Isolation of Mitochondria for Mitochondrial Supercomplex Analysis from Small Tissue and Cell Culture Samples

Raquel Moreno-Loshuertos; Patricio Fern&#225;ndez-Silva

doi:10.3791/66771

JoVE Journal > Biochemistry

Bioquímica

Isolation of Mitochondria for Mitochondrial Supercomplex Analysis from Small Tissue and Cell Culture Samples

Published: May 03, 2024

doi:

10.3791/66771

Raquel Moreno-Loshuertos^1,2, Patricio Fernández-Silva^1,2

¹Department of Biochemistry,University of Zaragoza, ²Institute for Biocomputation and Physics of Complex Systems,University of Zaragoza

Summary

This protocol describes a technique for the analysis of respiratory supercomplexes when only small amounts of samples are available.

Abstract

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.

Introduction

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.

Protocol

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.

Grow adherent cells to approximately 80% confluence or suspended cells to an adequate density.
Harvest cells by centrifugation at 500 × g for 5 min (directly from the cell suspension in the case of non-adherent cells or after standard trypsinization with a trypsin-EDTA solution of 0.05% trypsin and 0.02% EDTA in 1x PBS, in the case of adherent cells).
Wash cells 2x with cold 1x PBS and sediment them by centrifugation at 500 × g for 5 min.
NOTE: From this point, all the steps must be performed at 4 °C. Therefore, all the reagents must be cold and the tubes containing cells or mitochondria must be kept on ice.
After the second centrifugation, discard the supernatant, estimate the cell pellet volume (cpv), and freeze the cells at -80 °C for at least 15 min to facilitate membrane breaking in step 1.8.
NOTE: The protocol can be stopped at this point and the cells can be stored at -80 °C for weeks. Usually, 10 or 15 mL graduated tubes with conical bottoms are appropriate.
Let the cells thaw slowly on ice.
Resuspend the packed cell pellet in a volume of hypotonic buffer equal to 7x the cpv (10 mM MOPS, 83 mM sucrose, pH 7.2) (e.g., 100 µL of packed cells in 700 µL of buffer).
NOTE: The volume must be enough to obtain efficient pops (see NOTE at step 1.8).
Transfer the cell suspension into a Potter-Dounce homogenizer of the appropriate size and let the cells swell by incubating them in ice for 2 min. For example, for a volume of 700-800 µL of cell suspension, a 1 mL capacity homogenizer is adequate.
Break the cell membranes by performing eight to ten strokes in the homogenizer coupled to a motor-driven Teflon pestle rotating at 600 rpm.
NOTE: When making the strokes, it is important to create a vacuum that will increase the cell breakage efficiency along with the hypotonic and freezing actions for swelling and fragilizing the membranes, respectively. If it is well done, a "pop" sound will be heard when pulling down the homogenizer with a quick movement at each stroke.
Add the same volume of hypertonic buffer to the cell suspension (7x the cpv) (30 mM MOPS, 250 mM sucrose, pH 7.2) to generate an isotonic environment.
Transfer the homogenate into a 10-15 mL tube and centrifuge in a fixed rotor at 1,000 × g for 5 min at 4 °C.
NOTE: The original tube containing the unbroken cells can be used for this purpose.
Transfer the supernatant, which contains the mitochondrial fraction, into 1.5 mL polypropylene tubes.
OPTIONAL: To increase the yield of mitochondria, resuspend the pellet from step 1.10 in 7x the cell pellet volume of buffer A (10 mM Tris, 1 mM EDTA, 0.32 M sucrose, pH 7.4) by pipetting up and down and centrifuge at 1,000 × g for 5 min at 4 °C. Combine the new supernatant with the previous one before proceeding to step 1.13.
Centrifuge in a microfuge at 16,000 × g for 2 min at 4 °C to collect the mitochondrial crude fraction.
Discard the supernatant and resuspend each mitochondria-enriched pellet with 0.5 mL of buffer A combining the contents of two tubes into one and centrifuge under the same conditions as described in step 1.13. Repeat the same process until all the material is in only one tube.
Discard the supernatant, resuspend the final pellet with 300 µL of buffer A, and quantify mitochondrial protein concentration using the Bradford assay. Centrifuge again as before and proceed to section 3.

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.

