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 process, as they can be raised in large number over a short period of time and have a short lifespan. They also possess homologous structures to human organs, such as a heart, oenocytes (hepatocyte-like cells), fat bodies (functioning similarly as the liver and white adipose tissue), insulin producing cells (equivalent to β-pancreatic cells), as well as the hemolymph transporting metabolites (analogous to the blood of vertebrates)⁷. Moreover, the central pathways of intermediary metabolism (including the insulin/insulin-like growth factor-like signaling pathway and Target of Rapamycin-TOR pathways) are also highly conserved⁷. For these reasons, Drosophila have recently been exploited to describe the fundamental mechanisms that control metabolism, especially in pathological conditions inherent to human metabolic diseases such as diabetes⁸. A major component of the metabolism is the mitochondrion that integrates multiple pathways and performs one of life's most important biological functions, ATP production, via the oxidative phosphorylation process (OXPHOS). Considering their central role in organism's metabolism, it is not surprising that mitochondrial dysfunctions are involved in many diseases like Parkinson's⁹ and Alzheimer's diseases¹⁰, as well as in amyotrophic lateral sclerosis^11,12. They are also fundamental determinants of the aging process. Indeed, they are the main producers of reactive oxygen species (ROS) in the cell, which can be detrimental to the cell at high concentration through oxidative damages¹¹. Aging has also been associated to the accumulation of damaged or mutated mitochondrial DNA¹³, mitophagy dysfunctions^14,15 as well as impairment of mitochondrial biogenesis¹⁶. Mitochondria are also key determinants of the cell's homeostasis as they can utilize different substrates to adjust several cellular functions according to the abundance or scarcity of macronutrients^17,18.

Indeed, the different nutrients in the diet (carbohydrates, lipids and proteins) are digested, absorbed and transported in the cells. They are then transformed in the cytosol, and the derived substrates are transported into the mitochondrial matrix where they produce reducing equivalents, such as NADH and FADH₂¹⁹. These reducing equivalents are then oxidized by different enzymatic complexes of the electron transport system (ETS). These complexes are embedded in the mitochondrial inner membrane, such as Complex I and Complex II. Additionally, other enzymatic complexes such as the mitochondrial glycerol-3-phosphate dehydrogenase and the proline dehydrogenase represent alternate routes for the entry of electrons into the ETS^20,21. These 'alternative' complexes are particularly important in insects, as according to the species, they can actively participate to increase the respiration^20,22,23,21. Electrons from these ETS feeding systems are transferred to the ubiquinone and subsequently to Complex III, and then to Complex IV, until the final acceptor, molecular oxygen. This electron transfer generates a proton-motive force across the inner mitochondrial membrane driving the phosphorylation of ADP to ATP at Complex V (Figure 1). Considering the central role of mitochondria in cell's homeostasis, studying mitochondrial metabolism using the relevant model D. melanogaster represents a powerful tool to delineate the underlying mechanisms of various pathophysiological conditions or under cellular and environmental stresses. Surprisingly however, only a handful of studies actually measured mitochondrial respiration in Drosophila^24,25,26. Indeed, experiments aiming to evaluate mitochondrial oxygen consumption require the isolation of mitochondria. Although advantageous for the measurement of different mitochondrial functions (such as ROS production or P/O ratio as marker of mitochondrial efficiency^27,28), these isolations generally require rather large quantities of tissue from several individuals^24,29. This requirement for high amounts of tissue and individuals is an important limiting factor, especially considering that all individuals should be the same age and preferably of the same sex for the experiments, making the measure of respiration at different time points laborious at best. Moreover, while mitochondrial isolations can provide significant insight into the fundamental mechanisms governing mitochondrial metabolism, the methods used to isolate mitochondria have several drawbacks such as the difficulty to obtain replicable results, disruption of the mitochondrial network, and alteration of mitochondrial structure and function^29,30,31.

