Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.
Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.
Mitochondria are highly dynamic organelles that undergo multiple rounds of fission and fusion, are transported to sites of high energy demand and respond rapidly to physiological stimuli1. Since it is increasingly recognized that mitochondria within different tissues, even different cellular compartments, have distinct functional profiles, new methods are needed to identify these distinct mitochondrial subsets.
Microscopy provides a means whereby individual mitochondria can be visualized and the presence of a protein at or in mitochondria can be determined by immunofluorescence2. However, quantitative analysis by this method is labor intensive and is more suitable for experiments using immortalized or primary cell lines. The study of individual mitochondria derived from tissue is significantly more difficult and most methods do not allow for easy identification of mitochondrial subsets concurrently with the evaluation of mitochondrial function3.
In order to address this hurdle, a novel method to immunolabel mitochondria isolated from rodent tissues and subsequently analyzed by flow cytometry has been developed. This allows for the rapid detection and quantification of proteins localized to the mitochondrial outer membrane, which compared to analysis by microscopy, is much less labor intensive and permits the analysis of thousands of mitochondria in a single sample. This assay can be applied to monitor the fate and relative amount of mitochondrial outer membrane proteins that are thought to be constitutively present at the mitochondria, the recruitment of proteins to the mitochondrial surface, or the detection of proteins mislocalized to the mitochondria in pathological conditions. Moreover, the incorporation of conventional fluorescent indicator dyes permits the simultaneous evaluation of certain aspects of mitochondrial function in distinct mitochondrial subpopulations.
Animals used in this study were treated in strict accordance to a protocol (N08001CVsr) approved by the Centre de Recherche du Centre Hospitalier de l’Université de Montréal (CRCHUM) Institutional Committee for the Protection of Animals which follows national standards as outlined by the Canadian Council on Animal Care (CCAC).
Prepare all reagents required to perform this protocol (Table 1). All other details regarding equipment, supplies and suppliers can be found in List of Materials.
Table 1. Buffer Compositions.
1. Collection of Rat Spinal Cord
2. Isolation of Spinal Cord Mitochondria (Adapted from Vande Velde et al. 4)
3. Immunolabeling of Isolated Mitochondria for Flow Cytometry
4. Assaying Mitochondrial Transmembrane Potential and Mitochondrial Superoxide Production by Flow Cytometry
5. Acquisition and Analysis of Immunolabeled Isolated Mitochondria by Flow Cytometry
Mitochondria derived from rat spinal cords can be immunolabeled with an antibody targeted to Mitofusin2 (Mfn2), a protein implicated in the fusion of the outer membrane of mitochondria8. Following isolation and labeling with a Mfn2 specific antibody and a fluorescently conjugated secondary antibody, mitochondria are processed by flow cytometry (Figure 1). Following data acquisition, samples are analyzed using flow cytometry analysis software, by first visualizing all collected events on a dot plot (Figure 2A). Doublets and singlets are differentiated when the events are plotted in FSC, width (W) versus area (A) (Figure 2B). Once singlets are selected, gate the mitochondrial population via FSC/SSC (Figure 2C), and verify the number of events staining positive for mitochondria-specific dye by co-plotting a histogram of the unstained sample with a sample stained with the mitochondria-specific dye. To determine the mitochondrial events, place a gate at the intersection of the two peaks (Figure 2D). For spinal cord preparations, typically >90% of the events are positive for the mitochondria-specific dye. For other tissues such as liver, ~98% of the events will label with the dye (data not shown). After selecting only events that label positively for the mitochondria-specific dye, determine background labeling with the isotype control by selecting a gate (Mfn2+) that includes 1% or less isotype labeling (Figure 2E, left). Apply this gate uniformly to all samples labeled with antibody Mfn2 to determine the percentage of mitochondria with Mfn2 present on the outer mitochondrial membrane (Figure 2E, right). In this experiment, 30% of mitochondria derived from spinal cord label positive for Mfn2. It is important to note here, that although Mfn2 is considered to be a ubiquitously expressed mitochondrial outer membrane protein, it has not been previously quantified. Moreover, an immunocytochemical analysis of Mfn2 in cultured cells shows a non-homogenous labeling of individual mitochondria9. It is also possible that the Mfn2 epitope was unavailable due to post translational modifications or due to interactions with itself or other binding partners. Of note, the antibody in this study was generated using a synthetic peptide to the N-terminus (amino acids 38-55). There is a predicted splice variant of Mfn2 lacking the first 302 amino acids, although this variant has yet to be confirmed experimentally (UniProt database). Thus, this assay is unable to detect alternatively spliced Mfn2 lacking the N-terminal sequence, given the antibody used.
