Mitochondria are often referred to as the powerhouses of eukaryotic aerobic cells, but their functions go well beyond the generation of energy in the form of adenosine triphosphate (ATP). Mitochondria are also known to play roles in cell signaling, calcium homeostasis, immune responses (particularly those of the innate immune system), apoptosis, the biosynthesis of amino acids and phospholipids, and the pluripotency of stem cells. Because of these diverse roles, researchers now more than ever are profiling the role mitochondria play in a variety of diseases. Flow cytometry is proving to be a versatile and powerful platform for deciphering the functional profiles of mitochondria in different cell types. In this blog, we examine the fluid-structure and widespread roles of mitochondria, and how they are emerging as potential therapeutic targets.

The Structure of Mitochondria

Mitochondria typically range in size between 0.75 and 3µm, and unlike other organelles, they have two membranes that create different functional compartments within their structure. The outer membrane contains proteins such as cytochrome C that play major roles in energy conversion and apoptosis. In addition, the outer membrane contains various porin proteins that form channels that make this membrane highly permeable. Due to this porosity, there is no membrane potential across the outer mitochondrial membrane. This is in stark contrast with the highly impermeable inner mitochondrial membrane, which forms folds known as cristae that extend into the mitochondrial matrix (Fig. 1.). These cristae house the majority of the fully assembled complexes coordinating the electron transport chain for ATP synthesis. In tissues with very high energy demands, these cristae become highly stacked within the mitochondria- presumably to maximize the surface area for ATP production within each organelle. The compartment between the outer and inner membranes is known as the intermembrane space – this compartment has a low pH of 7.2 to 7.4, resulting in the creation of a proton gradient across the inner membrane.

The movement of ions and small molecules across this inner membrane is mediated by highly selective membrane transport proteins that function to generate an electrical membrane potential of ~180mV across the inner membrane – this is used to drive ATP synthesis. In fact, most adults generate ~50kg of ATP per day, a staggering feat of metabolism considering the size of these organelles. Within the inner membrane is the matrix compartment with a pH of 7.9-8.0- this high pH serves to maintain the transmembrane electrochemical gradient. The matrix is rich in proteins (up to 500mg/mL) and houses the enzymes of the citric acid cycle, as well the ribosomes and mitochondrial transcription factors that control the expression of proteins from the mitochondrial DNA. There are 37 genes contained within the mitochondrial genome, 13 of which encode various proteins of the electron transport chain.

Figure 1. Mitochondria are formed into 4 distinct compartments- the highly permeable outer membrane separates the mitochondrial from the cellular cytoplasm. The compartment between the outer and inner mitochondrial membranes is referred to as the intermembrane space. The proton gradient between the intermembrane space (pH 7.2-7.4) and the matrix (pH 7.9-8.0) drives the production of ATP within the cristae folds of the mitochondria.

The Fluid Morphology of Mitochondria

The morphology and cellular distribution of mitochondria vary widely between different types of cells, for example, fibroblast mitochondria are long filaments (1-10µm) of a distinct diameter (~700nm), whereas those observed in hepatocytes are ovoid or uniform spheres (Das, et. al. 2012). Within the endothelium, a network of mitochondria is frequently observed (Shenouda, et. al. 2011); although the functional significance of this is still unclear. Flow cytometry methods have identified three distinct forms of mitochondria from various tissues including kidneys, pancreatic acinar, and cardiac myocytes (Saunders, 2012). These mitochondrial populations appear to respond differently to cytosolic Ca2+ signals, indicating distinct functions within these cells (Park, et. al. 2001).

Mitochondrial morphology and cellular location have been shown to change during cell cycle progression within rat kidney epithelial cells; from isolated, distinct structures in G1 to a fused network surrounding the nucleus during the S phase (Mitra, et. al. 2009). This movement of mitochondria through the cytosol is thought to be mediated through the kinesin and dynein networks of proteins. This mobility, coupled with fusion and fission-mediating proteins within the mitochondria themselves all interact to create a diverse array of mitochondrial structures within cells. We are only now beginning to decipher these structures, but a growing number of studies link disease progression with changes in size and morphology of mitochondria in diabetes (Shenouda, et. al. 2011), pulmonary hypertension (Marsboom, et. al 2012), and ischemic reperfusion injury (Brooks, et. al. 2009).

