Cells can sense and transduce a broad range of mechanical forces into distinct sets of biochemical signals that ultimately regulate cellular processes, including adhesion, proliferation, differentiation, and apoptosis. Deciphering at the nanoscale the design principles by which sensory elements are integrated into structural protein motifs whose conformations can be switched mechanically is crucial to understand the process of transduction of force into biochemical signals that are then integrated to regulate mechanoresponsive pathways. While the major focus in the search for mechanosensory units has been on membrane proteins such as ion channels, integrins, and associated cytoplasmic complexes, a multimodular design of tandem repeats of various structural motifs is ubiquitously found among extracellular matrix proteins, as well as cell adhesion molecules, and among many intracellular players that physically link transmembrane proteins to the contractile cytoskeleton. Single-molecule studies have revealed an unexpected richness of mechanosensory motifs, including force-regulated conformational changes of loop-exposed molecular recognition sites, intermediate states in the unraveling pathway that might either expose cryptic binding or phosphorylation sites, or regions that display enzymatic activity only when unmasked by force. Insights into mechanochemical signal conversion principles will also affect various technological fields, from biotechnology to tissue engineering and drug development.

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. Cells are embedded within an extracellular matrix that regulates many cellular processes, including stem cell differentiation and cancer progression. Yet the underlying molecular mechanisms that mediate these processes remain unknown. Within the extracellular matrix of cells, super-molecular assemblies of fibronectin are dynamically stretched many times beyond their resting length by cell traction forces. Whether mechanical forces generated by cells can mechanically unfold fibronectin has been controversial. Clarification of this issue is important since fibronectin displays a large number of molecular recognition sites whose display might be altered by protein unfolding. Here, we used spectroscopic approaches to visualize whether this extracellular matrix protein is unfolded in cell culture. We show that indeed cell traction forces straighten fibronectin and unfold its modules. Fluorescence resonance energy transfer reveals the extent to which the extracellular matrix unfolds and thus potentially regulates cell signaling processes.