Mitochondrial involvement in genetically determined transition metal toxicity I. Iron toxicity

Iron that is not specifically chaperoned through its essential functional pathways is damaging to biological systems, in major part by catalyzing the production of reactive oxygen species. Iron serves in several essential roles in the mitochondrion, as an essential cofactor for certain enzymes of electron transport, and through its involvement in the assembly of iron-sulfur clusters and iron-porphyrin (heme) complexes, both processes occurring in the mitochondrion. Therefore, there are mechanisms that deliver iron specifically to mitochondria, although these are not well understood. Under normal circumstances the mitochondrion has levels of stored iron that are higher than other organelles, though lower than in cytosol, while in some disorders of iron metabolism, mitochondrial iron levels exceed those in the cytosol. Under these circumstances of excess iron, protective mechanisms are overwhelmed and mitochondrial damage ensues. This may take the form of acute oxidative stress with structural damage and functional impairment, but also may result in long-term damage to the mitochondrial genome. This review discusses the evidence that mitochondria do indeed accumulate iron in several genetic disorders, and are a direct target for iron toxicity when it is present in excess. We then consider two classes of genetic disorders involving iron and the mitochondrion. The first include defects in genes directly regulating mitochondrial iron metabolism that lead to Friedreich's ataxia and the various sideroblastic anemias, with excessive mitochondrial iron accumulation. Under the second class, we discuss various primary hemochromatoses that lead to direct mitochondrial damage, with reference to mutations in genes encoding HFE, hepcidin, hemojuvelin, transferrin receptor-2, ferroportin, transferrin, and ceruloplasmin.     

Electron tomographic and ultrastructural analysis of the Cryptosporidium parvum relict mitochondrion, its associated membranes, and organelles

Sporozoites of the apicomplexan Cryptosporidium parvum possess a small, membranous organelle sandwiched between the nucleus and crystalloid body. Based upon immunolabelling data, this organelle was identified as a relict mitochondrion. Transmission electron microscopy and tomographic reconstruction reveal the complex arrangement of membranes in the vicinity of this organelle, as well as its internal organization. The mitochondrion is enveloped by multiple segments of rough endoplasmic reticulum that extend from the outer nuclear envelope. In tomographic reconstructions of the mitochondrion, there is either a single, highly-folded inner membrane or multiple internal subcompartments (which might merge outside the reconstructed volume). The infoldings of the inner membrane lack the tubular "crista junctions" found in typical metazoan, fungal, and protist mitochondria. The absence of this highly conserved structural feature is congruent with the loss, through reductive evolution, of the normal oxidative phosphorylation machinery in C. parvum. It is proposed that the retention of a relict mitochondrion in C. parvum is a strategy for compartmentalizing away from the cytosol toxic ferrous iron and sulfide, which are needed for iron sulfur cluster biosynthesis, an essential function of mitochondria in all eukaryotes.     

The role of iron in mitochondrial function

BACKGROUND: Iron is an essential element for life, as it is a cofactor for enzymes involved in many metabolic processes, but it can also be harmful, since its excess is thought to enhance the production of reactive oxygen species and induce oxidative damage. Iron is transformed into its biologically available form in the mitochondrion by the iron-sulfur (Fe/S) cluster and heme synthesis pathways. During the past decade, substantial progress has been made in the elucidation of iron-linked mechanisms that occur in the mitochondrion, demonstrating the crucial role played by this organelle in maintaining cellular iron homeostasis. GENERAL SIGNIFICANCE: This review summarizes current knowledge of the mechanisms underlying iron trafficking in mitochondria and how it is handled inside the organelle. Relevant updates with regard to the Fe/S cluster and heme biosynthetic pathways, as well as the relationship between mitochondrial iron homeostasis impairment and related diseases, are also discussed.     

