The relevance of mitochondrial membrane topology to mitochondrial function

This review summarizes recent findings from electron tomography about the three-dimensional shape of mitochondrial membranes and its possible influence on a range of mitochondrial functions. The inner membrane invaginations called cristae are pleomorphic, typically connected by narrow tubular junctions of variable length to the inner boundary membrane. This design may restrict intra-mitochondrial diffusion of metabolites such as ADP, and of soluble proteins such as cytochrome c. Tomographic images of a variety of mitochondria suggest that inner membrane topology reflects a balance between membrane fusion and fission. Proteins that can affect cristae morphology include tBid, which triggers cytochrome c release in apoptosis, and the dynamin-like protein Mgm1, involved in inter-mitochondrial membrane fusion. In frozen-hydrated rat-liver mitochondria, the space between the inner and outer membranes contains 10-15 nm particles that may represent macromolecular complexes involved in activities that span the two membranes.     

Ultrastructure, peroxisomes and lipid peroxidation in reperfused myocardium

Reperfusion injury was studied in dog myocardium using a transmission electron microscope and 3,3'-diaminobenzidine (DAB) to locate areas of peroxidatic activity. Dark electron dense DAB reaction product was observed in peroxisomes and damaged mitochondria. These results suggest attack by reactive oxygen species on mitochondrial membranes, which might result in the formation of lipid peroxides and prostaglandin-like compounds. It is suggested that the release of lipid peroxide or prostaglandins from the injured cells may contribute to reactive hyperaemia, ventricular fibrillation and angina.     

Inter-mitochondrial complementation: Mitochondria-specific system preventing mice from expression of disease phenotypes by mutant mtDNA

Here we investigated the pathogenesis of deletion mutant mitochondrial (mt)DNA by generating mice with mutant mtDNA carrying a 4696-basepair deletion (DeltamtDNA4696), and by using cytochrome c oxidase (COX) electron micrographs to identify COX activity at the individual mitochondrial level. All mitochondria in tissues with DeltamtDNA4696 showed normal COX activity until DeltamtDNA4696 accumulated predominantly; this prevented mice from expressing disease phenotypes. Moreover, we did not observe coexistence of COX-positive and -negative mitochondria within single cells. These results indicate the occurrence of inter-mitochondrial complementation through exchange of genetic contents between exogenously introduced mitochondria with DeltamtDNA4696 and host mitochondria with normal mtDNA. This complementation shows a mitochondria-specific mechanism for avoiding expression of deletion-mutant mtDNA, and opens the possibility of a gene therapy in which mitochondria possessing full-length DNA are introduced.     

Localization of the ATP/ADP translocator in the inner membrane and regulation of contact sites between mitochondrial envelope membranes by ADP. A study on freeze-fractured isolated liver mitochondria.

The frequency of contacts between the mitochondrial envelope membranes was determined in freeze-fractured samples of isolated mitochondria by means of quantifying the frequency of fracture plane deflections between the two membranes. It was observed that the formation of contacts correlated with the concentration of free ADP despite of inhibition of electron transport by antimycin A. The activity of ATPase partially inhibited by oligomycin or depletion of membrane potential by K+ and valinomycin had no effect on the induction of the contacts by ADP. ATP was ineffective in creating contacts irrespective of the presence or absence of a membrane potential, whereas carboxyatractyloside induced the contacts under all conditions in a manner similar to ADP. These results suggest the involvement of the ATP/ADP translocator in regulation of contact sites. As a consequence, we analyzed its distribution in the inner membrane of kidney and liver mitochondria by binding of [3H]atractyloside to subfractions of this membrane. The experiments demonstrated that the translocator was located in the peripheral part of the inner membrane as well as in the portion which formed the cristae.     

A morphologic and morphometric study of the mitochondria in several hepatoma cell lines and in isolated hepatocytes.

