Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Podsumowanie

Mitochondria play central roles in the regulation of metabolism and homeostasis. Subtle changes in mitochondrial metabolism that affect organismal physiology could be difficult to detect in whole organism metabolomics studies. Here we describe an isolation method that enhances the detection of subtle metabolic shifts in Drosophila melanogaster.

Streszczenie

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often compromise the lives of those who suffer from these complex diseases. Detecting mitochondrial metabolic changes is vital to the understanding of mitochondrial disorders and mitochondrial responses to pharmacological agents. Although mitochondrial metabolism is at the core of metabolic regulation, the detection of subtle changes in mitochondrial metabolism may be hindered by the overrepresentation of other cytosolic metabolites obtained using whole organism or whole tissue extractions.

Here we describe an isolation method that detected pronounced mitochondrial metabolic changes in Drosophila that were distinct between whole-fly and mitochondrial enriched preparations. To illustrate the sensitivity of this method, we used a set of Drosophila harboring genetically diverse mitochondrial DNAs (mtDNA) and exposed them to the drug rapamycin. Using this method we showed that rapamycin modifies mitochondrial metabolism in a mitochondrial-genotype-dependent manner. However, these changes are much more distinct in metabolomics studies when metabolites were extracted from mitochondrial enriched fractions. In contrast, whole tissue extracts only detected metabolic changes mediated by the drug rapamycin independently of mtDNAs.

Wprowadzenie

The goal of this procedure is to develop enriched mitochondrial fractions that yield enough mitochondrial metabolites for metabolomics studies using Drosophila melanogaster. In our experience, metabolomics analysis using whole cellular extraction methods are unable to detect subtle mitochondrial metabolite changes in Drosophila. However, mitochondrial fractioning prior to metabolomics analysis increases the sensitivity to identify mitochondrial metabolite shifts.

Mitochondria are cellular organelles responsible for providing 90% of the energy that cells need for normal function¹. In recent years it has been recognized that mitochondria play a much more dynamic role in cellular and organismal function than merely producing adenosine triphosphate(ATP), and are now recognized as hubs for the regulation of metabolic homeostasis^2,3. Mitochondria are the result of an endosymbiotic process in which distinct microbial lineages merged ~1.5 billion years ago⁴. As mitochondria evolved into true organelles, genes from the endosymbiont were incorporated in the emerging nuclear genome. In animals today, approximately 1,500 mitochondrial proteins are nuclear-encoded while 37 genes remain in the mtDNA, 13 of which encode mitochondrial proteins that are subunits of the enzyme complexes of oxidative phosphorylation⁵. Coordination between mitochondria and nuclear compartments is needed to maintain proper mitochondrial function.

Using the methods described here we were able to detect mitochondrial metabolic changes in Drosophila that result from manipulation of the coordination between mitochondrial and nuclear genomes. We used a strain of Drosophila in which mtDNA from its sister species D. simulans was placed on a single D. melanogaster nuclear background⁶. This ‘disrupted’ mitonuclear genotype was compared to the ‘native’, or co-evolved mitonuclear genotype of D. melanogaster carrying the same nuclear genome with its native D. melanogaster mtDNA. The D. melanogaster and D. simulans mtDNAs differ by ~100 amino acids and >500 synonymous substitutions that affect mitonuclear communication^7,8. We generated whole fly extracts and mitochondrial enriched extracts to study metabolite shifts in response to pharmacological stress. Here we show that when using mitochondrial enriched fractions we detect pronounced shifts in mitochondrial metabolites between the native, co-evolved genotype carrying the D. melanogaster mtDNAs and the disrupted genotype carrying D. simulans mtDNA. In contrast, the metabolite changes between these two genotypes are subtle using normal methods that utilize whole fly extract. Therefore, this method provided a path to understand how mtDNAs mediate mitochondrial changes in response to different drugs.

Protokół

1. Reagents and Solutions

1. Preparation of fly food and media
   1. Heat fly ingredients 11% sugar, 2% autolyzed yeast, 5.2% cornmeal, agar 0.79% w/v in water in a hotplate set at 90 °C. Stir regularly until a homogenous slightly dense mixture is obtained. Prepare a final volume of 550 ml of fly food for a total of 100 vials with 5 ml fly food in each.
   2. Remove from the heating source, stir occasionally until the food cools down to 80 °C and add 0.2% tegosept-methyl 4-hydroxybenzoate dissolved in 95% ethanol.
   3. Split the food in two equal volumes. Add 1.1 ml of 50 mM rapamycin dissolved in ethanol to one volume and 1.1 ml of ethanol to the other volume. Stir the rapamycin and the ethanol solutions into the food. Be sure that the temperature decreases to 50 °C before adding drugs.
2. Isolation Buffer
   1. Prepare 500 ml of Isolation Buffer [225 mM Mannitol, 75 mM Sucrose, 10 mM 3-(N-morpholino) propanesulfonic acid (MOPS) and 1 mM ethylenediaminetetraacetic acid (EDTA) , 2.5 mg/ml bovine serum albumin (BSA)] in water.
   2. Adjust the pH to 7.2 using sodium hydroxide. The initial pH will be acidic.
   3. Use sterile filter storage bottles with a 0.22 µm pore size to sterilize solution and store it at 4 °C.
3. Wash Buffer
   1. Prepare 500 ml of wash buffer [225 mM Mannitol, 75 mM Sucrose, 10 mM KCl, 10 mM Tris HCl and 5 mM KH₂PO₄] in water.
   2. Adjust the pH to 7.2 using sodium hydroxide. The initial pH will be acidic.
   3. Use sterile filter storage bottles with a 0.22 µm pore size to sterilize solution and store it at 4 °C.

