Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

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.

Data availability

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

References

  1. Michaluk, P., Heller, J. P. & Rusakov, D. A. Rapid recycling of glutamate transporters on the astroglial surface. eLife 10, 64714 (2021).

    Article  Google Scholar 

  2. Zhang, J., Campbell, R. E., Ting, A. Y. & Tsien, R. Y. Creating new fluorescent probes for cell biology. Nat. Rev. Mol. Cell Biol. 3, 906–918 (2002).

    Article  CAS  PubMed  Google Scholar 

  3. Lippincott-Schwartz, J. & Patterson, G. H. Development and use of fluorescent protein markers in living cells. Science 300, 87–91 (2003).

    Article  CAS  PubMed  Google Scholar 

  4. Axelrod, D., Koppel, D. E., Schlessinger, J., Elson, E. & Webb, W. W. Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys. J. 16, 1055–1069 (1976).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Lippincott-Schwartz, J., Altan-Bonnet N. & Patterson G. H. Photobleaching and photoactivation: following protein dynamics in living cells. Nat. Cell Biol. Suppl, S7–14 (2003).

  6. Lambert, N. A. Uncoupling diffusion and binding in FRAP experiments. Nat. Methods 6, 183–184 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Choquet, D. & Triller, A. The role of receptor diffusion in the organization of the postsynaptic membrane. Nat. Rev. Neurosci. 4, 251–265 (2003).

    Article  CAS  PubMed  Google Scholar 

  8. Bannai, H., Levi, S., Schweizer, C., Dahan, M. & Triller, A. Imaging the lateral diffusion of membrane molecules with quantum dots. Nat. Protoc. 1, 2628–2634 (2006).

    Article  CAS  PubMed  Google Scholar 

  9. Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).

    Article  CAS  PubMed  Google Scholar 

  10. Rossier, O. et al. Integrins β1 and β3 exhibit distinct dynamic nanoscale organizations inside focal adhesions. Nat. Cell Biol. 14, 1057–1067 (2012).

    Article  CAS  PubMed  Google Scholar 

  11. Manley, S. et al. High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nat. Methods 5, 155–157 (2008).

    Article  CAS  PubMed  Google Scholar 

  12. Sydor, A. M., Czymmek, K. J., Puchner, E. M. & Mennella, V. Super-resolution microscopy: from single molecules to supramolecular assemblies. Trends Cell Biol. 25, 730–748 (2015).

    Article  CAS  PubMed  Google Scholar 

  13. Giannone, G. et al. Dynamic superresolution imaging of endogenous proteins on living cells at ultra-high density. Biophys. J. 99, 1303–1310 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Carion, O., Mahler, B., Pons, T. & Dubertret, B. Synthesis, encapsulation, purification and coupling of single quantum dots in phospholipid micelles for their use in cellular and in vivo imaging. Nat. Protoc. 2, 2383–2390 (2007).

    Article  CAS  PubMed  Google Scholar 

  15. Livet, J. et al. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450, 56–62 (2007).

    Article  CAS  PubMed  Google Scholar 

  16. Barnes, S. A. et al. Convergence of hippocampal pathophysiology in Syngap+/− and Fmr1−/y mice. J. Neurosci. 35, 15073–15081 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Politi, A. Z. et al. Quantitative mapping of fluorescently tagged cellular proteins using FCS-calibrated four-dimensional imaging. Nat. Protoc. 13, 1445–1464 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Heal, W. P., Wright, M. H., Thinon, E. & Tate, E. W. Multifunctional protein labeling via enzymatic N-terminal tagging and elaboration by click chemistry. Nat. Protoc. 7, 105–117 (2011).

    Article  PubMed  Google Scholar 

  19. Kamiyama, D. et al. Versatile protein tagging in cells with split fluorescent protein. Nat. Commun. 7, 11046 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Miesenbock, G., De Angelis, D. A. & Rothman, J. E. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394, 192–195 (1998).

