Red fluorescent CEPIA indicators for visualization of Ca dynamics in mitochondria.

Mitochondrial Ca dynamics are involved in the regulation of multifarious cellular processes, including intracellular Ca signalling, cell metabolism and cell death. Use of mitochondria-targeted genetically encoded Ca indicators has revealed intercellular and subcellular heterogeneity of mitochondrial Ca dynamics, which are assumed to be determined by distinct thresholds of Ca increases at each subcellular mitochondrial domain. The balance between Ca influx through the mitochondrial calcium uniporter and extrusion by cation exchangers across the inner mitochondrial membrane may define the threshold; however, the precise mechanisms remain to be further explored. We here report the new red fluorescent genetically encoded Ca indicators, R-CEPIA3mt and R-CEPIA4mt, which are targeted to mitochondria and their Ca affinities are engineered to match the intramitochondrial Ca concentrations. They enable visualization of mitochondrial Ca dynamics with high spatiotemporal resolution in parallel with the use of green fluorescent probes and optogenetic tools. Thus, R-CEPIA3mt and R-CEPIA4mt are expected to be a useful tool for elucidating the mechanisms of the complex mitochondrial Ca dynamics and their functions.