scholarly journals Cell surface distribution of fibronectin and vitronectin receptors depends on substrate composition and extracellular matrix accumulation.

1988 ◽  
Vol 106 (6) ◽  
pp. 2171-2182 ◽  
Author(s):  
I I Singer ◽  
S Scott ◽  
D W Kawka ◽  
D M Kazazis ◽  
J Gailit ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cell Surface ◽  
Cell Attachment ◽  
Cellular Migration ◽  
Surface Distribution ◽  
Fibronectin Receptor ◽  
Substrate Adhesion ◽  
Focal Contacts ◽  
Double Label ◽  
Coated Surfaces

We used antibodies against the alpha subunits of the human fibronectin receptor (FNR) and vitronectin receptor (VNR) to localize simultaneously FNR and VNR at major substrate adhesion sites of fibroblasts and melanoma cells with double-label immunofluorescence microscopy. In early (2-6-h) serum-containing cultures, both FNR and VNR coaccumulated in focal contacts detected by interference reflection microscopy. Under higher resolution immunoscanning electron microscopy, FNR and VNR were also observed to be distributed randomly on the dorsal cell surface. As fibronectin-containing extracellular matrix fibers accumulated beneath the cells at 24 h, FNR became concentrated at contacts with these fibers and was no longer detected at focal contacts. VNR was not observed at matrix contacts but remained strikingly localized in focal contacts of the 24-h cells. Since focal contacts represent the sites of strongest cell-to-substrate adhesion, these results suggest that FNR and VNR together play critical roles in the maintenance of stable contacts between the cell and its substrate. In addition, the accumulation of FNR at extracellular matrix contacts implies that this receptor might also function in the process of cellular migration along fibronectin-containing matrix cables. To define the factors governing accumulation of FNR and VNR at focal contacts, fibroblasts in serum-free media were plated on substrates coated with purified ligands. Fibronectin-coated surfaces fostered accumulation of FNR but not VNR at focal contacts. On vitronectin-coated surfaces, or substrata derivatized with a tridecapeptide containing the cell attachment sequence Arg-Gly-Asp, both FNR and VNR became concentrated at focal contacts. These observations suggest that the availability of ligand is critical to the accumulation of FNR and VNR at focal contacts, and that FNR might also recognize substrate-bound vitronectin.

scholarly journals Embryonic neural retinal cell response to extracellular matrix proteins: developmental changes and effects of the cell substratum attachment antibody (CSAT).

1987 ◽  
Vol 104 (3) ◽  
pp. 623-634 ◽  
Author(s):  
D E Hall ◽  
K M Neugebauer ◽  
L F Reichardt
Keyword(s):  
Extracellular Matrix ◽  
Cell Surface ◽  
Neurite Outgrowth ◽  
Cellular Response ◽  
Cell Attachment ◽  
Collagen Iv ◽  
Substrate Preference ◽  
Retinal Cell ◽  
Retinal Cells ◽  
Cell Surface Glycoprotein

Cell attachment and neurite outgrowth by embryonic neural retinal cells were measured in separate quantitative assays to define differences in substrate preference and to demonstrate developmentally regulated changes in cellular response to different extracellular matrix glycoproteins. Cells attached to laminin, fibronectin, and collagen IV in a concentration-dependent fashion, though fibronectin was less effective for attachment than the other two substrates. Neurite outgrowth was much more extensive on laminin than on fibronectin or collagen IV. These results suggest that different substrates have distinct effects on neuronal differentiation. Neural retinal cell attachment and neurite outgrowth were inhibited on all three substrates by two antibodies, cell substratum attachment antibody (CSAT) and JG22, which recognize a cell surface glycoprotein complex required for cell interactions with several extracellular matrix constituents. In addition, retinal cells grew neurites on substrates coated with the CSAT antibodies. These results suggest that cell surface molecules recognized by this antibody are directly involved in cell attachment and neurite extension. Neural retinal cells from embryos of different ages varied in their capacity to interact with extracellular matrix substrates. Cells of all ages, embryonic day 6 (E6) to E12, attached to collagen IV and CSAT antibody substrates. In contrast, cell attachment to laminin and fibronectin diminished with increasing embryonic age. Age-dependent differences were found in the profile of proteins precipitated by the CSAT antibody, raising the possibility that modifications of these proteins are responsible for the dramatic changes in substrate preference of retinal cells between E6 and E12.

