Name: preparation of mitochondrial enriched fractions for metabolic analysis in drosophila
Uploaded: 2015-09-30T14:00:00.000+00:00
Duration: 8 min 22 s
Description: Watch this Scientific Journal Video about Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila at JoVE.com

This work was supported by Adelphi University faculty development grant and grant R15GM113156 from NIGMS awarded to EVC, grant R01GM067862 from NIGMS and grant R01AG027849 from NIA awarded to DMR.

1. Reagents and Solutions
<ol>
	<li>Preparation of fly food and media
	<ol>
		<li>Heat fly ingredients 11% sugar, 2% autolyzed yeast, 5.2% cornmeal, agar 0.79% w/v in water in a hotplate set at 90 &#176;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.</li>
		<li>Remove from the heating source, stir occasionally until the food cools down to 80 &#176;C and add 0.2% tegosept-methyl 4-hydroxybenzoate dissolved in 95% ethanol.</li>
		<li>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 &#176;C before adding drugs.</li>
	</ol>
	</li>
	<li>Isolation Buffer
	<ol>
		<li>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.</li>
		<li>Adjust the pH to 7.2 using sodium hydroxide. The initial pH will be acidic.</li>
		<li>Use sterile filter storage bottles with a 0.22 &#181;m pore size to sterilize solution and store it at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Wash Buffer
	<ol>
		<li>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.</li>
		<li>Adjust the pH to 7.2 using sodium hydroxide. The initial pH will be acidic.</li>
		<li>Use sterile filter storage bottles with a 0.22 &#181;m pore size to sterilize solution and store it at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Rearing of the Drosophila Strains
<ol>
	<li>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.</li>
	<li>Remove parents after 48 hr to regulate egg density.</li>
	<li>Repeat steps 2.1 and 2.2 using the emerging F1 offspring, so two generations of this density control is achieved.</li>
	<li>To collect adults, anesthetize the flies using carbon dioxide (CO2) and separate by sex.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Transfer the remaining 150 flies to another 1 L cage. Do not overpopulate cages with more than 150 flies.</li>
	<li>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.</li>
	<li>Place cages at 25 &#176;C with cycle of 12 hr light and 12 hr dark.</li>
	<li>Provide fresh food every 2 or 3 days in a vial with 5 ml of food to maintain food quality.</li>
	<li>Maintain flies on food for 10 days. This step is needed for the rapamycin treatment7. Other treatments or conditions will differ.</li>
</ol>
3. Isolation of Mitochondrial Fractions
<ol>
	<li>Dump flies from one cage (150 flies) into a glass-teflon dounce homogenizer filled with 1 ml of chilled isolation buffer.</li>
	<li>Homogenize by moving the pestle up and down for 15 strokes. Keep the mortar on ice to keep mitochondria intact.</li>
	<li>Transfer homogenate to a 1.5 ml tube and centrifuge at 300 x g for 5 min at 4 &#176;C.</li>
	<li>Take the supernatant and centrifuge at 6,000 x g for 10 min at 4 &#176;C to enrich for mitochondria.</li>
	<li>As the supernatant contains the cytosolic fraction, discard the supernatant or use it for alternative experiments. Resuspend the pellet in 300 &#181;l of wash buffer.</li>
	<li>Repeat steps 3.1 - 3.5 for the second cage. Combine the resuspended pellets of both cages in a cryogenic microcentrifuge tube.</li>
	<li>Centrifuge at 6,000 x g for 10 min at 4 &#176;C.</li>
	<li>Discard the supernatant and flash-freeze the pellet in liquid nitrogen.</li>
	<li>Store the mitochondrial enriched fractions at -80 &#176;C or lower temperature.
	<ol>
		<li>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 &#176;C or lower temperature.</li>
	</ol>
	</li>
</ol>

