Förster resonance energy transfer efficiency of the vinculin tension sensor in cultured primary cortical neuronal growth cones

SIGNIFICANCE: Interaction of neurons with their extracellular environment and the mechanical forces at focal adhesions and synaptic junctions play important roles in neuronal development. AIM: To advance studies of mechanotransduction, we demonstrate the use of the vinculin tension sensor (VinTS) in primary cultures of cortical neurons. VinTS consists of TS module (TSMod), a Förster resonance energy transfer (FRET)-based tension sensor, inserted between vinculin’s head and tail. FRET efficiency decreases with increased tension across vinculin. APPROACH: Primary cortical neurons cultured on glass coverslips coated with poly-d-lysine and laminin were transfected with plasmids encoding untargeted TSMod, VinTS, or tail-less vinculinTS (VinTL) lacking the actin-binding domain. The neurons were imaged between day in vitro (DIV) 5 to 8. We detail the image processing steps for calculation of FRET efficiency and use this system to investigate the expression and FRET efficiency of VinTS in growth cones. RESULTS: The distribution of fluorescent constructs was similar within growth cones at DIV 5 to 8. The mean FRET efficiency of TSMod ([Formula: see text]) in growth cones was higher than the mean FRET efficiency of VinTS ([Formula: see text]) and VinTL ([Formula: see text]) ([Formula: see text]). While small, the difference between the FRET efficiency of VinTS and VinTL was statistically significant ([Formula: see text]), suggesting that vinculin is under low tension in growth cones. Two-hour treatment with the Rho-associated kinase inhibitor Y-27632 did not affect the mean FRET efficiency. Growth cones exhibited dynamic changes in morphology as observed by time-lapse imaging. VinTS FRET efficiency showed greater variance than TSMod FRET efficiency as a function of time, suggesting a greater dependence of VinTS FRET efficiency on growth cone dynamics compared with TSMod. CONCLUSIONS: The results demonstrate the feasibility of using VinTS to probe the function of vinculin in neuronal growth cones and provide a foundation for studies of mechanotransduction in neurons using this tension probe.