Genetically encoded calcium indicator with NTnC-like design and enhanced fluorescence contrast and kinetics

Abstract

Background: The recently developed genetically encoded calcium indicator (GECI), called NTnC, has a novel design with reduced size due to utilization of the troponin C (TnC) as a Ca²⁺-binding moiety inserted into the mNeonGreen fluorescent protein. NTnC binds two times less Ca²⁺ ions while maintaining a higher fluorescence brightness at the basal level of Ca²⁺ in neurons as compared with the calmodulin-based GECIs, such as GCaMPs. In spite of NTnC's high brightness, pH-stability, and high sensitivity to single action potentials, it has a limited fluorescence contrast (F^-Ca2+/F^+Ca2+) and slow Ca²⁺ dissociation kinetics.

Results: Herein, we developed a new NTnC-like GECI with enhanced fluorescence contrast and kinetics by replacing the mNeonGreen fluorescent subunit of the NTnC indicator with EYFP. Similar to NTnC, the developed indicator, named iYTnC2, has an inverted fluorescence response to Ca²⁺ (i.e. becoming dimmer with an increase of Ca²⁺ concentration). In the presence of Mg²⁺ ions, iYTnC2 demonstrated a 2.8-fold improved fluorescence contrast in vitro as compared with NTnC. The iYTnC2 indicator has lower brightness and pH-stability, but similar photostability as compared with NTnC in vitro. Stopped-flow fluorimetry studies revealed that iYTnC2 has 5-fold faster Ca²⁺ dissociation kinetics than NTnC. When compared with GCaMP6f GECI, iYTnC2 has up to 5.6-fold faster Ca²⁺ association kinetics and 1.7-fold slower dissociation kinetics. During calcium transients in cultured mammalian cells, iYTnC2 demonstrated a 2.7-fold higher fluorescence contrast as compared with that for the NTnC. iYTnC2 demonstrated a 4-fold larger response to Ca²⁺ transients in neuronal cultures than responses of NTnC. iYTnC2 response in neurons was additionally characterized using whole-cell patch clamp. Finally, we demonstrated that iYTnC2 can visualize neuronal activity in vivo in the hippocampus of freely moving mice using a nVista miniscope.

Conclusions: We demonstrate that expanding the family of NTnC-like calcium indicators is a promising strategy for the development of the next generation of GECIs with smaller molecule size and lower Ca²⁺ ions buffering capacity as compared with commonly used GECIs.

Keywords: Calcium imaging; Genetically encoded calcium indicator; Protein engineering.

Fig. 1

In vitro properties of the purified iYTnC2 indicator. a Absorbance spectra for iYTnC2 in Ca²⁺-bound or Ca²⁺-free states at indicated pH values. b Excitation and emission spectra for iYTnC2 in Ca²⁺-free state at pH 7.2. c Fluorescence intensity for iYTnC2 in Ca²⁺-free and Ca²⁺-bound states and their dynamic range as a function of pH. Error represents the standard deviation for the average of three records. d Ca²⁺ titration curves for iYTnC2 and GCaMP6f in the absence and in the presence of 1 mM MgCl₂. e Maturation curves for iYTnC2, NTnC in Ca²⁺-free state, and mEGFP. f Photobleaching curves for iYTnC2, NTnC in Ca²⁺-free state, and mEGFP. The power of light before objective lens was 7.3 mW/cm²

Fig. 2

Calcium association and dissociation kinetics for the iYTnC2 and GCaMP6f indicators studied using stopped-flow fluorimetry. a Calcium association kinetics curves for iYTnC2. b Observed Ca²⁺ association rate constants determined from association curves for iYTnC2 and control GCaMP6f GECIs. For the iYTnC2 indicator, fast (green) and slow (grey) exponents are shown. c Relative contribution of monoexponents A₁/(A₁ + A₂) and A₂/(A₁ + A₂) for the iYTnC2 indicator, where A₁ and A₂ are pre-exponential factors in the association curve equation ΔFlu(t) = A₁*exp.(-K_obs1*t)-A₂*exp.(-K_obs2*t). d Calcium dissociation kinetics for the iYTnC2, NTnC and GCaMP6f GECIs. Starting concentration of Ca²⁺ was 1000 nM

Fig. 3

Response of the iYTnC and iYTnC2 indicators to Ca²⁺ concentration changes in HeLa cells. a Confocal images of HeLa Kyoto cells co-expressing green iYTnC (a, left) and red R-GECO1 (a, right) calcium indicators. b-d The graphs illustrate changes in green fluorescence of iYTnC (b), NTnC (c) or iYTnC2 (d) indicators and in red fluorescence of the reference co-expressed R-GECO1 GECI in response to addition of 2 mM CaCl₂ and 2.5 μM ionomycin. The changes in panel b correspond to the area indicated with white circles in the panel a

Fig. 4

Response of iYTnC and iYTnC2 to Ca²⁺ variations as a result of spontaneous activity in cultured neurons. a Dissociated neuronal culture co-expressing iYTnC (a, left) and R-GECO1 (a, right) calcium indicators. b - d The graphs illustrate changes in red fluorescence of R-GECO1 (excitation 561 nm) and green fluorescence of iYTnC (b), NTnC (c) or iYTnC2 (d) (excitation 488 nm) as a result of spontaneous activity in neuronal culture. The graph on panel b illustrates changes in fluorescence in the area indicated with white circle in panel a. The minimal fluorescence values were normalized to the unit

Fig. 5

Fluorescence changes in GECI-expressing neurons in dissociated culture in response to intracellularly induced APs a Fluorescence changes in iYTnC2- and GCaMP6s- expressing cells to the train of 10 APs intracellularly induced with a frequency of 50 Hz. Ca²⁺ responses were averaged across representative recorded neurons in different wells (N = 6 for GCaMP6s and N = 10 for iYTnC2). Example of intracellular recording (grey) was taken from one representative cell. b Dependence of the amplitudes of responses induced by different numbers of APs in neurons expressing iYTnC2 and GCaMP6s. The linear regression was calculated for the 2–50 APs subset for both iYTnC2 and GCaMP6s. In the range of 2 to 50 APs the dependence is linear for both indicators while the amplitude of response to 100 APs in GCaMP6s-expressing neurons lies well below the linear regression line. At the same time response of iYTnC2 to 100APs is located directly on 2–50 regression line, i.e. dependence remains linear even for responses to strong stimulation. Values are shown as the means ± SEM

Fig. 6

Spontaneous calcium activity of neurons in hippocampus of freely behaving mouse visualized with iYTnC2 and nVista HD system. a Photo of nVista HD miniature microscope head-mounted to the mouse. b Detected calcium spikes and the average one; spikes exceeding 4 MAD threshold were aligned at the moment of the very start of the peak (0 s). c Spatial filters and sample traces obtained from an imaging session with a freely behaving mouse expressing iYTnC2. Stars over traces denote spikes that were counted as 4 MAD threshold crossings. The sensor was delivered to the hippocampus by means of rAAV (AAV-CAG-NES-iYTnC2) particles