Tubulin-Gene-Switch was provided by S. Pletcher. Elav-Gene-Switch was provided by H. Keshishian. S1106 was provided by L. Seroude. We transformed flies with pUAST plasmids containing Ndi1 (amplified by PCR from yeast genomic DNA) and performed seven rounds of backcrossing into a w1118 background.
Flies were collected under light nitrogen-induced anesthesia and housed at a density of 25-30 flies per vial. At least 6 vials were assayed per treatment. All flies were kept in a humidified, temperature-controlled incubator with 12 h on/off light cycle at 25 °C in vials containing standard cornmeal medium (Lewis 1960
). RU486 was administered by adding 200 μl of 0, 2, 10, or 50 μg/ml RU486 dissolved in ethanol to the top of the fly food, as previously described (Poirier et al. 2008
; Copeland et al. 2009
). Flies were flipped to fresh vials twice per week and scored for death.
Purification of Mitochondria
Mitochondria were isolated from adult flies as previously described (Walker et al. 2006
). Fifty flies were gently crushed in 1 ml chilled mitochondrial isolation medium (MIM: 250 mM sucrose, 10 mM Tris (pH 7.4), 0.15 mM MgCl2
) by using a glass-on-glass homogenizer, then spun twice at 1,000 × g for 5 min at 4°C to remove debris. The supernatant was then spun at 13,000 × g, for 5 min at 4°C. The pellet, containing the mitochondria, was washed with 1 ml MIM and resuspended in 50 μl MIM.
NADH-ubiquinone oxidoreductase Activity assay
Mitochondrial complex I (NADH-ubiquinone oxidoreductase) activity was measured spectrophotometrically at 600 nm in an incubation volume of 1 ml containing 25 mM potassium phosphate, 3.5 g/l BSA, 60 μM DCIP, 70 μM decylubiquinone, 1 μM antimycin A, and 0.2 mM NADH, pH 7.8. We preincubated an aliquot of 10 μl mitochondrial suspension, prepared as described above, at 25°C in 960 μl incubation mixture without NADH. After 3 minutes, 20 μl of 10 mM NADH was added, and the absorbance was measured at 30 sec intervals for 4 minutes at 25°C. After 4 minutes, we added 1 μl rotenone (1 mM) and measured the absorbance again at 30 sec intervals for 4 minutes. Complex I activity was expressed as a reduction rate of DCIP.
Analysis of CO2 release
The rate of CO2 emission was used to determine the metabolic rate of 8 groups of flies (10 individuals per group) from each treatment. We used flow through respirometry to measure CO2 release with Sable Systems (Las Vegas, NV, USA) data acquisition software logging data from an infrared CO2 analyzer (Li-Cor model 6251; Lincoln, NE, USA). During an experimental run, room air was pumped through two silica and one ascarite/drierite column to be scrubbed of water and CO2. Air then flowed through one of five 30 ml chambers attached to the multiplexor and subsequently passed into the CO2 analyzer. The chambers connected to the multiplexor included an empty control chamber used to collect baseline values, and four chambers containing flies from a particular treatment. The order in which each chamber was measured was controlled with SABLE system data acquisition software (Las Vegas, NV, USA). When a chamber was not being measured, it was still perfused with H20- and CO2 -free air at a rate equal to the regulated flow entering the measured chamber. Flow rate was adjusted using a mass flow controller (Sierra Instruments, Monterey, CA, USA) and was held at 100 ml/min.
An experimental run lasted a total of 75 minutes. Three five-minute baseline readings were recorded at the beginning, middle and end of the run and used to provide accurate zero values. The rate of CO2 emission of four fly groups was measured for 15 minutes within each run. The room temperature was maintained at 25 ± 1°C. The CO2 levels (ppm) were averaged and recorded once/second using Sable Systems data acquisition software. Sable Systems (Las Vegas, NV, USA) Expedata analysis software was used to process VCO2 measurements. CO2 levels were recorded in parts per million and, after data were zeroed using baseline values, converted to milliliters/minute.
Dihydroethidium (DHE) staining
Fly brains were dissected in PBS buffer (8 mM Na2HPO4, 150 mM NaCl, 2 mM KH2PO4, 3 mM KCl) and stained with 30 μM DHE solution for 10-15 minutes. Brains were washed 3 times in PBS buffer and visualized by confocal microscopy. The intensity of the brains was measured using the Scion Image Software (Scion Corp., Frederick, MD). To minimize variations in signal intensity, brains were mounted on the same slide and fluorescence intensities of five different brains of each treatment were averaged to give one data point.
Polyclonal rabbit antisera against NDI1 (1:2000), polyclonal rabbit antisera against dPorin1 (1:1000), and monoclonal mouse antibody against α-tubulin (1:5000 dilution, Cell Signalling). The primary antibodies were detected by HRP-conjugated anti-mouse or anti-rabbit IgG antibodies (1:10000 dilution, Sigma) and SuperSignal West Pico Chemiluminescent reagents (Thermo Scientific).
Mitochondrial respiration assay
Rate of oxygen consumption was measured polarographically using a Clark-type oxygen electrode connected to a computer-operated Oxygraph control unit (Hansatech Instruments) at 25°C. Freshly isolated mitochondria was prepared from 50 flies and then suspended in a respiration medium containing 20 mM Hepes-KOH (pH 7.1), 110 mM sucrose, 10 mM KH2PO4, 40mM KCl, 3 mM MgCl2 and 0.5 mM EGTA. The rate was measured continuously, with the following sequential additions: 5 mM glutamate plus 5 mM malate, 200 μM ADP, 0.5 μM rotenone, 5 mM succinate, 2.5 μM antimycin A, 1 mM ascorbate plus 0.25 mM N′-tetramethyl-1,4-phenylenediamine (TMPD).
