title: Quantifying intracellular protein binding thermodynamics during mechanotransduction based on FRET spectroscopy.
abstract: 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.
authors.affiliation: Department of Mechanical Engineering, Massachusetts Institute of Technology, 77 Mass Ave., Cambridge, MA 02139, United states.
authors.forename: Nur Aida
authors.initials: NA
authors.lastname: Abdul Rahim
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, 77 Mass Ave., Cambridge, MA 02139, United States; Department of Fundamental Microbiology, University of Lausanne, Biophore Building, Room 2406, CH-1015 Lausanne, Switzerland.
authors.forename: Serge
authors.initials: S
authors.lastname: Pelet
authors.affiliation: Department of Bioengineering, University of California Berkeley, 306 Stanley Hall MC #1762, Berkeley, CA 94720-1762, United States.
authors.forename: Mohammad R K
authors.initials: MR
authors.lastname: Mofrad
authors.affiliation: Department of Mechanical Engineering, Massachusetts Institute of Technology, 77 Mass Ave., Cambridge, MA 02139, United states; Department of Biological Engineering, Massachusetts Institute of Technology, 77 Mass Ave., Cambridge, MA 02139, United States; Laser Biomedical Research Center, A NIH NIBIB Research Resource, Massachusetts Institute of Technology, 77 Mass Ave., Cambridge, MA 02139, United States. Electronic address: ptso@mit.edu.
authors.forename: Peter T C
authors.initials: PT
authors.lastname: So
authors.affiliation: Department of Mechanical Engineering, Massachusetts Institute of Technology, 77 Mass Ave., Cambridge, MA 02139, United states; Department of Biological Engineering, Massachusetts Institute of Technology, 77 Mass Ave., Cambridge, MA 02139, United States.
authors.forename: Roger D
authors.initials: RD
authors.lastname: Kamm
mesh_terms: D000465:Algorithms; D000494:Allosteric Regulation; D000818:Animals; D002138:Calibration; D002417:Cattle; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D042783:Endothelial Cells / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D051417:Focal Adhesion Kinase 1 / Q000096:biosynthesis / Q000737:chemistry; D040542:Mechanotransduction, Cellular; D051419:Paxillin / Q000096:biosynthesis / Q000737:chemistry; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D059010:Single-Cell Analysis; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
chemical_list: D051419:Paxillin; D011993:Recombinant Fusion Proteins; D051417:Focal Adhesion Kinase 1
keywords: FCS; FLIM; FRET; Focal adhesion kinase; Mechanotransduction; Paxillin