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. Reactivos y Soluciones <ol><li> Preparación de alimentos con mosca y los medios de comunicación <ol><li> Calor ingredientes de la mosca 11% de azúcar, 2% de levadura autolizada, 5,2% de harina de maíz, agar 0,79% w / v en agua en una placa calefactora puesta a 90 ° C. Agitar regularmente hasta que se obtiene una mezcla ligeramente densa homogénea. Preparar un volumen final de 550 ml de la comida de la mosca para un total de 100 viales con 5 ml volar comida en cada uno. </li><li> Retirar de la fuente de calor, revuelva ocasionalmente hasta que la comida se enfría a 80 ° C y añadir 0,2% tegosept-4-hidroxibenzoato disuelto en etanol al 95%. </li><li> Divida la comida en dos volúmenes iguales. Añadir 1,1 ml de 50 mM rapamicina disolvieron en etanol a un volumen y 1,1 ml de etanol al otro volumen. Se agita la rapamicina y las soluciones de etanol en la comida. Asegúrese de que la temperatura desciende a 50 ° C antes de la adición de las drogas. </li></ol></li><li> Aislamiento Buffer <ol><li> Preparar 500 ml de Aislamiento Buffer [225 mM manitol, sacarosa 75 mM, 10 mM 3- (N &lt;/ em&gt; morfolino) propanosulfónico (MOPS) y 1 mM de ácido etilendiaminotetraacético (EDTA), 2,5 mg / ml de albúmina de suero bovino (BSA)] en agua. </li><li> Ajustar el pH a 7,2 utilizando hidróxido de sodio. El pH inicial será ácida. </li><li> Utilice botellas de almacenamiento de filtro estériles con un tamaño de poro de 0,22 micras para esterilizar la solución y almacenarlo a 4 ° C. </li></ol></li><li> Tampón de lavado <ol><li> Preparar 500 ml de tampón de lavado [225 mM manitol, sacarosa 75 mM, KCl 10 mM, Tris HCl 10 mM y 5 mM KH 2 PO 4] en agua. </li><li> Ajustar el pH a 7,2 utilizando hidróxido de sodio. El pH inicial será ácida. </li><li> Utilice botellas de almacenamiento de filtro estériles con un tamaño de poro de 0,22 micras para esterilizar la solución y almacenarlo a 4 ° C. </li></ol></li></ol> 2. Crianza de las cepas de Drosophila <ol><li> Para controlar la densidad de larvas durante el desarrollo, colocar 25 pares de los padres para una botella de cultivo y permitir que ponen huevos durante 48 horas. </li> &lt;li&gt; Eliminar padres después de 48 horas para regular la densidad de huevos. </li><li> Repita los pasos 2,1 y 2,2 mediante la descendencia F1 emergentes, por lo que dos generaciones de este control de la densidad se logra. </li><li> Para recoger los adultos, anestesiar las moscas utilizando dióxido de carbono (CO 2) y separados por sexo. <ol><li> Utilice un regulador de etapa única a entregado CO 2 puro a partir de un tanque de alta presión en un flujo continuo de 5 psi. Para evitar la electricidad estática, utilice un tubo de plástico para catalizar el CO 2 en 500 ml de filtrado matraz con agua. </li><li> Use un tapón de goma con un agujero para sellar el matraz. Utilice un tubo de plástico para conectar la abertura lateral del matraz a una almohadilla de CO 2. </li><li> Coloque