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

Marina A Ayad; Timothy Mahon; Mihir Patel; Marina M Cararo-Lopes; Ilker Hacihaliloglu; Bonnie L Firestein; Nada N Boustany

doi:10.1117/1.NPh.9.2.025002

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

Neurophotonics. 2022 Apr;9(2):025002. doi: 10.1117/1.NPh.9.2.025002. Epub 2022 May 30.

Authors

Marina A Ayad¹, Timothy Mahon¹, Mihir Patel², Marina M Cararo-Lopes², Ilker Hacihaliloglu¹, Bonnie L Firestein², Nada N Boustany¹

Affiliations

¹ Rutgers University, Department of Biomedical Engineering, Piscataway, New Jersey, United States.
² Rutgers University, Department of Cell Biology and Neuroscience, Piscataway, New Jersey, United States.

Abstract

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 ( $28.5 \pm 3.6 %$ ) in growth cones was higher than the mean FRET efficiency of VinTS ( $24.6 \pm 2 %$ ) and VinTL ( $25.8 \pm 1.8 %$ ) ( $p < 10^{- 6}$ ). While small, the difference between the FRET efficiency of VinTS and VinTL was statistically significant ( $p < 10^{- 3}$ ), 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.

Keywords: Förster resonance energy transfer; fluorescence microscopy; growth cones; neurons; vinculin tension sensor.