Methods to Measure Intracellular Ca2+ Fluxes with Organelle-Targeted Aequorin-Based ProbesCell-wide Metabolic Alterations Associated with Malignancy

The photoprotein aequorin generates blue light upon binding of Ca2+ ions. Together with its very low Ca2+-buffering capacity and the possibility to add specific targeting sequences, this property has rendered aequorin particularly suitable to monitor Ca2+ concentrations in specific subcellular compartments. Recently, a new generation of genetically encoded Ca2+ probes has been developed by fusing Ca2+-responsive elements with the green fluorescent protein (GFP). Aequorin has also been employed to this aim, resulting in an aequorin-GFP chimera with the Ca2+ sensitivity of aequorin and the fluorescent properties of GFP. This setup has actually solved the major limitation of aequorin, for example, its poor ability to emit light, which rendered it inappropriate for the monitoring of Ca2+ waves at the single-cell level by imaging. In spite of the numerous genetically encoded Ca2+ indicators that are currently available, aequorin based probes remain the method of election when an accurate quantification of Ca2+ levels is required. Here, we describe currently available aequorin variants and their use for monitoring Ca2+ waves in specific subcellular compartments. Among various applications, this method is relevant for the study of the alterations of Ca2+ homeostasis that accompany oncogenesis, tumor progression, and response to therapy.