Connexin/Innexin Channels in Cytoplasmic Organelles. Are There Intracellular Gap Junctions? A Hypothesis!

This paper proposes the hypothesis that cytoplasmic organelles directly interact with each other and with gap junctions forming intracellular junctions. This hypothesis originated over four decades ago based on the observation that vesicles lining gap junctions of crayfish giant axons contain electron-opaque particles, similar in size to junctional innexons that often appear to directly interact with junctional innexons; similar particles were seen also in the outer membrane of crayfish mitochondria. Indeed, vertebrate connexins assembled into hexameric connexons are present not only in the membranes of the Golgi apparatus but also in those of the mitochondria and endoplasmic reticulum. It seems possible, therefore, that cytoplasmic organelles may be able to exchange small molecules with each other as well as with organelles of coupled cells via gap junctions.

Complex V TMEM70 deficiency results in mitochondrial nucleoid disorganization.

Mutations in the TMEM70 gene are responsible for a familial form of complex V deficiency presenting with 3-methylglutaconic aciduria, lactic acidosis, cardiomyopathy and mitochondrial myopathy. Here we present a case of TMEM70 deficiency due to compound heterozygous mutations, who displayed abnormal mitochondria with whorled cristae in muscle. Immunogold electron microscopy and tomography shows for the first time that nucleoid clusters of mtDNA are disrupted in the abnormal mitochondria, with both nucleoids and mitochondrial respiratory chain complexes confined to the outer rings of the whorls. This could explain the differential effects on the expression and assembly of complex V in different tissues.

Mouse and human mitochondrial nucleoid--detailed structure in relation to function.

The independent mitochondrial genetic information is organized in so-called mitochondrial nucleoids that, in vertebrates, typically contain 5-7 genetic units. The total number of nucleoids per cell is several hundred in cultured cells. Mitochondrial nucleoids, similarly to the whole mitochondrial network, have recently been successfully and extensively visualized using fluorescent and confocal microscopy. In the present work, we show high-resolution micrographs of mouse and human mitochondrial nucleoids obtained by transmission electron microscopy. Position in the mitochondria, size, general appearance and other properties of the human nucleoids appear the same as those of mouse nucleoids, and all observations are also in full agreement with the results obtained in different laboratories using different approaches. Most of nucleoids are located inside mitochondrial tubes. However, we show directly that certain part of the nucleoids close to inner membrane is bound to the complex of molecules that crosscut both, the inner and the outer mitochondrial membranes. Nucleoids in cells starving for serum are mostly more dense than those in dividing cells. We discuss the position, appearance and other properties of the nucleoids in relation to functional stage. Other electron-dense structures inside mitochondria that could be erroneously considered to be mitochondrial nucleoids are also described.

Coupling factor B affects the morphology of mitochondria.

Ectopic expression of coupling factor B in animal cells resulted in altered mitochondrial morphology. Cells expressing factor B fused to green fluorescent protein (GFP) contained fragmented, balloon-shaped or thinned, filamentous mitochondria, terminating at one end with balloon-like structures. Ultrastructural analysis using transmission electron microscopy revealed changes in the organization of mitochondrial cristae in cells expressing factor B-GFP fusion protein.

Morphology and protein composition of the mitochondrial nucleoids in yeast cells lacking Abf2p, a high mobility group protein.

To elucidate the role of Abf2p, a major mitochondrial DNA-binding protein in the yeast Saccharomyces cerevisiae, we examined the morphology of the mitochondrial nucleoids (mt-nucleoids) in an ABF2-deficient mutant (Δabf2) in vivo and in vitro by 4',6-diamidino-2-phenylindole (DAPI) staining. The mt-nucleoids appeared as diffuse structures with irregular-size in Δabf2 cells that were grown to log phase in YPG medium containing glycerol, in contrast to the strings-of-beads appearance of mt-nucleoids in wild-type cells. In addition, DAPI-fluorescence intensity of the mt-nucleoids transmitted to the bud was significantly lower in Δabf2 cells than in wild-type cells at log phase. However, the lack of Abf2p did not affect the morphology or segregation of mitochondria. The protein composition of the mt-nucleoids isolated from Δabf2 cells grown to stationary phase in YPG medium was very similar to that of the mt-nucleoids isolated from wild-type cells cultured under the same conditions, except for the lack of Abf2p. These results together suggested that in log-phase cells, the lack of Abf2p influences not only the morphology of mt-nucleoids but also their transmission into the bud. On the other hand, our result suggested that in stationary-phase cells, the lack of Abf2p does not significantly alter the protein composition of the mt-nucleoids.

