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. Reagenzien und Lösungen <ol><li> Vorbereitung der Fliege Essen und Medien <ol><li> Wärme fly Inhaltsstoffe 11% Zucker, 2% autolysierte Hefe, 5,2% Maismehl, Agar 0,79% w / v in Wasser in einer Heizplatte bei 90 ° C eingestellt. Rühren regelmäßig, bis eine homogene leicht dichte Mischung erhalten wird. Bereiten Sie ein Endvolumen von 550 ml Fliege Nahrung für insgesamt 100 Fläschchen mit 5 ml Essen Fliegen in jedem. </li><li> Von der Wärmequelle zu entfernen, gelegentlich umrühren, bis das Essen kühlt auf 80 ° C abgekühlt und 0,2% tegosept-Methyl-4-hydroxybenzoat in 95% Ethanol gelöst. </li><li> Teilen Sie die Lebensmittel in zwei gleiche Volumina. Hinzuzufügen 1,1 ml 50 mM Rapamycin in Ethanol zu einem Volumen gelöst und 1,1 ml Ethanol zu dem Teil des Films. Rühren Sie die Rapamycin und die Ethanollösungen in das Lebensmittel. Seien Sie sicher, dass die Temperatur fällt auf 50 ° C, bevor Drogen. </li></ol></li><li> Isolierungspuffer <ol><li> Bereiten Sie 500 ml Isolationspuffer [225 mM Mannitol, 75 mM Sucrose, 10 mM 3- (N &lt;/ em&gt; morpholino) propansulfonsäure (MOPS) und 1 mM Ethylendiamintetraessigsäure (EDTA), 2,5 mg / ml Rinderserumalbumin (BSA)] in Wasser. </li><li> Den pH-Wert auf 7,2 unter Verwendung von Natriumhydroxid. Der anfängliche pH-Wert sauer sein. </li><li> Verwenden Sterilfilter Vorratsflaschen mit 0,22 um Porengröße, um Lösung zu sterilisieren, und speichern Sie sie bei 4 ° C. </li></ol></li><li> Wash Buffer <ol><li> Vorbereitung 500 ml Waschpuffer [225 mM Mannitol, 75 mM Saccharose, 10 mM KCl, 10 mM Tris HCl und 5 mM KH 2 PO 4] in Wasser. </li><li> Den pH-Wert auf 7,2 unter Verwendung von Natriumhydroxid. Der anfängliche pH-Wert sauer sein. </li><li> Verwenden Sterilfilter Vorratsflaschen mit 0,22 um Porengröße, um Lösung zu sterilisieren, und speichern Sie sie bei 4 ° C. </li></ol></li></ol> 2. Durchführung der Aufzucht der Drosophila-Stämmen <ol><li> Um Larvendichte während der Entwicklung zu steuern, legen Sie 25 Paare von Eltern zu einer Kulturflasche und es ihnen ermöglichen, um Eier für 48 Stunden lag. </li> &lt;li&gt; Eltern entfernen nach 48 h auf Eierdichte zu regulieren. </li><li> Wiederholen Sie die Schritte 2.1 und 2.2 unter Verwendung des Schwellen F1-Nachkommen, also zwei Generationen von dieser Dichtesteuerung erreicht wird. </li><li> Zu den Erwachsenen zu sammeln, zu betäuben die Fliegen unter Verwendung von Kohlendioxid (CO 2) und separatem nach Geschlecht. <ol><li> Verwenden Sie eine einzige Stufenregler geliefert reinen CO 2 von einem hohen Druck Tank bei einem kontinuierlichen Fluss von 5 psi. Um statische Elektrizität zu vermeiden, Kunststoffschlauch, um das CO 2 in einen 500 ml Saugflasche mit Wasser zu katalysieren. </li><li> Verwenden Sie einen Gummistopfen mit einem Loch, um den Kolben zu versiegeln. Verwenden Sie Kunststoffschlauch, um die seitliche Öffnung des Kolbens