Next Article in Journal
Comparative Analysis of Proteomes of a Number of Nosocomial Pathogens by KEGG Modules and KEGG Pathways
Next Article in Special Issue
High Sensitivity In Vivo Imaging of Cancer Metastasis Using a Near-Infrared Luciferin Analogue seMpai
Previous Article in Journal
Gastrointestinal Nematode-Derived Antigens Alter Colorectal Cancer Cell Proliferation and Migration through Regulation of Cell Cycle and Epithelial-Mesenchymal Transition Proteins
Previous Article in Special Issue
Luminescence of Cypridina Luciferin in the Presence of Human Plasma Alpha 1-Acid Glycoprotein
Open AccessArticle

RedquorinXS Mutants with Enhanced Calcium Sensitivity and Bioluminescence Output Efficiently Report Cellular and Neuronal Network Activities

1
Institut de Neurobiologie Alfred Fessard, UPR 3294, Centre National de la Recherche Scientifique (CNRS), Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
2
BioEmergences Unit, CNRS USR 3695, Université Paris-Saclay, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
3
Neuroscience Paris Seine—Institut de Biologie Paris Seine (NPS—IBPS), CNRS UMR8246, INSERM U1130, Sorbonne Université UM119, 75005 Paris, France
4
Photobiology Laboratory, Institute of Biophysics SB RAS, Federal Research Center “Krasnoyarsk Science Center SB RAS”, 660036 Krasnoyarsk, Russia
*
Authors to whom correspondence should be addressed.
These authors contributed equally to this work.
Int. J. Mol. Sci. 2020, 21(21), 7846; https://doi.org/10.3390/ijms21217846
Received: 8 October 2020 / Revised: 16 October 2020 / Accepted: 19 October 2020 / Published: 22 October 2020
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. View Full-Text
Keywords: bioluminescence; aequorin; calcium sensor; BRET; mutagenesis; GPCR assay; neuronal network imaging bioluminescence; aequorin; calcium sensor; BRET; mutagenesis; GPCR assay; neuronal network imaging
Show Figures

Figure 1

MDPI and ACS Style

Bakayan, A.; Picaud, S.; Malikova, N.P.; Tricoire, L.; Lambolez, B.; Vysotski, E.S.; Peyriéras, N. RedquorinXS Mutants with Enhanced Calcium Sensitivity and Bioluminescence Output Efficiently Report Cellular and Neuronal Network Activities. Int. J. Mol. Sci. 2020, 21, 7846. https://doi.org/10.3390/ijms21217846

AMA Style

Bakayan A, Picaud S, Malikova NP, Tricoire L, Lambolez B, Vysotski ES, Peyriéras N. RedquorinXS Mutants with Enhanced Calcium Sensitivity and Bioluminescence Output Efficiently Report Cellular and Neuronal Network Activities. International Journal of Molecular Sciences. 2020; 21(21):7846. https://doi.org/10.3390/ijms21217846

Chicago/Turabian Style

Bakayan, Adil; Picaud, Sandrine; Malikova, Natalia P.; Tricoire, Ludovic; Lambolez, Bertrand; Vysotski, Eugene S.; Peyriéras, Nadine. 2020. "RedquorinXS Mutants with Enhanced Calcium Sensitivity and Bioluminescence Output Efficiently Report Cellular and Neuronal Network Activities" Int. J. Mol. Sci. 21, no. 21: 7846. https://doi.org/10.3390/ijms21217846

Find Other Styles
Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Article Access Map by Country/Region

1
Search more from Scilit
 
Search
Back to TopTop