Integrin-Mediated Fibroblast Adhesion Strength: Role of the β1 Subunit

1993 ◽  
Vol 331 ◽  
Author(s):  
Kelly A. Ward ◽  
Jun-Lin Guan ◽  
Daniel A. Hammer
Keyword(s):  
Extracellular Matrix ◽  
Adhesion Strength ◽  
Cytoplasmic Domain ◽  
Integrin Receptors ◽  
Substrate Adhesion ◽  
Focal Contacts ◽  
Cell Substrate ◽  
Extracellular Matrix Molecules ◽  
Integrin Ligand ◽  
Cell Substrate Adhesion

AbstractCell-substratum adhesion is important in wound healing [4], embryogenic development [11], tissue architecture [6], and metastasis [7]. Integrins constitute a major class of heterodimeric cell-surface glycoproteins involved in receptor-mediated adhesion to the extracellular matrix (ECM). Focal contacts are regions of the cell-substratum adhesion in which clusters of integrin receptors connect the cytoskeleton to extracellular matrix molecules such as fibronectin. Focal contacts strengthen cell-substrate adhesion, and are sites of biochemical activity. Since cell adhesion strength in part depends on the cell's ability to cluster receptors and cytoskeleton into focal contacts, the integrity of the focal contact, and hence a cell's adhesive strength, will depend both on integrin-cytoskeletal binding as well as integrin-ligand binding.Using a centrifugation assay, we have quantified cell-substratum adhesion strength of mouse 3T3 cells transfected with the avian β1 integrin receptor (wild type), including various deletion mutants of its cytoplasmic domain, to surfaces containing varying concentrations of CSAT, a monoclonal antibody against the extracellular domain of the avian β1 subunit. For all the transfectants, adhesion strength decreases with decreasing CSAT concentration and increasing centrifugal strength. Different truncations of the cytoplasmic domain lead to different levels of adhesion. There is no simple correlation between the length of the cytoplasmic domain and the strength of adhesion.

scholarly journals Studies on cell adhesion and recognition. II. The kinetics of cell adhesion and cell spreading on surfaces coated with carbohydrate-reactive proteins (glycosidases and lectins) and fibronectin.

1981 ◽  
Vol 88 (1) ◽  
pp. 138-148 ◽  
Author(s):  
W G Carter ◽  
H Rauvala ◽  
S I Hakomori
Keyword(s):  
Cell Adhesion ◽  
Cell Surface ◽  
Cell Attachment ◽  
Cell Spreading ◽  
Galactose Oxidase ◽  
Cross Linking ◽  
Con A ◽  
Coated Surfaces ◽  
Plastic Surfaces ◽  
Kinetics Of

