Substrate-attached glycoproteins from normal and virus-transformed cells

Terry, Alan H.; Culp, Lloyd A.

doi:10.1021/bi00700a004

Cited by 115 publications

(61 citation statements)

References 24 publications

(22 reference statements)

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“…While this indicates the cables contained fibronectin, it of course does not mean that fibronectin was their only constituent (9,51,61,68). In submembranous regions of ECM sites, a-actinin labeling was generally present, but vinculin labeling occurred less frequently.…”

Section: Immunolabeling Patterns Of Contact Sitesmentioning

confidence: 96%

See 1 more Smart Citation

Immunoelectron microscopic studies of the sites of cell-substratum and cell-cell contacts in cultured fibroblasts.

Chen¹,

Singer²

1982

The Journal of Cell Biology

351

199

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Our object was to obtain information about the molecular structures present at cell-substratum and cell-cell contact sites formed by cultured fibroblasts. We have carried out double irnmunoelectron-microscopic labeling experiments on ultrathin frozen sections cut through such contact sites to determine the absolute and relative dispositions of the three proteins fibronectin, vinculin, and a-actinin with respect to these sites.(a) Three types of cell-substratum and cell-cell contact sites familiar from plastic sections could also be discriminated in the frozen sections by morphological criteria alone, i.e., the gap distances between the two surfaces, and the presence of submembranous densities. These types were: (i) focal adhesions (FA); (ii) close contacts (CC); and (iii) extracellular matrix contacts (ECM). This morphological typing of the contact sites allowed us to recognize and assign distinctive immunolabeling patterns for the three proteins to each type of site on the frozen sections.(b) FA sites were immunolabeled intracellularly for vinculin and a-actinin, with vinculin labeling situated closer to the membrane than a-actinin. Fibronectin was not labeled in the narrow gap between the cell surface and the substratum, or between two cells, at FA sites. Control experiments showed that this could not be ascribed to inaccessibility of the FA narrow gap to the immunolabeling reagents but indicated an absence or severe depletion of fibronectin from these sites.(c) CC sites were labeled intracellularly for a-actinin but not vinculin and were labeled extracellularly for fibronectin.(d) ECM sites were characterized by large separations (often >100 nm) between the cell and substratum or between two cells, which were connected by long cables of extracellular matrix components, including fibronectin. In late (24-36 h) cultures, ECM contacts predominated over the other types. ECM sites appeared to be of two kinds, one labeled intracellularly for both aactinin and vinculin, the other for a-actinin alone.(e) From these and other results, a coherent but tentative scheme is proposed for the molecular ultrastructure of these contacts sites, and specific functional roles are suggested for fibronectin, vinculin, and a-actinin in cell adhesion and in the linkage of intracellular microfilaments to membranes at the different types of contact sites.The nature and molecular structure of the sites of contact of fibroblastic cells with one another and with the substrata on which they grow are subjects of much recent interest. Three major types of investigation have been pursued in this area: (a) at the biochemical level, extracellular matrix molecules such as fibronectin (for review, see reference 42) and intracellular cytoskeletal molecules such as a-actinin (50,55) (12,26) have been isolated and implicated in the formation of such contacts; (b) at the light microscopic level, interference reflection microscopy has been used to define different sites of contact of cells to substrata (20,46,47), and immunofluorescence micro...

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Section: Immunolabeling Patterns Of Contact Sitesmentioning

confidence: 96%

“…It would appear that under our culture conditions there were one or more fibrous components present in the FA gaps but fibronectin was not one of them. These components may include glycosaminoglycans (61).…”

Section: Immunolabeling Patterns Of Contact Sitesmentioning

confidence: 99%

Immunoelectron microscopic studies of the sites of cell-substratum and cell-cell contacts in cultured fibroblasts.

Chen¹,

Singer²

1982

The Journal of Cell Biology

351

199

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“…4), but was not observed with Coomassie staining, and is probably a hyaluronate proteoglycan (6,16). Others of the glucosaminelabeled components that could be tentatively identified are reference polypeptides 2, 3, and 6 shown in Fig.…”

Section: Resultsmentioning

confidence: 93%

Polypeptides of cells transformed by RNA or DNA tumor viruses.

Strand¹,

August²

1977

Proc. Natl. Acad. Sci. U.S.A.

