Forberedelse om mitokondriel berigede Brøker for Metabolic Analyse i<em> Drosophila</em
Summary
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.
Abstract
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.
Introduction
Målet med denne procedure er at udvikle berigede mitokondriske fraktioner, der giver nok mitokondrie metabolitter for metabolomik undersøgelser med Drosophila melanogaster. Det er vores erfaring, metabolomics analyse ved hjælp hele cellulære udvindingsmetoder ikke er i stand til at opdage subtile mitokondrie metabolit ændringer i Drosophila. Men mitokondrie fraktionering før metabolomics analyse øger følsomheden for at identificere mitokondrie metabolit skift.
Mitokondrier er cellulære organeller, der er ansvarlige for at levere 90% af den energi, som cellerne har brug for en normal funktion 1. I de senere år er det blevet anerkendt, at mitokondrier spiller en langt mere dynamisk rolle i cellulær og organismal funktion end blot at producere adenosin trifosfat (ATP), og er nu anerkendt som knudepunkter for regulering af metaboliske homeostase 2,3. Mitokondrier er resultatet af en endosymbiotic proces, hvor disdistinkt mikrobielle slægter fusionerede ~ 1,5 milliarder år siden 4. Som mitokondrier udviklet sig til sande organeller, blev generne fra endosymbiont indarbejdet i det spirende nukleare genom. Hos dyr dag, ca. 1.500 mitokondrielle proteiner er nuklear-kodet mens 37 gener forblive i mtDNA, 13 som koder for mitokondrielle proteiner, der er underenheder af de enzymkomplekserne af oxidative fosforylering 5. Koordinering mellem mitokondrier og nukleare rum er nødvendig for at opretholde en ordentlig mitokondriefunktionen.
Hjælp af de metoder, der beskrives her var vi i stand til at opdage mitochondriske metaboliske ændringer i Drosophila, der skyldes manipulation af koordineringen mellem mitochondriale og nukleare genomer. Vi brugte en stamme af Drosophila hvor mtDNA fra sin søster arter D. simulans blev placeret på en enkelt D. melanogaster nukleare baggrund 6. Denne "forstyrret" mitonucleargenotype blev sammenlignet med den "indfødte", eller co-udviklet mitonuclear genotype D. melanogaster bærer samme kernegenomet med dets native D. melanogaster mtDNA. D. melanogaster og D. simulans mtDNAs afviger ~ 100 aminosyrer og> 500 synonyme substitutioner, som påvirker mitonuclear kommunikation 7,8. Vi genererede hele flyve ekstrakter og mitochondriske berigede ekstrakter til at studere metabolit forskydninger som reaktion på farmakologisk stress. Her viser vi, at når du bruger mitochondriske beriget fraktioner vi opdager markante skift i mitokondrie metabolitter mellem indfødte, co-udviklet genotype bærer D. melanogaster mtDNAs og de sprængte genotype bærer D. simulans mtDNA. I modsætning hertil metabolit ændringer mellem disse to genotyper er subtile anvendelse af normale metoder, der udnytter hele flyve ekstrakt. Derfor er denne fremgangsmåde en vej til at forstå, hvordan mtDNAs mægle mitokondrie ændringer ireaktion på forskellige lægemidler.
Protocol
Representative Results
Discussion
De mest kritiske trin i denne protokol er: 1) opdræt nok fluer i rigelige plads. Det er meget vigtigt ikke overpopulate de demografi bure med mere end 150 fluer hver; 2) ændring af fødevarer af burene ofte for at undgå konkurrence mad og ernæringsmæssig stress; og 3) at bevare alle prøver ved 4 ° C for at sikre integritet under isoleringen af mitochondriefraktionen. Det anbefales også til nedkøling isolation buffer, vaskebufferen, og glas-teflon dounce homogenisator før brug. For at reducere cytosolisk …
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 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 liter cage | Web Restaurant Store | 999RD32 | |
| 1 liter 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 |
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