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, 90 (5), 1790-6

A FRET-based Calcium Biosensor With Fast Signal Kinetics and High Fluorescence Change

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A FRET-based Calcium Biosensor With Fast Signal Kinetics and High Fluorescence Change

Marco Mank et al. Biophys J.

Abstract

Genetically encoded calcium biosensors have become valuable tools in cell biology and neuroscience, but some aspects such as signal strength and response kinetics still need improvement. Here we report the generation of a FRET-based calcium biosensor employing troponin C as calcium-binding moiety that is fast, is stable in imaging experiments, and shows a significantly enhanced fluorescence change. These improvements were achieved by engineering magnesium and calcium-binding properties within the C-terminal lobe of troponin C and by the incorporation of circularly permuted variants of the green fluorescent protein. This sensor named TN-XL shows a maximum fractional fluorescence change of 400% in its emission ratio and linear response properties over an expanded calcium regime. When imaged in vivo at presynaptic motoneuron terminals of transgenic fruit flies, TN-XL exhibits highly reproducible fluorescence signals with the fastest rise and decay times of all calcium biosensors known so far.

Figures

FIGURE 1
FIGURE 1
In vitro emission spectra of TN-L15, TN-L15 citrine cp174, and TN-L15 citrine cp158. Emission spectra of the various indicators are shown at 25 μM EGTA (dashed line) and 10 mM CaCl2 (solid line) in 100 mM KCL/10 mM MOPS pH 7.5. Emission was taken from 450 to 600 nm with an excitation at 432 nm.
FIGURE 2
FIGURE 2
In vitro characteristics of TN-XL and TN-L15 citrine cp174. (A) Scheme of the substitutions of amino acids in the C-terminal part of the calcium-binding moiety of TN-XL. Shown are the calcium-binding loops of EF-hands III and IV. (B) Calcium titration curves of TN-XL and TN-XL I131T. Apparent Kds are 2.5 μM for TN-XL and 1.7 μM for TN-XL I131T. (C) Emission spectra of TN-L15 citrine cp174 and TN-XL at 100 μM EDTA/25 μM EGTA (gray line), 1 mM MgCl2 (black dashed line), and 10 mM CaCl2/1 mM MgCl2 (black solid line). Note that the magnesium-induced change in FRET detectable in TN-L15 citrine cp174 is absent in TN-XL. (D) Dissociation kinetics of TN-XL. A stopped-flow chamber setup coupled to a fluorometer was used to mix calcium-saturated indicator with 10 mM MOPS/50 mM KCl/20 mM BAPTA (tetrapotassium salt) pH 7.5. The decay of citrine emission or the increase in CFP emission was followed over time and the ratio calculated. The inset shows the same ratio trace within a longer time period of up to 12 s. The in vitro decay time constant was obtained from three independent normalized measurements of 527/475 nm ratio.
FIGURE 3
FIGURE 3
Performance of TN-XL and TN-L15 in primary hippocampal neurons. (A) Traces obtained from a rat hippocampal neuron transfected with TN-XL. Upper trace shows the changes of the normalized ratio. The cell was repeatedly stimulated by high potassium (50 mM) followed by washout. Lower trace shows the corresponding changes in the intensities of the YFP (535/25 nm) and the CFP (485/35 nm) channel. Time axis for ratio and intensities has the same scale. (B) Responses of a primary hippocampal neuron transfected with TN-L15 treated like A. (C) Primary hippocampal neuron expressing TN-XL. The image shows the citrine cp174 emission at 535/25 nm upon excitation at 440/20 nm. Scale bar, 10 μm.
FIGURE 4
FIGURE 4
Rapid and linear changes of the TN-XL fluorescence in Drosophila in vivo. (A) TN-XL expression highlights presynaptic boutons at a transgenic Drosophila NMJ (raw intensity of citrine cp174 emission). Scale bar, 10 μm. (B) The TN-XL fluorescence exhibited staircase-like changes (ΔR/R) at all stimulation frequencies (20, 40, 80, and 160 Hz for 2.2 s, black traces). Increasing the external calcium concentration from 1.5 to 10 mM only moderately increased ΔR/R at 160 Hz stimulation (gray trace). The control experiments without stimulation as well as the time periods before and after stimulation show a remarkably stable TN-XL signal. Action potential (AP) rates of 20 and up to 160 Hz were reported by a fairly linear increase in ΔR/R (inset). (C) Comparison of TN-XL (blue trace) to six other GECI response kinetics using identical stimulus conditions (AP-frequency 40 Hz, 2.2 s). In TN-XL, the strong improvement in the kinetics of the rise achieved in TN-L15 is retained and combined to a dramatic shortening of the time necessary for the fluorescence signals to decay. The corresponding time constants are given in Table 2. Single fluorophore sensor traces show fractional fluorescence changes and ratiometric indicator traces fractional ratio changes, all normalized to the maximum value.

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