Quantification of fibronectin matrix assembly sites using a novel ELISA assay

Center for Cell Biology and Cancer Research, Albany Medical College, 47 New Scotland Avenue, Albany, New York 12208, USA.
Matrix Biology (Impact Factor: 5.07). 06/2007; 26(4):330-3. DOI: 10.1016/j.matbio.2006.12.004
Source: PubMed


Binding of the N-terminus of fibronectin to assembly sites on the cell surface is an essential step in fibronectin fibrillogenesis. Fibronectin matrix assembly sites have customarily been quantified using an iodinated 70 kDa N-terminal fibronectin fragment. The 125I-70 K fragment is a less than ideal reagent because its preparation requires large amounts of plasma fibronectin and it has a fairly short shelf life. An additional limitation is that the cells responsible for binding the 125I-70 K cannot be quantified or identified directly but must be assessed in parallel cultures. To overcome these disadvantages, we developed an ELISA-based assay using a recombinant HA-tagged 70 K fragment. This assay allows for the simultaneous quantification and localization of matrix assembly sites on the surface of adherent cells.

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    • "Human dermal fibroblasts (A1-F) were a gift from Dr. Lynn Allen-Hoffman (University of Wisconsin, Madison, WI) and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Fibronectin null (FN −/− ) mouse fibroblasts (5F) were a gift of Dr. Richard Hynes (Massachusetts Institute of Technology, Cambridge, MA) and maintained as previously described (Sottile et al., 1998; Zheng et al., 2007). Briefly, 5F cells were seeded onto collagen coated wells under serum-free conditions in a 1:1 mixture of Cellgro® (Mediatech, Herndon, VA) and Aim V (Invitrogen). "
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    ABSTRACT: Anastellin is an angiogenesis inhibitor derived from the first type III repeat of fibronectin (FN). Anastellin binds to fibronectin and promotes the polymerization of soluble fibronectin into a highly polymerized form termed superfibronectin. In addition, anastellin also causes remodeling of pre-existing fibronectin matrix and modulates cell signaling pathways in both endothelial cells and fibroblasts. In the present study, we address the relationship of anastellin's effects on fibronectin matrix to its effects on p38 MAP kinase (MAPK) activation. Using a mutant form of anastellin which binds to fibronectin matrix, but does not stimulate formation of superfibronectin, we demonstrate that the activation of p38 MAPK by anastellin is not dependent on the formation of superfibronectin. The mutant form of anastellin does stimulate matrix remodeling, but experiments using FN(-/-) cells show that the effect of anastellin on p38-MAPK activation is completely independent of fibronectin. Anastellin was able to activate p38 MAPK on cells in suspension as well as on cells null for beta1 integrins, suggesting that anastellin activity did not require ligation of integrins. These data suggest that the activation of p38 MAPK by anastellin is independent of anastellin's effects on fibronectin matrix organization.
    Matrix biology: journal of the International Society for Matrix Biology 02/2009; 28(2):101-9. DOI:10.1016/j.matbio.2009.01.003 · 5.07 Impact Factor
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    • "Studies indicate that active remodeling of contacts occurs and that the expected halflife of a focal contact can be as short as a few minutes (Edlund et al., 2001; Franco et al., 2006). Vitronectin has been shown to negatively regulate matrix assembly (Hocking et al., 1999; Zhang et al., 1999) through a mechanism requiring vitronectin integrin receptors (Zheng et al., 2007). This suggests that PAI-1 is relieving an inhibitory activity of the αvβ5 integrin. "
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    ABSTRACT: The plasminogen activation system regulates matrix remodeling through both proteolytic and non-proteolytic mechanisms. Studies were undertaken to determine the effects of the plasminogen activator inhibitor 1 (PAI1) on the assembly of the fibronectin matrix. The addition of PAI1 to MG-63 cells caused a 1.5- to threefold increase in the rate of fibronectin matrix assembly which was associated with an increase in beta integrin activation. PAI1 treatment led to a marked decrease in focal contacts and stress fibers, whereas tensin-containing matrix contacts remained unaffected. The effects of PAI1 on matrix assembly were independent of both urokinase-type plasminogen activator (uPA) and urokinase-type plasminogen activator receptor (uPAR), indicating that the stimulation of matrix assembly by PAI1 does not depend on its anti-proteolytic activity or on the association of uPAR with integrin receptors. Antagonists of the alphavbeta5 integrin mimicked the effect of PAI1 on cell morphology and fibronectin matrix deposition, indicating that stimulation of matrix assembly by PAI1 required disruption of the interaction between the alphavbeta5 integrin and vitronectin. Consistent with this conclusion, the Q123K PAI1 mutant which does not bind vitronectin had no effect on matrix assembly. Our data identify PAI1 as a novel regulator of fibronectin matrix assembly, and indicate that this regulation occurs through a previously undescribed crosstalk between the alphavbeta5 and alpha5beta1 integrins.
    Journal of Cell Science 06/2008; 121(Pt 10):1661-70. DOI:10.1242/jcs.020149 · 5.43 Impact Factor
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    ABSTRACT: The three-dimensional organization of the ubiquitous extracellular matrix glycoprotein fibronectin regulates cell fate and morphogenesis during development; in particular tubule formation that constitutes the vasculature, lung and kidney. Tenascin-C is a matrix protein with a restricted expression pattern; it is specifically up-regulated at sites of fibronectin fibril assembly during development and in remodeling adult tissues. Here we demonstrate that specific domains of tenascin-C inhibit fibronectin matrix assembly whereas full-length tenascin-C does not. These domains act via distinct mechanisms: TNfn1-8 blocks fibrillogenesis by binding to fibronectin fibrils and preventing intermolecular fibronectin interactions whilst FBG acts independently of binding to fibronectin and instead is internalized and causes cytoskeletal re-organization. We also show that TNfn1-8 disrupts epithelial cell tubulogenesis. Our data demonstrate that tenascin-C contains cryptic sites which can control tissue levels of fibrillar fibronectin either by preventing de novo fibril assembly or reducing levels of deposited fibronectin. Exposure of these domains during tissue remodeling may provide a novel means of controlling fibronectin assembly and tubulogenic processes dependent on the assembly of this matrix.
    Matrix biology: journal of the International Society for Matrix Biology 09/2010; 29(7):573-85. DOI:10.1016/j.matbio.2010.08.003 · 5.07 Impact Factor
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