Kryo-ET zeigt, wie Mitochondrien im Parkinson-Stoffwechselweg abgebaut und verschlungen werden

Parkinson's disease is genetically linked to defects in mitochondrial quality control, particularly through mutations in PINK1 and PRKN (Parkin). When mitochondria lose their membrane potential, PINK1 accumulates on the outer mitochondrial membrane, recruits and activates Parkin, and the damaged organelle is ultimately eliminated by mitophagy. Despite decades of biochemical work, the precise nanoscale structural changes occurring during this process inside intact cells had never been directly visualized — until now.

The research team engineered Parkin-expressing U2OS osteosarcoma cells co-expressing fluorescent reporters for mitochondria and Parkin, then treated them with oligomycin and antimycin A (OA) to selectively collapse mitochondrial membrane potential. Cryo-fluorescence microscopy guided the cryo-FIB milling process, producing ~200 nm lamellae from vitrified cells for cryo-ET. In total, 92 lamellae and 157 tomograms were reconstructed, providing an unprecedented in situ structural dataset of depolarized mitochondria.

Several striking structural consequences of depolarization were documented. Mitochondria fragmented from ~1.3 µm to ~0.7 µm average diameter. Cristae became sparser, and in some mitochondria, membrane-bound vesicles replaced the normal lamellar interior. Calcium phosphate clusters — normally abundant in the matrix — were completely absent after OA treatment. A subpopulation of F0F1 ATP synthase complexes, normally confined to cristae, was found mislocalized to the inner boundary membrane (IBM), the flat region of the inner membrane apposed to the outer membrane. About 17% of mitochondrial fragments were visibly targeted by phagophore-like double-membrane structures, with some captured at early, intermediate, and late (fully enveloped) stages. Critically, rod-like densities consistent with ATG2A/BLTP were identified bridging phagophore membranes to adjacent donor membranes, providing direct in-cell evidence for lipid transfer during phagophore expansion.

Perhaps the most novel structural finding involved the prohibitin complex, a dodecameric scaffold (six PHB1-PHB2 dimers) residing in the IMS on the inner mitochondrial membrane. Subtomogram averaging resolved the complex in both untreated and OA-treated cells at sufficient resolution to distinguish two conformational states — an open, splayed form and a closed, compact dome. OA treatment significantly enriched the closed conformation, suggesting that prohibitin undergoes a functionally relevant conformational switch in response to mitochondrial stress, a finding with potential implications for how cristae architecture and protein organization are regulated during mitophagy.

These data collectively establish a nanoscale structural baseline for PINK1-Parkin mitophagy in human cells. They demonstrate the power of correlative cryo-CLEM and cryo-ET for capturing transient mitochondrial remodeling events in situ and provide a framework for future studies dissecting the precise molecular machinery of selective autophagy in the context of neurodegeneration.