title: Application of strain and calibration of Förster Resonance Energy Transfer (FRET) emission for in vitro live cell response to cytoskeletal deformation.
abstract: Cellular mechanotransduction is an integral part of many crucial physiological processes, but non-invasive tools for quantifying intracellular strain in vivo are not available for complex tissues such as bone. As a first step to address this gap, we have utilized a novel, non-invasive approach to quantify cellular strain in vitro by employing a transfected alpha-actinin Förster Resonance Energy Transfer (FRET) sensor. Following validation experiments, mouse fibroblasts transfected to express FRET sensors were seeded to a silicone membrane and subjected to up to 10% tensile strain mounted on a multi-photon microscope. During tensile strain, fluorescent emission of acceptor (YFP) and donor (CFP) proteins was quantified. YFP/CFP ratio was normalized to the initial baseline (unstretched) ratio for each cell which demonstrates a negative linear correlation between the relative proximity ratio of emission spectra and cell strain, with a mean decrease of 1.017% normalized ratio for every percent strain experienced by the cell. The exciting implications of our findings are that the discovery of the stable correlation between loss of FRET and experimentally applied strain opens intriguing possibilities for future use of this technology with in vivo research, leading to discoveries improving disease treatments in mechanically sensitive tissues such as bone.
authors: Cincinnati Children׳s Hospital Medical Center, Cincinnati, OH, USA; Biomedical Engineering, University of Cincinnati, Cincinnati, OH, USA.
authors: Jacob M
authors: JM
authors: Knorr
authors: Biomedical Engineering, University of Cincinnati, Cincinnati, OH, USA.
authors: Joshua
authors: J
authors: Jackson
authors: Cincinnati Children׳s Hospital Medical Center, Cincinnati, OH, USA.
authors: Matthew R
authors: MR
authors: Batie
authors: Biomedical Engineering, University of Cincinnati, Cincinnati, OH, USA.
authors: Daria A
authors: DA
authors: Narmoneva
authors: Cincinnati Children׳s Hospital Medical Center, Cincinnati, OH, USA. Electronic address: donna.jones1@cchmc.org.
authors: Donna C
authors: DC
authors: Jones
mesh_terms: D000185:Actinin / Q000378:metabolism; D000818:Animals; D001696:Biomechanical Phenomena; D002138:Calibration; D002470:Cell Survival; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism*; D005260:Female; D031541:Fluorescence Resonance Energy Transfer; D008164:Luminescent Proteins / Q000378:metabolism; D040542:Mechanotransduction, Cellular; D051379:Mice; D013314:Stress, Mechanical
chemical_list: D008164:Luminescent Proteins; D000185:Actinin
keywords: AFM; Fibroblast; Mechanotransduction; Microscopy; Non-invasive strain measurement