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

Applied forces and the biophysical nature of the cellular microenvironment play a central role in determining cellular behavior. Specifically, forces due to cell contraction are transmitted into structural ECM proteins and these forces are presumed to activate integrin "switches." The mechanism of such switches is thought to be the partial unfolding of integrin-binding domains within fibronectin (Fn). However, integrin switches remain largely hypothetical due to a dearth of evidence for their existence, and relevance, in vivo. By using phage display in combination with the controlled deposition and extension of Fn fibers, we report the discovery of peptide-based molecular probes capable of selectively discriminating Fn fibers under different strain states. Importantly, we show that the probes are functional in both in vitro and ex vivo tissue contexts. The development of such tools represents a critical step in establishing the relevance of theoretical mechanotransduction events within the cellular microenvironment.

Conformational coupling of integrin and Thy-1 regulates Fyn priming and fibroblast mechanotransduction.

Progressive fibrosis is characterized by excessive deposition of extracellular matrix (ECM), resulting in gross alterations in tissue mechanics. Changes in tissue mechanics can further augment scar deposition through fibroblast mechanotransduction. In idiopathic pulmonary fibrosis, a fatal form of progressive lung fibrosis, previous work has shown that loss of Thy-1 (CD90) expression in fibroblasts correlates with regions of active fibrogenesis, thus representing a pathologically relevant fibroblast subpopulation. We now show that Thy-1 is a regulator of fibroblast rigidity sensing. Thy-1 physically couples to inactive αvß3 integrins via its RGD-like motif, altering baseline integrin avidity to ECM ligands and also facilitating preadhesion clustering of integrin and membrane rafts via Thy-1's glycophosphatidylinositol tether. Disruption of Thy-1-αvß3 coupling altered recruitment of Src family kinases to adhesion complexes and impaired mechanosensitive, force-induced Rho signaling, and rigidity sensing. Loss of Thy-1 was sufficient to induce myofibroblast differentiation in soft ECMs and may represent a physiological mechanism important in wound healing and fibrosis.