Weigh or estimate as closely as possible the amount (mg) of tissue.
Cut the tissue with a pair of scissors and make 3-4 washes in homogenization buffer with the help of a strainer taking care to avoid losing the smaller pieces.
NOTE: This step is more important in tissues like skeletal muscle or heart than in the brain or softer tissues whose cells can be easily disaggregated directly by the homogenization step.
Add fresh homogenization medium (4 mL per gram of liver, 10 mL per gram of heart, brown adipose tissue (BAT) or muscle, and 5 mL per gram of brain or kidney; see specific buffer composition for each case in Table 1).
Transfer the pieces of tissue with buffer to the homogenizer and follow the optimal homogenization and mitochondria isolation protocol depending on the selected tissue.
Isolation of mitochondria from liver, spleen, and kidney
1. Homogenize with four to six up-and-down strokes in the Elvehjem-Potter with a motor-driven Teflon pestle at 600 rpm.
2. Transfer the homogenized tissue to a sterile centrifuge tube. Centrifuge in a swinging rotor at 1,000 × g for 5 min at 4 °C.
3. Fill 1.5 mL polypropylene tubes with the supernatant obtained in the previous step. Centrifuge for 2 min at 16,000 × g in a microfuge at 4 °C and proceed hereafter as described before (steps 1.13 to 1.15)
Isolation of mitochondria from heart and muscle samples
1. Homogenize with six to eight strokes in the Elvehjem-Potter with a motor-driven Teflon pestle at 600 rpm. Transfer the homogenized tissue to a sterile centrifuge tube.
2. Centrifuge in a swinging rotor at 1,000 × g for 5 min at 4 °C. Pour the supernatant into clean 1.5 mL polypropylene tubes.
  NOTE: A second homogenization of the pellet obtained in step 2.6.2 can be performed with 4-5 additional strokes and ½ volume of homogenization buffer to increase the mitochondrial yield. The new supernatant (after centrifugation again at 1,000 × g for 5 min at 4 °C) can be combined with the previous one before proceeding to step 2.6.3. An alternative to increase the crude mitochondria yield is to dissociate heart and muscle tissue samples in trypsin solution before homogenization³⁶^,³⁷. In this case, the trypsin has to be efficiently inactivated/removed before the mitochondrial membranes are solubilized with digitonin (step 3.2).
3. Centrifuge at 16,000 × g in a microfuge for 2 min at 4 °C to obtain the crude mitochondrial fraction.
4. Discard the supernatant and resuspend each mitochondria-enriched pellet with 0.5 mL of buffer AT combining the contents of two tubes into one and centrifuge under the same conditions as described in step 2.6.3. Repeat the same process until all the material is in only one tube.
5. Discard the supernatant, resuspend the final pellet with 300 µL of buffer A (no BSA), and quantify mitochondrial protein concentration using the Bradford assay. Centrifuge again as before and proceed to section 3.
Isolation of mitochondria from brain
1. Homogenize the pieces of the brain with 10-15 strokes using a Dounce-type glass homogenizer with a manually driven glass pestle. Transfer the homogenized tissue to a sterile centrifuge tube.
2. Centrifuge in a swinging rotor at 1,000 × g for 5 min at 4 °C.
3. Collect the supernatant into a clean centrifuge tube.
4. Resuspend the pellet obtained in the previous centrifugation step in the same volume of medium AT used in the first homogenization. Re-homogenize the pellet by repeating the process described in step 2.7.1 using 5-10 passes and centrifuge the suspension in a swinging rotor at 1,000 × g for 5 min at 4 °C.
5. Remove the supernatant and add it into the tube prepared in step 2.7.3. Centrifuge at 10,000 × g in a fixed angle rotor for 10 min at 4 °C to obtain the crude mitochondrial fraction.
6. Discard the supernatant and resuspend the pellet into 1/2 the initial homogenization volume (step 2.7.1) of medium AT. Distribute the mitochondrial suspension into clean 1.5 mL polypropylene tubes and centrifuge at 16,000 × g in a microfuge for 2 min at 4 °C.
7. Discard the supernatant and resuspend each mitochondria-enriched pellet with 0.5 mL of buffer AT combining the contents of two tubes into one and centrifuge under the same conditions as described in step 2.7.6. Repeat the same process until all the material is in only one tube.
8. Discard the supernatant, resuspend the final pellet with 300 µL of buffer A (no BSA), and quantify mitochondrial protein concentration using the Bradford assay. Centrifuge again as before and proceed to section 3.
Isolation of mitochondria from BAT
1. Homogenize with eight to ten up-and-down strokes in the Elvehjem-Potter with a motor-driven Teflon pestle at 600 rpm. Transfer the homogenized tissue to a sterile centrifuge tube.
2. Centrifuge in a swinging rotor at 1,000 × g for 5 min at 4 °C.
3. Fill 1.5 mL polypropylene tubes with the supernatant obtained in the previous step avoiding the upper fat layer.
  NOTE: A second homogenization of the pellet obtained in step 2.8.2 can be performed to increase the mitochondrial yield. The new supernatant (after centrifugation again at 1,000 × g for 5 min at 4 °C) can be combined with the previous one before proceeding to step 2.8.4.
4. Centrifuge for 2 min at 16,000 × g for 2 min in a microfuge at 4 °C.
5. Discard the supernatant and resuspend each mitochondria-enriched pellet with 0.5 mL of buffer AT2 combining the contents of two tubes into one and centrifuge under the same conditions as described in step 2.8.4. Repeat the same process until all the material is in only one tube.
6. Discard the supernatant, resuspend the final pellet with 300 µL of buffer A (no BSA), and quantify mitochondrial protein concentration using the Bradford assay. Centrifuge again as before and proceed to section 3.

