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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.

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