This protocol describes a technique for the analysis of respiratory supercomplexes when only small amounts of samples are available.
Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial electron transport chain organization, giving rise to the proposal of the “plasticity model.” This model postulates the coexistence of different proportions of SCs and complexes depending on the tissue or the cellular metabolic status. The dynamic nature of the assembly in SCs would allow cells to optimize the use of available fuels and the efficiency of electron transfer, minimizing reactive oxygen species generation and favoring the ability of cells to adapt to environmental changes.
More recently, abnormalities in SC assembly have been reported in different diseases such as neurodegenerative disorders (Alzheimer’s and Parkinson’s disease), Barth Syndrome, Leigh syndrome, or cancer. The role of SC assembly alterations in disease progression still needs to be confirmed. Nevertheless, the availability of enough amounts of samples to determine the SC assembly status is often a challenge. This happens with biopsy or tissue samples that are small or have to be divided for multiple analyses, with cell cultures that have slow growth or come from microfluidic devices, with some primary cultures or rare cells, or when the effect of particular costly treatments has to be analyzed (with nanoparticles, very expensive compounds, etc.). In these cases, an efficient and easy-to-apply method is required. This paper presents a method adapted to obtain enriched mitochondrial fractions from small amounts of cells or tissues to analyze the structure and function of mitochondrial SCs by native electrophoresis followed by in-gel activity assays or western blot.
Supercomplexes (SCs) are supramolecular associations between individual respiratory chain complexes1,2. Since the initial identification of SCs and the description of their composition by the group of Schägger2,3, later confirmed by other groups, it was established that they contain respiratory complexes I, III, and IV (CI, CIII, and CIV, respectively) in different stoichiometries. Two main populations of SCs can be defined, those containing CI (and either CIII alone or CIII and CIV) and with very high molecular weight (MW, starting ~1.5 MDa for the smaller SC: CI + CIII2) and those containing CIII and CIV but not CI, with much smaller size (such as CIII2 + CIV with ~680 kDa). These SCs coexist in the inner mitochondrial membrane with free complexes, also in different proportions. Thus, while CI is mostly found in its associated forms (that is, in SCs: ~80% in bovine heart and more than 90% in many human cell types)3, CIV is very abundant in its free form (more than 80% in bovine heart), with CIII showing a more balanced distribution (~40% in its more abundant free form, as a dimer, in bovine heart).
While their existence is now generally accepted, their precise role is still under debate4,5,6,7,8,9,10. According to the plasticity model, different proportions of SCs and individual complexes can exist depending on the cell type or the metabolic status1,7,11. This dynamic nature of the assembly would allow cells to regulate the use of available fuels and the efficiency of the oxidative phosphorylation (OXPHOS) system in response to environmental changes4,5,7. SCs could also contribute to control the reactive oxygen species generation rate and participate in the stabilization and turnover of individual complexes4,12,13,14. Modifications of the SC assembly status have been described in association with different physiological and pathological situations15,16 and with the aging process17.
Thus, changes in the SC patterns have been described in yeast depending on the carbon source used for growth2 and in cultured mammalian cells when glucose is substituted by galactose4. Modifications have also been reported in mouse liver after fasting8 and in astrocytes when mitochondrial fatty acid oxidation is blocked18. In addition, a decrease or alterations in SCs and OXPHOS have been found in Barth syndrome19, heart failure20, several metabolic21 and neurological22,23,24 disorders, and different tumors25,26,27,28. Whether these alterations in SC assembly and levels are a primary cause or represent secondary effects in these pathological situations is still under investigation15,16. Different methodologies can give information about the assembly and function of SCs; these include activity measurements8,29, ultrastructural analysis30,31, and proteomics32,33. A useful alternative that is increasingly being employed and is the starting point for some of the previously mentioned methodologies is the direct determination of SC assembly status by Blue native (BN) electrophoresis developed for this purpose by Schägger's group34,35.
This approach requires reproducible and efficient procedures to obtain and solubilize mitochondrial membranes and can be complemented by other techniques such as in-gel activity analysis (IGA), second-dimension electrophoresis, and western blot (WB). A limitation in the studies on SC dynamics by BN electrophoresis can be the amount of starting cells or tissue samples. We present a series of protocols for the analysis of SC assembly and function, adapted from Schägger's group methods, that can be applied to fresh or frozen cell or tissue samples starting from as little as 20 mg of tissue.
NOTE: The composition of all culture media and buffers is specified in Table 1 and details related to all materials and reagents used in this protocol are listed in the Table of Materials.