zu einer CO 2-Pad zu verbinden. </li><li> Legen Sie die Fliegen in der Unterlage für nicht mehr als 10-15 Minuten. Lassen Sie sich von der Anästhesie während 24 Stunden zu erholen, bevor Sie sie in experimentellen Bedingungen. </li></ol></li><li> Um genügend Metaboliten für mitochondriale angereicherten Fraktionen zu erzeugen, verwenden Sie 300 Fliegen je Probe. Hier verwenden Weibchen, aber gender kann in anderen Experimenten abweichen. Übertragen Sie 150 Fliegen auf einen hausgemachten 1 L Demographie Käfig. Ermöglichen den Zugang zu 5 ml Fliege Essen in einem Fläschchen. </li><li> Übertragen Sie die verbleibenden 150 Fliegen auf einen anderen 1 L Käfig. Käfige übervölkern nicht mit mehr als 150 Fliegen. </li><li> Verwenden Sie sechs Parallelproben pro Versuchsbedingung. Seit gesamte tierische Extrakte liefern weitere Stoffwechselprodukte, zu erzeugen 1 Probe von Voll Tier isoliert, übertragen 50 weibliche fliegt in einem Käfig. Wie für die mitochondriale Isolate, verwenden sechs Parallelproben pro Versuchsbedingung. </li><li> Ort Käfigen bei 25 ° C mit dem Zyklus von 12 Stunden Licht und 12 Stunden Dunkelheit. </li><li> Für Frischnahrungs alle 2 oder 3 Tage in einem Fläschchen mit 5 ml von Lebensmitteln, um die Lebensmittelqualität aufrecht zu erhalten. </li><li> Pflegen Fliegen auf Nahrung für 10 Tage. Dieser Schritt wird für die Rapamycin-Behandlung 7 notwendig. Andere Behandlungen oder Bedingungen unterscheiden. </li></ol> 3. Isolierung der mitochondrialen Fraktionen <ol><li> Dump fliegt von einem Käfig (150 Linie) in eineGlas-Dounce-Homogenisator mit Teflon mit 1 ml gekühltem Isolationspuffer gefüllt. </li><li> Homogenisieren, indem Sie den Stößel nach oben und unten für 15 Schlägen. Halten Sie den Mörtel auf Eis, um Mitochondrien intakt zu halten. </li><li> Übertragungs Homogenisat in ein 1,5-ml-Röhrchen und zentrifugiert bei 300 g für 5 min bei 4 ° C. </li><li> Nehmen den Überstand und zentrifugieren bei 6.000 × g für 10 min bei 4 ° C für Mitochondrien zu bereichern. </li><li> Da der Überstand enthält die cytosolische Fraktion, den Überstand verwerfen oder verwenden sie für alternative Experimente. Das Pellet in 300 ul Waschpuffer. </li><li> Wiederholen Sie die Schritte 3,1-3,5 für den zweiten Käfig. Kombinieren Sie die resuspendierten Pellets beider Käfige in einer Tieftemperatur-Mikrozentrifugenröhrchen. </li><li> Zentrifuge bei 6000 × g für 10 min bei 4 ° C. </li><li> Überstand verwerfen und Flash-einfrieren das Pellet in flüssigem Stickstoff. </li><li> Bewahren Sie die mitochondriale angereicherten Fraktionen bei -80 ° C oder niedriger Temperatur. <ol><li> Alternativ können für ganze Tiervorgereitung, legen Sie die Erwachsenen in einer Tieftemperatur-Mikrozentrifugenröhrchen. Flash-einfrieren Die Durchstechflasche im flüssigen Stickstoff und speichern Erwachsenen bei -80 ° C oder niedriger Temperatur. </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>