Some characteristics of the mitochondria of hepatocytes and of three hepatoma cell lines have been compared. By means of stereologic analysis of electron micrographs of cross-sections through cells the volume of mitochondria per unit volume of cell cytoplasm and the surface areas of the mitochondrial envelope and cristae membranes have been measured. The relative mitochondrial volume in the cytoplasm decreases with increasing growth rate but the surface area of outer and cristae membranes per unit volume of mitochondria is not altered. The internal organization of hepatoma mitochondria, however, differs distinctly from that of normal liver mitochondria as evident from electron micrographs; the hepatoma cells contain mitochondria in which parallel cristae appear to cross the whole mitochondrial profile unlike the irregular, short cristae seen in normal liver mitochondria. Furthermore, in the fast-growing hepatoma cells the mitochondrial matrix appears less dense than in the hepatocyte. Hepatoma cells contain less organized rough endoplasmic reticulum than normal liver cells and the spatial relationship of the mitochondria to the rough cisternae, seen in the hepatocyte, is absent in the fast-growing hepatoma cell lines. It is concluded that hepatoma cells have fewer mitochondria than normal liver cells, but that the organelles have a normal content of inner membranes.     

Localization in a cell of a protein forming the ATP-dependent potassium-selective channels in the bilayer lipid membrane. An ultrastructural study

The localization in the cell of the protein forming the ATP-dependent potassium-selective channels in the bilayer lipid membrane has been studied. The electron microscope investigation of rat liver and heart tissue sections after their incubation with Abs against this protein and the visualization of the protein with secondary Abs conjugated with colloid gold were carried out. Colloid gold particles were observed both in mitochondrial membranes and in membranes of endoplasmic and sarcoplasmic reticulum. In heart mitochondria, these particles were significantly greater than in liver mitochondria. The localization of the channel protein both in mitochondria and reticulum, as well as the structural similarity between the mitochondrial channel and the precursor of calreticulin suggest that the channel protein belongs to the family of calreticulins. The possible function of the protein as a channel subunit of the mitochondrial ATP-dependent potassium channel is discussed.     

Localization in the cell of the protein forming ATP-dependent potassium-selective channels in a bilayer lipid membrane. An ultrastructural study

The localization in cell of the protein forming in BLM the ATP-dependent potassium-selective channels was studied. The electron-microscopic investigation of rat liver and heart tissue sections after their incubation with Abs against the studied protein and visualization of the protein with secondary Abs conjugated with colloid gold were carried out. The colloid gold particles were observed both in mitochondrial membranes and in membranes of endoplasmic and sarcoplasmic reticulum. In heart mitochondria these particles were significantly greater then in liver mitochondria. The detection of the channel-protein localization both in mitochondria and reticulum, as well as structural similarity between the mitochondrial channel and the precursor of calreticulin suggests that the channel protein belongs to the calreticulin family. The possible function of the studied protein as a channel subunit of the mitochondrial ATP-dependent potassium channel is discussed.     

Features of the intracellular repair of irradiated keratinocytes

By means of light and electron microscopy, intracellular reparation has been studied after a local x-ray radiation of the rat paws (7.74 X 10(-1) Ci/kg) using radioprotectors (mexamin, cysteamin, ionol) and other chemical compounds (including membranoprotective ones). Restoration of the intracellular structures after x-ray burns proceeds more slowly and more complexly than reparation of the epidermis as a tissue system. To the slowly repairing intracellular formations belong mitochondria and, especially, internal mitochondrial membrane, as well as intercellular contacts. Under radiation mitochondria increase their volume at the expense of their three-fold swelling. Preliminary treatment of the skin with some of the compounds mentioned above decreases or completely prevents these changes. By means of the membranoactive chemical compounds, as well as by means of the known radioprotectors it is possible essentially to normalize the process of intracellular reparation and physiological regeneration of the ultrastructures, and in some cases, to stimulate reparative processes in them.     