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	<li>Scheffler, I. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mitochondria (Scheffler, Mitochondria). , 484 (2007).</li><li>Guarente, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria--a+nexus+for+aging,+calorie+restriction,+and+sirtuins.">Mitochondria--a nexus for aging, calorie restriction, and sirtuins.</a> Cell. 132 (2), 171-176 (2008).</li><li>Raimundo, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+pathology:+stress+signals+from+the+energy+factory.">Mitochondrial pathology: stress signals from the energy factory.</a> Trends in molecular medicine. 20 (5), 282-292 (2014).</li><li>Embley, T. M., Martin, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eukaryotic+evolution,+changes+and+challenges.">Eukaryotic evolution, changes and challenges.</a> Nature. 440 (7084), 623-630 (2006).</li><li>Lane, N., , <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Power, Sex, Suicide: Mitochondria and the Meaning of Life. 368, (2006).</li><li>Montooth, K. L., Meiklejohn, C. D., Abt, D. N., Rand, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial-nuclear+epistasis+affects+fitness+within+species+but+does+not+contribute+to+fixed+incompatibilities+between+species+of+Drosophila.">Mitochondrial-nuclear epistasis affects fitness within species but does not contribute to fixed incompatibilities between species of Drosophila.</a> Evolution; international journal of organic evolution. 64 (12), 3364-3379 (2010).</li><li>Villa-Cuesta, E., Holmbeck, M. A., Rand, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapamycin+increases+mitochondrial+efficiency+by+mtDNA-dependent+reprogramming+of+mitochondrial+metabolism+in+Drosophila.">Rapamycin increases mitochondrial efficiency by mtDNA-dependent reprogramming of mitochondrial metabolism in Drosophila.</a> Journal of cell science. 127 (Pt 10), 2282-2290 (2014).</li><li>Zhu, C. -. T., Ingelmo, P., Rand, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G&#215;G&#215;E+for+Lifespan+in+Drosophila:+Mitochondrial,+Nuclear,+and+Dietary+Interactions+that+Modify+Longevity.">G&#215;G&#215;E for Lifespan in Drosophila: Mitochondrial, Nuclear, and Dietary Interactions that Modify Longevity.</a> PLoS genetics. 10 (5), e1004354 (2014).</li><li>Madala, N. E., Piater, L. A., Steenkamp, P. A., Dubery, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multivariate+statistical+models+of+metabolomic+data+reveals+different+metabolite+distribution+patterns+in+isonitrosoacetophenone-elicited+Nicotiana+tabacum+and+Sorghum+bicolor+cells.">Multivariate statistical models of metabolomic data reveals different metabolite distribution patterns in isonitrosoacetophenone-elicited Nicotiana tabacum and Sorghum bicolor cells.</a> SpringerPlus. 3, 254 (2014).</li><li>Hogeboom, G. H., Schneider, W. C., Pallade, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochemical+Studies+Of+Mammalian+Tissues+I+.+Isolation+Of+Intact+Mitochondria+From+Rat+Liver+Some+Biochemical+Properties+Of+Mitochondria+And+Submicroscopic+Particulate+Material.">Cytochemical Studies Of Mammalian Tissues I . Isolation Of Intact Mitochondria From Rat Liver Some Biochemical Properties Of Mitochondria And Submicroscopic Particulate Material.</a> Journal of Biological Chemistry. 172, 619-635 (1948).</li><li>Frezza, C., Cipolat, S., Scorrano, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle+isolation:+functional+mitochondria+from+mouse+liver,+muscle+and+cultured+fibroblasts.">Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts.</a> Nature protocols. 2 (2), 287-295 (2007).</li><li>Corcelli, A., Saponetti, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondria+isolated+in+nearly+isotonic+KCl+buffer:+focus+on+cardiolipin+and+organelle+morphology.">Mitochondria isolated in nearly isotonic KCl buffer: focus on cardiolipin and organelle morphology.</a> Biochimica et biophysica acta. 1798 (3), 681-687 (2010).</li><li>Roede, J. R., Park, Y., Li, S., Strobel, F. H., Jones, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detailed+mitochondrial+phenotyping+by+high+resolution+metabolomics.">Detailed mitochondrial phenotyping by high resolution metabolomics.</a> PloS one. 7 (3), e33020 (2012).</li></ol>

The authors have nothing to disclose.