Assembly of a chimeric respiratory chain from bovine heart submitochondrial particles and cytochrome bd terminal oxidase of Escherichia coli.

Cytochrome bd is a terminal quinol oxidase in Escherichia coli. Mitochondrial respiration is inhibited at cytochrome bc(1) (complex III) by myxothiazol. Mixing purified cytochrome bd oxidase with myxothiazol-inhibited bovine heart submitochondrial particles (SMP) restores up to 50% of the original rotenone-sensitive NADH oxidase and succinate oxidase activities in the absence of exogenous ubiquinone analogs. Complex III bypassed respiration and is saturated at amounts of added cytochrome bd similar to that of other natural respiratory components in SMP. The cytochrome bd tightly binds to the mitochondrial membrane and operates as an intrinsic component of the chimeric respiratory chain.

Leishmania major ascorbate peroxidase overexpression protects cells against reactive oxygen species-mediated cardiolipin oxidation.

Heme peroxidases are a class of multifunctional redox-active proteins found in all organisms. We recently cloned, expressed, and characterized an ascorbate peroxidase from Leishmania major (LmAPX) that was capable of detoxifying hydrogen peroxide. Localization studies using green fluorescent protein fusions revealed that LmAPX was localized within the mitochondria by its N-terminal signal sequence. Subcellular fractionation analysis of the cell homogenate by the Percoll density-gradient method and subsequent Western blot analysis with anti-LmAPX antibody further confirmed the mitochondrial localization of mature LmAPX. Submitochondrial fractionation analysis showed that the mature enzyme (~3.6 kDa shorter than the theoretical value of the whole gene) was present in the intermembrane space side of the inner membrane. Moreover, expression of the LmAPX gene was increased by treatment with exogenous H(2)O(2), indicating that LmAPX was induced by oxidative stress. To investigate the biological role of LmAPX we generated Leishmania cells overexpressing LmAPX in the mitochondria. Flow-cytometric analysis, thin-layer chromatography, and IC(50) measurements suggested that overexpression of LmAPX caused depletion of the mitochondrial ROS burden and conferred a protection against mitochondrial cardiolipin oxidation and increased tolerance to H(2)O(2). These results suggest that the single-copy LmAPX gene plays a protective role against oxidative damage.

Mitochondrial glycerol-3-P acyltransferase 1 is most active in outer mitochondrial membrane but not in mitochondrial associated vesicles (MAV).

Glycerol 3-phosphate acyltransferase-1 (GPAT1), catalyzes the committed step in phospholipid and triacylglycerol synthesis. Because both GPAT1 and carnitine-palmitoyltransferase 1 are located on the outer mitochondrial membrane (OMM) it has been suggested that their reciprocal regulation controls acyl-CoA metabolism at the OMM. To determine whether GPAT1, like carnitine-palmitoyltransferase 1, is enriched in both mitochondrial contact sites and OMM, and to correlate protein location and enzymatic function, we used Percoll and sucrose gradient fractionation of rat liver to obtain submitochondrial fractions. Most GPAT1 protein was present in a vesicular membrane fraction associated with mitochondria (MAV) but GPAT specific activity in this fraction was low. In contrast, highest GPAT1 specific activity was present in purified mitochondria. Contact sites from crude mitochondria, which contained markers for both endoplasmic reticulum (ER) and mitochondria, also showed high expression of GPAT1 protein but low specific activity, whereas contact sites isolated from purified mitochondria lacked ER markers and expressed highly active GPAT1. To determine how GPAT1 is targeted to mitochondria, recombinant protein was synthesized in vitro and its incorporation into crude and purified mitochondria was assayed. GPAT1 was rapidly incorporated into mitochondria, but not into microsomes. Incorporation was ATP-driven, and lack of GPAT1 removal by alkali and a chaotropic agent showed that GPAT1 had become an integral membrane protein after incorporation. These results demonstrate that two pools of GPAT1 are present in rat liver mitochondria: an active one, located in OMM and a less active one, located in membranes (ER-contact sites and mitochondrial associated vesicles) associated with both mitochondria and ER.

Mutagenicity of diesel exhaust particles mediated by cell-particle interaction in mammalian cells.