The kinetics of cell attachment and cell spreading on the coated surfaces of two classes of carbohydrate-reactive proteins, enzymes and lectins, have been compared with those on fibronectin-coated surfaces with the following results: (a) A remarkable similarity between the kinetics of cell attachment to fibronectin-coated and glycosidase-coated surfaces was found. In contrast, cell attachment kinetics induced by lectin- and galactose oxidase-coated surfaces, in general, were strikingly different from those on fibronectin and glycosidase surfaces. The distinction between fibronectin- or glycosidase- and lectin- or galactose oxidase (an enzyme with lectin-type characteristics)-coated surfaces was further supported by the finding that cytochalasin B and EDTA inhibited cell attachment to fibronectin- and glycosidase-coated surfaces but not lectin-coated surfaces. (b) Fibronectin, if labeled and added to a cell suspension, showed only low or negligible interaction with the cell surface. However, fibronectin absorbed on plastic surfaces showed a high cell-attaching activity. It is assumed that fibronectin coated on plastic surfaces may form polyvalent attachment sites in contrast to its lower valency in aqueous solution. (c) Various inhibitors of cell attachment to both fibronectin-, galactose oxidase-, and lectin-coated surfaces were effective only during the first few minutes of the adhesion assay, after which time the attached cells became insensitive to the inhibitors. It is suggested that the initial specific recognition on either lectin-type or fibronectin-type surfaces is followed by an active cell-dependent attachment process. The primary role of the adhesion surface is to stimulate the cell-dependent attachment response. (d) Cells attached on tetravalent concanavalin A (Con A) spread very rapidly and quantitatively, whereas divalent succinyl Con A and monovalent Con A were effective stimulators of cell attachment but not cell spreading. Cross-linking of succinyl Con A restored the cell spreading activity. Tetravalent Con A surfaces specifically bind soluble glycoproteins, whereas succinyl Con A has a greatly reduced ability to bind the same glycoproteins. These results suggest that cross-linking of cell surface glycoproteins by the multivalent adhesive surface may trigger the cellular reaction leading to cell spreading.

Hierarchical assembly of cell–matrix adhesion complexes

2004 ◽  
Vol 32 (3) ◽  
pp. 416-420 ◽  
Author(s):  
R. Zaidel-Bar ◽  
M. Cohen ◽  
L. Addadi ◽  
B. Geiger
Keyword(s):  
Extracellular Matrix ◽  
Cell Surface ◽  
Focal Adhesions ◽  
Leading Edge ◽  
Cellular Migration ◽  
Hierarchical Assembly ◽  
Surface Recognition ◽  
Cell Matrix ◽  
Molecular Events ◽  
Cell Matrix Adhesion

The adhesion of cells to the extracellular matrix is a dynamic process, mediated by a series of cell-surface and matrix-associated molecules that interact with each other in a spatially and temporally regulated manner. These interactions play a major role in tissue formation, cellular migration and the induction of adhesion-mediated transmembrane signals. In this paper, we show that the formation of matrix adhesions is a hierarchical process, consisting of several sequential molecular events. One of the earliest steps in surface recognition is mediated, in some cells, by a 1 μm-thick cell-surface hyaluronan coat, which precedes the establishment of stable, cytoskeleton-associated adhesions. The earliest forms of these integrin-mediated contacts are dot-shaped FXs (focal complexes), which are formed under the protrusive lamellipodium of migrating cells. These adhesions recruit, sequentially, different anchor proteins that are involved in binding the actin cytoskeleton to the membrane. Conspicuous in its absence from FXs is zyxin, which is recruited to these sites only on retraction of the leading edge and the transformation of the FXs into a focal adhesion. Continuing application of force to focal adhesions results in the formation of fibrillar adhesions and reorganization of the extracellular matrix. The formation of these adhesions depends on actomyosin contractility and matrix pliability.

scholarly journals Analysis of fibronectin receptor function with monoclonal antibodies: roles in cell adhesion, migration, matrix assembly, and cytoskeletal organization.

1989 ◽  
Vol 109 (2) ◽  
pp. 863-875 ◽  
Author(s):  
S K Akiyama ◽  
S S Yamada ◽  
W T Chen ◽  
K M Yamada
Keyword(s):  
Cell Adhesion ◽  
Cell Surface ◽  
Cell Attachment ◽  
Morphological Changes ◽  
Cell Spreading ◽  
Lung Fibroblasts ◽  
Dependent Manner ◽  
Matrix Assembly ◽  
Concentration Dependent Manner ◽  
Fibronectin Receptor

