Quantifying intracellular protein binding thermodynamics during mechanotransduction based on FRET spectroscopy

• Main Authors: Mofrad, Mohammad R.K (Author), Abdul Rahim, Nur Aida (Contributor), Pelet, Serge (Contributor), So, Peter T. C. (Contributor), Kamm, Roger Dale (Contributor)
• Other Authors: Massachusetts Institute of Technology. Department of Biological Engineering (Contributor), Massachusetts Institute of Technology. Department of Mechanical Engineering (Contributor), Massachusetts Institute of Technology. Laser Biomedical Research Center (Contributor)
• Format: Article
• Language: English
• Published: Elsevier, 2017-06-02T17:38:37Z.
• Subjects: Article
• Online Access: Get fulltext

Mechanical force modulates myriad cellular functions including migration, alignment, proliferation, and gene transcription. Mechanotransduction, the transmission of mechanical forces and its translation into biochemical signals, may be mediated by force induced protein conformation changes, subsequently modulating protein signaling. For the paxillin and focal adhesion kinase interaction, we demonstrate that force-induced changes in protein complex conformation, dissociation constant, and binding Gibbs free energy can be quantified by lifetime-resolved fluorescence energy transfer microscopy combined with intensity imaging calibrated by fluorescence correlation spectroscopy. Comparison with in vitro data shows that this interaction is allosteric in vivo. Further, spatially resolved imaging and inhibitor assays show that this protein interaction and its mechano-sensitivity are equal in the cytosol and in the focal adhesions complexes indicating that the mechano-sensitivity of this interaction must be mediated by soluble factors but not based on protein tyrosine phosphorylation.
United States. National Institutes of Health (9P41EB015871-26A1)
United States. National Institutes of Health (R01-EX017656)
United States. National Institutes of Health (5 R01 NS051320)
United States. National Institutes of Health (4R44EB012415-02)
National Science Foundation (U.S.) (CBET-0939511)