las moscas en la almohadilla por no más de 10-15 minutos. Permitir a recuperarse de la anestesia durante 24 horas antes de colocarlos en condiciones experimentales. </li></ol></li><li> Para generar suficientes metabolitos de fracciones enriquecidas mitocondriales, utilice 300 moscas por muestra. En este caso, utilizar las hembras, pero Gender puede diferir en otros experimentos. Moscas de transferencia 150 a un hecho en casa 1 L demografía jaula. Permitir el acceso a 5 ml de alimentos mosca en un vial. </li><li> Transferir los 150 moscas restantes a otra jaula de 1 l. No sobrepoblar jaulas con más de 150 moscas. </li><li> Utilice seis muestras replicadas por condición experimental. Desde extractos animales enteros producen más metabolitos, para generar 1 muestra de animal entero aísla, transferir 50 hembra vuela una jaula. En cuanto a los aislamientos mitocondriales, utilizar seis muestras replicadas por condición experimental. </li><li> Coloque las jaulas a 25 ° C con un ciclo de 12 horas de luz y 12 horas de oscuridad. </li><li> Proporcionar alimentos frescos cada 2 o 3 días en un vial con 5 ml de alimentos para mantener la calidad de los alimentos. </li><li> Mantener moscas en la comida durante 10 días. Este paso es necesario para el tratamiento rapamicina 7. Otros tratamientos o condiciones serán diferentes. </li></ol> 3. Aislamiento de fracciones mitocondriales <ol><li> Dump vuela de una jaula (150 moscas) en unvidrio-teflón homogeneizador Dounce llena con 1 ml de tampón de aislamiento frío. </li><li> Homogeneizar moviendo la mano del mortero hacia arriba y abajo de 15 golpes. Mantenga el mortero sobre hielo para mantener las mitocondrias intactas. </li><li> Transferencia homogeneizado a un tubo de 1,5 ml y centrifugar a 300 xg durante 5 min a 4 ° C. </li><li> Tome el sobrenadante y se centrifuga a 6000 xg durante 10 min a 4 ° C para enriquecer en las mitocondrias. </li><li> A medida que el sobrenadante contiene la fracción citosólica, descartar el sobrenadante o utilizarlo para experimentos alternativos. Resuspender el precipitado en 300 l de tampón de lavado. </li><li> Repita los pasos 3,1 a 3,5 para la segunda jaula. Combinar los sedimentos resuspendidos de ambas jaulas en un tubo de microcentrífuga criogénico. </li><li> Centrifugar a 6000 xg durante 10 min a 4 ° C. </li><li> Descartar el sobrenadante y flash-congelar el sedimento en nitrógeno líquido. </li><li> Guarde las fracciones enriquecidas mitocondriales a -80 ° C o temperatura inferior. <ol><li> Alternativamente, para pre animal completamenteparation, coloque los adultos en un tubo de microcentrífuga criogénico. Flash congelar el vial en nitrógeno y almacenar adultos líquido a -80 ° C o temperatura inferior. </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. 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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. 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Los autores no tienen nada que revelar.

