Summary
We present a protocol for a colorimetric assay of citrate synthase activity for quantification of intact mitochondrial mass in Drosophila tissue homogenates.
Abstract
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.
Introduction
Mitochondria are best known as the power-producing organelles in most eukaryotic organisms, which produce the energy currency, ATP, through the tricarboxylic acid cycle (i.e., Krebs cycle) and oxidative phosphorylation. Mitochondria are also found to play important roles in a lot of other physiological processes, such as regulation of apoptosis1, Ca2+ homeostasis2,3, reactive oxidation species (ROS) generation4, and endoplasmic reticulum (ER)-stress response5. Mitochondrial dysfunction can affect any organ in the body at any age and is a primary cause of metabolic, aging-related6, and neurodegenerative diseases7. Intact mitochondria are mechanistically related to mitochondrial function. Thus, proper quantification of intact mitochondrial mass is very important for evaluating mitochondrial function8. Citrate synthase, a rate-limiting enzyme in the first step of the tricarboxylic acid cycle9, is localized in the mitochondrial matrix within eukaryotic cells, and thus can be used as a quantitative marker for the presence of intact mitochondrial mass9,10. Citrate synthase activity can also be used as a normalization factor for intact mitochondrial proteins11,12.
The fruit fly, Drosophila melanogaster, is an excellent model system for studying mitochondrial function, as many molecules and pathways that play pivotal roles in mitochondria are evolutionarily conserved from Drosophila to humans13,14,15. Here, we present a protocol for a fast and simple method for measurement of citrate synthase activity by a colorimetric assay in Drosophila tissue homogenates16 in a 96 well plate format. In the citrate synthase activity assay, citrate synthase in Drosophila tissue homogenate catalyzes the reaction of oxaloacetate with acetyl coenzyme A (acetyl CoA) to form the citrate CoA-SH and H+. CoA-SH subsequently reacts with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to generate a colored product, 2-nitro-5-thiobenzoate (TNB), which can be easily measured spectrophotometrically at 412 nm. Citrate synthase activity can be reflected by the rate of color production.
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Protocol
1. Colorimetric Citrate Synthase Activity Assay for D. melanogaster16
- Collect ten adult flies for each sample. Collect at least triplicate samples for each genotype.
- Prepare 500 µL of ice-cold extraction buffer containing 20 mM HEPES (pH = 7.2), 1 mM EDTA, and 0.1% triton X-100 in a 1.5 mL test tube for each sample.
- Anesthetize adult flies with CO2 on an anesthesia pad and isolate the desired tissues. To isolate the adult fly thoraxes, for example, fix the fly thoraxes by a pair of forceps, and then isolate the fly abdomens by cutting along the border of the thorax and abdomen using a pair of scissors. Collect the fly thoraxes for the muscle citrate synthase activity assay.
NOTE: Determine the fresh weight of tissue at this step if using it to normalize citrate synthase activity. - Transfer 10 adult fly thoraxes to 100 µL of ice-cold extraction buffer immediately and homogenize with a pestle on ice. To keep the samples ice-cold, homogenize the samples for 5-10 s on ice, then have the samples sit on ice for 5 s, and repeat until all the tissues in the tube are homogenized completely.
NOTE: Tape the tube, check the homogenates to make sure that there are no clots in the homogenates and that the tissues in the tube are homogenized completely. All sample treatments should be performed on ice. - Take 10 µL of each homogenized sample in a new tube for protein content measurement. Keep the samples for protein measurement on ice.
NOTE: The protein samples can be frozen and stored at -80 °C for later analysis. The protein concentrations serve as an internal parameter for normalization of citrate synthase activities. - Add 400 µL of ice-cold extraction buffer to each remaining sample to make a total volume of 500 µL. Mix well by gently pipetting up and down at least 5x, avoiding bubble formation. For each reaction, add 1 µL of diluted cell lysate to 150 µL of the freshly prepared reaction solution (20 mM Tris-HCl (pH 8.0), 0.1 mM DTNB, 0.3 mM acetyl CoA, 1 mM oxaloacetic acid). Mix thoroughly and immediately by gently pipetting, avoiding bubble formation. Measure the absorbance at 412 nm every 10-30 s for 4 min at 25 °C using a plate reader capable of measuring absorbance at 412 nm at minimum intervals of 10 s.
NOTE: Change the tip before pipetting a different reagent and avoid forming bubbles in the wells. Incomplete mixing of the reagents may cause variations in the measurements. The citrate synthase activity assay should be done at room temperature immediately after the samples are collected, as this assay is used to quantify intact mitochondrial mass. The reaction buffer should be prepared immediately before use and should not be stored. - Plot data as optical density (OD) absorbance (abs) (Y-axis) versus time (in minutes) (X-axis). Then calculate the slope for the linear portion of the curve, where the reaction rate is
Divide the value by the sample protein concentration to normalize the citrate synthase activity. The citrate synthase activity is calculated as
NOTE: The sample protein concentration can be measured by a protein concentration assay kit.
