Project C03 (E) – Microscopic methods to study how GPCR dynamics and localization impact signaling specificity

Project of the 1st funding period (2020 -2023):
Our project address the question of how the subcellular localization of GPCRs affects their function: We will (i) describe the heterogeneous distribution of receptors, G-proteins and their dynamic coupling; (ii) analyze the dependence Receptors-, G proteins- and ligands-dependence on such heterogeneity (iii) investigate the effect of receptor-interacting proteins and local membrane curvature. As models we will use β-adrenergic receptors (β1 and β2AR), Neuropeptide Y-Receptors (NPY2R) und Muscarinic Acetylcholine Receptors (M2R). We will combine tools such as fluorescently labeled proteins and sensors with Single Molecule Tracking, Image Correlation Spectroscopy and Stochastic Localization Microscopy. We will then apply our approaches to study the compartmentalization of the receptors and their signals in Cardiac Myocytes.

Contact

Dr. Paolo Annibale (Project Leader)

Max Delbrück Center for Molecular Medicine
Robert-Rössle-Strasse 10, D-13125 Berlin

Phone +49 30 9406 1746
E-Mail
Web mdc-berlin.de/de/receptor-signaling

Prof. Dr. Martin Lohse (Project Leader)

Leipzig University, Medical Faculty
Rudolf-Boehm-Institute of Pharmacology and Toxicology
Härtelstrasse 16-18, D-04107 Leipzig

E-Mail

Iqra Sohail (PhD Student)

Max Delbrück Center for Molecular Medicine
Robert-Rössle-Strasse 10, D-13125 Berlin


Alexei Sirbu (PhD-Student)

Max Delbrück Center for Molecular Medicine
Robert-Rössle-Strasse 10, D-13125 Berlin

Resources & Techniques

  • Fluorescence microscopy
    Epifluorescent, confocal, super-resolution
  • FRET / BRET
    Electronic energy transfer through nonradiative dipole–dipole coupling
  • High-throughput screening
    Luminescence-based drug screening in microtiter plates
  • Nucleic acid and protein assays

Publications

Nagl M, Mönnich D, Rosier N, Schihada H, Sirbu A, Konar N, Reyes-Resina I, Navarro G, Franco R, Kolb P, Annibale P, Pockes S. Fluorescent Tools for the Imaging of Dopamine D2 -Like Receptors. Chembiochem. 2024 Jan 15;25(2):e202300659. doi: 10.1002/cbic.202300659 .

Rosier N, Mönnich D, Nagl M, Schihada H, Sirbu A, Konar N, Reyes-Resina I, Navarro G, Franco R, Kolb P, Annibale P, Pockes S. Shedding Light on the D1 -Like Receptors: A Fluorescence-Based Toolbox for Visualization of the D1 and D5 Receptors. Chembiochem. 2024 Jan 15;25(2):e202300658. doi: 10.1002/cbic.202300658 .

Lohse MJ, Bock A, Zaccolo M. G Protein-Coupled Receptor Signaling: New Insights Define Cellular Nanodomains. Annu Rev Pharmacol Toxicol. 2023 Sep 8. doi: 10.1146/annurev-pharmtox-040623-115054. Epub ahead of print. PMID: 37683278.

Anton SE, Kayser C, Maiellaro I, Nemec K, Möller J, Koschinski A, Zaccolo M, Annibale P, Falcke M, Lohse MJ, Bock A. Receptor-associated independent cAMP nanodomains mediate spatiotemporal specificity of GPCR signaling. Cell. 2022 Mar 31;185(7):1130-1142.e11. doi: 10.1016/j.cell.2022.02.011. Epub 2022 Mar 15. PMID: 35294858.

Boltz HH, Sirbu A, Stelzer N, de Lanerolle P, Winkelmann S, Annibale P. The Impact of Membrane Protein Diffusion on GPCR Signaling. Cells. 2022 May 17;11(10):1660. doi: 10.3390/cells11101660. PMID: 35626696.

Höpfner F, Paisdzior S, Reininghaus N, Sohail I, Scheerer P, Annibale P, Biebermann H, Kühnen P. Evaluation of Pharmacological Rescue of Melanocortin-4 Receptor Nonsense Mutations by Aminoglycoside. Life. 2022; 12(11):1793. https://doi.org/10.3390/life12111793

Nemec K, Schihada H, Kleinau G, Zabel U, Grushevskyi EO, Scheerer P, Lohse MJ, Maiellaro I. Functional modulation of PTH1R activation and signaling by RAMP2. Proc Natl Acad Sci U S A. 2022 Aug 9;119(32):e2122037119. doi: 10.1073/pnas.2122037119 . Epub 2022 Aug 1. PMID: 35914163.

Gmach P, Bathe-Peters M, Telugu N, Miller DC, Annibale P. Fluorescence Spectroscopy of Low-Level Endogenous β-Adrenergic Receptor Expression at the Plasma Membrane of Differentiating Human iPSC-Derived Cardiomyocytes. Int J Mol Sci. 2022 Sep 8;23(18):10405. doi: 10.3390/ijms231810405. PMID: 36142320; PMCID: PMC9499492.

