Article

Little, W. C., Smith, M. L., Ebneter, U. & Vogel, V. Assay to mechanically tune and optically probe fibrillar fibronectin conformations from fully relaxed to breakage. Matrix Biol. 27, 451-461

Department of Materials, ETH Zurich, CH-8093, Zürich, Switzerland.
Matrix Biology (Impact Factor: 3.65). 07/2008; 27(5):451-61. DOI: 10.1016/j.matbio.2008.02.003
Source: PubMed

ABSTRACT 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-protein interactions.

Download full-text

Full-text

Available from: Michael L Smith, Aug 01, 2015
0 Followers
 · 
95 Views
  • Source
    • "Finally, previous work from our group using FRET based fibronectin probes [14] has shown that the molecular conformations that fibronectin molecules assume within manually pulled fibers, which have been tuned by stretching/relaxing to experience a range of strain values from 0% to 500% as described above, is similar to the conformation range of fibronectin within cellderived fibrils in the extracellular matrix. The similarity in the molecular configuration of fibronectin on cell-derived and manually pulled fibers is intriguing if one takes into consideration the different processes involved during fibrillogenesis. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Fibronectin is a globular protein that circulates in the blood and undergoes fibrillogenesis if stretched or under other partially denaturing conditions, even in the absence of cells. Stretch assays made by pulling fibers from droplets of solutions containing high concentrations of fibronectin have previously been introduced in mechanobiology, particularly to ask how bacteria and cells exploit the stretching of fibronectin fibers within extracellular matrix to mechano-regulate its chemical display. Our electron microscopy analysis of their ultrastructure now reveals that the manually pulled fibronectin fibers are composed of densely packed lamellar spirals, whose interlamellar distances are dictated by ion-tunable electrostatic interactions. Our findings suggest that fibrillogenesis proceeds via an irreversible sheet-to-fiber transition as the fibronectin sheet formed at the air-liquid interface of the droplet is pulled off by a sharp tip. This far from equilibrium process is driven by the externally applied force, interfacial surface tension, shear-induced fibronectin self-association, and capillary force-induced buffer drainage. The ul-trastructural characterization is then contrasted with previous FRET studies that characterized the mo-lecular strain within these manually pulled fibers. Particularly relevant for stretch-dependent binding studies is the finding that the interior fiber surfaces are accessible to nanoparticles smaller than 10 nm. In summary, our study discovers the underpinning mechanism by which highly hierarchically structured fibers can be generated with unique mechanical and mechano-chemical properties, a concept that might be extended to other bio-or biomimetic polymers.
    Biomaterials 10/2014; 36. DOI:10.1016/j.biomaterials.2014.08.012 · 8.31 Impact Factor
  • Source
    • "Given the large size of Fn exceeding 100 nm, multiple donors and acceptors per dimer were used to increase the range of conformational changes that can be detected by FRET even though this prevents us from calculating distances from FRET efficiencies. Alexa Fluor 488 fluorophores (FRET donors) were randomly bound to amines via succinimidyl ester chemistry and Alexa Fluor 546 fluorophores (FRET acceptors) were specifically targeted to the cryptic cysteins on the Fn type III modules FnIII 7 and FnIII 15 via maleimide chemistry (Molecular Probes), as previously described (Baneyx et al., 2002; Smith et al., 2007; Little et al., 2008; Kubow et al., 2009; Legant et al., 2012). The donor/acceptor labeling ratio of this Fn-DA was determined by measuring the absorbance of at 280, 498 and 556 nm. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Early wound healing is associated with fibroblasts assembling a provisional fibronectin-rich extracellular matrix (ECM), which is subsequently remodeled and interlaced by type I collagen. This exposes fibroblasts to time-variant sets of matrices during different stages of wound healing. Our goal was thus to gain insight into the ECM-driven functional regulation of human foreskin fibroblasts (HFFs) being either anchored to a fibronectin (Fn) or to a collagen-decorated matrix, in the absence or presence of cyclic mechanical strain. While the cells reoriented in response to the onset of uniaxial cyclic strain, cells assembled exogenously added Fn with a preferential Fn-fiber alignment along their new orientation. Exposure of HFFs to exogenous Fn resulted in an increase in matrix metalloproteinase (MMP) expression levels, i.e. MMP-15 (RT-qPCR), and MMP-9 activity (zymography), while subsequent exposure to collagen slightly reduced MMP-15 expression and MMP-9 activity compared to Fn-exposure alone. Cyclic strain upregulated Fn fibrillogenesis and actin stress fiber formation, but had comparatively little effect on MMP activity. We thus propose that the appearance of collagen might start to steer HFFs towards homeostasis, as it decreased both MMP secretion and the tension of Fn matrix fibrils as assessed by Fluorescence Resonance Energy Transfer. These results suggest that HFFs might have a high ECM remodeling or repair capacity in contact with Fn alone (early event), which is reduced in the presence of Col1 (later event), thereby down-tuning HFF activity, a processes which would be required in a tissue repair process to finally reach tissue homeostasis.
    Matrix Biology 09/2014; 40. DOI:10.1016/j.matbio.2014.09.001 · 3.65 Impact Factor
  • Source
    • "Pulling of fibronectin fibers from the air-liquid interface Fibronectin is known to form stable, insoluble monolayers at airliquid interfaces [38] [39]. Based on previous work ([14] [16]), a fiber can be drawn by pulling a sharp tip away from a fibronectin monolayer that had formed at the surface of a droplet of concentrated plasma fibronectin (0.4 mg/ml) in PBS solution (Fig. 1A). At first, the droplet surface deforms due to capillary forces that tend to minimize the total liquid surface area. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Fibronectin is a globular protein that circulates in the blood and undergoes fibrillogenesis if stretched or under other partially denaturing conditions, even in the absence of cells. Stretch assays made by pulling fibers from droplets of solutions containing high concentrations of fibronectin have previously been introduced in mechanobiology, particularly to ask how bacteria and cells exploit the stretching of fibronectin fibers within extracellular matrix to mechano-regulate its chemical display. Our electron microscopy analysis of their ultrastructure now reveals that the manually pulled fibronectin fibers are composed of densely packed lamellar spirals, whose interlamellar distances are dictated by ion-tunable electrostatic interactions. Our findings suggest that fibrillogenesis proceeds via an irreversible sheet-to-fiber transition as the fibronectin sheet formed at the air-liquid interface of the droplet is pulled off by a sharp tip. This far from equilibrium process is driven by the externally applied force, interfacial surface tension, shear-induced fibronectin self-association, and capillary force-induced buffer drainage. The ultrastructural characterization is then contrasted with previous FRET studies that characterized the molecular strain within these manually pulled fibers. Particularly relevant for stretch-dependent binding studies is the finding that the interior fiber surfaces are accessible to nanoparticles smaller than 10 nm. In summary, our study discovers the underpinning mechanism by which highly hierarchically structured fibers can be generated with unique mechanical and mechano-chemical properties, a concept that might be extended to other bio- or biomimetic polymers.
    Biomaterials 01/2014; · 8.31 Impact Factor
Show more