Red fluorescent CEPIA indicators for visualization of Ca²⁺ dynamics in mitochondria

Mitochondrial Ca²⁺ dynamics are involved in the regulation of multifarious cellular processes, including intracellular Ca²⁺ signalling, cell metabolism and cell death. Use of mitochondria-targeted genetically encoded Ca²⁺ indicators has revealed intercellular and subcellular heterogeneity of mitochondrial Ca²⁺ dynamics, which are assumed to be determined by distinct thresholds of Ca²⁺ increases at each subcellular mitochondrial domain. The balance between Ca²⁺ 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 Ca²⁺ indicators, R-CEPIA3mt and R-CEPIA4mt, which are targeted to mitochondria and their Ca²⁺ affinities are engineered to match the intramitochondrial Ca²⁺ concentrations. They enable visualization of mitochondrial Ca²⁺ 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 Ca²⁺ dynamics and their functions.