    Article  CAS  PubMed  Google Scholar 

  21. Sankaranarayanan, S., De Angelis, D., Rothman, J. E. & Ryan, T. A. The use of pHluorins for optical measurements of presynaptic activity. Biophys. J. 79, 2199–2208 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Sankaranarayanan, S. & Ryan, T. A. Calcium accelerates endocytosis of vSNAREs at hippocampal synapses. Nat. Neurosci. 4, 129–136 (2001).

    Article  CAS  PubMed  Google Scholar 

  23. Matz, J., Gilyan, A., Kolar, A., McCarvill, T. & Krueger, S. R. Rapid structural alterations of the active zone lead to sustained changes in neurotransmitter release. Proc. Natl Acad. Sci. USA 107, 8836–8841 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Ashby, M. C. et al. Removal of AMPA receptors (AMPARs) from synapses is preceded by transient endocytosis of extrasynaptic AMPARs. J. Neurosci. 24, 5172–5176 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Ashby, M. C., Maier, S. R., Nishimune, A. & Henley, J. M. Lateral diffusion drives constitutive exchange of AMPA receptors at dendritic spines and is regulated by spine morphology. J. Neurosci. 26, 7046–7055 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Tanaka, K. et al. Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1. Science 276, 1699–1702 (1997).

    Article  CAS  PubMed  Google Scholar 

  27. Danbolt, N. C. Glutamate uptake. Progr. Neurobiol. 65, 1–105 (2001).

    Article  CAS  Google Scholar 

  28. Lehre, K. P. & Danbolt, N. C. The number of glutamate transporter subtype molecules at glutamatergic synapses: chemical and stereological quantification in young adult rat brain. J. Neurosci. 18, 8751–8757 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Diamond, J. S. & Jahr, C. E. Transporters buffer synaptically released glutamate on a submillisecond time scale. J. Neurosci. 17, 4672–4687 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Wadiche, J. I., Arriza, J. L., Amara, S. G. & Kavanaugh, M. P. Kinetics of a human glutamate transporter. Neuron 14, 1019–1027 (1995).

    Article  CAS  PubMed  Google Scholar 

  31. Lozovaya, N. A., Kopanitsa, M. V., Boychuk, Y. A. & Krishtal, O. A. Enhancement of glutamate release uncovers spillover-mediated transmission by N-methyl-d-aspartate receptors in the rat hippocampus. Neuroscience 91, 1321–1330 (1999).

    Article  CAS  PubMed  Google Scholar 

  32. Scimemi, A., Fine, A., Kullmann, D. M. & Rusakov, D. A. NR2B-containing receptors mediate cross talk among hippocampal synapses. J. Neurosci. 24, 4767–4777 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Zheng, K., Scimemi, A. & Rusakov, D. A. Receptor actions of synaptically released glutamate: the role of transporters on the scale from nanometers to microns. Biophys. J. 95, 4584–4596 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Henneberger, C. et al. LTP induction boosts glutamate spillover by driving withdrawal of perisynaptic astroglia. Neuron 108, 919–936 e911 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Maragakis, N. J. & Rothstein, J. D. Glutamate transporters: animal models to neurologic disease. Neurobiol. Dis. 15, 461–473 (2004).

    Article  CAS  PubMed  Google Scholar 

  36. Fontana, A. C. Current approaches to enhance glutamate transporter function and expression. J. Neurochem. 134, 982–1007 (2015).

    Article  CAS  PubMed  Google Scholar 

  37. Kruyer, A., Scofield, M. D., Wood, D., Reissner, K. J. & Kalivas, P. W. Heroin cue-evoked astrocytic structural plasticity at nucleus accumbens synapses inhibits heroin seeking. Biol. Psychiat. 86, 811–819 (2019).

    Article  CAS  PubMed  Google Scholar 

  38. Al Awabdh, S. et al. Neuronal activity mediated regulation of glutamate transporter GLT-1 surface diffusion in rat astrocytes in dissociated and slice cultures. Glia 64, 1252–1264 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Murphy-Royal, C. et al. Surface diffusion of astrocytic glutamate transporters shapes synaptic transmission. Nat. Neurosci. 18, 219–226 (2015).

