Cyclic Nucleotides signalling
Cyclic nucleotides are intracellular second messengers involved in a plethora of functions from the ancestral fight-or-flight response to control or memory formation, from regulation of inflammation and immunity to the modulation of metabolism.
The main approach of our laboratory is to use FRET-based reporters and other optical indicators to study signal transduction in intact, living cells. By real-time imaging of cells that carry these indicators we can monitor with high spatial and temporal resolution a number of biochemical events as they occur in the complex intracellular environment. We are particularly interested in understanding how modulation of signal transduction is achieved at the subcellular level and how compartment-specific signalling controls specific cellular functions.
By combining biochemical, molecular, genetic and imaging techniques we aim to elucidate molecular mechanisms that are critical for the temporal and spatial control of cyclic nucleotide signalling and their relevance to both physiological and disease conditions. By using targeted reporters we aim at dissecting the local modulation of signalling events, their integration into complex signaling networks and their role in the control of cell pathophysiology.
Compartmentalized cyclic nucleotide signals in cardiac myocytes
We are interested in the role of cyclic nucleotides in heart pathophysiology. We are investigating the role of phosphodiesterases, the enzymes that degrade cyclic nucleotides, in the regulation of compartmentalized signaling and their involvement in heart disease. We are especially interested in the interplay between cAMP and cGMP signalling and its role in heart pathophysiology. As cAMP plays a key role in the regulation of excitation-contraction coupling in the heart, we want to understand how local cAMP signals affect cardiac physiology and we aim at establishing how disruption of local control of signal propagation leads to pathological conditions such as cardiac hypertrophy and heart failure.
Quantitative measurements of cyclic nucleotides in intact cells
We are calibrating FRET sensor responses by microinfusion of known concentrations of cyclic nucleotides into living cells. With this „in cell“ calibration approach we intend to directly correlate FRET signals to cAMP concentrations. This will allow us to measure quantitatively the level of cyclic nucleotides in different subcellular compartments.
Current projects in the laboratory are focusing on:
cyclic nucleotide dynamics in subcellular compartments of cardiac myocytes
cAMP signalling at mitochondria
the role of centrosomal cAMP dynamics in cell cycle regulation
cAMP-mediated regulation of plasma membrane ion channels (e.g. CFTR)
local cAMP signals in endothelial cells
Adult Myocyte AKAP & Alpha-Actinin Merge
Confocal image of an adult ventricular myocyte expressing a reporter for cAMP targeted to endogenous AKAPs (green), the scaffolding proteins that anchor PKA in proximity of its targets. Immunostaining of a-actinin is shown in red. (Image by L. Fields)
Pseudocolour Image of Real time Cyclic-AMP measurement
Neonatal ventricular myocyte expressing a PKA-based targeted reporter for cAMP. The pseudocolored image shows the cAMP distribution in the cell and in specific compartments and is a snapshot of a real-time measurement of this living cell. Blue indicates a low, and red a high cAMP level. (Image by M. Zaccolo)
CHO-Cell expressing Mitochondria-targeted RFP
Targeted reporter used to monitor mitochondrial dynamics over time. It is a snapshot of a time-lapse observation of a CHO cell expressing a mitochondrial protein target with a Red Fluorescent Protein (RFP). (Image by N.C. Surdo)