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PMID: 18417335 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

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

Matrix biology : journal of the International Society for Matrix Biology ·Vol. 27 ·No. 5 ·2008-06-00 ·Pages 451-61

Little WC, Smith ML, Ebneter U, Vogel V

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-protein interactions.

MeSH Terms
Animals Elasticity Endothelial Cells/cytology,physiology Extracellular Matrix/chemistry,physiology Fibroblasts/cytology,physiology Fibronectins/chemistry,physiology Fluorescence Resonance Energy Transfer/methods Fluorescent Dyes/chemistry Humans Mechanotransduction, Cellular/physiology Mice Microscopy, Atomic Force Microscopy, Fluorescence NIH 3T3 Cells Peptide Fragments/chemistry Protein Conformation Protein Denaturation Protein Folding Protein Structure, Quaternary Protein Structure, Secondary Silicones/chemistry Stress, Mechanical Tensile Strength
Chemicals
Fibronectins Fluorescent Dyes Peptide Fragments Silicones fibroblast motility-stimulating factor
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Little William C
Department of Materials, ETH Zurich, CH-8093, Zürich, Switzerland.
Smith Michael L
Ebneter Urs
Vogel Viola
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Article Info
Journal
Matrix biology : journal of the International Society for Matrix Biology
Abbr.
Matrix Biol
ISSN
0945-053X
DOI
10.1016/j.matbio.2008.02.003
Published
2008-06-00
Epub
2008-00-21
Pages
451-61
Language
English
Region
Netherlands
NLM ID
9432592
PMCID
PMC5615104
Subset
IM
Grants
NEI NIH HHS · PN2 EY016586 · United States
External Links
PubMed source DOI: 10.1016/j.matbio.2008.02.003 PMC Full Text View journal details
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