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Development of Nonspecific BRET-Based Biosensors to Monitor Plasma Membrane Inositol Lipids in Living Cells

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Intracellular Lipid Transport

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1949))

Abstract

There are several difficulties to face when investigating the role of phosphoinositides. Although they are present in most organelles, their concentration is very low, sometimes undetectable with the available methods; moreover, their level can quickly change upon several external stimuli. Here we introduce a newly improved lipid sensor tool-set based on the balanced expression of luciferase-fused phosphoinositide recognizing protein domains and a Venus protein targeted to the plasma membrane, allowing us to perform Bioluminescence Resonance Energy Transfer (BRET) measurements that reflect phosphoinositide changes in a population of transiently transfected cells. This method is highly sensitive, specific, and capable of semiquantitative characterization of plasma membrane phosphoinositide changes with high temporal resolution.

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References

  1. Balla T (2013) Phosphoinositides: tiny lipids with giant impact on cell regulation. Physiol Rev 93:1019–1137

    Article  CAS  Google Scholar 

  2. Idevall-Hagren O, De Camilli P (2015) Detection and manipulation of phosphoinositides. Biochim Biophys Acta 1851:736–745

    Article  CAS  Google Scholar 

  3. Hokin LE, Hokin MR (1958) Phosphoinositides and protein secretion in pancreas slices. J Biol Chem 233:805–810

    CAS  PubMed  Google Scholar 

  4. Hokin MR, Hokin LE (1953) Enzyme secretion and the incorporation of P32 into phospholipides of pancreas slices. J Biol Chem 203:967–977

    CAS  PubMed  Google Scholar 

  5. Nakanishi S, Catt KJ, Balla T (1995) A wortmannin-sensitive phosphatidylinositol 4-kinase that regulates hormone-sensitive pools of inositolphospholipids. Proc Natl Acad Sci U S A 92:5317–5321

    Article  CAS  Google Scholar 

  6. Traynor-Kaplan AE, Harris AL, Thompson BL et al (1988) An inositol tetrakisphosphate-containing phospholipid in activated neutrophils. Nature 334:353–356

    Article  CAS  Google Scholar 

  7. Hsu FF, Turk J (2000) Characterization of phosphatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate by electrospray ionization tandem mass spectrometry: a mechanistic study. J Am Soc Mass Spectrom 11:986–999

    Article  CAS  Google Scholar 

  8. Kielkowska A, Niewczas I, Anderson KE et al (2014) A new approach to measuring phosphoinositides in cells by mass spectrometry. Adv Biol Regul 54:131–141

    Article  CAS  Google Scholar 

  9. Wenk MR, Lucast L, Di Paolo G et al (2003) Phosphoinositide profiling in complex lipid mixtures using electrospray ionization mass spectrometry. Nat Biotechnol 21:813–817

    Article  CAS  Google Scholar 

  10. Kraft ML, Klitzing HA (2014) Imaging lipids with secondary ion mass spectrometry. Biochim Biophys Acta 1841:1108–1119

    Article  CAS  Google Scholar 

  11. Wakelam MJ (2014) The uses and limitations of the analysis of cellular phosphoinositides by lipidomic and imaging methodologies. Biochim Biophys Acta 1841:1102–1107

    Article  CAS  Google Scholar 

  12. Golebiewska U, Kay JG, Masters T et al (2011) Evidence for a fence that impedes the diffusion of phosphatidylinositol 4,5-bisphosphate out of the forming phagosomes of macrophages. Mol Biol Cell 22:3498–3507

    Article  CAS  Google Scholar 

  13. Lipsky NG, Pagano RE (1983) Sphingolipid metabolism in cultured fibroblasts: microscopic and biochemical studies employing a fluorescent ceramide analogue. Proc Natl Acad Sci U S A 80:2608–2612

    Article  CAS  Google Scholar 

  14. Hammond GR, Dove SK, Nicol A et al (2006) Elimination of plasma membrane phosphatidylinositol (4,5)-bisphosphate is required for exocytosis from mast cells. J Cell Sci 119:2084–2094

    Article  CAS  Google Scholar 

  15. Hammond GR, Schiavo G, Irvine RF (2009) Immunocytochemical techniques reveal multiple, distinct cellular pools of PtdIns4P and PtdIns(4,5)P(2). Biochem J 422:23–35

    Article  CAS  Google Scholar 

  16. Varnai P, Gulyas G, Toth DJ et al (2017) Quantifying lipid changes in various membrane compartments using lipid binding protein domains. Cell Calcium 64:72–82

    Article  CAS  Google Scholar 

  17. Varnai P, Balla T (2008) Live cell imaging of phosphoinositides with expressed inositide binding protein domains. Methods 46:167–176