We have developed two rat mAbs that recognize different subunits of the human fibroblast fibronectin receptor complex and have used them to probe the function of this cell surface heterodimer. mAb 13 recognizes the integrin class 1 beta polypeptide and mAb 16 recognizes the fibronectin receptor alpha polypeptide. We tested these mAbs for their inhibitory activities in cell adhesion, spreading, migration, and matrix assembly assays using WI38 human lung fibroblasts. mAb 13 inhibited the initial attachment as well as the spreading of WI38 cells on fibronectin and laminin substrates but not on vitronectin. Laminin-mediated adhesion was particularly sensitive to mAb 13. In contrast, mAb 16 inhibited initial cell attachment to fibronectin substrates but had no effect on attachment to either laminin or vitronectin substrates. When coated on plastic, both mAbs promoted WI38 cell spreading. However, mAb 13 (but not mAb 16) inhibited the radial outgrowth of cells from an explant on fibronectin substrates. mAb 16 also did not inhibit the motility of individual fibroblasts on fibronectin in low density culture and, in fact, substantially accelerated migration rates. In assays of the assembly of an extracellular fibronectin matrix by WI38 fibroblasts, both mAbs produced substantial inhibition in a concentration-dependent manner. The inhibition of matrix assembly resulted from impaired retention of fibronectin on the cell surface. Treatment of cells with mAb 16 also resulted in a striking redistribution of cell surface fibronectin receptors from a streak-like pattern to a relatively diffuse distribution. Concomitant morphological changes included decreases in thick microfilament bundle formation and reduced adhesive contacts of the streak-like and focal contact type. Our results indicate that the fibroblast fibronectin receptor (a) functions in initial fibroblast attachment and in certain types of adhesive contact, but not in the later steps of cell spreading; (b) is not required for fibroblast motility but instead retards migration; and (c) is critically involved in fibronectin retention and matrix assembly. These findings suggest a central role for the fibronectin receptor in regulating cell adhesion and migration.

scholarly journals Development of cell surface linkage complexes in cultured fibroblasts.

1985 ◽  
Vol 100 (4) ◽  
pp. 1103-1114 ◽  
Author(s):  
W T Chen ◽  
E Hasegawa ◽  
T Hasegawa ◽  
C Weinstock ◽  
K M Yamada
Keyword(s):  
Monoclonal Antibody ◽  
Extracellular Matrix ◽  
Cell Surface ◽  
Protein Complex ◽  
Cell Attachment ◽  
Embryonic Fibroblasts ◽  
Cultured Fibroblasts ◽  
Contact Sites ◽  
Adhesion Sites ◽  
Microfilament Bundles

The possible role of a 140K membrane-associated protein complex (140K) in fibronectin-cytoskeleton associations has been examined. The 140K was identified by the monoclonal antibody JG22E. Monoclonal and polyclonal antibodies to the 140K showed identical patterns of binding to the cell membranes of fixed and permeabilized chicken embryonic fibroblasts; localization was diffuse, but with marked concentration in cell-to-extracellular matrix contact sites. Correlative localization with interference reflection microscopy and double-label or triple-label immunofluorescence showed that 140K co-distributed with extracellular fibronectin fibrils and intracellular alpha-actinin in microfilament bundles at extracellular matrix contact sites but tended not to co-localize with tropomyosin present in bundles at sites farther from adhesion sites. In addition, binding of antibodies to 140K, alpha-actinin, and fibronectin was excluded from vinculin-rich focal adhesion sites at the cellular periphery. A progressive development of cell surface alpha-actinin-140K-fibronectin associations was observed in early spreading cells. The anti-140K monoclonal antibody JG22E inhibited the attachment and spreading of both normal and Rous sarcoma virus-transformed chicken embryonic fibroblasts to a fibronectin substratum. However, the anti-140K monoclonal antibody became a positive mediator of cell attachment and spreading if it was adsorbed or cross-linked to the substratum. Our results provide the first description of a membrane-associated protein complex that co-localizes with fibronectin and microfilament bundles, and they suggest that the 140K complex may be part of a cell surface linkage between fibronectin and the cytoskeleton.

scholarly journals The distribution of distinct integrins in focal contacts is determined by the substratum composition

