Bioluminescent Low-Affinity Ca Indicator for ER with Multicolor Calcium Imaging in Single Living Cells.

The sarco/endoplasmic reticulum (SR/ER) is the foremost intercellular Ca store (at submillimolar concentrations), playing a crucial role in controlling intracellular Ca levels. For the investigation of SR/ER Ca dynamics in cells, fluorescent protein-based genetically encoded calcium indicators (GECIs) with low Ca 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 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 = 110 μM) intensiometric bioluminescent GECI, which enables monitoring of the Ca dynamics in the ER of HeLa cells and the SR of C2C12-derived myotubes. To facilitate the broad concentration range of Ca in cellular organelles, we additionally developed an intermediate affinity ( K = 18 μM), orange color, and bioluminescent GECI, which enables monitoring of Ca 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 imaging in three distinct organelles (nuclei, mitochondria, and ER) simultaneously. The multicolor, live, bioluminescent Ca imaging demonstrated here can be used to stably reveal the ER Ca homeostasis and cooperative Ca regulation among organelles. This will lead to the further understanding of Ca-related physiological functions and pathophysiological mechanisms.