In situ cryo-ET visualization of mitochondrial depolarization and mitophagic engulfment
Published July 30, 2025
Output Details
Preprint April 1, 2025
Published July 30, 2025
Description
Defective mitochondrial quality control in response to loss of mitochondrial membrane polarization is implicated in Parkinson’s disease by mutations in *PINK1* and *PRKN*. Application of *in situ* cryo-electron tomography (cryo-ET) made it possible to visualize the consequences of mitochondrial depolarization at higher resolution than heretofore attainable. Parkin-expressing U2OS cells were treated with the depolarizing agents oligomycin and antimycin A (OA), subjected to cryo-FIB milling, and mitochondrial structure was characterized by *in situ* cryo-ET. Phagophores were visualized in association with mitochondrial fragments. Bridge-like lipid transporter (BLTP) densities potentially corresponding to ATG2A were seen connected to mitophagic phagophores. Mitochondria in OA-treated cells were fragmented and devoid of matrix calcium phosphate crystals. The intermembrane gap of cristae was narrowed and the intermembrane volume reduced, and some fragments were devoid of cristae. A subpopulation of ATP synthases re-localized from cristae to the inner boundary membrane (IBM) apposed to the outer membrane (OMM). The structure of the dome-shaped prohibitin complex, a dodecamer of PHB1-PHB2 dimers, was determined *in situ* by sub-tomogram averaging in untreated and treated cells and found to exist in open and closed conformations, with the closed conformation is enriched by OA treatment. These findings provide a set of native snapshots of the manifold nano-structural consequences of mitochondrial depolarization and provide a baseline for future *in situ* dissection of Parkin-dependent mitophagy.
Identifier (DOI)
10.1073/pnas.2511890122
