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Monitoring cell membrane recycling dynamics of proteins using whole-cell fluorescence recovery after photobleaching of pH-sensitive genetic tags

Nature Protocols volume 17, pages 3056–3079 (2022)Cite this article

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Abstract

Population behavior of signaling molecules on the cell surface is key to their adaptive function. Live imaging of proteins tagged with fluorescent molecules has been an essential tool in understanding this behavior. Typically, genetic or chemical tags are used to target molecules present throughout the cell, whereas antibody-based tags label the externally exposed molecular domains only. Both approaches could potentially overlook the intricate process of in–out membrane recycling in which target molecules appear or disappear on the cell surface. This limitation is overcome by using a pH-sensitive fluorescent tag, such as Super-Ecliptic pHluorin (SEP), because its emission depends on whether it resides inside or outside the cell. Here we focus on the main glial glutamate transporter GLT1 and describe a genetic design that equips GLT1 molecules with SEP without interfering with the transporter’s main function. Expressing GLT1-SEP in astroglia in cultures or in hippocampal slices enables monitoring of the real-time dynamics of the cell-surface and cytosolic fractions of the transporter in living cells. Whole-cell fluorescence recovery after photobleaching and quantitative image-kinetic analysis of the resulting time-lapse images enables assessment of the rate of GLT1-SEP recycling on the cell surface, a fundamental trafficking parameter unattainable previously. The present protocol takes 15–20 d to set up cell preparations, and 2–3 d to carry out live cell experiments and data analyses. The protocol can be adapted to study different membrane molecules of interest, particularly those proteins whose lifetime on the cell surface is critical to their adaptive function.

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Fig. 1: Assessing membrane protein turnover using the pH-sensitive SEP tag and whole-cell FRAP: fist principles.
Fig. 2: Analyzing whole-cell FRAP kinetics.
Fig. 3: A full sequence of experiments in one cell: an example.
Fig. 4: Analyzing whole-cell FRAP data in one cell: an example.
Fig. 5: Anticipated results: an example.

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Data availability

The original experimental data are available as Source Data files in the supporting primary research article1.

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Acknowledgements

The study was supported by: Wellcome Trust (212251_Z_18_Z), MRC (MR/W019752/1), ERC (323113) and European Commission NEUROTWIN (857562) to D.A.R.; National Science Centre Poland (2017/26/D/NZ3/01017) to P.M.

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Authors and Affiliations

  1. UCL Queen Square Institute of Neurology, University College London, London, UK

    Piotr Michaluk & Dmitri A. Rusakov

  2. BRAINCITY, Laboratory of Neurobiology, Nencki Institute of Experimental Biology PAS, Warsaw, Poland

    Piotr Michaluk

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P.M. suggested and implemented genetic designs, planned and carried out experiments, and analyzed the results; D.A.R. narrated the study, designed imaging methods and performed theoretical data analyses; D.A.R. and P.M. wrote the manuscript.

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Correspondence to Piotr Michaluk or Dmitri A. Rusakov.

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Michaluk, P. et al. eLife 10, 64714 (2021): https://doi.org/10.7554/eLife.64714

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Michaluk, P., Rusakov, D.A. Monitoring cell membrane recycling dynamics of proteins using whole-cell fluorescence recovery after photobleaching of pH-sensitive genetic tags. Nat Protoc 17, 3056–3079 (2022). https://doi.org/10.1038/s41596-022-00732-4

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