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.
Highlights
The biochemical mechanisms involved in Ca2+ regulation of a large number of physiological processes have been elucidated [1,2,3] in addition to other methods with the help of the Ca2+sensitive photoprotein aequorin [4] and synthetic fluorescent probes [5]
We previously reported the fusion of red fluorescent protein mRFP1.2 and aequorin [18], not spectrally identical to mRFP1-aequorin (RA) described by Curie et al [19]
The hybrid proteins did not carry targeting signals, transfection in HeLa cells or neurons in culture resulted in a uniform fluorescence in both the cytoplasm and the nucleus with no sign of aggregation or toxicity, as shown with other FPaequorin fusions [13,18,19]
Summary
The biochemical mechanisms involved in Ca2+ regulation of a large number of physiological processes have been elucidated [1,2,3] in addition to other methods with the help of the Ca2+sensitive photoprotein aequorin [4] and synthetic fluorescent probes [5]. Luminometry and fluorometry provided a good time resolution at the cost of spatial information, but have since been superseded by imaging techniques given the development of sensitive detectors on the one hand and a large palette of fluorescent proteins (FPs) on the other [6]. The latter has allowed the development of fluorescent recombinant Ca2+ indicators by fusing FPs and various Ca2+-binding proteins [7]. Aequorin-based methods offer the following features: (i) bioluminescence does not require excitation light, avoiding problems like phototoxicity, photobleaching and autofluorescence, and making it minimally-invasive with high signal/noise; (ii) availability of coelenterazines with different Ca2+-affinities [9] and mutated low Ca2+-affinity aequorin [10] allow measuring [Ca2+] from 1027 to 1023 M; (iii) aequorin is almost insensitive to changes in pH; (iv) it can be molecularly targeted to different subcellular compartments to report local [Ca2+] [10,11]; (v) it is not present in mammalian cells, and it showed little, if any, toxicity during development in transgenic mice [12]; (vi) aequorin signals are difficult to image because of a combination of low emission quantum yield and low protein stability [13]; each molecule only performs one emission cycle upon Ca2+ binding (recharging with the cofactor is relatively slow) [14]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
