Abstract
Caveolae are bulb-shaped invaginations of the plasma membrane that are enriched in specific lipids including cholesterol, phosphatidylserine and sphingolipids. Caveolae have many described cellular roles and functions, including endocytic transport, transcytosis, mechanosensing, and serving as a buffer against plasmalemmal stress. Caveola are formed through interactions between integral membrane proteins (Caveolin) and a cavin family of peripheral proteins (Cavins). Nearly half of the human proteome resides within or at the surface of membranes. Studying protein–protein interactions, especially of transmembrane domain containing proteins can be challenging. Fortunately, sophisticated biophysical methods allow for the monitoring of protein interactions in intact cells. Here, we describe the principles of Förster resonance energy transfer, fluorescence lifetime, and how their properties can be used to assess protein–protein interactions. Additionally, we discuss and demonstrate how fluorescence lifetime can be monitored microscopically thereby providing caveolin–cavin interaction data from living cells.
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Acknowledgments
Original work in the laboratory of G.D.F. is supported by the Canadian Institutes of Health Research (Grants: PJT166010 and PJT165968) and the Natural Sciences and Engineering Research Council of Canada. Figures 1–3 were generated using BioRender.com.
Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Nagwekar, J., Di Ciano-Oliveira, C., Fairn, G.D. (2022). Monitoring Transmembrane and Peripheral Membrane Protein Interactions by Förster Resonance Energy Transfer Using Fluorescence Lifetime Imaging Microscopy. In: Heit, B. (eds) Fluorescent Microscopy. Methods in Molecular Biology, vol 2440. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2051-9_4
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