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Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy
Published on: April 9, 2017
Quantification of GPCR internalization by single-molecule microscopy in living cells
Arnauld Sergé1, Sandra de Keijzer, Freek Van Hemert
1Physics of Life Processes, Leiden Institute of Physics, Leiden University, P.O. Box 9504, Leiden, The Netherlands.
Summary
Single-molecule imaging reveals how Dictyostelium cAMP receptors (cAR1) internalize after stimulation. This phosphorylation-dependent process is crucial for cell development and offers a new way to study G-protein-coupled receptors (GPCRs).
Area of Science:
- Cell Biology
- Biophysics
- Developmental Biology
Background:
- Receptor internalization is vital for cellular signaling and environmental sensing.
- Visualizing G-protein-coupled receptor (GPCR) trafficking in vivo is challenging due to autofluorescence.
- Single-molecule fluorescence microscopy offers high-resolution tracking of cellular processes.
Purpose of the Study:
- To visualize and quantify the real-time internalization of Dictyostelium discoideum cAMP receptors (cAR1) using single-molecule fluorescence microscopy.
- To investigate the role of phosphorylation in cAR1 internalization.
- To determine the involvement of cAR1 internalization in Dictyostelium multicellular development.
Main Methods:
- Utilized single-molecule fluorescence microscopy to track eYFP-tagged cAR1 in living Dictyostelium cells.
- Employed phosphorylation-deficient cAR1 mutants and antagonists to study the phosphorylation dependence of internalization.
- Observed cAR1 internalization dynamics in mound-stage Dictyostelium cells.
Main Results:
- Demonstrated that persistent agonist stimulation leads to an increase in cytosolic receptors and subsequent degradation.
- Confirmed that cAR1 internalization is phosphorylation-dependent, as evidenced by mutant studies and antagonist treatments.
- Showed that phosphorylation-induced cAR1 internalization is essential for proper multicellular development in Dictyostelium.
Conclusions:
- This study provides the first visualization of phosphorylation-dependent single cAR1 molecule internalization in living cells.
- cAR1 internalization is a critical process for Dictyostelium multicellular development.
- The sensitive imaging technique developed can be broadly applied for pharmacological characterization of GPCRs.
