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

Authors
  • Subach, Oksana M1
  • Barykina, Natalia V2
  • Chefanova, Elizaveta S3
  • Vlaskina, Anna V1
  • Sotskov, Vladimir P4
  • Ivashkina, Olga I1, 2, 4
  • Anokhin, Konstantin V2, 4
  • Subach, Fedor V1
  • 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.
Type
Published Article
Journal
International Journal of Molecular Sciences
Publisher
MDPI AG
Publication Date
Dec 24, 2020
Volume
22
Issue
1
Identifiers
DOI: 10.3390/ijms22010111
PMID: 33374320
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

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.

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