Arabidopsis thaliana ferrochelatase-I and -II are not imported into Arabidopsis mitochondria

Using in vitro import assays into purified mitochondria and chloroplasts we found that Arabidopsis ferrochelatase-I and ferrochelatase-II were not imported into mitochondria purified from Arabidopsis (or several other plants) but were imported into pea leaf chloroplasts. Other dual targeted proteins could be imported into purified mitochondria from Arabidopsis. As only two ferrochelatase genes are present in the completed Arabidopsis genome, the presence of ferrochelatase activity in plant mitochondria needs to be re-evaluated. Previous reports of Arabidopsis ferrochelatase-I import into pea mitochondria are due to the fact that pea leaf (and root) mitochondria appear to import a variety, but not all chloroplast proteins. Thus pea mitochondria are not a suitable system to either study dual targeting, or to distinguish between isozymes present in mitochondria and chloroplasts.     

Effects of glucose starvation on mitochondrial subpopulations in the meristematic and submeristematic regions of maize root

Mitochondria isolated from 3-mm long maize (Zea mays L. var Dea) root tips were found to be heterogeneous on Percoll density gradients. The ultrastructure of these isolated mitochondria correlated well with that of mitochondria observed in situ and was consistent with the existence of mitochondria at different stages of maturation during cell development. The mitochondria of higher density presented an ultrastructure with many cristae and a dense matrix. These mitochondria showed classic respiratory properties, although with low ADP/O ratios. In contrast, the mitochondria of lower density showed few cristae and a clear matrix and did not seem to be fully functional because their rate of respiration was low and showed weak respiratory control. Lower- and higher- density mitochondria were shown to be differentially affected during the first stages of glucose starvation. The higher-density mitochondria from glucose-starved maize root tips retained the ultrastructure and most of the respiratory properties of nonstarved mitochondria, whereas lower- and intermediate-density mitochondria were absent in the mitochondrial preparations from glucose-starved maize root tips and were not observed in situ. Quantitatively, there was a decrease of the total mitochondrial pool when expressed as the amount of mitochondrial protein per root tip. However, this decrease affected low- and intermediate-density mitochondria, but not higher-density mitochondria. Thus, it was shown that a significant pool of functional mitochondria is maintained in maize root tips during the first stages of glucose starvation. The reasons for these apparently selective effects of glucose starvation on mitochondria are discussed in relation to effects on mitotic and differentiation processes.     

Preparation and properties of mitochondria derived from synaptosomes.

A method has been developed whereby a fraction of rat brain mitochondria (synaptic mitochondria) was isolated from synaptosomes. This brain mitochondrial fraction was compared with the fraction of "free" brain mitochondria (non-synaptic) isolated by the method of Clark & Nicklas (1970). (J. Biol. Chem. 245, 4724-4731). Both mitochondrial fractions are shown to be relatively pure, metabolically active and well coupled. 2. The oxidation of a number of substrates by synaptic and non-synaptic mitochondria was studied and compared. Of the substrates studied, pyruvate plus malate was oxidized most rapidly by both mitochondrial populations. However, the non-synaptic mitochondria oxidized glutamate plus malate almost twice as rapidly as the synaptic mitochondria. 3. The activities of certain tricarboxylic acid-cycle and related enzymes in synaptic and non-synaptic mitochondria were determined. Citrate synthase (EC 4.1.3.7), isocitrate dehydrogenase (EC 1.1.1.41) and malate dehydrogenase (EC 1.1.1.37) activities were similar in both fractions, but pyruvate dehydrogenase (EC 1.2.4.1) activity in non-synaptic mitochondria was higher than in synaptic mitochondria and glutamate dehydrogenase (EC 1.4.1.3) activity in non-synaptic mitochondria was lower than that in synaptic mitochondria. 4. Comparison of synaptic and non-synaptic mitochondria by rate-zonal separation confirmed the distinct identity of the two mitochondrial populations. The non-synaptic mitochondria had higher buoyant density and evidence was obtained to suggest that the synaptic mitochondria might be heterogeneous. 5. The results are also discussed in the light of the suggested connection between the heterogeneity of brain mitochondria and metabolic compartmentation.     

Spatiotemporally controlled initiation of Parkin-mediated mitophagy within single cells

Mitophagy, the selective removal of mitochondria through the autophagic pathway, is involved in cellular mitochondria quality control. Dysfunctional mitochondria can be selectively eliminated through Parkin-mediated mitophagy. Parkin is a ubiquitin E3 ligase that selectively translocates onto impaired mitochondria to initiate mitophagy, and mutations in Parkin have been identified in autosomal recessive forms of Parkinson disease. Here with the use of a genetically encoded, mitochondria-matrix targeting photosensitizer, we established a robust strategy that allows for spatiotemporally controlled initiation of Parkin-mediated mitophagy in single cells with light. The method can specifically target varying numbers of mitochondria into the Parkin-mediated mitophagy pathway for clearance. Combined with live cell imaging, we demonstrated that mitochondria can be cleared by Parkin-mediated mitophagy without juxtanuclear mito-aggresome formation. Autophagy proceeded with the asynchronous appearance of small LC3B-coated structures on Parkin-labeled mitochondria subsections in a nucleation-expansion manner. Our method allows for quantitative measurement on the Parkin-mediated mitophagy process, and can be multiplexed in imaging for higher throughput studies.     

