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

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.

Advances in Imaging Plant Cell Dynamics

“…LSFM also can track physiological processes such as calcium fluxes by using ratiometric calcium indicators in the intact and freely moving nematode Caenorhabditis elegans (Ardiel et al, 2017) or during the neuronal activity of zebrafish larvae (Barykina et al, 2017). In the first case, fast exposure (2.5-4.5 ms) was translated to a 5-Hz volumetric acquisition frame rate, allowing imaging of the entire animal.…”

FGCaMP7, an Improved Version of Fungi-Based Ratiometric Calcium Indicator for In Vivo Visualization of Neuronal Activity

“…It had a bimodal excitation spectrum peaking at 415 and 494 nm, and the relative intensity of green fluorescence (at 511 and 517 nm) at 494 and 415 nm excitation was changed by about 10-fold between Ca 2+ -saturated and Ca 2+ -free forms. The GEX-GECO1 ratiometric green GECI based on GFP fused to mammalian M13-peptide and CaM had a fluorescence ratio change of 18-26-fold [3,19]. However, both forms of GEX-GECO1 have substantially lower brightness than EGFP.…”

Presynaptic GCaMP expression decreases vesicle release probability at the calyx of Held

“…Calcium as a secondary messenger plays a significant role in cellular signaling and is intimately involved in the regulation of a variety of physiological activities (Barykina et al, ; Lin & Schnitzer, ). When voltage‐gated calcium channels open following membrane depolarization, intracellular calcium ([Ca 2+ ] i ) in neurons, and other excitable cells is raised.…”

“…GECIs have a typical calcium binding domain, such as calmodulin or troponin C, and undergo conformational changes when bound to calcium, resulting in increased fluorescence of the fused protein probe (Barykina et al, ). The GCaMP family of proteins, in particular, is a widely utilized tool for calcium imaging (Akerboom et al, ; Badura, Sun, Giovannucci, Lynch, & Wang, ; Chen et al, ; T. W. Chen et al, ; Chisholm, Khovanov, Lopes, LaRussa, & McMahon, ; Xing & Wu, ).…”