Force Required to Break α5β1Integrin-Fibronectin Bonds in Intact Adherent Cells Is Sensitive to Integrin Activation State

Garcı́a, Andrés J.; Huber, François; Boettiger, David

doi:10.1074/jbc.273.18.10988

Cited by 140 publications

(179 citation statements)

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“…Figure 1B shows a plot of the 50 , or adhesion strength, for replicate analyses at different fibronectin densities. The linear relationship between 50 and ligand density has been shown for ␣5␤1 on other cells and for other integrin-ligand combinations (Garcia et al, 1998a;Garcia and Boettiger, 1999;Boettiger et al, 2001). This relationship provides a quantitative measure of adhesion strength that is directly related to the number of ␣5␤1-fibronectin bonds.…”

mentioning

confidence: 96%

Section: Proportion Of Ligand-bound ␣5␤1 Integrin As a Function Of Fimentioning

confidence: 99%

“…Cell adhesion to adsorbed fibronectin was measured using a spinning disk device (Garcia et al, 1998a). HT1080 cells were incubated in serum-free DMEM medium at 37°C for 15-18 h, dissociated with EDTA, washed, and resuspended in complete PBS plus 2 mM glucose.…”

Section: Quantitative Adhesion Assaymentioning

confidence: 99%

“…Fibronectin coating was done by incubation with different concentrations of fibronectin for 30 min at room temperature followed by incubation with 1% heat-inactivated BSA for 30 min. Levels of adsorbed fibronectin were determined using 125 I-labeled fibronectin (Garcia et al, 1998a). Ethidium homodimer was obtained from Molecular Probes (Eugene, OR).…”

Section: Cells and Reagentsmentioning

confidence: 99%

“…Control experiments have demonstrated that i) cross-linked integrins are preferentially localized to focal adhesion-like structures, ii) crosslinked integrins contribute directly to an increase in adhesion strength, iii) cross-linking requires an activated form of ␣5␤1 that can bind fibronectin; and iv) cross-linking requires that the integrin be bound to its ligand. (Enomoto-Iwamoto et al, 1993;Garcia et al, 1998a;Garcia and Boettiger, 1999).Chemical cross-linking of HT1080 cells at 60 min after plating on fibronectin substrates demonstrated a linear relationship between the number of receptor-ligand bonds and the proportion of ␣5 (Figure 2A) and ␤1 ( Figure 2B), which could be cross-linked. Therefore the proportion of ␣5 and ␤1 integrin that can be cross-linked is a measure of the relative number of ␣5␤1-fibronectin bonds.…”

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confidence: 99%

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A Novel Mode for Integrin-mediated Signaling: Tethering Is Required for Phosphorylation of FAK Y397

Shi

Boettiger

2003

MBoC

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The common model for integrin mediated signaling is based on integrin clustering and the potential for that clustering to recruit signaling molecules including FAK and src. The clustering model for transmembrane signaling originated with the analysis of the EGF receptor signaling and remains the predominant model. The roles for substrate-bound ligand and ligand occupancy in integrin-mediated signaling are less clear. A kinetic model was established using HT1080 cells in which there was a linear relationship between the strength of adhesion, the proportion of ␣5␤1 integrin that could be chemically cross-linked, and the number of receptor-ligand bonds. This graded signal produced a similarly graded response measured by the level of specific phosphorylation of FAK Y397. FAK Y397 phosphorylation could also be induced by antibody bound to the substrate. In contrast, clustering of ␣5␤1 on suspended cells with either antibody to ␤1 or by clustering of soluble ligand bound to ␣5␤1 induced the phosphorylation of FAK Y861 but not Y397. There were no differences in signaling when activating antibodies were compared with blocking antibodies, presence or absence of ligand. Only tethering of ␣5␤1 to the substrate was required for induction of FAK Y397 phosphorylation. INTRODUCTIONThere are several classes of transmembrane receptors that are involved in the transmission of signals from the extracellular space across the plasma membrane. It is a logical requirement of these systems that ligand binding to the extracellular domain results in a change in the cytoplasmic domain. How the signal is transferred from the extracellular domain is basic to the generation of the intracellular downstream signals. Allosteric proteins have been described in which the occupation of a binding site on one side of the protein can result in a conformational change that affects other binding sites. The interposition of a lipid bilayer between the receiving and effector domains of the transmembrane receptors poses limitations in the application of the allosteric model. Although a few of the receptor tyrosine kinase receptor systems have been analyzed in detail, the mechanism for transmembrane signal transduction by other receptor classes is less well understood.The EGF receptor is the best understood model. Receptor dimerization is initiated by the binding of EGF to extracellular domain 1 altering the conformation of the extracellular domain to generate a dimerization of receptors mediated through domain 2. This dimerization brings the cytoplasmic domains of two EGF receptors into proximity and allows the cross-phosphorylation of cytoplasmic domains by the encoded tyrosine kinase (Schlessinger, 2000). The generation of signals through receptor dimerization is a general theme. Among the tyrosine kinase receptors the mechanisms of generating the dimer vary from the use of bivalent ligands for growth hormone and erythropoietin (Kossiakoff and de Vos, 1998;Jiang and Hunter, 1999), to dimeric ligands for PDGF and VEGF (Wiesmann et al., 1997), to complexe...

