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Visualizing Neurons Under Tension In Vivo with Optogenetic Molecular Force Sensors

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Mechanobiology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2600))

Abstract

The visualization of mechanical stress distribution in specific molecular networks within a living and physiologically active cell or animal remains a formidable challenge in mechanobiology. The advent of fluorescence-resonance energy transfer (FRET)-based molecular tension sensors overcame a significant hurdle that now enables us to address previously technically limited questions. Here, we describe a method that uses genetically encoded FRET tension sensors to visualize the mechanics of cytoskeletal networks in neurons of living animals with sensitized emission FRET and confocal scanning light microscopy. This method uses noninvasive immobilization of living animals to image neuronal β-spectrin cytoskeleton at the diffraction limit, and leverages multiple imaging controls to verify and underline the quality of the measurements. In combination with a semiautomated machine-vision algorithm to identify and trace individual neurites, our analysis performs simultaneous calculation of FRET efficiencies and visualizes statistical uncertainty on a pixel by pixel basis. Our approach is not limited to genetically encoded spectrin tension sensors, but can also be used for any kind of ratiometric imaging in neuronal cells both in vivo and in vitro.

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Acknowledgments

We thank the ICFO SLN facility for the generous use of their microscopes. M.K. acknowledges financial support from the Spanish Ministry of Economy and Competitiveness through the Plan Nacional (PGC2018-097882-A-I00), “Severo Ochoa” program for Centres of Excellence in R&D (CEX2019-000910-S; RYC-2016-21062), from Fundació Privada Cellex, Fundació Mir-Puig, and from Generalitat de Catalunya through the CERCA and Research program (2017 SGR 1012), in addition to funding through ERC (MechanoSystems) and HFSP (CDA00023/2018), la Caixa Foundation (ID 100010434, LCF/BQ/DI18/11660035), and MINECO (FPIPRE2019-088840 funded by MCIN/AEI/10.13039/501100011033 and ESF “Investing in your future” to N.S.). M.B.G. is supported by NIH Grant R35105092 and A.R.D. by NIH grant 1R35GM130332-01.

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Authors and Affiliations

  1. Neurophotonics and Mechanical Systems Biology, ICFO, Institut de Ciències Fotòniques, ICFO, Castelldefels, Spain

    Neus Sanfeliu-Cerdán, Li-Chun Lin & Michael Krieg

  2. Department of Chemical Engineering, Stanford University, Stanford, CA, USA

    Alexander R. Dunn

  3. Molecular and Cellular Physiology, Stanford University, Stanford, CA, USA

    Miriam B. Goodman

Authors
  1. Neus Sanfeliu-Cerdán
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  2. Li-Chun Lin
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  3. Alexander R. Dunn
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  4. Miriam B. Goodman
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  5. Michael Krieg
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Corresponding author

Correspondence to Michael Krieg .

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Editors and Affiliations

  1. Department of Cell and Developmental Biology, Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

    Ronen Zaidel-Bar

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Sanfeliu-Cerdán, N., Lin, LC., Dunn, A.R., Goodman, M.B., Krieg, M. (2023). Visualizing Neurons Under Tension In Vivo with Optogenetic Molecular Force Sensors. In: Zaidel-Bar, R. (eds) Mechanobiology. Methods in Molecular Biology, vol 2600. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2851-5_16

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  • DOI: https://doi.org/10.1007/978-1-0716-2851-5_16

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  • Publisher Name: Humana, New York, NY

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