Cell surface-associated structural proteins in connective tissue cells.

Börnstein, Paul; Ash, J. F.

doi:10.1073/pnas.74.6.2480

Cited by 117 publications

(45 citation statements)

References 39 publications

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“…The mechanism of these two effects is unknown. Fibroblast cultures contain fibrils which stain for both FN and collagen when examined by immunofluorescence microscopy (17). The basis for this congruence of location may be the recently described noncovalent interactions of FN with other FN molecules (44) and with collagen (18).…”

Section: Discussionmentioning

confidence: 99%

Synthesis of fibronectin by cultured human endothelial cells.

Jaffe¹,

Mosher²

1978

The Journal of Experimental Medicine

448

113

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Plasma fibronectin is probably the major nonimmune particulate opsonin in blood and is cross-linked to fibrin during the final stage of blood coagulation. Fibronectin also occurs in an insoluble form in basement membranes especially those underlying endothelial cells and in loose connective tissue. Fibronectin was demonstrated in cultured human endothelial cells and in the surrounding extracellular matrix by immunofluorescence microscopy by using antibody to human plasma fibronectin. Cultured human endothelial cells released fibronectin into the culture medium which was immunologically identical to the fibronectin in human plasma. Cultured human endothelial cells were labeled with [3H] leucine. The radioactive fibronectin present in the endothelial postculture medium and in urea extracts of cellular monolayers was isolated with either anti-fibronectin coupled to Protein A-Sepharose or double antibody immunoprecipitation and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When reduced, the [3H] fibronectin synthesized by cultured endothelial cells had the same mol wt (approximately 200,000) as plasma fibronectin. Unreduced, the [3H] fibronectin synthesized by endothelial cells migrated as a dimer, as did plasma fibronectin. Fibronectin accounted for approximately 15% of the protein synthesized and released by endothelial cells into the culture medium. Thus, cultured endothelial cells synthesize fibronectin, secrete it into the culture medium, and incorporate it into extracellular matrix. The results suggest that the endothelial cell is potentially a major site of synthesis of circulating plasma fibronectin. In addition, fibronectin derived from endothelial cells may be an important structural component of the subendothelium.

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Section: Discussionmentioning

confidence: 99%

Synthesis of fibronectin by cultured human endothelial cells.

Jaffe¹,

Mosher²

1978

The Journal of Experimental Medicine

448

113

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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: 99%

“…However, ECM cables such as are involved at these sites most likely contain other fbrous components besides fibronectin, such as glycosaminoglycans (61) and collagen (9,51,68), which may be more directly bound to the external surface of the cell at those sites; the involvement of fibronectin at these ECM-1 sites could therefore be totally indirect (see above, Fig. 9).…”

Section: Localization Of Fibronectinmentioning

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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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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“…Fibronectin may have provided a template for the proper deposition of GP140 through noncovalent interactions, followed by the intermolecular disulfide bonding of matrix GP140 . A number of reports (4,5,7,(31)(32)(33) have indicated that type I and III collagens and/or procollagens also co-distribute with fibronectin in the extracellular matrix .…”

Section: Assembly Of Gp140 In the Extracellular Matrixmentioning

confidence: 99%

The role of intermolecular disulfide bonding in deposition of GP140 in the extracellular matrix.

Carter¹

1984

The Journal of Cell Biology

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Human WI-38 fibroblasts in cultures synthesized at least three molecular forms of the major, extracellular matrix glycoprotein (GP), GP140 : (a) cytoplasmic GP140 (1 .2 ng of GPl40/pg of cell protein) was detergent-soluble, underglycosylated, and possessed detectable levels of intermolecular disulfide bonding ; (b) matrix GP140 (3.6 ng of GP140/Ag of cell protein) was detergent-insoluble, more highly glycosylated and polymerized by intermolecular disulfide bonding, and co-distributed in the extracellular matrix with fibronectin ; and (c) released GP140 (2 ng of GP140/Ag of cell protein per 24 h) was recovered in the conditioned culture media and lacked intermolecular disulfide bonding . Cytoplasmic GP140 was the immediate biosynthetic precursor of the matrix form of GP140 . In addition, various human adult and fetal tissues contained a form of GP140 that resembled the fibroblast matrix GP140 in the degree of intermolecular disulfide bonding, relative molecular mass, and immunological reactivity. Analysis of the sequence of events in assembly of GP140 and fibronectin in the extracellular matrix detected the following : (a) fibronectin was first to appear in the extracellular matrix; (b) GP140 accumulated in the cytoplasm, then deposited in the extracellular matrix and co-aligned with the established fibronectin ; and (c) maturation of the extracellular matrix proceeded by continued intermolecular disulfide bonding. To evaluate possible roles for intermolecular disulfide bonding in cell interactions, a unique assay system was utilized based on the ability of labeled cells to incorporate radioactive matrix components into a biotinylated exogenous matrix. Precipitation of the biotinylated matrix from extracts of the cultures using avidin indicated : (a) disulfide bonding of radioactive GP140 and fibronectin into the exogenous biotinylated matrix required cell contact with the matrix. The newly deposited GP140 and fibronectin derived from the cells and not from GP140 and fibronectin present in the conditioned culture media. (b) Pro-a1 and Pro-a2 procollagens, present in the culture media, bound to the exogenous matrix in a noncovalent manner and were independent of cell contact . (c) SV40 transformed cells (WI-38 VA13) synthesized released form GP140 but did not deposit GP140 into the biotinylated matrix .

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Cell surface-associated structural proteins in connective tissue cells.

Abstract: Collagen and/or procollagen was demonstrated on the surface of monolayers of fibroblasts from normal rat kidney by indirect immunofluorescence with affinity-purified antibodies to collagen.

Cited by 117 publications

References 39 publications

Synthesis of fibronectin by cultured human endothelial cells.

Synthesis of fibronectin by cultured human endothelial cells.

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

The role of intermolecular disulfide bonding in deposition of GP140 in the extracellular matrix.

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