The aim of this study is to present a robust protocol to measure mitochondrial oxygen consumption in Drosophila using only a minimal amount of tissue from very few individuals. This protocol consists of measuring mitochondrial oxygen consumption in situ using permeabilized muscle fibers²⁹ from Drosophila thoraxes in combination with high-resolution respirometry^32,33,34,35. This method has also additional advantages compared to the classic mitochondrial isolation method since the interactions with the other components of the cell as well as mitochondrial structure and function are more preserved in permeabilized fibers^29,31,36, which makes this approach more physiologically relevant. With this protocol, mitochondrial functions can be accurately evaluated using high-resolution respirometry in only three thoraxes of Drosophila, with substrates allowing the determination of oxygen consumption at several different steps of the ETS. Therefore, this protocol could help answer key questions about the fundamental mechanisms that control metabolism in the context of many environmental or pathophysiological conditions by taking advantage of the Drosophila model.

To measure the oxygen consumption at several different steps of the ETS and evaluate how different substrates contribute to the respiration, different substrates (Figure 1), uncoupler, and inhibitors are used³⁰ after permeabilization of the tissue. Specifically, sequential additions of different substrates are performed to stimulate the entry of electrons through different complexes of the ETS. An uncoupler, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), is then added at optimal concentration to measure the non-coupled respiration, i.e., the non-phosphorylating respiration stimulated to maximal oxygen consumption. Sequential inhibitions of complexes I, II, and III are then performed to monitor the residual oxygen consumption that is due to non-ETS oxidation reactions. Finally, complex IV maximal respiration capacity can be evaluated by the injection of N,N,N',N,-Tetramethyl-p-phenylenediamine (TMPD), an artificial electron provider, and ascorbate. It is important to note that the experiments are conducted at 24 °C since it is the temperature at which the flies are raised.

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, 5 mM KH₂PO₄, 3 mM HEPES, 1 mM EGTA, 1 mM MgCl₂, and 0.2% BSA (w/v), pH 7.2.
   2. Prepare substrates, uncoupler, inhibitors. Dissolve all substrates in H₂O, and dissolve the uncoupler FCCP as well as the inhibitors in ethanol absolute (except malonate which is diluted in H₂O). Neutralize most of the substrates as well as malonate (see Table of Materials). All concentrations given in the protocol are final concentrations inside 2 mL chambers.

2. Preparation and Calibration of the High-Resolution Respirometer.

1. Wash the chambers, caps, and stoppers of the respirometer three times with 70% ethanol and three times with distilled water.
2. Calibrate at zero oxygen concentration: pipet 2.3 mL of respiration medium in the chambers and add 10 mg of sodium dithionite. Although the maximal volume of the chamber is 2 mL, 2.3 mL are added to ensure that no air bubble stays under the stopper when closing the chambers. Close the chambers with the stoppers and measure the oxygen concentration for 10-20 min. Calibrate the electrodes at zero oxygen concentration with the resulting oxygen consumption rate.
3. Wash the chambers with distillated water, incubate in absolute ethanol for 10 min, and then rinse with distilled water.
4. Calibrate at air saturation: pipet 2.3 mL of respiration medium in the chambers, insert the stoppers, aspirate the excess medium on top of the stoppers, and then lift the stoppers with the spacer. Excess respiration medium is pipetted in the chambers (volume of 2 mL total) to avoid the presence of air bubbles after closing the chambers.
5. Monitor the oxygen consumption for 45 min to an hour, until the oxygen concentration (blue trace) is stable around 250 nM at 24 °C.

3. Dissection of Flies and Permeabilization of Tissue

1. Make sure all steps are performed on ice.
2. Anaesthetize six male flies of the same strain, same age and raised on the same diet on ice to facilitate dissection of the flies (wild-type w¹¹¹⁸, 15 days old, fed on cornmeal medium).
3. Using a scalpel and fine-tipped forceps, dissect the flies (remove the heads and abdomens) to keep only the thoraxes containing the flight muscles. This step can be done with the naked eye. Handle the resulting thoraxes with fine-tipped forceps.
4. Transfer three dissected thoraxes in a 25 mm Petri dish containing 2 mL of ice-cold preservation solution using the forceps.
5. With the fine-tipped forceps, mechanically permeabilize the thoraxes by inserting the tip of the forceps into the thoraxes and repeatedly tear apart the tissue to obtain a loosely connected network.
6. In a 24-well plate, fill two wells with 12.5 µL of the saponin solution and 1 mL of preservation solution to obtain a final concentration of 62.5 µg/mL of saponin. Fill the two adjacent wells with 1 mL of respiration medium.
7. With the fine-tipped forceps, transfer three permeabilized thoraxes into the diluted saponin solution and incubate with mild agitation on an orbital shaker, on ice, for 20 min.
8. After the latter incubation, transfer the fibers into the adjacent wells filled with the respiration medium with mild agitation, on ice, for 5 min to rinse off the saponin.

