JoVE Encyclopedia of Experiments
Immunology
0 views • 3:39 min • July 8th, 2025
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.
On day one, pick L4 larvae of transgenic animals expressing both DCT-1::GFP and DsRed::LGG-1 in body wall muscle cells and place them onto an OP50-seeded nematode growth media plate. Place 5 to 10 worms onto each 3.5-cen
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