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BackgroundActivation by extracellular ligands of G protein-coupled (GPCRs) and tyrosine kinase receptors (RTKs), results in the generation of second messengers that in turn control specific cell functions. Further, modulation/amplification or inhibition of the initial signalling events, depend on the recruitment onto the plasma membrane of soluble protein effectors.High throughput methodologies to monitor quantitatively second messenger production, have been developed over the last years and are largely used to screen chemical libraries for drug development. On the contrary, no such high throughput methods are yet available for the other aspect of GPCRs regulation, i.e. protein translocation to the plasma membrane, despite the enormous interest of this phenomenon for the modulation of receptor downstream functions. Indeed, to date, the experimental procedures available are either inadequate or complex and expensive.ResultsHere we describe the development of a novel conceptual approach to the study of cytosolic proteins translocation to the inner surface of the plasma membrane. The basis of the technique consists in: i) generating chimeras between the protein of interests and the calcium (Ca2+)-sensitive, luminescent photo-protein, aequorin and ii) taking advantage of the large Ca2+concentration [Ca2+] difference between bulk cytosolic and the sub-plasma membrane rim.ConclusionThis approach, that keeps unaffected the translocation properties of the signalling protein, can in principle be applied to any protein that, upon activation, moves from the cytosol to the plasma membrane.Thus, not only the modulation of GPCRs and RTKs can be investigated in this way, but that of all other proteins that can be recruited to the plasma membrane also independently of receptor activation.Moreover, its automated version, which can provide information about the kinetics and concentration-dependence of the process, is also applicable to high throughput screening of drugs affecting the translocation process.

Mitochondria rapidly accumulate Ca2+ through a low-affinity uptake system (the mitochondrial Ca2+ uniporter, MCU) because they are exposed to high [Ca2+] microdomains generated by the opening of ER Ca2+ channels. These rapid [Ca2+] changes stimulate Ca2+-sensitive dehydrogenases of the mitochondrial matrix, and hence rapidly upregulate ATP production in stimulated cells. Ca2+ also sensitizes to cell death mediators, e.g. ceramide. Accordingly, we demonstrated that Bcl-2 reduces the state of filling of ER Ca2+ stores, and this alteration is effective in reducing the sensitivity to apoptotic challenges. I will discuss our recent discovery of the molecular identity of the MCU, i.e. the key molecule of mitochondrial Ca2+ homeostasis. I will present the strategy, and the experiments, that allowed to identify the protein, that remained elusive for 50 years. Then, I will present data that clarify the composition and the regulatory mechanisms of this highly sophisticated signaling machinery. Finally, I will show that molecular targeting of MCU allows novel insight into the regulation of cellular metabolism and cell death processes.ReferenceD. De Stefani, A. Raffaello, E. Teardo, I. Szabo, R. Rizzuto (2011) A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter. Nature 476:336-340