Project C03 – Microscopic methods to study how GPCR dynamics and localization impact signaling specificity
Our project will 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.
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
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.