Real-Time Imaging of Mu Opioid Receptors by Total Internal Reflection Fluorescence Microscopy

  • Cristina Roman-Vendrell
  • Guillermo Ariel Yudowski
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1230)

Abstract

Receptor trafficking and signaling are intimately linked, especially in the Mu opioid receptor (MOR) where ligand-dependent endocytosis and recycling have been associated with opioid tolerance and dependence. Ligands of MOR can induce receptor endocytosis and recycling within minutes of exposure in heterologous systems and cultured neurons. Endocytosis removes desensitized receptors after their activation from the plasma membrane, while recycling promotes resensitization by delivering functional receptors to the cell surface. These rapid mechanisms can escape traditional analytical methods where only snapshots are obtained from highly dynamic events.

Total internal reflection fluorescence (TIRF) microscopy is a powerful tool that can be used to investigate, in real time, surface trafficking events at the single molecule level. The restricted excitation of fluorophores located at or near the plasma membrane in combination with high sensitivity quantitative cameras makes it possible to record and analyze individual endocytic and recycling event in real time. In this chapter, we describe a TIRF microscopy protocol to investigate in real time, the ligand-dependent MOR trafficking in Human Embryonic Kidney 293 cells and dissociated striatal neuronal cultures. This approach can provide unique spatio-temporal resolution to understand the fundamental events controlling MOR trafficking at the plasma membrane.

Key words

G protein-coupled receptor MOR TIRF Live cell imaging Endocytosis Recycling Resensitization 

References

  1. 1.
    Koch T, Widera A, Bartzsch K et al (2005) Receptor endocytosis counteracts the development of opioid tolerance. Mol Pharmacol 67: 280–287PubMedCrossRefGoogle Scholar
  2. 2.
    Koch T, Höllt V (2008) Role of receptor internalization in opioid tolerance and dependence. Pharmacol Ther 117:199–206PubMedCrossRefGoogle Scholar
  3. 3.
    Whistler JL, Chuang HH, Chu P et al (1999) Functional dissociation of mu opioid receptor signaling and endocytosis: implications for the biology of opiate tolerance and addiction. Neuron 23:737–746PubMedCrossRefGoogle Scholar
  4. 4.
    Bushell T, Endoh T, Simen AA et al (2002) Molecular components of tolerance to opiates in single hippocampal neurons. Mol Pharmacol 61:55–64PubMedCrossRefGoogle Scholar
  5. 5.
    Bailey CP, Couch D, Johnson E et al (2003) Mu-opioid receptor desensitization in mature rat neurons: lack of interaction between DAMGO and morphine. J Neurosci 23:10515–10520PubMedGoogle Scholar
  6. 6.
    Haberstock-Debic H, Kim K-A, Yu YJ et al (2005) Morphine promotes rapid, arrestin-dependent endocytosis of mu-opioid receptors in striatal neurons. J Neurosci 25:7847–7857PubMedCrossRefGoogle Scholar
  7. 7.
    Grecksch G, Bartzsch K, Widera A et al (2006) Development of tolerance and sensitization to different opioid agonists in rats. Psychopharmacology 186:177–184PubMedCrossRefGoogle Scholar
  8. 8.
    Enquist J, Kim J, Bartlett S (2011) A novel knock-in mouse reveals mechanistically distinct forms of morphine tolerance. J Pharmacol 338: 633–640Google Scholar
  9. 9.
    Schmoranzer J, Goulian M, Axelrod D et al (2000) Imaging constitutive exocytosis with total internal reflection fluorescence microscopy. J Cell Biol 149:23–32PubMedCrossRefPubMedCentralGoogle Scholar
  10. 10.
    Steyer JA, Almers W (2001) A real-time view of life within 100 nm of the plasma membrane. Nat Rev Mol Cell Biol 2:268–275PubMedCrossRefGoogle Scholar
  11. 11.
    Wennmalm S, Simon SM (2007) Studying individual events in biology. Annu Rev Biochem 76:419–446PubMedCrossRefGoogle Scholar
  12. 12.
    Roman-Vendrell C, Yu YJ, Yudowski GA (2012) Fast modulation of μ-opioid receptor (MOR) recycling is mediated by receptor agonists. J Biol Chem 287:14782–14791PubMedCrossRefPubMedCentralGoogle Scholar
  13. 13.
    Yu YJ, Dhavan R, Chevalier MW et al (2010) Rapid delivery of internalized signaling receptors to the somatodendritic surface by sequence-specific local insertion. J Neurosci 30:11703–11714PubMedCrossRefPubMedCentralGoogle Scholar
  14. 14.
    Soohoo AL, Puthenveedu MA (2013) Divergent modes for cargo-mediated control of clathrin-coated pit dynamics. Mol Biol Cell 24:1725–1734PubMedCrossRefPubMedCentralGoogle Scholar
  15. 15.
    Henry AG, Hislop JN, Grove J et al (2012) Regulation of endocytic clathrin dynamics by cargo ubiquitination. Dev Cell 23:519–532PubMedCrossRefPubMedCentralGoogle Scholar
  16. 16.
    Miesenbock G, De Angelis DA, Rothman JE (1998) Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394:192–195PubMedCrossRefGoogle Scholar
  17. 17.
    Sankaranarayanan S, De Angelis D, Rothman JE et al (2000) The use of pHluorins for optical measurements of presynaptic activity. Biophys J 79:2199–2208PubMedCrossRefPubMedCentralGoogle Scholar
  18. 18.
    Sage D, Neumann FR, Hediger F et al (2005) Automatic tracking of individual fluorescence particles: application to the study of chromosome dynamics. IEEE Trans Image Process 14: 1372–1383PubMedCrossRefGoogle Scholar
  19. 19.
    Saffarian S, Cocucci E, Kirchhausen T (2009) Distinct dynamics of endocytic clathrin-coated pits and coated plaques. PLoS Biol 7: e1000191PubMedCrossRefPubMedCentralGoogle Scholar
  20. 20.
    Flores-Otero J et al (2014) Ligand-specific endocytic dwell times control functional selectivity of the cannabinoid receptor 1. Nat Commun 5:4589 doi:10.1038/ncomms5589
  21. 21.
    Herskowitz I (1987) Functional inactivation of genes by dominant negative mutations. Nature 329:219–222PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Cristina Roman-Vendrell
    • 1
    • 2
    • 3
  • Guillermo Ariel Yudowski
    • 1
    • 2
  1. 1.Department of Anatomy and Neurobiology, School of MedicineUniversity of Puerto RicoSan JuanPuerto Rico
  2. 2.Institute of NeurobiologyUniversity of Puerto RicoSan JuanPuerto Rico
  3. 3.Department of Physiology, School of MedicineUniversity of Puerto RicoSan JuanPuerto Rico

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