The cell-type specificity of mitochondrial dynamics

Recent advances in mitochondrial imaging have revealed that in many cells mitochondria can be highly dynamic. They can undergo fission/fusion processes modulated by various mitochondria-associated proteins and also by conformational transitions in the inner mitochondrial membrane. Moreover, precise mitochondrial distribution can be achieved by their movement along the cytoskeleton, recruiting various connector and motor proteins. Such movement is evident in various cell types ranging from yeast to mammalian cells and serves to direct mitochondria to cellular regions of high ATP demand or to transport mitochondria destined for elimination. Existing data also demonstrate that many aspects of mitochondrial dynamics, morphology, regulation and intracellular organization can be cell type-/tissue-specific. In many cells like neurons, pancreatic cells, HL-1 cells, etc., complex dynamics of mitochondria include fission, fusion, small oscillatory movements of mitochondria, larger movements like filament extension, retraction, fast branching in the mitochondrial network and rapid long-distance intracellular translocation of single mitochondria. Alternatively, mitochondria can be rather fixed in other cells and tissues like adult cardiomyocytes or skeletal muscles with a very regular organelle organization between myofibrils, providing the bioenergetic basis for contraction. Adult cardiac cells show no displacement of mitochondria with only very small-amplitude rapid vibrations, demonstrating remarkable, cell type-dependent differences in the dynamics and spatial arrangement of mitochondria. These variations and the cell-type specificity of mitochondrial dynamics could be related to specific cellular functions and demands, also indicating a significant role of integrations of mitochondria with other intracellular systems like the cytoskeleton, nucleus and endoplasmic reticulum (ER).     

Production of reactive oxygen by mitochondria from normoxic and hypoxic rat heart tissue.

Reactive oxygen species (ROS), which may be involved in ischemic or reperfusion heart injury, can be produced by mitochondria. Previous work indicated that coupled mitochondria from ischemic heart tissue incubated in calcium-free medium produced less ROS than normal. The effects of calcium, which may be elevated in hypoxic or ischemic tissue, were not examined. The relative production of ROS by mitochondria from normoxic or hypoxic rat heart tissue was estimated by measuring the oxidation of dichlorofluorescin to the fluorescent compound, dichlorofluorescein. ROS were detectable during succinate-stimulated State 4 respiration. In the absence of calcium, mitochondria from hypoxic (60 min) heart tissue produced less ROS than mitochondria from normoxic heart tissue. In the presence of 0.1, 1 or 10 microM calcium, ROS produced by hypoxic mitochondria were increased to normoxic levels. While function was depressed in mitochondria from hypoxic tissue, the presence of 0.1 and 1 microM calcium had no further effect. Respiration was uncoupled in the presence of 10 microM calcium in mitochondria from both normoxic and hypoxic heart tissue. ROS production was increased in mitochondria from hypoxic tissue with both increasing concentrations of calcium and increasing duration of exposure. ROS production in mitochondria from normoxic heart tissue was only stimulated after 200 or more seconds of exposure to 1 or 10 microM calcium. Production of ROS in mitochondria from hypoxic tissue in the presence of 1 microM calcium was inhibited by rotenone (80%), ruthenium red (69%), and a combination of these agents (96%). In contrast, ruthenium red had no effect on ROS production by mitochondria from normoxic heart tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spatiotemporally controlled initiation of Parkin-mediated mitophagy within single cells

Mitophagy, the selective removal of mitochondria through the autophagic pathway, is involved in cellular mitochondria quality control. Dysfunctional mitochondria can be selectively eliminated through Parkin-mediated mitophagy. Parkin is a ubiquitin E3 ligase that selectively translocates onto impaired mitochondria to initiate mitophagy, and mutations in Parkin have been identified in autosomal recessive forms of Parkinson disease. Here with the use of a genetically encoded, mitochondria-matrix targeting photosensitizer, we established a robust strategy that allows for spatiotemporally controlled initiation of Parkin-mediated mitophagy in single cells with light. The method can specifically target varying numbers of mitochondria into the Parkin-mediated mitophagy pathway for clearance. Combined with live cell imaging, we demonstrated that mitochondria can be cleared by Parkin-mediated mitophagy without juxtanuclear mito-aggresome formation. Autophagy proceeded with the asynchronous appearance of small LC3B-coated structures on Parkin-labeled mitochondria subsections in a nucleation-expansion manner. Our method allows for quantitative measurement on the Parkin-mediated mitophagy process, and can be multiplexed in imaging for higher throughput studies.     