4. Determination of Dry Weight

1. Dry the permeabilized thoraxes on an absorbent surface and flip 3-4 times using the fine-tipped forceps to make sure all moisture is removed.
2. Weigh the three thoraxes together on a microbalance. Note the obtained weight as it will be used to normalize the mitochondrial oxygen consumption rates.
3. Transfer the tissues immediately in a drop of respiration medium on ice.

5. Oxygen Consumption Rates Determination

1. Open the chambers of the respirometer (remove the stoppers) and add 10 mM of pyruvate and 2 mM of malate into each chamber.
2. Add the permeabilized tissues directly into the chambers filled with the respiration medium.
3. Replace the stoppers using the spacer.
4. With a 60 mL syringe, collect oxygen directly from an oxygen tank and inject 2-5 mL of oxygen through the stopper capillary of each chamber to make sure oxygen diffusion through the tissue will not limit oxygen consumption.
5. Insert a Hamilton syringe into the capillary of each stopper to make sure the thoraxes remain in the chamber.
6. Close the chambers completely when the oxygen concentration is around 400 nM, achieving oxygen levels above air saturation (around 160% air saturation) inside the chambers.
7. Stop and restart the stirrers to check for air bubbles in the chambers.
8. Enter the mass of tissue weighed previously to normalize respiration rates by mass of tissue (mg), displaying mass-specific oxygen consumption (pmol O₂ consumed.s^-1.mg^-1 of tissue).
9. Once the oxygen consumption is stabilized (red trace), add 5 mM ADP with a Hamilton syringe.
10. After each injection, rinse the syringes with distilled water, 70% ethanol, and distilled water again.
11. Once the oxygen consumption is stable again, inject 5 µL of a 4 mM of cytochrome c.
12. Proceed to the sequential injections of the following substrates to evaluate the mitochondrial oxygen consumption at different steps of the ETS:
    1. Add 5 µL of a 2 M Proline.
    2. Add 10 µL of a 1 M succinate.
    3. Add 30 µL of a 1 M glycerol-3-phosphate.
13. Inject the uncoupler FCCP in a titration manner by steps of 0.5-1 µM (2 µL of a 1 mM solution) until the optimal concentration is reached, i.e., the concentration to attain the highest oxygen consumption possible without inhibition (CAUTION: see Table of Materials).
14. Inject the following inhibitors sequentially to entirely inhibit the electron flux in the ETS in order to measure the residual oxygen consumption (ROX) i.e. non-respiratory reactions such as oxygenase reactions among others:
    1. Add 1 µL of a 1 mM rotenone (CAUTION: see Table of Materials).
    2. Add 5 µL of a 2 M malonate (CAUTION: see Table of Materials).
    3. Add 1 µL of a 5 mM antimycin A (CAUTION: see Table of Materials).
15. Add 0.2 mM ascorbate and 0.5 mM TMPD sequentially into the chambers, starting with the ascorbate to measure the oxygen consumption by complex IV.
16. Add 20 mM of sodium azide to inhibit complex IV (CAUTION: see Table of Materials).
17. Once the signal is stable, open the chambers with the spacer, re-oxygenate the chambers by injecting 2 mL of pure oxygen and close the chambers when the concentration is around 250-300 nmol.mL^-1.
18. Measure the signal for 10-15 min and calculate the chemical background, i.e., the oxygen consumption due to autoxidation of TMPD.

6. Cleaning of the Respirometer

1. After the measurements, rinse the chambers with distilled water once and rinse the chambers three times in 70% ethanol.
2. Incubate in absolute ethanol for 10 min to inactivate the inhibitors.
3. Rinse three times with distilled water, fill the chambers with 70% ethanol and replace the stoppers and the caps until next utilization.

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.