Genetically encoded probe for calcium imaging in lifetime domain

Session

Stream 6 (Frontiers): Quantifying Dynamic Movement in Living Cells

Authors

Tatiana Simonyan (1), Anastasia Mamontova (1), Aleksander Shakhov (2), Konstantin Lukyanov (1), Alexey Bogdanov (3)

Affiliations

1. Center of Life Sciences, Skolkovo Institute of Science and Technology
2. Semenov Federal Research Center for Chemical Physics
3. Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry

Abstract text

Tatiana R. Simonyan¹, Anastasia V. Mamontova¹, Aleksander M. Shakhov², Konstantin A. Lukyanov¹, Alexey M. Bogdanov^3*

¹Center of Life Sciences, Skolkovo Institute of Science and Technology, Moscow 121205, Russia;

²Semenov Federal Research Center for Chemical Physics, Moscow 119991, Russia;

³Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow 117997, Russia;

^*Correspondence: [email protected] 

Categories: Quantitative SPM for Biology, Biomedicine, and Bioinspired Technologies

Preferable type of presentation: oral presentation

This work was supported by the Russian Foundation for Basic Research (grant 19-34-60019).

Abstract

Calcium cation (Ca²⁺) is the common secondary messenger in a cellular signaling. It provides regulation of many biological processes such as muscle contraction, neuronal transmission, fertilization, hormonal secretion, etc. Although a number of fluorescent calcium indicators has been described to date, most of them allow detecting the relative values/changes of the intracellular calcium concentration. Here we describe GCaMP6s-BrUSLEE – a genetically encoded calcium indicator that demonstrates pronounced changes in its fluorescence lifetime depending on calcium concentration, and thus potentially suitable for quantitative calcium imaging. 

Introduction

As one of the most abundant and multifunctional secondary messengers in cell biology, calcium is known not only to control contraction of the striated and vascular smooth muscle, but also to regulate such cellular processes as growth, proliferation, transcription, exocytosis, apoptosis, and to provide neuronal activity [1]. Calcium imaging is, therefore, of enormous importance in studying of diverse intracellular cascades. Among various types of calcium indicators, one can highlight the group of genetically encoded GFP-based molecules, which is especially attractive for live cell and in vivo applications and in turn a high performance GCaMP family within this group [2, 3, 4].

Usually, fluorescent indicators change the intensity of fluorescence in response to a shift in the analytes concentration. This principle allows detecting only the relative concentration value, but not the absolute one. The fluorescence lifetime is an intrinsic value and can be used for calibration measurements, which opens up the possibility to quantify the parameter under study.

One of the promising GFP variants capable of changing the fluorescence lifetime in a wide range is BrUSLEE [5]. It is characterized by both high brightness (~80% of EGFP) and a short fluorescence lifetime of 820 ps. This combination of fluorescence characteristics makes BrUSLEE an attractive label for FLIM imaging. Moreover, we have shown that, depending on the conditions, the lifetime of BrUSLEE fluorescence can vary smoothly in the range of 0.5-7 ns (unpublished data), which probably indicates a high conformational mobility of the chromophore, which is important for sensing in the time domain. Therefore, we decided to use BrUSLEE as a fluorescent core for one of the popular calcium indicators GCaMP6s.

Methods/Materials

For calcium titrations, GCaMP6s and  GCaMP6s-BrUSLEE were first diluted 1:100 in triplicate in 30 mM MOPS, pH 7.2, 100 mM KCl with either 10 mM EGTA (zero free calcium) or 10 mM CaEGTA (~39 μM free calcium). These 2 solutions were mixed in various ratios to give 11 different free calcium    concentrations (Calcium Calibration Buffer Kit #1, Life Technologies). For fluorescence excitation-emission spectra measurements, Cary 100 UV/VIS spectrophotometer and Cary Eclipse fluorescence spectrophotometer (Varian) were used. The fluorescence lifetime was measured using Mini-tau Lifetime TCSPC Spectrometer (Edinburg Instruments).

Results 

We engineered circularly permuted BrUSLEE, inserted it into the GCaMP6s instead of the cpEGFP and compared the original GCaMP6s and GCaMP6s-BrUSLEE in the spectral and time domains.

In the spectral domain, GCaMP6s-BrUSLEE shows a tenfold contrast which is slightly less than contrast of the original indicator (Fig. 1 A, B). 

In vitro measurements of the indicators in the time domain showed that GCaMP6s is characterized by a mono-exponential fluorescence decay with a lifetime of about 2.8 ns, which was insensitive to a change in calcium concentration (Fig.1 C). In contrast to it, GCaMP6s-BrUSLEE is characterized by a complex fluorescence decay fitted best with three lifetime components. The longest third component shows nearly linear dependence of lifetime value on calcium concentration within the range of 10-200 nM with a fourfold contrast. (Fig. 1 D)  

FFig. 1. Dependence of fluorescence intensity (A, B) and fluorescence lifetime (C, D) GCaMP6s and GCaMP6s-BrUSLEE on calcium concentration. 

These in vitro calibration data allows us to hope that the GCaMP6s-BrUSLEE can be used for quantitative measurements of calcium in live cells.

References

[1] Zhong et al. Front. Med., 2019

[2] Mollinedo-Gajate et al. Spring. Nat. Switz. AG, 2019

[3] Zhang et al. Janelia Research Campus, 2020

 [4] Dana et al. bioRxiv, 2018

 [5] Mamontova et al., Sci. Rep., 2018

References

[1] Zhong et al. Front. Med., 2019

[2] Mollinedo-Gajate et al. Spring. Nat. Switz. AG, 2019

[3] Zhang et al. Janelia Research Campus, 2020

 [4] Dana et al. bioRxiv, 2018

 [5] Mamontova et al., Sci. Rep., 2018