Name: preparation of mitochondrial enriched fractions for metabolic analysis in drosophila
Uploaded: 2015-09-30T14:00:00
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. Reagentes e Soluções <ol><li> Preparação do alimento mosca e mídia <ol><li> Mosca calor ingredientes de açúcar 11%, 2% de levedura autolisada, 5,2% de farinha de milho, agar de 0.79% w / v em água em uma placa fixada em 90 ° C. Agita-se regularmente até uma mistura ligeiramente densa homogénea. Preparar um volume final de 550 ml de alimento mosca para um total de 100 tubos de ensaio com 5 ml em cada mosca alimentos. </li><li> Remover a partir da fonte de calor, agita-se ocasionalmente até que o alimento...

<ol>
	<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. 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Os passos mais críticos neste protocolo são: 1) criação de moscas suficientes no espaço abundante. É muito importante para as gaiolas não overpopulate demografia com mais de 150 moscas cada; 2) mudando a comida das gaiolas com freqüência para evitar a concorrência alimentar e estresse nutricional; e 3) a manutenção de todas as amostras a 4 ° C para assegurar a integridade durante o isolamento da fracção mitocondrial. Também é recomendado para relaxar o tampão de isolamento, o tampão de lavagem, e o ho...

O objetivo deste procedimento é desenvolver frações mitocondriais enriquecidos que produzem metabólitos mitocondriais suficientes para metabolômica estudos utilizando Drosophila melanogaster. Em nossa experiência, metabolômica análise usando métodos de extração celular inteiras são incapazes de detectar mudanças sutis metabólitos mitocondriais em Drosophila. No entanto, fracionamento mitocondrial antes da análise metabolômicas aumenta a sensibilidade para identificar mudanças de metabolitos mitocondriais. As mitocôndrias são organelas celulares responsáveis ​​pelo fornecimento de 90% da energia que as células precisam para a função normal 1. Nos últimos anos, tem sido reconhecido que as mitocôndrias desempenhar um papel muito mais dinâmico na função celular e do organismo do que simplesmente produzir trifosfato de adenosina (ATP), e são agora reconhecidos como hubs para a regulação da homeostase metabólica 2,3. As mitocôndrias são o resultado de um processo em que endosymbiotic dislinhagens microbianas tinct fundiu ~ 1,5 bilhões de anos atrás 4. Como as mitocôndrias evoluíram para verdadeiras organelas, genes da endosymbiont foram incorporados no genoma nuclear emergente. Em animais, hoje, cerca de 1.500 proteínas mitocondriais são codificados nuclear enquanto 37 genes permanecem na mtDNA, 13 dos quais codificam proteínas mitocondriais que são subunidades dos complexos de enzimas de fosforilação oxidativa 5. A coordenação entre as mitocôndrias e compartimentos nucleares é necessária para manter a função mitocondrial adequada. Usando os métodos descritos aqui, fomos capazes de detectar alterações metabólicas mitocondriais em Drosophila que resultam da manipulação da coordenação entre os genomas mitocondriais e nucleares. Nós usamos uma linhagem de Drosophila em que mtDNA de suas espécies irmãs D. simulans foi colocada sobre um único D. melanogaster fundo nuclear 6. Este mitonuclear &#39;interrompido&#39;genótipo foi comparado com o genótipo mitonuclear &quot;nativo&quot;, ou co-evoluiu de D. melanogaster transportando a mesma com o seu genoma nuclear nativa D. melanogaster mtDNA. O D. melanogaster e D. simulans mtDNAs diferem por ~ 100 aminoácidos e&gt; 500 substituições sinônimas que afetam mitonuclear 7,8 comunicação. Geramos extratos mosca integrais e extratos enriquecidos mitocondriais para estudar mudanças de metabolitos em resposta ao estresse farmacológico. Aqui nós mostramos que, ao usar frações mitocondriais enriquecido detectamos mudanças acentuadas em metabólitos mitocondriais entre o genótipo nativa, co-evoluído levando a D. mtDNAs melanogaster e as genótipo rompidas transportando D. simulans mtDNA. Em contraste, as modificações do metabolito entre estes dois genótipos são subtis usando métodos normais que utilizam extracto mosca todo. Por conseguinte, este método forneceu um caminho para entender como mtDNAs mediar alterações mitocondriais emresposta a diferentes drogas.

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	<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.

Usando o protocolo acima referido, foi realizada a análise fracções enriquecidas em metabolômico mitocondriais e extractos de animais inteiros para testar o efeito do fármaco rapamicina em mtDNAs divergentes 7. Obtivemos 200 uM de rapamicina por dissolução da droga no alimento mosca. As moscas foram expostos a rapamicina durante 10 dias. Os metabolitos de extractos de mosca inteiras e extractos de mitocondriais foram obtidos utilizando um espectrómetro de massa de...

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.

Preparação de mitocondriais frações enriquecidas para Análise metabólica em<em&gt; Drosophila</em

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

Research

JoVE Journal

Developmental Biology

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

Análise de Zebrafish larvas Músculo Esquelético Integrity com 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 ...

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Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments

Preparação em média<em&gt; Drosophila</em&gt; Extractos de embrião para experiências proteômicas

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

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

Correlativa Super-resolução e microscopia eletrônica para resolver a localização da proteína na Retina de Zebrafish

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

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

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

Dissecção e imunofluorescência coloração de cogumelo corpo e fotoreceptor neurônios em cérebros adultos Drosophila 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

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

Preparando a desenvolver tecido olfativo periférico para Molecular e análise imuno-histoquímica em 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

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

Preparação de amostras de larvas de Drosophila por cromatografia gasosa / espectrometria de massa (GC-MS)-com base em metabolómica

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

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

Dissecação da Retina Pupal drosófila para imuno-histoquímica, análise ocidental e isolamento de RNA

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

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Imagens ao vivo e análise de contrações musculares no embrião de Drosophila

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

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Descongelamento, cultivo e Cryopreserving linhas de células de Drosophila

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

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

Preparação de Drosophila Larval e Pupal Testes para Análise de Divisão Celular em Tecido Vivo e Intacto

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