Mitochondria are double membrane enclosed cell organelles found in the cell cytoplasm. It has many functions but by far the most important role that it possesses is as the cells power plant. It provides energy in the form of ATP from the breaking down of sugar molecules. Within the mitochondria lie compartments separated by membranes. These compartments and membranes each have their own unique role in the metabolic pathway. Apart from being the cell's power plant, mitochondria are also important in other biochemical reactions such as apoptosis and calcium storage. A mutation within the mitochondrial DNA can lead to dysfunctions of the mitochondria and will get worse if not treated. This review will mainly discuss the structures, functions, and dysfunctions of the mitochondria.
Introducing the Mitochondria
In order to survive and thrive, organisms require energy. Organisms need energy in order to maintain homeostasis (inner cell environment stability), ensuring a well-working metabolism, and to keep the body's essential processes working . Mitochondria (mitochondrion = singular) is well known as the power plant of the cell. Its name was derived by the Greek word "mitos" (thread) & "chondros" (granule). Therefore, the name mitochondria mean "thread-like granule" .
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The first recorded detection of mitochondria was first detected back in 1857 by a Swiss physiologist and anatomist, Albert von Kolliker as "granule-like" structures present in muscle cells. During this time, microscopes were very simple so scientists that time can only see and observe the different organelles in a cell due to very rough morphology observations. This means scientists that time have yet to discover the importance of mitochondria in cell life. In later years, German pathologist and histologist Richard Altmann employed a dye technique in order to stain the structures to make them easier to visualize under a light microscope. This method turned out to be successful and he was able to distinguish the early mitochondria from other cell organelles. Altmann then named this organelle the "bioblast" and he presumed this organelle to be essential in cellular activity. Later in 1898, German scientist Carl Benda eventually patented the name mitochondria to replace bioblast. Further researches concerning the functions, roles, mechanisms, and dysfunctions of the mitochondria in cells are still continuing even until today . The discovery of mitochondria was named one of the greatest science discoveries of all time according to Kendall Haven's book, "100 Greatest Science Discoveries of All Time" .
Figure1. Albert von Kolliker, the man who first noticed the existence of the mitochondria .
Mitochondria are double membrane-enclosed organelles, meaning it is covered with an independent membrane. It has an outer membrane and also an inner membrane. These membranes are made up of a bilayer of phospholipids. These two membranes then enclose two compartments which are the intermembrane space and the mitochondrial matrix (for further information, see the following section). The shape of mitochondria resembles a kidney or a sausage. They are approximately 1-10 Âµm long which makes them the largest organelle found free in the cytoplasm .
Mitochondria can be found in almost all eukaryotic cells and their numbers vary in each cell. The number of mitochondria in a cell depends on how much energy the cell needs. It can range from just only one to a few thousand per cell. If a specific cell needs more energy than another cell, the number of mitochondria within the specific cell would be higher than that of the other cell. For example, muscle cells require more energy than kidney cells because muscle cells execute more work than kidney cells due to their contraction quantities and strength. The mitochondria can occasionally be found situated between the myofibrils of muscles or at the base of a sperm cell's flagellum . By using an electron micrograph, mitochondria can be seen forming a complex 3 dimensional branching network inside the cell with the cytoskeleton .
Due to its independent bacteria-like DNA and ribosomes, mitochondria are presumed to be once an external bacterial symbiote which was then engulfed by a larger procaryotic cell. This bacterial symbiote then wasn't broken down; in fact, it was kept alive within the larger cell which then undergoes a mutualism symbiosis with the symbiote. The symbiote provides energy for the larger cell and the larger cell provides protection and a suitable living environment for the much smaller symbiote cell. This particular occurrence was thought by many scientists as the birth of modern eukaryotic cells. This particular theory is called the endosymbiosis theory. American biologist Lynn Margulis was the person who invented and outspreaded the theory worldwide. Because of its independent DNA & ribosomes, the replication of mitochondria can also be done independent from the mother cell; this means its replication can be done independently from the normal nuclei replication that mostly occurs .
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Figure2. The scheme of endosymbiosis theory evolution of mitochondria in eukaryotes .
