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

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.

The mRubyFT Protein, Genetically Encoded Blue-to-Red Fluorescent Timer

“…Mammalian live cell imaging was performed as described earlier [ 28 ]. Briefly, transient transfection of the HeLa Kyoto cells was performed in a 24-well format using lipofectamine reagent according to the manufacturer’s protocol.…”

Optogenetics for light control of biological systems

“…D) The soluble enzyme bPAC 62 and the rhodopsin-guanyl-cyclase CaRhGC 68 produce cAMP and cGMP following illumination whereas the non-bleaching opsins mOPN4 48 , eOPN3 54 , PPO 53 and JellyOP 47 activate different G-protein pathways. e: Genetically encoded sensors with diverse excitation spectra (depicted on the x axis) can be used to monitor changes in Ca2+ voltage, and pH, such as GCaMP and R-CaMP 156 and FRCaMP 263 for Ca 2+ , ASAP3 264 , Voltron 265 , VARNAM 266 , Quasar 205 and Archon 267 for voltage, and pHluorin 268 for pH. In experiments combining sensors and actuators, both need to be chosen carefully to minimize optical crosstalk.…”

Fluorescent indicators for live-cell and in vitro detection of inorganic cadmium dynamics

“…EGFP (QY 0.60, pH 7.4) [20] was used as a control to calculate the quantum yield of N0C0. Similarly, the quantum yield of N1C1 was measured and calculated using the Brightness and Fluorescence Changes [21].…”

“…It causes great harm to the human body and has aroused widespread concern [2][3][4]. The intake of cadmium can adversely affect the kidneys, lungs, bone, and nervous system, with a biological half-life in the range of [17][18][19][20][21][22][23][24][25][26][27][28][29][30] years in the human body [5]. The long-term presence of cadmium causes renal dysfunction, calcium metabolism disorders, and an increased incidence of various diseases [6,7].…”