Red Fluorescent Protein-Aequorin Fusions as Improved Bioluminescent Ca2+ Reporters in Single Cells and Mice

Abstract

Bioluminescence recording of Ca2+ signals with the photoprotein aequorin does not require radiative energy input and can be measured with a low background and good temporal resolution. Shifting aequorin emission to longer wavelengths occurs naturally in the jellyfish Aequorea victoria by bioluminescence resonance energy transfer (BRET) to the green fluorescent protein (GFP). This process has been reproduced in the molecular fusions GFP-aequorin and monomeric red fluorescent protein (mRFP)-aequorin, but the latter showed limited transfer efficiency. Fusions with strong red emission would facilitate the simultaneous imaging of Ca2+ in various cell compartments. In addition, they would also serve to monitor Ca2+ in living organisms since red light is able to cross animal tissues with less scattering. In this study, aequorin was fused to orange and various red fluorescent proteins to identify the best acceptor in red emission bands. Tandem-dimer Tomato-aequorin (tdTA) showed the highest BRET efficiency (largest energy transfer critical distance R0) and percentage of counts in the red band of all the fusions studied. In addition, red fluorophore maturation of tdTA within cells was faster than that of other fusions. Light output was sufficient to image ATP-induced Ca2+ oscillations in single HeLa cells expressing tdTA. Ca2+ rises caused by depolarization of mouse neuronal cells in primary culture were also recorded, and changes in fine neuronal projections were spatially resolved. Finally, it was also possible to visualize the Ca2+ activity of HeLa cells injected subcutaneously into mice, and Ca2+ signals after depositing recombinant tdTA in muscle or the peritoneal cavity. Here we report that tdTA is the brightest red bioluminescent Ca2+ sensor reported to date and is, therefore, a promising probe to study Ca2+ dynamics in whole organisms or tissues expressing the transgene.

GFP-Aequorin Protein Sensor for Ex Vivo and In Vivo Imaging of Ca 2+ Dynamics in High-Ca 2+ Organelles

“…On the other hand, given the recent expansion of hues in fluorescent proteins (Shaner et al, 2004), future efforts will focus on the engineering of GAP variants with different colors (cyan, yellow, orange, or red) that enable simultaneous imaging of Ca 2+ in various locations. Fusions of aequorin to red fluorescent proteins such as mRFP1 (Manjarres et al, 2008) or tdtomato (Bakayan et al, 2011) have been previously reported as bioluminescent sensors. Substitution of the GFP moiety of GAP for mRFP1 rendered a protein insensitive to calcium (P.N.-N., unpublished data).…”

Improving the luminescence properties of aequorin by conjugating to CdSe/ZnS quantum dot nanoparticles: Red shift and slowing decay rate

“…Due to the presence and proximity of both GFP and aequorin inside the jellyfish, the blue light, which is emitted by aequorin as the donor molecule is transferred to GFP as the acceptor molecule via BRET phenomenon, and it results in a change in the emission wavelength to green. Based on this natural model, researchers design different BRET systems in which some fluorescent proteins [35,37,38] or chromophores [36,39] are used as energy accepters. In this study, aequorin is modified through attaching quantum dots (QDs) nanoparticles to it in order to achieve the longer emission wavelength.…”

“…In these systems, photoprotein acts as a donor, whereas fluorescent proteins [35,37,38], fluorophores [36,39] can act as acceptors. One of the surprising synthetic fluorescent molecules, which acts as the BRET acceptor is QDs [40][41][42][43].…”

Engineered cells as biosensing systems in biomedical analysis

“…Therefore, detection of OH-PCBs in physiological samples may serve as a means of evaluating exposure to PCBs. Furthermore, OH-PCBs, although less toxic than PCBs, have significant adverse health effects, given that they can act as endocrine disruptors [37] and increase the formation of reactive oxygen species [1]. This emphasizes the importance of OH-PCB detection in physiological samples.…”

“…Mercury can exist in three major forms, elemental, inorganic, and organic. Approximately 80% of mercury released from human activities is in the elemental form [37]. Dental amalgams contain 50% mercury, with silver, tin, and copper [40].…”