3. Preparation of samples for Blue Native analysis

Resuspend the mitochondrial fractions (obtained from mammalian cell cultures or tissues) in BN sample buffer (50 mM NaCl, 50 mM Imidazole, 5 mM Aminocaproic acid, 4 mM PMSF) to obtain a protein concentration of around 10 mg/mL. See Table 2 for expected yields for the different types of samples.
Solubilize mitochondrial membranes by adding digitonin (10% stock solution) to obtain a ratio of 4 g of digitonin/g of mitochondrial protein (4 µL of digitonin 10% stock for 10 µL of the mitochondrial suspension prepared in step 3.1).
NOTE: The detergent-to-protein ratio (g/g) must be optimized for each type of sample to obtain reproducible results; 2-8 g of digitonin/g of mitochondrial protein was found to be optimal for most tissues³⁴^,³⁵.
Mix by gently pipetting up and down. Incubate samples on ice for 5 min.
NOTE: After this step, the mitochondrial suspension should become clearer (shift from opaque to translucent); otherwise, it could indicate that the amount of detergent is insufficient for correct membrane solubilization.
Centrifuge at full speed in a microfuge (20,000 × g approx.) for 25 min at 4 °C to remove insoluble material.
Collect the supernatant in a fresh tube. Add to the supernatant a volume of 5% G-250 (Coomassie Blue G-250 5% in 0.75 M aminocaproic acid) equivalent to 1/3 of the initial resuspension volume (step 3.1, this would correspond to a final proportion of 1.6 g of Coomassie/g of protein) and mix by pipetting.
Keep on ice before loading on the gel or freeze aliquots at -80 °C.

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.

Load the upper and lower chambers with the cold cathode A and anode buffers, respectively. Alternatively, load the samples in the wells before carefully filling the upper chamber with cathode buffer A.
NOTE: Commercial electrophoresis buffers are used in our laboratory, but they can be prepared as indicated in Table 1.
Load between 30 and 100 µg of mitochondrial protein.
NOTE: Usually, 30-50 µg of protein (for WB), or 40-100 µg of protein (for IGA) are loaded per well in a 10 lane gel (0.5 x 0.15 cm wells). Cathode A buffer contains Coomassie Blue G-250 and has an intense blue color that makes it difficult to see the gel wells for sample loading. It is convenient to mark the center of each well, with a red marker, for example, to help in placing the pipette tip during this step. Native molecular weight markers can be useful when setting up the BN-PAGE technique to confirm that the gradient gels are properly formed and that the complexes and SCs pattern is the correct one.
Run at 80-100 V for 25-30 min and then at 160-180 V, limiting the current to 12 mA/gel, until the dye reaches the bottom of the gel (~125-165 min in total).
If in-gel activity assays (IGAs) are to be performed, substitute the cathode buffer A with cathode buffer B when the dye front is in the middle of the gel (~1 h after the beginning of the run).
NOTE: Although the gels can be documented without any staining just after the run since some bands are visible (mainly complex V, which can be considered an "internal" maker with a MW of ~600 kDa) due to their binding to G-250 dye, usually, SCs and individual complexes will not be visible without staining or IGA assays. The gels to be used in IGA assays or for WB should not be stained or fixed.
Disassemble the gel from the cassette and continue with Coomassie blue staining (section 5), IGA analysis (section 6), or WB immunodetection (section 7).

