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. Reaktifler ve Çözümleri <ol><li> Sinek gıda ve medya hazırlanması <ol><li> Isı uçucu bileşenler 90 ° C &#39;ye ayarlanmış bir ocak su içinde ağırlık / hacim olarak% 0.79 agar% 11 şeker,% 2 otolize uğramış mayası,% 5.2 mısır unu. Homojen biraz yoğun karışım elde edilinceye kadar düzenli olarak karıştırın. Her bir yiyecek kalkan 5 ml ile 100 şişelerde bir toplam uçucu gıda 550 ml&#39;lik bir nihai hacim hazırlayın. </li><li> Isıtma cihazından çıkar Gıda aşağı 80 ° C&#39;ye gelene kadar zaman karışması ve% 95 etanol,% 0.2 tegosept-metil 4-hidroksibenzoat ekleyin. </li><li> Iki eşit hacimlerde gıda bölün. Diğer hacim etanol, 50 mM bir hacme kadar etanol rapamisinin 1,1 ml 1,1 ml ilave edilir. Rapamisini ve gıda içine etanol çözümleri karıştırın. Sıcaklık ilaçlar eklemeden önce 50 ° C&#39;ye düşer emin olun. </li></ol></li><li> İzolasyon Tampon <ol><li> İzolasyon Tamponu [225 mM Mannitol, 75 mM sakaroz, 10 mM 3- (N &lt;500 ml hazırlayın/ em&gt; morfolino) propansülfonik asit (MOPS) ve 1 mM etilendiamintetraasetik asit (EDTA), 2.5 mg / ml sığır serumu albümini (BSA)] su içinde. </li><li> Sodyum hidroksit kullanarak pH&#39;ı 7.2&#39;ye ayarlayın. Başlangıç ​​pH&#39;ı asidik olacaktır. </li><li> Solüsyonu sterilize etmek ve 4 ° C de saklamak için 0.22 um gözenek boyutuna sahip olan steril filtre depolama şişeler kullanın. </li></ol></li><li> Yıkama Tamponu <ol><li> Yıkama tampon maddesi [225 mM Mannitol, 75 mM sakaroz, 10 mM KCI, 10 mM Tris HCI ve 5 mM KH 2 PO 4] su içinde 500 ml hazırlayın. </li><li> Sodyum hidroksit kullanarak pH&#39;ı 7.2&#39;ye ayarlayın. Başlangıç ​​pH&#39;ı asidik olacaktır. </li><li> Solüsyonu sterilize etmek ve 4 ° C de saklamak için 0.22 um gözenek boyutuna sahip olan steril filtre depolama şişeler kullanın. </li></ol></li></ol> Drosophila SUŞLARINDA 2. Yetiştirme <ol><li> , Gelişim sırasında larva yoğunluğu kontrol bir kültür şişe ebeveynlerin 25 çift yerleştirmek ve onları 48 saat boyunca yumurtalarını bırakmak için izin vermek için. </li> &lt;li&gt; Yumurta yoğunluğunu düzenlemek için 48 saat sonra anne çıkarın. </li><li> Yineleyin ortaya çıkan F1 yavru kullanarak 2.1 ve 2.2 adımları, bu nedenle bu yoğunluk kontrolü iki kuşak elde edilir. </li><li> Yetişkin toplamak için, karbondioksit (CO2) ve cinsiyete göre ayrı kullanarak sinekler uyutmak. <ol><li> 5 psi sürekli bir akış bir yüksek basınçlı tanktan teslim saf CO 2 tek aşamalı regülatör kullanın. Statik elektriği önlemek için, su ile balonun filtreleme 500 ml CO 2 katalize etmek için plastik boru kullanılır. </li><li> Şişeyi kapatmak için tek delikle bir lastik tıpa kullanın. CO2 pede şişenin yanal açıklığı bağlamak için plastik boru kullanın. </li><li> 10-15 dk daha fazlası için ped sinek yerleştirin. Deneysel koşullar koymadan önce 24 saat anestezi kurtarmak için izin verin. </li></ol></li><li> Mitokondriyal zenginleştirilmiş kesirler için yeterli metabolitleri oluşturmak numune başına 300 sinekler kullanın. Burada, kadın kullanın, ancak gender, diğer deneylerde farklı olabilir. Bir ev yapımı 1 L demografi kafesine 150 sinekler aktarın. Bir şişe içinde uçucu gıda 5 ml erişimine izin verir. </li><li> Başka bir 1 L kafesine geri kalan 150 sinekler aktarın. 150&#39;den fazla sinekler kafeslere overpopulate etmeyin. </li><li> Deneysel koşul başına altı çoğaltma örnekleri kullanın. Bütün hayvan ekstreleri fazla metabolitleri verim yana, bütün hayvan 1 örnek izolatlarının oluşturmak 50 kadın, bir kafes uçar aktarmak için. Mitokondriyal izolatların gelince, deneysel koşul başına altı çoğaltma örnekleri kullanın. </li><li> 12 saat ışık ve 12 saat karanlık çevrimi ile 25 ° C &#39;de yer kafesleri. </li><li> Gıda kalitesini korumak için gıda 5 ml bir şişe içinde her 2 ya da 3 gün taze yiyecek sağlamak. </li><li> 10 gün boyunca gıda sinekler koruyun. Bu adım, rapamisin tedavisi 7 için gereklidir. Diğer tedaviler veya şartlar farklı olacaktır. </li></ol> Mitokondriyal Kesirler 3. İzolasyonu <ol><li> Damperli bir içine bir kafes (150 sinekler) çıkışlı uçarCam-Teflon Dounce homojenleştirici soğutulmuş yalıtım 1 ml tampon ile doldurulmuştur. </li><li> Aşağı 15 vuruş için havaneli yukarı ve hareket ettirerek homojenleştirin. Sağlam mitokondri tutmak için buz üzerinde harç tutmak. </li><li> 4 ° C&#39;de 5 dakika boyunca 300 x g&#39;de, 1.5 ml tüp ve santrifüj transfer homojenat. </li><li> Mitokondri için zenginleştirmek için 4 ° C&#39;de 10 dakika boyunca 6000 x g&#39;de santrifüj süpernatan ve al. </li><li> Süpernatant sitozolik fraksiyonu içerdiğinden, süpernatant atın veya alternatif deneyler için kullanabilirsiniz. Yıkama tamponu 300 ul pelletini. </li><li> İkinci kafes için 3,5 - 3,1 tekrarlayın adımları. Bir kriyojenik mikrosantrifüj tüpü içinde yeniden süspansiyon haline getirilmiş, her iki kafesleri pelet birleştirin. </li><li> 4 ° C&#39;de 10 dakika boyunca 6000 x g&#39;de santrifüjleyin. </li><li> Süpernatantı atın ve sıvı azot içinde pelet flash dondurma. </li><li> -80 ° C veya daha düşük bir sıcaklıkta mitokondriyal zenginleştirilmiş kısımlar saklayın. <ol><li> Alternatif olarak, bütün hayvan öncesi içinTerkip, bir kriyojenik mikrosantrifüj tüp içinde yetişkin yerleştirin. C veya daha düşük sıcaklıkta -80 ° sıvı azot ve mağaza erişkinlerde şişeyi Flash dondurma. </li></ol></li></ol>

