scholarly journals Inhibition of binding of fibronectin to matrix assembly sites by anti-integrin (alpha 5 beta 1) antibodies.

1990 ◽  
Vol 111 (2) ◽  
pp. 699-708 ◽  
Author(s):  
F J Fogerty ◽  
S K Akiyama ◽  
K M Yamada ◽  
D F Mosher
Keyword(s):  
Cell Adhesion ◽  
Direct Interaction ◽  
Fibroblast Cell ◽  
Inhibitory Antibody ◽  
Matrix Assembly ◽  
Fibronectin Receptor ◽  
Amino Terminal ◽  
Fab Fragments ◽  
Cell Layers ◽  
Fibronectin Binding

Fibroblasts have cell surface sites that mediate assembly of plasma and cellular fibronectin into the extracellular matrix. Cell adhesion to fibronectin can be mediated by the interaction of an integrin (alpha 5 beta 1) with the Arg-Gly-Asp-Ser (RGDS)-containing cell adhesion region of fibronectin. We have attempted to elucidate the role of the alpha 5 beta 1 fibronectin receptor in assembly of fibronectin in matrices. Rat monoclonal antibody mAb 13, which recognizes the integrin beta 1 subunit, completely blocked binding and matrix assembly of 125I-fibronectin as well as binding of the 125I-70-kD amino-terminal fragment of fibronectin (70 kD) to fibroblast cell layers. Fab fragments of the anti-beta 1 antibody were also inhibitory. Antibody mAb 16, which recognizes the integrin alpha 5 subunit, partially blocked binding of 125I-fibronectin and 125I-70-kD. When cell layers were coincubated with fluoresceinated fibronectin and either anti-beta 1 or anti-alpha 5, anti-beta 1 was a more effective inhibitor than anti-alpha 5 of binding of labeled fibronectin to the cell layer. Inhibition of 125I-fibronectin binding by anti-beta 1 IgG occurred within 20 min. Inhibition of 125I-fibronectin binding by anti-beta 1 Fab fragments or IgG could not be overcome with increasing concentrations of fibronectin, suggesting that anti-beta 1 and exogenous fibronectin may not compete for the same binding site. No beta 1-containing integrin bound to immobilized 70 kD. These data indicate that the beta 1 subunit plays an important role in binding and assembly of exogenous fibronectin, perhaps by participation in the organization, regeneration, or cycling of the assembly site rather than by a direct interaction with fibronectin.

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.

A novel role for alpha 3 beta 1 integrins in extracellular matrix assembly

1995 ◽  
Vol 108 (6) ◽  
pp. 2511-2523 ◽  
Author(s):  
C. Wu ◽  
A.E. Chung ◽  
J.A. McDonald
Keyword(s):  
Extracellular Matrix ◽  
Cell Adhesion ◽  
Binding Activity ◽  
Integrin Subunit ◽  
Pericellular Matrix ◽  
Matrix Assembly ◽  
Amino Terminal ◽  
Beta 1 Integrin ◽  
Fibronectin Deposition

To study the biological role of alpha 3 beta 1 integrins in cell adhesion, migration, and in the deposition of extracellular matrix, we stably expressed the human alpha 3 integrin subunit in the alpha 4, alpha 5 integrin deficient CHO cell line B2. The expression of alpha 3 beta 1 integrins enhanced cell adhesion on entactin (also known as nidogen), but not on fibronectin. Using recombinant GST-fusion proteins that span the entire length of the entactin molecule, we located cell adhesive activity to the G2 domain of entactin. These results suggest that the alpha 3 beta 1 integrin functions as an adhesion receptor interacting with the G2 domain of entactin. On the other hand, the expression of alpha 3 beta 1 integrins did not confer the ability to migrate on entactin. Strikingly, the expression of alpha 3 beta 1 dramatically increased the deposition of entactin and fibronectin into the pericellular matrix. This was accompanied by increased binding activity of the 29 kDa amino-terminal domain of fibronectin. Thus, similar to alpha 5 beta 1 integrins, alpha 3 beta 1 integrins can play an important role in modulating the assembly of pericellular matrices. However, unlike fibronectin deposition supported by alpha 5 beta 1, alpha 3 beta 1 supported fibronectin deposition into pericellular matrix was not inhibited by antibodies binding to the RGD containing cell adhesion domain of fibronectin, demonstrating that the two processes are mechanistically distinct. The role of alpha 3 beta 1 in pericellular matrix assembly potentially implicates this receptor in the assembly and/or recognition of entactin-containing pericellular matrices, an observation consistent with its apparent role in the renal glomerulus of the mammalian kidney.

