Red fluorescent CEPIA indicators for visualization of Ca 2+ dynamics in mitochondria.

Mitochondrial Ca ²⁺ dynamics are involved in the regulation of multifarious cellular processes, including intracellular Ca ²⁺ signalling, cell metabolism and cell death. Use of mitochondria-targeted genetically encoded Ca ²⁺ indicators has revealed intercellular and subcellular heterogeneity of mitochondrial Ca ²⁺ dynamics, which are assumed to be determined by distinct thresholds of Ca ²⁺ increases at each subcellular mitochondrial domain. The balance between Ca ²⁺ 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 ²⁺ indicators, R-CEPIA3mt and R-CEPIA4mt, which are targeted to mitochondria and their Ca ²⁺ affinities are engineered to match the intramitochondrial Ca ²⁺ concentrations. They enable visualization of mitochondrial Ca ²⁺ 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 ²⁺ dynamics and their functions.