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The cell. 6. No vesicular

MITOCHONDRIA

Mitochondria are almost ubiquitous organelles in eukaryote cells. However, they have not been found in Archezoa because these organelles were probably lost during evolution. Mitochondria already were regarded as a key element for eukaryote cells in the late XIX century. Altmann (1890), by using fuchsin staining, observed tiny cellular structures that he named as bioblasts. Bioblasts were later identified in all eukaryotic cells. In 1914, it was already known that mitochondria (bioblasts) were able to adopt many forms, such as rods, long cords, and reticular structures. With help of the electron microscope, it was observed that they were delimited by two membranes, inner and outer membranes. In 1962, it was proposed that mitochondria could grow and then split by fission in two new mitochondria. Thus, the morphology and number of mitochondria are dynamic. Currently, mitochondria dynamics can be studied in vivo by using fluorescent molecules, and it has been uncovered the ability of mitochondria to fuse between each other.

Mitochondria are organelles that evolved from bacteria that got associated with other cell (most probably an archaea) to form eukaryote cells. It has been proposed that this happened about 2 billion years ago. The ongoing endosymbiosis during all this time of evolution transformed bacteria into the current mitochondria.

1. Morphology

The shape of mitochondria is variable, from branched to small ellipsoids. It could be said that there is no mitochondria as individual organelles but a mitochondrial network, and that some pieces can be transiently come off. As a network or isolated, mitochondria are made up of an outer membrane, an inner membrane, intermembrane space and the mitochondrial matrix enclosed by the inner membrane (Figures 1 and 2).

 Mitochondria
Figure 1. Mitochondria show a wide variety of forms, from long branched to short unbranched morphologies. At ultrastructural level, mitochondria have an outer membrane, intermembrane space, an inner membrane and the mitochondrial matrix. The inner membrane has many invaginations known as mitochondrial cristae. The mitochondrial matrix contains the DNA and the enzymes that carry out many metabolic processes.
 Mitochondria
Figure 2. Transmission electron microscopy images. A) Mitochondria from hepatocytes. White arrow indicates mitochondrial cristae. It can be observed that mitochondria, as well as cristae, show a highly variable morphology. B) Higher magnification image showing the continuity of the inner mitochondrial membrane with the mitochondrial cristae (white arrows). Black arrow points to the outer mitochondrial membrane. C) The black arrow indicates a long mitochondrion located inside a dendrite of a neuron. Bars: A and C: 0.4 µm, and C: 50 nm.

The outer mitochondrial membrane is highly permeable and contains many copies of the protein aquaporin, which is an aqueous channel. This membrane is like a kind of sieve allowing the pass of all those molecules smaller than 5000 Dalton, including small proteins.

On the other hand, the inner mitochondrial membrane is impermeable to ions and small molecules. Mitochondria have to make the inner membrane impermeable enough to make possible a stable proton gradient. It could be done by increasing the cholesterol concentration in the membrane, which increases hydrophobicity. However, mitochondria lack cholesterol and, in addition, cholesterol would decrease membrane fluidity. Inner membrane needs both hydrophobicity and fluidity at the same time to properly perform its function. Mitochondria synthesize cardiolipin, a phospholipid with highly unsaturated fatty acid chains that is translocated to the inner membrane, providing both hydrophobicity and fluidity.

Mitochondrial matrix contains those molecules that can cross both membranes after selective transport from the cytosol. Inner membrane is heavily folded toward the matrix. Each fold is called mitochondrial crista (cristae in plural). There are three morphological types of cristae: discoidal, tubular and flattened. The number and form of cristae is the result of the mitochondrial activity. Cristae form a distinct compartment in the inner membrane because they contain a particular set of proteins. The inner membrane regions near the outer membrane, known as inner boundary membranes, are involved in exchanging lipids between both membranes, and importing and assembling proteins of the respiratory chain. Functional protein complexes of the respiratory chain and ATPase are moved to cristae. The physical connection between cristae and inner boundary membranes is mediated by thin tubular-like structures that limit the lateral diffusion of transmembrane proteins. Cristae are actually a way to increase enormously the membrane surface to accommodate a large amount to respiratory chain complexes and ATPases. In a hepatic cell, the inner mitochondrial membrane is about 1/3 of the total cell membranes.

DNA, ribosomes, and many enzymes are located in the mitochondrial matrix, where several metabolic pathways take place. Mitochondrial DNA is located in places referred as nucleoids, where enzymes for replication and repairing can be found. Some nucleoids may contain more than one DNA molecule. DNA is condensed by TFAM proteins. DNA contains about 16500 paired bases and around 37 genes that, in humans, code for 13 proteins of the respiratory chain, 2 ribosomal RNA and 22 transference RNA. So they can synthesize proteins. Mitochondrial DNA replication is not coupled to the cell cycle and it can be replicated at any time in the life of a cell. Nucleoids are associated to the inner mitochondrial membrane through the MitOS protein complex.

