Quantum Dot Toolbox in Membrane Neurotransmitter Transporter Research

  • Lucas B. Thal
  • Danielle M. Bailey
  • Oleg Kovtun
  • Sandra J. Rosenthal
Protocol
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Quantum dot-based fluorescence techniques enable multi-scale molecular profiling ranging from real-time single molecule dynamics to expression trends in million-cell populations. In comparison to currently available probes, quantum dots are particularly well suited for such studies by virtue of their unique photophysical properties. We discuss in this chapter methodological components of what makes up the “Quantum Dot Toolbox” in neurotransmitter transporter studies along with specific work our group has published. First, we describe ensemble analysis of subcellular transporter localization and provide visualization of transporter residence in distinct cellular surface features. Second, we provide discussion on high content analysis of changes in transporter surface levels and give insight into the advantages of using quantum dot probes in flow cytometry. Third, we review the fundamental principles of subdiffraction-limit fluorescence microscopy and single molecule analysis of transporter surface dynamics. Included in this chapter are three protocols with experimental considerations specific to each technical section.

Keywords

Flow cytometry Neurotransmitter transporters Quantum dot-based fluorescence Quantum dots Subcellular transporter localization 

References

  1. 1.
    Bruchez M, Moronne M, Gin P, Weiss S, Alivisatos AP (1998) Semiconductor nanocrystals as fluorescent biological labels. Science 281:2013–2016CrossRefPubMedGoogle Scholar
  2. 2.
    Chan WCW, Nie S (1998) Quantum dot bioconjugates for ultrasensitive nonisotopic detection. Science 281:2016–2018CrossRefPubMedGoogle Scholar
  3. 3.
    Dahan M et al (2003) Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking. Science 302:442–445CrossRefPubMedGoogle Scholar
  4. 4.
    Bouzigues C, Morel M, Triller A, Dahan M (2007) Asymmetric redistribution of GABA receptors during GABA gradient sensing by nerve growth cones analyzed by single quantum dot imaging. Proc Natl Acad Sci 104:11251–11256CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Rosenthal SJ, Chang JC, Kovtun O, McBride JR, Tomlinson ID (2011) Biocompatible quantum dots for biological applications. Chem Biol 18:10–24CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Frischknecht R et al (2009) Brain extracellular matrix affects AMPA receptor lateral mobility and short-term synaptic plasticity. Nat Neurosci 12:897–904CrossRefPubMedGoogle Scholar
  7. 7.
    Chang JC et al (2011) A fluorescence displacement assay for antidepressant drug discovery based on ligand-conjugated quantum dots. J Am Chem Soc 133:17528–17531CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Chang JC et al (2012) Single molecule analysis of serotonin transporter regulation using antagonist-conjugated quantum dots reveals restricted, p38 MAPK-dependent mobilization underlying uptake activation. J Neurosci 32:8919–8929CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Kovtun O et al (2011) Visualization of the cocaine-sensitive dopamine transporter with ligand-conjugated quantum dots. ACS Chem Nerosci 2:370–378CrossRefGoogle Scholar
  10. 10.
    Kovtun O, Ross EJ, Tomlinson ID, Rosenthal SJ (2012) A flow cytometry-based dopamine transporter binding assay using antagonist-conjugated quantum dots. Chem Commun 48:5428–5430CrossRefGoogle Scholar
  11. 11.
    Chang JC, Rosenthal SJ (2013) A bright light to reveal mobility: single quantum dot tracking reveals membrane dynamics and cellular mechanisms. J Phys Chem Lett 4:2858–2866CrossRefGoogle Scholar
  12. 12.
    Chang JC, Rosenthal SJ (2013) In: Sandra Rosenthal J, David Wright W (eds) NanoBiotechnology protocols. Humana Press, New York, pp 71–84Google Scholar
  13. 13.
    Kovtun O et al (2015) Single-quantum-dot tracking reveals altered membrane dynamics of an attention-deficit/hyperactivity-disorder-derived dopamine transporter coding variant. ACS Chem Nerosci 6:526–534CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  • Lucas B. Thal
    • 1
  • Danielle M. Bailey
    • 1
    • 2
    • 3
  • Oleg Kovtun
    • 1
  • Sandra J. Rosenthal
    • 1
    • 2
    • 3
    • 4
    • 5
    • 6
    • 7
  1. 1.Department of ChemistryVanderbilt UniversityNashvilleUSA
  2. 2.Department of PharmacologyVanderbilt UniversityNashvilleUSA
  3. 3.Department of Interdisciplinary Materials ScienceVanderbilt UniversityNashvilleUSA
  4. 4.Department of Chemical and Biomolecular EngineeringVanderbilt UniversityNashvilleUSA
  5. 5.Department of Physics and AstronomyVanderbilt UniversityNashvilleUSA
  6. 6.Vanderbilt Institute of Nanoscale Science and EngineeringVanderbilt UniversityNashvilleUSA
  7. 7.Materials Science and Technology DivisionOak Ridge National LaboratoryOak RidgeUSA

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