Mitochondrial transmembrane potential (ΔΨm) and superoxide production can be assessed in this assay. Across the inner mitochondrial membrane, there is a separation of charge which drives ATP production via oxidative phosphorylation. TMRM is a cationic dye that accumulates within the mitochondria in a membrane potential dependent manner5, and therefore can be used as a reporter of mitochondrial transmembrane potential. The majority of mitochondria (95%) are TMRM positive after staining, compared to the unstained control (Figure 3A). However, when the uncoupler CCCP is added there is a significant decrease in the number of mitochondria able to retain TMRM (Figure 3A). CCCP allows the free passage of ions across the inner mitochondrial membrane, essentially destroying the separation of charge and depolarizing the membrane.
Mitochondria release superoxide as a normal byproduct of oxidative phosphorylation from complex I and III of the electron transport chain. Mitochondrial superoxide can be measured via a membrane permeable dye that is targeted to mitochondria and becomes fluorescent following a reaction with superoxide7. Functional mitochondria produce a basal amount of superoxide compared to unstained samples (Figure 3B). Addition of the complex III inhibitor Antimycin A yields an increase in superoxide, as seen by a rightward shift in florescence and a higher number of mitochondria (typically ~10%) that are fluorescent with this mitochondrial superoxide indicator dye (Figure 3B).
Figure 1. Schematic of isolation, immunolabeling and analysis of mitochondria. Step 1: Collect tissue and homogenize. Step 2: The isolation procedure contains eight centrifugation steps. Myelin, a major containment of central nervous system tissue is removed by diluting mitochondria in Iodixanol (density gradient medium) and centrifuging, resulting in the myelin floating to the top of the tube, while the mitochondria are pelleted. Step 3: Following isolation and quantification, mitochondria are blocked, labeled with primary antibody and washed. A secondary antibody conjugated to a fluorophore is then added. Unbound antibody is washed out. Step 4: At this point fluorescent dyes that report on mitochondrial purity, or mitochondrial function can be added. Step 5: Mitochondria are now ready to be analyzed by flow cytometry.
Figure 2. Strategy for the analysis of isolated mitochondria by flow cytometry. The Forward Scatter (FSC) and Side Scatter (SSC) voltages must be adjusted for small events, using both parameters in logarithmic mode. FSC width (FSC-W) data must be collected to exclude doublets. Note, on most flow cytometers, the default setting for FSC and SSC is linear mode. (A) Visualize all collected events on a dot plot. (B) Doublets, two mitochondria passing by the laser at the same time, can be distinguished from singlets by plotting FSC versus FSC-W (linear mode). Events are excluded if the FSC-W value is more than twice the mean FSC-W value of the majority of events, i.e., those that are part of the dense cloud. Events under this threshold are gated as singlets. (C) Again, visualize the events in a dot plot, and gate on the remaining events. (D) Plot a histogram of the unstained sample (solid, grey, filled) and sample stained with a mitochondrial specific dye (MSD: solid, green). Gate the events staining positive for the MSD (MSD+). (E) Histogram of the isotype control, rabbit IgG (dashed, black) and Mfn2 labeled sample (solid, pink). Set the gate so as to yield ≤ 1% Mfn2+ on the isotype control peak and apply this same gate to experimental sample to determine the percentage of events labeling positive for Mfn2 (Mfn2+). Please click here to view a larger version of this figure.
Figure 3. Assaying mitochondrial transmembrane potential (ΔΨm) and superoxide production in isolated mitochondria by flow cytometry. (A) Under basal conditions all active mitochondria will label with TMRM (solid, black), compared to unstained control (solid, grey, filled) because the dye accumulates in mitochondria with a transmembrane potential. Addition of the protonophore CCCP (dashed, blue) dissipates the transmembrane potential causing the mitochondria to depolarize and retain less dye compared to basal conditions. Data is reported as the delta mean fluorescent intensity (ΔMFI), which is the mean fluorescent intensity of the unstained control subtracted from the mean fluorescent intensity of the sample. (B) Under basal conditions, mitochondria produce superoxide as a by-product of oxidative phosphorylation. This mitochondrial source of superoxide can be assayed with a mitochondrial superoxide indicator, (MitoO2-: solid, black) compared to unstained control (solid, grey, filled). Addition of the complex III inhibitor, Antimycin A (dashed, orange) results in increased superoxide production, compared with basal levels. Data is reported as percent of cells staining positive for the MitoO2- indicator. Please click here to view a larger version of this figure.
It is increasingly evident that mitochondria are key players in both normal physiology and disease. While immunoblotting can determine which proteins are found within mitochondria or at the mitochondrial surface in a certain condition, this method reports on the average of the entire population. This method cannot yield information about relative abundances of mitochondrial subpopulations or subsets. While it has been previously assumed that all mitochondria are created equally, the field is increasingly recognizing that mitochondria within a cell have extensive variability in terms of morphology and/or function10.
