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

Abstract

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.

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

“…Circular dichroism spectroscopy of FRET-labeled Fn confirmed that refolding occurs in physiological buffer (Smith et al, 2007).…”

“…Variations of the pulling rate by which the fibers were drawn out of solution, from 1.0 mm/s to 0.2 mm/s, had no major impact on the conformational distribution (data not shown). The width of the histogram originates from the pixel-to-pixel variations of the local averaged Fn conformation and from instrument noise (as further discussed in Smith et al, 2007). The widths of the histograms taken from Fn-d/a in solution (1 M GdnHCl; data not shown) and in manually deposited fibers (Fig.…”

“…On the extracellular side, fibronectin (Fn) can be unfolded by cell generated tension which causes at least a partial loss of secondary structure of its domains (Baneyx et al, 2002;Smith et al, 2007). Fn exists in a broad range of conformations, once it is harvested by cells from solution and incorporated into their own matrix as revealed by fluorescence resonance energy transfer (FRET) (Baneyx et al, 2002;Barker et al, 2005;Smith et al, 2007).…”

“…Fn exists in a broad range of conformations, once it is harvested by cells from solution and incorporated into their own matrix as revealed by fluorescence resonance energy transfer (FRET) (Baneyx et al, 2002;Barker et al, 2005;Smith et al, 2007).…”

Mechanism of Membrane Interaction and Disruption by α-Synuclein

“…They offer an attractive model for the study of protein aggregation on membranes due to their simplicity compared to cell membranes and their ease of formation on solid supports, making them ideal for use with surface sensitive imaging techniques. Furthermore labeling with donor and acceptor fluorophores allows inter-protein, 29 intra-protein 30 and also protein-lipid interactions 31 to be studied by Förster resonance energy transfer (FRET). Traditional microscopy techniques cannot easily distinguish between fluorescence from surface-bound molecules and background signal from fluorophores in the solution.…”