Flow Cytometry Analysis of Mitochondria

Flow cytometry techniques have been successfully applied to quantitatively interrogate mitochondria within cells, as well as ex vivo. These techniques rely on the use of fluorescent probes, that selectively discriminate different features of mitochondria, and their use is helping researchers understand how mitochondria respond to different stimuli, and profile mitochondria in different cell types and disease states.

Early examples included studies using Rhodamine-123 (Rh123), 3,3′-dihexyloxacarbocyanine iodide (DiOC6(3)), or 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide for the evaluation of mitochondrial membrane potential in various cell types, however, these probes display high levels of non-specific binding and may interfere with mitochondrial function and should therefore be carefully titrated.

The stain 10-nonyl acridine orange interacts directly with the cardiolipin in the inner mitochondrial membrane and can be used in combination with tetramethylrhodamine-methyl-ester and 1,1′,3,3,3′,3′-hexamethylindodicarbocyanine iodide to measure mitochondrial membrane potential with high specificity (Mattiasson, 2004).

More recently, the family of MitoTracker probes has been popular for studying mitochondrial systems in cells. They are highly specific, easy to use, and work across many different cell types without inhibiting cellular processes, making them the go-to mitochondrial probes for flow cytometry applications.

MitoTracker Green is a stain used to measure the mitochondrial mass (otherwise known as mitochondrial load) of cells under typically physiological conditions. This probe selectively binds covalently to mitochondrial proteins through cysteine residues, staining the matrix compartment. The MFI is proportional to the mitochondrial load of the cells. The reaction is considered to be completely independent of the mitochondrial membrane potential. However, there is some evidence that it MitoTracker Green binding may be altered by oxidative and nitrosative stress, and it is therefore important to understand the reactive oxygen species (ROS) and reactive nitrogen species (RNS) state of the mitochondria before employing MitoTracker Green for measuring mitochondrial mass (Doherty, 2017).

Mitotracker Red is a stain used to evaluate mitochondrial function. This probe is taken up by polarized, negatively charged mitochondria, and is therefore dependent on the mitochondrial membrane potential (ΔΨ_m). Dysfunctional mitochondria displaying a collapsed or compromised membrane potential display reduced labeling by MitoTracker Red.

Using a combination of MitoTracker Green and MitoTracker Red staining, we can apply flow cytometry to evaluate how different drugs can modify the levels of respiratory and dysfunctional mitochondria within distinct cell populations. The MFIs from each of these dyes can provide a quantitative measure of mitochondrial mass and membrane potential.

MitoSOX Red is a positively charged probe that can rapidly accumulate in mitochondria. Upon exposure to superoxide, the molecule becomes hydroxylated and exhibits a fluorescence excitation peak of ~400nm, an emission peak of ~590nm that is not seen in the oxidation products generated by other ROS. Mitochondria are the major source of cellular ROS, mainly in the form of superoxide, but these molecules can pass into the cytosol, and therefore to measure only mitochondrial-generated superoxide, the MitoSOX should be titrated on the cell population of interest. MitoSOX Red staining can be incorporated into cell cycle analysis as outlined in Fig. 2c.

Mitochondrial function plays a central role in immune cell function and the inflammatory response (Breda, et. al 2019), and using some of these cell stains has enabled researchers to examine changes in mitochondrial profiles in near real-time. The role mitochondria play in T-cell activation is now well characterized opening the possibility for new strategies to restore T-cell vigilance by increasing mitochondrial function coupled with enhanced purigenic feedback mechanisms (Ledderose, 2015).

