Probing the Folded State of Fibronectin Type III Domains in Stretched Fibrils by Measuring Buried Cysteine Accessibility

Abstract

Fibronectin (FN) is an extracellular matrix protein that is assembled into fibrils by cells during tissue morphogenesis and wound healing. FN matrix fibrils are highly elastic, but the mechanism of elasticity has been debated: it may be achieved by mechanical unfolding of FN-III domains or by a conformational change of the molecule without domain unfolding. Here, we investigate the folded state of FN-III domains in FN fibrils by measuring the accessibility of buried cysteines. Four of the 15 FN-III domains (III-2, -3, -9, and -11) appear to unfold in both stretched fibrils and in solution, suggesting that these domains spontaneously open and close even in the absence of tension. Two FN-III domains (III-6 and -12) appear to unfold only in fibrils and not in solution. These results suggest that domain unfolding can at best contribute partially to the 4-fold extensibility of fibronectin fibrils. Fibronectin (FN)2 is a dimer of a 250-kDa protein that forms insoluble fibrils that are a critical component of the extracellular matrix. It is made up of a series of three domain types connected end-to-end (see Fig. 1A) (1). The N terminus of FN consists of nine FN-I and two FN-II domains, each of which contains an internal disulfide bond. The C terminus of FN consists of three FN-I domains and contains intermolecular disulfides that link the two FN monomers to form the dimer. The central portion of the FN molecule is made up of 15 tandem FN-III domains; these domains each consist of seven ␤-strands folded into a ␤-sandwich structure and contain no internal disulfides (see Fig. 1B). FN in solution adopts a compact conformation in which the second to fourth FN-III domains (III-2-4) of one subunit interact with the 12 th to 14 th FN-III domains (III-12-14) of the other, causing the FN dimer to fold onto itself (see Fig. 1A) (2).FN interacts with both cells and other extracellular matrix proteins. The N-terminal domains contain binding sites for the extracellular matrix proteins collagen and fibrin, whereas the ninth and tenth FN-III domains bind to integrins at the cell surface (3-6). Upon attachment to cells, FN is stretched and assembled into fibrils by cell contraction: matrix assembly is inhibited if cell contractility is inhibited (7) or if tension within the matrix is released (8). Although the exact mechanism of FN fibril formation is still unknown, studies have shown that FN fibrils are stretched to up to 4 times their resting length by cells and are highly elastic structures (9 -12). This elasticity requires cell contraction; disruption of cytoskeletal contraction relaxes stretched fibrils (10).Two different mechanisms have been proposed to explain the elasticity of FN matrix fibrils (see Fig. 1A) (13). One theory contends that the elasticity is a function of FN-III domains unfolding under tension (see Fig. 1A, mechanism 2) (14, 15). These domains contain no internal disulfides and can be mechanically unfolded by atomic force microscopy (16, 17). It is unclear how relevant these atomic force microscopy pulling force...

Mechanotransduction Dynamics at the Cell-Matrix Interface

“…However, our model clearly demonstrates a maximal fourfold elasticity of fibrils, which is consistent with experimental observations (34). Previous work by the senior author quantified the force needed to open a single Type III domain and argued that the force needed to stretch each domain fourfold is greater than physiologically relevant forces (59). However, this calculation only examined a single FN molecule and did not consider an entire FN fibril, as our current model does.…”

Finding the weakest link – exploring integrin-mediated mechanical molecular pathways

“…Upon stretching, fibronectin fibers can extend up to eightfold before breaking (Klotzsch et al, 2009), which leads to unfolding of the FNIII domains and exposure of cryptic sites. This unfolding, however, might account for only a fraction of the fibronectin stretch, which could be mostly determined by a global conformational change (Lemmon et al, 2011). In any case, stretching of FNIII domains is a form of mechanotransduction, because the exposure of the cryptic sites leads to downstream effects, such as the regulation of the fibrillogenesis of fibronectin (Sechler et al, 2001), stimulation of cell growth and contractility (Hocking and Kowalski, 2002), and stimulation of the digestion of gelatin, type IV collagen, a-and bcasein and insulin b-chain (Schnepel and Tschesche, 2000).…”

Studying early stages of fibronectin fibrillogenesis in living cells by atomic force microscopy

“…Important insight into FNIII domain unfolding has been provided by fluorescence resonance energy transfer (FRET) spectroscopy in cell culture studies and in vitro stretching experiments (Baneyx et al, 2001(Baneyx et al, , 2002Smith et al, 2007). Free cysteine labeling experiments support domain unfolding of in vitro-stretched fibrils (Bradshaw and Smith, 2011) but indicate that only a limited subset of FNIII domains may unfold in cell-derived fibrils (Lemmon et al, 2011).…”