This study investigates mitophagy in transgenic nematodes expressing fluorescently tagged proteins. The methodology involves using a mitophagy-inducing drug to observe the clearance of damaged mitochondria through fluorescence microscopy.
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-centimeter plate using at least three plates. When finished, incubate the nematodes at 20 degrees Celsius.
Next, on day 5, synchronize the nematodes by selecting 15 to 20 L4 transgenic larvae and transferring them onto fresh OP50-seeded plates. Use at least 5 plates for each experimental condition. On day seven, prepare some of the vehicle plates for mitophagy-affecting drugs of interest and some to use as positive controls.
Next, expose E. coli-seeded plates to UV light for 15 minutes at an intensity of 222 microwatts per centimeter squared to ensure the mitophagy-inducing compounds on the plates are not metabolized by bacteria. Now, add the compound of interest in 10 microliters to the top of the seeded plates, and add an equivalent amount of compound vehicle to the vehicle plates as a negative control.
For positive control of mitophagy induction, add paraquat, a mitochondrial toxicant. Gently swirl the plates until the solution cuts the entire surface, and allow the plates to dry with the lids closed at room temperature for at least one hour. Once dry, transfer 10 to 20 2-day-old adult transgenic animals to the plates. Incubate them at 20 degrees Celsius for two days.
On day 9, prepare 2% agarose pads, and add 1 droplet of 20 millimolar levamisole in M9 per pad. Immobilize the transgenic animals for imaging by placing them in the M9-levamisole drop. Then, gently place a coverslip on the top of the drop. Place the sample on a confocal microscope stage and image the single body wall muscle cells by taking Z-Stack images under 63 times magnification.
繁體中文
