Coexisting conformations of fibronectin in cell culture imaged using fluorescence resonance energy transfer

Abstract

Fluorescence resonance energy transfer (FRET) between fluorophores attached to single proteins provides a tool to study the conformation of proteins in solution and in cell culture. As a protein unfolds, nanometer-scale increases in distance between donor and acceptor fluorophores cause decreases in FRET. Here we demonstrate the application of FRET to imaging coexisting conformations of fibronectin (Fn) in cell culture. Fn is a flexible 440-kDa extracellular matrix protein, with functional sites that are regulated by unfolding events. Fn was labeled with multiple donor and acceptor fluorophores such that intramolecular FRET could be used to distinguish a range of Fn conformations. The sensitivity of FRET to unfolding was tested by progressively denaturing labeled Fn using guanidium chloride. To investigate Fn conformation changes during cell binding and matrix assembly, we added labeled Fn to the culture medium of NIH 3T3 fibroblasts. Coexisting conformations of Fn were visualized using fluorescence microscopy, and spectra from specific features were measured with an attached spectrometer. Using FRET as an indicator of Fn conformation, Fn diffusely bound to cells was in a compact state, whereas Fn in matrix fibrils was highly extended. Matrix fibrils exhibited a range of FRET that suggested some degree of unfolding of Fn's globular modules. Fn in cell-associated clusters that preceded fibril formation appeared more extended than diffuse cell-bound Fn but less extended than fibrillar Fn, suggesting that Fn undergoes extension after cell binding and before polymerization. FRET thus provides an approach to gain insight into the integrin-mediated pathway of Fn fibrillogenesis. Imaging tools that discriminate between protein conformations in cell culture are essential for insight into how changes in protein structure modulate cell signaling. Fluorescence resonance energy transfer (FRET) has emerged to fill a void in techniques that provide high-resolution structural information on proteins adsorbed to surfaces or in cell culture. FRET is the nonradiative transfer of excitation energy from a fluorescent molecule, called the donor, to another molecule, called the acceptor (1, 2). It is generally detected as an increase in acceptor emission with a concomitant decrease in donor emission. Energy transfer from donor to acceptor fluorophore decays rapidly with increasing distance (the rate is inversely proportional to the sixth power of the separation), resulting in nanometer-scale sensitivity for distances up to 10 nm. By using fluorescently labeled proteins, FRET has been applied to detect protein-protein association on cell surfaces (3, 4) as well as unfolding transitions of proteins in solution (5, 6). Here we demonstrate FRET applied to imaging coexisting protein conformations in cell culture.Fibronectin (Fn) was chosen as a model protein because it undergoes dramatic conformational changes that control its biological activity, yet the underlying molecular mechanisms are largely unknown. In blood and when secreted by c...

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

“…These data on the total range of extensibility of manually deposited Fn fibers further support recent conclusions that native, cell-derived Fn matrices are unfolded by cell traction forces (Baneyx et al, 2001;Baneyx et al, 2002;Smith et al, 2007). FRET measurements of duallabeled Fn matrices supported a model whereby Fn fiber elasticity involves unfolding of typeIII modules (Baneyx et al, 2001;Baneyx et al, 2002).…”

“…Native, cell-derived Fn matrices, however, are not ideal systems for investigating forceinduced potentially altered structure-function relationships. A key complication is the complex and interwoven nature of ECM fibers, where the conformation of Fn can vary from fiber to fiber and even within fibers as they are not freely suspended (Baneyx et al, 2001;Baneyx et al, 2002;Chen et al, 1997;Peters et al, 1998).…”

Differential Contributions of Conformation Extension and Domain Unfolding to Properties of Fibronectin Nanotextiles

“…10,16 In the second model, relaxed FN matrix fibers contain FN molecules that are already in an extended conformation, and elasticity is due to individual FN III domains that unfold and refold within the fibril. [17][18] We report results in support of the first model and found that FN elastically deforms at low extensions (λ < 2) due to conformational unfolding. Beyond this threshold, FN plastically deforms due to domain unfolding.…”

“…In the second model, relaxed FN matrix fibers contain polymerized FN that is already in an extended conformation, where elasticity is due to individual FN III domains that unfold and refold within the fibril. [17][18] We reasoned FN fibers would display different and distinguishable mechanical properties during conformational and domain unfolding. To test our hypothesis, we manufactured FN fabrics by a previously published method that requires assembly of protein fabrics on a surface with microcontact printing and then its subsequent release.…”

“…1,[17][18][22][23] We measured the temporal changes in fluorescence resonance energy transfer (FRET) intensity before, during, and after the labeled fabrics were released from the PIPAAm substrate (Figure 2a). We observed an exponential increase in the ratio between the acceptor and donor emission intensities (I A /I D ) until fabrics were freed from the PIPAAm substrate and contraction was completed (t= 560 sec, Figure 2b We asked how the secondary structural changes of FN affect the bulk mechanical properties of the released FN fabrics.…”