Measuring Ca2+ Levels in Subcellular Compartments with Genetically Encoded GFP-Based Indicators.

Abstract

Ca2+ homeostasis is crucial for the entire life of eukaryotic cells from the beginning to the end. Mishandling in Ca2+ 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 Ca2+ indicators have been developed allowing to accurately measure both basal Ca2+ concentration and agonist-induced Ca2+ signals in a wide spectrum of organelles. Among these, the genetically encoded GFP-based indicators are routinely used to measure Ca2+ transients thanks to their ability to change their spectral properties in response to Ca2+ 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 Ca2+ homeostasis in different cell populations and experimental settings, representing a precious tool to unravel the pathological pathways leading to pathogenic conditions.