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

Summary

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.

Abstract

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.

Introduction

The fruit fly, Drosophila melanogaster, has been used as a model organism for genetic research for over a century¹. The study of this organism has not only led to significant fundamental knowledge about sex-linked inheritance², mutation rate³, the development of neural system and the cell fate determination⁴, but has also recently emerged as a valuable tool to study the mechanisms inherent to several diseases such as Alzheimer's and Parkinson's^5,6. Moreover, it is a popular model to study the aging ....

Protocol

1. Reagents Preparation

1. Prepare the following solutions for dissection and permeabilization of the tissue.
   1. Prepare preservation solution: 2.77 mM CaK₂EGTA, 7.23 mM K₂EGTA, 5.77 mM Na₂ATP, 6.56 mM MgCl₂, 20 mM taurine, 15 mM Na₂phosphocreatine, 20 mM imidazole, 0.5 mM dithiothreitol, and 50 mM K-MES, pH 7.1 (can be stored at -20 °C).
   2. Prepare saponin solution: 5 mg of saponin in 1 mL of preservation solution (prepare fresh daily).
2. Prepare the following solutions for the measurement of the respiration.
   1. Prepare respiration medium: 120 mM KCl, ....

Results

A representative trace of mitochondrial oxygen consumption using the protocol described above is provided in Figure 2. The pyruvate and malate injected in the chambers along with the permeabilized muscle fibers are referred to the CI-LEAK respiration, i.e., when the complex I of the ETS is stimulated by the NADH produced through oxidation of pyruvate and malate via the tricarboxylic acid cycle (CI). During this respiration rate, the mitochondrial oxy.......

Discussion

In this study, a method for sample preparation prior to the measurements of mitochondrial oxygen consumption in Drosophila is described. This method was developed to overcome different problems related to the protocols using mitochondrial isolations, notably in terms of duration and number of individuals required. Instead of working with mitochondrial isolations usually requiring large amount of tissues obtained from several individuals, this experiment is performed on permeabilized muscle fibers from thoraxes of few Dro.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
High-resolution respirometer Oxygraph O2K	Oroboros Instruments, Innsbruck, Austria	10022-02	Startup O2K respirometer kit
O2K-Titration Set	Oroboros Instruments, Innsbruck, Austria	20820-03	Hamilton syringes with different volumes
Datlab software	Oroboros Instruments, Innsbruck, Austria	20700	Software for data acquisition and analysis
Fine-tipped antimagnetic forceps	VWR	82027-400
Secura225D-1S-DQE	Sartorius AG, Goettingen, Germany		Semi-micro balance (distributed by several companies)
Drosophila melanogaster wild-type w1118	Bloomington Drosophila stock Center, IN, USA		Storage Condition: 24 °C
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid	Sigma-Aldrich	E4378	EGTA Storage Condition: RT
KOH	Sigma-Aldrich	P1767	CAUTION: corrosive to metals, acute toxicity, skin corrosion, serious eye damage, acute aquatic toxicity. Storage Condition: RT
CaCO3	Sigma-Aldrich	C4830	Storage Condition: RT
Na2ATP	Sigma-Aldrich	A2383	Storage Condition: -20 °C
MgCl2.6H2O	Sigma-Aldrich	M9272	Storage Condition: RT
Taurine	Sigma-Aldrich	T0625	Storage Condition: RT
Na2Phosphocreatine	Sigma-Aldrich	P7936	Storage Condition: -20 °C
Imidazole	Sigma-Aldrich	I5513	Storage Condition: RT
Dithiothreitol	Sigma-Aldrich	D0632	Storage Condition: 2-8 °C
MES hydrate	Sigma-Aldrich	M8250	Storage Condition: RT
Saponin from quillaja bark	Sigma-Aldrich	S7900	Saponin Storage Condition: RT Solution Preparation: 5 mg in 1 mL of preservation solution. Prepare fresh daily.
KCl	Sigma-Aldrich	P9541	Storage Condition: RT
KH2PO4	Sigma-Aldrich	P9791	Storage Condition: RT
HEPES	Sigma-Aldrich	H3375	Storage Condition: RT
BSA	Sigma-Aldrich	05470	Storage Condition: 2-8 °C
Na2S2O4	Sigma-Aldrich	157953	Sodium dithionite. CAUTION: self-heating substances and mixtures, acute toxicity, acute aquatic toxi chronic aquatic toxicity. Storage Condition: RT
Sodium pyruvate	Sigma-Aldrich	P2256	Pyruvate Storage Condition: 2-8 °C Solution Preparation: In MilliQ water. Prepare fresh daily.
L-(-)-Malic acid	Sigma-Aldrich	M1000	Malate Storage Condition: RT Solution Preparation: In MilliQ water. Neutralize with KOH and store at -20 °C.
Adenosine 5'-diphosphate monopotassium salt hydrate	Sigma-Aldrich	A5285	ADP Storage Condition: -20 °C Solution Preparation: In MilliQ water. Neutralize with KOH and store at -80 °C.
Cytochrome c from equine heart	Sigma-Aldrich	C7752	Cytochrome c Storage Condition: -20 °C Solution Preparation: In MilliQ water. Store at -20 °C.
L-Proline	Sigma-Aldrich	P0380	Proline Storage Condition: RT Solution Preparation: In MilliQ water. Store at -20 °C.
Sodium succinate dibasic hexahydrate	Sigma-Aldrich	S2378	Succinate Storage Condition: RT Solution Preparation: In MilliQ water. Neutralize with HCl and store at -20 °C.
sn-Glycerol 3-phosphate bis(cyclohexylammonium) salt	Sigma-Aldrich	G7886	Glycerol-3-phosphate Storage Condition: -20 °C Solution Preparation: In MilliQ water. Neutralize with HCl and store at -80 °C.
Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone	Sigma-Aldrich	C2920	FCCP. CAUTION: acute toxicity, skin sensitisation, chronic aquatic toxicity. Storage Condition: RT Solution Preparation: In absolute ethanol. Store in glass vials at -20 °C.
Rotenone	Sigma-Aldrich	R8875	CAUTION: acute toxicity, skin irritation, eye irritation, specific target organ toxicity (respir sytem), acute aquatic toxicity, chronic aquatic toxicity. Solution Preparation: In absolute ethanol. Store in dark vials at -20 °C.
Malonic acid	Sigma-Aldrich	M1296	Malonate. CAUTION: acute toxicity, serious eye damage. Storage Condition: RT Solution Preparation: In MilliQ water. Neutralize with KOH. Prepare fresh daily.
Antimycin A from Streptomyces sp.	Sigma-Aldrich	A8674	Antimycin A. CAUTION: acute toxicity, acute aquatic toxicity, chronic aquatic toxicity. Storage Condition: -20 °C Solution Preparation: In absolute ethanol. Store at -20 °C.
N,N,N′,N′-Tetramethyl-p-phenylenediamine	Sigma-Aldrich	T7394	TMPD Storage Condition: RT Solution Preparation: In MilliQ water. Store in dark vials at  -20 °C.
(+)-Sodium L-ascorbate	Sigma-Aldrich	A4034	Ascorbate Storage Condition: RT Solution Preparation: In MilliQ water. Store in dark vials at  -20 °C.
NaN3	Sigma-Aldrich	S2002	Sodium azide. CAUTION: acute toxicity (oral and dermal), specific target organ toxicity (brain), aquatic toxicity, chronic aquatic toxicity.  Solution Preparation: In MilliQ water. Store at -20 °C.