NTnC-like genetically encoded calcium indicator with a positive and enhanced response and fast kinetics

Abstract

The NTnC genetically encoded calcium indicator has an advantageous design because of its smaller size, GFP-like N- and C-terminal ends and two-fold reduced number of calcium binding sites compared with widely used indicators from the GCaMP family. However, NTnC has an inverted and modest calcium response and a low temporal resolution. By replacing the mNeonGreen fluorescent part in NTnC with EYFP, we engineered an NTnC-like indicator, referred to as YTnC, that had a positive and substantially improved calcium response and faster kinetics. YTnC had a 3-fold higher calcium response and 13.6-fold lower brightness than NTnC in vitro. According to stopped-flow experiments performed in vitro, YTnC had 4-fold faster calcium-dissociation kinetics than NTnC. In HeLa cells, YTnC exhibited a 3.3-fold lower brightness and 4.9-fold increased response to calcium transients than NTnC. The spontaneous activity of neuronal cultures induced a 3.6-fold larger ΔF/F response of YTnC than previously shown for NTnC. On patched neurons, YTnC had a 2.6-fold lower ΔF/F than GCaMP6s. YTnC successfully visualized calcium transients in neurons in the cortex of anesthetized mice and the hippocampus of awake mice using single- and two-photon microscopy. Moreover, YTnC outperformed GCaMP6s in the mitochondria and endoplasmic reticulum of cultured HeLa and neuronal cells.

Figure 1

In vitro properties of the purified YTnC indicator. (a) A scheme of the original library for optimization of linkers in YTnC indicator and crystal structure of NTnC*2Ca²⁺ complex (pdb 5MWC) for the sensor having a similar design. (b) Absorbance spectra for YTnC in Ca²⁺-free and Ca²⁺-bound states at different pH values. (c) Excitation and emission spectra for YTnC in Ca²⁺-free and Ca²⁺-bound states. (d) Fluorescence intensity for YTnC in Ca²⁺-free and Ca²⁺-bound states as a function of pH. (e) Ca²⁺ titration curves for YTnC in the absence and presence of 1 mM MgCl₂. (f) Maturation curves for YTnC in Ca²⁺-saturated state and mEGFP. (g) Photobleaching curves for YTnC in Ca²⁺-free state and mEGFP. The power of light before objective lens was 7.3 mW/cm². (h) Fast protein liquid chromatography of YTnC (14 mg/ml) in 40 mM Tris-HCl (pH 7.5) and 200 mM NaCl buffer supplemented with 5 mM CaCl₂. Error bars represent the standard deviation for the average of three records.

Figure 2

Calcium-association and -dissociation kinetics for the YTnC indicator investigated using stopped-flow fluorimetry. (a–c) Calcium-association kinetics curves for YTnC, GCaMP6f and NTnC, respectively. (d) Observed Ca²⁺-association rate constants determined from association curves for YTnC and control GCaMP6f and NTnC GECIs. For the YTnC indicator, fast (green) and slow (grey) exponents are shown. The solid lines represent the fitting curves to the equation k_obs = k_on × [Ca²⁺]ⁿ + k_off. (e) Relative contribution of monoexponents A₁/(A₁ + A₂) and A₂/(A₁ + A₂) for the YTnC indicator, where A₁ and A₂ are the pre-exponential factors in the association curve equation ΔFlu(t) = A₁ × exp(−k^on_obs1 × t)−A₂ × exp(−k^on_obs2 × t). The dots correspond to the A₁ and A₂ values, which were approximated by a linear equation (R² = 5.083). (f) Calcium-dissociation kinetics for YTnC, GCaMP6f and NTnC GECIs. Starting concentration of free Ca²⁺ was 1000 nM. Three replicates were averaged for analysis. Whiskers correspond to SD error.