Evaluation of structuro-functional heterogeneity of isolated mitochondria from the normal and the ischemic myocardium

The structural and functional heterogeneity of mitochondria isolated from intact and ischemic (after 60 min exposure at 37 degrees C) rabbit myocardium was evaluated. In the presence of cytochrome c. a relatively high (260 +/- 26 ng at O/min . mg of protein) rate of rotenone-sensitive NADH oxidation was observed, which was increased in ischemia. Cytochrome c stimulated the increase of NADH oxidation in mitochondria of normal and ischemic myocardium by the factors of 3.5 and 3.4, respectively. Succinate oxidation in the presence of bromthymol blue in normal and ischemic myocardium mitochondria was activated by cytochrome c 3.3- and 2.9-fold, respectively. The percentage of mitochondria with both structurally damaged membranes was 15% and 25% in normal and ischemic myocardium preparations, respectively. In the absence of ADP, cytochrome c contributed to the increase of the succinate oxidase activity in ischemic mitochondria; that in the 3rd state was inhibited in ischemia and normalized by cytochrome c. A principle was proposed for estimating the percentage of mitochondria with damaged outer membranes, the indices being equal to 34% in control and to 56% in ischemic myocardium. Evidence was obtained suggesting that this mitochondrial fraction was characterized by lowered coupling and absence of rotenone-sensitive NADH: oxidase activity. The percentage of intact mitochondria, in which succinate oxidation is inhibited by bromthymol blue and does not need exogenous cytochrome c, is 51% in control and 19% in ischemic myocardium mitochondria.     

Synthesis of proteins in heart mitochondria during postnatal development of the rat

Proteins of inner mitochondrial membranes of the albino rat myocardium during postnatal development of 1, 3 and 6 months old animals were electrophoretically separated in 10% polyacrylamide gel. The rate of 14C-amino acids incorporation into examined proteins was determined in vitro. Specific radioactivity of the total mitochondrial fraction decreased in the course of the postnatal development. That of outer membranes remained unchanged, though it sharply increased in inner membranes of mature animals as compared with animals aged one month. Levels of radioactive precursor incorporation in separate protein fractions of inner membranes of the myocardium mitochondria were estimated.     

Role of membrane contact sites in protein import into mitochondria

Mitochondria import more than 1,000 different proteins from the cytosol. The proteins are synthesized as precursors on cytosolic ribosomes and are translocated by protein transport machineries of the mitochondrial membranes. Five main pathways for protein import into mitochondria have been identified. Most pathways use the translocase of the outer mitochondrial membrane (TOM) as the entry gate into mitochondria. Depending on specific signals contained in the precursors, the proteins are subsequently transferred to different intramitochondrial translocases. In this article, we discuss the connection between protein import and mitochondrial membrane architecture. Mitochondria possess two membranes. It is a long‐standing question how contact sites between outer and inner membranes are formed and which role the contact sites play in the translocation of precursor proteins. A major translocation contact site is formed between the TOM complex and the presequence translocase of the inner membrane (TIM23 complex), promoting transfer of presequence‐carrying preproteins to the mitochondrial inner membrane and matrix. Recent findings led to the identification of contact sites that involve the mitochondrial contact site and cristae organizing system (MICOS) of the inner membrane. MICOS plays a dual role. It is crucial for maintaining the inner membrane cristae architecture and forms contacts sites to the outer membrane that promote translocation of precursor proteins into the intermembrane space and outer membrane of mitochondria. The view is emerging that the mitochondrial protein translocases do not function as independent units, but are embedded in a network of interactions with machineries that control mitochondrial activity and architecture.     

Cnm1 mediates nucleus–mitochondria contact site formation in response to phospholipid levels

Mitochondrial functions are tightly regulated by nuclear activity, requiring extensive communication between these organelles. One way by which organelles can communicate is through contact sites, areas of close apposition held together by tethering molecules. While many contacts have been characterized in yeast, the contact between the nucleus and mitochondria was not previously identified. Using fluorescence and electron microscopy in S. cerevisiae, we demonstrate specific areas of contact between the two organelles. Using a high-throughput screen, we uncover a role for the uncharacterized protein Ybr063c, which we have named Cnm1 (contact nucleus mitochondria 1), as a molecular tether on the nuclear membrane. We show that Cnm1 mediates contact by interacting with Tom70 on mitochondria. Moreover, Cnm1 abundance is regulated by phosphatidylcholine, enabling the coupling of phospholipid homeostasis with contact extent. The discovery of a molecular mechanism that allows mitochondrial crosstalk with the nucleus sets the ground for better understanding of mitochondrial functions in health and disease.     

