Biophysical Society Thematic Meeting | Bucharest 2026

Biophysics of Membrane Reactions in Brian

Wednesday Speaker Abstracts

4D FLUORESCENCE INTENSITY FLUCTUATION SPECTROMETRY MONITORS THE LOCALIZATION AND INTERACTIONS OF LIGAND-ACTIVATED MUSCARINIC ACETYLCHOLINE RECEPTORS AMONG THEMSELVES AND WITH DOWNSTREAM SIGNALING EFFECTORS Thomas D Killeen 1 ; Michael R Stoneman 1 ; Sanam Bista 1 ; Ionel Popa 1 ; Qiuyan Chen 2 ; Valerica Raicu 1,3 ; 1 University of Wisconsin-Milwaukee, Department of Physics and Astronomy, Milwaukee, WI, USA 2 Indiana University School of Medicine, Department of Biochemistry and Molecular Biology, Indianapolis, IN, USA 3 University of Wisconsin-Milwaukee, Milwaukee, Department of Electrical Engineering, Milwaukee, WI, USA G Protein-Coupled Receptor (GPCR) homo-interactions, as well as interactions with ligands, heterotrimeric G proteins, and β -arrestins play crucial roles in cell signaling in the brain. Obtaining quantitative information on the intricacies of these interactions presents numerous challenges, motivating our development of fluorescence methods that analyze both mean emission and intensity fluctuations from tagged proteins. One of our methods, two-dimensional Fluorescence Intensity Fluctuation (2D FIF) Spectrometry, analyzes pixel-to-pixel spatial intensity fluctuations in fluorescence micrographs to determine the number of proteins in the complex. I will begin this talk with a brief overview of our published results using this and other methods to quantify homo-oligomerization of three important members of the GPCR family – the M1, M2, and M3 muscarinic acetylcholine receptors (i.e., M1R, M2R, and M3R) – in cell cultures and in mouse brain. I will then introduce a major evolution of 2D FIF spectrometry into a four-dimensional technique, 4D FIF spectrometry, which leverages an advanced laser-scanning fluorescence micro-spectroscope to simultaneously capture and deconvolute the intensity fluctuations of two fluorescent protein populations with different emission spectra. This dual color capability allows us to visualize, in live cell membranes, the localization and interaction dynamics of M2R and non-visual β -arrestins and, separately, M2R and G proteins, following activation of the receptor with agonist ligand. We believe this method is a powerful tool for deciphering intermolecular interaction events and untangling different signaling pathways of brain GPCRs mediated by G proteins or β -arrestins.

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