Structure, Visualization, and Isolation of Mitochondria
A mitochondria structure is generally consist of the outer membrane, the inner membrane, the intermembrane space, cristae, and the mitochondrial matrix. Each of these parts has their own specific functions and cannot operate without the presence of the others. In this section, the functions and roles of different parts of the mitochondria will be described. Following it, will be discussed about the visualization and isolation method of mitochondria.
Figure3. An overview of the structure of the mitochondria .
The outer membrane is a smooth complex of phospholipid bilayer which is freely permeable to small molecules (with a size <5000 Dalton) and ions. This layer has a hydrophobic tail made up of fatty acids that are sandwiched between hydrophilic heads. Due to this, it gives the membrane its fluid properties. The outer membrane contains many integral proteins called porins to help transport small molecules simultaneously into the mitochondrial intermembrane space. Porins are - barrel proteins and can be found in all Gram negative bacteria and some Gram positive ones besides in the mitochondria .
Figure4. A visualization of the Phospholipid bilayer complex containing its hydrophobic tails and hydrophilic heads .
Figure5. A Computer generated image of Porin, a Î²-barrel transmembrane protein . Porin allows instantaneous diffusion of small molecules and ions from the cytoplasm to the interior of the mitochondria which to be more precise, is the intermembrane space that is located between the two mitochondrial membranes.
The mitochondrial inner membrane is also a bilayer of phospholipids just like the outer membrane but is more compact. Unlike the outer membrane, the inner membrane is impermeable to most small molecules and ions, including protons (H+) and its surface. The inner membrane of the mitochondria is the region where the essential components of the respiratory chain and ATP synthase are located. The respiratory chain present here are the respiratory electron carriers of Complexes I until IV . The inner mitochondrial membrane is where electron transport and proton (H+) pumping takes place .
Foldings formed within the inner membrane of the mitochondria is called the cristae. The cristae contain various kinds' proteins, such as ATP-synthase (F0F1) and numerous cytochromes. The cristae are folded to increase the surface area of the inner membrane of the mitochondria. An increase of surface area can lead to an induced metabolism because the electron transfer phase which occurs here contributes as the largest ATP producer. The size of the cristae (surface area of the mitochondrial inner membrane) in each cell varies. Cells that have a high work rate like the heart and muscle cells tend to have more cristae than that of other cells that have a less work rate like liver and kidney cells. This is so because it needs more electron transfer chain systems to fulfill its energy (in this case ATP) requirement .
The intermembrane space (short IMS) of the mitochondria is the region that lies between the outer and inner membrane of the mitochondria. This is the place where the oxidative phosphorylation takes place .
The mitochondrial matrix is another compartment in the mitochondria which is enclosed by the mitochondrial outer and inner membrane apart from the intermembrane space. Though similar, but the matrix is much larger than the narrow intermembrane space. This compartment contains a concentrated liquid mixture of numerous enzymes that break down carbohydrates and in effect, carry out cellular respiration . The mitochondrial matrix is the region where the Kreb's Cycle or the Citric Acid Cycle takes place. Apart from the enzymes, the mitochondrial matrix is also the home of the mitochondrial DNA and ribosomes, which both have a crucial role for mitochondrial replication. Here are some major components of the mitochondrial matrix: Pyruvate dehydrogenase complex, Citric Acid Cycle Enzymes, Fatty Acid Î²-oxidation enzymes, mtDNA (mitochondrial DNA), mtribosomes (mitochondrial ribosomes), ATP, ADP, Pi (inorganic phosphate), Mg2+, Ca2+, K+, etc .
Isolation and Visualization of Mitochondria
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Isolation of mitochondria is necessary if a morphological observation of mitochondria is needed. By isolating the mitochondria, observation can be much easier because the isolated sample consists only with mitochondria. Using this, finding the mitochondria will be much easier. The Isolation of mitochondria can be done from both cells and tissue cultures.
There are 3 key steps towards the isolation of mitochondria. These steps are: 1) Cell rupture; 2) Low speed differential centrifugation; and 3) High speed centrifugation .
