Nanoscale pharmacology and biochemistry using advanced fluorescence microscopies

Fluorescent labeling of individual proteins in organisms and individual cells has allowed to quantitatively monitor molecular processes and biological signaling within living cells. Over the last decades, fluorescence microscopy methods have greatly developed, allowing increasing spatial and temporal resolutions, until the climax of super-resolution optical microscopies, which allow to ‘break’ the diffraction limit.

In particular, some of these techniques can be used to extract precise information on the dynamics as well as oligomerization state of specific molecules, namely the receptors of extracellular stimuli present on cell membranes. A prominent family of such receptors, is represented by G Protein-Coupled Receptors, a family of approximately 800 members, mediating a large number of extracellular stimuli, and thus a prominent pharmacological target.

Cell signaling, in particular that mediated by G Protein-Coupled Receptors (GPCRs), entails multiple levels, starting from ligand binding, proceeding with receptor activation, coupling to downstream signaling partners and finally generation of second messenger molecules. Each of these steps, can be finely modulated by the local environment that the key signaling proteins are experiencing within the cell. In multiple instances, this subcellular modulation is responsible for surprisingly diverse responses to apparently similar stimuli.

The molecular mechanisms responsible for this heterogeneous subcellular response can be visualized and investigated by a toolbox of high spatial and temporal resolution fluorescence spectroscopy methods (Scarselli, Annibale et al. 2016, Annibale and Lohse 2020). We present here three examples: the use of molecular brightness to probe the functional oligomerization of the C-X-C chemokine receptor 4 (CXCR4) (Isbilir, Moller et al. 2020); the use of fluorescence correlation spectroscopy to observe the heterogeneous localization of 𝛃-adrenergic receptors in single adult cardiomyocytes; and finally the use of spatial-temporal image correlations to unravel the dynamics and local modulation of the second messenger cAMP by cell phosphodiesterases (Bock, Annibale et al. 2020).

-Weiss P.S., Nobel Prizes for Super-Resolution Imaging, ACS Nano 2014, 8, 10, 9689–9690
-Annibale, P. and M. J. Lohse (2020). "Spatial heterogeneity in molecular brightness." Nat Methods 17(3): 273-275.
-Bock, A., P. Annibale, C. Konrad, A. Hannawacker, S. E. Anton, I. Maiellaro, U. Zabel, S. Sivaramakrishnan, M. Falcke and M. J. Lohse (2020). "Optical Mapping of cAMP Signaling at the Nanometer Scale." Cell 182(6): 1519-1530 e1517.
-Isbilir, A., J. Moller, M. Arimont, V. Bobkov, C. Perpina-Viciano, C. Hoffmann, A. Inoue, R. Heukers, C. de Graaf, M. J. Smit, P. Annibale and M. J. Lohse (2020). "Advanced fluorescence microscopy reveals disruption of dynamic CXCR4 dimerization by subpocket-specific inverse agonists." Proc Natl Acad Sci U S A.
-Scarselli, M., P. Annibale, P. J. McCormick, S. Kolachalam, S. Aringhieri, A. Radenovic, G. U. Corsini and R. Maggio (2016). "Revealing G-protein-coupled receptor oligomerization at the single-molecule level through a nanoscopic lens: methods, dynamics and biological function." FEBS J 283(7): 1197-1217.

Short bio

Dr. Paolo Annibale is a cell biologist and biophysicist with over ten years of experience as a research microscopist, first using scanning probe methods, then fluorescence fluctuations and superresolution techniques. This expertise builds on top of a training in solid state physics, molecular electronics and semiconducting thin films matured working in Italy, Switzerland the US and now Germany. He is currently leading a small team researching fundamental molecular mechanisms modulating G protein-coupled Receptors (GPCR) signaling and the underlying pharmacology, within the context of a grant of the German Research Foundation.

The Institute of Advanced Studies and the Collegio Superiore jointly promote a series of lectures – called Battistini Lectures – to commemorate Professor Andrea Battistini, internationally reknown Italianist, Professor of Italian Literature and first Director of the Collegio Superiore (1997-2000).