Mitochondrial DNA in anucleate human blood cells

Homogeneous populations of human blood platelets or erythrocytes were lysed in alkaline EDTA, bound to nitrocellulose and hybridized to a radioactive mtDNA probe. By comparison to standards of known mtDNA concentration, we determined that platelets contained 4 mtDNA molecules per cell. Rhodamine 123 staining revealed an average of 4 mitochondria per platelet indicating that each mitochondrion contains a single mtDNA molecule. No detectable mtDNA was found in erythrocyte lysates. Using the same procedure, we found that in nucleated cells, mitochondria contained multiple mtDNAs per mitochondrion.     

Artemisinin Directly Targets Malarial Mitochondria through Its Specific Mitochondrial Activation

The biological mode of action of artemisinin, a potent antimalarial, has long been controversial. Previously we established a yeast model addressing its mechanism of action and found mitochondria the key in executing artemisinin''s action. Here we present data showing that artemisinin directly acts on mitochondria and it inhibits malaria in a similar way as yeast. Specifically, artemisinin and its homologues exhibit correlated activities against malaria and yeast, with the peroxide bridge playing a key role for their inhibitory action in both organisms. In addition, we showed that artemisinins are distributed to malarial mitochondria and directly impair their functions when isolated mitochondria were tested. In efforts to explore how the action specificity of artemisinin is achieved, we found strikingly rapid and dramatic reactive oxygen species (ROS) production is induced with artemisinin in isolated yeast and malarial but not mammalian mitochondria, and ROS scavengers can ameliorate the effects of artemisinin. Deoxyartemisinin, which lacks an endoperoxide bridge, has no effect on membrane potential or ROS production in malarial mitochondria. OZ209, a distantly related antimalarial endoperoxide, also causes ROS production and depolarization in isolated malarial mitochondria. Finally, interference of mitochondrial electron transport chain (ETC) can alter the sensitivity of the parasite towards artemisinin. Addition of iron chelator desferrioxamine drastically reduces ETC activity as well as mitigates artemisinin-induced ROS production. Taken together, our results indicate that mitochondrion is an important direct target, if not the sole one, in the antimalarial action of artemisinins. We suggest that fundamental differences among mitochondria from different species delineate the action specificity of this class of drugs, and differing from many other drugs, the action specificity of artemisinins originates from their activation mechanism.     

Organization of multiple nucleoids and DNA molecules in mitochondria of a human cell.

Mitochondrial nucleoids (mt-nucleoids) of the A2780 line of cultured human cells were stained with DAPI and observed using an epifluorescence microscope. The mt-nucleoids appeared to be organized compactly in mitochondria. Numbers of mt-nucleoids per mitochondrion ranged from 1 to more than 10, and 70% were "multinucleated" mitochondria. Intensities of fluorescence of mt-nucleoids in each mitochondrion were measured by a video-intensified microscope system (VIM system) and copy numbers of mitochondrial DNA (mtDNA) in each mitochondria were determined. The copy numbers of mtDNA per mitochondrion ranged from 1 to 15, and the average was 4.6. Because the cells had 107 mitochondria on average, the copy number of mtDNA per cell was estimated to be about 500.     

Morphological changes of giant mitochondria in the unicellular to multicellular phase during parthenogenesis of Ulva partita (Ulvophyceae) revealed by expression of mitochondrial targeting GFP and PEG transformation

A giant mitochondrion that branches and connects as a single mitochondrion in a cell has been observed during specific phases of the cell cycle of unicellular green algae, but has not been observed in multicellular algae. The genus Ulva is a green macroalga in which the haploid and diploid phases are isomorphic and its gametes develop parthenogenetically. The existence or absence of the giant mitochondrion, and its behavior in Ulva partita, were investigated using a parthenogenesis system. To observe the parthenogenesis of gametes and the dynamics of mitochondria by fluorescence microscopy, we developed an experimental system for culturing and observing U. partita on cover slips: gametes were suspended in 6‐well plates filled with artificial seawater, and cover slips were placed on the well bottoms. The gametes settled on the cover slips as spherical cells (1‐cell S phase). These cells grew into larger cells, losing their eyespot (1‐cell L phase), and developed into multicellular thalli. Gene introduction using the polyethylene glycol (PEG) method is available with transformation efficiencies of 9.0–15.1%. Transformation was performed using a plasmid encoding green fluorescent protein (GFP) fused to the mitochondrial targeting sequence, and mitochondria were labeled by GFP fluorescence. This revealed a string‐shaped giant mitochondrion in a cell of the 1‐cell S phase. In the 1‐cell L phase, a reticular mitochondrion was observed. After the initiation of cell division, the reticular mitochondrion was fragmented, and small oval mitochondria were observed in the 5‐cell phase.     

