Fibronectin forms the most extensible biological fibers displaying switchable force-exposed cryptic binding sites

Abstract

Rather than maximizing toughness, as needed for silk and muscle titin fibers to withstand external impact, the much softer extracellular matrix fibers made from fibronectin (Fn) can be stretched by cell generated forces and display extraordinary extensibility. We show that Fn fibers can be extended more than 8-fold (>700% strain) before 50% of the fibers break. The Young's modulus of single fibers, given by the highly nonlinear slope of the stressstrain curve, changes orders of magnitude, up to MPa. Although many other materials plastically deform before they rupture, evidence is provided that the reversible breakage of force-bearing backbone hydrogen bonds enables the large strain. When tension is released, the nano-sized Fn domains first contract in the crowded environment of fibers within seconds into random coil conformations (molten globule states), before the force-bearing hydrogen bond networks that stabilize the domain's secondary structures are reestablished within minutes (double exponential). The exposure of cryptic binding sites on Fn type III modules increases steeply upon stretching. Thus fiber extension steadily up-regulates fiber rigidity and cryptic epitope exposure, both of which are known to differentially alter cell behavior. Finally, since stress-strain relationships cannot directly be measured in native extracellular matrix (ECM), the stress-strain curves were correlated with stretch-induced alterations of intramolecular fluorescence resonance energy transfer (FRET) obtained from trace amounts of Fn probes (mechanical strain sensors) that can be incorporated into native ECM. Physiological implications of the extraordinary extensibility of Fn fibers and contraction kinetics are discussed.fibrillogenesis ͉ matrix biology ͉ mechanotransduction ͉ multimodular proteins ͉ supramolecular assembly

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

“…A recent report suggested that FN nanofibers formed from a droplet exhibited a complete elastic recoil, even after large extensions (λ = 3.5); 22 however, we argue that this observation is dependent on the fabrication method. Due to the nature of their synthesis, these fibers pulled from a droplet 22 experience a pre-stretch of λ = 2.4, as they are deposited prior to mechanical testing.…”

“…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.…”

“…Due to the nature of their synthesis, these fibers pulled from a droplet 22 experience a pre-stretch of λ = 2.4, as they are deposited prior to mechanical testing. According to our molecular model and experimental data, the pre-stretch initiated during deposition is enough to induce a plastic deformation on the pulled FN fibers prior to any mechanical testing.…”

Phage-based molecular probes that discriminate force-induced structural states of fibronectin in vivo

“…In particular, forces emanating from contractile cells such as pathogenic myofibroblasts have been hypothesized to partially unfold ECM proteins like Fn, thus engaging/disengaging theorized integrin switches (25). Despite considerable in silico and in vitro evidence for the extensibility of Fn within fibers and Fn type III domain unfolding (26)(27)(28), there is still no direct evidence that such molecular events occur in vivo, a fact that perpetuates the debate regarding the validity of such observations.…”

Controlled Assembly of Fibronectin Nanofibrils Triggered by Random Copolymer Chemistry

“…20,21 Other ways to induce the formation of fibronectin fibrils on material surface, mostly triggered by force, have been reported elsewhere. 20,[23][24][25] Here, a series of copolymers with controlled ethyl acrylate/methyl acrylate (EA/MA) ratio were synthetised seeking to modulate the degree of FN fibrillogenesis. We investigated FN adsorption and conformation as a function of the amount of EA in the copolymers.…”