Yujie Zhu

Mitochondrial Ca ²⁺ (mitoCa ²⁺ ) simultaneously implicates respiration, mitochondrial physiology, and cell signaling, which prevents the disentanglement of mitoCa ²⁺ from those complex processes. Although mitochondria have long been recognized as temporary Ca ²⁺ buffer, how mitoCa ²⁺ 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 mitoCa ²⁺ transients, without any extra-mitochondrial Ca ²⁺ involved. The two-photon excitation to complex I and II accelerates the entire electron transport chain (ETC) and promotes ATP synthesis. Free mitoCa ²⁺ is thus released from the Ca ²⁺ -phosphate ion (Pi) complexes at complex V in mitochondrial matrix during ATP synthesis to form mitoCa ²⁺ transients there. The abnormal mitoCa ²⁺ signaling by knockdown of ATP synthase subunit affects cell proliferation, apoptosis, and mitophagy. Our results reveal mitoCa ²⁺ 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 Ca ²⁺ buffer to temporarily deposit abnormal cytosolic Ca ²⁺ for cellular Ca ²⁺ homeostasis (Garbincius and Elrod, 2022; Kirichok et al., 2004; Lambert et al., 2019). However, the Ca ²⁺ entry into mitochondria definitely influence those mitochondrial processes and functions (Garbincius and Elrod, 2022). The acute and direct consequences of Ca ²⁺ entry into mitochondria are found as depolarization of mitochondrial membrane potential (MMP). If the cellular Ca ²⁺ 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, Ca ²⁺ in mitochondrial matrix (mitoCa ²⁺ ) 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 mitoCa ²⁺ (Glancy and Balaban, 2012; Wescott et al., 2019). Technically, perturbation to mitoCa ²⁺ affects most mitochondrial processes and physiology, which makes it quite difficult to interrogate how Ca ²⁺ is regulated and released in mitochondria. So far, the regulation of mitoCa ²⁺ 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 mitoCa ²⁺ is released from Ca ²⁺ -phosphate ion (Pi) complexes by ATP synthesis at complex V in mitochondria. These results clarify the mechanism of mitoCa ²⁺ regulation and provide further insights in the relationship between mitoCa ²⁺ and respiration.

Dysregulation of the PINK1/Parkin-mediated mitophagy is essential to Parkinson’s disease. Although important progress has been made in previous researches, the biochemical reagents that induce global and significant mitochondrial damage may still hinder deeper insights into the mechanisms of mitophagy. The origin of PINK1/Parkin pathway activation in mitophagy remains elusive. In this study, we develop an optical method, ultra-precise laser stimulation (UPLaS) that delivers a precise and noninvasive stimulation onto a submicron region in a single mitochondrial tubular structure. UPLaS excites localized mitochondrial Ca²⁺ (mitoCa²⁺) oscillations with tiny perturbation to mitochondrial membrane potential (MMP) or mitochondrial reactive oxygen species. The UPLaS-induced mitoCa²⁺ oscillations can directly induce PINK1 accumulation and Parkin recruitment on mitochondria. The Parkin recruitment by UPLaS requires PINK1. Our results provide a precise and noninvasive technology for research on mitophagy, which stimulates target mitochondria with little damage, and reveal mitoCa²⁺ oscillation directly initiates the PINK1-Parkin pathway for mitophagy without MMP depolarization.

Pulsed lasers at the near infrared (NIR) range have been widely used in dermatology. Ultrashort pulsed picosecond lasers are found with the specific ability of very effective activation of skin repair and remodeling along with significant photodamage. Femtosecond lasers, with a shorter pulse width, may be a promising alternative to current NIR lasers in clinic. In this study, we performed optical micromachining by a femtosecond laser at 1,030 nm to skin of live mice in two modes of scanning of focused laser and direct irradiation by unfocused laser. The acute and one-day delayed immune molecular responses of the skin to the micromachining are studied by immunofluorescence microscopy of the skin sections. Our data shows the focused laser can activate remodeling of skin without any significant immune responses. In contrast, the direct irradiation by the unfocused laser activate significant immune responses in the deep dermis with high regulation of interleukin. Those results suggest focused femtosecond laser is of good promising potential in activation of skin remodeling and repairing with little immune or physical damage.

Presents corrections to author byline and affiliation information to the above named paper.

Keratins play critical roles in intermediate filament formation, inflammatory responses and cellular signaling in epithelium. While keratins is a major epidermal fluorophore, the mechanisms underlying the autofluorescence (AF) of keratins and its biomedical implications have remained unknown. Our study used mouse skin as a model to study these topics, showing that UV dose-dependently induced increases in green AF at the spinous layer of the epidermis of mouse within 6 hr of the UV exposures, which may be used for non-invasive prediction of UV-induced skin damage. The UV-induced AF appears to be induced by cysteine protease-mediated keratin 1 proteolysis: 1) UV rapidly induced significant keratin 1 degradation; 2) administration of keratin 1 siRNA largely decreased the UV-induced AF; and 3) administration of E-64, a cysteine protease inhibitor, significantly attenuated the UV-induced AF and keratin 1 degradation. Our study has also suggested that the UV-induced keratin 1 proteolysis may be a novel crucial pathological factor in UV-induced skin damage, which is supported by both the findings that indicate critical biological roles of keratin 1 in epithelium and our observation that prevention of UV-induced keratin 1 proteolysis can lead to decreased UV-induced skin damage. Collectively, our study has suggested that UV-induced keratin 1 proteolysis may be a novel and valuable target for diagnosis, prevention and treatment of UV-induced skin damage.