Die Autoren haben nichts zu offenbaren.

Die wichtigsten Schritte in diesem Protokoll sind: 1) die Aufzucht genug Fliegen in reichlich Platz. Es ist sehr wichtig, um die Demographie Käfigen mit mehr als 150 Fliegen je nicht übervölkern; 2) Veränderung der Lebensmittel der Käfige häufig Nahrungskonkurrenz und Ernährungs Stress zu vermeiden; und 3) die Beibehaltung aller Proben bei 4 ° C, um die Integrität während der Isolierung der mitochondrialen Fraktion zu gewährleisten. Es wird auch empfohlen, den Isolationspuffer, der Waschpuffer und das Glas-Do...

Das Ziel dieses Verfahrens ist es, angereicherten mitochondriale Fraktionen, die genug mitochondrialen Metaboliten für Metabolomics-Studien mit Drosophila melanogaster Ausbeute zu entwickeln. Nach unserer Erfahrung Metabolomics-Analyse unter Verwendung von Vollzellextraktionsverfahren sind nicht imstande, subtile mitochondrialen Stoffwechselveränderungen in Drosophila zu erkennen. , Mitochondriale Fraktionierung vor der Metabolomics-Analyse erhöht jedoch die Empfindlichkeit auf der mitochondrialen Metaboliten Verschiebungen zu identifizieren. Mitochondrien sind Organellen zur Bereitstellung 90% der Energie, die Zellen brauchen für die normale Funktion 1 verantwortlich. In den letzten Jahren hat man erkannt, dass Mitochondrien spielen in zellulären und organismischen Funktion eine viel dynamischere Rolle als nur die Herstellung Adenosintriphosphat (ATP) und sind nun als Knotenpunkte für die Regulierung der metabolischen Homöostase 2,3 angesetzt. Mitochondrien sind das Ergebnis eine endosymbiontische Verfahren, bei dem distinct mikrobiellen Linien verschmolzen ~ Vor 1500000000 Jahre 4. Als Mitochondrien in wahre Organellen entwickelt wurden Gene aus der Endosymbionten in der entstehenden Kerngenom integriert. Bei Tieren heute sind ca. 1500 mitochondriale Proteine ​​kerncodierten während 37 Gene in der mtDNA, von denen 13 kodieren mitochondriale Proteine, die Untereinheiten der Enzymkomplexe der oxidativen Phosphorylierung 5 bleibt. Die Koordinierung zwischen den Mitochondrien und Kernfächer benötigt wird, um die ordnungsgemäße Funktion der Mitochondrien zu halten. Mit den hier beschriebenen Methoden konnten wir mitochondriale Stoffwechselveränderungen in Drosophila, die von Manipulation der Abstimmung zwischen mitochondrialer und nuklearer Genome führen zu erkennen. Wir verwendeten einen Stamm von Drosophila, in der mtDNA von ihrer Schwesterarten D. simulans wurde an einem einzigen D. platziert melanogaster Kern Hintergrund 6. Diese &#39;gestört&#39; mitonuclearGenotyp wurde auf die &quot;native&quot;, oder co-entwickelt mitonuclear Genotyp D. Vergleich melanogaster, die dasselbe Kerngenom mit seiner nativen D. melanogaster mtDNA. Die D. melanogaster und D. simulans mtDNAs unterscheiden sich um ~ 100 Aminosäuren und&gt; 500 auch Substitutionen, die mitonuclear Kommunikations 7,8 beeinflussen. Wir erzeugten gesamten Fliegenextrakten und mitochondriale angereicherte Extrakte Metabolit Verschiebungen als Reaktion auf pharmakologische Stress zu studieren. Hier zeigen wir, dass bei der Verwendung der mitochondrialen angereicherten Fraktionen wir erkennen ausgeprägte Veränderungen in der mitochondrialen Metaboliten zwischen dem nativen, co-entwickelt Genotyp tragenden D. melanogaster mtDNAs und die aufgebrochenen Genotyp tragen D. simulans mtDNA. Im Gegensatz dazu sind die Stoffwechselveränderungen zwischen diesen beiden Genotypen subtile Verwendung normaler Verfahren, die gesamte fly Extrakt zu verwenden. Daher, sofern diese Methode einen Weg, um zu verstehen, wie mtDNAs vermitteln mitochondrialen VeränderungenReaktion auf verschiedene Medikamente.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>0.2% tegosept -methyl 4-hydroxybenzoate&#160;</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-(N-morpholino) propanesulfonic acid (MOPS)</td>
			<td>Sigma-Aldrich</td>
			<td>M1254&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>38057&#160;</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&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>RES3098T-B7</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-Aldrich</td>
			<td>1551139</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 pads to anesthetize flies</td>
			<td>Tritech Research</td>
			<td>MINJ-DROS-FP</td>
			<td></td>
		</tr>
		<tr>
			<td>1 liter cage&#160;</td>
			<td>Web Restaurant Store</td>
			<td>999RD32</td>
			<td></td>
		</tr>
		<tr>
			<td>1 liter cage lid&#160;</td>
			<td>Web Restaurant Store</td>
			<td>999LRD</td>
			<td></td>
		</tr>
		<tr>
			<td>a glass-teflon dounce homogenizer&#160;</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&#160;</td>
			<td>LC Laboratories&#160;</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&#8482; BCA Protein Assay Kit&#160;</td>
			<td>Thermo Scientific&#160;</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 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.