Unstimulated immune cells such as macrophages contain mitochondria in a highly polarized state with a stable negative membrane potential across the inner mitochondrial membrane. Upon stimulation with LPS for example, we see a loss in ΔΨ_m through a decrease in the MitoTracker Red signal, whereas no change in the mitochondrial mass is observed, with consistent staining with MitoTracker Green (Fig. 2a). In fact, when macrophages are activated with LPS, they undergo metabolic reprogramming from oxidative phosphorylation to glycolysis leading to mitochondrial dysfunction (Eddit, et. al. 2017). The proportion of dysfunctional mitochondria across different cell populations can also be profiled effectively by flow cytometry (Fig. 2b).

Figure 2. Adapted from Monteiro et. al. 2020

1. Analysis of mitochondrial mass and membrane potential in bone-marrow-derived macrophages (BMDM). Cells were treated with 100ng/mL LPS for 6 hours before staining with the macrophage surface maker F4/80. Live/Dead and the mitochondrial probes MitoTracker Green and MitoTracker Red. Representative MFIs for mitochondrial mass (Mitotracker Green) and ΔΨ_m (Mitotracker Red) demonstrate that the mitochondrial mass is unchanged, whereas the ΔΨ_m is significantly reduced in the presence of LPS.

1. Flow cytometric quantification of the percentage of functional versus dysfunctional mitochondria in macrophages treated with PBS (control) and LPS. BMDMs were treated with 10ng/mL LPS for 6 hours before staining with F4/80 to label macrophages, live/dead, and the mitochondrial probes MitoTracker Green and MitoTracker Red.

   Functional mitochondria (MitoTracker Green ^high/Mitotracker Red ^high)

   Dysfunctional mitochondria (Mitotracker Green ^high/Mitotracker Red ^low).

1. Mitochondrial superoxide production by M1 macrophages can be evaluated using the MitoSOX stain. BMDMs were treated with 20ng/mL IFN-γ + 100nm/mL LPS for 6 hours- this induces an M1 phenotype within the macrophage population. Staining the cells with the macrophage surface marker CD11b, Live/Dead and MitoSOX Red enables the macrophages to be gated out and the mitochondrial superoxide production in the M0 and M1 populations to be compared.

The Role of Mitochondria in Apoptosis

Apoptosis is characterized by both nuclear and cytosolic condensation and the fragmentation of the cell contents into membrane-bound apoptotic bodies that are phagocytosed by other cells. All of this occurs without an inflammatory response and with minimal disruption to surrounding tissues. It is a highly orchestrated process or ATP-dependent steps including caspase activation, enzymatic hydrolysis of cellular components, chromatin condensation, and apoptotic body formation. Caspases are central to apoptosis; they exist as inactive proenzymes that when cleaved activated the apoptotic pathways. There are two major apoptotic pathways: extrinsic and intrinsic (Fig. 3). Essentially the extrinsic pathway commences outside of the cell and is triggered when extracellular conditions determine that the cell must die. Typically, this involves ligand binding to a death receptor such as TNF-α to TNFR1. In contrast, the intrinsic apoptosis pathway is triggered when an event occurs within the cell, resulting in stresses that activated apoptosis. It is this intrinsic pathway that at least in mammalian systems, involves the interaction of anti-apoptotic (such as BCL-2) and proapoptotic proteins within the mitochondria. Mitochondrial cytochrome C is a component of the activation complex of the apoptosis initiator caspase-9; this triggers the release of Smac (second mitochondria-derived activator of caspase) and Omi from mitochondria into the cytosol and nucleus where these proteins bind to inhibitors of apoptosis (IAPs) and relieve their inhibition. Mitochondrial permeability transition appears to be a pivotal event in apoptosis and a point of no return. This transition is associated with several features including changes in membrane potential, a decrease in ATP:ADP, an increase in calcium within the mitochondrial matrix, and an increased level of oxidative stress and mitochondrial permeability. These can be tracked specifically within the mitochondria using probes such as Rhod-2AM to monitor calcium levels within mitochondria and calcein to track changes in mitochondrial permeability.

