Abstract
Involvement of parkin in mitophagy is well studied. Translocation of parkin from cytosol to depolarized mitochondria triggers ubiquitination-dependent mitophagy. In this study, we used Airyscan super-resolution microscopy and correlative light-electron microscopy (CLEM) to characterize parkin association during photodamage-induced mitophagy, a form of phosphoinositide 3-kinase-independent or Type 2 mitophagy. After photodamage with 488-nm light to small groups of mitochondria within GFP-LC3 transgenic mouse hepatocytes, U-shaped crescents of GFP-LC3 fluorescence formed around damaged, depolarized mitochondria beginning after 31 ± 18 min. Fluorescence of crescents progressively intensified, but their length remained unchanged. Subsequently, LC3-containing puncta merged with damaged mitochondria containing crescents to form a continuous ring. Adenovirally expressed mCherry-TOMM20 (outer membrane marker) labeled the surface of all mitochondria. After photodamage, gaps of mCherry-TOMM20 surface labeling appeared as a result of mitochondrial swelling and outer membrane rupture. Crescents of GFP-LC3 fluorescence formed over these gaps not containing mCherry-TOMM20. Brightfield microscopy confirmed mitochondrial swelling after photodamage, leading to outer membrane rupture and herniation of the inner membrane. CLEM then revealed that GFP-LC3 labeling occurred at the bare surfaces of herniated inner membranes at early stages of photodamage-induced mitophagy. Adenoviral expression of mCherry-parkin showed that parkin did not associate with photodamaged mitochondria at any stage after up to 2 h. In parkin KO mouse hepatocytes adenovirally expressing GFP-LC3 and mCherry-TOMM20, the same mitophagy pattern was observed. Thus, mitochondrial swelling, outer membrane rupture, and inner membrane herniation after photodamage exteriorizes bare surfaces of mitochondrial inner membranes that become the proximate site for LC3 but not parkin association and the initiation of mitophagy (AA021191, AA025379, DK073336).
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