Verwendung des Protokolls oben erläutert, metabolomic Analyse führten wir auf die mitochondriale angereicherten Fraktionen und ganzen Tierextrakten, um die Wirkung des Medikaments auf Rapamycin abweichenden mtDNAs 7 testen. Wir lieferten 200 uM von Rapamycin durch Lösen des Arzneistoffs in der Flug Nahrung. Fliegen wurden Rapamycin für 10 Tage ausgesetzt. Stoffwechselprodukte aus Vollfliegenextrakten und von mitochondrialen Extrakte wurden mit Hilfe der Gaschromatogra...

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.

Vorbereitung der Mitochondrial angereicherten Fraktionen für die Stoffwechselanalyse in<em&gt; Drosophila</em

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

Research

JoVE Journal

Developmental Biology

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

9.5K Aufrufe.  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. 

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

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

Analyse von Zebrafisch-Larven Skelettmuskel Integrität mit Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Forschung

Entwicklungsbiologie

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

Analysis of protein-protein interactions (PPIs) has become an indispensable approach to study biological processes and mechanisms, such as cell signaling, organism development, and disease. It is often desirable to obtain PPI information using in vivo material, to gain the most natural and unbiased view of the interaction networks. The fruit fly Drosophila melanogaster is an excellent platform to study PPIs in vivo, and lends itself to straightforward approaches to isolating material for biochemical experiments. In particular, fruit fly embryos represent a convenient type of tissue to study PPIs, due to the ease of collecting animals at this developmental stage and the fact that the majority of proteins are expressed in embryogenesis, thus providing a relevant environment to reveal most PPIs. Here we present a protocol for collection of Drosophila embryos at medium scale (0.5-1 g), which is an ideal amount for a wide range of proteomic applications, including analysis of PPIs by affinity purification-mass spectrometry (AP-MS). We describe our designs for 1 L and 5 L cages for embryo collections that can be easily and inexpensively set up in any laboratory. We also provide a general protocol for embryo collection and protein extraction to generate lysates that can be directly used in downstream applications such as AP-MS. Our goal is to provide an accessible means for all researchers to carry out the analyses of PPIs in vivo.

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

The goal of this protocol is to provide a straightforward and inexpensive approach to collecting Drosophila embryos at medium scale (0.5-1 g) and preparing protein extracts that can be used in downstream proteomic applications, such as affinity purification-mass spectrometry (AP-MS).

Mittelmaßnahme Vorbereitung von<em&gt; Drosophila</em&gt; Embryoextrakte für Proteomversuche

Analysis of protein-protein interactions (PPIs) has become an indispensable approach to study biological processes and mechanisms, such as cell signaling, ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Dissektion, Immunofluorescent Färbung der Pilz Körper und Photorezeptor Neuronen im Gehirn Erwachsener Taufliege melanogaster

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and behavioral evolution. Drosophila olfactory tissues house the olfactory receptor neurons (ORNs) that detect volatile chemical cues in addition to hydro- and thermo-sensory neurons. In this protocol, we describe the dissection of developing peripheral olfactory tissue of the adult Drosophila species. We first describe how to stage and age Drosophila larvae, followed by the dissection of the antennal disc from early pupal stages, followed by the dissection of the antennae from mid-pupal stages and adults. We also show methods where preparations can be utilized in molecular techniques, such as the RNA extraction for qRT-PCR, RNAseq, or immunohistochemistry. These methods can also be applied to other Drosophila species after species-specific pupal development times are determined, and respective stages are calculated for appropriate aging.

