Mito-Kaede photoactivation and chase experiment for mitophagy: mitophagy flux response toward various stimulations

Mitophagy, a crucial mitochondrial quality control system for cellular stress adaptation, is a key focus in pathophysiology and drug discovery. Developing a simple and versatile mitophagy flux assay is vital for advancing our understanding of cellular responses. Addressing a gap in systematic methods, we employ the photoactivatable fluorescent protein mito-Kaede in C2C12 myocytes, demonstrating its remarkable versatility in quantifying mitophagy flux responses under various stimuli, including carbonyl cyanide m-chlorophenyl hydrazone (CCCP), TNF-α, lipopolysaccharide (LPS), and hypoxia. This study underscores the validity and distinctive advantages of the mito-Kaede assay through comparative analysis with conventional assays including Western blotting (WB), potentially providing valuable insights for both mitophagy flux analysis and drug development. The expression of mito-Kaede, which is suitable for monitoring mitochondrial kinetics, was successfully confirmed in the new C2C12 cell line. Mitophagy flux kinetics were measured under four different inducing conditions: CCCP, TNF-α, LPS, and hypoxia. These results were then compared to those obtained from a Western blotting flux-blockade experiment. Additionally, the mito-Kaede assay was evaluated against the mito-QC cell reporter assay. The findings suggested that mito-Kaede provided a more quantitative analysis. Mitophagy is essential for mitochondrial quality control and is implicated in diverse diseases, including sepsis, neurodegeneration, and muscle wasting. Current mitophagy assays often fail to capture the full dynamic flux, especially during the induction phase. This study addresses the gap by evaluating the mito-Kaede photoactivation system for monitoring mitophagy flux in live muscle cells. C2C12 myocytes were genetically engineered to stably express mito-Kaede, a photoswitchable fluorescent protein that transitions from green to red upon UV activation. Time-lapse fluorescence imaging tracked mitochondrial turnover following stimulation by CCCP, TNF-α, LPS, or hypoxia, with or without mitophagy inhibitors (E64d/pepstatin-A or CQ). Mitochondrial fractions were validated for purity and used for corroborative Western blotting to quantify LC3 levels under stimulated and blocked conditions. All four stimulants (CCCP, TNF-α, LPS, hypoxia) induced significant mitophagy flux, as evidenced by signal decay in mito-Kaede time-lapse imaging. Mitophagy flux was specifically blocked by lysosomal inhibitors, confirming the specificity of the assay. Western blot analysis of LC3 in mitochondrial fractions aligned with imaging data, further validating the mito-Kaede assay’s reliability. Mito-Kaede provided greater sensitivity than conventional Western blotting in detecting early-phase mitophagy dynamics. This study demonstrates that the mito-Kaede system robustly captures mitophagy flux from initiation to degradation phases, unlike pH-dependent reporters (e.g., mito-QC, mito-Keima). The assay is highly responsive to physiologically relevant stimuli, not just artificial mitochondrial uncouplers. Mito-Kaede enables real-time, noninvasive, quantitative monitoring of mitochondrial clearance dynamics. The system holds promise for use in drug screening and understanding disease-specific mitophagy defects. Potential adaptations may allow analysis of mitophagy responses in patient serum or primary cells, pending improvements in assay accessibility beyond transgenic expression systems.