Figure 3

Response of the YTnC indicator to Ca²⁺ variations in HeLa Kyoto cells and cultured neurons. (a) Confocal images of HeLa Kyoto cells co-expressing green YTnC (panel a, left) and red R-GECO1 (panel a, right) calcium indicators. (b, c) The graphs illustrate changes in green fluorescence of YTnC (panel b) or control NTnC (panel c) indicators and in red fluorescence of the reference co-expressed R-GECO1 GECI in response to the addition of 2 mM CaCl₂ and 2.5 μM ionomycin. The changes in panel b correspond to the area indicated with white circles in panel a. One example of three repeats is shown. (d) Dissociated neuronal culture co-expressing YTnC (panel d, left) and R-GECO1 (panel d, right) calcium indicators. (e,f) The graphs illustrate changes in red fluorescence of R-GECO1 (excitation 561 nm) and green fluorescence of YTnC (excitation 488 nm) (panel e) or control NTnC (panel f) as a result of spontaneous activity in neuronal culture. The graph in panel e illustrates changes in fluorescence in the area indicated with white circle in panel d. (b–c, e–f) The minimal fluorescence values were normalized to one.

Figure 4

Fluorescence changes in neurons expressing YTnC and GCaMP6s indicators in dissociated culture in response to intracellularly induced APs. (a) Fluorescence changes in GECI-expressing cells to the train of 10 APs intracellularly induced with a frequency of 50 Hz. Ca²⁺ responses were averaged across representative recorded neurons from different wells (N = 6 and 7 neurons from 3 and 4 cultures for GCaMP6s and YTnC, respectively). Example of intracellular recording (grey) was obtained from one representative cell. (b) Dependence of the amplitudes of responses induced by different numbers of APs in neurons expressing YTnC and GCaMP6s. The linear regression shown in the figure was calculated for the 2–50 AP subset for both YTnC (R² = 0.9959) and GCaMP6s (R² = 0.9981). Note that in the range of 2 to 50 APs, dependence is linear for both indicators, whereas the amplitude of response to 100 APs in GCaMP6s-expressing neurons lies well below the linear regression line. At the same time, the response of YTnC to 100APs is located directly on the 2–50 regression line, i.e., dependence remains linear even for responses to strong stimulation. Values are shown as the means ± SEM.

Figure 5

In vivo drifting grating-evoked neuronal activity in the mouse cortex visualized with YTnC and GCaMP6s calcium indicators using two-photon microscopy. (a) Example of the 3D reconstruction of YTnC-positive cells in the V1 visual cortex area using 960 nm excitation light. Block size, 317 × 317 × 350 μm. (b) Averaged ΔF/F responses corresponding to spontaneous (non-specific) and grating-evoked (specific) activity across neurons (n = 9, YTnC; n = 5, GCaMP6s and n = 3, YTnC; n = 2, GCaMP6s, respectively) in the V1 area for the YTnC and GCaMP6s indicators, respectively. (c) Two-photon images of two regions from V1 layer 2/3 neurons captured during presentation of drifting grating to the mouse for YTnC and GCaMP6s indicators. Averaged ΔF/F responses during presentation of drifting gratings (eight directions, 10 repetitions) are shown for the selected neurons. The directions of the drifting gratings (blue lines) are shown with arrows (in black). Grey and red lines correspond to the individual and averaged traces across ten repetitions, respectively. Black horizontal lines correspond to the time of grating presentation.

Figure 6

In vivo neuronal Ca²⁺ activity in hippocampus of freely behaving mice visualized with calcium indicators YTnC, GCaMP6f, GCaMP6s and an nVista HD system. (a) Photo of an nVista HD miniature microscope mounted to the head of mice. (b) Spatial filters and sample traces obtained from a 10-min imaging session with freely behaving mice expressing GCaMP6f, YTnC and GCaMP6s. (c) Mean spikes for calcium indicators GCaMP6f, YTnC and GCaMP6s; spikes exceeding the 4 MAD threshold were aligned at the moment of the very start of the peak (3 s). (d) Photo of O-shaped track with landmarks. (e) Circular plot of mouse trajectory during the exploration of circular track, synchronized with the spikes of a place cell (green triangles). The sensors GCaMP6f, YTnC and GCaMP6s were delivered to the hippocampus with rAAV (AAV-CAG-NES-GCaMP6f, AAV-CAG-NES-YTnC and AAV-CAG-GCaMP6s, respectively) particles.

Figure 7

Localization of YTnC and GCaMP6s calcium indicators targeted to the different compartments of HeLa and neuronal cells. Confocal images of cells transiently expressing the listed fusions. The DNAs coding YTnC and GCaMP6s sensors were delivered to the HeLa and neuronal cells using transient transfection with lipofectamine or calcium-phosphate precipitation, respectively.