Los pasos más críticos en este protocolo son: 1) la cría de moscas suficientes en abundante espacio. Es muy importante que no se pueblan la demografía jaulas con más de 150 moscas cada uno; 2) el cambio de la comida de las jaulas con frecuencia para evitar la competencia de alimentos y el estrés nutricional; y 3) el mantenimiento de todas las muestras a 4 ° C para garantizar la integridad durante el aislamiento de la fracción mitocondrial. También se recomienda para enfriar el tampón de aislamiento, el tampón...

El objetivo de este procedimiento es desarrollar fracciones enriquecidas mitocondriales que producen metabolitos mitocondriales suficientes para estudios de metabolómica utilizando Drosophila melanogaster. En nuestra experiencia, la metabolómica análisis utilizando métodos de extracción celular enteros son incapaces de detectar sutiles cambios de metabolitos mitocondriales en Drosophila. Sin embargo, fraccionamiento mitocondrial antes del análisis metabolómica aumenta la sensibilidad para identificar los cambios de metabolitos mitocondriales. Las mitocondrias son orgánulos celulares responsables para proporcionar 90% de la energía que las células necesitan para la función normal 1. En los últimos años se ha reconocido que las mitocondrias juegan un papel mucho más dinámico en la función celular y del organismo que simplemente la producción de trifosfato de adenosina (ATP), y ahora son reconocidos como centros para la regulación de la homeostasis metabólica 2,3. Las mitocondrias son el resultado de un proceso en el que endosimbiótica dislinajes microbianos Tinct fusionaron ~ Hace 1,5 millones de años 4. Como las mitocondrias evolucionaron hasta convertirse en verdaderos orgánulos, genes de la endosymbiont fueron incorporados en el genoma nuclear emergente. En los animales hoy en día, aproximadamente 1.500 proteínas mitocondriales son codificada en el núcleo, mientras que 37 genes permanecen en el ADNmt, 13 de los cuales codifican proteínas mitocondriales que son subunidades de los complejos de enzimas de la fosforilación oxidativa 5. Es necesaria la coordinación entre las mitocondrias y compartimentos nucleares para mantener la función mitocondrial adecuada. Utilizando los métodos descritos aquí hemos sido capaces de detectar cambios metabólicos mitocondriales en Drosophila que resultan de la manipulación de la coordinación entre los genomas mitocondriales y nucleares. Se utilizó una cepa de Drosophila en el que el ADNmt de su especie hermana D. simulans se colocó en una sola D. melanogaster fondo nucleares 6. Este mitonuclear &#39;perturbado&#39;genotipo se comparó con el genotipo mitonuclear &#39;nativa&#39;, o co-evolucionado de D. melanogaster que lleva el mismo genoma nuclear con su D. nativa melanogaster ADNmt. El D. melanogaster y D. simulans ADNmts difieren de ~ 100 aminoácidos y&gt; 500 sinónimo sustituciones que afectan a 7,8 comunicación mitonuclear. Hemos generado extractos de moscas enteras y extractos enriquecidos mitocondriales para estudiar los cambios de metabolitos en respuesta al estrés farmacológico. Aquí nos muestran que al utilizar fracciones mitocondriales enriquecida detectamos cambios pronunciados en metabolitos mitocondriales entre el genotipo co-evolucionado nativo que lleva el D. ADNmts melanogaster y el genotipo perturbado llevan D. simulans ADNmt. En contraste, los cambios de metabolitos entre estos dos genotipos son sutiles utilizando métodos normales que utilizan extracto de mosca conjunto. Por lo tanto, este método proporciona un camino para entender cómo ADNmts median cambios mitocondriales enrespuesta a diferentes fármacos.

<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.

Utilizando el protocolo explicado anteriormente, se realizó un análisis metabolomic en fracciones enriquecidas mitocondriales y extractos animales enteros para probar el efecto de la droga rapamicina sobre ADNmts divergentes 7. Hemos entregado 200 M de rapamicina disolviendo el fármaco en la comida de la mosca. Las moscas se expusieron a la rapamicina durante 10 días. Los metabolitos a partir de extractos de moscas enteras y a partir de extractos mitocondriales se obt...

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.

Preparación de mitocondriales enriquecidos fracciones de Análisis metabólico en<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

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

9.5K vistas.  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.

Medición de consumo de oxígeno mitocondrial en las fibras Permeabilized de Drosophila utilizando cantidades mínimas de tejido

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

Investigación

Bioquímica

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.

Un ensayo colorimétrico de la actividad de citrato sintasa en Drosophila Melanogaster

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.

Preparación de muestras de larvas de Drosophila para cromatografía de gases-espectrometría de masas (GC-MS)-base metabolómica

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

Biología del Desarrollo

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.

Purificación de las mitocondrias de las células de levadura

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

Biología

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, ...

Comportamiento

Análisis de la fisiología mitocondrial y la bioenergética en moscas de la fruta

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.

Autor destacado: Explorando la función mitocondrial y la toxicidad química usando 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.

Análisis de la función mitocondrial en un mutante PINK1B9-nulo de Drosophila melanogaster mediante respirometría de alta resolución

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.

Análisis metabólico del cerebro adulto y Larval de Drosophila melanogaster

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

Neurociencias

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.

Purificación de las transcripciones y los metabolitos de<em&gt; Drosophila</em&gt; Cabezales

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

Preparación de mitocondriales enriquecidos fracciones de Análisis metabólico en

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

Análisis de la función mitocondrial en un mutante PINK1^B9-nulo de Drosophila melanogaster mediante respirometría de alta resolución

Análisis de la función mitocondrial en un mutante PINK1^B9-nulo de Drosophila melanogaster mediante respirometría de alta resolución

Análisis de la función mitocondrial en un mutante PINK1^B9-nulo de Drosophila melanogaster mediante respirometría de alta resolución

Análisis metabólico del cerebro adulto y Larval de Drosophila melanogaster

Purificación de las transcripciones y los metabolitos de