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RedquorinXS Mutants with Enhanced Calcium Sensitivity and Bioluminescence Output Efficiently Report Cellular and Neuronal Network Activities / A. Bakayan, S. Picaud, N. P. Malikova [et al.] // Int. J. Mol. Sci. - 2020. - Vol. 21, Is. 21. - Ст. 7846, DOI 10.3390/ijms21217846. - Cited References:53. - This work was supported by grants from Centre National de la Recherche Scientifique (AAP Prematuration CNRS 2016, to A.B. and N.P.; equipment transfer to S.P. and B.L.), from Agence Nationale de la Recherche (AAP Prematuration FCS/IDEX Paris Saclay, to A.B. and N.P., France BioImaging infrastructure ANR-10-INBS-04, ANR-11-EQPX-029 to N.P.), from Fondation pour la Recherche sur le Cerveau/Rotary Club de France (B.L.), and from RFBR (project number 20-04-00085 to N.P.M. and E.S.V.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. . - ISSN 1422-0067

РУБ Biochemistry & Molecular Biology + Chemistry, Multidisciplinary
Рубрики:
IN-VIVO
   PHOTOPROTEIN AEQUORIN
   CA2+-REGULATED PHOTOPROTEINS
   SPREADING
Кл.слова (ненормированные):
bioluminescence -- aequorin -- calcium sensor -- BRET -- mutagenesis -- GPCR -- assay -- neuronal network imaging
Аннотация: 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.

WOS
Держатели документа:
Ctr Natl Rech Sci CNRS, Inst Neurobiol Alfred Fessard, UPR 3294, Ave Terrasse, F-91198 Gif Sur Yvette, France.
Univ Paris Saclay, BioEmergences Unit, CNRS, USR 3695, Ave Terrasse, F-91198 Gif Sur Yvette, France.
Sorbonne Univ, Inst Biol Paris Seine NPS IBPS, INSERM, Neurosci Paris Seine,CNRS,UMR8246,U1130,UM119, F-75005 Paris, France.
Inst Biophys SB RAS, Fed Res Ctr, Photobiol Lab, Krasnoyarsk Sci Ctr SB RAS, Krasnoyarsk 660036, Russia.

Доп.точки доступа:
Bakayan, Adil; Picaud, Sandrine; Malikova, Natalia P.; Tricoire, Ludovic; Lambolez, Bertrand; Vysotski, Eugene S.; Peyrieras, Nadine; Vysotski, Eugene; Centre National de la Recherche ScientifiqueCentre National de la Recherche Scientifique (CNRS); Agence Nationale de la RechercheFrench National Research Agency (ANR) [ANR-10-INBS-04, ANR-11-EQPX-029]; Fondation pour la Recherche sur le Cerveau/Rotary Club de France; RFBRRussian Foundation for Basic Research (RFBR) [20-04-00085]

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Redquorinxs mutants with enhanced calcium sensitivity and bioluminescence output efficiently report cellular and neuronal network activities / A. Bakayan, S. Picaud, N. P. Malikova [et al.] // Int. J. Mol. Sci. - 2020. - Vol. 21, Is. 21. - Ст. 7846. - P1-22, DOI 10.3390/ijms21217846 . - ISSN 1661-6596
Кл.слова (ненормированные):
Aequorin -- Bioluminescence -- BRET -- Calcium sensor -- GPCR assay -- Mutagenesis -- Neuronal network imaging
Аннотация: 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. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.

Scopus
Держатели документа:
Institut de Neurobiologie Alfred Fessard, UPR 3294, Centre National de la Recherche Scientifique (CNRS), Avenue de la Terrasse, Gif?sur?Yvette, 91198, France
BioEmergences Unit, CNRS USR 3695, Universite Paris?Saclay, Avenue de la Terrasse, Gif?sur?Yvette, 91198, France
Neuroscience Paris Seine ? Institut de Biologie Paris Seine (NPS ? IBPS), CNRS, UMR8246, INSERM U1130, Sorbonne Universite UM119, Paris, 75005, France
Photobiology Laboratory, Institute of Biophysics SB RAS, Federal Research Center “Krasnoyarsk Science Center SB RAS”, Krasnoyarsk, 660036, Russian Federation

Доп.точки доступа:
Bakayan, A.; Picaud, S.; Malikova, N. P.; Tricoire, L.; Lambolez, B.; Vysotski, E. S.; Peyrieras, N.

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