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Representative Results
Figure 1 presents an example of the kinetic curves for the OD absorbance at 412 nm over time obtained using the citrate synthase activity colorimetric assay to measure the Drosophila thorax tissue homogenates of different genotypes. It is well known that PGC-1α is a master regulator of mitochondrial biogenesis. PGC-1α is functionally conserved between Drosophila and humans. Drosophila RNF34 (dRNF34) is an E3 ubiquitin ligase for Drosophila PGC-1α, dPGC-1, and promotes dPGC-1 protein degradation17. Transmission electron microscopy and mitochondrial DNA qPCR have shown that knockdown of dRNF34 in Drosophila muscle increases mitochondrial content, which is suppressed by knockdown of dPGC-117. Based on these results we hypothesized that knockdown of dRNF34 in Drosophila muscle would increase mitochondrial citrate synthase activity, which should be reversed by knockdown of dPGC-1. Indeed, using the colorimetric assay of citrate synthase activity described here, we found that knockdown of dRNF34 in Drosophila muscle increased mitochondrial citrate synthase activity, which was reversed by knockdown of dPGC-1. Specifically using the method described here, an initial linear enzymatic rate was established for each assay (Figure 1). The trend lines with their formulas and coefficient of determination (R2) are shown in the graph (Figure 1). The slopes of the trend lines represent the maximal reaction rates, which are equivalents of the maximal citrate synthase activities of the different genotypes. The slopes for the different genotypes are different (Figure 1). The coefficient of determination is closer to 1; the formulas of the trend lines are more reliable. The protein concentration normalized maximal citrate synthase activities of different genotypes were calculated from the Figure 1 data (Figure 2). The maximal citrate synthase activity of fly thoraxes with muscle-specific dRNF34 knockdown increased, which was reversed by muscle-specific dPGC-1 knockdown (Figure 2).
Figure 1: An example of the kinetic curves for the colorimetric assay of citrate synthase activity. The adult fly thorax homogenates of (a) 24B-Gal4>+ (b) 24B-Gal4>dRNF34RNAi(II), and (c) 24B-Gal4>dRNF34RNAi(II),dPGC-1RNAi were subjected to the colorimetric assay of citrate synthase activity. The horizontal axes represent reaction times, and the vertical axes represent OD absorbencies at 412 nm. A linear enzymatic rate was established for each genotype. The trend lines were drawn and the formulas and R2 of the trend lines are shown in the graph. Please click here to view a larger version of this figure.
Figure 2: The maximal citrate synthase activities calculated from the kinetic curves and normalized by protein concentration. The maximal citrate synthase activity of fly thoraxes with muscle-specific dRNF34 knockdown increased, which was reversed by muscle-specific dPGC-1 knockdown. All the data are represented as means ± SEM (*p < 0.01, by ANOVA test, and Tukey's test for multiple comparisons, n = 3, 10 thoraxes per replicate). This figure is modified from Wei et al.17. Please click here to view a larger version of this figure.
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Discussion
Metabolic studies using Drosophila as a model must take into consideration the genetic background, diet, and stock maintenance of the flies18. To avoid the effects of different genetic backgrounds on the measurement of citrate synthase activity, different strains of Drosophila should be backcrossed to the control strain for 10 generations. The genetic background of all Drosophila strains used in our experiments is w1118, so we used w1118 as a control. Generally, we think w1118 is a good control, as it is the genetic background for most Drosophila strains and is much easier to backcross than Canton-S. The diet and stock maintenance must be exactly the same for all the experimental groups. In our experiments, flies are normally maintained at 25 °C and 50-60% relative humidity. The sample preparation step should also be performed with caution. The crosses that are set up for the experiment should be in a similar and appropriate scale. To set up a cross, 3-4 female virgin adult flies are kept with 2-3 male adult flies in a 4 cm diameter, 15 cm high vial supplied with fresh food. The food recipe can vary (e.g., high fat diet, normal diet, high sugar diet), depending on the experimental design. Two days later, the parental generation of flies is transferred to a new vial with fresh food. Caring for the larvae hatched in the food includes adding some water if necessary. When the filial generation of flies starts to hatch, the vials are flipped every 24 h, the flies of the desired genotype are collected in a new vial for the experiment, and the hatch date of the flies is marked on each vial. Approximately 20-30 collected flies of the same genotype, gender, and age are kept in a vial with food. The collected flies must be transferred to fresh vials with appropriate food every two days until the experiments are performed. To avoid the effect of age on citrate synthase activity, the flies of the different groups tested should hatch on the same day. In addition, the gender of the flies should be matched. Usually the body size of females is larger than that of males, thus the protein concentration of female bodies is higher than that of male bodies.
