Mitochondria Dyes and Probes

Mitochondria Organelle Structure and Function

Mitochondria are approximately 2 μm long and are found in the cytoplasm of most eukaryotic cells, one exception being mature red blood cells. Other simple organisms have been found to function with either mutated mitochondria structures or no mitochondria at all e.g. Monocercomonoides.

Mitochondria found in present day mammals arose out of a symbiotic relationship with a bacterial mitochondrion ancestor and have their own distinctive set of DNA: mitochondrial DNA or mtDNA. The mitochondrial DNA encodes 13 proteins for the electron transport chain (also known as the respiratory chain) and some mitochondrial tRNAs, but most of the mitochondrial proteins are encoded by human nuclear genes. Mitochondrial DNA is inherited through the maternal line and so does not follow the rules of Mendelian inheritance.

Mitochondria are double membrane-bound cell organelles, comprising inner and outer membranes that separate the intermembrane space and the matrix. The outer membrane contains a large number of enzymes, proteins and transporters involved in metabolite transport, elongation of fatty acids and calcium signaling, among other functions. One important transporter found in the outer membrane is the voltage-dependent anion channel (VDAC). This channel is the main transporter of nucleotides, ions and metabolites between the cytosol and the intermembrane space. The inner mitochondrial membrane contains proteins involved in the electron transport chain and ATP synthase. The inner membrane of the mitochondrion forms folds or cristae; these compartmentalized folds provide greatly increased surface area for increased ATP production. Cells with higher ATP demand, such as muscle cells have many more cristae folds. The matrix contains all the enzymes involved in the Krebs cycle, mitochondrial ribosomes, tRNA and mtDNA.

The main function of mitochondria is cellular respiration and the generation of ATP, by oxidizing sugars, proteins and fats. However, mitochondria also play a central role in cell signaling, stem cell differentiation, the cell cycle, calcium signaling and cell death. The composition of mitochondrial cristae and the cellular location of mitochondria are tightly regulated to ensure a constant mitochondrial population with the correct morphology corresponding to the needs of each specific cell type. This is achieved through biogenesis, autophagy, fission and fusion.

Figure 1: Mitochondria structure and function. Top: shows mitochondria location within the cell's cytoplasm. Left: shows basic mitochondria structure. Right: shows location of key enzymes and transporters.

Mitochondria Bioenergetics

Mitochondria metabolize the main metabolic products of glucose, namely pyruvate and nicotinamide adenine dinucleotide (NADH), through the process of cellular respiration, also known as aerobic respiration.

Sugars undergo glycolysis in the cells' cytosol and enter the mitochondria as pyruvate, where it is converted into acetyl-CoA by pyruvate dehydrogenase. Acetyl-CoA then enters the Krebs cycle, also known as the citric cycle or tricarboxylic acid (TCA) cycle. Fatty acids are converted into acetyl-CoA in the mitochondria via β oxidation, while there are several mechanisms and a series of different enzymes for conversion of amino acids into pyruvate and acetyl-CoA.

In the Krebs cycle, acetyl-CoA goes through a sequence of enzymatic steps. Excess carbon atoms are removed as carbon dioxide, while electrons (or energy) are passed to NADH and flavin adenine dinucleotide (FADH₂), which then enter the electron transport chain (ETC).

NADH and FADH₂ undergo redox reactions powering the pumping of protons from the matrix to the intermembrane space against the concentration gradient. This generates a mitochondrial membrane potential at 200 mV across the inner mitochondrial membrane, which is used to drive the synthesis of ATP by ATP synthase. Oxidative phosphorylation of NADH and FADH₂ produces 3 and 2 molecules of ATP, respectively.

Five Steps of the Electron Transport Chain

1. NADH₂ undergoes redox reactions; electrons go to complex I (NADH dehydrogenase) and four protons (H⁺) are pumped into the intermembrane space.
2. FADH₂ undergoes redox reactions; at complex II (succinate dehydrogenase), electrons are transferred to the lipid soluble redox carrier coenzyme Q and transported to complex III (cytochrome c reductase). (Unique among the respiratory chain complexes, complex II is completely encoded by nuclear DNA and does not pump any protons across the intermembrane.)
3. Complex III oxidizes coenzyme Q and transfers electrons to two molecules of cytochrome c. Four protons are pumped into the intermembrane space, two derived from coenzyme Q and two translocated from the mitochondrial matrix (cytochrome c plays a critical role in apoptosis)
4. Redox of complex IV (cytochrome c oxidase); two protons are pumped into the intermembrane space and the electrons that are passed along the chain, drive the formation of water from O₂ and the four electrons that were donated from cytochrome c.
5. Complex V (ATP synthase) carries out the final steps of mitochondrial oxidative phosphorylation, the formation of ATP. Complex V acts as a rotary molecular motor; protons pass through complex V and power the rotation of the stalk portion of the enzyme, which creates an energetically favorable conformation for phosphorylating ADP to create ATP. Each rotation produces three ATP molecules.

