Assay to mechanically tune and optically probe fibrillar fibronectin conformations from fully relaxed to breakage

Abstract

In response to growing needs for quantitative biochemical and cellular assays that address whether the extracellular matrix (ECM) acts as a mechanochemical signal converter to co-regulate cellular mechanotransduction processes, a new assay is presented where plasma fibronectin fibers are manually deposited onto elastic sheets, while force-induced changes in protein conformation are monitored by fluorescence resonance energy transfer (FRET). Fully relaxed assay fibers can be stretched at least 5-6 fold, which involves Fn domain unfolding, before the fibers break. In native fibroblast ECM, this full range of stretch-regulated conformations coexists in every field of view confirming that the assay fibers are physiologically relevant model systems. Since alterations of protein function will directly correlate with their extension in response to force, the FRET vs. strain curves presented herein enable the mapping of fibronectin strain distributions in 2D and 3D cell cultures with high spatial resolution. Finally, cryptic sites for fibronectin's N-terminal 70-kD fragment were found to be exposed at relatively low strain, demonstrating the assay's potential to analyze stretch-regulated protein-rotein interactions.

Impact of substrate elasticity on human hematopoietic stem and progenitor cell adhesion and motility

“…To promote cell adhesion, the hydrogels were functionalized with fibronectin isolated from human plasma as described elsewhere (Little et al, 2008). For this purpose the carboxylic acid groups of the gels were converted into active esters.…”

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

“…Fn fibrillogenesis can be activated by force, independent of whether the force originates from cell-generated tension (13)(14)(15) or is applied by other external means (16)(17)(18). As an alternative to using native ECM fibers, Fn fibers with a more homogeneous diameter can be pulled with a needle from a droplet of concentrated Fn and deposited onto stretchable substrates (18).…”

“…As an alternative to using native ECM fibers, Fn fibers with a more homogeneous diameter can be pulled with a needle from a droplet of concentrated Fn and deposited onto stretchable substrates (18). As revealed by fluorescence resonance energy transfer (FRET), manually deposited and externally stretched Fn fibers are physiologically relevant models (18).…”

“…As an alternative to using native ECM fibers, Fn fibers with a more homogeneous diameter can be pulled with a needle from a droplet of concentrated Fn and deposited onto stretchable substrates (18). As revealed by fluorescence resonance energy transfer (FRET), manually deposited and externally stretched Fn fibers are physiologically relevant models (18). When the single deposited fibers were gradually stretched, from totally relaxed to highly unfolded, they display all conformations that are seen in the ECM fibers of living cell cultures which were differentially stretched by cell-generated tension yielding a highly heterogeneous distribution of Fn conformations (15,18,19).…”

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

“…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.…”