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 &#176;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 cytosolic contamination of the enriched mitochondrial factions, the mitochondrial pellet from step 3.5 could be washed with wash buffer.
The extraction of metabolites from enriched mitochondrial fractions requires a high number of replicate samples (6 replicates/experimental condition). In total, 24 cages are used for Figures 1 and 2. We recommend performing the extraction of all the samples the same day to decrease experimental variability. Although feasible, this protocol entails careful planning and preparation of materials needed ahead of time. Labeling of the tubes ahead of time, and designing a plan to ensured consistency of waiting times between samples is needed to preserve sample quality and to decrease variation between replicates.
The buffers normally used to extract mitochondria contain sugars10,11; hence, the analysis of particular sugar metabolites may be affected by the extraction method. Although we have not used them in our analysis, KCl extraction buffers may be an alternative to mitochondrial isolation buffers that contain sugars12.
Due to the homeostatic nature of metabolic networks and a complex system of cellular signals that mediated this response, changes in mitochondrial metabolism could be detected by shifts in whole cell metabolite pools. In the case of mitochondrial regulation of such systems, subtle metabolite shifts may be hindered unless enough metabolites are extracted from mitochondrial enriched fractions. In model systems where plenty of mitochondria can be isolated from tissue, high resolution metabolomics has successfully detected changes in mitochondrial phenotypes13. However, in Drosophila melanogaster, harboring enough quality metabolites for a good composition of a sample may present a challenge. Using this protocol we obtained high quantity, quality and variety of metabolites to demonstrate that different extraction protocols reveal distinct metabolite shifts for whole-fly vs. mitochondrial fractions. Although the Western blot of mitochondrial and cytosolic proteins suggests little contamination of proteins in the two fractions, to confirm detailed quantitative measurements of mitochondrial metabolites, additional steps to limit contamination of cytosolic metabolites in the mitochondrial fraction could be applied. Measurement of metabolites before freezing the sample and comparison of these metabolites to those obtained after the freeze/thaw step may provide a more accurate quantification of mitochondrial specific metabolites. However, in this model where we manipulate the mitochondrial genome, the procedures we have used to prepare samples are particularly informative and may point to novel pathways of metabolic reprogramming mediated through mitochondria.

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 homeostasis2,3. 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 &#8216;disrupted&#8217; mitonuclear genotype was compared to the &#8216;native&#8217;, 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 &#62;500 synonymous substitutions that affect mitonuclear communication7,8. 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.

<table><tbody><tr><td>0.2% tegosept-methyl 4-hydroxybenzoate</td><td>VWR</td><td>AAA14289</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>792799</td><td></td></tr><tr><td>Mannitol</td><td>Sigma-Aldrich</td><td>M4125</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S9378</td><td></td></tr><tr><td>3-(&lt;em&gt;N&lt;/em&gt;-morpholino) propanesulfonic acid (MOPS)</td><td>Sigma-Aldrich</td><td>M1254&amp;nbsp;</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich</td><td>38057&amp;nbsp;</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma-Aldrich</td><td>5470</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P9333</td><td></td></tr><tr><td>Tris HCl</td><td>Sigma-Aldrich</td><td>RES3098T-B7</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>1551139</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; pads to anesthetize flies</td><td>Tritech Research</td><td>MINJ-DROS-FP</td><td></td></tr><tr><td>1 L cage</td><td>Web Restaurant Store</td><td>999RD32</td><td></td></tr><tr><td>1 L cage lid</td><td>Web Restaurant Store</td><td>999LRD</td><td></td></tr><tr><td>a glass-teflon dounce homogenizer&amp;nbsp;</td><td>Fisher Scientific</td><td>NC9661231</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>S8045</td><td></td></tr><tr><td>rapamycin&amp;nbsp;</td><td>LC Laboratories&amp;nbsp;</td><td>R-5000</td><td></td></tr><tr><td>anti-porin</td><td>MitoSciences</td><td>MSA03</td><td></td></tr><tr><td>anti-alpha tubulin</td><td>Developmental Studies Hybridoma Bank</td><td>12G10</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit&amp;nbsp;</td><td>Thermo Scientific&amp;nbsp;</td><td>23225</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; pad</td><td>Tritech Research, Inc</td><td>MINJ-DROS-FP</td><td></td></tr><tr><td>filter flask</td><td>enasco</td><td>SB08184M</td><td></td></tr><tr><td>rubber stopper</td><td>enasco</td><td>S08512M</td><td></td></tr></tbody></table>

preparation of mitochondrial enriched fractions for metabolic analysis in drosophila

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.

