2026-10-10T20:43:57Z https://api.figshare.com/v2/oai

oai:figshare.com:article/33986593 2026-09-24T15:35:55Z category_1 category_4 category_915 category_7 category_12 category_14 category_16 category_21 category_873 category_61 category_64 portal_63 item_type_2 month_year_09_2026

Precise Alternation between Image-Forming Sample Planes Enables Quantitative Monitoring of Receptor-Arrestin Interaction Dynamics at the Plasma Membrane of Live Cells Thomas D. Killeen (25104661) Michael R. Stoneman (20667457) Ionel Popa (1634974) Qiuyan Chen (5601662) Valerică Raicu (9581023) Biophysics Biochemistry Physical Sciences not elsewhere classified Medicine Cell Biology Molecular Biology Physiology Biotechnology Chemical Sciences not elsewhere classified Developmental Biology Cancer forming sample planes enhanced vertical resolution complex molecular mechanisms 20 nanometer repeatability cellular cross sections interactions remains challenging cytoplasmic arr2 toward arrestin interaction dynamics live cells investigations 2 </ sub live cells membrane cross numerous cells living cells individual cells quantitative analysis pronounced redistribution precision alternation precise alternation plasma membrane nonvisual arrestins multiphoton microscope high precision g protein following stimulation enabling high dynamic recruitment cell variability basolateral membrane based signaling agonist ligand 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. 2026-09-24T00:00:00Z Dataset Media 10.1021/acs.jpcb.6c02313.s004 https://figshare.com/articles/media/Precise_Alternation_between_Image-Forming_Sample_Planes_Enables_Quantitative_Monitoring_of_Receptor-Arrestin_Interaction_Dynamics_at_the_Plasma_Membrane_of_Live_Cells/33986593 CC BY-NC 4.0