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mentioning

confidence: 96%

Section: Proportion Of Ligand-bound ␣5␤1 Integrin As a Function Of Fimentioning

confidence: 99%

Section: Quantitative Adhesion Assaymentioning

confidence: 99%

Section: Cells and Reagentsmentioning

confidence: 99%

mentioning

confidence: 99%

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A Novel Mode for Integrin-mediated Signaling: Tethering Is Required for Phosphorylation of FAK Y397

Shi

Boettiger

2003

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Biology Lessons for Engineering Surfaces for Controlling Cell–Material Adhesion

Lee

Garcı́a

2012

Biomimetic Approaches for Biomaterials Development

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IntroductionIn surface aspects, we discuss important biology lessons that are required for understanding cell-material adhesion. This chapter discusses the basics of the extracellular matrix (ECM), protein structure and adsorption to surfaces, and cellular communication and signaling proteins. It finally gives a review of current engineered cell adhesion technologies. These lessons are critical to understanding how to engineer biomaterial surfaces for use in both in vitro and in vivo applications. The Extracellular MatrixThis section provides a brief overview of the ECM and its functions. This overview provides knowledge important to understand the composition of the ECM and engineer synthetic ECMs to tune cell-fate-based processes.ECMs are composed of a complex, insoluble, three-dimensional mixture of secreted macromolecules, including collagens and noncollagenous proteins, such as elastin and fibronectin (FN), glycosaminoglycans, and proteoglycans, which are present between cells [1]. In addition, provisional fibrin-based networks constitute specialized matrices for wound healing and tissue repair. The ECM's function is to provide structure and order to the extracellular space and regulate multiple functions associated with the establishment, maintenance, and remodeling of differentiated tissues [2]. Matrix components such as FN and laminin mediate adhesive interactions that support cell anchorage, migration, and tissue organization. These all serve to activate signaling pathways that direct cell survival, proliferation, and differentiation.ECM components can interact with growth and differentiation factors, chemokines, and other soluble factors that regulate cell cycle progression and differentiation to control their availability and activity. By using various strategies to immobilize and order these proteins (ligands), ECMs control the spatial and Biomimetic Approaches for Biomaterials Development, First Edition. Edited by João F. Mano.

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A Novel Approach to Observing Synergy Effects of PHSRN on Integrin–RGD Binding Using Intelligent Surfaces

et al. 2008

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Force Required to Break α5β1Integrin-Fibronectin Bonds in Intact Adherent Cells Is Sensitive to Integrin Activation State

Cited by 140 publications

References 57 publications

A Novel Mode for Integrin-mediated Signaling: Tethering Is Required for Phosphorylation of FAK Y397

A Novel Mode for Integrin-mediated Signaling: Tethering Is Required for Phosphorylation of FAK Y397

Biology Lessons for Engineering Surfaces for Controlling Cell–Material Adhesion

A Novel Approach to Observing Synergy Effects of PHSRN on Integrin–RGD Binding Using Intelligent Surfaces

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