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

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

1. I reagenti e soluzioni <ol><li> Preparazione di volare cibo e dei media <ol><li> Calore mosca gli ingredienti 11% di zucchero, 2% lievito Autolyzed, 5,2% farina di mais, agar 0,79% w / v in acqua in una piastra fissata a 90 ° C. Mescolare regolarmente fino ad ottenere una miscela omogenea leggermente densa. Preparare un volume finale di 550 ml di mosca cibo per un totale di 100 flaconcini con 5 ml volare alimentare in ogni. </li><li> Rimuovere dalla fonte di calore, agitare occasionalmente finché il cibo si raffredda a 80 ° C e aggiungere 0,2% tegosept-metil 4-idrossibenzoato disciolto in 95% di etanolo. </li><li> Dividere il cibo in due volumi uguali. Aggiungere 1,1 ml di 50 mM rapamicina sciolti in etanolo ad un volume e 1,1 ml di etanolo per l&#39;altro volume. Mescolare il le soluzioni di etanolo nel cibo e rapamicina. Assicurarsi che la temperatura scende a 50 ° C prima di aggiungere farmaci. </li></ol></li><li> Isolamento Buffer <ol><li> Preparare 500 ml di isolamento Buffer [225 mM Mannitolo, saccarosio 75 mm, 10 mM 3- (N &lt;/ em&gt; -morpholino) propanosulfonico (MOPS) e 1 mm di acido etilendiamminotetraacetico (EDTA), 2,5 mg / ml di albumina sierica bovina (BSA)] in acqua. </li><li> Portare il pH a 7,2 utilizzando idrossido di sodio. Il pH iniziale sarà acida. </li><li> Utilizzare bottiglie di archiviazione filtro sterili con dimensione dei pori di 0,22 micron per sterilizzare soluzione e conservarlo a 4 ° C. </li></ol></li><li> Tampone di lavaggio <ol><li> Preparare 500 ml di tampone di lavaggio [225 mM Mannitolo, saccarosio 75 mm, KCl 10 mm, 10 mM Tris HCl e 5 KH mM PO 2 4] in acqua. </li><li> Portare il pH a 7,2 utilizzando idrossido di sodio. Il pH iniziale sarà acida. </li><li> Utilizzare bottiglie di archiviazione filtro sterili con dimensione dei pori di 0,22 micron per sterilizzare soluzione e conservarlo a 4 ° C. </li></ol></li></ol> 2. Governo dei ceppi di Drosophila <ol><li> Per controllare la densità larvale durante lo sviluppo, inserire 25 coppie di genitori in una bottiglia cultura e di permettere loro di deporre le uova per 48 ore. </li> &lt;li&gt; Rimuovi genitori dopo 48 ore di regolare la densità di uova. </li><li> Ripetere i punti 2.1 e 2.2 con la prole F1 emergente, così due generazioni di questo controllo della densità è raggiunto. </li><li> Per raccogliere gli adulti, anestetizzare linea utilizzando l&#39;anidride carbonica (CO 2) e separati per sesso. <ol><li> Utilizzare un unico regolatore fase consegnato CO 2 pura da un serbatoio ad alta pressione in un flusso continuo di 5 psi. Per evitare l&#39;elettricità statica, utilizzare tubi di plastica per catalizzare la CO 2 in un matraccio da 500 ml di filtraggio con acqua. </li><li> Utilizzare un tappo di gomma con un foro per sigillare nel pallone. Utilizzare tubi di plastica per collegare l&#39;apertura laterale del pallone di un pad di CO 2. </li><li> Posizionare le mosche in tampone per non più di 10-15 minuti. Permette di recuperare da anestesia per 24 ore prima di metterli in condizioni sperimentali. </li></ol></li><li> Per generare abbastanza metaboliti frazioni arricchito mitocondriali, utilizzare 300 mosche per campione. Qui, utilizzare femmine, ma gender può differire in altri esperimenti. Trasferire 150 mosche per un 1 L demografia gabbia fatta in casa. Consentire l&#39;accesso a 5 ml di mosca alimentare in una fiala. </li><li> Trasferire i restanti 150 mosche in un altro 1 L gabbia. Non sovrappopolare gabbie con più di 150 mosche. </li><li> Utilizzare sei campioni replicati per ogni condizione sperimentale. Poiché estratti animali interi resa più metaboliti, di generare 1 campione di tutto l&#39;animale isolati, trasferire 50 femminile vola a una gabbia. Per quanto riguarda gli isolati mitocondriali, utilizzare sei campioni replicati per ogni condizione sperimentale. </li><li> Inserire gabbie a 25 ° C con ciclo di 12 ore di luce e 12 ore scuro. </li><li> Fornire cibo fresco ogni 2 o 3 giorni in una fiala con 5 ml di cibo per mantenere la qualità del cibo. </li><li> Mantenere mosche sul cibo per 10 giorni. Questo passo è necessario per il trattamento rapamicina 7. Altri trattamenti o condizioni saranno diverse. </li></ol> 3. Isolamento di mitocondriali Frazioni <ol><li> Dump vola da una gabbia (150 mosche) in unvetro-teflon Dounce omogeneizzatore riempita con 1 ml di tampone isolamento refrigerati. </li><li> Omogeneizzare spostando il pestello su e giù per 15 colpi. Tenere il mortaio su ghiaccio per mantenere intatto mitocondri. </li><li> Trasferimento omogenato in una provetta da 1,5 ml e centrifugare a 300 xg per 5 minuti a 4 ° C. </li><li> Prendere il surnatante e centrifugare a 6000 xg per 10 min a 4 ° C per arricchire per mitocondri. </li><li> Come il surnatante contiene la frazione citosolica, scartare il surnatante o usarlo per esperimenti alternativi. Risospendere il pellet in 300 ml di tampone di lavaggio. </li><li> Ripetere i passaggi 3,1-3,5 per il secondo gabbia. Unire i pellet risospeso di entrambe le gabbie in una provetta criogenica. </li><li> Centrifugare a 6000 xg per 10 min a 4 ° C. </li><li> Scartare il surnatante e flash-congelare il pellet in azoto liquido. </li><li> Conservare le frazioni arricchite mitocondriali a -80 ° C o temperature inferiori. <ol><li> In alternativa, per tutta la pre animaleparazione, posizionare le adulti in una provetta criogenica. Flash-congelare la fiala in azoto e conservare adulti liquido a -80 ° C o la temperatura più bassa. </li></ol></li></ol>