    Article  CAS  PubMed  Google Scholar 

  40. Sylantyev, S. & Rusakov, D. A. Sub-millisecond ligand probing of cell receptors with multiple solution exchange. Nat. Protoc. 8, 1299–1306 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Shen, Y., Rosendale, M., Campbell, R. E. & Perrais, D. pHuji, a pH-sensitive red fluorescent protein for imaging of exo- and endocytosis. J. Cell Biol. 207, 419–432 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Liu, A. Y. et al. pHmScarlet is a pH-sensitive red fluorescent protein to monitor exocytosis docking and fusion steps. Nat. Commun. 12, 1413 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Lazarenko, R. M., DelBove, C. E., Strothman, C. E. & Zhang, Q. Ammonium chloride alters neuronal excitability and synaptic vesicle release. Sci. Rep. 7, 5061 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Reits, E. A. J. & Neefjes, J. J. From fixed to FRAP: measuring protein mobility and activity in living cells. Nat. Cell Biol. 3, E145–E147 (2001).

    Article  CAS  PubMed  Google Scholar 

  45. Jensen, T. P., Zheng, K., Tyurikova, O., Reynolds, J. P. & Rusakov, D. A. Monitoring single-synapse glutamate release and presynaptic calcium concentration in organised brain tissue. Cell Calcium 64, 102–108 (2017).

    Article  CAS  PubMed  Google Scholar 

  46. Jensen, T. P. et al. Multiplex imaging relates quantal glutamate release to presynaptic Ca2+ homeostasis at multiple synapses in situ. Nat. Commun. 10, 1414 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Gabriel, L., Stevens Z. & Melikian H. Measuring plasma membrane protein endocytic rates by reversible biotinylation. J. Vis. Exp. https://doi.org/10.3791/1669 (2009).

  48. Turvy, D. N. & Blum J. S. Biotin labeling and quantitation of cell-surface proteins. Curr. Protoc. Immunol. Chapter 18, Unit 18 17 (2001).

  49. Holton, K. L., Loder, M. K. & Melikian, H. E. Nonclassical, distinct endocytic signals dictate constitutive and PKC-regulated neurotransmitter transporter internalization. Nat. Neurosci. 8, 881–888 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Scott, D. B., Michailidis, I., Mu, Y., Logothetis, D. & Ehlers, M. D. Endocytosis and degradative sorting of NMDA receptors by conserved membrane-proximal signals. J. Neurosci. 24, 7096–7109 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Rizzolio, S. & Tamagnone, L. Antibody-feeding assay: a method to track the internalization of neuropilin-1 and other cell surface receptors. Methods Mol. Biol. 1493, 311–319 (2017).

    Article  CAS  PubMed  Google Scholar 

  52. Chiu, A. M., Barse L., Hubalkova P. & Sanz-Clemente A. An antibody feeding approach to study glutamate receptor trafficking in dissociated primary hippocampal cultures. J. Vis. Exp. https://doi.org/10.3791/59982 (2019).

  53. Kim, S., Bell, K., Mousa, S. A. & Varner, J. A. Regulation of angiogenesis in vivo by ligation of integrin α5β1 with the central cell-binding domain of fibronectin. Am. J. Pathol. 156, 1345–1362 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Zilberstein, A., Snider, M. D., Porter, M. & Lodish, H. F. Mutants of vesicular stomatitis virus blocked at different stages in maturation of the viral glycoprotein. Cell 21, 417–427 (1980).

    Article  CAS  PubMed  Google Scholar 

  55. Pepperkok, R. et al. Imaging platforms for measurement of membrane trafficking. Methods Enzymol. 404, 8–18 (2005).

    Article  CAS  PubMed  Google Scholar 

  56. Passafaro, M., Piech, V. & Sheng, M. Subunit-specific temporal and spatial patterns of AMPA receptor exocytosis in hippocampal neurons. Nat. Neurosci. 4, 917–926 (2001).

