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

Piotr Michaluk, Dmitri A. Rusakov

Published: 2022-09-04 DOI: 10.1038/s41596-022-00732-4

pH-sensitive fluorescent tag

cell membrane recycling

fluorescence recovery after photobleaching

GLT1 transporter

live-cell imaging

AI 解读

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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
Introduction
Materials
Procedure
Troubleshooting
Timing
Anticipated results
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References
ACKNOWLEDGMENTS
ACKNOWLEDGMENTS

Michaluk, P., Heller, J. P. & Rusakov, D. A. Rapid recycling of glutamate transporters on the astroglial surface. eLife 10, 64714 (2021).
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).
Lippincott-Schwartz, J. & Patterson, G. H. Development and use of fluorescent protein markers in living cells. Science 300, 87–91 (2003).
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).
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).
Lambert, N. A. Uncoupling diffusion and binding in FRAP experiments. Nat. Methods 6, 183–184 (2009).
Choquet, D. & Triller, A. The role of receptor diffusion in the organization of the postsynaptic membrane. Nat. Rev. Neurosci. 4, 251–265 (2003).
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).
Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).
Rossier, O. et al. Integrins β1 and β3 exhibit distinct dynamic nanoscale organizations inside focal adhesions. Nat. Cell Biol. 14, 1057–1067 (2012).
Manley, S. et al. High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nat. Methods 5, 155–157 (2008).
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).
Giannone, G. et al. Dynamic superresolution imaging of endogenous proteins on living cells at ultra-high density. Biophys. J. 99, 1303–1310 (2010).
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).
Livet, J. et al. Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450, 56–62 (2007).
Barnes, S. A. et al. Convergence of hippocampal pathophysiology in Syngap+/− and Fmr1−/y mice. J. Neurosci. 35, 15073–15081 (2015).
Politi, A. Z. et al. Quantitative mapping of fluorescently tagged cellular proteins using FCS-calibrated four-dimensional imaging. Nat. Protoc. 13, 1445–1464 (2018).
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).
Kamiyama, D. et al. Versatile protein tagging in cells with split fluorescent protein. Nat. Commun. 7, 11046 (2016).
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).
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).
Sankaranarayanan, S. & Ryan, T. A. Calcium accelerates endocytosis of vSNAREs at hippocampal synapses. Nat. Neurosci. 4, 129–136 (2001).
Kaech, S. & Banker, G. Culturing hippocampal neurons. Nat. Protoc. 1, 2406–2415 (2006).
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).
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).
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).
Tanaka, K. et al. Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1. Science 276, 1699–1702 (1997).
Danbolt, N. C. Glutamate uptake. Progr. Neurobiol. 65, 1–105 (2001).
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).
Diamond, J. S. & Jahr, C. E. Transporters buffer synaptically released glutamate on a submillisecond time scale. J. Neurosci. 17, 4672–4687 (1997).
Wadiche, J. I., Arriza, J. L., Amara, S. G. & Kavanaugh, M. P. Kinetics of a human glutamate transporter. Neuron 14, 1019–1027 (1995).
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).
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).
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).
Henneberger, C. et al. LTP induction boosts glutamate spillover by driving withdrawal of perisynaptic astroglia. Neuron 108, 919–936 e911 (2020).
Maragakis, N. J. & Rothstein, J. D. Glutamate transporters: animal models to neurologic disease. Neurobiol. Dis. 15, 461–473 (2004).
Fontana, A. C. Current approaches to enhance glutamate transporter function and expression. J. Neurochem. 134, 982–1007 (2015).
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).
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).
Murphy-Royal, C. et al. Surface diffusion of astrocytic glutamate transporters shapes synaptic transmission. Nat. Neurosci. 18, 219–226 (2015).
Sylantyev, S. & Rusakov, D. A. Sub-millisecond ligand probing of cell receptors with multiple solution exchange. Nat. Protoc. 8, 1299–1306 (2013).
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).
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).
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).
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).
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).
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).
Gabriel, L., Stevens Z. & Melikian H. Measuring plasma membrane protein endocytic rates by reversible biotinylation. J. Vis. Exp. http://doi.org/10.3791/1669 (2009).
Turvy, D. N. & Blum J. S. Biotin labeling and quantitation of cell-surface proteins. Curr. Protoc. Immunol. Chapter 18, Unit 18 17 (2001).
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).
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).
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).
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. http://doi.org/10.3791/59982 (2019).
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).
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).
Pepperkok, R. et al. Imaging platforms for measurement of membrane trafficking. Methods Enzymol. 404, 8–18 (2005).
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).
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).
Hopp, T. P. et al. A short polypeptide marker sequence useful for recombinant protein identification and purification. Nat. Biotechnol. 6, 1204–1210 (1988).
Leake, M. C. et al. Stoichiometry and turnover in single, functioning membrane protein complexes. Nature 443, 355–358 (2006).
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).
Luo, N., Yan, A. & Yang, Z. B. Measuring exocytosis rate using corrected fluorescence recovery after photoconversion. Traffic 17, 554–564 (2016).
Chen, X., Zaro, J. L. & Shen, W. C. Fusion protein linkers: property, design and functionality. Adv. Drug Deliv. Rev. 65, 1357–1369 (2013).
Beaudoin, G. M. 3rd et al. Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex. Nat. Protoc. 7, 1741–1754 (2012).
Lein, P. J., Barnhart, C. D. & Pessah, I. N. Acute hippocampal slice preparation and hippocampal slice cultures. Methods Mol. Biol. 758, 115–134 (2011).
Jiang, M. & Chen, G. High Ca2+-phosphate transfection efficiency in low-density neuronal cultures. Nat. Protoc. 1, 695–700 (2006).
Chen, C. C. et al. Patch-clamp technique to characterize ion channels in enlarged individual endolysosomes. Nat. Protoc. 12, 1639–1658 (2017).
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).
Savtchenko, L. P. et al. Disentangling astroglial physiology with a realistic cell model in silico. Nat. Commun. 9, 3554 (2018).
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).
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).
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).
Nifosi, R. & Luo, Y. Predictions of novel two-photon absorption bands in fluorescent proteins. J. Phys. Chem. B 111, 14043–14050 (2007).
Rueden, C. T. et al. ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinformatics 18, 529 (2017).

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

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