1989 ◽  
Vol 92 (1) ◽  
pp. 67-75 ◽  
Author(s):  
K.R. Fath ◽  
C.J. Edgell ◽  
K. Burridge
Keyword(s):  
Cell Surface ◽  
Cell Line ◽  
Culture Conditions ◽  
Fibronectin Receptor ◽  
Vitronectin Receptor ◽  
Normal Culture ◽  
Focal Contacts ◽  
Cytoplasmic Face ◽  
Predominant Form

The distribution of two integrins, the fibronectin receptor and the vitronectin receptor, has been explored in an endothelium-derived cell line plated onto various substrata. On a fibronectin substratum, in the presence of serum, these cells develop focal contacts that contain the fibronectin receptor, whereas the vitronectin receptor is diffusely distributed over the cell surface. Conversely, cells plated onto vitronectin-coated coverslips concentrate only the vitronectin receptor within focal contacts. The accumulation of the vitronectin receptor within focal contacts also occurs when the cells are plated on uncoated coverslips but in the presence of serum. Therefore, we conclude that under normal culture conditions (i.e. in serum-containing media), the vitronectin receptor is the predominant form of integrin involved in substratum adhesion. This conclusion is supported by experiments in which cells were cultured on fibronectin-coated coverslips in the presence of serum. Initially these cells developed focal contacts containing only the fibronectin receptor. Within several hours, however, there was a progressive replacement of focal contacts containing the fibronectin receptor by focal contacts expressing the vitronectin receptor. After approximately 12 h in culture, most cells contained focal contacts expressing only the vitronectin receptor. Focal contacts containing either the fibronectin or vitronectin receptor were both associated with the termini of stress fibres and contained the proteins talin and vinculin. These observations lead us to propose that the cell does not discriminate between these different integrins when assembling the cytoskeletal components at the cytoplasmic face of focal contacts.

scholarly journals The alpha v beta 1 integrin functions as a fibronectin receptor but does not support fibronectin matrix assembly and cell migration on fibronectin

1993 ◽  
Vol 122 (1) ◽  
pp. 235-242 ◽  
Author(s):  
Z Zhang ◽  
AO Morla ◽  
K Vuori ◽  
JS Bauer ◽  
RL Juliano ◽  
...  
Keyword(s):  
Cell Migration ◽  
Cell Attachment ◽  
Focal Adhesions ◽  
Cho Cell ◽  
Matrix Assembly ◽  
Fibronectin Receptor ◽  
Fibronectin Matrix ◽  
Beta 1 Integrin ◽  
Fibronectin Binding ◽  
Coated Surfaces

The fibronectin receptor, alpha 5 beta 1, has been shown to be required for fibronectin matrix assembly and plays an important role in cell migration on fibronectin. However, it is not clear whether other fibronectin binding integrins can take the place of alpha 5 beta 1 during matrix assembly and cell migration. To test this, we expressed the human alpha v subunit in the CHO cell line CHO-B2 that lacks the alpha 5 subunit. We found that the human alpha v combined with CHO cell beta 1 to form the integrin alpha v beta 1. Cells that expressed alpha v beta 1 attached to and spread well on fibronectin-coated dishes, but did so less well on vitronectin-coated dishes. This, along with other data, indicated that alpha v beta 1 functions as a fibronectin receptor in CHO-B2 cells. The alpha v beta 1-expressing cells failed to produce a fibronectin matrix or to migrate on fibronectin, although the same cells transfected with alpha 5 do produce a matrix and migrate on fibronectin. The affinity of the alpha v beta 1-expressing cells for fibronectin was fourfold lower than that of the alpha 5 beta 1-expressing cells. In addition, alpha v beta 1 was distributed diffusely throughout the cell surface, whereas alpha 5 beta 1 was localized to focal adhesions when cells were seeded onto fibronectin-coated surfaces. Thus, of the two fibronectin receptors, alpha v beta 1 and alpha 5 beta 1, only alpha 5 beta 1 supports fibronectin matrix assembly and promotes cell migration on fibronectin in the CHO-B2 cells. Possible reasons for this difference in the activities of alpha v beta 1 and alpha 5 beta 1 include the lower affinity of alpha v beta 1 for fibronectin and the failure of this integrin to localize in adhesion plaques on a fibronectin substrate. These results show that two integrins with similar ligand specificities and cell attachment functions may be quite different in their ability to support fibronectin matrix assembly and cell motility on fibronectin.

