Red fluorescent CEPIA indicators for visualization of Ca2+ 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-CEPIA3 mt and R-CEPIA4 mt , 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-CEPIA3 mt and R-CEPIA4 mt are expected to be a useful tool for elucidating the mechanisms of the complex mitochondrial Ca 2+ dynamics and their functions.