1. Mitochondria isolation from cell culture
NOTE: The minimum volume of cells assayed has been ~30-50 µL of packed cells (step 1.4). This can correspond approximately to at least two or three 100 mm cell culture plates or to one 150 mm plate at 80-90% of confluence, depending on the cell type (between 5 × 106 and 107 cells in L929-derived cells or MDA-MB-468). Efficient cell breakage is a critical step.
2. Isolation of mitochondria from small amounts of animal tissues
NOTE: The minimum amount of tissue to apply this protocol with a certain confidence depends on the cell type and its mitochondrial abundance but could be ~20-30 mg of tissue for most cases. The tissue samples can be fresh material or frozen samples. In the latter case, allow the samples to thaw in homogenization buffer placed on ice before starting the procedure.
3. Preparation of samples for Blue Native analysis
4. Blue Native gel electrophoresis
NOTE: The electrophoresis is performed in the cold room (4-8 °C). If there is no cold room facility, a cooling block can be introduced in the electrophoresis tank. Commercial 3-13% native polyacrylamide gels29 are used, but homemade gels of the desired gradient concentrations can also be used38,39.
5. Gel staining
6. In gel-activity (IGA) assays
7. Western blot analysis
The yields of mitochondria obtained following the above-described protocols vary depending on several factors such as the cell line or tissue type, the nature of the samples (i.e., if fresh or frozen tissues are used), or the efficiency of the homogenization process. Expected yields of mitochondria from different cell lines and tissues are collected in Table 2. Once the mitochondrial fractions have been obtained, the next step is the analysis of respiratory SCs pattern, which is performed after the crude mitochondrial sample solubilization and electrophoretic separation by BN-PAGE followed by IGA-analysis or WB immunodetection. Figure 1 shows the aspect of unstained gel lanes just after the run and the pattern of bands after standard Coomassie staining of a cultured cell line and a mouse liver mitochondrial sample.
In Figure 2A,B, clear differences between the SC assembly patterns in human and mouse cells after using IGA assays can be observed. Thus, free complex I is observed in mouse cells whereas it is not detectable in human mitochondria. Complex IV-IGA pattern is very similar in both human cell lines (Figure 2B) while presenting differences between the two mouse cell lines analyzed in Figure 2A. These differences are due to the fact that BL6 cells express a mutant variant of SCAF18.
The SC patterns, obtained with the protocol for mitochondrial isolation described here to work with small samples, are maintained with respect to the "conventional" protocols used for larger samples. This can be seen in Figure 2C, by comparing lanes 1 and 6 (obtained after mitochondrial isolation using a conventional protocol from 3 g of liver and 1.1 g of BAT) with, respectively, lanes 2 and 5 (obtained using the protocols here presented from around 0.1 g of both tissues). As proposed in the plasticity model, the relative proportion of respiratory complexes and SCs varies depending on the cell type and the metabolic state. As shown in Figure 2C,D, the different tissues show different SC assembly patterns. Thus, brain mitochondria show very low levels of free complex I and a higher proportion of SCs when compared with the other tissues, and BAT is characterized by its low levels of CV (Figure 2A) and high amounts of SC III + IV (Figure 2D). Heart mitochondria present high levels of SCs and CV. CV assembly patterns are very similar between the cultured cell line and a mouse liver mitochondrial sample (Figure 2E).
SC assembly can also be analyzed by WB as shown in Figure 3 for two samples obtained from spleen control and tumor cells. In this case, 30-50 µg of mitochondrial protein are usually enough to detect the different complexes and SCs with the specific antibodies. Figure 4 shows some of the main pitfalls that can happen during the application of these protocols. Wrong gel gradients can produce abnormal and undesired migration patterns as in Figure 4A, lane 1, where the SCs remain close to the well in the upper part of the lane due to a higher-than-normal acrylamide concentration in the gel. Incubation of the samples, even for relatively short times, at high temperatures (here 37 °C and 40 °C for 10 min) causes degradation of SCs and CI (Figure 4A lanes 2-4). Likewise, the detergent-to-protein ratio is critical for good results and adequate gel resolution. Below a ratio of 2 g of digitonin/g of protein, the band resolution is progressively reduced and at the lower concentration (0.5 g/g) only a smear in the area corresponding to SCs is visible (Figure 4B).