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Le fasi più critiche di questo protocollo sono: 1) l&#39;allevamento abbastanza mosche nello spazio abbondante. E &#39;molto importante non sovrappopolare gabbie demografia con più di 150 mosche ciascuna; 2) cambiare il cibo delle gabbie frequentemente per evitare la concorrenza cibo e stress nutrizionale; e 3) il mantenimento di tutti i campioni a 4 ° C per garantire l&#39;integrità durante l&#39;isolamento della frazione mitocondriale. Si raccomanda inoltre di raffreddare il buffer di isolamento, il tampone di lav...

L&#39;obiettivo di questa procedura è quello di sviluppare le frazioni mitocondriali arricchito che producono sufficienti metaboliti mitocondriali per metabolomica studi che utilizzano Drosophila melanogaster. Nella nostra esperienza, la metabolomica analisi che utilizzano interi metodi di estrazione cellulari sono in grado di rilevare i cambiamenti sottili metaboliti mitocondriali in Drosophila. Tuttavia, frazionamento mitocondriale prima dell&#39;analisi metabolomica aumenta la sensibilità per identificare spostamenti metaboliti mitocondriali. I mitocondri sono organelli cellulari responsabili della fornitura del 90% dell&#39;energia che le cellule hanno bisogno per la normale funzione 1. Negli ultimi anni è stato riconosciuto che i mitocondri hanno un ruolo molto più dinamico in funzione cellulare e organismal che soltanto la produzione di adenosina trifosfato (ATP), e sono ora riconosciuti come hub per la regolazione del metabolismo omeostasi 2,3. I mitocondri sono il risultato di un processo in cui endosimbiontica dislignaggi microbiche stinti fuse ~ 1,5 miliardi di anni fa 4. Come si sono evoluti in veri mitocondri organelli, i geni della endosimbionte sono stati incorporati nel genoma nucleare emergente. Negli animali oggi, circa 1.500 proteine ​​mitocondriali sono codificate dal nucleo, mentre 37 geni rimangono nel mtDNA, 13 dei quali codificano proteine ​​mitocondriali che sono subunità dei complessi enzimatici della fosforilazione ossidativa 5. Coordinamento tra mitocondri e compartimenti nucleari è necessaria per mantenere una corretta funzione mitocondriale. Utilizzando i metodi descritti qui siamo stati in grado di rilevare i cambiamenti metabolici mitocondriali in Drosophila che derivano dalla manipolazione del coordinamento tra genomi mitocondriali e nucleari. Abbiamo utilizzato un ceppo di Drosophila in cui mtDNA dalle specie sorelle D. simulans è stato posto su un singolo D. melanogaster sfondo nucleare 6. Questo mitonuclear &#39;interrotto&#39;il genotipo è stato confrontato con il &#39;nativo&#39;, o co-evoluti genotipo mitonuclear di D. melanogaster portando lo stesso genoma nucleare con la sua nativa D. melanogaster mtDNA. Il D. melanogaster e D. simulans mtDNA differiscono di ~ 100 amminoacidi e&gt; 500 sostituzioni sinonime che influenzano mitonuclear 7,8 comunicazione. Abbiamo generato estratti mosca integrali e estratti arricchiti mitocondriali per studiare cambiamenti metabolici in risposta allo stress farmacologico. Qui mostriamo che quando si utilizza mitocondriali arricchito frazioni rileviamo cambiamenti pronunciati in metaboliti mitocondriali tra i nativi, genotipo co-evoluta portando la D. mtDNA melanogaster e il genotipo perturbato che trasportano D. simulans mtDNA. Al contrario, le modifiche dei metaboliti tra questi due genotipi sono sottili utilizzando normali metodi che utilizzano estratto intero volo. Pertanto, questo metodo ha fornito un percorso per capire come mtDNA mediare i cambiamenti mitocondriali inrisposta ai farmaci diversi.