Diesel exhaust particle (DEP) has been identified as a class 2A human carcinogen and closely related to the increased incidence of respiratory allergy, cardiopulmonary morbidity and mortality, and risk of lung cancer. However, the molecular mechanisms of DEP mutagenicity/carcinogenicity are still largely unknown. In the present study, we focused on the mutagenicity of DEPs in human-hamster hybrid (A(L)) cells and evaluated the role of cell-particle interaction in mediating mutagenic process. We found that DEPs formed micron-sized aggregates in the medium and located mainly in large cytoplasmic vacuoles of cells by 24h treatment. The cellular granularity was increased by DEP treatment in a dose-dependent manner. DEPs resulted in a dose-dependent increase of mutation yield at CD59 locus in A(L) cells, while inflicting minimal cytotoxicity. There was a more than two-fold increase of mutation yield at CD59 locus in A(L) cells exposed to DEPs at a dose of 50mug/ml. Such induction was significantly reduced by concurrent treatment with phagocytosis inhibitors, cytochalasin B and ammonium chloride (p<0.05). These results provided direct evidence that DEPs was mutagenic in mammalian cells and that cell-particle interaction played an essential role in the process.

The peripheral stalk of the mitochondrial ATP synthase.

The peripheral stalk of F-ATPases is an essential component of these enzymes. It extends from the membrane distal point of the F1 catalytic domain along the surface of the F1 domain with subunit a in the membrane domain. Then, it reaches down some 45 A to the membrane surface, and traverses the membrane, where it is associated with the a-subunit. Its role is to act as a stator to hold the catalytic alpha3beta3 subcomplex and the a-subunit static relative to the rotary element of the enzyme, which consists of the c-ring in the membrane and the attached central stalk. The central stalk extends up about 45 A from the membrane surface and then penetrates into the alpha3beta3 subcomplex along its central axis. The mitochondrial peripheral stalk is an assembly of single copies of the oligomycin sensitivity conferral protein (the OSCP) and subunits b, d and F6. In the F-ATPase in Escherichia coli, its composition is simpler, and it consists of a single copy of the delta-subunit with two copies of subunit b. In some bacteria and in chloroplasts, the two copies of subunit b are replaced by single copies of the related proteins b and b' (known as subunits I and II in chloroplasts). As summarized in this review, considerable progress has been made towards establishing the structure and biophysical properties of the peripheral stalk in both the mitochondrial and bacterial enzymes. However, key issues are unresolved, and so our understanding of the role of the peripheral stalk and the mechanism of synthesis of ATP are incomplete.

Structure and dynamics of the mitochondrial inner membrane cristae.

Three-dimensional images of mitochondria provided by electron tomography reveal that the micro-compartments (cristae) defined by the inner membrane are connected to the periphery of this membrane by narrow tubular junctions, which likely restrict diffusion. The tomograms also strongly suggest that inner membrane topology represents a balance between membrane fusion and fission processes. The hypothesis being developed is that inner membrane topology is a regulated property of mitochondria. This review summarizes the evidence about how inner membrane shape influences mitochondrial function and, conversely, what is known about the factors that determine this membrane's topology.

Evaluation of copper toxicity in isolated human peripheral blood mononuclear cells and it's attenuation by zinc: ex vivo.

Copper and zinc act as a cofactor of over 300 mammalian proteins. Both have same electronic configuration therefore they are antagonist at higher individual concentration. The present study was designed with the aim to investigate the mechanisms pertaining to toxic effects of copper on human peripheral blood mononuclear cells (PBMCs) and to evaluate the cytoprotective effect of zinc on copper-induced cytotoxicity. The copper uptake into PBMCs was progressively increased with increasing concentration of metal in the growth medium. However, no significant effect on copper uptake was observed in the presence of zinc. Cell proliferation rate was decreased with increasing copper concentration. Interestingly, the proliferation rate of zinc treated PBMCs remained nearly the same as that of control cells. LD(50) of copper (115 microM) was increased six times (710 microM) in presence of zinc for PBMCs. At higher concentrations of copper (> 100 microM) decrease level of GSH was noticed. Increased levels of metallothionein in PBMCs were observed in response to zinc. DNA fragmentation studies also showed that copper produced DNA fragmentation at LD(50) (115 microM). Subsequently, zinc showed protection against DNA fragmentation caused by copper. Cell structure of PBMCs at LD(50) (115 microM copper) showed membrane bound cystic spaces and mitochondria having disrupted cristae and few myelin figures. In presence of zinc at LD(50) of copper (115 microM) cells showed improvement in mitochondrial structure and membrane bound cystic spaces. Taken together, the results of our study demonstrates that zinc play an important role in prevention of copper toxicity in peripheral blood mononuclear cells.