Superoxide production was assayed, in isolated mitochondria, in respiration medium with 5 mM glutamate, 5 mM malate and 50 μM MitoSox (Invitrogen) at 510 nm excitation/580 nm emission.
Five flies were homogenized in 100 μl of 6 M guanidine-HCl in extraction buffer (100 mM Tris and 4 mM EDTA, pH 7.8) and boiled for 3 minutes. The samples were then centrifuged to collect the supernatant, which was then diluted (1/750) with extraction buffer. The luminescence was measured by a luminometer using autoinjection of the luminescent solution (Invitrogen), and the results were compared to the standards. The ATP level was then calculated by dividing the luminescence by the total protein concentration, which was determined by the Bradford method.
Quantitative Real-Time PCR
Total RNA was extracted from heads of female flies using Trizol reagent. RNA concentration was measured with a Nanodrop spectrophotometer, and 1 μg RNA was used for qRT-PCR measurements. Actin5C was the reference gene used to normalize amplicon amounts. The following primer pairs were used: Act57B: GTGCTATGTTGCCCTGGACT, GCTGGAAGGTGGACAGAGAG; Ndi1: GGTGGGCCTACTGGTGTAGA, CAATGGCGAAAATGTTGTTG. cDNA synthesis and qRT-PCR were combined into one step using Power SYBR Green RNA-to-CT 1-Step kit (Applied Biosystems) and DNA amount was monitored during the 40-cycle PCR by using a 7300 thermal cycler (Applied Biosystems).
Blue native-polyacrylamide gel electrophoresis (BN-PAGE) and In-gel enzyme activity staining
BN-PAGE was performed using Novex Native PAGE Bis-Tris Gel System followed the manufacturer's (Invitrogen) procedure. In brief, 80 μg of the purified mitochondria were resuspended in 25 μl of 1× Native PAGE Sample buffer (Invitrogen) with 1% digitonin and protease inhibitors (Roche) and incubated on ice for 15 min. After centrifugation at 20,000 × g for 30 min, the 25 μl of supernatant was resuspended with 1.25 μl of 5% G-250 sample additive and 10 μl of 4× Native PAGE Sample Buffer (Invitrogen). The final volume was adjusted to 40 μl by addition of double-distilled H2O. Samples were loaded on 3-12% Bis-Tris Native PAGE gels and electrophoresed using 1× Native PAGE Running buffer system (Invitrogen). The upper buffer included 1× Cathode Buffer Additive (Invitrogen). NativeMark Protein standard (Invitrogen) was used as the molecular weight marker.
For in-gel activity assay, the gels were rinsed briefly and equilibrated in the appropriate reaction buffer without chromogenic reagents for 10 minutes. The gels were then incubated in fresh reaction buffer plus chromogenic reagents for varying lengths of time. For complex I, the reaction buffer contains 5 mM Tris-HCl (pH 7.4), 2.5 mg/ml NTB (nitrotetrazolium blue) and 0.1 mg/ml NADH. For Complex II, the reaction buffer contains 5 mM Tris-HCl (pH 7.4), 20 mM sodium succinate, 0.2 mM phenazine methosulfate, and 2.5 mg/ml NTB. For complex III and IV, the reaction buffer contains 50mM sodium phosphate (pH 7.2), 0.05% 3,3′-diaminobenzidine tetrahydrochloride, 50 mM horse heart cytochrome c. All activity staining was carried out at room temperature. The reactions were stopped by fixing the gels in 45% (v/v) methanol and 10% (v/v) acetic acid after color development. The band intensities of both protein contents and activity staining were estimated by optical densitometry.
Female flies were aged on ethanol (diluent) or RU486-supplemented media for 10 days, starved for 6 h and then transferred to vials containing two 2.4cm glass fiber filter circles (Whatman) wetted with 150μl of 30 mM paraquat (Sigma) in 5% sucrose solution. Flies were kept in the dark at all times, except for scoring death and the daily addition of 150μl 30mM paraquat solution.
female flies were aged on ethanol (diluent) or RU486-supplemented media for 10 days and then transferred to 250 μM rotenone-supplemented medium with diluent or RU486. Survival rate was measured every 2 days, with survivors transferred into fresh vials every 4 days.
Female flies were aged on ethanol (diluent) or RU486-supplemented medium for 10 days and then transferred to 1% agar solution with 200 μl diluent or RU486. Percentage survival was measured every day, with survivors transferred to fresh vials every 3-4 days.
Four individual crosses were established each with five virgin females that developed on either 0 μg/ml or 2 μg/ml RU486 and five wild-type (Canton-S) males. Crosses were transferred twice per week to fresh food containing either 0 μg/ml or 10 μg/ml RU486. Adult progeny from each vial were recorded and averaged to generate the fertility over time.
30 adult female flies were placed in a Drosophila activity meter (TriKinetics Inc., Massachusetts). Movements were recorded continuously under normal culturing conditions for 24 hours on a 12:12-hour dark:light cycle. Triplicate samples were used for each activity measurements.