Fibronectin (Fn), a major component of the extracellular matrix (ECM) of developing tissues and healing wounds, is a large, dimeric protein consisting of more than 50 repeating subunits (for review see [1
]). Fn displays a number of surface-exposed molecular recognition sites for cells, including integrin binding sites such as the RGD loop, PHSRN synergy site, and LDV sequence, and binding sites for other ECM components, including collagen, heparin, and fibrin. Together these binding sites provide Fn with a diverse array of scaffolding and cell recognition functions. In addition, a number of cryptic binding sites, sequences normally buried in the equilibrium fold of the protein, and surface-exposed binding sites have been proposed to be exposed or deactivated, respectively, as a result of force-dependent conformational change (as reviewed in [2
]). It has therefore been hypothesized that in addition to other physical properties of the ECM such as substrate rigidity and matrix composition, matrix unfolding may alter outside-in cell signaling.
Soluble Fn in physiological buffer has a compact, folded quaternary structure (A) stabilized through intermonomer ionic interactions between III2–3
of one arm and III12–14
of the opposing arm [7
]. Low concentrations of chemical denaturants first destabilize these ionic interactions, leading to separation of the crossed-over arms (extended structure; B), and increasing denaturant concentrations finally unfold Fn (C). Erickson originally proposed that module unfolding constituted the mechanism for fibril extensibility by estimating the free energy of denaturation and extension of Fn type III (FnIII) modules in comparison to the force generated by single myosin or kinesin motor proteins [8
]. Yet the conformation of Fn within fibrillar ECM is still debated [8
]. Two structural models to explain the several-fold, force-induced extension of Fn within fibrils have been proposed:
Schematic Sketch of Putative Fn Conformations in Solution and within ECM Fibrils
The quaternary structure model proposes that Fn within fully relaxed fibrils assumes a compact structure with crossed-over Fn arms similar to that found in solution (D; [12
]). Under the influence of cell traction, tensile force would first separate the Fn arms, and finally align them along the force vector (E) with still intact tertiary/secondary structures of individual modules (F; [12
]). This model postulates that the fibrils break before Fn starts unfolding.
The unfolding model for Fn elongation proposes that fully relaxed fibrils are composed of Fn in an already extended conformation where the dimeric arms are already separated (F) and that fibril extension originates from the unfolding of FnIII modules (G). Type I and II modules are stabilized by intramodular disulfide bonds, and therefore only FnIII modules can be completely unfolded by force. FnIII modules have been shown to unfold by passing through several intermediate states [13
While previous fluorescence resonance energy transfer (FRET) studies indicated that cell contractility is sufficient to unfold fibrillar Fn [9
], the alternative quaternary structural model was proposed based on the following two observations. First, a single-molecule Fn–green fluorescent protein (GFP) study [12
] showed that the mechanical stabilities of GFP and the FnIII module are similar over the range of pulling velocities tested (50 to 1,745 nm/s). Second, if Fn-GFP is assembled by cells into micro-sized ECM fibers, stretched Fn-GFP fibrils are reported to maintain a constant integrated level of fluorescence even after contraction to 1/3 or 1/4 their starting length [11
]. Combining this information derived from single-molecule mechanics with spectroscopic data obtained from a densely packed Fn-GFP fiber, it was concluded that FnIII modules do not unfold in Fn fibrils under the influence of cell traction forces since GFP fluorescence was independent of the extent to which the Fn fibril was strained [11
]. However, it is unclear whether Fn-GFP embedded in a densely packed fiber may have an altered mechanical stability compared to single-molecule Fn-GFP.
Determining whether Fn is indeed unfolded in ECM fibrils in vivo by cell contractile forces is essential to understand the molecular mechanism of Fn fibrillogenesis and whether exposure of the numerous molecular recognition and cryptic sites alters Fn function in a strain-dependent manner [1
]. Since no experimental techniques were available to directly probe the loss of tertiary/secondary structure of Fn in cell culture, intramolecular FRET was used to gain conformational information [9
]. Here, two Fn labeling schemes for FRET were utilized. Amine/cysteine FRET-labeled Fn (amine/cys Fn-DA) was produced by labeling plasma Fn on all four free cysteines within III7
(yellow modules in ) with Alexa 546 acceptors and on seven random amines with Alexa 488 donors. In the second labeling approach, only the free cysteines per Fn dimer were randomly labeled with two donors and two acceptors (cysteine/cysteine FRET-labeled Fn [cys/cys Fn-DA]). The Förster radius of this fluorophore pair is ~6 nm (from Invitrogen); therefore, energy transfer is limited to within 12 nm of III7
(yellow fading spheres in ). Amine/cys Fn-DA provides sensitivity to the full conformational range since energy transfer can occur between the crossed arms (intermonomer FRET; A and D) and along the arms (intramonomer FRET; B, C, and 1E–1G). The crossover of Fn arms in the fully compact conformation brings the fluorophores attached to III7
of opposing arms into close proximity (see A and D). 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
]. The spatially resolved ratio of acceptor to donor intensities (IA
) was quantified using both labeling schemes within the ECM of living fibroblasts cultured on glass.