Year-end Sale % OFF 
Abstract
Considerable efforts have been focused on shifting the wavelength of aequorin Ca2+-dependent blue bioluminescence through fusion with fluorescent proteins. This approach has notably yielded the widely used GFP-aequorin (GA) Ca2+ sensor emitting green light, and tdTomato-aequorin (Redquorin), whose bioluminescence is completely shifted to red, but whose Ca2+ sensitivity is low. In the present study, the screening of aequorin mutants generated at twenty-four amino acid positions in and around EF-hand Ca2+-binding domains resulted in the isolation of six aequorin single or double mutants (AequorinXS) in EF2, EF3, and C-terminal tail, which exhibited markedly higher Ca2+ sensitivity than wild-type aequorin in vitro. The corresponding Redquorin mutants all showed higher Ca2+ sensitivity than wild-type Redquorin, and four of them (RedquorinXS) matched the Ca2+ sensitivity of GA in vitro. RedquorinXS mutants exhibited unaltered thermostability and peak emission wavelengths. Upon stable expression in mammalian cell line, all RedquorinXS mutants reported the activation of the P2Y2 receptor by ATP with higher sensitivity and assay robustness than wt-Redquorin, and one, RedquorinXS-Q159T, outperformed GA. Finally, wide-field bioluminescence imaging in mouse neocortical slices showed that RedquorinXS-Q159T and GA similarly reported neuronal network activities elicited by the removal of extracellular Mg2+. Our results indicate that RedquorinXS-Q159T is a red light-emitting Ca2+ sensor suitable for the monitoring of intracellular signaling in a variety of applications in cells and tissues, and is a promising candidate for the transcranial monitoring of brain activities in living mice.
Highlights
Intracellular Ca2+ is a critical signal in many important physiological processes and pathophysiological conditions across living species, and measurements of intracellular Ca2+ concentration are widely used in virtually all domains of biology [1]
As for other Ca2+-dependent photoproteins, the bioluminescence from wild-type aequorin is emitted at a slow rate at low Ca2+ concentrations, the light emission intensity below 1 μM Ca2+ being less than one-thousandth of that obtained in 1 mM Ca2+ [6,9,14]
AequorinXS mutations enhanced all aspects of Redquorin Ca2+ sensitivity, with some Redquorin mutants exhibiting CitA-like properties (e.g., RedquorinQD+AT for sensitivity to low Ca2+, and RedquorinQT+AD for light emission rate at high Ca2+ in CLZ-f condition), and others being close to GA (e.g., RedquorinQ159T, notably in CLZ-f condition)
Summary
Intracellular Ca2+ is a critical signal in many important physiological processes and pathophysiological conditions across living species, and measurements of intracellular Ca2+ concentration are widely used in virtually all domains of biology [1]. As for other Ca2+-dependent photoproteins, the bioluminescence from wild-type (wt) aequorin is emitted at a slow rate at low Ca2+ concentrations, the light emission intensity below 1 μM Ca2+ being less than one-thousandth of that obtained in 1 mM Ca2+ [6,9,14] This low bioluminescence output significantly limits the spatiotemporal precision of aequorin as a reporter of intracellular Ca2+ signals, which are in the 0.1–0.5 μM range in most mammalian cell types. Apo-aequorin mutants showing extra sensitivity to Ca2+ (AequorinXS) were used to generate six Redquorin mutants that were characterized in vitro and in cellulo Four of these mutants, named RedquorinXS, were stably expressed in CHO cell lines and tested for their sensitivity to physiological Ca2+ signals. A RedquorinXS Ca2+ sensor was expressed in a mouse brain slices through viral transfer and validated for the imaging of neuronal network activities
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.
