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Article

FRCaMP, a Red Fluorescent Genetically Encoded Calcium Indicator Based on Calmodulin from Schizosaccharomyces Pombe Fungus

1
Complex of NBICS Technologies, National Research Center “Kurchatov Institute”, 123182 Moscow, Russia
2
Laboratory for Neurobiology of Memory, P.K. Anokhin Research Institute of Normal Physiology, 125315 Moscow, Russia
3
Department of NBIC-Technologies, Moscow Institute of Physics and Technology, 123182 Moscow, Russia
4
Institute for Advanced Brain Studies, Lomonosov Moscow State University, 119991 Moscow, Russia
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2021, 22(1), 111; https://doi.org/10.3390/ijms22010111
Received: 4 December 2020 / Revised: 22 December 2020 / Accepted: 22 December 2020 / Published: 24 December 2020
Red fluorescent genetically encoded calcium indicators (GECIs) have expanded the available pallet of colors used for the visualization of neuronal calcium activity in vivo. However, their calcium-binding domain is restricted by calmodulin from metazoans. In this study, we developed red GECI, called FRCaMP, using calmodulin (CaM) from Schizosaccharomyces pombe fungus as a calcium binding domain. Compared to the R-GECO1 indicator in vitro, the purified protein FRCaMP had similar spectral characteristics, brightness, and pH stability but a 1.3-fold lower ΔF/F calcium response and 2.6-fold tighter calcium affinity with Kd of 441 nM and 2.4–6.6-fold lower photostability. In the cytosol of cultured HeLa cells, FRCaMP visualized calcium transients with a ΔF/F dynamic range of 5.6, which was similar to that of R-GECO1. FRCaMP robustly visualized the spontaneous activity of neuronal cultures and had a similar ΔF/F dynamic range of 1.7 but 2.1-fold faster decay kinetics vs. NCaMP7. On electrically stimulated cultured neurons, FRCaMP demonstrated 1.8-fold faster decay kinetics and 1.7-fold lower ΔF/F values per one action potential of 0.23 compared to the NCaMP7 indicator. The fungus-originating CaM of the FRCaMP indicator version with a deleted M13-like peptide did not interact with the cytosolic environment of the HeLa cells in contrast to the metazoa-originating CaM of the similarly truncated version of the GCaMP6s indicator with a deleted M13-like peptide. Finally, we generated a split version of the FRCaMP indicator, which allowed the simultaneous detection of calcium transients and the heterodimerization of bJun/bFos interacting proteins in the nuclei of HeLa cells with a ΔF/F dynamic range of 9.4 and a contrast of 2.3–3.5, respectively. View Full-Text
Keywords: genetically encoded calcium indicator; protein engineering; calcium imaging; Schizosaccharomyces pombe; FRCaMP; GECI; red fluorescent; fluorescent protein; split; bJun/bFos genetically encoded calcium indicator; protein engineering; calcium imaging; Schizosaccharomyces pombe; FRCaMP; GECI; red fluorescent; fluorescent protein; split; bJun/bFos
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MDPI and ACS Style

Subach, O.M.; Barykina, N.V.; Chefanova, E.S.; Vlaskina, A.V.; Sotskov, V.P.; Ivashkina, O.I.; Anokhin, K.V.; Subach, F.V. FRCaMP, a Red Fluorescent Genetically Encoded Calcium Indicator Based on Calmodulin from Schizosaccharomyces Pombe Fungus. Int. J. Mol. Sci. 2021, 22, 111. https://doi.org/10.3390/ijms22010111

AMA Style

Subach OM, Barykina NV, Chefanova ES, Vlaskina AV, Sotskov VP, Ivashkina OI, Anokhin KV, Subach FV. FRCaMP, a Red Fluorescent Genetically Encoded Calcium Indicator Based on Calmodulin from Schizosaccharomyces Pombe Fungus. International Journal of Molecular Sciences. 2021; 22(1):111. https://doi.org/10.3390/ijms22010111

Chicago/Turabian Style

Subach, Oksana M., Natalia V. Barykina, Elizaveta S. Chefanova, Anna V. Vlaskina, Vladimir P. Sotskov, Olga I. Ivashkina, Konstantin V. Anokhin, and Fedor V. Subach 2021. "FRCaMP, a Red Fluorescent Genetically Encoded Calcium Indicator Based on Calmodulin from Schizosaccharomyces Pombe Fungus" International Journal of Molecular Sciences 22, no. 1: 111. https://doi.org/10.3390/ijms22010111

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