The first step, cell rupture is the most basic phase. To able to isolate mitochondria, the mitochondria need to be excavated out of the cell. In order to release the mitochondria from the cell, the cell must be ruptured or damaged. By rupturing the cells, the organelles of a cell can be released out into its solution environment. When examining a plant, this process is often used with an ice-cold grinding buffer to grind of the cells' cell wall and membrane .
The next step is the differential low speed centrifugation. This centrifugation step is done to separate cell debris and large organelles from the mitochondria. The speed of this centrifugation is roughly above 1000 rpm and will only take no more than approximately 5 minutes. When this centrifugation is finished, the cell debris and large organelles would become the pellet while the mitochondria will be mixed with smaller cell organelles as the supernatant of the after spin. So, the supernatant of this after spin will be collected and transferred into a new tube for the final step .
The final step is the high speed centrifugation. In this step, the mitochondria will be isolated for good from the rest of the cell components. This step usually uses a high speed spinning (over 10000 rpm) for a longer period of time than the first centrifugation (approximately more than 30 minutes). After centrifuge is done, the pellet of this after spin would be the mitochondrial suspension and the supernatant would be the other unnecessary components. The pellet should be rather brownish due to the presence of cytochromes within the mitochondria. The supernatant can be discarded and the pellet can be resuspended in an assay buffer .
There are many methods to visualize mitochondria. Electron microscopes can always be used . One of the most common way to visualize mitochondria is using immunofluorescence microscopy . The sample that is about to be observed will be stained with a certain dye like for example Mitotracker. Mitotracker is a potentiometric dye that is taken up exclusively by living mitochondria because of their membrane potential . Besides Mitotracker, numerous proteins can also be used as a dye for example Green Fluorescence Protein (GFP).
Figure6. A Human Mitochondria. The immunofluorescence microscopy image above is used by a scanning fluorescence confocal micrograph of a cultured human myoblast with the mitochondria stained with Mitotracker .
Mitochondria as the Power Plant of Modern Eukaryotes
As mentioned above, the mitochondria's main role is to produce energy for the cells so it can do it will be able to do cellular activities depending on the type of cells. The mitochondria accomplish this task by oxidizing pyruvate and NADH, which are the products of glycolysis in the cell cytosol . This preliminary breakdown through glycolysis though only yields a modest amount of energy, which is not enough to sustain the organisms' entire cellular activity. For every digestion of a glucose (C6H12O6) molecule within the cytosol, glycolysis yields 2 ATP molecules of energy with an extra 6 more ATPs in the form of NADH+H+.
Figure7. An overview of the three stages in cellular respiration; Glycolysis, Kreb's Cycle, and Electron Transport Chain .
The pyruvic acid products of glycolysis are then drawn into the mitochondrial matrix where it will be fully degraded to carbon dioxide and water in the Citric Acid Cycle. In this process, up to 30 more ATPs can be generated to sum up a total value of 38 ATP molecules generated from the complete breakdown of one glucose molecule .
The Citric Acid Cycle originally doesn't produce any ATP molecules, but only high energy electrons. These high energy electrons are then directed to the inner mitochondrial membrane by using electron carriers NADH+H+ and FADH2. Present in the inner mitochondrial membrane is an array of other electron carriers and enzymes which are grouped into several complexes and anchored in place by transmembrane proteins. This electron transport chain (ETC) will convert energy from the electrons of NADH+H+ and FADH2 into ATP. NADH+H+ can be converted to approximately or equivalent to 3 ATPs while FADH2 can be converted to approximately 2 ATPs .
File:Citric acid cycle with aconitate 2.svg
Figure8. An overview of the Kreb's or Citric Acid Cycle . It is a cycle that starts with a transfer of two carbon-acetyl group from acetyl-CoA to the four-carbon acceptor compound oxaloacetate to form a six-carbon compound (citrate). Citrate then undergoes a series of chemical transformations. During the cycle, various components are created and various enzymes are used to generate the transformation processes. At the end of the cycle, the four-carbon oxaloacetate has been regenerated and the cycle will go back to the beginning again with the fusion of acetyl-CoA with oxaloacetate all over again. Because glycolysis results in two pyruvic acids, the breakdown of one glucose molecule will generate two Kreb's Cycles simultaneously. Therefore, the products of one glucose molecule breakdown through the Kreb's Cycle are: 2 GTP, 6 NADH, 2 FADH, and 4 Co2.
