Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

The overall goal of the following experiment is to obtain enriched mitochondrial fractions that yield enough mitochondrial metabolites for metabolomic studies using drosophila melanogaster. This is achieved by first raising a sufficient amount of flies to generate enough metabolites for mitochondrial enriched fractions. As a second step, flies are homogenized into a glass Teflon downs homogenizer, which breaks down the cellular membrane without damaging mitochondrial integrity.

Next homogenates are subjected to differential centrifugation in order to generate enriched mitochondrial samples. The results show that pronounced mitochondrial metabolic changes were detected using this protocol, which were undetected using wholely based metabolomics analysis on mitochondrial enriched fractions and whole animal extracts. The main advantage of this procedure versus a whole fly metabolomic analysis is that isolation of mitochondria prior to metabolomic analysis allows for a increased sensitivity and detection of mitochondrial metabolite shift.

To begin the experiment, heat the fly food ingredients on a hot plate at 90 degrees Celsius. Stir the mixture regularly until it is homogenous and slightly dense. Remove the food from the heating source and stir it occasionally when the food cools down to 80 degrees Celsius at 0.2%TE decept methyl four hydroxy benzoate dissolved in 95%Ethanol next, split the food into two equal volumes.

Ensure that the food temperature has decreased to 50 degrees Celsius and stir 1.1 milliliter of 50 millimolar rapamycin dissolved in ethanol into one volume and 1.1 milliliter of untreated ethanol into the second volume. To collect adults use a single stage regulator to deliver pure carbon dioxide from a high pressure tank. At a continuous flow of five PS, I avoid static electricity by using a plastic tube to pipe the carbon dioxide into a 500 milliliter filtering flask with tubing to bubble the carbon dioxide through water.

To do that, use a rubber stopper that has one hole to seal the flask. Connect plastic tubing to the lateral aperture of the flask to a carbon dioxide pad to control larval density during development. Place 25 pairs of parents into a culture bottle and allow them to lay eggs for 48 hours.

Then remove the parents to regulate egg density and repeat these steps using the emerging F1 offspring to achieve two generations of this density control. After two generations under density controlled conditions, separate the flies by sex, placing the flies on the pad for no more than 10 to 15 minutes. Allow the flies to recover from anesthesia for 24 hours before placing them in experimental conditions.

Designate 300 flies per sample to generate enough metabolites for mitochondrial enriched fractions. Transfer 150 flies to a homemade one liter cage. Then transfer the remaining 150 flies to a separate one liter cage and do not overpopulate cages with more than 150 flies.

Use six replicate samples per experimental condition and place the cages at 25 degrees Celsius in a 12 hour light dark cycle to finish drosophila rearing. Provide the flies with fresh food every two or three days in a vial with five milliliters of food to maintain food quality for a total of 10 days. Start isolation by dumping flies from one cage into a glass Teflon down homogenizer filled with one milliliter of chilled isolation buffer.

Next, place some mortar on ice to keep the mitochondria intact and homogenize the flies with 15 strokes. Taking care not to twist the pestle, transfer the homogenate to a 1.5 milliliter tube and centrifuge the tube at 300 times G for five minutes at four degrees Celsius. Transfer the supernatant to a different tube and centrifuge the supernatant at 6, 000 times G for 10 minutes at four degrees Celsius to enrich the mitochondria.

After centrifugation, discard the supernatant containing the cytosolic fraction. Reese bend the pellet in 300 microliters of wash buffer by pipetting up and down. Gently repeat the previous steps for the second cage gyro.

Then combine the resuspended pellets of both cages in a cryogenic micro centrifuge tube and centrifuge the combined pellets at 6, 000 times G for 10 minutes at four degrees Celsius. Discard the supernatant and flash freeze the pellet in liquid nitrogen. Finally store the mitochondrial enriched fractions at minus 80 degrees Celsius.

This western blot displays an enrichment of the mitochondrial membrane transport protein poin, which serves as an indicator of the effective separation of mitochondria in mitochondrial fractions. Poin protein is undetectable in cytosolic fractions while tubulin protein, A cytosolic microtubule protein is not detected in the mitochondrial fraction demonstrating the minimal contamination of cytosolic proteins. Principle components analyses or PCA were generated using the values generated from the mass spec analysis that were normalized to Bradford protein concentration log transformed and amputated with minimal observed values for each compound.

PCA plots were generated using complete metabolite. Profiles from native and disrupted strains of flies are bring different mitochondrial DNA metabolites obtained with a standard whole fly extract can be compared against A PC.A plot from metabolites in mitochondrial enriched extracts the metabolite profiles from wholely separate along PC axis two and reflect the main effects of rapamycin treatment. There are only subtle differences between the alternative M-T-D-N-A genotypes.

In contrast, the metabolite profiles from mitochondrial enriched extracts show a separation between the native M mt DNA genotype on normal food and the other three samples, the native MTD NA on rapamycin, and the alternative MTD NA on either normal or rapamycin food. This implies that the effect of the alternative MTD NA shifts metabolite profiles in a manner analogous to the effects of rapamycin treatment. While doing this protocol, it's important, it is recommended to actually do all the distractions the same day, to decrease variability between replicate.

I will recommend to have a plan prepared ahead of time and labeling of the vials, and keep track of the waiting records between replicates to improve the quality of our sample preparation.