine hybridoma B cells were placed in test tubes and periodically inverted at fixed time intervals, causing the beads to settle through the suspension under creeping flow conditions. Capture was dependent upon interception: the captured cells must have traveled along streamlines that brought them to within a cell radius of the bead surface. B cells attached to 99-micrometer beads (maximum shear rate 8.1 s-1) were captured with greater efficiency but in lesser quantity than those attached to 170-micrometer beads (maximum shear rate 13.9 s-1). Cell capture unexpectedly reached a plateau in less than 2 h, a phenomenon that appears to involve changes in both the cells and the beads. Capture of cells was effective out to dilutions of 1:10 000 with purity in the captured population of better than 74%. This method allows for the study of physical parameters important for cell attachment and capture as well as for practical separation of rare cells.     

Continuous flow magnetic cell fractionation based on antigen expression level

Cell separation is important in medical and biological research and plays an increasingly important role in clinical therapy and diagnostics, such as rare cancer cell detection in blood. The immunomagnetic labeling of cells with antibodies conjugated to magnetic nanospheres gives rise to a proportional relationship between the number of magnetic nanospheres attached to the cell and the cell surface marker number. This enables the potential fractionation of cell populations by magnetophoretic mobility (MM). We exploit this feature with our apparatus, the Dipole Magnet Flow Fractionator (DMFF), which consists of an isodynamic magnetic field, an orthogonally-oriented thin ribbon of cell suspension in continuous sheath flow, and ten outlet flows. From a sample containing a 1:1 mixture of immunomagnetically labeled (label+) and unlabeled (label-) cells, we achieved an increase in enrichment of the label+ cell fraction with increasing outlet numbers in the direction of the magnetic field gradient (up to 10-fold). The total recovery of the ten outlet fractions was 90.0+/-7.7%. The mean MM of label+ cells increased with increasing outlet number by up to a factor of 2.3. The postulated proportionality between the number of attached magnetic beads and the number of cell surface markers was validated by comparison of MM measured by cell tracking velocimetry (CTV) with cell florescence intensity measured by flow cytometry.     

A comparison of cAMP phosphodiesterases in normal,malignant, and somatic cell hybrids

Hybrids (PCM) between a malignant mouse lymphoma suspension cell line (P388F-36) and a normal Chinese hamster fibroblastic cell line (Ch23) have already been isolated in this laboratory. Investigations were carried out on the cAMP phospodiesterases of the parents and two of these hybrids--PCM2 and PCM3. PCM3 shows a rather unusual growth characteristic in that a considerable proportion of the cells exist at any one time either in suspension or only loosely attached to the substratum, the remaining cell population existing in a monolayer form. It was found that each cell line exhibited multiple forms of the enzyme with varying affinities for cAMP. Both parents, although different, contained high-, low-, and extra-high apparent Km forms of the enzyme. The hybrids exhibited characteristics of both parental systems but were different from each other. Neither hybrid exhibited a high-Km enzyme, but both exhibited two low-Km forms. There was also a slight variation between monolayer and suspension cells of PCM3 hybrid. An attempt has been made to explain these phenomena with respect to hybridization and the growth characteristics of the cells.     

Changes in the distribution of a major fibroblast protein, fibronectin, during mitosis and interphase

The distribution of a major fibroblast protein, fibronectin, was studied by immunofluorescence and immunoscanning electron microscopy in cultures of human and chicken fibroblasts during different phases of the cell cycle. The main findings were: (a) In interphase cells, the intensity of surface-associated fibronectin fluorescence correlated with that of intracellular fibronectin fluorescence. (b) The intensity of the fluorescence of both surface-associated and intracellular fibronectins was not changed in cells that were synthesizing DNA. (c) Mitotic cells had reduced amounts of surface-associated but not of intracellular fibronectin. The surface fibronectin that remained on meta-, ana-, or telophase cells had a distinct punctate distribution and was also localized to strands attaching the cells to the substratum. Fibronectin strands first reappeared on the surface of flattening cytoplasmic parts of telophase cells. (d) Fibronectin was also detected in extracellular fibrillar material on the growth substratum, particularly around dividing cells. Thus, surface-associated fibrillar fibronectin was present during G(1), S, and G(2) but in cells undergoing mitosis the distribution was altered and the amount appeared to be reduced. The observations on the distribution of surface-associated fibronectin suggest that rather than being involved in growth control this fibronectin plays a structural role in interactions of cells with the environment.     

Distribution of the cell substratum attachment (CSAT) antigen on myogenic and fibroblastic cells in culture

Previous studies (Neff et al., 1982, J. Cell. Biol. 95:654-666; Decker et al., 1984. J. Cell. Biol. 99:1388-1404) have described a monoclonal antibody (CSAT Mab) directed against a complex of three integral membrane glycoproteins of 120,000-160,000 mol wt (CSAT antigen [ag]) involved in the cell matrix adhesion of myoblasts and fibroblasts. In localization studies on fibroblasts presented here, CSAT ag has a discrete, well-organized distribution pattern. It co-aligns with portions of stress fibers and is enriched at the periphery of, but not directly beneath vinculin-rich focal contacts. In this last location, it co-distributes with fibronectin, consistent with the suggestion that the CSAT ag participates in the mechanism by which fibroblasts attach to fibronectin. In prefusion myoblasts, which are rapidly detached by CSAT Mab, CSAT ag is distributed diffusely as are vinculin, laminin, and fibronectin. After fusion, myotubes become more difficult to detach with CSAT Mab. The CSAT ag and vinculin are organized in a much more discrete pattern on the myotube surface, becoming enriched at microfilament bundle termini and in lateral lamellae which appear to attach myotubes to the substratum. These results suggest that the organization of CSAT ag-adhesive complexes on the surface of myogenic cells can affect the stability of their adhesive contacts. We conclude from the sum of the studies presented that, in both myogenic and fibroblastic cells, the CSAT ag is localized in sites expected of a surface membrane mediator of cell adhesion to extracelluon of CSAT ag-adhesive complexes on the surface of myogenic cells can affect the stability of their adhesive contacts. We conclude from the sum of the studies presented that, in both myogenic and fibroblastic cells, the CSAT ag is localized in sites expected of a surface membrane mediator of cell adhesion to extracellular matrix. The results from studies that use fibroblasts in particular suggest the involvement of CSAT ag in the adhesion of these cells to fibronectin.