Red-Shifted Aequorin-Based Bioluminescent Reporters for in Vivo Imaging of Ca ²⁺ Signaling

Abstract

Real-time visualization of calcium (Ca(2+)) dynamics in the whole animal will enable important advances in understanding the complexities of cellular function. The genetically encoded bioluminescent Ca(2+) reporter green fluorescent protein-aequorin (GA) allows noninvasive detection of intracellular Ca(2+) signaling in freely moving mice. However, the emission spectrum of GA is not optimal for detection of activity from deep tissues in the whole animal. To overcome this limitation, two new reporter genes were constructed by fusing the yellow fluorescent protein (Venus) and the monomeric red fluorescent protein (mRFP1) to aequorin. Transfer of aequorin chemiluminescence energy to Venus (VA) is highly efficient and produces a 58 nm red shift in the peak emission spectrum of aequorin. This substantially improves photon transmission through tissue, such as the skin and thoracic cage. Although the Ca(2+)-induced bioluminescence spectrum of mRFP1-aequorin (RA) is similar to that of aequorin, there is also a small peak above 600 nm corresponding to the peak emission of mRFP1. Small amounts of energy transfer between aequorin and mRFP1 yield an emission spectrum with the highest percentage of total light above 600 nm compared with GA and VA. Accordingly, RA is also detected with higher sensitivity from brain areas. VA and RA will therefore improve optical access to Ca(2+) signaling events in deeper tissues, such as the heart and brain, and offer insight for engineering new hybrid molecules.

Red and green aequorins for simultaneous monitoring of Ca2+ signals from two different organelles

“…3 legend for details on derivation). The CRET efficiency in the RFP-AEQ fusion prepared by Curie et al [15] was also low, with a wide spectrum consistent with the low red/green ratios found here (Fig. 3b).…”

“…The chemiluminescence emission spectrum of a RFP-AEQ chimera very similar to the one prepared here has been published recently [15] domains are generated in HEK cells during activation of capacitative calcium entry. These high Ca 2+ microdomains would feed mitochondrial uptake, but dissipate soon, once capacitative Ca 2+ entry is deactivated when ER begins to fill [23].…”

“…Calibrations in [Ca 2+ ] were done using the constant values published before [11]. Neither the fusion of aequorin to GFP, RFP nor to any of the targeting sequences affected the affinity for Ca 2+ ( [15,21] and data not shown).…”

“…The wide dynamic range and steep Ca 2+ dependence of AEQ favor sharp definition of high Ca 2+ microdomains [3], but the low light emission severely limits spatiotemporal resolution in imaging experiments [12]. To circumvent this problem, we have targeted two spectrally distinct AEQs, one fused to GFP [13] and the other to the monomeric red fluorescent protein (mRFP) [14,15], to different subcellular locations and co-expressed them in the same cells. The real-time signals coming from both probes were monitored independently.…”

Organellar calcium signalling mechanisms inDrosophilaepithelial function

“…In order to improve detection of light from deep areas in mammalian tissues, the GFP-aequorin probe has also been engineered by fusing either enhanced yellow fluorescent protein (Venus) or monomeric red fluorescent protein (mRFP1) to aequorin. Light transmission through skin and thoracic cage is enhanced by using the Venus-aequorin probe whereas the emission spectrum by mRFP1-aequorin allows the detection of calcium signals in brain tissue (Curie et al, 2007).…”