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 &#181;M 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 chromatography-tandem mass spectrometry&#160;(LC/MS/MS) using standard solvent extraction methods.
Figure 1 shows an enrichment of the membrane transport protein porin as indicator of mitochondrial enrichment in the mitochondrial fractions. Porin protein was undetectable in the cytosolic fractions. Conversely, tubulin protein was not detected in the mitochondrial fraction, suggesting little contamination of cytosolic proteins. Figure 2 and Figure 3 show principle components analysis (PCA) of the complete metabolite profiles from the &#8216;native&#8217; D. melanogaster strain (OreR) and the &#8216;disrupted&#8217; strain of flies harboring mtDNA from D. simulans and nuclear DNA from D. melanogaster strain OreR (sm21). Both strains were exposed to the drug rapamycin. The data are presented as a bi-plot of principle component axes 1 and 2 to visualize the effect of treatment and mtDNAs7,9. Metabolites obtained using a standard whole fly extract are shown in Figure 2A. Figure 2B shows a similar PCA plot for metabolites in the mitochondrial enriched extracts. For whole fly extracts (Figure 2A), rapamycin treatment sifts the samples to significantly lower values of PC2. Yet, changes in metabolites due to mtDNA genotype are subtle, since OreR and sm21 mtDNAs exposed to rapamycin or control vehicle are closely adjacent or overlap in PCA space. However, when using enriched mitochondrial extracts, mtDNAs show distinct rapamycin effects on mitochondrial metabolite profiles. In particular, rapamycin has a strong effect on the metabolite profiles of the OreR strain, shown by the displacement along the PC1 axis. But, on the &#8216;disrupted&#8217; sm21 mtDNA genotype, the metabolite profile for the control-food condition is displaced from that of the native OreR strain (red and black polygons, respectively, in Figure 2B). As a result, the rapamycin treatment has noticeably less of an effect on the shift of the metabolite landscapes in the disrupted mtDNA genotype, sm21 (compare red and blue polygons in Figure 2B).
Figure 3 shows PCA plots of metabolites involved in energy homeostasis (a subset of all metabolites, limited to cofactors, vitamins and Krebs cycle intermediates), often located inside of the mitochondria. Here again, metabolite shifts from enriched mitochondrial fractions show a different behavior than those from whole animal extracts.
These results illustrate how metabolic information obtained by mitochondrial enriched extract and whole animal extracts differ but complement each other, with the mitochondrial enriched extract being more sensitive to metabolic shifts in the mitochondria.
<img alt="Figure 1" src="/files/ftp_upload/53149/53149fig1.jpg" /> 
Figure 1. Western blot analysis showing effective separation of mitochondria in mitochondrial fractions. Antibodies used were against porin (a mitochondrial outer membrane protein) and tubulin (a cytosolic protein). Protein abundance of mitochondrial and cytosolic fractions were quantified using bicinchoninic acid (BCA) assay. 30 &#181;g of total protein were loaded in each lane. Two independent extractions were performed.
<img alt="Figure 2" src="/files/ftp_upload/53149/53149fig2.jpg" /> 
Figure 2. Principle component analysis (PCA) of metabolites obtained using whole animal samples vs. mitochondrial enriched extracts on flies carrying OreR and sm21 mitochondrial haplotypes. (A) PCA of 210 metabolites identified in whole animal samples. (B) PCA of 230 metabolites detected on the mitochondrial extracts. Polygons surrounding points are intended to aid the visualization of the six replicate samples for each treatment. <a href="https://www.jove.com/files/ftp_upload/53149/53149fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53149/53149fig3.jpg" /> 
Figure 3. Principal component analysis of metabolites involved in energy homeostasis on flies carrying OreR and sm21 mitochondrial haplotypes. (A) PCA of 12 energy metabolites from whole fly extracts and 13 energy metabolites from mitochondrial extracts. (B) Polygons surrounding points are intended to aid the visualization of the six replicate samples for each treatment. <a href="https://www.jove.com/files/ftp_upload/53149/53149fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila at JoVE.com

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

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.

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often ...

preparation-mitochondrial-enriched-fractions-for-metabolic-analysis

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

9.5K Views.  Adelphi University. 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. 

Video: Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human organs, possess highly conserved metabolic pathways and have a relatively short lifespan that allows the study of different fundamental mechanisms in a short period of time. It is, however, surprising that one of the mechanisms essential for cellular metabolism, the mitochondrial respiration, has not been thoroughly investigated in this model. It is likely because the measure of the mitochondrial respiration in Drosophila usually requires a very large number of individuals and the results obtained are not highly reproducible. Here, a method allowing the precise measurement of mitochondrial oxygen consumption using minimal amounts of tissue from Drosophila is described. In this method, the thoraxes are dissected and permeabilized both mechanically with sharp forceps and chemically with saponin, allowing different compounds to cross the cell membrane and modulate the mitochondrial respiration. After permeabilization, a protocol is performed to evaluate the capacity of the different complexes of the electron transport system (ETS) to oxidize different substrates, as well as their response to an uncoupler and to several inhibitors. This method presents many advantages compared to methods using mitochondrial isolations, as it is more physiologically relevant because the mitochondria are still interacting with the other cellular components and the mitochondrial morphology is conserved. Moreover, sample preparations are faster, and the results obtained are highly reproducible. By combining the advantages of Drosophila as a model for the study of metabolism with the evaluation of mitochondrial respiration, important new insights can be unveiled, especially when the flies are experiencing different environmental or pathophysiological conditions.