Figure 2: Electron Transport Chain and Krebs Cycle. Enzymes in the Krebs cycle generate high energy molecules including GTP, NADH and FADH₂. NADH and FADH₂ are used to drive the electron transport chain and oxidative phosphorylation, ultimately generating ATP.

Mitochondria Morphology: Fission and Fusion Events

The morphology of mitochondria is linked to its ability to produce ATP and is governed by a balance of fission and fusion events. Fission is the main mechanism by which old or damaged mitochondria are removed through mitophagy, a specialized form of autophagy. Defective mitochondrial kinase PINK1, can cause dysregulation of mitophagy and is linked with Parkinson's disease.

Dysregulation of these events can lead to mitochondria with uneven mtDNA distribution, impaired ability to respond to ATP production demand, increased tendency to produce reactive oxygen species and make the cells more susceptible to apoptosis.

Mitochondrial Dysfunction and Diseases

Errors in mitochondrial DNA and mitochondria dysfunction are linked to numerous diseases including mitochondrial myopathy, Leigh syndrome and mitochondrial DNA depletion syndrome. Furthermore, mitochondria function is often altered in endocrine-related disorders including diabetes.

Atherosclerosis, diabetes, neurodegenerative diseases, and cancer, have all been associated with oxidative stress caused by Reactive Oxygen Species (ROS). These molecules can form from electrons leaking from the electron transport chain complexes, which erroneously pass to oxygen molecules, rather than forming water at complex V during the last step of oxidative phosphorylation.

Mitochondria as Targets in Cancer

One of the major mitochondrial enzymes that feeds into the Krebs cycle is glutamate dehydrogenase (GDH), which converts glutamate to α-ketoglutarate (α-KG), an essential intermediate in the Krebs cycle. Blocking GDH attenuates the use of glutamine in the Krebs cycle, which in glioblastoma causes cells to become sensitized to glucose withdrawal.

Another major mitochondrial enzyme cancer target is the mutant form of isocitrate dehydrogenase (mIDH), because of its role in hypermethylation of CpG islands in cancers such as glioblastomas and acute myeloid leukemia (AML). Visit our cancer metabolism page for more information and to view our product range.

Figure 3: Mitochondria Targets in Cancer. Adapted from our Cancer Metabolism Poster. Download or request a copy for more information.

Mitochondria and Calcium Signaling

Calcium signaling between the cytosol and matrix coordinates the energy demands of the cell and the levels of oxidative phosphorylation in mitochondria.

Increased cellular energy demands are associated with a rise in cytosolic Ca²⁺ concentration. Mitochondria express the mitochondrial calcium uniporter (MCU) in the inner mitochondrial membrane, so when there is a local increase in the Ca²⁺ levels, it is promptly taken up by the MCU and transported into the matrix, activating pyruvate, α-ketoglutarate and NAD-isocitrate dehydrogenases, which in turn increases the rate of NAPH production. ATP synthase is also upregulated in response to increased matrix Ca²⁺, increasing the rate of ATP production.

A rise in cytosolic Ca²⁺ also leads to activation of the glutamate/aspartate transporter (ARALAR), which increases substrate supply across the inner mitochondrial membrane, increasing the capacity of the mitochondria to rapidly and efficiently increase ATP production.

The removal of matrix Ca²⁺ is carried out by Na⁺/Ca²⁺ exchangers and is a comparatively slow process when compared to cytosolic Ca²⁺ removal. The metabolic response of increased calcium levels therefore tracks matrix Ca²⁺ levels not cytosolic Ca²⁺.

Mitochondria and Apoptosis

Mitochondria-induced apoptosis is essential in developmental biology, as well as the removal of damaged cells. Apoptosis can occur through one of two major pathways, the extrinsic or the intrinsic pathway. The extrinsic pathway involves cell surface receptor activation, which results in the eventual activation of caspase 8. The intrinsic pathway involves permeabilizing the outer membrane of the mitochondria. In this latter pathway, cell death signals cause specific mitochondrial proteins to move from the intermembrane space into the cytosol. Once there these proteins activate caspases to induce cell death or block cytosolic inhibitors of cell death.

