Red fluorescent CEPIA indicators for visualization of Ca2+ dynamics in mitochondria
Red fluorescent CEPIA indicators for visualization of Ca2+ dynamics in mitochondria
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DOI：
10.1038/s41598-020-59707-8
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发表时间：
2020-02-18
影响因子：
4.6
通讯作者：
Iino, Masamitsu
中科院分区：
文献类型：
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作者：
Kanemaru, Kazunori;Suzuki, Junji;Iino, Masamitsu
线粒体钙动力学参与调节多种细胞过程，包括细胞内钙信号、细胞代谢和细胞死亡。线粒体基因编码的钙指示剂的使用揭示了线粒体钙动力学的细胞间和亚细胞间的异质性，这被认为是由每个亚细胞线粒体域的不同的钙增加阈值决定的。钙离子通过线粒体钙单转运体的内流和阳离子交换器通过线粒体内膜的排泄之间的平衡可能决定了这一阈值；然而，其确切的机制仍有待进一步探讨。我们在这里报告了新的红色荧光基因编码的钙指示剂R-CEPIA3mt和R-CEPIA4mt，它们针对线粒体，它们的钙亲和力被设计成与线粒体内的钙浓度相匹配。它们能够在使用绿色荧光探针和光遗传工具的同时，以高时空分辨率可视化线粒体钙动态。因此，R-CEPIA3mt和R-CEPIA4mt有望成为阐明线粒体复杂的钙动力学机制及其功能的有用工具。
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.