Fluorescent microscopy approaches do take into account the heterogeneity of mitochondria. However, quantification of this type of data is labor intensive. Furthermore, this approach is better suited to studies using cultured cells, as labeling of mitochondria in vivo/in situ is difficult due to the excessive number of mitochondria present, making differentiation of individual organelles inherently difficult. In most immunocytochemistry protocols, it is also not possible to simultaneously label outer mitochondrial membrane proteins and assess mitochondrial function due to the cellular permeabilization step required for antibody labeling. The current method works with isolated mitochondria, and thus does not require a permeabilization step. In addition, the visualization of individual mitochondria is only possible via electron microscopy, which is not amenable to the analysis of mitochondrial function. That being said, we recognize that isolation of mitochondria from tissue leads to disruption of the mitochondrial network and this could affect some elements of mitochondrial function. However, comparisons of functional aspects of isolated mitochondria from tissues that are processed similarly remain valid.
This method of immunolabeling of tissue-derived isolated mitochondria and subsequent analysis by flow cytometry allows for a rapid and quantifiable method to detect and monitor the presence of a protein located on the outer membrane. Detection of a highly abundant mitochondrial protein (like Mfn2) is possible with this technique. Similarly, this method can detect low abundance proteins that are only deposited on the mitochondrial membrane in disease, like misfolded SOD1 in the context of Amyotrophic Lateral Sclerosis (ALS)11. In addition, this method could be useful to monitor the proteins that transiently associate with the mitochondrial surface as part of their normal function. Examples include Dynamin-Related protein-1 (Drp1), a cytosolic protein that is recruited to the mitochondria to promote mitochondrial fission12 and tumor necrosis factor receptor-associated factor 6 (TRAF6), which translocates to mitochondria to augment mitochondrial reactive oxygen species as a part of an innate immune response13.
At present this technique is amenable only to proteins located at the mitochondrial surface, as standard permeabilization protocols for intracellular labeling require a detergent that disrupts the structural integrity of mitochondria. While a number of possible reagents have been tried (unpublished), further optimization of the immunolabeling protocol is still needed to make this protocol amenable to detecting intra-mitochondrial components.
This technique has broad applications and can be used to detect the presence or recruitment of one or more proteins to the mitochondria under different experimental paradigms. Furthermore, mitochondria can be co-labeled with two different antibodies, as well as with fluorescent indicators11. Other fluorescent probes could also be incorporated to characterize additional aspects of mitochondrial function. For example, commercially available dyes to monitor mitochondrial pH14, calcium uptake with Calcium green-5N15, and ATP levels16 are possible.
The authors have nothing to disclose.
We thank Laurie Destroismaisons and Sarah Peyrard for outstanding technical support and Dr.Alexandre Prat for access to the flow cytometer. We would also like to acknowledge Dr. Timothy Miller for his contribution regarding the removal of myelin from the preparations. This work was supported by the Canadian Institutes of Health Research (CIHR) Neuromuscular Research Partnership, Canadian Foundation for Innovation, ALS Society of Canada, the Frick Foundation for ALS Research, CHUM Foundation and Fonds de la Recherche en Santé du Québec (C.V.V.). Both C.V.V. and N.A. are Research Scholars of the Fonds de la Recherche en Santé du Québec and CIHR New Investigators. S.P. is supported by the Tim Noël Studentship from the ALS Society of Canada.
Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
Rats | Charles River | Strain code 400 | Adult (9 weeks to 18 weeks) male or female rats can be used for the isolation protocol. Weight of rats is dependent on gender and age (males between 300 to 500 g and females between 200 to 350 g) are typically used. |
Dounce homogenizer | Kontes Glass Co. | 885450-0022 | Duall 22 |
Microcentrifuge | Thermo Scientific | Sorvall Legend Micro 17 R | |
Ulara-Clear Ultracentrifuge tubes | Beckman Coultier | 344057 | Transparent, thin walled |
Sorvall Ultracentrifuge | Thermo Scientific | Sorvall WX UltraSeries | |
AH-650 rotor and buckets | Thermo Scientific | ||
Opti-prep | Axis-Shield | 1114542 | Iodixanol, density gradient medium |
Fatty acid free Bovine Serum Albumin | Sigma | A8806 | Must be fatty acid free for mitochondria |
Sodium succinate dibasic hexahydrate | Sigma | S9637 | |
Rabbit anti-Mitofusin2 antibody | Sigma | M6319 | |
Rabbit IgG | Jackson Immuno Research | 011-000-003 | |
Anti-Rabbit IgG PE | eBioscience | 12-4739-81 | |
Micro titer tube | Bio-Rad | 223-9391 | For sample acquisition by flow cytometry |
MitoTrackerGreen (MTG) | Invitrogen | M7514 | 100 nM: Ex490 nm/Em516 nm |
TMRM | Invitrogen | T668 | 100 nM: Ex548 nm/Em574 nm |
CCCP | Sigma | C2759 | |
MitoSOX Red | Invitrogen | M36008 | 5 µM: Ex540 nm/Em600 nm |
Antimycin A | Sigma | A8874 | |
LSR II flow cytometer | BD | ||
BS FACSDiva Software | BD | ||
FlowJo | TreeStar Inc. | Software used for analysis | |
BCA protein assay kit | Pierce/Thermo Scientific | 23225 | Bradford assay is not recomended as it is not compatible with high concentrations of SDS |