Local two-photon excitation reveals the mechanism of mitochondrial Ca2+ release

Mitochondrial Ca2+ (mitoCa2+) simultaneously implicates respiration, mitochondrial physiology, and cell signaling, which prevents the disentanglement of mitoCa2+ from those complex processes. Although mitochondria have long been recognized as temporary Ca2+ buffer, how mitoCa2+ is regulated and released remains unclear. In this study, we report a specific photochemical excitation to flavoproteins in single-mitochondrion tubulars by a tightly-focused femtosecond laser that triggers local mitoCa2+ transients, without any extra-mitochondrial Ca2+ involved. The two-photon excitation to complex I and II accelerates the entire electron transport chain (ETC) and promotes ATP synthesis. Free mitoCa2+ is thus released from the Ca2+-phosphate ion (Pi) complexes at complex V in mitochondrial matrix during ATP synthesis to form mitoCa2+ transients there. The abnormal mitoCa2+ signaling by knockdown of ATP synthase subunit affects cell proliferation, apoptosis, and mitophagy. Our results reveal mitoCa2+ is released and regulated by ETC and ATP synthesis rather than the reverse. Mitochondria are multi-functional units to simultaneously produce energy and coordinate molecular signaling for cells. The key process of metabolism, tricarboxylic acid (TCA) cycle, is continuously ongoing in mitochondrial matrix and generates reducing equivalents for subsequent electron transfer (Martinez-Reyes and Chandel, 2020; Martinez-Reyes et al., 2016). The respiration is accomplished by electron transport chain (ETC) in the inner mitochondrial membrane (IMM) (Marreiros et al., 2016). Mitochondria regulate a series of signaling cascades to mediate apoptosis, autophagy, and cell senescence (Galluzzi et al., 2014; Rasola and Bernardi, 2011; Ziegler et al., 2021). Biosynthesis of some amino acids and nucleotides also takes place in mitochondria (Ahn and Metallo, 2015; Li and Hoppe, 2023). Those processes are involved with each other through complex crosstalk and feedbacks. Mitochondria have long been recognized as Ca2+ buffer to temporarily deposit abnormal cytosolic Ca2+ for cellular Ca2+ homeostasis (Garbincius and Elrod, 2022; Kirichok et al., 2004; Lambert et al., 2019). However, the Ca2+ entry into mitochondria definitely influence those mitochondrial processes and functions (Garbincius and Elrod, 2022). The acute and direct consequences of Ca2+ entry into mitochondria are found as depolarization of mitochondrial membrane potential (MMP). If the cellular Ca2+ level is too high, mitochondria may become damaged and dysfunctional. Mitophagy/autophagy and even apoptosis are initiated (Galluzzi et al., 2014; Lou et al., 2020; Rasola and Bernardi, 2011). Moreover, Ca2+ in mitochondrial matrix (mitoCa2+) takes an essential role in quite a few physiological processes including mitochondrial fission and fusion, cell development, and proliferation (Doonan et al., 2014; Singh and Mabalirajan, 2021; Steffen and Koehler, 2018). The respiration is also believed to be related to mitoCa2+ (Glancy and Balaban, 2012; Wescott et al., 2019). Technically, perturbation to mitoCa2+ affects most mitochondrial processes and physiology, which makes it quite difficult to interrogate how Ca2+ is regulated and released in mitochondria. So far, the regulation of mitoCa2+ remains in mist. In this study, we report a single-mitochondrion photochemical process by tightly-focused femtosecond laser that specifically excites complex I and II by two-photon excitation and accelerate ETC. Free mitoCa2+ is released from Ca2+-phosphate ion (Pi) complexes by ATP synthesis at complex V in mitochondria. These results clarify the mechanism of mitoCa2+ regulation and provide further insights in the relationship between mitoCa2+ and respiration.