2. Rearing of the Drosophila Strains

1. To control larval density during development, place 25 pairs of parents in to a culture bottle and allow them to lay eggs for 48 hr.
2. Remove parents after 48 hr to regulate egg density.
3. Repeat steps 2.1 and 2.2 using the emerging F1 offspring, so two generations of this density control is achieved.
4. To collect adults, anesthetize the flies using carbon dioxide (CO₂) and separate by sex.
   1. Use a single stage regulator to delivered pure CO₂ from a high pressured tank at a continuous flow of 5 psi. To avoid static electricity, use plastic tubing to catalyze the CO₂ into a 500 ml filtering flask with water.
   2. Use a rubber stopper with one hole to seal the flask. Use plastic tubing to connect the lateral aperture of the flask to a CO₂ pad.
   3. Place the flies in the pad for no more than 10-15 min. Allow to recover from anesthesia for 24 hr before placing them in experimental conditions.
5. To generate enough metabolites for mitochondrial enriched fractions, use 300 flies per sample. Here, use females, but gender may differ in other experiments. Transfer 150 flies to a homemade 1 L demography cage. Allow access to 5 ml of fly food in a vial.
6. Transfer the remaining 150 flies to another 1 L cage. Do not overpopulate cages with more than 150 flies.
7. Use six replicate samples per experimental condition. Since whole animal extracts yield more metabolites, to generate 1 sample of whole animal isolates, transfer 50 female flies to one cage. As for the mitochondrial isolates, use six replicate samples per experimental condition.
8. Place cages at 25 °C with cycle of 12 hr light and 12 hr dark.
9. Provide fresh food every 2 or 3 days in a vial with 5 ml of food to maintain food quality.
10. Maintain flies on food for 10 days. This step is needed for the rapamycin treatment⁷. Other treatments or conditions will differ.

3. Isolation of Mitochondrial Fractions

1. Dump flies from one cage (150 flies) into a glass-teflon dounce homogenizer filled with 1 ml of chilled isolation buffer.
2. Homogenize by moving the pestle up and down for 15 strokes. Keep the mortar on ice to keep mitochondria intact.
3. Transfer homogenate to a 1.5 ml tube and centrifuge at 300 x g for 5 min at 4 °C.
4. Take the supernatant and centrifuge at 6,000 x g for 10 min at 4 °C to enrich for mitochondria.
5. As the supernatant contains the cytosolic fraction, discard the supernatant or use it for alternative experiments. Resuspend the pellet in 300 µl of wash buffer.
6. Repeat steps 3.1 - 3.5 for the second cage. Combine the resuspended pellets of both cages in a cryogenic microcentrifuge tube.
7. Centrifuge at 6,000 x g for 10 min at 4 °C.
8. Discard the supernatant and flash-freeze the pellet in liquid nitrogen.
9. Store the mitochondrial enriched fractions at -80 °C or lower temperature.
   1. Alternatively, for whole animal preparation, place the adults in a cryogenic microcentrifuge tube. Flash-freeze the vial in liquid nitrogen and store adults at -80 °C or lower temperature.

Wyniki

Using the protocol explained above, we performed metabolomic analysis on mitochondrial enriched fractions and whole animal extracts to test the effect of the drug rapamycin on divergent mtDNAs ⁷. We delivered 200 µM of rapamycin by dissolving the drug in the fly food. Flies were exposed to rapamycin for 10 days. Metabolites from whole fly extracts and from mitochondrial extracts were obtained by using gas chromatography mass spectrometry (GC/MS) and liquid chromatogra...

Dyskusje

The most critical steps in this protocol are: 1) rearing enough flies in abundant space. It is very important to not overpopulate the demography cages with more than 150 flies each; 2) changing the food of the cages frequently to avoid food competition and nutritional stress; and 3) maintaining all samples at 4 °C to ensure integrity during the isolation of the mitochondrial fraction. It is also recommended to chill the isolation buffer, the wash buffer, and the glass-teflon dounce homogenizer before use. To reduce ...

Materiały

Name	Company	Catalog Number	Comments
0.2% tegosept-methyl 4-hydroxybenzoate	VWR	AAA14289
Ethanol	Sigma-Aldrich	792799
Mannitol	Sigma-Aldrich	M4125
Sucrose	Sigma-Aldrich	S9378
3-(N-morpholino) propanesulfonic acid (MOPS)	Sigma-Aldrich	M1254
Ethylenediaminetetraacetic acid (EDTA)	Sigma-Aldrich	38057
Bovine serum albumin (BSA)	Sigma-Aldrich	5470
KCl	Sigma-Aldrich	P9333
Tris HCl	Sigma-Aldrich	RES3098T-B7
KH2PO4	Sigma-Aldrich	1551139
CO2 pads to anesthetize flies	Tritech Research	MINJ-DROS-FP
1 L cage	Web Restaurant Store	999RD32
1 L cage lid	Web Restaurant Store	999LRD
a glass-teflon dounce homogenizer	Fisher Scientific	NC9661231
Sodium hydroxide	Sigma-Aldrich	S8045
rapamycin	LC Laboratories	R-5000
anti-porin	MitoSciences	MSA03
anti-alpha tubulin	Developmental Studies Hybridoma Bank	12G10
Pierce BCA Protein Assay Kit	Thermo Scientific	23225
CO2 pad	Tritech Research, Inc	MINJ-DROS-FP
filter flask	enasco	SB08184M
rubber stopper	enasco	S08512M