Recombinant aequorin and green fluorescent protein as valuable tools in the study of cell signalling

Luminous proteins include primary light producers, such as aequorin, and se condary photoproteins that in some organisms red-shift light emission for b etter penetration in space. When expressed in heterologous systems, both ty pes of proteins may act as versatile reporters capable of monitoring phenom ena as diverse as calcium homoeostasis, protein sorting, gene expression, a nd so on. The Ca2+-sensitive photoprotein aequorin was targeted to defined intracellular locations (organelles, such as mitochondria, endoplasmic reti culum, sarcoplasmic reticulum, Golgi apparatus and nucleus, and cytoplasmic regions. such as the bulk cytosol and the subplasmalemmal rim). and was us ed to analyse Ca2+ homoeostasis at the subcellular level. We will discuss t his application, reviewing its advantages and disadvantages and the experim ental procedure. The applications of green fluorescent protein (GFP) are ev en broader. Indeed, the ability to molecularly engineer and recombinantly e xpress a strongly fluorescent probe has provided a powerful tool for invest igating a wide variety of biological events in live cells (e.g, tracking of endogenous proteins, labelling of intracellular structures, analysing prom oter activity etc.). More recently, the demonstration that, using appropria te mutants and/or fusion proteins, GFP fluorescence can become sensitive to physiological parameters or activities (ion concentration, protease activi ty. etc.) has further expanded its applications and made GFP the favourite probe of cell biologists. We will here present two applications in the fiel d of cell signalling, i.e, the use of GFP chimaeras for studying the recrui tment of protein kinase C isoforms and the activity of intracellular protea ses.