Measuring Ca2+ inside intracellular organelles

Abstract

Interest on Ca2+ signaling is moving from cytosol to organelles, as proper organellar functioning requires proper control of their luminal Ca2+ homeostasis. Subcellular Ca2+ signals in organelles are best monitored using targeted genetically encoded calcium indicators (GECI). Aequorins (AEQ) were the first GECI used and novelties including low affinity AEQ will be reviewed. Fluorescent GECI include several families, mostly derived from calmodulin and troponin, and they have been widely engineered for changes in affinity, brightness or gain. We shall focus on a new GECI family, GAP, based on the fusion of GFP and AEQ. GAP exhibits a unique combination of features: dual-excitation ratiometric imaging capability, high dynamic range, good signal-to-noise ratio, insensitivity to pH and Mg2+, uncomplicated calibration, and targetability to at least five different organelles. Low affinity GAP variants were engineered for measuring in high [Ca2+] compartments, such as ER or Golgi. As neither AEQ nor GFP are present in mammalian cells, perturbations by interactions with endogenous ligands are prevented and transgenic animals expressing ER-targeted GAP did not show phenotypic alterations and GAP was expressed in a variety of organs and tissues including nervous system. We shall provide proof of concept for the suitability of the new biosensors to monitor Ca2+ dynamics inside intracellular organelles, including ex vivo and in vivo measurements in transgenic animals. The new sensors fill a gap in the actual repertoire of Ca2+ indicators and open an avenue to explore complex Ca2+ signaling in animal models of health and disease.