Imaging early signaling events in T lymphocytes with fluorescent biosensors

Many recent advances in our understanding of T lymphocyte functions in adaptive immunity are derived from sophisticated imaging techniques used to visualize T lymphocyte behavior in vitro and in vivo. A current challenge is to couple such imaging techniques with methods that will allow researchers to visualize signaling phenomenon at the single-cell level. Fluorescent biosensors, either synthetic or genetically encoded, are emerging as important tools for revealing the spatio-temporal regulation of intracellular biochemical events, such as specific enzyme activities or fluctuations in metabolites. In this review, we revisit the development of fluorescent Ca(2+) sensors with which the first experiments visualizing T lymphocyte activation at the single-cell were performed, and which have since become routine tools in immunology. We then examine a number of examples of how fluorescence resonance energy transfer (FRET)-based biosensors have been developed and applied to T lymphocyte migration, adhesion and T-cell receptor (TCR)-mediated signal transduction. These include the study of small GTPases such as RhoA, Rac and Rap1, the tyrosine kinases Lck and ZAP-70, and metabolites such as cAMP and Ca(2+) . Future development and use of biosensors should allow immunologists to reconcile the vast wealth of existing biochemical data concerning T-cell functions with the power of dynamic live-cell imaging.