Abstract
The mechanism of assembly of the extracellular matrix protein fibronectin (FN) into elastic, insoluble fibrils is still poorly understood. FN fibrillogenesis requires cell-generated forces, which expose cryptic FN-FN binding sites buried in FN Type III domains. The number and location of cryptic binding sites have been debated, but experimental evidence suggests multiple domains may contain FN-FN binding sites. The requirement of cell-dependent forces to generate FN fibrils restricts investigation of the mechanism of assembly. To address this, we use a recently developed biophysical model of fibrillogenesis to test competing hypotheses for the location and number of cryptic FN-FN binding sites and quantify the effect of these molecular alterations on assembled FN fibril properties. Simulations predict that a single FN-FN binding site facilitates either negligible fibrillogenesis or produces FN fibrils that are neither robust nor physiological. However, inclusion of multiple FN-FN binding sites predicts robust fibrillogenesis, which minimally depends on individual domain properties. Multiple FN-FN binding site models predict a heterogeneous fibril population that contains two distinct phenotypes with unique viscoelastic properties, which we speculate may play a key role in generating heterogeneous mechanical signaling in the extracellular matrix of developing and regenerating tissues.
Highlights
Earlier studies demonstrating that FN fibrils only assemble when FN molecules are subjected to cell contractile forces suggests that there is a buried cryptic binding site in FN molecules that is only exposed when under tension[13,14]
Our results demonstrate that a single binding site located N-terminal of or at III10 facilitates negligible FN fibrillogenesis
These findings provide a mechanistic explanation of previous experimental studies that suggest that multiple Type III domains contribute to robust fibronectin fibrillogenesis[20,21]
Summary
Earlier studies demonstrating that FN fibrils only assemble when FN molecules are subjected to cell contractile forces suggests that there is a buried cryptic binding site in FN molecules that is only exposed when under tension[13,14]. Other studies have demonstrated binding between the 70 kDa fragment of FN and III1019, III12–1424, and III4–525 This suggests that multiple Type III domains may be capable of binding to soluble FN and facilitating fibrillogenesis. Steered molecular dynamics (SMD) simulations have indicated that stretched FN Type III domains have a stable intermediate conformation in which β-strands along the edges of the domain are extended and exposed[32,33]. This conformation would be capable of binding other proteins by β-strand addition. Note that integrin springs are connected in parallel with springs representing FN Type III domains
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