Measuring Ca Levels in Subcellular Compartments with Genetically Encoded GFP-Based Indicators.

Ca homeostasis is crucial for the entire life of eukaryotic cells from the beginning to the end. Mishandling in Ca homeostasis is indeed linked with a large number of pathological conditions. Thus, the possibility to specifically monitor cellular calcium fluxes in different subcellular compartments represents a key tool to deeply understand the mechanisms involved in cellular dysfunctions. To cope with this need, several Ca indicators have been developed allowing to accurately measure both basal Ca concentration and agonist-induced Ca signals in a wide spectrum of organelles. Among these, the genetically encoded GFP-based indicators are routinely used to measure Ca transients thanks to their ability to change their spectral properties in response to Ca binding. In this chapter, we will describe a protocol that utilizes the GCaMP6f probe targeted to mitochondria (4mtGCaMP) to measure mitochondrial calcium levels in resting conditions in HeLa cells. This method allows to easily and quickly register alterations of mitochondrial Ca homeostasis in different cell populations and experimental settings, representing a precious tool to unravel the pathological pathways leading to pathogenic conditions.