In this paper, a method to measure oxygen consumption using high-resolution respirometry in permeabilized thoraxes of Drosophila is described. This technique requires a minimal amount of tissue compared to the classic mitochondrial isolation technique and the results obtained are more physiologically relevant.

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human ...

Biochemistry

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of physiological processes. Mitochondrial dysfunction is a primary cause of a number of metabolic and neurodegenerative diseases. Intact mitochondria are critical for their proper functioning. The enzyme citrate synthase is localized in the mitochondrial matrix and thus can be used as a quantitative enzyme marker of intact mitochondrial mass. Given that many molecules and pathways that have important functions in mitochondria are highly conserved between humans and Drosophila, and that an array of powerful genetic tools are available in Drosophila, Drosophila serves as a good model system for studying mitochondrial function. Here, we present a protocol for fast and simple measurement of citrate synthase activity in tissue homogenate from adult flies without isolating mitochondria. This protocol is also suitable for measuring citrate synthase activity in larvae, cultured cells, and mammalian tissues.

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

We present a protocol for a colorimetric assay of citrate synthase activity for quantification of intact mitochondrial mass in Drosophila tissue homogenates.

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of ...

Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry (GC-MS)-based Metabolomics

Recent advances in the field of metabolomics have established the fruit fly Drosophila melanogaster as a powerful genetic model for studying animal metabolism. By combining the vast array of Drosophila genetic tools with the ability to survey large swaths of intermediary metabolism, a metabolomics approach can reveal complex interactions between diet, genotype, life-history events, and environmental cues. In addition, metabolomics studies can discover novel enzymatic mechanisms and uncover previously unknown connections between seemingly disparate metabolic pathways. In order to facilitate more widespread use of this technology among the Drosophila community, here we provide a detailed protocol that describes how to prepare Drosophila larval samples for gas chromatography-mass spectrometry (GC-MS)-based metabolomic analysis. Our protocol includes descriptions of larval sample collection, metabolite extraction, chemical derivatization, and GC-MS analysis. Successful completion of this protocol will allow users to measure the relative abundance of small polar metabolites, including amino acids, sugars, and organic acids involved in glycolysis and the TCA cycles.

Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry GC-MS-based Metabolomics

This protocol describes how to prepare Drosophila larvae for GC-MS-based metabolomic analysis.

Recent advances in the field of metabolomics have established the fruit fly Drosophila melanogaster as a powerful genetic model for studying animal ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Biology

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

The usefulness of Drosophila as a model organism for the study of human diseases, behaviors and basic biology is unquestionable. Although practical, Drosophila research lacks popularity in developing countries, possibly due to the misinformed idea that establishing a lab and performing relevant experiments with such tiny insects is difficult and requires expensive, specialized apparatuses. Here, we describe how to build an affordable flylab to quantitatively analyze a myriad of behavioral parameters in D. melanogaster, by 3D-printing many of the necessary pieces of equipment. We provide protocols to build in-house vial racks, courtship arenas, apparatuses for locomotor assays, etc., to be used for general fly maintenance and to perform behavioral experiments using adult flies and larvae. We also provide protocols on how to use more sophisticated systems, such as a high resolution oxygraph, to measure mitochondrial oxygen consumption in larval samples, and show its association with behavioral changes in the larvae upon the xenotopic expression of the mitochondrial alternative oxidase (AOX). AOX increases larval activity and mitochondrial leak respiration, and accelerates development at low temperatures, which is consistent with a thermogenic role for the enzyme. We hope these protocols will inspire researchers, especially from developing countries, to use Drosophila to easily combine behavior and mitochondrial metabolism data, which may lead to information on genes and/or environmental conditions that may also regulate human physiology and disease states.

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

We provide protocols for anyone with a "maker culture" mind to start building a flylab for quantitative analysis of a myriad of behavioral parameters in Drosophila melanogaster, by 3D-printing many of the necessary pieces of equipment. We also describe a high resolution respirometry protocol using larvae to combine behavioral and mitochondrial metabolism data.

