TY - JOUR
T1 - Förster resonance energy transfer efficiency of the vinculin tension sensor in cultured primary cortical neuronal growth cones
AU - Ayad, Marina A.
AU - Mahon, Timothy
AU - Patel, Mihir
AU - Cararo-Lopes, Marina M.
AU - Hacihaliloglu, Ilker
AU - Firestein, Bonnie L.
AU - Boustany, Nada N.
N1 - Funding Information:
This paper is based on results presented at the 2021 SPIE conference on Neural Imaging and Sensing.55 We thank Dr. Brenton D. Hoffman and his laboratory for plasmids used for instrument calibration and for pertinent discussions getting this project started. We thank Audrey Menaesse and Daniel Sumetsky for their assistance with instrument calibration and David Lee for assistance with data analysis. MMC-L was supported by a Predoctoral Fellowship from the Coordination for the Improvement of Higher Education Personnel (CAPES; #88881.175866/ 2018-01), and MVP was supported by NJCBIR Predoctoral Fellowship #CBIR15FEL009. This study was supported by the National Science Foundation (NSF) under grant no. CMMI-1825433. Any opinions, findings and conclusions or recommendations expressed in this material do not necessarily reflect the views of the NSF.
Publisher Copyright:
© The Authors. Published by SPIE under a Creative Commons Attribution 4.0 International License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.
PY - 2022/4/1
Y1 - 2022/4/1
N2 - 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 ± 3.6 %) in growth cones was higher than the mean FRET efficiency of VinTS (24.6 ± 2 %) and VinTL (25.8 ± 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.
AB - 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 ± 3.6 %) in growth cones was higher than the mean FRET efficiency of VinTS (24.6 ± 2 %) and VinTL (25.8 ± 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.
KW - Förster resonance energy transfer
KW - fluorescence microscopy
KW - growth cones
KW - neurons
KW - vinculin tension sensor
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U2 - 10.1117/1.NPh.9.2.025002
DO - 10.1117/1.NPh.9.2.025002
M3 - Article
AN - SCOPUS:85133592085
SN - 2329-4248
VL - 9
JO - Neurophotonics
JF - Neurophotonics
IS - 2
M1 - 025002
ER -