Mitochondria, or portions of the mitochondrial network, are moved through the cytoplasm by the cytoskeleton. They show an extraordinary motility and can be moved to energy- or calcium-demanding regions of the cell (see below). The distance traveled by mitochondria may be very long. For example, in neurons, mitochondria move from the soma toward the distant dendrites and axons and come back to the soma. The movement is not continuous, but saltatory, which means that the movement is stalled from time to time. Long distance movements are ruled by microtubules, whereas local movements depend more on actin filaments. Sometimes both types of cytoskeleton filaments cooperate to either move mitochondria or keep them motionless. In the axons, the speed of the mitochondrial movement along microtubules is around 0.1 to 1.4 µm/s. However, 50 µm/s has been observed in growing axons.

2. Fusion and fission

Mitochondria frequently divide and fuse between each other mixing their mitochondrial DNA. It can be said that there is a mitochondrial network containing many DNA molecules, and sometimes there are isolated fragments of this network having a DNA molecule. The advantage of this behavior may be sharing the synthesized products by separated parts of the mitochondrial network, reduce local damages, or share the mitochondrial DNA. If two cells are fused between each other, the mitochondrial network becomes homogeneous in 8 hours. Fusion and fission of mitochondria are complex mechanisms because the two mitochondrial membranes are involved, and they must be opened and sealed correctly. The number of mitochondria in a cell may change because of this behavior. However, it has been measured the total mitochondrial volume of some cellular types, and it has been observed that the increase in mitochondrial volume is related to the increase in the cell volume.

Division of mitochondria is mediated by DRP proteins (dynamin related proteins) (Figure 3). Dynamins are protein involved in vesicle formation. In mammal cells, the endoplasmic reticulum is found at the region where mitochondrial division is going to happen. Before division, and after recruiting DRP proteins, there is an initial constriction produced by actin filaments. Nucleoids are found near the constriction points. The role of endoplasmic reticulum appears to be related to the proper segregation of nucleoids, and to lipid transfer between the endoplasmic reticulum and mitochondrial membranes. This lipid exchange appears to be important for synthesizing some lipids like phosphatidylethanolamine and cardiolipin in mitochondria.

 Mitochondria
Figure 3. Drawing that shows the distribution of mitochondria in a fibroblast. Different components are involved in mitochondrial fission: endoplasmic reticulum, actin filaments, and DRP (dynamin related proteins) proteins. DRP are also involved in fusion processes (adapted from Friedman and Nunnari 2014).

3. Functions

One of the most important function of mitochondria is synthesizing ATP, the fuel for most cellular processes. Another function is β-oxidation, which is part of fatty acid metabolism. They also work as compartments for calcium storing. Mitochondria are involved in apoptosis, cancer, aging, and pathologies like Parkinson disease and diabetes. Furthermore, comparative studies of the mitochondrial DNA are very useful in anthropology for deciphering genealogies and in other phylogenetic studies because mitochondrial DNA is inherited by maternal lineage, and recombination does not occur in mitochondrial DNA during sexual reproduction.

ATP synthesis

Most ATP in eukaryotic cells (non photosynthetic eukaryotes) is synthesized in mitochondria. The citric acid metabolic cycle uses Acetyl-CoA to produce CO2 and NADH. Electrons from NADH are transferred to the electron transport chain of proteins located in the inner mitochondrial membrane. Electrons are finally transferred to oxygen to give water. The transport process of electrons through these chain of proteins is able to move protons across the inner mitochondrial membrane, from mitochondrial matrix to the intermembrane space. A gradient of protons is generated, which is used by the ATP synthase to produce ATP. ATP synthase is a transmembrane protein located in the inner mitochondrial membrane. This metabolic process is referred as oxidative phosphorylation because it chemically joins inorganic phosphorus and ADP into ATP. In anaerobic bacteria, which do not have mitochondria, this process takes place in the cell membrane.

Electron transport chain proteins and ATP synthases are located in the cristae, the invaginations of the inner mitochondrial membrane. These folds are a way to increase the amount of membrane for including more proteins, and therefore increase the ATP production. There are so many copies of these proteins that they are up to 80% of the total weight of the inner mitochondrial membrane.

The set of proteins that carries out the transport of electrons from NADH to water is known as electron transport chain or respiratory chain (Figure 4). It is made up of around 40 proteins, 15 of them are for electron transport. Most of these proteins are grouped in 3 complexes: NADH dehydrogenase complex, b-c1 cytochrome complex, and cytochrome oxidase complex. All have mechanisms to extrude protons from the mitochondrial matrix to the intermembrane space across the inner mitochondrial membrane, process coupled to the transport of electrons.