<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. 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>

Yazarlar ifşa hiçbir şey yok.

Bu protokolde en kritik adımlar şunlardır: 1) bol uzayda yeterli sinekler yetiştirme. 150&#39;den fazla sinekler her biri demografi kafesleri overpopulate değil çok önemlidir; 2) sık sık gıda rekabeti ve beslenme stresi önlemek için kafes gıda değişen; ve 3), 4 ° C &#39;de, tüm numuneler muhafaza mitokondriyal fraksiyonunun izolasyonu sırasında bütünlüğünü sağlamak için. Ayrıca yalıtım tamponu, yıkama tamponu ve kullanımdan önce kullanılan cam teflon homojenleştirici soğuk Dounce tavs...

Bu prosedürün amacı, Drosophila melanogaster&#39;in kullanılmasının metabolomiks çalışmalar için yeterli mitokondriyal metabolitleri elde zenginleştirilmiş mitokondriyal fraksiyonlar geliştirmektir. Bizim tecrübelerimize göre, tüm hücresel çıkarma yöntemlerini kullanarak metabolomik analiz Drosophila ince mitokondriyal metabolit değişikliklerini tespit edemiyoruz. Ancak önceki metabolomik analiz mitokondriyal fraksiyon mitokondriyal metabolit vardiya tanımlamak için hassasiyetini artırır. Mitokondri hücreleri normal fonksiyon 1 için gereken enerjinin% 90 sağlanmasından sorumlu hücresel organellerdir. Son yıllarda bu mitokondrinin sadece adenozin trifosfat (ATP) üretmek daha hücresel ve organizma işlevi çok daha dinamik bir rol oynayabilir ve şimdi metabolik homeostazı 2,3 düzenlenmesi için hub olarak tanınan kabul edilmiştir. Mitokondri dis olan bir endosimbiyotik işleminin sonucudurTinct mikrobik soy 1,5 milyar yıl önce 4 ~ birleşti. Mitokondri gerçek organellere haline gibi, endosymbiont gelen genler ortaya çıkan nükleer genomu dahil edildi. 37 gen oksidatif fosforilasyon 5 enzim komplekslerinin alt birimleri olan mitokondriyal proteinleri kodlayan 13 olan mtDNA, kalırken hayvanlarda bugün yaklaşık 1.500 mitokondriyal proteinlerin, nükleer kodlanmış bulunmaktadır. Mitokondri ve nükleer bölmeleri arasında koordinasyon düzgün mitokondriyal fonksiyonunu korumak için gereklidir. Burada açıklanan yöntemleri kullanarak mitokondriyal ve nükleer genomlarındaki arasındaki koordinasyon manipülasyon sonucu Drosophila mitokondriyal metabolik değişiklikleri tespit etmek mümkün. Biz hangi mtDNA D. kardeş türünden Drosophila bir yük kullanılan simulans tek D. yerleştirildi Nükleer arka plan 6 melanogaster. Bu &quot;kesintiye &#39;mitonuclearGenotip D. &#39;doğal&#39;, ya da ko-evrimleşmiş mitonuclear genotip ile karşılaştırılmıştır kendi doğal D. ile aynı nükleer genomu taşıyan melanogaster mtDNA melanogaster. D. melanogaster ve D simulans mtDNAs 100 ~ tarafından amino asitler ve mitonuclear iletişim 7,8 etkileyen&gt; 500 eşanlamlı değiştirmelerin farklıdır. Biz farmakolojik strese yanıt olarak metabolit vardiya çalışmak için tüm sinek özleri ve mitokondriyal zenginleştirilmiş özleri üretti. Burada mitokondrial zenginleştirilmiş kesirler kullanırken biz D. taşıyan yerli, eş-evrim genotip arasındaki mitokondriyal metabolitleri belirgin değişimler tespit olduğunu göstermektedir melanogaster mtDNAs ve D. taşıyan kesintiye genotip simulans mtDNA. Buna karşılık, bu iki genotipleri arasındaki metabolit değişiklikleri tüm sinek özü kullanan, normal yöntemlerle ince olur. Bu nedenle, bu yöntem mtDNAs mitokondriyal değişiklikler arabuluculuk nasıl anlamak için bir yol sağladıFarklı ilaçlara yanıt.