    Article  CAS  Google Scholar 

  18. Balla T, Varnai P (2009) Visualization of cellular phosphoinositide pools with GFP-fused protein-domains. Curr Protoc Cell Biol 24(24):24

    Google Scholar 

  19. Stauffer TP, Ahn S, Meyer T (1998) Receptor-induced transient reduction in plasma membrane PtdIns(4,5)P2 concentration monitored in living cells. Curr Biol 8:343–346

    Article  CAS  Google Scholar 

  20. Hammond GR, Balla T (2015) Polyphosphoinositide binding domains: key to inositol lipid biology. Biochim Biophys Acta 1851:746–758

    Article  CAS  Google Scholar 

  21. Stahelin RV, Scott JL, Frick CT (2014) Cellular and molecular interactions of phosphoinositides and peripheral proteins. Chem Phys Lipids 182:3–18

    Article  CAS  Google Scholar 

  22. van der Wal J, Habets R, Varnai P et al (2001) Monitoring agonist-induced phospholipase C activation in live cells by fluorescence resonance energy transfer. J Biol Chem 276:15337–15344

    Article  Google Scholar 

  23. Toth DJ, Toth JT, Gulyas G et al (2012) Acute depletion of plasma membrane phosphatidylinositol 4,5-bisphosphate impairs specific steps in endocytosis of the G-protein-coupled receptor. J Cell Sci 125:2185–2197

    Article  CAS  Google Scholar 

  24. Hamdan FF, Percherancier Y, Breton B et al (2006) Monitoring protein-protein interactions in living cells by bioluminescence resonance energy transfer (BRET). Curr Protoc Neurosci 5(5):23

    PubMed  Google Scholar 

  25. Toth JT, Gulyas G, Toth DJ et al (2016) BRET-monitoring of the dynamic changes of inositol lipid pools in living cells reveals a PKC-dependent PtdIns4P increase upon EGF and M3 receptor activation. Biochim Biophys Acta 1861:177–187

    Article  CAS  Google Scholar 

  26. Kuo MS, Auriau J, Pierre-Eugene C et al (2014) Development of a human breast-cancer derived cell line stably expressing a bioluminescence resonance energy transfer (BRET)-based phosphatidyl inositol-3 phosphate (PIP3) biosensor. PLoS One 9:e92737

    Article  Google Scholar 

  27. Szymczak AL, Workman CJ, Wang Y et al (2004) Correction of multi-gene deficiency in vivo using a single ‘self-cleaving’ 2A peptide-based retroviral vector. Nat Biotechnol 22:589–594

    Article  CAS  Google Scholar 

  28. Woo J, von Arnim AG (2008) Mutational optimization of the coelenterazine-dependent luciferase from Renilla. Plant Methods 4:23

    Article  Google Scholar 

  29. Gulyas G, Radvanszki G, Matuska R et al (2017) Plasma membrane phosphatidylinositol 4-phosphate and 4,5-bisphosphate determine the distribution and function of K-Ras4B but not H-Ras proteins. J Biol Chem 292:18862–18877

    Article  CAS  Google Scholar 

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Acknowledgments

This work was supported by the Hungarian National Research, Development and Innovation Fund Grants NKFIH K105006 (to PV), NVKP 16-1-2016-0039 (to LH). The technical assistance of Kata Szabolcsi and Dániel Nagy is highly appreciated.

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Authors and Affiliations

  1. Faculty of Medicine, Department of Physiology, Semmelweis University, Budapest, Hungary

    József T. Tóth, László Hunyady & Péter Várnai

  2. Faculty of Medicine, Department of Anesthesiology and Intensive Therapy, Semmelweis University, Budapest, Hungary

    József T. Tóth

  3. Faculty of Medicine, Department of Physiology Semmelweis University, Budapest, Hungary

    Gergő Gulyás

  4. MTA-SE Laboratory of Molecular Physiology, Budapest, Hungary

    László Hunyady & Péter Várnai

Authors
  1. József T. Tóth
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  2. Gergő Gulyás
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  3. László Hunyady
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  4. Péter Várnai
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Corresponding author

Correspondence to Péter Várnai .

Editor information

Editors and Affiliations

  1. Institut de Pharmacologie Moléculaire et Cellulaire, Université Côte d’Azur and CNRS, Valbonne, France

    Guillaume Drin

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Tóth, J.T., Gulyás, G., Hunyady, L., Várnai, P. (2019). Development of Nonspecific BRET-Based Biosensors to Monitor Plasma Membrane Inositol Lipids in Living Cells. In: Drin, G. (eds) Intracellular Lipid Transport. Methods in Molecular Biology, vol 1949. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9136-5_3

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  • DOI: https://doi.org/10.1007/978-1-4939-9136-5_3

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9135-8

  • Online ISBN: 978-1-4939-9136-5

  • eBook Packages: Springer Protocols

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