The Compact Conformation of Fibronectin Is Determined by Intramolecular Ionic Interactions

Johnson, Kamin J.; Sage, Harvey J.; Briscoe, Gina; Erickson, Harold P.

doi:10.1074/jbc.274.22.15473

Cited by 171 publications

(223 citation statements)

References 34 publications

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“…Additional decreases in FRET at Ͼ2 M Gdn⅐HCl indicated further Fn unfolding. In these stronger denaturing conditions, it has been shown that Fn modules progressively unfold (17). Thus, decreases in FRET at Ͼ2 M Gdn⅐HCl were likely the result of donor-acceptor separation caused by module unfolding.…”

Section: Resultsmentioning

confidence: 99%

“…The first model proposes that Fn dimers within fibrils are structured such that their N-terminal regions are aligned with the fibril axis, and C-terminal regions fold on themselves normal to the fibril axis. Mechanical tension pulls the molecule into alignment with the fibril axis, causing the fibril to stretch (14,17). According to this model, the level of FRET in fibrillar Fn-D͞A under tension should be larger or equal to that of Fn-D͞A extended with its FnIII modules intact-the level of FRET in 0.5 M to 2 M Gdn⅐HCl, 60% to 75% (Fig.…”

Section: Discussionmentioning

confidence: 99%

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Fibronectin extension and unfolding within cell matrix fibrils controlled by cytoskeletal tension

Baneyx

Baugh

Vogel

2002

Proc. Natl. Acad. Sci. U.S.A.

333

306

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Evidence is emerging that mechanical stretching can alter the functional states of proteins. Fibronectin (Fn) is a large, extracellular matrix protein that is assembled by cells into elastic fibrils and subjected to contractile forces. Assembly into fibrils coincides with expression of biological recognition sites that are buried in Fn's soluble state. To investigate how supramolecular assembly of Fn into fibrillar matrix enables cells to mechanically regulate its structure, we used fluorescence resonance energy transfer (FRET) as an indicator of Fn conformation in the fibrillar matrix of NIH 3T3 fibroblasts. Fn was randomly labeled on amine residues with donor fluorophores and site-specifically labeled on cysteine residues in modules FnIII7 and FnIII15 with acceptor fluorophores. Intramolecular FRET was correlated with known structural changes of Fn in denaturing solution, then applied in cell culture as an indicator of Fn conformation within the matrix fibrils of NIH 3T3 fibroblasts. Based on the level of FRET, Fn in many fibrils was stretched by cells so that its dimer arms were extended and at least one FnIII module unfolded. When cytoskeletal tension was disrupted using cytochalasin D, FRET increased, indicating refolding of Fn within fibrils. These results suggest that cell-generated force is required to maintain Fn in partially unfolded conformations. The results support a model of Fn fibril elasticity based on unraveling and refolding of FnIII modules. We also observed variation of FRET between and along single fibrils, indicating variation in the degree of unfolding of Fn in fibrils. Molecular mechanisms by which mechanical force can alter the structure of Fn, converting tensile forces into biochemical cues, are discussed. E xtracellular matrices (ECM) are complex supramolecular assemblies that control cell signaling and behavior. While many ECM proteins have been characterized, little is known about how matrix assembly alters their structure and confers functions not present in individual proteins. Less is known about mechanisms by which cell contractile forces applied to protein assemblies regulate protein function. Many ECM proteins, such as fibronectin (Fn), laminin, and thrombospondin, are large (Ͼ100 kDa), multifunctional proteins composed of repeating, structurally defined modules often less than 100 aa each. The multimodular structure may serve as a convenient way to integrate multiple functions in one molecule. For example, some modules carry cell adhesion sites, whereas others carry sites for binding other proteins and for self-assembly. Exposure of functional sites may be controlled by the organization of such sites in extracellular matrices, and by the application of force by cells (1-3). However, the molecular mechanisms regulating ECM protein assembly and the exposure of binding sites remain unclear.Fn is an ECM protein that undergoes cell-mediated assembly into insoluble, elastic fibrils. In blood and when secreted by cells such as fibroblasts, Fn exists as a soluble dimer. The two strand...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Fibronectin extension and unfolding within cell matrix fibrils controlled by cytoskeletal tension

Baneyx

Baugh

Vogel

2002

Proc. Natl. Acad. Sci. U.S.A.

333

306

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“…The compact conformation of FN dimers is maintained by intramolecular interactions involving the type III 12-14 repeats of FN. 53,54 The type III [12][13][14] repeats, also known as heparin II binding domain, interact with the HS chains from various members of the Sdc family. 55 Binding of Sdc1 with heparin II may facilitate unfolding of dimeric FNs and expose FN self-assembly (FN-binding) sites, thus promoting FN deposition and fibrillogenesis.…”

Section: Discussionmentioning

confidence: 99%

Syndecan-1 in Breast Cancer Stroma Fibroblasts Regulates Extracellular Matrix Fiber Organization and Carcinoma Cell Motility