The citrate synthase activity assay can also be used for measuring the citrate synthase activity in larvae. To this end, each sample can consist of five wandering third instar larvae. The third instar larvae can be distinguished by a dark orange ring at the tip of their posterior spiracles, which is lacking or weakly present in the second instar larvae.
In step 6, the dilution ratios of samples may vary for different samples. For the samples that are first measured, several dilution ratios have to be tried to determine an appropriate dilution ratio to establish a linear enzymatic rate in the assay. The total measuring time for the maximal enzyme activity that establishes a linear enzymatic rate in the assay varies depending on the enzyme-substrate ratio. If the substrate is extremely overdosed compared to the enzyme, then the enzyme activity or the reaction rate reaches maximal, which allows the establishment of a linear enzymatic rate in the assay. As the reaction goes on, the amount of the substrate decreases, and the enzyme activity or the reaction rate slows down. If a linear enzymatic rate cannot be plotted, which means that the substrate is not overdosed compared to the enzyme, further dilute the tissue homogenates with reaction solution or shorten the interval time for the 412 nm readings until a linear enzymatic rate plot is established. The interval time for the 412 nm reading should be no more than 30 s. The duration for the 412 nm reading is based on the reaction rate and time. The spectrophotometer reading should stop when no further color change is observed for all reactions.
The citrate synthase activity can be normalized by the sample protein concentration, sample fresh weight, or cell number. The sample fresh weight can be measured before homogenizing as in step 3. The cell number can also be used to normalize the lysate of cultured cells, but the cells must be completely lysed. DNA content is not a good option for an internal control, as the DNA extraction procedure may introduce variations in the samples, particularly for samples containing a small amount of DNA.
If no color change is observed after the reaction, several different troubleshooting steps can be taken:
1. Check the sample preparation step, making sure to handle the samples properly, keep the samples on ice, and avoid multiple freeze-thaw cycles.
2. Try to use a higher protein concentration, because some samples might have much lower expression levels of citrate synthase that are undetectable by the citrate synthase activity assay.
3. Note that some commercially available kits for citrate synthase activity assay cannot be used for Drosophila, because these kits determine mitochondrial citrate synthase activity based on immunocapture of mammalian citrate synthase and the mammalian citrate synthase antibody may not recognize the Drosophila citrate synthase.
Likewise, if there is a discrepancy between samples:
1. Make sure there are no bubbles in the wells.
2. Examine whether this is caused by poor pipetting technique.
In contrast to measuring citrate synthase activity by a colorimetric assay in purified mitochondria, which requires almost 200 flies for the purification of mitochondria of each genotype19, we present a protocol for a fast and simple assay for measurement of citrate synthase activity by a colorimetric method in tissue homogenate or in whole cell extracts16. For each sample, only ten flies hatched on the same day are needed; for triplicate samples of each genotype, 30 flies are needed. The protocol described is applicable not only to Drosophila but also to other systems, such as cultured cells and mammalian tissues.
Besides the citrate synthase activity assay, other methods can be used to quantify mitochondrial mass. Compared to other commonly used quantitative methods of mitochondria, such as measurement of mitochondrial DNA content by qPCR and quantification of mitochondrial proteins by Western blotting, which detect all the mitochondria regardless of their function, the citrate synthase activity assay is used to quantify the presence of intact mitochondrial mass. Unlike the mitochondrial respiration assay, which needs mitochondrial purification and specialized assay equipment16, the citrate synthase activity assay allows researchers to carry out rapid measurements by spectrophotometry with simple sample preparation, such as whole cell extract or tissue homogenate, and no need for mitochondrial isolation. Thus, the citrate synthase activity assay is a rapid and economical assay for quantification of intact mitochondrial mass.
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Disclosures
The authors declare no conflicts of interest.
Acknowledgments
This work was supported by the grants from the National Natural Science Foundation of China (31401013 and 31471010), the Science and Technology Commission of Shanghai Municipality, Shanghai Pujiang Program (14PJ1405900), and Natural Science Foundation of Shanghai (19ZR1446400).
Materials
| Name | Company | Catalog Number | Comments |
| 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) | Sigma-Aldrich | V900477 | |
| 2-Amino-2-(hydroxymethyl)-1,3-propanediol (TRIZMA Base) | Sigma-Aldrich | V900483 | |
| Acetyl-CoA | Sigma-Aldrich | A2181 | |
| Dithio-bis-nitrobenzoic acid (DTNB) | Sigma-Aldrich | D8130 | |
| Ethylenediaminetetraacetic acid (EDTA) | Sigma-Aldrich | V900106 | |
| Oxaloacetate | Sigma-Aldrich | O4126 | |
| Pellet pestle | Sangon | F619072 | |
| Pellet pestle motor | Tiangen | OSE-Y10 | |
| Plate reader | BioTek | Eon | |
| Protein BCA Assay kit | Beyotime | P0010S | |
| Scissors | WPI | 14124 | |
| Triton X-100 | Sangon | A110694-0100 |
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