<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.

Utilizzando il protocollo spiegato in precedenza, abbiamo effettuato l&#39;analisi metabolomica sulle frazioni arricchito mitocondriali ed estratti di animali interi per testare l&#39;effetto del farmaco rapamicina sul mtDNA divergenti 7. Abbiamo consegnato 200 mM di rapamicina sciogliendo il farmaco nel cibo mosca. Le mosche sono stati esposti a rapamicina per 10 giorni. I metaboliti da estratti interi fly e da estratti mitocondriali sono stati ottenuti usando la spettro...

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.

Preparazione di mitocondriali arricchiti frazioni per l&#39;analisi metabolica 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 Views.  Adelphi University. Mitochondria play central roles in the regulation of metabolism and homeostasis. Subtle changes in mitochondrial metabolism that affect organismal physiology could be difficult to detect in whole organism metabolomics studies. Here we describe an isolation method that enhances the detection of subtle metabolic shifts in Drosophila melanogaster. 

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

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

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

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

Misura del consumo di ossigeno mitocondriale nelle fibre Permeabilized di Drosophila utilizzando la minima quantità di tessuto

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

Ricerca

Biochimica

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 saggio colorimetrico dell'attività Citrate Synthase in 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.

Preparazione di campioni larvale della drosofila per spettrometria della cromatografia-massa del Gas (GC-MS)-base di metabolomica

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

Biologia dello sviluppo

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.

Purificazione dei mitocondri da cellule di lievito

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

Biologia

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, ...

Comportamento

Analisi della fisiologia mitocondriale e della bioenergetica nei moscerini della frutta

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.

Autore in primo piano: Esplorazione della funzione mitocondriale e della tossicità chimica con 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.

Analisi della funzione mitocondriale in un mutante PINK1B9-null di Drosophila melanogaster utilizzando la respirometria ad alta risoluzione

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.

Analisi metabolica di Drosophila melanogaster Larval e cervello adulto

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

Neuroscienze

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.

Purificazione di Trascrizioni e metaboliti<em&gt; Drosophila</em&gt; Teste

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

Preparazione di mitocondriali arricchiti frazioni per l'analisi metabolica in

Riepilogo

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Un saggio colorimetrico dell'attività Citrate Synthase in Drosophila Melanogaster

Preparazione di campioni larvale della drosofila per spettrometria della cromatografia-massa del Gas (GC-MS)-base di metabolomica

Purificazione dei mitocondri da cellule di lievito

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

Analisi della funzione mitocondriale in un mutante PINK1^B9-null di Drosophila melanogaster utilizzando la respirometria ad alta risoluzione

Analisi della funzione mitocondriale in un mutante PINK1^B9-null di Drosophila melanogaster utilizzando la respirometria ad alta risoluzione

Analisi della funzione mitocondriale in un mutante PINK1^B9-null di Drosophila melanogaster utilizzando la respirometria ad alta risoluzione

Analisi metabolica di Drosophila melanogaster Larval e cervello adulto

Purificazione di Trascrizioni e metaboliti