Yang

Mosher

Seo

et al. 2011

The American Journal of Pathology

125

126

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Stromal fibroblasts of breast carcinomas frequently express the cell surface proteoglycan syndecan-1 (Sdc1). In human breast carcinoma samples, stromal Sdc1 expression correlates with an organized, parallel, extracellular matrix (ECM) fiber architecture. To examine a possible link between stromal Sdc1 and the fiber architecture, we generated bioactive cell-free three-dimensional ECMs from cultures of Sdc1-positive and Sdc1-negative murine and human mammary fibroblasts (termed ECM-Sdc1 and ECM-mock, respectively). Indeed, ECM-Sdc1 showed a parallel fiber architecture that contrasted markedly with the random fiber arrangement of ECM-mock. When breast carcinoma cells were seeded into the fibroblast-free ECMs, ECM-Sdc1, but not ECM-mock, promoted their attachment, invasion, and directional movement. We further evaluated the contribution of the structural/compositional modifications in ECM-Sdc1 on carcinoma cell behavior. By microcontact printing of culture surfaces, we forced the Sdc1-negative fibroblasts to produce ECM with parallel fiber organization, mimicking the architecture observed in ECM-Sdc1. We found that the fiber topography governs carcinoma cell migration directionality. Conversely, an elevated fibronectin level in ECM-Sdc1 was responsible for the enhanced attachment of the breast carcinoma cells. These observations suggest that Sdc1 expression in breast carcinoma stromal fibroblasts promotes the assembly of an architecturally abnormal ECM that is permissive to breast carcinoma directional migration and invasion. Epithelial-stromal interactions play crucial roles in directing mammary gland development and in maintaining normal tissue homeostasis. Conversely, during tumorigenesis, the stroma accelerates carcinoma growth and progression. The predominant cell type within the stromal compartment is the fibroblast, which synthesizes, organizes, and maintains a three-dimensional (3D) network of glycoproteins and proteoglycans known as the extracellular matrix (ECM). Normal stromal fibroblasts and their ECM are believed to exert an inhibitory constraint on tumor growth and progression. 1,2 Major alterations occur in the stromal fibroblasts and ECM during neoplastic transformation, giving rise to a permissive and supportive microenvironment for carcinomas. Compared with their quiescent normal counterpart, carcinoma-associated fibroblasts display an activated phenotype, which is characterized by the expression of smooth muscle markers, an enhanced proliferative and migratory potential, and altered gene expression profiles. Carcinoma-associated fibroblasts produce and deposit elevated amounts and abnormal varieties of ECM components. 3-5 Recent evidence 6,7 indicates that not only ECM composition but also ECM architecture are altered in carcinomas and that these changes may promote tumor progression. However, the contribution of these stromal modifications to tumor development and the molecular mechanisms and signaling events underlying these alterations are incompletely understood.

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“…Under physiological conditions, FN adheres more strongly in its compact conformation on hydrophobic surfaces 39 , which may also cause subsequent denaturation of the FN secondary structure to increase substrate binding affinity 40 . The compact conformation of FN is stabilized by intermolecular bonds 41 but can be disrupted by interacting surface groups, particularly hydrophilic and negatively charged surfaces, causing the protein to adopt a more unfolded conformation 25,42 . In this conformation, the protein can interact via different domain regions that are specific to various ECM proteins (e.g.…”

Section: Resultsmentioning

confidence: 99%

Quantifying fibronectin adhesion with nanoscale spatial resolution on glycosaminoglycan doped polypyrrole using Atomic Force Microscopy

Gelmi

Higgins

Wallace

2013

Biochimica et Biophysica Acta (BBA) - General Subjects

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The interaction of ECM proteins is critical in determining the performance of materials used in biomedical applications such as tissue regeneration, implantable bionics and biosensing. Methods: To improve our understanding of ECM protein-conducting polymer interactions, we have used Atomic Force Microscopy (AFM) to elucidate the interactions of fibronectin (FN) on polypyrrole (PPy) doped with different glycosaminoglycans. Results: We were able to classify four main types of FN interactions, including those related to 1) non-specific adhesion, 2) protein unfolding and subsequent unbinding from the surface, 3) desorption and 4) interactions with no adhesion. FN adhesion on PPy/hyaluronic acid showed a significantly lower density of surface adhesion with the adhesion restricted to nodule structures, as opposed to their peripheries, of the polymer morphology. In contrast, PPy/chondroitin sulfate showed a significantly higher density of surface adhesion to the point where the distribution of adhesion effectively masked the topography. Through conductive AFM imaging, we found that the conductive regions correlated with regions of FN adhesion. Conclusions: Given that the conductivity requires doping of the polymer, these findings suggest that FN adhesion is mediated by interactions with chondroitin sulfate and hyaluronic acid at the polymer surface and may be indicative of specific interactions due to contributions from electrostatic attraction between the FN and sulfate/anionic groups of the dopants. General significance: This study demonstrates the ability of AFM to resolve the protein-conducting polymer interactions at the molecular and nanoscale level, which will be important for interfacing these polymer materials with biological systems. This article is part of a Special Issue entitled Organic Bioelectronics -Novel Applications in Biomedicine.

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The Compact Conformation of Fibronectin Is Determined by Intramolecular Ionic Interactions

Cited by 171 publications

References 34 publications

Fibronectin extension and unfolding within cell matrix fibrils controlled by cytoskeletal tension

Fibronectin extension and unfolding within cell matrix fibrils controlled by cytoskeletal tension

Syndecan-1 in Breast Cancer Stroma Fibroblasts Regulates Extracellular Matrix Fiber Organization and Carcinoma Cell Motility

Quantifying fibronectin adhesion with nanoscale spatial resolution on glycosaminoglycan doped polypyrrole using Atomic Force Microscopy

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