The usefulness of Drosophila as a model organism for the study of human diseases, behaviors and basic biology is unquestionable. Although practical, ...

Behavior

Analyzing Mitochondrial Physiology and Bioenergetics in Fruit Flies

Analyzing Mitochondrial Function in a Drosophila melanogaster PINK1B9-Null Mutant Using High-resolution Respirometry

Neurodegenerative diseases, including Parkinson&#39;s Disease (PD), and cellular disturbances such as cancer are some of the disorders that disrupt energy metabolism with impairment of mitochondrial functions. Mitochondria are organelles that control both energy metabolism and cellular processes involved in cell survival and death. For this reason, approaches to evaluate mitochondrial function can offer important insights into cellular conditions in pathological and physiological processes. In this regard, high-resolution respirometry (HRR) protocols allow evaluation of the whole mitochondrial respiratory chain function or the activity of specific mitochondrial complexes. Furthermore, studying mitochondrial physiology and bioenergetics requires genetically and experimentally tractable models such as Drosophila melanogaster.
This model presents several advantages, such as its similarity to human physiology, its rapid life cycle, easy maintenance, cost-effectiveness, high throughput capabilities, and a minimized number of ethical concerns. These attributes collectively establish it as an invaluable tool for dissecting complex cellular processes. The present work explains how to analyze mitochondrial function using the Drosophila melanogaster PINK1B9-null mutant. The pink1 gene is responsible for encoding PTEN-induced putative kinase 1, through a process recognized as mitophagy, which is crucial for the removal of dysfunctional mitochondria from the mitochondrial network. Mutations in this gene have been associated with an autosomal recessive early-onset familial form of PD. This model can be used to study mitochondrial dysfunction involved in the pathophysiology of PD.

Author Spotlight: Exploring Mitochondrial Function and Chemical Toxicity Using Drosophila melanogaster 

Here we present a high-resolution respirometry protocol to analyze bioenergetics in PINK1B9-null mutant fruit flies. The method uses the Substrate-Uncoupler-Inhibitor-Titration (SUIT) protocol.

Neurodegenerative diseases, including Parkinson&#39;s Disease (PD), and cellular disturbances such as cancer are some of the disorders that disrupt energy ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

Neuroscience

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Fluorescence microscopy is an indispensable tool for the direct assessment of mitochondrial structure and function in live cells and for studying the mitochondrial structure-function relationship, which is primarily modulated by the molecules governing fission and fusion events between mitochondria. This paper describes and demonstrates specific methods for studying mitochondrial structure and function in live as well as in fixed tissue in the model organism Drosophila melanogaster. The tissue of choice here is the Drosophila ovary, which can be isolated and made amenable for ex vivo live confocal microscopy. Furthermore, the paper describes how to genetically manipulate the mitochondrial fission protein, Drp1, in Drosophila ovaries to study the involvement of Drp1-driven mitochondrial fission in modulating the mitochondrial structure-function relationship. The broad use of such methods is demonstrated in already-published as well as in novel data. The described methods can be further extended towards understanding the direct impact of nutrients and/or growth factors on the mitochondrial properties ex vivo. Given that mitochondrial dysregulation underlies the etiology of various diseases, the described innovative methods developed in a genetically tractable model organism, Drosophila, are anticipated to contribute significantly to the understanding of the mechanistic details of the mitochondrial structure-function relationship and to the development of mitochondria-directed therapeutic strategies.

Studying Mitochondrial Structure and Function in Drosophila Ovaries

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial functionality. Specific methods for studying mitochondrial structure and function in live and fixed Drosophila ovaries are described and demonstrated in this paper.

Analysis of the mitochondrial structure-function relationship is required for a thorough understanding of the regulatory mechanisms of mitochondrial ...

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Summary

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Chapters in this video

Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry (GC-MS)-based Metabolomics

Purification of Mitochondria from Yeast Cells

An Affordable and Efficient "Homemade" Platform for Drosophila Behavioral Studies, and an Accompanying Protocol for Larval Mitochondrial Respirometry

Analyzing Mitochondrial Function in a Drosophila melanogaster PINK1^B9-Null Mutant Using High-resolution Respirometry

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

Purification of Transcripts and Metabolites from Drosophila Heads

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Studying Mitochondrial Structure and Function in Drosophila Ovaries