Electron transport complexes contain peptide chains that incorporate heme (a prosthetic group of an iron atom contained in the center of a large heterocyclic organic ring called a porphyrin) and iron-sulfur structures which are crucial to the transfer of electrons in the ETS. There are several types of heme like heme A, heme B, heme C, heme O, etc. However, the most common heme that occurred in the ETS is heme C. The organs that are mainly involved in the synthesis of heme are theÂ liverÂ and theÂ bone marrow .
Figure9. The molecular structure of Heme C . The iron atom core can be seen clearly in the middle.
Figure10. An overview of the process of heme synthesis . Here, the pathway described from the mitochondria to the cytoplasm vice-versa are mentioned. As seen, heme is produced from inside the mitochondria which is when transported to the cytoplasm, will combine with globin chains that will make hemoglobin, the oxygen carrier in red blood cells.
Complex I is the NADH dehydrogenase complex, which is the largest complex of the ETC. It contains more than 40 peptide chains. The electrons from NADH pass first to the flavin (a biomolecule produced from riboflavin) then it will go through 7 iron-sulfur centers to the ubiquinone or coenzyme Q-10 .
Complex II is the succinate dehydrogenase. Here, additional electrons are sent into the quinone pool (Q) .
Complex III is the cytochrome b-c1 complex. It contains 22 protein chains with 6 heme groups in cytochromes and an addition of 2 iron-sulfur centers. Its duty is to pass electrons to cytochrome c .
Complex IV is the cytochrome oxidase complex. In this particular complex, 26 protein chains including 4 cytochromes each attached with 2 copper atoms are contained. Complex IV has the duty to receive electrons from cytochrome c one at a time. It will then pass them four at a time to oxygen to produce water. The copper atoms present her bind to peptide chains and transfer electrons one at a time. Copper is incorporated into heme a3 where electrons gather at the end of the ETC .
Flavin mononucleotide (FMN) receives electrons from NADH and transports them to Complex 1. When electrons pass through flavin, protons are drawn along. The electrons then traverse a series of iron-sulfur boxes. Ubiquinone or coenzyme Q-10 transports electrons from Complex I to Complex III, which is the smallest electron carrier. Electrons pass along parallel pathways through complex III. The heme group of cytochrome c is the one responsible to transport electron pairs to Complex IV. After transported to Complex IV, the spent electrons accumulate at heme a3. Meanwhile, molecules of oxygen (O2) diffuse into the cell. The oxygen molecules are then captured by the iron copper core at heme a3. Oxygen is very attracted to electrons, but adding fewer than four electrons at a time makes oxygen unstable. So, heme a3 holds the apart until four electrons accumulate. When these four once high-energy electrons have accumulated at heme a3, they are all fed at once to oxygen. Therefore, by taking these, the result is water (H2O) .
Meanwhile, protons or hydrogen ions (H+) have been drawn out and accumulated out of the watery medium of the mitochondrial matrix. The protons are trapped in the intermembrane space between the inner membrane and outer membrane of the mitochondria by their attraction to high energy electrons. The inner mitochondrial membrane is impermeable to protons; therefore the difference in hydrogen ion (H+) concentration between the mitochondrial matrix and the intermembrane space may result in a great pH difference. After water is produced (previous paragraph), the number of hydrogen ions trapped in the intermembrane space are approximately ten times higher than inside the mitochondrial matrix. When this occurs, the ATP Synthase will make its move .
