Ms. Marina M. Cararo-Lopes Profile

Marina M. Cararo-Lopes

at Rutgers The State Univ of New Jersey

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Publications (2)

SPIE Journal Paper | 30 May 2022 Open Access

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

Marina Ayad, Timothy Mahon, Mihir Patel, Marina Cararo-Lopes, Ilker Hacihaliloglu, Bonnie Firestein, Nada Boustany

NPh, Vol. 9, Issue 02, 025002, (May 2022) https://doi.org/10.1117/12.10.1117/1.NPh.9.2.025002

KEYWORDS: Fluorescence resonance energy transfer, Neurons, Image segmentation, Sensors, Resonance energy transfer, Energy efficiency, Neurophotonics, Image registration, Glasses, Optical filters

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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.

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Proceedings Article | 5 March 2021 Presentation + Paper

Vinculin tension probe in neurons

Marina Ayad, Timothy Mahon, Daniel Sumetsky, Marina Cararo-Lopes, Bonnie Firestein, Nada Boustany

Proceedings Volume 11629, 1162921 (2021) https://doi.org/10.1117/12.2578501

KEYWORDS: Neurons, Fluorescence resonance energy transfer, Sensors, Glasses, Neurogenesis, Head, Energy efficiency, Brain

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