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

Here, we present a protocol to stage and dissect developing olfactory tissue from Drosophila species. The dissected tissue can later be used for molecular analyses, such as quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) or RNA sequencing (RNAseq), as well as in vivo analyses such as immunohistochemistry or in situ hybridization.

Vorbereitung, Entwicklung von peripheren olfaktorische Gewebe für Molekulare und immunhistochemische Analysen in Drosophila

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and ...

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.

Vorbereitung von Drosophila Larven Proben für Gaschromatographie-Massenspektrometrie (GC-MS)-basierte Metabolomik

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

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a reliable protocol for the dissection of the delicate Drosophila pupal retina. Our surgical approach utilizes readily-available microdissection tools to open pupae and precisely extract eye-brain complexes. These can be fixed, subjected to immunohistochemistry, and retinas then mounted onto microscope slides and imaged if the goal is to detect cellular or subcellular structures. Alternatively, unfixed retinas can be isolated from brain tissue, lysed in appropriate buffers and utilized for protein gel electrophoresis or mRNA extraction (to assess protein or gene expression, respectively). Significant practice and patience may be required to master the microdissection protocol described, but once mastered, the protocol enables relatively quick isolation of mainly undamaged retinas.

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and RNA-extraction.&#160;

Dissektion der Drosophila Pupal Netzhaut Immunohistochemistry, westlichen Analyse und RNA-Isolation

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits are required to control this behavior. Failure to produce the rhythmic pattern of contractions can be indicative of neurological abnormalities. We previously found that defects in protein O-mannosylation, a posttranslational protein modification, affect the axon morphology of sensory neurons and result in abnormal coordinated muscle contractions in embryos. Here, we present a relatively simple method for recording and analyzing the pattern of peristaltic muscle contractions by live imaging of late stage embryos up to the point of hatching, which we used to characterize the muscle contraction phenotype of protein O-mannosyltransferase mutants. Data obtained from these recordings can be used to analyze muscle contraction waves, including frequency, direction of propagation and relative amplitude of muscle contractions at different body segments. We have also examined body posture and taken advantage of a fluorescent marker expressed specifically in muscles to accurately determine the position of the embryo midline. A similar approach can also be utilized to study various other behaviors during development, such as embryo rolling and hatching.

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Here, we present a method to record embryonic muscle contractions in Drosophila embryos in a non-invasive and detail-oriented manner.

Live Imaging und Analyse von Muskelkontraktionen im Drosophila-Embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the number of novel cell lines is expected to increase. The DGRC aims to familiarize researchers with using Drosophila cell lines as an experimental tool to complement and drive their research agenda. Procedures for working with a variety of Drosophila cell lines with distinct characteristics are provided, including protocols for thawing, culturing, and cryopreserving cell lines. Importantly, this publication demonstrates the best practices required to work with Drosophila cell lines to minimize the risk of contaminations from adventitious microorganisms or from other cell lines. Researchers who become familiar with these procedures will be able to delve into the many applications that use Drosophila cultured cells including biochemistry, cell biology and functional genomics.

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Drosophila cell lines are important reagents for both fundamental and biomedical research. This article provides protocols for thawing, subculturing, and the cryopreservation of commonly used Drosophila cell lines to assist researchers in incorporating the use of these reagents in their research.

