Tutorial: fluorescence lifetime microscopy of membrane mechanosensitive Flipper probes

Chloé Roffay¹, Juan Manuel García-Arcos¹, Pierrik Chapuis¹, Javier López-Andarias^{2

3}, Falk Schneider⁴, Adai Colom^{5

6

7}, Caterina Tomba⁸, Ilaria Di Meglio¹, Katia Barrett⁹, Valentin Dunsing⁹, Stefan Matile^{2

3}, Aurélien Roux^{10

11}, Vincent Mercier^{12

13}

Affiliations

¹ Department of Biochemistry, University of Geneva, Geneva, Switzerland.
² Department of Organic Chemistry, University of Geneva, Geneva, Switzerland.
³ National Center of Competence in Research in Chemical Biology, University of Geneva, Geneva, Switzerland.
⁴ Translational Imaging Center, University of Southern California, Los Angeles, CA, USA.
⁵ Biofisika Institute (CSIC, UPV/EHU), Leioa, Spain.
⁶ Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, Campus Universitario, University of the Basque Country (UPV/EHU), Leioa, Spain.
⁷ IKERBASQUE, Basque Foundation for Science, Bilbao, Spain.
⁸ CNRS, INSA Lyon, Ecole Centrale de Lyon, Universite Claude Bernard Lyon 1, CPE Lyon, INL, Villeurbanne, France.
⁹ Aix-Marseille Université & CNRS, IBDM-UMR7288 & Turing Centre for Living Systems, Parc Scientifique de Luminy, Marseille, France.
¹⁰ Department of Biochemistry, University of Geneva, Geneva, Switzerland. aurelien.roux@unige.ch.
¹¹ National Center of Competence in Research in Chemical Biology, University of Geneva, Geneva, Switzerland. aurelien.roux@unige.ch.
¹² Department of Biochemistry, University of Geneva, Geneva, Switzerland. vincent.mercier@unige.ch.
¹³ National Center of Competence in Research in Chemical Biology, University of Geneva, Geneva, Switzerland. vincent.mercier@unige.ch.

PMID: 39210094
DOI: 10.1038/s41596-024-01027-6

Review

Tutorial: fluorescence lifetime microscopy of membrane mechanosensitive Flipper probes

Chloé Roffay et al. Nat Protoc. 2024 Dec.

. 2024 Dec;19(12):3457-3469.

doi: 10.1038/s41596-024-01027-6. Epub 2024 Aug 29.

Authors

Affiliations

¹ Department of Biochemistry, University of Geneva, Geneva, Switzerland.
² Department of Organic Chemistry, University of Geneva, Geneva, Switzerland.
³ National Center of Competence in Research in Chemical Biology, University of Geneva, Geneva, Switzerland.
⁴ Translational Imaging Center, University of Southern California, Los Angeles, CA, USA.
⁵ Biofisika Institute (CSIC, UPV/EHU), Leioa, Spain.
⁶ Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, Campus Universitario, University of the Basque Country (UPV/EHU), Leioa, Spain.
⁷ IKERBASQUE, Basque Foundation for Science, Bilbao, Spain.
⁸ CNRS, INSA Lyon, Ecole Centrale de Lyon, Universite Claude Bernard Lyon 1, CPE Lyon, INL, Villeurbanne, France.
⁹ Aix-Marseille Université & CNRS, IBDM-UMR7288 & Turing Centre for Living Systems, Parc Scientifique de Luminy, Marseille, France.
¹⁰ Department of Biochemistry, University of Geneva, Geneva, Switzerland. aurelien.roux@unige.ch.
¹¹ National Center of Competence in Research in Chemical Biology, University of Geneva, Geneva, Switzerland. aurelien.roux@unige.ch.
¹² Department of Biochemistry, University of Geneva, Geneva, Switzerland. vincent.mercier@unige.ch.
¹³ National Center of Competence in Research in Chemical Biology, University of Geneva, Geneva, Switzerland. vincent.mercier@unige.ch.

PMID: 39210094
DOI: 10.1038/s41596-024-01027-6

Abstract

Measuring forces within living cells remains a technical challenge. In this Tutorial, we cover the development of hydrophobic mechanosensing fluorescent probes called Flippers, whose fluorescence lifetime depends on lipid packing. Flipper probes can therefore be used as reporters for membrane tension via the measurement of changes in their fluorescence lifetime. We describe the technical optimization of the probe for imaging and provide working examples for their characterizations in a variety of biological and in vitro systems. We further provide a guideline to measure biophysical parameters of cellular membranes by fluorescence lifetime imaging microscopy using Flipper probes, providing evidence that flippers can report long range forces in cells, tissues and organs.

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Conflict of interest statement

Competing interests: The authors declare no competing interests.

References

1. Roca-Cusachs, P., Conte, V. & Trepat, X. Quantifying forces in cell biology. Nat. Cell Biol. 19, 742–751 (2017). - PubMed
1. Abella, M., Andruck, L., Malengo, G. & Skruzny, M. Actin-generated force applied during endocytosis measured by Sla2-based FRET tension sensors. Dev. Cell 56, 2419–2426.e4 (2021). - PubMed
1. Déjardin, T. et al. Nesprins are mechanotransducers that discriminate epithelial–mesenchymal transition programs. J. Cell Biol. 219, e201908036 (2020). - PubMed - PMC
1. Li, W. et al. A membrane-bound biosensor visualizes shear stress-induced inhomogeneous alteration of cell membrane tension. iScience 7, 180–190 (2018). - PubMed - PMC
1. Páez-Pérez, M., López-Duarte, I., Vyšniauskas, A., Brooks, N. J. & Kuimova, M. K. Imaging non-classical mechanical responses of lipid membranes using molecular rotors. Chem. Sci. 12, 2604–2613 (2021).

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Tutorial: fluorescence lifetime microscopy of membrane mechanosensitive Flipper probes

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Tutorial: fluorescence lifetime microscopy of membrane mechanosensitive Flipper probes

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