scholarly journals Activation of Distinct α5β1-mediated Signaling Pathways by Fibronectin's Cell Adhesion and Matrix Assembly Domains

1998 ◽  
Vol 141 (1) ◽  
pp. 241-253 ◽  
Author(s):  
Denise C. Hocking ◽  
Jane Sottile ◽  
Paula J. McKeown-Longo
Keyword(s):  
Cell Adhesion ◽  
Amino Terminus ◽  
Type I ◽  
New Paradigm ◽  
Matrix Assembly ◽  
Rgd Peptides ◽  
Surface Receptors ◽  
Amino Terminal ◽  
Dynamic Cell ◽  
Α5β1 Integrin

The interaction of cells with fibronectin generates a series of complex signaling events that serve to regulate several aspects of cell behavior, including growth, differentiation, adhesion, and motility. The formation of a fibronectin matrix is a dynamic, cell-mediated process that involves both ligation of the α5β1 integrin with the Arg-Gly-Asp (RGD) sequence in fibronectin and binding of the amino terminus of fibronectin to cell surface receptors, termed “matrix assembly sites,” which mediate the assembly of soluble fibronectin into insoluble fibrils. Our data demonstrate that the amino-terminal type I repeats of fibronectin bind to the α5β1 integrin and support cell adhesion. Furthermore, the amino terminus of fibronectin modulates actin assembly, focal contact formation, tyrosine kinase activity, and cell migration. Amino-terminal fibronectin fragments and RGD peptides were able to cross-compete for binding to the α5β1 integrin, suggesting that these two domains of fibronectin cannot bind to the α5β1 integrin simultaneously. Cell adhesion to the amino-terminal domain of fibronectin was enhanced by cytochalasin D, suggesting that the ligand specificity of the α5β1 integrin is regulated by the cytoskeleton. These data suggest a new paradigm for integrin-mediated signaling, where distinct regions within one ligand can modulate outside-in signaling through the same integrin.

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 The function of multiple extracellular matrix receptors in mediating cell adhesion to extracellular matrix: preparation of monoclonal antibodies to the fibronectin receptor that specifically inhibit cell adhesion to fibronectin and react with platelet glycoproteins Ic-IIa.

1988 ◽  
Vol 107 (5) ◽  
pp. 1881-1891 ◽  
Author(s):  
E A Wayner ◽  
W G Carter ◽  
R S Piotrowicz ◽  
T J Kunicki
Keyword(s):  
Extracellular Matrix ◽  
Monoclonal Antibodies ◽  
Cell Adhesion ◽  
Fibroblast Cell ◽  
Fibronectin Receptor ◽  
Functional Roles ◽  
Late Antigen ◽  
Specific Tissue ◽  
Coated Surfaces ◽  
Extracellular Matrix Receptors

We have identified monoclonal antibodies that inhibit human cell adhesion to collagen (P1H5), fibronectin (P1F8 or P1D6), and collagen and fibronectin (P1B5) that react with a family of structurally similar glycoproteins referred to as extracellular matrix receptors (ECMRs) II, VI, and I, respectively. Each member of this family contains a unique alpha subunit, recognized by the antibodies, and a common beta subunit, each of approximately 140 kD. We show here that ECMR VI is identical to the fibronectin receptor (FNR), very late antigen (VLA) 5, and platelet glycoproteins Ic-IIa and shall be referred to as FNR. Monoclonal antibodies to FNR inhibit lymphocyte, fibroblast, and platelet adhesion to fibronectin-coated surfaces. ECMRs I, II, and FNR were differentially expressed in platelets, resting or activated lymphocytes, and myeloid, epithelial, endothelial, and fibroblast cell populations, suggesting a functional role for the receptors in vascular emigration and selective tissue localization. Tissue staining of human fetal skin localized ECMRs I and II to the basal epidermis primarily, while monoclonal antibodies to the FNR stained both the dermis and epidermis. Experiments carried out to investigate the functional roles of these receptors in mediating cell adhesion to complex extracellular matrix (ECM) produced by cells in culture revealed that complete inhibition of cell adhesion to ECM required antibodies to both the FNR and ECMR II, the collagen adhesion receptor. These results show that multiple ECMRs function in combination to mediate cell adhesion to complex EMC templates and predicts that variation in ECM composition and ECMR expression may direct cell localization to specific tissue domains.