Local oxygen gradients near isolated mitochondria.

The oxygen concentration in tissue can vary on several length scales. The basic scale of variation is determined by capillary spacing. It is this scale that is manifest in the simplest Krogh cylinder model. A second, smaller scale of variation is associated with the consumption of oxygen by mitochondria. This paper gives a theoretical analysis of these smaller-scale oxygen variations near an isolated mitochondrion. To illustrate the effects of shape, we have carried out the calculations for prolate spheroids as well as for spheres. The principal result is that the local drop in oxygen pressure around a consuming mitochondrion is of the order of (gamma/3K) (3V/4 pi)2/3, where gamma is the oxygen consumption rate per unit mitochondrial volume, K is the Krogh oxygen diffusivity of the surrounding tissue, and V is the mitochondrial volume. The theory is applied to skeletal muscle in vivo and to hepatocytes in cell suspension experiments. In both cases, we find that local oxygen variations produced by oxygen consumption are much smaller than the cell-wide variations produced by the collective effect of all the mitochondria. For example, in maximally consuming skeletal muscle, the drop in oxygen pressure around a consuming mitochondrion is only of the order of 0.03 Torr.     

Identification of two discrete states of energized mitochondria: Experiments on single mitochondria

Dynamic phase microscopy was used to measure the refractivity of a single mitochondrion. Our previous studies showed that application of an electric potential to artificial and natural mitochondrial membranes sharply increases their refractivity. Under the conditions of proton pump activity, the refractivity of a single mitochondrion is 2 to 4 times higher than an average refractivity of deenergized mitochondria. This study demonstrates that the membrane potential of energized mitochondria varies depending on environmental conditions and is controlled by the mitochondrial osmoregulation system. The refractivity of energized mitochondria, i.e., the difference between the refraction indexes of a single mitochondrion and the medium, is 0.02 ± 0.01, i.e., several times lower than that of the energized mitochondria whose membranes bear an electric charge. Earlier it was shown that refractivity of model multilayer systems formed from purified natural lecithin depends linearly on the electric field strength. These data point to a relationship between the refractivity of a single mitochondrion and the membrane potential generated during operation of the proton pump. Under normal conditions (250 mOsm), the mitochondrion behaves as a dynamic system oscillating on a minute scale between two functional states with different values of the refractivity index and different membrane potentials. The transition time is 10–30 s; the lifetime of both states is several minutes. The histograms reflecting the distribution of refractivities of single energized mitochondria within a population (n = 20–30) revealed the presence of two independent peaks (fractions II and III) with average refractivity values of 0.05 ± 0.01 and 0.09 ± 0.01, respectively; these fractions correspond to two long-lived states of mitochondria. However, under hypotonic conditions (120 mOsm), only one (“static”) state was identified, in which oscillations were absent and the refractivity of the overall mitochondrial population does not exceed 0.05 ± 0.01 (fraction II). Studies on mitoplast showed that values of refractivity are related to the inner mitochondrial membrane. It is inferred from these data that there exist two discrete states of mitochondria. Analysis of low-amplitude fluctuations of the refractivity of single mitochondria revealed the presence of frequency components at 1–3 Hz, presumably generated in response to non-uniform functioning of mitochondrial proton pumps. It is suggested that frequency components at 1.8-2.6 Hz are more characteristic of the ATPase pump, while the 1–1.3 Hz frequencies predominate during the functioning of respiratory proton pumps.     

