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Vinculin is a known key regulator of focal adhesions; it undergoes tension in the locations of attachment to the extracellular matrix. In this study, we explore the use of a vinculin tension FRET probe to investigate vinculin tension within neurons. A critical component of neuronal growth is migration, which is dependent on the mechanical cues between the cells and the extracellular matrix. An understanding of tension variation within the neuron may help us understand mechanisms of neurogenesis. To study these forces, we use a previously developed molecular tension sensor, which consists of an elastic linker, TSMod, a 40-amino-acid-long peptide inserted between teal fluorescent protein (mTFP1) and mVenus. The vinculin tension sensor, VinTS, consists of TSMod embedded between the Vinculin head and tail. When under tension, VinTS will exhibit a lower fluorescence resonance energy transfer (FRET) efficiency between mTFP1 and mVenus. Cortical neurons were isolated from embryonic rat brains and cultured on glass coverslips coated with poly-D-lysine and laminin. The neurons were transfected with TSMod (the unloaded tension sensor) or VinTS. Neurons expressing TSMod are used as the experiment’s control group since TSMod on its own is not affected by vinculin tension. The mean FRET efficiency of 171 TSMod and 127 VinTS expressing neurons was 27.08 ± 4.98%, and 22.86 ± 3.98%, respectively. The FRET efficiency of VinTS was significantly lower than that of TSMod (p = 6.6e15 by Welch’s t-test). These results support the feasibility of using the VinTS probe in neurons and provide a first assessment of VinTS FRET efficiency in neurons. The lower FRET efficiency of VinTS compared with TSMod suggests that VinTS may be under tension in neurons. However, additional studies are required to further characterize these results. 

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 τ D = 3.11 ± 0.02 ns in the absence of acceptor. For modulation frequencies, ω, such that (ω · τ D ) < 1.1, the phase lifetime of mTFP1in the presence of acceptor (mVenus), τ ϕ D A , was directly related to the amplitude-weighted lifetime τ a v e D A inferred from the known FRET efficiency ( E FRET true ) of the constructs. A linear fit to a plot of ( ω · τ ϕ D A )   v s .   ( ω · τ a v e D A )   yielded a slope of 0.79 ± 0.05 and intercept of 0.095 ± 0.029 (R 2 = 0.952). Thus, our results suggest that a linear relationship exists between the apparent E FRET app based on the measured phase lifetime and E FRET true for frequencies such that (ω · τ D ) < 1.1. We had previously reported a similar relationship between E FRET app and E FRET true 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 τ ϕ D A and τ a v e D A has the potential to simplify significantly data acquisition and interpretation in fluorescence lifetime measurements of FRET constructs.