Green fluorescent genetically encoded calcium indicator based on calmodulin/M13-peptide from fungi.

Natalia V Barykina,Erica E Jung,Konstantin V Anokhin,Fedor V Subach,Andrey O Bogorodskiy,Edward S Boyden,Grigori N Enikolopov,Galina E Pozmogova,Oksana M Subach,Ivan V Smirnov,Valentin I Borshchevskiy,Aleksey Y Malyshev,Anna M Varizhuk,Kiryl D Piatkevich

doi:10.1371/journal.pone.0183757

Abstract

Currently available genetically encoded calcium indicators (GECIs) utilize calmodulins (CaMs) or troponin C from metazoa such as mammals, birds, and teleosts, as calcium-binding domains. The amino acid sequences of the metazoan calcium-binding domains are highly conserved, which may limit the range of the GECI key parameters and cause undesired interactions with the intracellular environment in mammalian cells. Here we have used fungi, evolutionary distinct organisms, to derive CaM and its binding partner domains and design new GECI with improved properties. We applied iterative rounds of molecular evolution to develop FGCaMP, a novel green calcium indicator. It includes the circularly permuted version of the enhanced green fluorescent protein (EGFP) sandwiched between the fungal CaM and a fragment of CaM-dependent kinase. FGCaMP is an excitation-ratiometric indicator that has a positive and an inverted fluorescence response to calcium ions when excited at 488 and 405 nm, respectively. Compared with the GCaMP6s indicator in vitro, FGCaMP has a similar brightness at 488 nm excitation, 7-fold higher brightness at 405 nm excitation, and 1.3-fold faster calcium ion dissociation kinetics. Using site-directed mutagenesis, we generated variants of FGCaMP with improved binding affinity to calcium ions and increased the magnitude of FGCaMP fluorescence response to low calcium ion concentrations. Using FGCaMP, we have successfully visualized calcium transients in cultured mammalian cells. In contrast to the limited mobility of GCaMP6s and G-GECO1.2 indicators, FGCaMP exhibits practically 100% molecular mobility at physiological concentrations of calcium ion in mammalian cells, as determined by photobleaching experiments with fluorescence recovery. We have successfully monitored the calcium dynamics during spontaneous activity of neuronal cultures using FGCaMP and utilized whole-cell patch clamp recordings to further characterize its behavior in neurons. Finally, we used FGCaMP in vivo to perform structural and functional imaging of zebrafish using wide-field, confocal, and light-sheet microscopy.

Highlights

Calcium ions are universal second messengers involved in the regulation of physiological responses in a wide range of organisms
The sensory module is located on the N- or C-terminus of the Genetically encoded calcium indicators (GECIs) and includes mammalian calmodulin (CaM) that undergoes Ca2+-dependent binding with the M13 peptide of myosin light chain kinase (CaM/M13) fused to the opposite end of the indicator
The cloned CaMs from Aspergillus niger fungus and Komagataella pastoris yeast were further used to assemble four bacterial libraries composed of CaMs, cpEGFP from GCaMP6f variant [16] and M13-like peptides or calcineurins (Fig 1A)

Summary

Introduction

Calcium ions are universal second messengers involved in the regulation of physiological responses in a wide range of organisms. The sensory module is located on the N- or C-terminus of the GECI and includes mammalian calmodulin (CaM) that undergoes Ca2+-dependent binding with the M13 peptide of myosin light chain kinase (CaM/M13) fused to the opposite end of the indicator. These features vary slightly among the many types of GECI that currently exist. Two current calcium indicators, Camgaroo 1 and 2, do not contain the M13 peptide, and the mammalian CaM is inserted inside enhanced yellow FP (EYFP) [3,4]. The GECIs described and current publicationsindicate a potential issue, that the diversity of domains in the sensory modules are limited to proteins from metazoa such as mammals, birds, and teleosts

Methods

Results

Conclusion