Figure 1: Pattern of mitochondrial complexes and supercomplexes after BNGE separation of digitonin-permeabilized mitochondria from a culture cell line (L929Balbc) and mouse liver. The lanes on the left (1 and 2) represent the aspect of the unstained gel just after the run, where only CV is detectable, while the lanes on the right (3 and 4) show the pattern of bands after Coomassie staining. Abbreviations: BNGE = Blue Native gel electrophoresis; CV = complex V; SCs = supercomplexes. Please click here to view a larger version of this figure.
Figure 2: IGA-analysis of mitochondria isolated from different cell lines and mouse tissues. (A,B) In-gel activity of the indicated complexes in mitochondria isolated from (A) mouse or (B) human cell lines. L929Balbc is a transmitochondrial cell line generated in our laboratory by transferring mitochondria from Balb/cJ mouse platelets to ρ°L929neo cells as described previously40,41. BL6 fibroblasts are an immortalized cell line generated in our lab from an ear biopsy of a BL6 mouse. (C) CI-IGA patterns from mouse mitochondria isolated using either a conventional method starting with 3 g of liver (lane 1) and with 1.1 g of BAT (lane 6) or the protocol presented here and starting with around 0.1 g of tissue in all cases (lane 2, liver; lane 3, heart; lane 4, brain, and lane 5 BAT). (D) CIV-IGA patterns (performed after CI-IGA shown in panel 2C, lanes 2-5) in mouse liver, heart, brain, and BAT mitochondria. (E) CV-IGA activity analyzed in mouse cultured cells and liver. Around 60-75 µg of mitochondrial protein per lane were loaded. Abbreviations: IGA = in-gel activity; SCs = supercomplexes; CI = complex I; BAT = brown adipose tissue. Please click here to view a larger version of this figure.
Figure 3: WB immunodetection of the different mitochondrial complexes and supercomplexes in spleen samples. After Blue Native PAGE separation of mitochondrial complexes and SCs obtained from spleen control (Spleen M9) and tumor (Tumor M9) samples, they were transferred to a PVDF membrane and sequentially hybridized with antibodies that recognize the indicated CI, CIII, CIV, and CII subunits (panels A–D, respectively). Around 50 µg of mitochondrial protein were loaded per lane. Asterisks indicate the signal remaining from a previous western blot. Abbreviations: SC = supercomplex; CI = complex I; CIII = complex III; CIV = complex IV; CII = complex II. Please click here to view a larger version of this figure.
Figure 4: Pitfalls in SC analysis by Blue-Native PAGE. CI IGA-analysis of mitochondria isolated from (A) fresh mouse liver and (B) frozen rat liver under different solubilization conditions. (A) Lane 1, wrong gel gradient concentration (8-13% instead of the normal 3-13%). Lanes 2-4, samples incubated for 10 min on ice or at 37 °C and 40 °C before loading, respectively. (B) Pattern after IGA for CI of mitochondria obtained from frozen rat liver using different digitonin/protein ratios for solubilization. Abbreviations: SC = supercomplex; PAGE = polyacrylamide gel electrophoresis; CI = complex I; IGA = in-gel activity. Please click here to view a larger version of this figure.
Medium | Composition | ||
Hypotonic buffer | 10 mM MOPS, 83 mM sucrose, pH 7.2 | ||
Hypertonic buffer | 30 mM MOPS, 250 mM sucrose, pH 7.2 | ||
Homogenization Buffer A | 10 mM Tris, 1 mM EDTA, 0.32 M sucrose, pH 7.4 | ||
Homogenization Buffer AT | 225 mM mannitol, 1 mM EGTA, 75 mM sucrose, 0.01% BSA pH 7.4 | ||
Homogenization Buffer AT2 | 225 mM mannitol, 1 mM EGTA, 75 mM sucrose, 0.02% BSA pH 7.4 | ||
BN sample buffer | 50 mM NaCl, 50 mM Imidazole, 2 mM Aminocaproic acid, 1 mM EDTA pH 7.0 | ||
Digitonin solution | 10% digitonin in 50 mM NaCl, 50 mM Imidazole, 5 mM Aminocaproic, 4 mM PMSF | ||
BN loading buffer | 5% Coomassie Blue G in 0.75 M aminocaproic acid | ||
BN cathode buffer A | 50 mM tricine, 15 mM Bis-Tris pH =7.0 (4 °C), 0.02% G-250 (Coomassie brilliant blue G-250) | ||
BN cathode buffer B | 50 mM tricine, 15 mM Bis-Tris pH=7.0 (4 °C), 0.002% G-250 | ||
BN anode buffer | 50 mM Bis-Tris, pH=7.0 | ||
Complex I IGA substrate | 5 mM Tris-HCl pH 7.4, 0.1 mg/mL NADH, 2.5 mg/mL Nitroblue Tetrazolium (NBT) | ||
Complex II IGA substrate | 50 mM Potassium phosphate buffer pH 7.4, 20 mM sodium succinate, 0.2 mM phenazine methosulfate (PMS), 2.5 mg/mL NBT | ||
Complex III IGA substrate | 50 mM Potassium phosphate buffer pH 7.2, 0.05% diaminobenzidine (DAB) | ||
Complex IV IGA substrate | Same solution as CIII plus 50 μM cytochrome c | ||
Complex V IGA substrate | 35 mM Tris, 270 mM glycine pH 8.3, 14 mM MgSO4, 0.2% Pb(NO3)2, 8 mM ATP | ||
Staining solution | Coomassie dye R-250 at 0.25% in 40% methanol and 10% Acetic acid | ||
Destaining/fixing solution | 40% Methanol, 10% Acetic Acid | ||
Transfer buffer | 48 mM Tris, 39 mM Glycine, 20% methanol |
Table 1: Buffers and media composition.