In addition to being an essential protein in the electron transport chain, cytochrome c is an apoptogenic protein. When cytochrome c is released from the mitochondria, it binds to Apaf-1, which then binds ATP, oligomerizes and forms an apoptosome, which in turn binds caspase 9 and initiates apoptosis.

Mitochondria Under the Microscope: Fluorescent Probes and Indicators for Mitochondria Staining

Mitochondria can encompass a broad spectrum of morphologies in different cell types, from small spheres and tubules with a diameter less than a micrometer (μm) to reticular networks across the cell. Fluorescent mitochondrial staining and high-resolution fluorescence microscopy techniques are commonly used for imaging these intricate and dynamic intracellular organelles, to study their structure, function and location. Our catalog contains standard dyes including: MitoMark Green I (Cat. No. 6444) and MitoMark Red I (Cat. No. 6445) for mitochondria staining; MitoPY1 (Cat. No. 4428) for imaging hydrogen peroxide (H₂O₂) in mitochondria of living cells; and Mito-HE (Cat. No. 7641) for imaging superoxide in the mitochondria of living cells.

Related Products: Inhibitors and Activators for Investigating Mitochondrial Function

In addition to our fluorescent probes for mitochondria staining, Tocris offers a comprehensive range of other tools for investigating mitochondria function, including oxidative-phosphorylation inhibitors such as BAY 87-2243 (Cat. No. 6980), a mitochondrial complex I inhibitor and the oxidative phosphorylation uncoupler CCCP (Cat. No. 0452); Cytochrome P450 inhibitors such as Metyrapone (Cat. No. 3292); fluorescent P450 substrates including 7-Ethoxyresorufin (Cat. No. 6204); Mitochondrial calcium uniporter tools e.g. MCU i4 (Cat. No. 7195), a negative modulator of mitochondrial Ca²⁺ uniporter; and mitochondrial permeability transition pore inhibitors including Cyclosporin A (Cat. No. 1101), which inhibits MPTP opening.

Mitochondria Markers from Bio-Techne Brands

Visit our sister brand Novus Biologicals for a comprehensive list of mitochondria markers including common markers ATP5A, Prohibitin, VDAC1 and many more. They also provide Apoptosis markers, oxidative phosphorylation markers, and markers for mitochondrial dynamics.

R&D Systems provide the highest quality antibodies giving you repeatedly reliable results. Among their vast catalog of antibodies for mitochondria markers, are Human/Mouse/Rat AIF Antibodies, Human/Mouse/Rat HSP60 Antibodies and Human/Mouse COX4 Antibodies. Search for your target at rndsystems.com.

Literature for Mitochondria Dyes and Probes

Tocris offers the following scientific literature for Mitochondria Dyes and Probes to showcase our products. We invite you to request* your copy today!

*Please note that Tocris will only send literature to established scientific business / institute addresses.

Fluorescent Dyes and Probes Research Product Guide

This product guide provides a background to the use of Fluorescent Dyes and Probes, as well as a comprehensive list of our:

• Fluorescent Dyes, including dyes for flow cytometry
• Fluorescent Probes and Stains, including our new MitoBrilliant^TM mitochondria stains
• Tissue Clearing Kits and Reagents
• Aptamer-based RNA Imaging Reagents
• Fluorescent Probes for Imaging Bacteria
• TSA Vivid^TM Fluorophore Kits
• TSA Reagents for Enhancing IHC, ICC & FISH Signals

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MitoBrilliant Research Product Guide

This product guide provides a background, protocols and data from use in different research applications for our Mitobrilliant™ fluorescent mitochondrial probes:

• MitoBrilliant™ 646
• MitoBrilliant™ Live 646
• MitoBrilliant™ Live 549

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Novel Live-cell and Fixable Mitochondrial Probes Poster

Fluorescent probes that accumulate in the mitochondria are useful tools to explore mitochondrial function and cell health. This poster reports the development inhouse and characterization of novel fluorescent probes, known as MitoBrilliant that specifically localize to mitochondria due to mitochondrial membrane potential. The probes are suitable for live or fixed cell staining. The data demonstrate the specificity and enhanced properties of our MitoBrilliant dyes and their use in a range of applications. This poster was presented at CYTO 2022, June 2022, Philadelphia, USA.

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