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

Oksana M. Subach,Fedor V. Subach

doi:10.3390/ijms21186883

Abstract

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 ΔF/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 ΔF/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 ΔF/F response in the range of 0–20 APs and in this range demonstrated a 1.4-fold larger ΔF/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.

Highlights

Encoded calcium indicators (GECIs) are an indispensable tool for the visualization of calcium dynamics in living cells [1,2]
To address some of the abovementioned limitations, in this paper, we developed a version of the GAF-CaMP2–superfolder green fluorescent protein (sfGFP) indicator, called GAF-CaMP3–sfGFP
As a result of this directed selection, we found a final variant of GAF-CaMP2–sfGFP, named GAF-CaMP3–sfGFP

Summary

Introduction

Encoded calcium indicators (GECIs) are an indispensable tool for the visualization of calcium dynamics in living cells [1,2]. Efforts have been made to expand the palette of available fluorescence colors of GECIs to the near-infrared region [3,4]. Two versions of near-infrared GECIs, called NIR-GECO1 [3] and GAF-CaMP2 [4], were recently published. Both indicators are based on bacterial phytochromes in which fluorescence arises from a biliverdin (BV) chromophore. Endogenous BV is present in mammalian cells as an intermediate of heme degradation, externally added BV usually enhances the brightness of fluorescent proteins based on BV. NIR-GECO1 has been successfully applied to monitor neuronal calcium activity in cultured cells, primary neurons, and acute brain slices, and the GAF-CaMP2–superfolder green fluorescent protein (sfGFP) fusion has been used to robustly monitor calcium transient in cultured cells

Methods

Results

Conclusion