Mitochondrial localization of cellular prion protein (PrPC) invokes neuronal apoptosis in aged transgenic mice overexpressing PrPC.

Recent studies suggest that the disease isoform of prion protein (PrPSc) is non-neurotoxic in the absence of cellular isoform of prion protein (PrPC), indicating that PrPC may participate directly in the neurodegenerative damage by itself. Meanwhile, transgenic mice harboring a high-copy-number of wild-type mouse (Mo) PrPC develop a spontaneous neurological dysfunction in an age-dependent manner, even without inoculation of PrPSc and thus, investigations of these aged transgenic mice may lead to the understanding how PrPC participate in the neurotoxic property of PrP. Here we demonstrate mitochondria-mediated neuronal apoptosis in aged transgenic mice overexpressing wild-type MoPrPC (Tg(MoPrP)4053/FVB). The aged mice exhibited an aberrant mitochondrial localization of PrPC concomitant with decreased proteasomal activity, while younger littermates did not. Such aberrant mitochondrial localization was accompanied by decreased mitochondrial manganese superoxide dismutase (Mn-SOD) activity, cytochrome c release into the cytosol, caspase-3 activation, and DNA fragmentation, most predominantly in hippocampal neuronal cells. Following cell culture studies confirmed that decrease in the proteasomal activity is fundamental for the PrPC-related, mitochondria-mediated apoptosis. Hence, the neurotoxic property of PrPC could be explained by the mitochondria-mediated neuronal apoptosis, at least in part.

Alternative translation initiation generates a novel isoform of insulin-degrading enzyme targeted to mitochondria.

IDE (insulin-degrading enzyme) is a widely expressed zinc-metallopeptidase that has been shown to regulate both cerebral amyloid beta-peptide and plasma insulin levels in vivo. Genetic linkage and allelic association have been reported between the IDE gene locus and both late-onset Alzheimer's disease and Type II diabetes mellitus, suggesting that altered IDE function may contribute to some cases of these highly prevalent disorders. Despite the potentially great importance of this peptidase to health and disease, many fundamental aspects of IDE biology remain unresolved. Here we identify a previously undescribed mitochondrial isoform of IDE generated by translation at an in-frame initiation codon 123 nucleotides upstream of the canonical translation start site, which results in the addition of a 41-amino-acid N-terminal mitochondrial targeting sequence. Fusion of this sequence to the N-terminus of green fluorescent protein directed this normally cytosolic protein to mitochondria, and full-length IDE constructs containing this sequence were also directed to mitochondria, as revealed by immuno-electron microscopy. Endogenous IDE protein was detected in purified mitochondria, where it was protected from digestion by trypsin and migrated at a size consistent with the predicted removal of the N-terminal targeting sequence upon transport into the mitochondrion. Functionally, we provide evidence that IDE can degrade cleaved mitochondrial targeting sequences. Our results identify new mechanisms regulating the subcellular localization of IDE and suggest previously unrecognized roles for IDE within mitochondria.

Hyperglycaemia and intramitochondrial glycogen granules in the brain of mice. Ultrastructural study.

The mechanism of cytotoxic effects of hyperglycaemia on the brain has not yet been explained and the proposed hypotheses are not fully convincing. In the present study, we aimed to assess the effect of high doses of glucose on the ultrastructure of the mice brain. The results, which are in agreement with the literature data, show that the administration of a single high dose of glucose, as well as its chronic application, leads to accumulation of glycogen granules in the cytoplasm of astrocytes. A new observation is the detection of glycogen granules in the ultrastructurally changed mitochondria of astrocytes as well as in the mitochondria of some synapses. Our hypothesis assumes that excess of glucose may cause an increase in the vulnerability of the brain mitochondria. This in turn may enable glucose and cytoplasmic enzymes to penetrate into the mitochondria and they therein synthesise glycogen. Mitochondrial dysfunction may in turn lead to neurodegeneration by apoptotic process.

