Abstract
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.