The ATP Synthase has 2 subunits. There's the FO and F1. The FO subunit lies within the inner membrane while the F1 subunit is above the membrane - inside the mitochondrial matrix. The FO subunit is more like rotor. It works by spinning, thus moving up protons from the intermembrane space towards the mitochondrial matrix. This ability is done by the energy in the form of protons moving down an electrochemical gradient. This contraption of such an enzyme is powered by the pH eagerness of protons to return to the mitochondrial matrix, and provides a channel for them to do so. Its rotation produces ATP by combining ADP and an inorganic phosphate. Hence, the full reaction would be: ADP + Pi ïƒ ATP. However, this mechanism doesn't work all the time. There will be a time where this mechanism is inhibited to stop transportation of protons down the electrochemical gradient when is not necessary. The antibiotic oligomycin inhibits the rotor movement of the FO subunit of the ATP Synthase. If this mechanism is inhibited, the reaction of ATP production will not take place .
Figure11. An overview scheme of the electron transport mechanism through the Electron Transport Chain (ETC) . ETC produces a total of 32 ATPs from the conversion of NADH, FADH, and GTP molecules.
Figure12. A scheme of ATP Synthase . The ATP synthase has two subunits, FO and F1. FO as seen above lies within the phospholipid bilayer of the mitochondrial inner membrane and forms some kind of a rotor mechanism. The F1 subunit lies above or below the FO subunit in the mitochondrial matrix. The scheme above also describes the ATP synthase in a fully active mode and an inhibited or inactive mode.
Other Functions and Roles of Mitochondria
Apart from being the major power plants of cells, mitochondria also have several other functions and roles within the cell. Mitochondria also possess a crucial role in apoptosis and calcium ions storage.
Mitochondria in Apoptosis
Apoptosis is defined as programmed cell death. Apoptosis serves as major defense mechanism in the regulation of cell numbers to prevent harmful and unwanted cells to grow. Apoptosis was first observed in amphibians' metamorphosis. Scientists back then studied the disappearance of the tails and gills of juvenile frogs growing to adulthood. The advantages of apoptosis include membrane blebbing, cell shrinkage, chromatine condensation, DNA cleavage, cell fragmentation, and also the phagocytosis of a cell .
Apoptosis can be divided into three major phases; the activation phase, the execution phase, and the destruction phase. The first phase is the activation phase. During this phase, multiple signaling pathways lead from the various death-triggering signals to the central control of the cell death machinery and then activate it. The next phase is the execution phase. Here, the activated machinery acts on multiple cell targets. Then, the final phase is the destruction phase. During this final stage, the targeted dead or dying cell is then broken down and degraded by degrading enzymes and engulfed by phagositosic cells .
Genetic studies have led to the discovery of so called "cell death genes" (ced). One of the most crucial ced in apoptosis are ced-3, ced-4, and ced-9. Ced-3 and ced-4 are essential for cell death, while ced-9 works antagonizing the ced-3 & ced-4 because it inhibits the two's activity. Hence, ced-9 protects the cells that should survive whenever there is a sudden accidental activation of the cell death mechanism .
Mitochondria in Calcium Ions Storage
Calcium ions concentration in the cell is crucial. An optimum concentration of Ca2+ ions can regulate an array of biochemical reactions and important in a cell's signal transduction. Mitochondria possess the ability to store calcium by rapidly taking up Ca2+ ions and store them for later release whenever it's necessary. This means that mitochondria harness a substantial role in calcium ions homeostasis within a cell [27, 28]. Recent studies show that the endoplasmic reticulum (ER) is the most significant calcium storage site and there is also a significant relationship between the ER and mitochondria concerning calcium uptakes . Calcium ions are taken up by the mitochondrial matrix by a calcium uniporter integral protein on the inner mitochondrial membrane . This flow is mainly initiated by the mitochondrial membrane potential.
The accumulation of calcium ions within the mitochondria regulates mitochondrial metabolism and has the ability to cause a transient depolarization of mitochondrial membrane potential.
Mitochondrial Dysfunctions and Diseases
Like any organelle, a dysfunction of the mitochondria will lead into a mitochondrial disorder or disease. As a vital organelle in the metabolic system, even a slight disorder in the function of the mitochondria can be harmful to the cell. Mitochondrial dysfunctions happen when a mutation occurs in the mitochondrial DNA (mtDNA). Apart from mutations, mitochondrial diseases can also be attained by inheritance. The cells that are the most affected when a mitochondrial disorder occurred are the muscle and brain cells. These cells require a lot of energy to induce their activity .
