Assay to mechanically tune and optically probe fibrillar fibronectin conformations from fully relaxed to breakage.
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-protein interactions.
Little
William C
WC
Department of Materials, ETH Zurich, CH-8093, Zürich, Switzerland.
Smith
Michael L
ML
Ebneter
Urs
U
Vogel
Viola
V
D000818:Animals; D004548:Elasticity; D042783:Endothelial Cells / Q000166:cytology / Q000502:physiology; D005109:Extracellular Matrix / Q000737:chemistry / Q000502:physiology*; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D005353:Fibronectins / Q000737:chemistry / Q000502:physiology*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D040542:Mechanotransduction, Cellular / Q000502:physiology*; D051379:Mice; D018625:Microscopy, Atomic Force; D008856:Microscopy, Fluorescence; D041681:NIH 3T3 Cells; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D012828:Silicones / Q000737:chemistry; D013314:Stress, Mechanical; D013718:Tensile Strength
D005353:Fibronectins; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D012828:Silicones; C107678:fibroblast motility-stimulating factor