An In Vivo Assay to Detect Mitophagy in Transgenic Nematodes
Transcripción
Take transgenic nematodes, with body-wall muscle cells expressing green fluorophore-tagged DCT-1, an outer mitochondrial membrane protein, and a red fluorophore-tagged LGG-1, a phagophore membrane protein.
Place the worms on top of a growth medium containing a mitophagy-inducing drug.
The drug damages the cellular mitochondria, initiating mitophagy or selective clearance of damaged mitochondria.
DCT-1 on damaged mitochondria undergoes ubiquitination and phosphorylation, facilitating interaction with LGG-1 on the phagophore membrane.
The phagophore expands around the mitochondria to form an autophagosome targeted for lysosomal degradation.
Place the nematodes in an anesthetic on a microscope slide to immobilize them, and place a coverslip.
Under a fluorescence microscope, visualize yellow spots within the muscle cells, indicating the co-localization of DCT-1 emitting green fluorescence and LGG-1 displaying red fluorescence. This confirms the occurrence of mitophagy.