Dynamics of mitochondrial ultrastructure in small pieces of cardiac tissue under long-term anoxic incubation

Anoxic incubation of isolated small pieces of cardiac tissue for 72 h caused emergence of an unusual population of mitochondria, referred to as "mitochondrion inside mitochondrion". We studied dynamics of the origin of this event. In the most part of a mitochondrial population after a 6 h anoxic incubation of myocardial tissue, a local increase in some region of the intermembrane space was observed. Some regions of matrix with adjoined inner membrane move into these regions of intermembrane space, to be constricted eventually. After 12 h of incubation densely neighbouring layers of membrane are observed in these structures. By 24 h of incubation, inside new-formed structures well-distinguished concentric layers of membrane appear. Between these layers some electron-dense material ultrastructurally identical to mitochondrial matrix is seen. By 72 h of anoxic incubation, in cardiomyocytes of the experimental tissue structures with well-marked morphological features of mitochondria appear, which we called "mitochondrion inside mitochondrion". Results of our study are discussed in terms of a conception of changes that occur in the structure of mitochondrial reticulum during apoptosis.     

寄生原生动物线粒体与适应性进化

真核生物的线粒体一般具有一定的典型的结构和功能。然而,在单细胞的寄生原生动物中却不断发现从数量、结构到功能均与典型线粒体明显不同的线粒体,表现出线粒体的巨大可塑性和丰富的多样性。该文对寄生原生动物中这些多样的线粒体进行了概述,并对形成这种多样性的根本原因,即这些生物对寄生生活微氧或无氧环境线粒体所发生的种种适应性进化进行了分析探讨。     

Coupling membranes as energy-transmitting cables. I. Filamentous mitochondria in fibroblasts and mitochondrial clusters in cardiomyocytes

An hypothesis considering mitochondria as intracellular power-transmitting protonic cables was tested in human fibroblasts where mitochondria are thin and long and in rat cardiomyocytes where they show cluster organization. Mitochondria in the cell were specifically stained with fluorescent-penetrating cation ethylrhodamine, which electrophoretically accumulates in the mitochondrial matrix. A 40-micron-long mitochondrial filament of fibroblast was illuminated by a very narrow (less than or equal to 0.5 micron) laser beam to induce local damage of the mitochondrial membranes. Such a treatment was found to induce quenching of the ethylrhodamine fluorescence in the entire filament. According to the electron microscope examination, the laser-treated filament retained its continuity after the laser illumination. Other mitochondrial filaments (some of which were localized at a distance less than 10 micron from the laser-treated one) remained fluorescent. In a cell where mitochondrial filaments seemed to be united in a network, laser illumination of one filament resulted in fluorescence quenching in the whole network, whereas fluorescence of small mitochondria not connected with the network was unaffected. The illumination of cardiomyocyte was found to result in the fluorescence quenching not only in a laser-illuminated mitochondrion but also in a large cluster of organelles composed of many mitochondria. Electron microscopy showed that all the mitochondria in the cluster change from the orthodox to the condensed state. It was also found that mitochondria in the cluster are connected to one another with specific junctions. If a mitochondrion did not form junctions with a quenched cluster, its fluorescence was not decreased even when this mitochondrion was localized close to an illuminated one. The size of the mitochondrial cluster may be as long as 50 micron. The cluster is formed by branched chains of contacting mitochondria, which may be defined as Streptio mitochondriale. In the cardiomyocyte there are several mitochondrial clusters or, alternatively, the quenched cluster is a result of decomposition of a supercluster uniting all the mitochondria of the cell. Cluster organization of mitochondria could also be revealed when a single mitochondrion was punctured in situ with a microcapillary. The obtained data are in agreement with the idea that mitochondrial junctions are H+ permeable so that, within the cluster, delta psi may be transmitted from one mitochondrion to another. The above results are consistent with the assumption that mitochondrial filaments or networks represent a united electrical system.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural changes in L-cell mitochondria exposed to cyanide