The mtDNA is a circular DNA, much like to the plasmids of bacterial cells. The human mtDNA is approximately 16 kbp long and encode 37 genes. Human mtDNA are commonly used and more preferred than nuclei DNA to construct evolutionary trees or ancestry tracing. There are several reasons why people use mtDNA to do this instead of using nuclei DNA: 1) mtDNAs have a higher rate of substitutions (single nucleotide mutations) than nuclei DNA that makes them easier to resolve the differences between closely related individuals; 2) mtDNA is only inherited from the mother rather than the combination of mother and father, which allows tracing of a direct genetic line; 3) mtDNAs don't recombine .
Inheritance of mitochondrial diseases in a human being is transferred maternally (inherited from one's biological mother). Why can't it be transferred paternally? Before the fertilization of an ovum (egg cell) by a sperm cell, these two parental cells each have mitochondria. In a sperm cell, the mitochondria are located at the base of the tail or flagellum. Sperm cells need to obtain lots of energy because it swims independently and competes with millions of other sperm cells towards an ovum, therefore, require a lot of energy for their movement. Men semen contains sucrose sugar to supply the sperm cells food to obtain enough energy to do its journey down the oviduct. However, during fertilization, the sperm cell will lose its tail or will discard its tail while it drills and penetrates into the wall of an ovum. This means that the mitochondria containing tail did not penetrate the ovum, resulting in no traces of mitochondria from the sperm cell (father). Therefore, a human mitochondrial disease transferred paternally is theoretically impossible .
Figure13. The gene coding regions of human mtDNA (left) and the sites within the mtDNA gene in which certain illness occur (right) .
In the following are some examples of mitochondrial diseases that are present among human beings.
MELAS (Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke) Syndrome is a chronic progressive neurodegenerative disorder. The nervous system seems to have broken down and degenerated.Â MELAS is associated with mutations in mtDNA and also possibly a biochemical deficiency of ETC Complex I. Early symptoms of MELAS include muscle weakness, joint pain, recurrent headaches, appetite loss, frequent vomiting, and seizures. Further symptoms include stroke-like episodes along with vision abnormalities and severe headaches. Extremely ill patients have been known to lose sight, moving problems, and decreasing intellectual .
MERRF is short for Myoclonic Epilepsy with Ragged Red Fibers. It is an extremely rare mitochondrial disease. This disease is caused by a mutation in one of the mitochondrial transfer RNA (tRNA) genes. It is characterized by a decrease in synthesis of the mitochondrial proteins required for the ETC and ATP synthesis. Patients suffering from this disease will experience muscle weakness or cardiac problems, epilepsy, and dementia.
File:Ragged red fibers in MELAS.jpg
Figure14. An example of ragged red fiber cells from an MERRF patient .
Mitochondrial Neuro-Gastrointestinal Encephalopathy (MNGIE) syndrome is a type of autosomal recessive mitochondrial disease. Unlike other typical mitochondrial diseases caused by mtDNA mutations, MNGIE is caused by mutations in theÂ TYMPÂ gene, which encodes theÂ enzymeÂ thymidine phosphorylase. MNGIE is a multi-system disorder with symptoms such as; malabsorption of the gastro-intestinal tract, diarrhea, constipation, gastroparesis (delayed gastric emptying), vomiting, weight loss, peripheral neuropathy, and retinal degeneration .
Mitochondrial research has been a very interesting board of interest over the past few decades. It is always very interesting to follow the development of mitochondrial research because there still remain many unsolved mysteries, mysteries that have yet to be even discovered. Nowadays, there are many companies and societies worldwide that dedicate themselves to mitochondrial research exclusively. Many of these companies and societies are even non-profit. Adding to this, numerous numbers of articles and scientific journals referring exclusively to mitochondria are published everyday and are still growing.
Through modern technology, people now know that mitochondrial DNAs can even be used to trace people's ancestors and map the ancestry puzzle between modern and early man. Possibly even to trace as back as the earliest man. With a vast and rapid growth in modern science and technology, new and fascinating new discoveries of mitochondria could be attained really soon.