Precise Alternation between Image-Forming Sample Planes Enables Quantitative Monitoring of Receptor-Arrestin Interaction Dynamics at the Plasma Membrane of Live Cells.
- 1Department of Physics & Astronomy, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin53211, United States.
- 2Department of Biochemistry, Molecular Biology and Pharmacology, Indiana University School of Medicine, Indianapolis, Indiana46202, United States.
- 3Department of Electrical Engineering, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin53211, United States.
Abstract
Investigations of G protein-coupled receptors (GPCRs) interactions with nonvisual arrestins in living cells are essential to understanding the complex molecular mechanisms of GPCR-based signaling. Quantitative analysis of these interactions remains challenging in live cells, particularly when attempting to repeatedly image distinct cellular regions with high precision. Here, we describe the implementation of an optical imaging stabilization approach that integrates the recently developed Focal Readjustment for Enhanced Vertical Resolution (FREVR) technology into a multiphoton microscope, enabling high-precision alternation between image-forming sample planes with <20 nanometer repeatability and stability over time. Using this setup, we monitored the dynamic recruitment of arrestin-2 (Arr2) to the plasma membrane of HEK-293 cells expressing muscarinic acetylcholine M2 receptors (M2R) by alternately imaging distinct planes of interest, the basolateral membrane and a membrane cross-section. Following stimulation of M2R by agonist ligand, we observed a pronounced redistribution of cytoplasmic Arr2 toward the plasma membrane in both cellular cross sections and at the basolateral membrane. This method enables direct comparison of receptor and arrestin dynamics across regions of individual cells with very high precision, eliminating the need for averaging over numerous cells in order to denoise biologically relevant signals, and thereby capturing physiological cell-to-cell variability with accuracy.
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BibTeX
@article{killeen2026precise,
title = {{Precise Alternation between Image-Forming Sample Planes Enables Quantitative Monitoring of Receptor-Arrestin Interaction Dynamics at the Plasma Membrane of Live Cells.}},
author = {Thomas D Killeen and Michael R Stoneman and Ionel Popa and Qiuyan Chen and Valerică Raicu},
journal = {The journal of physical chemistry. B},
year = {2026},
month = oct,
doi = {10.1021/acs.jpcb.6c02313},
url = {https://europepmc.org/article/MED/42916714},
note = {CC BY},
}
RIS
TY - JOUR
TI - Precise Alternation between Image-Forming Sample Planes Enables Quantitative Monitoring of Receptor-Arrestin Interaction Dynamics at the Plasma Membrane of Live Cells.
AU - Thomas D Killeen
AU - Michael R Stoneman
AU - Ionel Popa
AU - Qiuyan Chen
AU - Valerică Raicu
PY - 2026
DA - 2026/10/01
JO - The journal of physical chemistry. B
DO - 10.1021/acs.jpcb.6c02313
UR - https://europepmc.org/article/MED/42916714
AB - Investigations of G protein-coupled receptors (GPCRs) interactions with nonvisual arrestins in living cells are essential to understanding the complex molecular mechanisms of GPCR-based signaling. Quantitative analysis of these interactions remains challenging in live cells, particularly when attempting to repeatedly image distinct cellular regions with high precision. Here, we describe the implementation of an optical imaging stabilization approach that integrates the recently developed Focal Readjustment for Enhanced Vertical Resolution (FREVR) technology into a multiphoton microscope, enabling high-precision alternation between image-forming sample planes with <20 nanometer repeatability and stability over time. Using this setup, we monitored the dynamic recruitment of arrestin-2 (Arr2) to the plasma membrane of HEK-293 cells expressing muscarinic acetylcholine M2 receptors (M2R) by alternately imaging distinct planes of interest, the basolateral membrane and a membrane cross-section. Following stimulation of M2R by agonist ligand, we observed a pronounced redistribution of cytoplasmic Arr2 toward the plasma membrane in both cellular cross sections and at the basolateral membrane. This method enables direct comparison of receptor and arrestin dynamics across regions of individual cells with very high precision, eliminating the need for averaging over numerous cells in order to denoise biologically relevant signals, and thereby capturing physiological cell-to-cell variability with accuracy.
SN - 1520-6106
SN - 1520-5207
ER -