Cell type/tissue | Starting amount | Expected yield (µg mitochondrial protein) | Number of lanes (50-60 µg/lane) |
Cultured cells | |||
MDA-MB-468 | 100 µL vpc | 300-400 µg | 6-9 |
143B-derived | 100 µL vpc | 200-350 µg | 4-7 |
Mouse Fibroblasts | 100 µL vpc | 150-200 µg | 3-5 |
Heart | 100 mg | 400-500 µg | 10-12* |
Skeletal muscle | 100 mg | 300-400 µg | 8-10* |
Liver | 100 mg | 400-500 µg | 8-10 |
Spleen | 50 mg | 150-250 µg | 3-5 |
BAT | 100 mg | 200-300 µg | 4-6 |
Brain | 100 mg | 250-300 µg | 5-8 |
Table 2: Expected yields of mitochondria from different cell types and mouse tissues. The mitochondrial fractions obtained from heart or skeletal muscle are purer than those obtained from other tissues. Thus, smaller amounts can be loaded in the gels (*).
The methodological adaptations introduced in the protocols described here are intended to avoid losses and increase the yield while maintaining mitochondrial complex activities (which is crucial when the availability of enough amounts of samples is compromised) and reproduce the tissue's or cell line's expected pattern of SCs (see Figure 2C). With this purpose and since a high mitochondrial purity is not required to properly detect the SCs, the number of steps, times, and volumes have been reduced whenever this was possible.
Thus, after homogenization and removal of nuclei and unbroken cells by low-speed centrifugation, the supernatants containing the mitochondria are transferred to 1.5 mL polypropylene tubes, and the subsequent centrifugations to obtain the crude mitochondrial fractions are performed in a microfuge by increasing the speed and reducing the times and volumes with respect to conventional protocols. This allows the processing of several samples in a relatively short time: a close estimation is that 10 samples can be processed in ~2-2.5 h (from the start of the homogenization (protocol step 1.6. or equivalent for tissues) to the obtention of the SCs fraction ready to be loaded in a gel (protocol step 3.7) and minimizes the losses while keeping activities. Another change with respect to the original Schägger's group protocols is to substitute, after membrane solubilization for the obtention of the SCs fraction, an ultracentrifugation step by a simple microfuge centrifugation (protocol step 3.4).
There are some critical steps in the procedure. First, the efficiency of cell breakage, especially in the case of cultured cells (see note after protocol step 1.8) and particularly in the case of small cells. Tissues and cell lines present a variable difficulty degree in cell breakage: for example, in the heart and muscle, this step is usually less efficient than in the liver or spleen, but it is compensated in yield because the former have a higher mitochondrial content. One alternative to improve mitochondrial yield when isolated from muscle or heart would be the treatment of samples with trypsin before homogenization. The brain is another tissue that presents low efficiency in cell breakage because of the manual homogenization, which is milder and less efficient and probably also because of the higher variation in cell types in this tissue, as it has been reported previously 42. This can be partially compensated by a second homogenization step as suggested in our protocol (step 2.7.4). On the other hand, if cell breakage is excessive, the final sample containing SCs could be contaminated with nuclear DNA fragments that may interfere with the electrophoretic running. This problem is shown as an increase in the viscosity of the sample when digitonin is added to solubilize the mitochondrial membranes and can be solved by treatment with DNAse I.