<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.

Protokol Yukarıda açıklandığı kullanarak, farklı mtDNAs 7 rapamisin ilacın etkisini test etmek için, mitokondriyal zenginleştirilmiş fraksiyonları ve bütün hayvansal özler üzerine metabolomic analizi yapıldı. Biz sinek gıda ilaç çözerek rapamisinin 200 mcM teslim etti. Sinekler için 10 gün rapamisin maruz bırakılmıştır. Bütün uçucu ekstrelerinden ve mitokondriyal ekstrelerinden metabolitleri standart çözücü çıkarma yöntemleri kullana...

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.

Metabolik Analiz Mitokondriyal Zenginleştirilmiş Kesirler Hazırlanması<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 San Sebastian

Research

JoVE Journal

Developmental Biology

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

9.5K Görüntüleme.  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.

Doku çok az miktarda kullanarak Drosophila Permeabilized liflerinin mitokondrial oksijen harcama ölçme noktalar

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

Biyokimya

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.

Drosophila Melanogaster'de Sitrat Synthase Aktivitesinin Kolorimetrik Tsöplini

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.

Gaz kromatografi-kütle spektrometresi (GC-MS) Drosophila larva örnekleri hazırlanması-Metabolomics dayalı

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

Gelişim Biyolojisi

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.

Maya hücreleri Mitokondri saflaştırılması

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

Biyoloji

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, ...

Davranış

Meyve Sineklerinde Mitokondriyal Fizyoloji ve Biyoenerjetik Analizi

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.

Yazar Spotlight: Drosophila melanogaster Kullanarak Mitokondriyal Fonksiyonu ve Kimyasal Toksisiteyi Keşfetmek 

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.

Yüksek Çözünürlüklü Respirometri Kullanarak Bir Drosophila melanogaster PINK1B9-Null Mutantında Mitokondriyal Fonksiyonun Analizi

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.

Drosophila melanogaster Larval ve yetişkin beyin metabolik Analizi

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

Nörobilim

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.

Transkript ve Metabolitler gelen saflaştırılması<em&gt; Drosophila</em&gt; Kafaları

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

Metabolik Analiz Mitokondriyal Zenginleştirilmiş Kesirler Hazırlanması

Özet

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Doku çok az miktarda kullanarak Drosophila Permeabilized liflerinin mitokondrial oksijen harcama ölçme noktalar

Drosophila Melanogaster'de Sitrat Synthase Aktivitesinin Kolorimetrik Tsöplini

Gaz kromatografi-kütle spektrometresi (GC-MS) Drosophila larva örnekleri hazırlanması-Metabolomics dayalı

Maya hücreleri Mitokondri saflaştırılması

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

Yüksek Çözünürlüklü Respirometri Kullanarak Bir Drosophila melanogaster PINK1^B9-Null Mutantında Mitokondriyal Fonksiyonun Analizi

Yüksek Çözünürlüklü Respirometri Kullanarak Bir Drosophila melanogaster PINK1^B9-Null Mutantında Mitokondriyal Fonksiyonun Analizi

Yüksek Çözünürlüklü Respirometri Kullanarak Bir Drosophila melanogaster PINK1^B9-Null Mutantında Mitokondriyal Fonksiyonun Analizi

Drosophila melanogaster Larval ve yetişkin beyin metabolik Analizi

Transkript ve Metabolitler gelen saflaştırılması