scholarly journals Cell-to-substrate contacts in an adhesion-defective mutant of Balb/c3T3 cells

1981 ◽  
Vol 52 (1) ◽  
pp. 183-196
Author(s):  
J.R. Yates ◽  
C.S. Izzard
Keyword(s):  
Cell Surface ◽  
Close Contact ◽  
Cell Types ◽  
Separation Distance ◽  
Specific Substrate ◽  
Substrate Adhesion ◽  
Focal Contacts ◽  
Defective Mutant ◽  
Mutant Cells ◽  
Cell Thickness

The cell-to-substrate contacts of the adhesion-defective mutant, AD6, have been examined by interference reflexion microscopy and compared with those of the wild-type Balb/c3T3 cell. The 2 cell-types differed in their ability to produce focal contacts with the substrate (10-15 nm separation distance). Only 10% of AD6 versus 92% of Balb/c3T3 cells formed these contacts. When present in AD6 the focal contacts were smaller and fewer in number per cell than in Balb/c3T3. Close contact with the substrate (approx. 30 nm separation distance) was formed by both cell types. The absence of the stronger focal contacts accounts for the reduced substrate adhesion of the mutant cells and for other phenotypic characteristics such as reduced spreading, rounded shape, altered pattern of movement, and absence of stress fibres. The reduced adhesion of the mutant has been attributed to a change in the pattern of glycoproteins exposed at the cell surface, which results from a defect in glycosylation. This suggests that normal glycosylation and correct exposure of one or more cell surface components are required for the formation of a specific substrate adhesion, the focal contact, and offers the possibility of identifying individual surface components involved in formation of this adhesion. Attention is drawn to zero-order minima originating from cell thickness and not cell-to-substrate separation distance in the interference reflexion image.

scholarly journals Tenascin mediates cell attachment through an RGD-dependent receptor.

1989 ◽  
Vol 108 (3) ◽  
pp. 1149-1155 ◽  
Author(s):  
M A Bourdon ◽  
E Ruoslahti
Keyword(s):  
Extracellular Matrix ◽  
Cell Attachment ◽  
Human Tumor ◽  
Surface Protein ◽  
Cell Types ◽  
Glioma Cell Line ◽  
Fibronectin Receptor ◽  
Cell Matrix ◽  
Undifferentiated Tumors ◽  
Cell Matrix Interactions

Tenascin is an extracellular matrix glycoprotein expressed in association with mesenchymal-epithelial interactions during development and in the neovasculature and stroma of undifferentiated tumors. This selective expression of tenascin indicates a specific role in cell matrix interactions. We now show that tenascin can support the adhesion of a variety of cell types, including various human tumor cells, normal fibroblasts, and endothelial cells, all of which can attach to a substrate coated with tenascin. Detailed studies on the mechanism of the tenascin-promoted cell attachment were carried out with the human glioma cell line U251MG. The attachment of these cells and others to tenascin were inhibited specifically by peptides containing the RGD cell attachment signal. Affinity chromatography procedures similar to those that have been used to isolate other adhesion receptors yielded a heterodimeric cell surface protein which bound to a tenascin affinity matrix in an RGD-dependent fashion. One of the subunits of this putative tenascin receptor comigrates with the beta subunit of the fibronectin receptor in SDS-PAGE and cross reacts with antibodies prepared against the fibronectin receptor in immunoblotting. These results identify the tenascin receptor as a member of the fibronectin receptor family within the integrin superfamily of receptors. The cell attachment response on tenascin is distinctly different from that seen on fibronectin, suggesting that cell adhesion and motility may be modulated at those sites where tenascin is expressed in the extracellular matrix.

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