Using light and electron microscopy, as well as stereological analysis, a study was made of structural changes in mitochondria of cultured L-cells acted upon with 1 mM cyanide. After 23 hours of incubation, in cells cultured in the presence of cyanide the volume density of mitochondria, the volume of the average mitochondrion and the surface area of its membranes increased nearly twice as much. Concurrently, the number of mitochondria decrease also practically two-fold without any reliable changes in the surface area of membranes per unit of mitochondrion volume. A shorter (7 hours) incubation of L-cells in the presence of cyanide results in a decreased volume density of mitochondria in the cytoplasm, decreased volume of the average mitochondrion, without any significant changes in other above mentioned parameters. It is supposed that the changes in mitochondria under a prolonged cyanide treatment of cells are conditioned by the fusion of mitochondria as well as by a compensatory increase in the total surface of mitochondrial membranes.     

Deregulation of Mitochondrial Membrane Potential by Mitochondrial Insertion of Granzyme B and Direct Hax-1 Cleavage

The cytoplasm and the nucleus have been identified as activity sites for granzyme B (GrB) following its delivery from cytotoxic lymphocyte granules into target cells. Here we report on the ability of exogenous GrB to insert into and function within a proteinase K-resistant mitochondrial compartment. We identified Hax-1 (HS-1-associated protein X-1), a mitochondrial protein involved in the maintenance of mitochondrial membrane potential, as a GrB substrate within the mitochondrion. GrB cleaves Hax-1 into two major fragments: an N-terminal fragment that localizes to mitochondria and a C-terminal fragment that localizes to the cytosol after being released from GrB-treated mitochondria. The N-terminal Hax-1 fragment major cellular impact is on the regulation of mitochondrial polarization. Overexpression of wild-type Hax-1 or its uncleavable mutant form protects the mitochondria against GrB or valinomycin-mediated depolarization. The N-terminal Hax-1 fragment functions as a dominant negative form of Hax-1, mediating mitochondrial depolarization in a cyclophilin D-dependent manner. Thus, induced expression of the N-terminal Hax-1 fragment results in mitochondrial depolarization and subsequent lysosomal degradation of such altered mitochondria. This study is the first to demonstrate GrB activity within the mitochondrion and to identify Hax-1 cleavage as a novel mechanism for GrB-mediated mitochondrial depolarization.     

Mitochondrial Fusion Is Essential for Steroid Biosynthesis

Although the contribution of mitochondrial dynamics (a balance in fusion/fission events and changes in mitochondria subcellular distribution) to key biological process has been reported, the contribution of changes in mitochondrial fusion to achieve efficient steroid production has never been explored. The mitochondria are central during steroid synthesis and different enzymes are localized between the mitochondria and the endoplasmic reticulum to produce the final steroid hormone, thus suggesting that mitochondrial fusion might be relevant for this process. In the present study, we showed that the hormonal stimulation triggers mitochondrial fusion into tubular-shaped structures and we demonstrated that mitochondrial fusion does not only correlate-with but also is an essential step of steroid production, being both events depend on PKA activity. We also demonstrated that the hormone-stimulated relocalization of ERK1/2 in the mitochondrion, a critical step during steroidogenesis, depends on mitochondrial fusion. Additionally, we showed that the SHP2 phosphatase, which is required for full steroidogenesis, simultaneously modulates mitochondrial fusion and ERK1/2 localization in the mitochondrion. Strikingly, we found that mitofusin 2 (Mfn2) expression, a central protein for mitochondrial fusion, is upregulated immediately after hormone stimulation. Moreover, Mfn2 knockdown is sufficient to impair steroid biosynthesis. Together, our findings unveil an essential role for mitochondrial fusion during steroidogenesis. These discoveries highlight the importance of organelles’ reorganization in specialized cells, prompting the exploration of the impact that organelle dynamics has on biological processes that include, but are not limited to, steroid synthesis.     