Bathe-Peters M, Gmach P, Boltz HH, Einsiedel J, Gotthardt M, Hübner H, Gmeiner P, Lohse MJ, Annibale P. Visualization of β-adrenergic receptor dynamics and differential localization in cardiomyocytes. Proc Natl Acad Sci U S A. 2021 Jun 8;118(23):e2101119118. doi: 10.1073/pnas.2101119118. PMID: 34088840. 

Boltz HH, Sirbu A, Stelzer N, Lohse MJ, Schütte C, Annibale P. Quantitative spectroscopy of single molecule interaction times. Opt Lett. 2021; 46:1538-1541. https://doi.org/10.1364/OL.413030

Işbilir A, Serfling R, Möller J, Thomas R, De Faveri C, Zabel U, Scarselli M, Beck-Sickinger AG, Bock A, Coin I, Lohse MJ, Annibale P. Determination of G-protein–coupled receptor oligomerization by molecular brightness analyses in single cells. Nat Protoc. 2021. https://doi.org/10.1038/s41596-020-00458-1.

Annibale P, Lohse MJ. Spatial heterogeneity in molecular brightness. Nat Methods. 2020 Mar;17(3):273-275. doi: 10.1038/s41592-020-0732-0. Epub 2020 Feb 10. PMID: 32042187.

Bathe-Peters M, Gmach P, Annibale P, Lohse MJ. Linescan microscopy data to extract diffusion coefficient of a fluorescent species using a commercial confocal microscope. Data Brief. 2020 Jan 2;29:105063. doi: 10.1016/j.dib.2019.105063. eCollection 2020 Apr.

Bock A, Annibale P, Konrad C, Hannawacker A, Anton SE, Maiellaro I, Zabel U, Sivaramakrishnan S, Falcke M, Lohse MJ. Optical Mapping of cAMP Signaling at the Nanometer Scale. Cell. 2020 Aug 20:S0092-8674(20)30943-0. doi: 10.1016/j.cell.2020.07.035. Epub ahead of print. PMID: 32846156.

Paisdzior S, Dimitriou IM, Schöpe PC, Annibale P, Scheerer P, Krude H, Lohse MJ, Biebermann H, Kühnen P. Differential Signaling Profiles of MC4R Mutations with Three Different Ligands. Int J Mol Sci. 2020 Feb 12;21(4). pii: E1224. doi: 10.3390/ijms21041224. PMID: 32059383

Sungkaworn T, Jobin M-L, Burnecki K, Weron A, Lohse MJ, Calebiro D. Single-molecule imaging reveals receptor-G protein interactions at cell surface hot spots. 2017; 550:543-547.

Calebiro D, Rieken F, Wagner J, Sungkaworn T, Zabel U, Borzi A, Cocucci E, Zürn A, Lohse MJ. Single-molecule analysis of fluorescently labeled GPCRs reveals receptor-specific complexes with distinct dynamics and organization. Proc Natl Acad Sci USA. 2013; 110:743-8.

Nikolaev VO, Bünemann M, Schmitteckert E, Lohse MJ, Engelhardt S. cAMP imaging in cardiac myocytes reveals far-reaching β1– but confined β2-adrenergic receptor-mediated signaling. Circ Res. 2006; 99:1084-91.

Nikolaev VO, Gambaryan S, Lohse MJ. Fluorescent sensors for rapid monitoring of intracellular cGMP. Nat Meth. 2006; 3:23-25.

Vilardaga JP, Bünemann M, Krasel C, Castro M, Lohse MJ. Measurement of the millisecond activation switch of G-protein-coupled receptors in living cells. Nature Biotech. 2003; 21:807-12.

Di Rienzo C, Annibale P. Visualizing the molecular mode of motion from a correlative analysis of localization microscopy datasets. Opt Lett. 2016; 41:4503-6.

Scarselli M, Annibale P, McCormick P, Kolachalam S, Aringhieri S, Radenovic A, Corsini GU, Maggio R. Revealing G-protein-coupled receptor oligomerization at the single-molecule level through a nanoscopic lens: methods, dynamics and biological function. FEBS Journal. 2016; 283:1197-217.

Annibale P, Dvornikov A, Gratton E Electrical Tunable Lens Speeds-up Orbital Tracking. Biomed Opt Express. 2015; 6:2181-90.

Scarselli M, Annibale P, Radenovic A. Cell-type-specific β2 adrenergic receptor clusters identified using photo-activated localization microscopy are not lipid raft related, but depend on actin cytoskeleton integrity. J Biol Chem. 2012; 287:16768-80.

Annibale P, Vanni S, Scarselli M, Rothlisberger U, Radenovic A. Identification of clustering artifacts in PhotoActivated Localization Microscopy. Nature Meth. 2011; 8:527-528.