Auftauen, Kultivierung und Cryopreserving Drosophila -Zell-Linien

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with development, tissue homeostasis, and disease processes. Drosophila spermatocytes undergoing meiosis are ideal for this analysis because (1) whole Drosophila testes containing spermatocytes are relatively easy to prepare for microscopy, (2) the spermatocytes&#8217; large size makes them well suited for high resolution imaging, and (3) powerful Drosophila genetic tools can be integrated with in vivo analysis. Here, we present a readily accessible protocol for the preparation of whole testes from Drosophila third instar larvae and early pupae. We describe how to identify meiotic spermatocytes in prepared whole testes and how to image them live by time-lapse microscopy. Protocols for fixation and immunostaining whole testes are also provided. The use of larval testes has several advantages over available protocols that use adult testes for spermatocyte analysis. Most importantly, larval testes are smaller and less crowded with cells than adult testes, and this greatly facilitates high resolution imaging of spermatocytes. To demonstrate these advantages and the applications of the protocols, we present results showing the redistribution of the endoplasmic reticulum with respect to spindle microtubules during cell division in a single spermatocyte imaged by time-lapse confocal microscopy. The protocols can be combined with expression of any number of fluorescently tagged proteins or organelle markers, as well as gene mutations and other genetic tools, making this approach especially powerful for analysis of cell division mechanisms in the physiological context of whole tissues and organs.

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

The goal of this protocol is to analyze cell division in intact tissue by live and fixed cell microscopy using Drosophila meiotic spermatocytes. The protocol demonstrates how to isolate whole, intact testes from Drosophila larvae and early pupae, and how to process and mount them for microscopy.

Vorbereitung von Drosophila Larval und Pupal Hoden für die Analyse der Zellteilung in Leben, intaktes Gewebe

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with ...

Use of Bisection to Reduce Mitochondrial DNA in the Bovine Oocyte

Interspecies somatic cell nuclear transfer (iSCNT) may be used to rescue endangered species, but two distinct populations of mitochondrial DNA (mtDNA) exist within the reconstructed embryo: one within the recipient ooplasm and one within the donor somatic cell. This mitochondrial heteroplasmy can lead to developmental issues in the embryo and the fetus. Handmade cloning protocols include oocyte bisection, which can be used to decrease the mtDNA copy number, reducing the degree of mitochondrial heteroplasmy in a reconstructed embryo. Centrifugation of denuded, mature bovine oocytes produced a visible mitochondria-dense fraction at one pole of the oocyte. Oocytes&#39; zonae pellucidae were removed by exposure to a pronase solution. Bisection was performed using a microblade to remove the visible mitochondria fraction. qPCR was used to quantify the mtDNA present in DNA samples extracted from whole oocytes and bisected ooplasts, providing a comparison of mtDNA copy numbers before and after bisection. Copy numbers were calculated using cycle threshold values, a standard curve&#39;s regression line formula, and a ratio that included the respective sizes of mtDNA PCR products and genomic PCR products. One bovine oocyte had an average mtDNA copy number (&#177; standard deviation) of 137,904 &#177; 94,768 (n = 38). One mitochondria-depleted ooplast had an average mtDNA copy number of 8,442 &#177; 13,806 (n = 33). Average mtDNA copies present in a mitochondria-rich ooplast were 79,390 &#177; 58,526 mtDNA copies (n = 28). The differences between these calculated averages indicate that the centrifugation and subsequent bisection can significantly decrease the mtDNA copy numbers present in the mitochondria-depleted ooplast when compared to the original oocyte (P &#60; 0.0001, determined by one-way ANOVA). The reduction in mtDNA should decrease the degree of mitochondrial heteroplasmy in a reconstructed embryo, possibly fostering standard embryonic and fetal development. Supplementation with mitochondrial extract from the somatic donor cell may also be essential to achieve successful embryonic development.

Here, we present a protocol to significantly reduce the mitochondrial DNA copy numbers in a bovine oocyte (P &lt; 0.0001). This method utilizes centrifugation and bisection to substantially reduce oocyte mitochondria and may allow for an increased chance of development in the reconstructed interspecies somatic cell nuclear transfer embryos.

Verwendung von Bisektion zur Reduzierung der mitochondrialen DNA in der Rinderzelle

Interspecies somatic cell nuclear transfer (iSCNT) may be used to rescue endangered species, but two distinct populations of mitochondrial DNA (mtDNA) ...