Membrane contacts of the endoplasmic reticulum and their possible functions in the plant cell

Close contacts of the endoplasmic reticulum membrane and plasmalemma have been visualized inside plant cells by means of electron microscopy. The qualitative similarity of these contacts to high-permeable intercellular contacts in animals has been shown. New data confirming the hypothesis of the identity of stromules, i.e., dynamic tubular protuberances of the plastid membrane of the plant cell, and tubular elements of the endoplasmic reticulum have been presented. New possible functions of the contacts of the endoplasmic reticulum membrane with other membranes inside the cell have been discussed on the basis of this hypothesis.     

Interactions between the endoplasmic reticulum, mitochondria, plasma membrane and other subcellular organelles

Several recent works show structurally and functionally dynamic contacts between mitochondria, the plasma membrane, the endoplasmic reticulum, and other subcellular organelles. Many cellular processes require proper cooperation between the plasma membrane, the nucleus and subcellular vesicular/tubular networks such as mitochondria and the endoplasmic reticulum. It has been suggested that such contacts are crucial for the synthesis and intracellular transport of phospholipids as well as for intracellular Ca²⁺ homeostasis, controlling fundamental processes like motility and contraction, secretion, cell growth, proliferation and apoptosis. Close contacts between smooth sub-domains of the endoplasmic reticulum and mitochondria have been shown to be required also for maintaining mitochondrial structure. The overall distance between the associating organelle membranes as quantified by electron microscopy is small enough to allow contact formation by proteins present on their surfaces, allowing and regulating their interactions. In this review we give a historical overview of studies on organelle interactions, and summarize the present knowledge and hypotheses concerning their regulation and (patho)physiological consequences.     

Alterations in myocardial ultrastructure and protein expression after a single injection of isoproterenol

Immunochemical and electron microscopic characterization of rat myocardium was conducted 2 h and 3 weeks after a single injection of isoproterenol in rats. The relative content of several myospecific proteins (KRP – kinase-related protein, desmin), cytoskeletal proteins (tubulin, vinculin, myosin light chain kinase – MLCK) and extracellular matrix protein fibronectin was determined by immunoblotting. Two hours after injection of 50 mg/kg isoproterenol a destruction of some cardiomyocytes, contracture of myofibrils and mild edema of intercellular space was observed. The content of all the studied proteins except KRP decreased below control levels. This situation sustained 3 weeks after injection and paralleled alterations in cardiomyocyte ultrastructure. Areas of myofibrillar contracture and lysis were noted, glycogen granules were sparse; mitochondria contained arrow-like inclusions that are characteristic for calcium overload, also huge mitochondria contacting each other by specialized intermitochondrial contacts were detected. Clumps of unripe elastic fibers in enlarged intercellular space were combined with increased deposition of collagens type I and III forming areas of fibrosis. The smaller dosage of isoproterenol (10 mg/kg) rendered no significant damage in the acute postinjection period but 3 weeks later it induced the thickening of extracellular matrix around cardiac cells and the increase in KRP and tubulin content by 26 and 32%, correspondingly. MLCK levels remained depressed throughout the experiment. The rise in KRP expression was also observed after the addition of isoproterenol to cultured chicken embryo cardiomyocytes. Obtained results indicate that even a single injection of isoproterenol creates long lasting structural alterations in cardiac muscle accompanied by the increased expression of extracellular matrix proteins and several sarcoplasmic proteins apparently involved in hypertrophic response of cardiomyocytes.     

Contact sites between mitochondrial membranes: comments.

Extensive contact sites between the two mitochondrial membranes are visualized particularly when tissues are prepared by freeze-substitution methods for electron microscopy. Such contacts rapidly diminish if animals are asphyxiated even for a relatively short period of 8 min. Thus contacts between the mitochondrial membranes are likely to be labile.     

Plasma membranes from Candida tropicalis grown on glucose or hexadecane. I. Isolation, identification and purification.

