Transmembrane Linkage of Fibronectin to Intracellular Actin‐containing Filaments in Cultured Human Fibroblasts*

Heggeness, Michael H.; Ash, J. F.; Singer, S. J.

doi:10.1111/j.1749-6632.1978.tb16822.x

Cited by 115 publications

(54 citation statements)

References 9 publications

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“…Furthermore, the correlation between a disruption of the microfilament network of spread fibroblasts by CB and the subsequent, although not immediate, loss of the fibronectin network suggests that the maintenance and possible establishment of the fibronectin matrix may be dependent on the presence of the microfilament bundles. This hypothesis is supported by studies which show the colinearity of these two structural networks (15,17,26) and suggest linkage through the membrane via specific membrane proteins (5,27). Although the loss of substrate adhesion in transformed cells has been attributed both to a loss of fibronectin matrix (21) and to an alteration of microfilament bundles (7,25,29), neither of these transformationrelated changes accounts for the difference in morphology of normal and RSV-transformed cells in CB.…”

Section: Resultssupporting

confidence: 52%

“…Also, the addition of fibronectin to chondroblast floater cells induces both attachment and spreading of the cells (32). Cooperative effects of actin and fibronectin in the determination of cell shape have been suggested by double immunofluorescence labeling experiments which demonstrate that the fibronectin fibers are coincident with microfilament bundles within the cell (15,17,26). It has been postulated that the fibronectin fibers may be cross-linked to the microfilament bundles via…”

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confidence: 99%

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Altered cell spreading in cytochalasin B: a possible role for intermediate filaments.

Menko¹,

Toyama²,

Boettiger³

et al. 1983

Mol. Cell. Biol.

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Trypsinized chicken embryo dermal fibroblasts plated in the presence of cytochalasin B (CB) quickly attached to the substrate and within 24 h obtained an arborized morphology. This morphology is the result of the pushing out of pseudopodial processes along the substrate from the round central cell body. There were no microfilament bundles in the processes of these cells plated in the presence of CB; however, the processes were packed with highly oriented, parallelaligned intermediate filaments. Rous sarcoma virus which is temperature sensitive for pp60src, formed arborized cells with properties similar to those of uninfected fibroblasts when plated in the presence of CB at the nonpermissive temperature (41°C). At the permissive temperature for transformation (36°C), the cells attached to the substrate but remained round. These round cells were not only deficient in microfilament bundles but also lacked the highly organized intermediate filaments found in the processes of the arborized cells at 41°C. Although both microfilament bundles and the fibronectin matrix were decreased after transformation with Rous sarcoma virus, neither was involved in the formation of processes in normal cells plated in CB. Therefore, the inability of the transformed cells to form or maintain processes in CB must be the result of another structural alteration in the transformed cells, such as that of the intermediate filaments.When trypsinized fibroblasts are plated in culture, they attach to and spread on the substrate within several hours. This spreading process involves cell movement and is accompanied by the establishment of an actin-containing microfilament network within the cytoplasm just under the plasma membrane. These microfilaments are implicated as an essential element in cell spreading (13) and are required for many cell movements, including cytokinesis and translational movement. The extracellular matrix proteins are another major element in cell spreading, particularly fibronectin, which is laid down as a network on the substrate under the spreading cell (see reference 33 for a review). There are conflicting data on the association of the fibronectin component of the extracellular matrix with the microfilaments and the microfilamentassociated proteins. One line of evidence indicates that the fibronectin matrix is present all along the plasma membrane leading up to but not including the area of adhesion plaques (5), whereas another group of investigators have evidence of a transmembrane linkage of fibronectin and vinculin fibers at the adhesion plaques (27). These fibronectin-vinculin complexes are aligned with the microfilament bundles found along the substrate within the cell.The role of fibronectin in cell spreading is demonstrated by the ability of exogenously added fibronectin to increase the spreading of poorly spread, transformed cells (34). Also, the addition of fibronectin to chondroblast floater cells induces both attachment and spreading of the cells (32). Cooperative effects of actin and fibronectin in the det...

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Section: Resultssupporting

confidence: 52%

mentioning

confidence: 99%

Altered cell spreading in cytochalasin B: a possible role for intermediate filaments.

Menko¹,

Toyama²,

Boettiger³

et al. 1983

Mol. Cell. Biol.

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show abstract

“…In this manner, parallel arrays of fibronectin filaments on the exterior cell surface and microfilaments on the interior would be linked together. This could provide one way (ECM-2 sites providing another, see below) to account for immunofluorescent (41,44) and immuno¢lectron microscopic (56) observations of colinear distributions of fibronectin and actin bundles lying parallel to fibroblast membranes, at either the ventral or dorsal surfaces. On the exterior cell surface at FA sites, some unknown serum factor (62) and/or some non-fibronectin fibrous component (61) (Fig.…”

Section: On the Transmembrane Ultrastructure Of Cellsubstratum Contacmentioning

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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“…Because fibronectin (FN) is a pericellular glycoprotein that apparently mediates the adhesion of cells to substrates (25,36,39,50), and because it is coincident with actin microfilament bundles on the ventral cell surface (30,33), it was reasonable to hypothesize that FN is a constituent of focal contacts (33). In addition, a very close (8-20 nm) transmembrane relationship between individual fibronectin fibrils and actin microfilaments termed the fibronexus has been demonstrated with electron microscopy (44).…”

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Association of fibronectin and vinculin with focal contacts and stress fibers in stationary hamster fibroblasts.

Singer¹

1982

The Journal of Cell Biology

139

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We have recently observed a transmembrane association between extracellular fibronectin (FN) fibers and elongated focal patches or fibers of vinculin (VN) in G,-arrested stationary Nil 8 hamster fibroblasts, with double-label immunofluorescence microscopy (Singer and Paradiso, 1981, Cell . 24:481-492) . We hypothesized that these FN-VN complexes might correspond to focal contacts, the membrane sites that are probably mainly responsible for attaching cells to their substrata, because vinculin is often localized in focal contacts . However, because fibronectin-vinculin associations may not be restricted to the substrate adhesive surface of the cell, it became necessary to determine whether some or all of the various kinds of FN-VN complexes which we described are in proximity to the substrate. Using interference reflection optics and double-label immunofluorescence microscopy for fibronectin and vinculin, many elongated (up to 38 p.m) FN-VN associations were found to be strikingly coincident with focal contacts in the perinuclear area of extremely flattened arrested Nil 8 fibroblasts in 0.3% fetal bovine serum (FBS) . In addition, the long FN-VN adhesion complexes were precisely aligned with the major phase-dense stress fibers observed at the ventral surfaces of these stationary cells with phase contrast microscopy . Fibronectin was neither associated with vinculin-containing focal contacts of Nil 8 cells cultured in medium with 5% FBS nor with vinculin-negative focal contacts located at the extreme edges of stationary cells arrested in 0.3% FBS. Our time-course experiments suggest that early FN-VN lacking-focal contacts, which form at the cellular margins, develop into mature substrate adhesion complexes containing both fibronectin and vinculin, localized in the major stress fibers at the centers of sessile fibroblasts .

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Transmembrane Linkage of Fibronectin to Intracellular Actin‐containing Filaments in Cultured Human Fibroblasts*

Cited by 115 publications

References 9 publications

Altered cell spreading in cytochalasin B: a possible role for intermediate filaments.

Altered cell spreading in cytochalasin B: a possible role for intermediate filaments.

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

Association of fibronectin and vinculin with focal contacts and stress fibers in stationary hamster fibroblasts.

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