Bioluminescent Low-Affinity Ca2+ Indicator for ER with Multicolor Calcium Imaging in Single Living Cells

摘要

The sarco/endoplasmic reticulum (SR/ER) is the foremost intercellular Ca~(2+) store (at submillimolar concentrations), playing a crucial role in controlling intracellular Ca~(2+) levels. For the investigation of SR/ER Ca~(2+) dynamics in cells, fluorescent protein-based genetically encoded calcium indicators (GECIs) with low Ca~(2+) affinity have been used. Recently, bioluminescent protein-based GECIs with high brightness have been reported to counter the constraints of fluorescence imaging, such as phototoxicity. However, their Ca~(2+) affinity is high and limited for imaging in the cytosol, nucleus, or mitochondria. In this study, we developed a novel cyan color, low-affinity (K _(d) = 110 μM) intensiometric bioluminescent GECI, which enables monitoring of the Ca~(2+) dynamics in the ER of HeLa cells and the SR of C2C12-derived myotubes. To facilitate the broad concentration range of Ca~(2+) in cellular organelles, we additionally developed an intermediate affinity (K _(d) = 18 μM), orange color, and bioluminescent GECI, which enables monitoring of Ca~(2+) dynamics in the mitochondria of HeLa cells. With these indicators, in conjunction with an existing high-affinity, green, bioluminescent GECI, we succeeded in multicolor bioluminescent Ca~(2+) imaging in three distinct organelles (nuclei, mitochondria, and ER) simultaneously. The multicolor, live, bioluminescent Ca~(2+) imaging demonstrated here can be used to stably reveal the ER Ca~(2+) homeostasis and cooperative Ca~(2+) regulation among organelles. This will lead to the further understanding of Ca~(2+)-related physiological functions and pathophysiological mechanisms.