Biosensors for the Detection of Calcium and pH

The wide diffusion of molecular biology techniques, with the possibility of modifying and expressing in virtually all cell types exogenous cDNAs, has been responsible for the large expansion in the use of protein probes in cell biology. Two groups of reporter proteins are currently employed: the chemiluminescent proteins (e.g. aequorin of Aequorea victoria), and the fluorescent proteins (e.g. green fluorescent protein GFP of Aequorea victoria). The study of isolated mitochondria, dating back to the 60ties, has provided a wealth of information on the biochemical routes allowing these organelles, deriving from the adaptation of primordial symbionts, to couple oxidation of substrates to the production of ATP. Moreover, recent work highlighted the role of signals reaching the mitochondria in the activation of apoptosis. In this context, it is an exciting task to study mitochondrial function in living cells. For this purpose, new tools are needed, that combine a specific mitochondrial distribution to the sensitivity to the parameter of interest. Recombinant reporter proteins are emerging as the tools of choice, as targeting sequences can be appended that direct them to the compartment of interest. In particular, we will describe the development, and use, of protein chimeras (deriving from proteins naturally present in the medusa Aequorea victoria) specifically targeted to the mitochondria, either to the matrix or to the intermembrane space. Aequorin, the pioneer of the targeted recombinant probes, is a Ca2+-sensitive photoprotein, that emits light upon binding of Ca2+ to three high affinity binding sites. Then, we will describe the use of mutants of GFP as Ca2+ and pH probes