    Article  CAS  PubMed  Google Scholar 

  57. Hein, L., Ishii, K., Coughlin, S. R. & Kobilka, B. K. Intracellular targeting and trafficking of thrombin receptors. A novel mechanism for resensitization of a G protein-coupled receptor. J. Biol. Chem. 269, 27719–27726 (1994).

    Article  CAS  PubMed  Google Scholar 

  58. Hopp, T. P. et al. A short polypeptide marker sequence useful for recombinant protein identification and purification. Nat. Biotechnol. 6, 1204–1210 (1988).

    Article  CAS  Google Scholar 

  59. Leake, M. C. et al. Stoichiometry and turnover in single, functioning membrane protein complexes. Nature 443, 355–358 (2006).

    Article  CAS  PubMed  Google Scholar 

  60. Willig, K. I., Rizzoli, S. O., Westphal, V., Jahn, R. & Hell, S. W. STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature 440, 935–939 (2006).

    Article  CAS  PubMed  Google Scholar 

  61. Luo, N., Yan, A. & Yang, Z. B. Measuring exocytosis rate using corrected fluorescence recovery after photoconversion. Traffic 17, 554–564 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Chen, X., Zaro, J. L. & Shen, W. C. Fusion protein linkers: property, design and functionality. Adv. Drug Deliv. Rev. 65, 1357–1369 (2013).

    Article  CAS  PubMed  Google Scholar 

  63. Beaudoin, G. M. 3rd et al. Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex. Nat. Protoc. 7, 1741–1754 (2012).

    Article  CAS  PubMed  Google Scholar 

  64. Kaech, S. & Banker, G. Culturing hippocampal neurons. Nat. Protoc. 1, 2406–2415 (2006).

    Article  CAS  PubMed  Google Scholar 

  65. Lein, P. J., Barnhart, C. D. & Pessah, I. N. Acute hippocampal slice preparation and hippocampal slice cultures. Methods Mol. Biol. 758, 115–134 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Jiang, M. & Chen, G. High Ca2+-phosphate transfection efficiency in low-density neuronal cultures. Nat. Protoc. 1, 695–700 (2006).

    Article  CAS  PubMed  Google Scholar 

  67. Chen, C. C. et al. Patch-clamp technique to characterize ion channels in enlarged individual endolysosomes. Nat. Protoc. 12, 1639–1658 (2017).

    Article  CAS  PubMed  Google Scholar 

  68. Rathje, M. et al. AMPA receptor pHluorin-GluA2 reports NMDA receptor-induced intracellular acidification in hippocampal neurons. Proc. Natl Acad. Sci. USA 110, 14426–14431 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Savtchenko, L. P. et al. Disentangling astroglial physiology with a realistic cell model in silico. Nat. Commun. 9, 3554 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  70. Drobizhev, M., Makarov, N. S., Tillo, S. E., Hughes, T. E. & Rebane, A. Two-photon absorption properties of fluorescent proteins. Nat. Methods 8, 393–399 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Hafner, G. et al. Mapping brain-wide afferent inputs of parvalbumin-expressing GABAergic neurons in barrel cortex reveals local and long-range circuit motifs. Cell Rep. 28, 3450–3461 e3458 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Valenti, M. T. et al. The effect of bisphosphonates on gene expression: GAPDH as a housekeeping or a new target gene? BMC Cancer 6, 49 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  73. Nifosi, R. & Luo, Y. Predictions of novel two-photon absorption bands in fluorescent proteins. J. Phys. Chem. B 111, 14043–14050 (2007).

    Article  CAS  PubMed  Google Scholar 

  74. Rueden, C. T. et al. ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinformatics 18, 529 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

Contributions

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.

Corresponding authors

Correspondence to Piotr Michaluk or Dmitri A. Rusakov.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Protocols thanks Michael Ashby, Alex Verkhratsky and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key reference using this protocol

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

Rights and permissions

Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-022-00732-4

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing