Espino 2016 Abstract Mito Xmas Meeting Innsbruck

Mitochondrial [Ca2+] measurements using a novel very-low Ca2+ affinity aequorin-based probe.

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Espino J, De Stefani D, Rizzuto R (2016)

Event: Mito Xmas Meeting 2016 Innsbruck AT

Aequorin is a 22-kDa photoprotein produced by the jellyfish Aequorea victoria that has been long utilised for the study of Ca²⁺ signaling [1]. It has been also engineered to induce its specific targeting to various cell regions so as to monitor [Ca²⁺] in different subcellular comparments, e.g., mitochondrial matrix [2]. Nevertheless, its potential applicability is somewhat limited owing to consumption or saturation of aequorin throughout the experiment as well as stability of aequorin at physiological temperature. Herein, in an attempt to overcome the aforementioned disadvantages, we have developed a mitochondria-targeted triple-mutated form (Asp119Ala, Gln168Arg and Leu170Ile) of the photoprotein aequorin that enables measurement of [Ca²⁺] in the millimolar range. In fact, it is shown that addition of extramitochondrial Ca²⁺ to permeabilized HeLa cells triggers an increase in mitochondrial [Ca²⁺] up to approximately 2 mM. In intact cells, the novel probe allows recording agonist-stimulated mitochondrial [Ca²⁺] rises without problems derived from aequorin saturation and/or consumption. Notably, in addition to the increased dynamic range, the Gln168Arg and Leu170Ile mutations endowed this new aequorin-based probe with an increased lifetime at 37°C. This also allowed the generation of a cell line stably expressing the probe at very high levels.

Labels:

Organism: Human, Molluscs  Tissue;cell: HeLa  Preparation: Intact cells, Permeabilized cells 

Regulation: Calcium 

Event: Poster 

Affiliations

Espino J, De Stefani D, Rizzuto R

1. Dept Biomedical Sc, Univ Padova, Italy

Reference

1. Shimomura O, Johnson FH, Saiga Y (1962) Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol 59:223–39.
2. Bonora M, Giorgi C, Bononi A, Marchi S, Patergnani S, Rimessi A, Rizzuto, R, Pinton P (2013) Subcellular calcium measurements in mammalian cells using jellyfish photoprotein aequorin-based probes. Nat Protoc 8:2015-8.