Figure 3. Schematic summary of the extrinsic and intrinsic apoptosis pathways. In both instances, the loss of mitochondrial integrity amplifies the cascade driving apoptosis.

The Role of Mitochondria in Stem Cell Regulation

Even though many types of stem cells have a low mitochondrial mass (Rafalski et. al. 2012), there is a growing body of evidence demonstrating the central role that mitochondria have in stem cell activation, pluripotency, dissociation, and defense against senescence. These factors are leading research towards mitochondrial therapeutic targeting for regenerative medicine.

Different types of stem cells rely on different metabolic pathways for energy, for example, cancer stem cells rely on glycolysis (Chen. 2016), whereas hematopoietic progenitors use both anabolic glycolysis and oxidative phosphorylation (Piccoli, et. al. 2005). However, stem cells can also utilize mitochondrial fatty acid oxidation in addition to glycolysis during self-renewal. Inhibition of this process alters the asymmetric division properties of stem cells resulting in a loss of their ability to maintain a reserve pool during an expansion (Ito et. al. 2012). Indeed, asymmetrical stem cell division provides a mechanism to clear away older mitochondria from this reserve pool and help maintain the ‘stemminess’ of the stem cell population. Interestingly, during conversion, induced pluripotent stem cells (iPSCs) display a transition from oxidative phosphorylation to glycolysis that correlates with a change in mitochondrial morphology to a more immature state (Prigione et. al. 2010).

Mitochondria have also been shown to program stem cell fate through the movement of mitochondrial-derived TCA cycle intermediate metabolites such as acetyl-CoA, α-ketoglutarate, and citrate into the nucleus where they impose epigenetic regulation of DNA and histones. Furthermore, the generation of reactive oxygen species (ROS) by mitochondria has also been shown to regulate somatic stem cell fate, with an increase in ROS being associated with a decrease in the regeneration potential of human mesenchymal stem cells, correlating with progenitor commitment and differentiation.

Flow cytometry represents one of the few platforms that can comprehensively interrogate the mitochondrial status of stem cells and describe the mitochondrial mass, ROS, mtDNA copy number, and mitochondrial volume (Liang, et. al. 2021). This type of research has important implications for how stem cells such as hematopoietic stem cells are expanded ex vivo for clinical applications to maximize their self-renewal properties and more fully understand the mechanisms underlying stem cell fate decision-making (Vannini et. al 2016).

Final Thoughts

Mitochondrial abnormalities are now known to be associated with the pathogenesis of a wide range of cellular-degenerative – and hyperproliferative diseases. As such, they are now being evaluated for early diagnostic applications and as therapeutic targets across many areas of medicine.

Therapeutic intervention at the mitochondrial level has been envisioned for several neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, Huntingdon’s disease, and Amyotrophic lateral sclerosis (ALS). Although these diseases have different underlying roots, they all display mitochondrial dysfunction that increases ROS production leading to unmanageable oxidative stress and cell damage. Therapeutic intervention with Dimebon, an antihistamine that appears to stabilize mitochondria, and mitochondrial-targeted metabolic antioxidants (creatine, α-lipoic acid, N-acetyl-carnitine, and coenzyme Q10) and S-S peptides have all shown some promise in managing disease progression in animal models and small clinical trials (Moreira, 2010)

Similarly, the cardiovascular disease involves mitochondrial function disruption that results in a bioenergetic mismatch between energy supply and demand in cardiac tissues, coupled with an increase in ROS generation resulting in cellular damage. Several drug-based approaches to managing mitochondrial redox regulation and ion transport have been shown to improve cardiac function and metabolism in patients with heart failure.

We know more about mitochondria than any other organelle, and it is clear that they support a diverse array of cellular processes. Flow cytometry is enabling researchers to characterize mitochondria within different cell populations and interrogate their complex functions. This type of research is revealing the role of mitochondria in cellular health, and now mitochondrial-targeted therapies appear to hold great promise, particularly for neurological and metabolic diseases with very few therapeutic options.

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