Development of a Novel Blue Fluorescent Gene-encoded Calcium Indicator Modified from GCaMP3
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Development of a Novel Blue Fluorescent Gene-encoded Calcium Indicator Modified from GCaMP3 Chorok Yum 1 & Taekyoung Ahn 1 & Won-Sik Shim 1,2,3 Received: 4 July 2020 / Accepted: 11 August 2020 # Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract Intracellular calcium can be monitored by various calcium-specific fluorescent dyes including gene-encoded calcium indicators (GECI). GCaMP is a widely-used GECI that emits green fluorescence proportional to the level of intracellular calcium. However, since many tagging proteins also emit green fluorescence, GCaMP cannot be used with another green fluorescent protein. Therefore, it would be ideal to develop a GECI that has a distinct color profile other than green. In this regard, we developed a novel blue fluorescent calcium indicator modified from GCaMP called Ser222-Ala229-Cys330-BCaMP3. Specifically, a simple threonine to histidine substitution to a green fluorescent Cys330-GCaMP3 successfully changed its fluorescence to blue (Cys330-BCaMP3, B for blue). Furthermore, a couple of additional amino acid substitutions resulted in more enhanced blue fluorescence intensity. Among other Cys330-BCaMP3 variants, it was found that Ser222-Ala229-Cys330-BCaMP3 exhibited the strongest blue fluorescence intensity. When Ser222-Ala229Cys330-BCaMP3 was co-expressed with TRPA1 – a non-selective cation channel – in HEK293T cells, it showed moderate blue fluorescence. One of the drawbacks of Ser222-Ala229-Cys330-BCaMP3 was that the fluorescence intensity was not enough when cells were cultured under 37°C. However, this limitation was circumvented by lowering cell culture temperature to 28°C, allowing much more enhanced blue fluorescence. Although Ser222-Ala229-Cys330-BCaMP3 mandates further optimization, the present study has found a promising blue fluorescent GECI that is derived from GCaMP3. Keywords Calcium dye . Genetically encoded calcium indicators . GCaMP3 . Green fluorescent protein . Blue fluorescent protein
Introduction Calcium, as a second messenger, plays a significant role in various signaling pathways at cellular level [1]. As cellular responses are often initiated by abrupt changes in intracellular calcium levels, measuring the calcium changes are vital to understand intricate signaling cascades in cells. Fortunately, measurement of calcium levels can be easily achieved by calcium-specific fluorescent indicators known as “calcium dyes”. These calcium indicators share common characteristics Chorok Yum and Taekyoung Ahn contributed equally to this work. * Won-Sik Shim [email protected] 1
College of Pharmacy, Gachon University, Hambakmoeiro 191, Yeonsu-gu Incheon 21936, Republic of Korea
2
Gachon Institute of Pharmaceutical Sciences, Hambakmoeiro 191, Yeonsu-gu Incheon 21936, Republic of Korea
3
College of Pharmacy, Gachon University, Incheon 406-799, South Korea
that (a) fluorescence is only emitted in the presence of intracellular calcium, and (b) the intensity of fluorescence is proportional to the calcium levels. Most
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