Second, if the detergent-to-protein ratio is too low, that would result in less efficient membrane solubilization and the possibility of aggregate formation. This could show in the gels as poor resolution in high molecular weight SCs (see Figure 4) or as CIV giving multiple bands corresponding to oligomers43. If the ratio is too high, it could lead to disaggregation of SCs (mainly CI-SCs). Third, it is important to avoid heating samples during the whole procedure (see Figure 4) and avoid repeated cycles of freezing-thawing since they may affect SC assembly status.
In Table 2, a summary of expected yields is presented for several cell types and tissue samples. The values are approximate and will depend on the cell breakage efficiency and the nature of the source. In the case of tissues, if the starting material is frozen it tends to give a higher yield than with fresh samples. An estimation of the number of lanes (considering a load between 50 and 60 µg of mitochondrial protein per lane) that can be loaded is presented.
The protocols presented here cover a variety of tissues and cell types with small variations, they can be used with fresh or frozen samples and require simple equipment and a relatively low cost, not involving the use of expensive purification kits or gradients. However, they may need some adaptations to be used with particular cell types or samples, as well as when specific mitochondrial populations, such as brain synaptic mitochondria or muscle subsarcolemmal mitochondria, for example, are the object of analysis.
Since the precise physiological roles as well as the potential involvement of SCs in pathological situations remains to be defined, the analysis of their dynamics and the factors affecting their assembly, stability, and function is needed. Our protocols can represent valuable tools that allow to obtain reproducible results on the SCs patterns with small sample sizes and from a variety of cell types and tissues, particularly when multiple experimental conditions need to be compared29.
The authors have nothing to disclose.
This work was supported by grant number "PGC2018-095795-B-I00" from Ministerio de Ciencia e Innovación (https://ciencia.sede.gob.es/) and by grants “Grupo de Referencia: E35_17R” and grant number “LMP220_21” from Diputación General de Aragón (DGA) (https://www.aragon.es/) to PF-S and RM-L.
Acetic acid | PanReac | 131008 | |
Aminocaproic acid | Fluka Analytical | 7260 | |
ATP | Sigma-Aldrich | A2383 | |
Bis Tris | Acrons Organics | 327721000 | |
Bradford assay | Biorad | 5000002 | |
Coomassie Blue G-250 | Serva | 17524 | |
Coomassie Blue R-250 | Merck | 1125530025 | |
Cytochrome c | Sigma-Aldrich | C2506 | |
Diamino benzidine (DAB) | Sigma-Aldrich | D5637 | |
Digitonin | Sigma-Aldrich | D5628 | |
EDTA | PanReac | 131669 | |
EGTA | Sigma-Aldrich | E3889 | |
Fatty acids free BSA | Roche | 10775835001 | |
Glycine | PanReac | A1067 | |
Homogenizer Teflon pestle | Deltalab | 196102 | |
Imidazole | Sigma-Aldrich | I2399 | |
K2HPO4 | PanReac | 121512 | |
KH2PO4 | PanReac | 121509 | |
Mannitol | Sigma-Aldrich | M4125 | |
Methanol | Labkem | MTOL-P0P | |
MgSO4 | PanReac | 131404 | |
Mini Trans-Blot Cell | BioRad | 1703930 | |
MOPS | Sigma-Aldrich | M1254 | |
MTCO1 Monoclonal Antibody | Invitrogen | 459600 | |
NaCl | Sigma-Aldrich | S9888 | |
NADH | Roche | 10107735001 | |
NativePAGE 3 to 12% Mini Protein Gels | Invitrogen | BN1001BOX | |
NativePAGE Cathode Buffer Additive (20x) | Invitrogen | BN2002 | |
NativePAGE Running Buffer (20x) | Invitrogen | BN2001 | |
NDUFA9 Monoclonal Antibody | Invitrogen | 459100 | |
Nitroblue tetrazolium salt (NBT) | Sigma-Aldrich | N6876 | |
Pb(NO3)2 | Sigma-Aldrich | 228621 | |
PDVF Membrane | Amersham | 10600023 | |
Phenazine methasulfate (PMS) | Sigma-Aldrich | P9625 | |
Pierce ECL Substrate | Thermo Scientific | 32106 | |
PMSF | Merck | PMSF-RO | |
SDHA Monoclonal Antibody | Invitrogen | 459200 | |
Sodium succinate | Sigma-Aldrich | S2378 | |
Streptomycin/penicillin | PAN biotech | P06-07100 | |
Sucrose | Sigma-Aldrich | S3089 | |
Tris | PanReac | A2264 | |
UQCRC1 Monoclonal Antibody | Invitrogen | 459140 | |
XCell SureLock Mini-Cell | Invitrogen | EI0001 |