Plasma membranes from Candida tropicalis grown on glucose or hexadecane were isolated using a method based on the difference in surface charge of mitochondria and plasma membranes. After mechanical disruption of the cells, a fraction consisting of mitochondrial and plasma membrane vesicles was obtained by differential centrifugation. Subsequently the mitochondria were separated from the plasma membrane vesicles by aggregation of the mitochondria at a pH corresponding to their isoelectric point. Additional purification of the isolated plasma membrane vesicles was achieved by osmolysis. Surface charge densities of mitochondria and plasma membranes were determined and showed substrate-dependent differences. The isolated plasma membranes were morphologically characterized by electron microscopy and, as a marker enzyme, the activity of Mg2+-dependent ATPase was determine. By checking for three mitochondrial marker enzymes the plasma membrane fractions were estimated to be 94% pure with regard to mitochondrial contamination.     

Plant mitochondrial dynamics

Higher plant mitochondria are dynamic, pleomorphic organelles. The higher plant chondriome (all mitochondria in a cell collectively) is typically composed of numerous, physically discrete, mitochondria. However, frequent inter-mitochondrial fusion, enabling the mixing and recombination of mtDNA, ensures that the higher plant chondriome functions, at least genetically, as a discontinuous whole. Nothing is known about the genes controlling mitochondrial fusion in plants; there are no plant homologues of most of the genes known to be involved in fusion in other organisms. In contrast, the mitochondrial fission apparatus is generally conserved. Higher plant mitochondria use dynamin-like and Fis-type proteins for division; like yeast and animals, higher plants have lost the mitochondrial-specific form of the prokaryote-derived protein, FtsZ. In addition to being providers of energy for life, mitochondria provide a trigger for death. The role of mitochondrial dynamics in the initiation and promulgation of cell death is conserved in higher plants although there are specific differences in the genes and mechanisms involved relative to other higher eukaryotes.     

Plant mitochondrial dynamics

Higher plant mitochondria are dynamic, pleomorphic organelles. The higher plant chondriome (all mitochondria in a cell collectively) is typically composed of numerous, physically discrete, mitochondria. However, frequent inter-mitochondrial fusion, enabling the mixing and recombination of mtDNA, ensures that the higher plant chondriome functions, at least genetically, as a discontinuous whole. Nothing is known about the genes controlling mitochondrial fusion in plants; there are no plant homologues of most of the genes known to be involved in fusion in other organisms. In contrast, the mitochondrial fission apparatus is generally conserved. Higher plant mitochondria use dynamin-like and Fis-type proteins for division; like yeast and animals, higher plants have lost the mitochondrial-specific form of the prokaryote-derived protein, FtsZ. In addition to being providers of energy for life, mitochondria provide a trigger for death. The role of mitochondrial dynamics in the initiation and promulgation of cell death is conserved in higher plants although there are specific differences in the genes and mechanisms involved relative to other higher eukaryotes.     

Lateral phase separations and structural integrity of the inner membrane of rat-liver mitochondria: Effect of compression. Implications in the centrifugation of these organelles

When maintained in the vicinity of the lower transition temperature of their membrane lipids, rat-liver mitochondria undergo lysis as shown by the release of malate dehydrogenase, (an enzyme located within the mitochondrial matrix), in the surrounding medium.Structural changes take place in the membranes of mitochondria subjected to increasing pressure at 0°C, when the pressure reaches 750 kg/cm². Freeze-fracture electron microscopy shows the appearance of smooth areas devoid of particles in fracture faces of mitochondrial membranes, together with zones, where aggregated particles can be seen. Concurrently, a suppression of the malate dehydrogenase structure-linked latency is observed. These structural changes can be prevented by increasing the temperature at which compression is performed. The freeze-etching observations suggest that lateral phase separations occur in mitochondrial membranes subjected to high pressure. This can be explained by supposing that pressure promotes the gel-phase appearance in a lipid system and raises the transition temperature since the transition liquid crystal → gel is accompanied by a decrease in volume. The deterioration of mitochondria subjected to high pressure is interpreted as a result of the lateral phase separation induced by compression in the membranes.These results are discussed with respect to our interpretation of the damaging effects that hydrostatic pressure, generated by centrifugation, exerts on rat-liver mitochondria.