Retrospective on the development of aequorin and aequorin‐based imaging to visualize changes in intracellular free [Ca²⁺]

Abstract

SUMMARYIn this review, we take a retrospective look at the discovery and utilization of the Ca 2þ -sensitive bioluminescent protein complex, aequorin. We do consider the contribution it has made to our understanding of the natural phenomenon of bioluminescence, but it is in the application of extracted and purified aequorin as a reporter of Ca 2þ dynamics in living cells, which is arguably its major contribution to biological and biomedical science. Following its extraction, purification, and subsequent availability in the mid-1960s, aequorin became the intracellular reporter of choice until it was replaced in the late 1970s by easier-to-use fluorescence-based reporters. From the mid-1980s onwards, however, aequorin-based Ca 2þ imaging underwent a renaissance following the cloning of the aequorin gene and the emergence of routine techniques to target and express it exogenously in plant and animal systems. The development of aequorin as a tool continues as spectral varieties are being developed that allow simultaneous imaging of Ca 2þ dynamics in different cellular organelles and microdomains. We predict that further developments in the use of aequorin, as well as other bioluminescent proteins, will continue, especially in the areas of regenerative medicine and whole organism imaging.Mol. Reprod. Dev. 82: 563À586, 2015. ß

Calcium and Cell Response to Heavy Metals: Can Yeast Provide an Answer?

“…This was made possible by the isolation of aequorin, a Ca 2+ -binding photoprotein, isolated from the luminescent jellyfish, Aequorea victoria. Aequorin consists of two distinct units, the apoprotein apoaequorin (22 kDa) and the prosthetic group, coelenterazine, which reconstitute spontaneously in the presence of molecular oxygen, forming the functional protein [72][73][74]. Aequorin has become a useful instrument for the measurement of intracellular Ca 2+ levels, since it has binding sites for Ca 2+ ions responsible for protein conformational changes that convert through oxidation its prosthetic group, coelenterazine, into excited coelenteramide and CO 2 ( Figure 3A).…”

“…If the contamination is not excessive, the cations would probably get stuck at this level, due to the mannoproteins that compose the outer layer of the cell wall (alongside of β-glucans and chitin) which are heavily phosphorylated and carboxylated, decorating the cell façade with a negatively charged shield prone to bind to positively charged species, such as the metal cations [83]. Excess metal ions which escape the negatively charged groups on the cell wall surface penetrate the porous cell [72][73][74]. Cells expressing apo-aequorin are first incubated with the cell-permeant coelenterazine to produce functional aequorin.…”

Assessment of a Silicon-Photomultiplier-Based Platform for the Measurement of Intracellular Calcium Dynamics with Targeted Aequorin

“…Techniques for Ca 2+ sensing include fluorescence and bioluminescence assays. , In particular, fluorescent synthetic Ca 2+ probes like Fura-2 and Fluo-4 have become popular due to their ease of use and the wide distribution of fluorescent microscopes. On the other hand, genetically encoded calcium indicators (GECIs) offer a reliable mean to detect and measure Ca 2+ in specific subcellular locations.…”

“…These probes include fluorescent probes (e.g., the GFP-derived GCaMP, and GeCO), − fluorescence resonance energy transfer (FRET)-based Ca 2+ probes (e.g., cameleons and their analogues), , luminescent Ca 2+ probes (e.g., AEQ-based indicators), and bioluminescence resonance energy transfer (BRET)-based Ca 2+ indicators (the last two reviewed in ref ). Among these techniques, aequorin bioluminescence offers outstanding advantages. , Aequorin (AEQ) is a 21 kDa bioluminescent protein that can be used as a calcium reporter without being externally excited. More in detail, it is an enzyme that, upon the binding of three Ca 2+ ions, catalyzes the oxidation of its substrate coelenterazine, ultimately producing a blue light emission (peaked at 465 nm): …”

“…Among these techniques, aequorin bioluminescence offers outstanding advantages. 8,16 Aequorin (AEQ) is a 21 kDa bioluminescent protein that can be used as a calcium reporter without being externally excited. More in detail, it is an enzyme that, upon the binding of three Ca 2+ ions, catalyzes the oxidation of its substrate coelenterazine, ultimately producing a blue light emission (peaked at 465 nm):…”