Mitochondria attached to desmosomes in the ciliary epithelia of human,monkey, and rabbit eyes

In the normal ciliary epithelia of the rhesus monkey, owl monkey, albino rabbit, and human eye, a previously unreported relationship exists between mitochondria and certain desmosomes. At these sites, two mitochondria appear like "sentinels" attached to the cytoplasmic surfaces of their respective sides of a desmosome. In other instances, only one side of the junction may be afforded an associated mitochondrion. In each case the cytoplasmic filaments of the desmosome are seen to blend with the outer membrane of the mitochondrion. The relationship between desmosomes and mitochondria in the ciliary epithelium is unique among ocular tissues. A survey of ocular epithelia in the various species examined, failed to give any evidence of similar junctional/organelle complexes. Various functional roles for this relationship are discussed including the possibility that the mitochondria could control the cytoplasmic calcium ion concentration in the microenvironment of their associated desmosomal junctions.     

An ADP/ATP-specific mitochondrial carrier protein in the microsporidian Antonospora locustae

The mitochondrion is one of the defining characteristics of eukaryotic cells, and to date, no eukaryotic lineage has been shown to have lost mitochondria entirely. In certain anaerobic or microaerophilic lineages, however, the mitochondrion has become severely reduced that it lacks a genome and no longer synthesizes ATP. One example of such a reduced organelle, called the mitosome, is found in microsporidian parasites. Only a handful of potential mitosomal proteins were found to be encoded in the complete genome of the microsporidian Encephalitozoon cuniculi, and significantly no proteins of the mitochondrial carrier family were identified. These carriers facilitate the transport of solutes across the inner mitochondrial membrane, are a means of communication between the mitochondrion and cytosol, and are abundant in organisms with aerobic mitochondria. Here, we report the characterization of a mitochondrial carrier protein in the microsporidian Antonospora locustae and demonstrate that the protein is heterologously targeted to mitochondria in Saccharomyces cerevisiae. The protein is phylogenetically allied to the NAD⁺ transporter of S. cerevisiae, but we show that it has high specificity for ATP and ADP when expressed in Escherichia coli. An ADP/ATP carrier may provide ATP for essential ATP-dependent mitosomal processes such as Hsp70-dependent protein import and export of iron-sulfur clusters to the cytosol.     

Thread-grain transition of mitochondrial reticulum as a step of mitoptosis and apoptosis

Association of mitochondrial population to a mitochondrial reticulum is typical of many types of the healthy cells. This allows the cell to organize a united intracellular power-transmitting system. However, such an association can create some difficulties for the cell when a part of the reticulum is damaged or when mitochondria should migrate from one cell region to another. It is shown that in these cases decomposition of extended mitochondria to small roundish organelles takes place (the thread-grain transition). As an intermediate step of this process, formation of beads-like mitochondria occurs when several swollen parts of the mitochondrial filament are interconnected with thin thread-like mitochondrial structures. A hypothesis is put forward that the thread-grain transition is used as a mechanism to isolate a damaged part of the mitochondrial system from its intact parts. If the injury is not repaired, spherical mitochondrion originated from the damaged part of the reticulum is assumed to convert to a small ultracondensed and presumably dead mitochondrion (this process is called 'mitoptosis'). Then the dead mitochondrion is engulfed by an autophagosome. Sometimes, an ultracondensed mitoplast co-exists with a normal mitoplast, both of them being surrounded by a common outer mitochondrial membrane. During apoptosis, massive thread-grain transition is observed which, according to Youle et al. (S. Frank et al., Dev Cell 1: 515, 2002), is mediated by a dynamin-related protein and represents an obligatory step of the mitochondria-mediated apoptosis. We found that there is a lag phase between addition of an apoptogenic agent and the thread-grain transition. When started, the transition occurs very fast. It is also found that this event precedes complete de-energization of mitochondria and cytochrome c release to cytosol. When formed, small mitochondria migrate to (and in certain rare cases even into) the nucleus. It is suggested that small mitochondria may serve as a transportable form of organelles ('cargo boats' transporting some apoptotic proteins to their nuclear targets).