Monitoring oligomerization dynamics of individual human neurotensin receptors one in living cells and in SMALP nanodiscs

Lukas Spantzel; Iván Pérez; Thomas Heitkamp; Anika Westphal; Stefanie Reuter; Ralf Mrowka; Michael Börsch

doi:10.1117/12.2648222

25 April 2023 Monitoring oligomerization dynamics of individual human neurotensin receptors one in living cells and in SMALP nanodiscs

Lukas Spantzel, Iván Pérez, Thomas Heitkamp, Anika Westphal, Stefanie Reuter, Ralf Mrowka, Michael Börsch

Author Affiliations +

Proceedings Volume 12384, Multiphoton Microscopy in the Biomedical Sciences XXIII; 1238407 (2023) https://doi.org/10.1117/12.2648222
Event: SPIE BiOS, 2023, San Francisco, California, United States

Abstract

The human neurotensin receptor one (NTSR1) is a G protein-coupled receptor. The receptor is activated by a small peptide ligand neurotensin. NTSR1 can be expressed in HEK cells by stable transfection. Previously we used the fluorescent protein markers mRuby3 or mNeonGreen fused to NTSR1 for EMCCD-based Structured Illumination Microscopy (SIM) in living HEK cells. Ligand binding induced conformational changes in NTSR1 which triggered the intracellular signaling processes. Recent single-molecule studies revealed a dynamic monomer/dimer equilibrium of this receptor in artificial lipid bilayers. Here we report on the oligomerization state of human NTSR1 from living cells by trapping them into lipid nanodiscs. Briefly, SMALPs (styrene-maleic acid copolymer lipid nanoparticles) were produced directly from the plasma membranes of living HEK293T FlpIn cells. SMALPs with a diameter of 15 nm were soluble and stable. NTSR1 in SMALPs were analyzed by single-molecule intensity measurements one membrane patch at a time using a custom-built confocal anti-Brownian electrokinetic trap (ABEL trap) microscope. We found oligomerization changes before and after stimulation of the receptor with its ligand neurotensin.

Conference Presentation

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Lukas Spantzel, Iván Pérez, Thomas Heitkamp, Anika Westphal, Stefanie Reuter, Ralf Mrowka, and Michael Börsch "Monitoring oligomerization dynamics of individual human neurotensin receptors one in living cells and in SMALP nanodiscs", Proc. SPIE 12384, Multiphoton Microscopy in the Biomedical Sciences XXIII, 1238407 (25 April 2023); https://doi.org/10.1117/12.2648222

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