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

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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GAF-CaMP3–sfGFP, An Enhanced Version of the Near-Infrared Genetically Encoded Positive Phytochrome-Based Calcium Indicator for the Visualization of Neuronal Activity

International Journal of Molecular Sciences ◽

10.3390/ijms21186883 ◽

2020 ◽

Vol 21 (18) ◽

pp. 6883

Author(s):

Oksana M. Subach ◽

Fedor V. Subach

Keyword(s):

Calcium Ions ◽

Near Infrared ◽

Fluorescent Protein ◽

Dynamic Range ◽

Fluorescent Proteins ◽

Spectral Characteristics ◽

Confocal Imaging ◽

Neuronal Cultures ◽

Decay Kinetics ◽

Calcium Indicator

The first generation of near-infrared, genetically encoded calcium indicators (NIR-GECIs) was developed from bacterial phytochrome-based fluorescent proteins that utilize biliverdin (BV) as the chromophore moiety. However, NIR-GECIs have some main drawbacks such as either an inverted response to calcium ions (in the case of NIR-GECO1) or a limited dynamic range and a lack of data about their application in neurons (in the case of GAF-CaMP2–superfolder green fluorescent protein (sfGFP)). Here, we developed an enhanced version of the GAF-CaMP2–sfGFP indicator, named GAF-CaMP3–sfGFP. The GAF-CaMP3–sfGFP demonstrated spectral characteristics, molecular brightness, and a calcium affinity similar to the respective characteristics for its progenitor, but a 2.9-fold larger DF/F response to calcium ions. As compared to GAF-CaMP2–sfGFP, in cultured HeLa cells, GAF-CaMP3–sfGFP had similar brightness but a 1.9-fold larger DF/F response to the elevation of calcium ions levels. Finally, we successfully utilized the GAF-CaMP3–sfGFP for the monitoring of the spontaneous and stimulated activity of neuronal cultures and compared its performance with the R-GECO1 indicator using two-color confocal imaging. In the cultured neurons, GAF-CaMP3–sfGFP showed a linear DF/F response in the range of 0–20 APs and in this range demonstrated a 1.4-fold larger DF/F response but a 1.3- and 2.4-fold slower rise and decay kinetics, respectively, as compared to the same parameters for the R-GECO1 indicator.

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