. 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 ( ) in growth cones was higher than the mean FRET efficiency of VinTS ( ) and VinTL ( ) ( ). While small, the difference between the FRET efficiency of VinTS and VinTL was statistically significant ( ), 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.

We utilize a cost-effective frequency-domain fluorescence lifetime imaging microscope to measure the phase lifetime of mTFP1 in mTFP1-mVenus fluorescence resonance energy transfer (FRET) constructs relevant to the VinTS molecular tension probe. Our data were collected at 15 modulation frequencies ω/2π selected between 14 and 70 MHz. The lifetime of mTFP1 was τD = 3.11 ± 0.02 ns in the absence of acceptor. For modulation frequencies, ω, such that (ω · τD) < 1.1, the phase lifetime of mTFP1in the presence of acceptor (mVenus), τϕDA, was directly related to the amplitude-weighted lifetime τaveDA inferred from the known FRET efficiency (EFRETtrue) of the constructs. A linear fit to a plot of (ω·τϕDA) vs. (ω·τaveDA) yielded a slope of 0.79 ± 0.05 and intercept of 0.095 ± 0.029 (R2 = 0.952). Thus, our results suggest that a linear relationship exists between the apparent EFRETapp based on the measured phase lifetime and EFRETtrue for frequencies such that (ω · τD) < 1.1. We had previously reported a similar relationship between EFRETapp and EFRETtrue at 42 MHz. Our current results provide additional evidence in support of this observation, but further investigation is still required to fully characterize these results. A direct relationship between τϕDAand τaveDA has the potential to simplify significantly data acquisition and interpretation in fluorescence lifetime measurements of FRET constructs.

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