Calibration and testing of FRET efficiency in a vinculin tension probe

As interest grows in the analysis of cellular level mechanical forces, including cell adhesion, cellular extension, and other microscopic-level physical phenomena, our goal is to use a previously developed Vinculin tension probe, VinTS, in conjunction with Fluorescence Resonance Energy Transfer (FRET) microscopy to quantify the mechanical forces that arise in growth cones during neuronal development. The VinTS tension probe consists of a donor and an acceptor fluorophore connected by an elastic linker inserted between the head and tail of vinculin. The work presented here aims to establish a protocol to properly correct, calibrate and convert the imaging data to FRET efficiency which is instrument-independent, and reports on nanometric changes in the distance between the donor and acceptor. For this calibration, we used control FRET constructs, with known FRET efficiency that we expressed in immortalized baby mouse kidney (iBMK) cells, bovine aortic endothelial cells (BAEC), and isolated cortical neurons. The calibration constructs consisted of either our donor, mTFP1, our acceptor, mVenus, or both connected by a long or short linker. The long linker protein was TRAF (TNF receptor associated factor) while the short linker was the amino acid sequence (GGSGGS)2. These constructs were transfected into the selected cell types using Lipofectamineâ LTX according to well-established protocols. All of the constructs were tested in the aforementioned cell lines. The methods involved imaging using a 3-channel “sensitized emission” FRET methodology, with the results analyzed in a lab-developed MATLAB Script. The collected images were also corrected for background. The calibrated system was then utilized to measure the FRET efficiency in the VinTS tension probe and comparing its efficiency to that of the unloaded tension module (TSMod). The efficiency of VinTS and TSMod was measured in iBMK cells and neurons. We investigated segmentation methods to isolate the focal adhesions so that the FRET efficiency could be measured in solely those areas. So far, the investigation has allowed for calibration within the BAEC and iBMK cells, with positive results matching published FRET efficiency values for the calibration constructs. The VinTS and TSMod constructs also appear to produce expected values within the target range in iBMK cells. Current data from neuronal cells appear to have significantly more variability than the other cells which have an epithelial cell morphology. Additional data acquisition and analysis in neuronal cells is currently underway and will pave the way for further study of neuronal growth cones on substrates with various surface properties.M.S.Includes bibliographical reference