High-resolution spatiotemporal analysis of receptor dynamics by single-molecule fluorescence microscopy

Titiwat Sungkaworn, Finn Rieken, Martin J. Lohse, Davide Calebiro

Research output: Contribution to journalArticlepeer-review

9 Citations (Scopus)

Abstract

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., β2-adrenergic and γ-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

Original languageEnglish
Article numbere51784
JournalJournal of Visualized Experiments
Issue number89
DOIs
Publication statusPublished - 25 Jul 2014

Keywords

  • Bioengineering
  • Dimerization
  • Issue 89
  • Live-cell imaging
  • Microscopy
  • Pharmacology
  • Protein-protein interactions
  • Receptor
  • Single-molecule
  • Total internal reflection fluorescence
  • Tracking

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Immunology and Microbiology(all)
  • Biochemistry, Genetics and Molecular Biology(all)

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