THE THEODOR BU ¨ CHER LECTURE Investigating signal transduction with genetically encoded fluorescent probes Delivered on 22 October 2002 at the 28th FEBS Meeting in Istanbul Tullio Pozzan 1,2 , Marco Mongillo 2 and Ru ¨ diger Rudolf 1 1 Department of Biomedical Sciences, CNR Institute of Neurosciences, University of Padua, Italy; 2 Venetian Institute for Molecular Medicine, Padua, Italy Ca 2+ and cAMP are ubiquitous second messengers in euk- aryotes and control numerous physiological responses ran- ging from fertilization to cell death induction 1 . To distinguish between these different responses, their subtle regulation in time, space and amplitude is needed. Therefore, the char- acterization of the signalling process requires measurement of second messengers with tools of precise localization, high dynamic range and as little disturbance of cell physiology as possible. Recently, fluorescent proteins of marine jellyfish have given rise to a set of genetically encoded biosensors which fulfil these criteria and which have already led to important new insights into the subcellular handling of Ca 2+ and cAMP. The use of these probes in combination with new microscopical methods such as two-photon micro- scopy now enables researchers to study second messenger signalling in intact tissues. In this review, the genetically encoded measurement probes and their origin are briefly introducedandsomerecentinsightsintothespatio-temporal complexity of both Ca 2+ and cAMP signalling obtained with these tools are discussed. Second messengers and the encoding paradigm Cells are structures of immense spatio-temporal complexity. Myriadsofdifferentproteins,fattyacids,carbohydrates,and other organic and inorganic compounds are being produced, degraded or transported at each moment. To enhance their reliability and efficiency, these processes are organized in a highly complex and dynamic system of subcellular compart- ments. These compartments not only include membrane- enclosed organelles such as the endoplasmic reticulum (ER), mitochondria, etc., but are also generated through the discrete localization of the signalling processes within the apparently homogeneous cytoplasmic environment. In addition, cells are continuously bombarded by signals released by closely located or even distant cells. To translate these extracellular signals into intracellular responses, all eukaryotic cells utilize complex transductory machineries. Such machineries are assembled with transmembrane recep- tors that sense external stimuli and a series of intracellular relay proteins, the sequential activation of which leads to the modulation of the levels of a few compounds named second messengers. These are typically very small molecules such as inositol 1,4,5-triphosphate (IP3), Ca 2+ or cyclic nucleotides. Given that numerous extracellular signals modulate the level of the same intracellular second messenger a problem of specificity obviously arises. Specificity of the signalling through second messengers is achieved by a complex encoding of (a) the localization of second messenger molecule release (compartmentalization); (b) the number of molecules released (amplitude); and (c) its temporal release pattern (frequency). In this way, a plethora of input signals, which may come from hormones, neurotransmitters, cell surface molecules, growth factors or other signalling mole- cules, is computed intracellularly to give rise to very specific cellular responses, ranging from fertilization to induction of cell death 2 , and including a vast amount of physiological processes such as secretion, contraction, growth and prolif- eration, to name only a few. Obviously, to exert all these processes the signalling must be finely tuned in a highly dynamicmanner.Thus,tostudysecondmessengersignalling it is crucial to measure their distribution in vivo and under physiological conditions. Furthermore, as signal compart- mentalization plays an important role, one ideally would need to analyse levels of second messengers simultaneously, in different subcellular compartments and with a high spatial and temporal resolution. No such ideal system is presently available, but the new tools that have been introduced in the last few years have enormously expanded our possibility to monitor the dynamics of intracellular events in the living cell. In the following paragraphs we will briefly discuss the characteristics of a few genetically engineered probes gener- ated to monitor second messenger levels in living cells, and discuss some of the newest information on cell physiology that has been obtained with these new tools. Correspondence to T. Pozzan, Department of Biomedical Sciences, University of Padua, Viale G. Colombo, 3, 35121 Padova, Italy. Fax: + 39 049 827 6049, Tel.: + 39 049 827 6070, E-mail: tullio.pozzan@unipd.it Abbreviations: AEQ, aequorin; cAMP, adenosine cyclic-3¢,5¢-mono- phosphate; cGMP, guanosine cyclic-3¢,5¢-monophosphate; ER, endoplasmic reticulum; FRET, fluorescence resonance energy trans- fer; GFP, green fluorescent protein; IP3, inositol 1,4,5-triphosphate; PKA, protein kinase A. (Received 24 February 2003, revised 7 April 2003, accepted 10 April 2003) Eur. J. Biochem. 270, 2343–2352 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03615.x