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Proteins solubilized from normal BALB/3T3 cells and BALB/3T3 transformed by simian virus 40 or Kirsten sarcoma virus have been analyzed by two-dimensional gel electrophoresis and tryptic peptide mapping. A large fraction of the polypeptides of the virus-transformed cells, about 30%, were different from normal cells. In contrast to the marked differences between normal and transformed cells, the polypeptides of the DNA and RNA virus-transformed cells were almost identical. These findings were observed with polypeptides stained by Coomassie Blue, or labeled with ['4C]glucosamine or [35Sjmethionine. Pulse-chase analysis showed that most of the polypeptides were stable during 20 hr of incubation. The identity of several polypeptides was confirmed by tryptic peptide mapping. Analysis of the protein content of transformed as compared to normal cells has been directed chiefly at studies of cell surface glycoproteins (for a review see ref. 1). Several proteins are found to be reduced in amount or in their association with the membrane. Chief among these are a glycoprotein of about 250,000 daltons (LETS) and actin (2-4). Other major differences between the glycoproteins of normal and transformed cells have been shown by the binding of iodine-labeled lectins (5), and by cell surface proteins identified as substrate-attached components (6).The present study was initiated to analyze the extent and specificity of change in the protein content of cells following oncogenic transformation. For these studies, we have compared the total cellular proteins of BALB/3T3 cells infected by simian virus 40 (a DNA virus) and the proteins of the same cell transformed by a completely different virus, Kirsten murine sarcoma type-C virus (an RNA virus). The results obtained by twodimensional gel electrophoresis indicate that transformation is accompanied by complex changes in the type, concentration, or properties of a large number of cellular polypeptides and that the great majority of these changes are the same irrespective of whether the cell is transformed by a DNA or RNA tumor virus. MATERIALS AND METHODSCells. The clonal line (AS1) of BALB/3T3 (7), and a derivative subclone K-234, ACi/ml (445 Ci/mmol) and without fetal bovine serum. Following incubation for 1 hr at 370 the monolayers were washed twice with warm growth medium and the cells were then either lysed (pulse) or incubated with 10 ml of growth medium at 370 for various time periods (chase).Preparation of Cell Extracts. Cells were harvested by removing the cell culture fluid and washing the monolayer twice with 10 ml each of warm Dulbecco's phosphate-buffered saline (Gibco). Labeled cells were only washed once. They were suspended in 10 ml of Dulbecco's phosphate-buffered saline containing 1 mM EDTA by incubation at 370 for 5 min, collected by centrifugation at 2000 X g for 5 min, and lysed by suspension in 0.6 ml of extraction buffer [5 mM Tris-HCl, pH 9.2/1 mM EDTA/400 mM KCI/1% (vol/vol) Triton X-100/1 mM phenylmethylsulfonyl fluoride (PMSF)]. The lysed cel...

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“…It is not apparent why 3T3 cells attach more efficiently to SVT2 SAM, even though SAM from normal cells and SAM from transformed cells appear to be highly similar qualitatively (1). Perhaps serum components bind to different types of SAM and this affects the cellular attachment processes; serum components have recently been identified on the surface of the substrate (5).…”

Section: Discussionmentioning

confidence: 99%

“…It has been postulated that substrate-attached material (SAM) plays an important role in adhesion of cells to the substrate, and its study may suggest mechanisms for cell-tocell adhesion (1). This substrate-attached material may be a component of the cellular "microexudates" reported by others (4)(5)(6)(7)(8).…”

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confidence: 99%

Substrate-Attached Glycoproteins Mediating Adhesion of Normal and Virus-Transformed Mouse Fibroblasts

Culp

1974

The Journal of Cell Biology

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118

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When BALB/c 3T3, simian virus 40 (SV40)-transformed 3T3 (SVT2), and revertant variants of the transformed cells are removed by EGTA treatment from the substrate on which they were grown, they leave behind a layer of glycoprotein which has been characterized biochemically (Terry, A. H. and L. A. Culp. 1974. B i o c h e m i s t r y . 13:414.)--substrate-attached material (SAM). The influence of SAM from normal and from transformed cells on cellular attachment to the substrate, morphology, movement, and growth has been examined. All three cell types displayed a 30% higher plating efficiency when grown on 3T3 SAM. The morphology of SVT2 colonies and of individual SVT2 cells was dramatically affected by growth on 3T3 S A M --t h e cells (a) were more highly spread on the substrate, (b) resisted crawling over neighboring cells, and (c) resisted movement away from the edge of colonies; SVT2 S A M was not effective in causing these changes. A ceU-to-substrate attachment assay using thymidine-radiolabeled cells and untreated or SAM-coated cover slips was developed. SVT2 cells attached to 3T3 SAM-or SVT2 SAM-coated cover slips with a faster initial rate and to a higher saturation level than to untreated substrate, whereas 3T3 and revertant cells exhibited no preference; there was no species specificity in these cell-substrate attachment phenomena. Trypsin-released cells attached much more slowly than EGTA-released cells. 3T3 SAM, however, was not effective in lowering the saturation density of mass cultures of virus-transformed cells. These experiments suggest that the substrate-attached glycoproteins of'normal cells affect the cellular adhesivity, morphology, movement, and perhaps growth patterns of virus-transformed cells--i.e., causing partial reversion of these properties of transformed cells to those found in contact-inhibited fibroblasts. A model for the involvement of substrate-attached glycoproteins in cell-to-substrate adhesion, and possibly cell-to-cell adhesion, has been proposed.When normal or SV40-transformed mouse fibroblasts are removed from the plastic or glass substrate on which they are grown by the Ca +÷-specific chelating agent EGTA, 1 they leave a layer Abbreviations used in this paper." con A, concanavalin of glycoprotein firmly bound to the substrate (1, 2). The morphologically flat, contact-inhibited 3T3 A; EDTA, (ethylenedinitrilo) tetraacetic acid; EGTA, ethylenebis (oxyethylenenitrilo) tetraacetic acid; MEM × 4, Eagle's minimal essential medium supplemented

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Substrate-attached glycoproteins from normal and virus-transformed cells

Cited by 115 publications

References 24 publications

Immunoelectron microscopic studies of the sites of cell-substratum and cell-cell contacts in cultured fibroblasts.

Immunoelectron microscopic studies of the sites of cell-substratum and cell-cell contacts in cultured fibroblasts.

Polypeptides of cells transformed by RNA or DNA tumor viruses.

Substrate-Attached Glycoproteins Mediating Adhesion of Normal and Virus-Transformed Mouse Fibroblasts

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