Single molecule fluorescence studies of surface-adsorbed fibronectin

Antia, Meher; Islas, León D; Boness, David; Baneyx, Gretchen; Vogel, Viola

doi:10.1016/j.biomaterials.2005.06.014

Cited by 33 publications

(33 citation statements)

References 42 publications

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“…Obviously, this relies upon having differently reactive cysteines which is not always possible [50]. If structural information is available, then it may be possible to selectively label a cysteine following selection of different conformations [4]. A relatively new approach takes advantage of the fact that cysteine also bind to metal ions.…”

Section: Direct Labeling With Organic Fluorophoresmentioning

confidence: 99%

Fluorescent labeling and modification of proteins

2013

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This review provides an outline for fluorescent labeling of proteins. Fluorescent assays are very diverse providing the most sensitive and robust methods for observing biological processes. Here, different types of labels and methods of attachment are discussed in combination with their fluorescent properties. The advantages and disadvantages of these different methods are highlighted, allowing the careful selection for different applications, ranging from ensemble spectroscopy assays through to single-molecule measurements.

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Section: Direct Labeling With Organic Fluorophoresmentioning

confidence: 99%

Fluorescent labeling and modification of proteins

2013

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“…Therefore, cys/cys Fn-DA is sensitive to intermonomer energy transfer in the compact conformation. However, energy transfer between opposing arms cannot occur when the arms are separated [24,25]. The spatially resolved ratio of acceptor to donor intensities ( I A / I D ) was quantified using both labeling schemes within the ECM of living fibroblasts cultured on glass.…”

Section: Introductionmentioning

confidence: 99%

Force-Induced Unfolding of Fibronectin in the Extracellular Matrix of Living Cells

et al. 2007

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Whether mechanically unfolded fibronectin (Fn) is present within native extracellular matrix fibrils is controversial. Fn extensibility under the influence of cell traction forces has been proposed to originate either from the force-induced lengthening of an initially compact, folded quaternary structure as is found in solution (quaternary structure model, where the dimeric arms of Fn cross each other), or from the force-induced unfolding of type III modules (unfolding model). Clarification of this issue is central to our understanding of the structural arrangement of Fn within fibrils, the mechanism of fibrillogenesis, and whether cryptic sites, which are exposed by partial protein unfolding, can be exposed by cell-derived force. In order to differentiate between these two models, two fluorescence resonance energy transfer schemes to label plasma Fn were applied, with sensitivity to either compact-to-extended conformation (arm separation) without loss of secondary structure or compact-to-unfolded conformation. Fluorescence resonance energy transfer studies revealed that a significant fraction of fibrillar Fn within a three-dimensional human fibroblast matrix is partially unfolded. Complete relaxation of Fn fibrils led to a refolding of Fn. The compactly folded quaternary structure with crossed Fn arms, however, was never detected within extracellular matrix fibrils. We conclude that the resting state of Fn fibrils does not contain Fn molecules with crossed-over arms, and that the several-fold extensibility of Fn fibrils involves the unfolding of type III modules. This could imply that Fn might play a significant role in mechanotransduction processes.

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“…First indications also exist that downstream cell signaling is impacted by the conformation of Fn Lan et al, 2005). When adsorbed from solution to biomaterial surfaces, its conformation is for example impacted by the underlying surface chemistry (Altroff et al, 2003;Antia et al, 2006;Baugh and Vogel, 2004;Garcia et al, 1999;Halter et al, 2005) which can then alter integrin binding . The conformations Fn assumes on synthetic surfaces, however, are rather distinct from those of native Fn in soluble and fibrillar forms (reviewed in Antia et al, 2006;Baugh and Vogel, 2004;Halter et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

“…When adsorbed from solution to biomaterial surfaces, its conformation is for example impacted by the underlying surface chemistry (Altroff et al, 2003;Antia et al, 2006;Baugh and Vogel, 2004;Garcia et al, 1999;Halter et al, 2005) which can then alter integrin binding . The conformations Fn assumes on synthetic surfaces, however, are rather distinct from those of native Fn in soluble and fibrillar forms (reviewed in Antia et al, 2006;Baugh and Vogel, 2004;Halter et al, 2005). Fn polymerization into fibers is thought to proceed as cryptic self-assembly sites are exposed by cell contractility (Baneyx and Vogel, 1999;Mao and Schwarzbauer, 2005;Ohashi et al, 1999;Zhong et al, 1998).…”

Section: Introductionmentioning

confidence: 99%

Assay to mechanically tune and optically probe fibrillar fibronectin conformations from fully relaxed to breakage

et al. 2008

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In response to growing needs for quantitative biochemical and cellular assays that address whether the extracellular matrix (ECM) acts as a mechanochemical signal converter to co-regulate cellular mechanotransduction processes, a new assay is presented where plasma fibronectin fibers are manually deposited onto elastic sheets, while force-induced changes in protein conformation are monitored by fluorescence resonance energy transfer (FRET). Fully relaxed assay fibers can be stretched at least 5-6 fold, which involves Fn domain unfolding, before the fibers break. In native fibroblast ECM, this full range of stretch-regulated conformations coexists in every field of view confirming that the assay fibers are physiologically relevant model systems. Since alterations of protein function will directly correlate with their extension in response to force, the FRET vs. strain curves presented herein enable the mapping of fibronectin strain distributions in 2D and 3D cell cultures with high spatial resolution. Finally, cryptic sites for fibronectin's N-terminal 70-kD fragment were found to be exposed at relatively low strain, demonstrating the assay's potential to analyze stretch-regulated protein-rotein interactions.

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Single molecule fluorescence studies of surface-adsorbed fibronectin

Cited by 33 publications

References 42 publications

Fluorescent labeling and modification of proteins

Fluorescent labeling and modification of proteins

Force-Induced Unfolding of Fibronectin in the Extracellular Matrix of Living Cells

Assay to mechanically tune and optically probe fibrillar fibronectin conformations from fully relaxed to breakage

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