Measuring Baseline Ca2+ Levels in Subcellular Compartments Using Genetically Engineered Fluorescent Indicators

Intracellular Ca2+ signaling is involved in a series of physiological and pathological processes. In particular, an intimate crosstalk between bioenergetic metabolism and Ca2+ homeostasis has been shown to determine cell fate in resting conditions as well as in response to stress. The endoplasmic reticulum and mitochondria represent key hubs of cellular metabolism and Ca2+ signaling. However, it has been challenging to specifically detect highly localized Ca2+ fluxes such as those bridging these two organelles. To circumvent this issue, various recombinant Ca2+ indicators that can be targeted to specific subcellular compartments have been developed over the past two decades. While the use of these probes for measuring agonist-induced Ca2+ signals in various organelles has been extensively described, the assessment of basal Ca2+ concentrations within specific organelles is often disregarded, in spite of the fact that this parameter is vital for several metabolic functions, including the enzymatic activity of mitochondrial dehydrogenases of the Krebs cycle and protein folding in the endoplasmic reticulum. Here, we provide an overview on genetically engineered, organelle-targeted fluorescent Ca2+ probes and outline their evolution. Moreover, we describe recently developed protocols to quantify baseline Ca2+ concentrations in specific subcellular compartments. Among several applications, this method is suitable for assessing how changes in basal Ca2+ levels affect the metabolic profile of cancer cells.