 Synthesis of ATP
Figure 4. The production of energy in mitochondria is a two steps process: first, a proton gradient across the inner mitochondrial membrane is generated by the electron transport chain of proteins, and second, ATP synthase uses the proton gradient for synthesizing ATP. Both steps take place in the inner membrane, more precisely in the mitochondrial cristae.

The journey of the electrons begins when a hydride ion is donated by NADH. The ion is split into two electrons and one proton. This process happens in the NADH dehydrogenase complex that also gets the two electrons. By means of ubiquinones, the two electrons are transferred to the b-c1 cytochrome oxidase complex. The delivering of electrons by the two complexes is coupled to the extrusion across the inner mitochondrial membrane of two protons, one per complex, from the mitochondrial matrix to the intermembrane space. From the b-c1 cytochrome complex, the two electrons are taken by cytochrome C and transferred to the cytochrome C complex. In this third complex, another proton is moved through the inner mitochondrial membrane when the electrons are finally captured by oxygen molecules.

The electron transfer is like in batteries, where electrons move from one side with lower affinity to other with higher affinity. This movement releases energy that is used to transport protons against concentration gradient. Weakly bound electrons jump from NADH to the NADH complex, and from this complex to the next because electrons are attracted more strongly. Without the protein complexes of the respiratory chain, the electrons are lost and the energy is released as heat energy. Protein complexes are able to create a concentration of protons ten times higher in the intermembrane space than in the mitochondrial matrix. In addition, a more negative global charge is generated in the mitochondrial matrix as a consequence of proton extrusion to the intermembrane space. In this way, protons are pushed by electro-chemical grandient to enter the mitochondrial matrix again.

ATP sinthase enzyme has a passage that allows protons to cross the inner mitochondrial membrane to balance the concentration gradient created by the electron transport chain. This flux of protons toward the mitochondrial matrix is coupled to the synthesis of ATP. ATP synthase is a well-preserved enzyme during evolution so that it is present in bacteria, chloroplasts, and in mitochondria. It is a large protein made up of many subunits. The molecular mechanism for ATP synthesis is not well-known but every synthesized ATP molecule needs 3 protons passing through the ATP synthase. It has the ability to synthesize more than 100 ATPs per second. It is interesting that this enzyme is able to carry out the opposite process, i.e., it uses ATP to pump protons from the matrix to the intermembrane space.

ATP synthesis is not the only purpose of the proton gradient. Other electrically charged molecules such as pyruvate, ADP, and inorganic phosphorus, must be pumped into the mitochondrial matrix, whereas ATP, which is synthesized in the inner membrane toward the mitochondrial matrix, must be transferred to the cytosol. Pyruvate and inorganic phosphorus enter coupled to the inward flux of protons. However, ADP enters coupled to the ATP exit, by antiport transport.

Lipid metabolism

An major amount of lipid synthesis happens in mitochondria. They synthesize lysophosphatidic acid, from which triglycerides are synthesized. Phosphatidic acid and phosphatidylglycerol are also synthesized in mitochondria. Phosphatidylglycerol is needed for the synthesis of cardiolipin and phosphatidylethanolamine.

Others

In some species, mitochondria have evolved into organelles with particular functions. For example, hydrogenosomes are related to hydrogen metabolism and mitosomes are involved in sulfur metabolism. These organelles do not contain DNA. Recently, mitochondria, together with the endoplasmic reticulum, have been involved in the generation of peroxisomes.

Although it was thought that mitochondria may protect against aging because of their oxidative metabolism, there is not strong evidence linking oxidative radicals with aging, so that antioxidants do not appear to be very important for preventing aging. In addition, mutations in the mitochondrial DNA in transgenic mice do not clearly produce aging. Sometimes, those mice can even live longer.

4. Protein import

Mitochondria have a very low number of genes compared to the large number of different types of proteins they contain. Yeast mitochondria contain around 1000 different proteins, whereas it is around 1500 in humans. Only a few proteins are coded by mitochondrial DNA, so that most of mitochondrial proteins must be imported from the cytosol. Importing proteins is a complex process since there are several compartments in the mitochondria where proteins can be targeted: external membrane, inner membrane and mitochondrial matrix. The imported proteins contain amino acid sequences that are like a mailing address, informing to mitochondrial transporters where these proteins must be targeted.

Bibliography

Kiefel BR, Gilson PR, Beech PL. 2006. Cell biology of mitochondrial dynamics. International review of cytology. 254: 151-213.

MacAskill AF, Kittler JT. 2010. Control of mitochondrial transport and localization in neurons. Trends in cell biology. 20: 102-112.

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