Complex protein composition of isolated focal adhesions: A two‐dimensional gel and database analysis

Niederreiter, M; Gimona, Mario; Streichsbier, Franz; Celis, Julio E.; Small, J. Victor

doi:10.1002/elps.1150150169

Cited by 16 publications

(9 citation statements)

References 26 publications

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“…In contrast, Signal Integration in Focal Adhesions ECM-dependent activation of chemical signaling events occurs within minutes after integrin binding Shattil and Brugge, 1991;Kornberg et al, 1992;Lipfert et al, 1992;Sorisky et al, 1992;Cybulsky et al, 1993; (Niederreiter et al, 1994). Although these methods permit compositional analysis, the amount of protein isolated is extremely low and thus, study of associated enzyme activities is significantly limited.…”

Section: Discussionmentioning

confidence: 99%

Convergence of integrin and growth factor receptor signaling pathways within the focal adhesion complex.

Plopper

McNamee

Dike

et al. 1995

MBoC

479

315

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Extracellular matrix controls capillary endothelial cell sensitivity to soluble mitogens by binding integrin receptors and thereby activating a chemical signaling response that rapidly integrates with growth factor-induced signaling mechanisms. Here we report that in addition to integrins, growth factor receptors and multiple molecules that transduce signals conveyed by both types of receptors are immobilized on the cytoskeleton (CSK) and spatially integrated within the focal adhesion complex (FAC) at the site of integrin binding. FACs were rapidly induced in round cells and physically isolated from the remainder of the CSK after detergent-extraction using magnetic microbeads coated with fibronectin or a synthetic RGD-containing peptide. Immunofluorescence microscopy revealed that multiple signaling molecules (e.g., pp60csrc, ppl25FAKphosphatidylinositol-3-kinase, phospholipase C-'y, and Na+/H+ antiporter) involved in both integrin and growth factor receptor signaling pathways became associated with the CSK framework of the FAC within 15 min after binding to beads coated with integrin ligands. Recruitment of tyrosine kinases to the FAC was also accompanied by a local increase in tyrosine phosphorylation, as indicated by enhanced phosphotyrosine staining at the site of integrin binding. In contrast, neither recruitment of signaling molecules nor increased phosphotyrosine staining was observed when cells bound to beads coated with a control ligand (acetylated low density lipoprotein) that ligates transmembrane scavenger receptors, but does not induce FAC formation. Western blot analysis confirmed that FACs isolated using RGD-beads were enriched for pp6Ocsrc, pp125FAK, phospholipase G-y, and the Na+ /H+ antiporter when compared with intact CSK or basal cell surface preparations that retained lipid bilayer. Isolated FACs were also greatly enriched for the high affinity fibroblast growth factor receptor flg. Most importantly, isolated FACs continued to exhibit multiple chemical signaling activities in vitro, including protein tyrosine kinase activities (pp60csrc and pp125FAK) as well as the ability to undergo multiple sequential steps in the inositol lipid synthesis cascade. These data suggest that many of the chemical signaling events that are induced by integrins and growth factor receptors in capillary cells may effectively function in a "solid-state" on insoluble CSK scaffolds within the FAC and that the FAC may represent a major site for signal integration between these two regulatory pathways. Future investigations into the biochemical and biophysical basis of signal transduction may be facilitated by this method, which results in isolation of FACs that retain the CSK framework as well as multiple associated chemical signaling activities.

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Section: Discussionmentioning

confidence: 99%

Convergence of integrin and growth factor receptor signaling pathways within the focal adhesion complex.

Plopper

McNamee

Dike

et al. 1995

MBoC

479

315

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“…Vinculin present in the focal adhesion, as opposed to the diffusible cellular fraction, is resistant to Triton X-100 extraction (Niederiter et al, 1994). Treatment with this detergent is used as a means to retain only the FA vinculin.…”

Section: Eliminating the Causes Of Variation-the Specific Labelling Amentioning

confidence: 99%

Focal adhesion quantification - A new assay of material biocompatibility? : Review

Do²,

Gwynn³

et al. 2005

eCM

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The development of novel synthetic biomaterials is necessitated by the increasing demand for accelerated healing of tissues following surgical intervention. Strict testing of such materials is necessary before application. Currently, before any material can be marketed, approval by regulatory organisations such as the FDA is required. Presently, in vitro testing is performed as a prerequisite to in vivo evaluation. The in vitro techniques currently employed do not reflect the progress in our understanding of extra and intra-cellular processes, with far more sensitive in vitro evaluations now available. Obtaining quantifiable data is increasingly relevant to evaluating events occurring in vivo. Quantifying cell adhesion to surfaces provides some of this data as an initial assessment method. Major developments in this field are occurring but many investigators still use less than optimal methods for assessing biomaterials. The relevance of using cell adhesion assays to help determine biomaterial biocompatibility is reviewed. Additionally, current in vitro methods of evaluating biomaterials are discussed in the context of novel testing concepts developed by the authors.

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“…Analysis of extracts from amniotic mesenchymal cells by two-dimensional electrophoresis has demonstrated the presence of ␤1-integrin receptors in SAM. 25 In the present study, we fluorescently labeled fibronectin receptor in SAM from skin fibroblasts. In contrast to the diffuse distribution of the integrins observed in SAM prepared on cpTi, a higher concentration could be detected in the SAM on TPS, closely following the outlines of the cells.…”

Section: Discussionmentioning

confidence: 99%

Quantifying the adherence of fibroblasts to titanium and its enhancement by substrate-attached material

Derhami

Wolfaardt

Wennerberg

et al. 2000

J. Biomed. Mater. Res.

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Normal human skin fibroblasts were cultured on tissue culture polystyrene and on commercially pure titanium. In addition, substrate-attached material that remained on the surfaces after detachment of fibroblasts with a chelating agent was examined. The force required to detach 50% of the fibroblasts from each substrate was assessed by centrifugation. The results showed a time-dependent decrease in the force required to detach fibroblasts from titanium not seen on tissue culture polystyrene. Nearly all cells detached from the titanium surfaces at 7.85 x 10(x3) dynes/cell after 3 or 5 days in culture, whereas few cells detached from tissue culture polystyrene. Cells freshly seeded onto titanium substrates that had been coated with substrate-attached material by prior culture of fibroblasts for 3 or 5 days showed an approximately sixfold increased adherence. The results of immunofluorescence staining for fibronectin and its receptor suggest that the nature of the interaction between this extracellular matrix ligand and the substrate may be important in determining cellular stiffness at the cell-extracellular matrix interface.

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Complex protein composition of isolated focal adhesions: A two‐dimensional gel and database analysis

Cited by 16 publications

References 26 publications

Convergence of integrin and growth factor receptor signaling pathways within the focal adhesion complex.

Convergence of integrin and growth factor receptor signaling pathways within the focal adhesion complex.

Focal adhesion quantification - A new assay of material biocompatibility? : Review

Quantifying the adherence of fibroblasts to titanium and its enhancement by substrate-attached material

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