This protocol describes how to induce autophagy in the Drosophila melanogaster larval fat body via amino acid depletion and how to analyze the differences in autophagy among mutant clones. As autophagy is highly sensitive to nutrition availability in culture conditions, clonal analysis, a method that analyzes mutant cells versus wild type cells in the same tissue, has an advantage in dissecting autophagy defects. Begin by introducing three males and 15 female adult flies into a culture vial with standard cornmeal or molasses or agar Drosophila media at 25 degrees Celsius for mating.
At 48 hours post-induction, tap the mating vial until the flies are stunned and drop down onto the media at the base of the vial. Unplug the mating vial and cover its mouth with an unplugged inverted culture vial with fresh media. Then flip and tap the vials to transfer the flies to the fresh culture vial.
Plug the fresh culture vial and discard the old vial. Place the fresh culture vial in a 25 degree Celsius incubator for egg laying on the fresh media. Remove the flies after six hours of egg laying and discard the flies by dumping them into a flask containing 75%ethanol.
Incubate the vial with embryos in a 37 degree Celsius water bath for one hour to induce flippase expression. Subsequently, place them in a 25 degree Celsius incubator and allow the embryos to continue to develop. Using a laboratory spatula, scoop out the media containing the developing larvae into a Petri dish 75 hours post egg laying.
Add 1X PBS to the dish and gently separate out the culture media and the larvae using long forceps. Then select 10 to 15 early third instar larvae. Fill the wells of a nine-well glass depression spot plate with 1X PBS and place the separated third instar larvae in the wells using long forceps.
Wash the larvae thoroughly to remove all the media residues. Take five milliliters of 20%sucrose solution in an empty vial and place the clean third instar larvae in this solution using long forceps. Incubate this vial in a 25 degree Celsius incubator for six hours before harvesting them for dissection.
Add 1X PBS into each well of the nine-well glass depression spot plate and transfer the larvae into the wells with long forceps. Place one larvae in a well with the dorsal side facing up. Grip the cuticle of the larva with two 5 forceps in the middle of the larval trunk and gently tear open the cuticles.
The exposed fat bodies, along with other larval internal tissues, will still be attached to the carcass of the larva. Pull enough to expose the internal organs as much as possible. Repeat this step for all the larvae.
Transfer the larval carcass into a 1.5 milliliter microcentrifuge tube containing 500 microliters of 4%PFA and incubate for 30 minutes at 25 degrees Celsius without shaking the tubes. After 30 minutes, pipette out the PFA solution. Add 500 microliters of 1X PBS into the tube.
Gently shake the tube on a flat rotator for 10 minutes and then discard the 1X PBS solution. Repeat this three times. Using long forceps, transfer the fixed and washed larval carcass to a well in the nine depression spot plate filled with 1X PBS.
Remove all the non-fat body tissues with 5 forceps. Finally, mount the pieces of the fat body on a microscope slide using 80%glycerol as the mounting medium and lay a coverslip on top. Mutant one and mutant two are two independent lethal mutants on the X chromosome and isogenized yellow white FRT 19A flies serve as a control here.
GFP-Atg8a patterns were analyzed in the control, mutant one, or mutant two mosaic larval fat bodies, and the mutant clones or control clones were negatively marked by RFP. In the control clones, the patterns of GFP-Atg8a Puncta were similar to those in the surrounding RFP positive cells. In mutant one mutant clones, the GFP-Atg8a Puncta were greatly reduced.
And in mutant two mutant clones, the numbers and size of GFP-Atg8a Puncta were increased. The larvae developmental stage is critical here. The time for development and the duration of starvation need to be modified according to each lab's culture medium recipes.
This procedure can be applied to explore selective autophagy. For example, mitophagy can be monitored in fly fat bodies by attaching a fluorescent protein to a mitochondrial targeting sequence.