Investigation of Au SAMs Photoclick Derivatization by PM-IRRAS

2019 ◽  
Author(s):  
Mark Workentin ◽  
François Lagugné-Labarthet ◽  
Sidney Legge
Keyword(s):  
Cell Adhesion ◽  
Self Assembly ◽  
Materials Science ◽  
Fibroblast Cell ◽  
Fine Tuning ◽  
Chemical System ◽  
Infrared Reflection ◽  
Assembled Monolayers ◽  
One Step ◽  
High Degree

In this work we present a clean one-step process for modifying headgroups of self-assembled monolayers (SAMs) on gold using photo-enabled click chemistry. A thiolated, cyclopropenone-caged strained alkyne precursor was first functionalized onto a flat gold substrate through self-assembly. Exposure of the cyclopropenone SAM to UV-A light initiated the efficient photochemical decarbonylation of the cyclopropenone moiety, revealing the strained alkyne capable of undergoing the interfacial strain-promoted alkyne-azide cycloaddition (SPAAC). Irradiated SAMs were derivatized with a series of model azides with varied hydrophobicity to demonstrate the generality of this chemical system for the modification and fine-tuning of the surface chemistry on gold substrates. SAMs were characterized at each step with polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS) to confirm successful functionalization and reactivity. Furthermore, to showcase the compatibility of this approach with biochemical applications, cyclopropenone SAMs were irradiated and modified with azide-bearing cell adhesion peptides to promote human fibroblast cell adhesion, then imaged by live cell fluorescence microscopy. Thus, the “photoclick” methodology reported here represents an improved, versatile, catalyst-free protocol that allows for a high degree of control over the modification of material surfaces, with applicability in materials science as well as biochemistry.<br>

scholarly journals Fibronectin's cell-adhesive domain and an amino-terminal matrix assembly domain participate in its assembly into fibroblast pericellular matrix.

1987 ◽  
Vol 262 (7) ◽  
pp. 2957-2967 ◽  
Author(s):  
J.A. McDonald ◽  
B.J. Quade ◽  
T.J. Broekelmann ◽  
R. LaChance ◽  
K. Forsman ◽  
...  
Keyword(s):  
Pericellular Matrix ◽  
Matrix Assembly ◽  
Amino Terminal ◽  
Cell Adhesive

Polarization and interaction of adhesion molecules P-selectin glycoprotein ligand 1 and intercellular adhesion molecule 3 with moesin and ezrin in myeloid cells

Blood ◽  
2000 ◽  
Vol 95 (7) ◽  
pp. 2413-2419 ◽  
Author(s):  
José L. Alonso-Lebrero ◽  
Juan M. Serrador ◽  
Carmen Domı́nguez-Jiménez ◽  
Olga Barreiro ◽  
Alfonso Luque ◽  
...  
Keyword(s):  
Adhesion Molecules ◽  
Adhesion Molecule ◽  
Direct Interaction ◽  
Intercellular Adhesion ◽  
Actin Binding ◽  
Intercellular Adhesion Molecule ◽  
Human Neutrophils ◽  
Binding Assays ◽  
Glutathione S Transferase ◽  
Amino Terminal

Abstract In response to the chemoattractants interleukin 8, C5a,N-formyl-methionyl-leucyl-phenylalanine, and interleukin 15, adhesion molecules P-selectin glycoprotein ligand 1 (PSGL-1), intercellular adhesion molecule 3 (ICAM-3), CD43, and CD44 are redistributed to a newly formed uropod in human neutrophils. The adhesion molecules PSGL-1 and ICAM-3 were found to colocalize with the cytoskeletal protein moesin in the uropod of stimulated neutrophils. Interaction of PSGL-1 with moesin was shown in HL-60 cell lysates by isolating a complex with glutathione S-transferase fusions of the cytoplasmic domain of PSGL-1. Bands of 78- and 81-kd were identified as moesin and ezrin by Western blot analysis. ICAM-3 and moesin also coeluted from neutrophil lysates with an anti-ICAM-3 immunoaffinity assay. Direct interaction of the cytoplasmic domains of ICAM-3 and PSGL-1 with the amino-terminal domain of recombinant moesin was demonstrated by protein-protein binding assays. These results suggest that the redistribution of PSGL-1 and its association with intracellular molecules, including the ezrin-radixin-moesin actin-binding proteins, regulate functions mediated by PSGL-1 in leukocytes stimulated by chemoattractants.

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