Localization of an alkyl-acetyl-glycerol-CDP-choline: cholinephosphotransferase activity in submitochondrial fractions of Tetrahymena pyriformis.

Tetrahymena pyriformis contains platelet-activating factor (PAF) as a minor lipid, which is biosynthesized de novo. A dithiothreitol-insensitive CDP-choline:cholinephosphotransferase (AAG-CPT), which utilizes alkyl-acetyl-glycerol as a substrate, had been detected in both the mitochondrial and microsomal fractions of the protozoan. In the present report, localization of this enzyme in submitochondrial fractions was studied. Cell fractionation was evaluated with enzyme and morphological markers. In this respect, succinate dehydrogenase, NADPH:cytochrome c reductase, glucose-6-phosphatase, alkaline phosphatase, monoaminoxidase, and cytochrome c oxidase activities were investigated. In the presence of antimycin A, mitochondrial activity of NADPH-cytochrome c reductase, was increased, while the microsomal one was reduced. Cardiolipin was distributed in the inner mitochondrial membrane. Alkaline phosphatase was found exclusively in the cytosol of the protozoan. The main portion of the dithiothreitol-insensitive AAG-CPT was localized in the inner mitochondrial membrane. Our data indicate that mitochondria are able to produce PAF, which might be associated with their function.

[Electron microscope study of haemolymph cells of Decticus verrucivorus (Orthoptera, Tettigoniidae) in larva and imago stages]. / Elektonno-mikroskopicheskoe issledovanie kletok gemolimfy skakuna serogo Decticus verrucivorus (Orthoptera, Tettigoniidae) na stadiiakh lichinok i imago.

The haemolymph of larvae and imago stages of Decticus verrucirus was studied with electron and light microscope. PAS-positive and PAS-negative granules were detected in haemocytes. On the electronograms, granulocytes were recognized as the only type of haemocytes. In the cytoplasm of granulocytes, granules of two types were found: those of mitochondrial origin, and originating from the Golgi apparatus, respectively. The discharge of a secret is realized by the merocrine way. Four stages of granulocyte development have been distinguished: 1) granule formation and organelle development, 2) granule formation and accumulation, 3) active secretion, and 4) cell destruction.

Novel mitochondrial creatine transport activity. Implications for intracellular creatine compartments and bioenergetics.

Immunoblotting of isolated mitochondria from rat heart, liver, kidney, and brain with antibodies made against N- and C-terminal peptide sequences of the creatine transporter, together with in situ immunofluorescence staining and immunogold electron microscopy of adult rat myocardium, revealed two highly related polypeptides with molecular masses of approximately 70 and approximately 55 kDa in mitochondria. These polypeptides were localized by immunoblotting of inner and outer mitochondrial membrane fractions, as well as by immunogold labeling in the mitochondrial inner membrane. In addition, a novel creatine uptake via a mitochondrial creatine transport activity was demonstrated by [(14)C]creatine uptake studies with isolated mitochondria from rat liver, heart, and kidney showing a saturable low affinity creatine transporter, which was largely inhibited in a concentration-dependent manner by the sulfhydryl-modifying reagent NEM, as well as by the addition of the above anti-creatine transporter antibodies to partially permeabilized mitochondria. Mitochondrial creatine transport was to a significant part dependent on the energetic state of mitochondria and was inhibited by arginine, and to some extent also by lysine, but not by other creatine analogues and related compounds. The existence of an active creatine uptake mechanism in mitochondria indicates that not only creatine kinase isoenzymes, but also creatine transporters and thus a certain proportion of the creatine kinase substrates, might be subcellularly compartmentalized. Our data suggest that mitochondria, shown here to possess creatine transport activity, may harbor such a creatine/phosphocreatine pool.

The many shapes of mitochondrial membranes.

The roles of mitochondria in cell death and in aging have generated much excitement in recent years. At the same time, however, a quiet revolution in our thinking about mitochondrial ultrastructure has begun. This revolution started with the use of vital dyes and of green fluorescent protein fusion proteins, showing that mitochondria are very dynamic structures that constantly move, divide and fuse throughout the life of a cell. More recently, some of the first proteins contributing to these various processes have been discovered. Our view of the internal structures of mitochondria has also changed. Three-dimensional reconstructions obtained with high voltage electron microscopy show that cristae are often connected to the mitochondrial inner membrane by thin tubules. These new insights are brought to bear on the wealth of data collected by conventional electron microscopic analysis.