title: Measuring baseline Ca(2+) levels in subcellular compartments using genetically engineered fluorescent indicators.
abstract: Intracellular Ca(2+) signaling is involved in a series of physiological and pathological processes. In particular, an intimate crosstalk between bioenergetic metabolism and Ca(2+) 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 Ca(2+) signaling. However, it has been challenging to specifically detect highly localized Ca(2+) fluxes such as those bridging these two organelles. To circumvent this issue, various recombinant Ca(2+) 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 Ca(2+) signals in various organelles has been extensively described, the assessment of basal Ca(2+) 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 Ca(2+) probes and outline their evolution. Moreover, we describe recently developed protocols to quantify baseline Ca(2+) concentrations in specific subcellular compartments. Among several applications, this method is suitable for assessing how changes in basal Ca(2+) levels affect the metabolic profile of cancer cells.
authors.affiliation: Department of Cell and Developmental Biology, Consortium for Mitochondrial Research, University College London, London, United Kingdom.
authors.forename: Julia M
authors.initials: JM
authors.lastname: Hill
authors.affiliation: Department of Biomedical Sciences, CNR Neuroscience Institute, University of Padua, Padua, Italy.
authors.forename: Diego
authors.initials: D
authors.lastname: De Stefani
authors.affiliation: Department of Cell and Developmental Biology, Consortium for Mitochondrial Research, University College London, London, United Kingdom.
authors.forename: Aleck W E
authors.initials: AW
authors.lastname: Jones
authors.affiliation: Department of Neurosciences, University of the Basque Country (UPV/EHU), Achúcarro Basque Center for Neuroscience-UPV/EHU, Leioa, Spain; Instituto de Salud Carlos III, Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas, (CIBERNED), Madrid, Spain.
authors.forename: Asier
authors.initials: A
authors.lastname: Ruiz
authors.affiliation: Department of Biomedical Sciences, CNR Neuroscience Institute, University of Padua, Padua, Italy.
authors.forename: Rosario
authors.initials: R
authors.lastname: Rizzuto
authors.affiliation: Department of Cell and Developmental Biology, Consortium for Mitochondrial Research, University College London, London, United Kingdom; Department of Biomedical Sciences, CNR Neuroscience Institute, University of Padua, Padua, Italy. Electronic address: g.szabadkai@ucl.ac.uk.
authors.forename: Gyorgy
authors.initials: G
authors.lastname: Szabadkai
mesh_terms: D002118:Calcium / Q000378:metabolism*; D002451:Cell Compartmentation; D005456:Fluorescent Dyes / Q000378:metabolism*; D005818:Genetic Engineering; D015335:Molecular Probes; D013347:Subcellular Fractions / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D015335:Molecular Probes; D002118:Calcium
keywords: Endoplasmic reticulum; FRET; Genetically encoded Ca(2+) indicator; Mitochondria; Organelle targeting; Recombinant fluorescent Ca(2+) probe