5. Gel staining

Stain the gel for 10-15 min at room temperature (RT) using Coomassie blue dye solutions (Coomassie dye R-250 at 0.25% in 40% methanol, 10% acetic acid; stain for 10-15 min).
Destain with several washes (typically 4-5 x 15 min) in 40% methanol plus 10% acetic acid at RT.
Document the gel.

6. In gel-activity (IGA) assays

Prepare IGA solutions for the analysis of the different respiratory complexes before the end of the electrophoresis and maintain them in the dark (by using dark-colored plastic boxes or covering them with aluminum foil, for example). The composition of each buffer is detailed in Table 1.
Place the gel or the lanes to be used in a plastic box as small as possible to accommodate it.
Add enough volume of the appropriate solution to cover the gel (usually 5 or 10 mL for 5 or 10 gel lanes, respectively) and incubate at RT with gentle shaking (60-80 rpm) and away from light.
NOTE: The time of incubation depends on the complex to be analyzed and on the nature and amount of sample loaded; CI and CIV are the easiest and fastest to give results. Thus, CI activity will usually start to become visible after a few minutes, while CII and CIV need ~30 min to be observed and CV ~1.5-2 h. The reaction can continue for hours in all cases and the rate of signal intensification can be reduced by moving the incubation to a cold room (4-6 °C), for example, for an overnight (ON) incubation. CIII activity works only for clear native electrophoresis (CN), not for BN³⁴.
When the appropriate bands have developed, stop the reactions by removing the assay solutions, washing 2x with distilled water, and fixing the gel with 40% methanol plus 10% acetic acid (except for CV which is fixed only with methanol) for 30 min.
1. To analyze CIV activity after CI detection by IGA, wash the gel 2x with distilled water, document CI activity, incubate the gel (without fixing it!) with 50 mM potassium phosphate buffer (pH 7.2) 2x for 30 min, and then with the complete CIV reaction buffer until the complex IV bands appear (usually by incubating ON at RT or 4 °C).
Document the gels.
NOTE: For ATPase activity, as the developed bands are white, when documenting the gel, place it on a dark background so that the clear bands would be visible.

7. Western blot analysis

Prepare transfer buffer according to Table 1 and keep at 4 °C until the end of the electrophoresis.
Place the gel in a tray and add transfer buffer. Incubate at RT for 10-15 min.
Cut a piece of PVDF membrane of the same size as the gel and activate it in methanol for 10 s under agitation. Wash several times with distilled water and add transfer buffer. Incubate at RT for 10-15 min with gentle shaking.
Prepare the transfer sandwich, from bottom to top, avoiding bubbles between the gel and the membrane, in the following order: black side of cassette-sponge-blotting paper-gel-membrane-blotting paper- sponge-clear side of cassette.
Close and lock the cassette and put the sandwich in the transfer tank in the correct orientation for transfer (black side towards the negative pole).
Fill the transfer tank with transfer buffer, add a magnetic stirrer to agitate the buffer, and connect the power supply at 80 V for 2 h or at 100 V for 1 h. Perform the transfer between 4 and 8 °C (for example in the cold room) and with a cooling block in the transfer system.
Retrieve the membrane and continue with a standard western blot protocol, using specific antibodies for the different respiratory complexes to be detected²⁹.

Representative Results

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 (L929^Balbc) 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. L929^Balbc 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 (*).

Discussion

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.

Declarações

The authors have nothing to disclose.

Acknowledgements

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.

Materials

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

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Moreno-Loshuertos, R., Fernández-Silva, P. Isolation of Mitochondria for Mitochondrial Supercomplex Analysis from Small Tissue and Cell Culture Samples . J. Vis. Exp. (207), e66771, doi:10.3791/66771 (2024).