Lipidg Peroxidation in Liver and Ehrlich Ascites Cell Mitochondria

Ehrlich ascites cell mitochondria are highly resistant to lipid peroxidation as compared to liver mitochondria from host animals. Succinate protects mitochondria from peroxidative damage, proteins from crosslinks, enzymes from inactivation of the enzymes and membranes from permeability changes. The sensitivity of Ehrlich ascites cell mitochondrial membranes to lipid peroxidation is significantly increased in sub-mitochondrial particles. Lipid peroxidation in tumour mitochondrial membranes can not be diminished by succinate as effectively as in liver mitochondria. Ascites cell mitochondria seems to be protected very efficiently from peroxidative damage by a glutathione-dependent mechanism.     

Germ-line mitochondria exhibit suppressed respiratory activity to support their accurate transmission to the next generation

Mitochondria are accurately transmitted to the next generation through a female germ cell in most animals. Mitochondria produce most ATP, accompanied by the generation of reactive oxygen species (ROS). A specialized mechanism should be necessary for inherited mitochondria to escape from impairments of mtDNA by ROS. Inherited mitochondria are named germ-line mitochondria, in contrast with somatic ones. We hypothesized that germ-line mitochondria are distinct from somatic ones. The protein profiles of germ-line and somatic mitochondria were compared, using oocytes at two different stages in Xenopus laevis. Some subunits of ATP synthase were at a low level in germ-line mitochondria, which was confirmed immunologically. Ultrastructural histochemistry using 3,3′-diaminobenzidine (DAB) showed that cytochrome c oxidase (COX) activity of germ-line mitochondria was also at a low level. Mitochondria in one oocyte were segregated into germ-line mitochondria and somatic mitochondria, during growth from stage I to VI oocytes. Respiratory activity represented by ATP synthase expression and COX activity was shown to be low during most of the long gametogenetic period. We propose that germ-line mitochondria that exhibit suppressed respiration alleviate production of ROS and enable transmission of accurate mtDNA from generation to generation.     

Lipid Composition in Synaptic and Nonsynaptic Mitochondria from Rat Brains and Effect of Aging

The cholesterol, phospholipid, and fatty acid compositions in synaptic and nonsynaptic mitochondria from rat brains and the effect of aging were studied. Both cholesterol and phospholipid contents were found to be significantly different in synaptic compared to nonsynaptic mitochondria. In both types of brain mitochondria, aging decreases the cholesterol content by 27% and the phospholipid content by approximately 12%. The difference between these decreases observed in the organelles causes decreases in the cholesterol/phospholipid molar ratios for synaptic and nonsynaptic mitochondria of 17 and 19%, respectively. Also, the phospholipid composition is significantly different in synaptic compared to nonsynaptic mitochondria. Among phospholipids, only the cardiolipin fraction showed a significant decrease (26%) in nonsynaptic mitochondria from the brains of aged rats. Instead, the fatty acid composition was not significantly different in synaptic compared to nonsynaptic mitochondria. The 21% aging decrease in linoleic acid (18:2), observed only in nonsynaptic mitochondria, may be related to a decrease in cardiolipin, which contains a large amount of this fatty acid.     

Regulation of the number and function of mitochondria during artificial increase of their mass in fish embryos

The mechanisms of mitochondrial mass reduction were investigated by microinjection of mitochondria in developing loach embryos. This reduction can be due to the degradation of the injected mitochondria or to the triggering of regulatory mechanisms. In the latter case the decrease of mitochondrial excess should be caused by exogenous and endogenous mitochondria of the embryos. When the protein-labelled mitochondria were injected into unlabelled eggs or the unlabelled mitochondria were injected into the eggs containing labelled mitochondria, the label content in the mitochondrial protein was decreased 2-fold within 12 hours and then remained unchanged at later stages of embryogenesis. After injection of 3H-labelled mitochondria into the 14C-labelled eggs the 3H/14C ratio in the mitochondrial protein during embryogenesis remained unchanged. These data suggest that the restoration of the normal amount of the mitochondrial mass is caused by the triggering of regulatory mechanisms. Oxygen uptake in the embryos with the artificially increased amount of mitochondria is maintained at a control level or even below control, i. e. undergoes regulation. In the homogenates of these embryos the regulatory control is absent and oxygen uptake is proportional to the amount of mitochondria.     

Lithium desensitizes brain mitochondria to calcium, antagonizes permeability transition, and diminishes cytochrome C release

Among the numerous effects of lithium on intracellular targets, its possible action on mitochondria remains poorly explored. In the experiments with suspension of isolated brain mitochondria, replacement of KCl by LiCl suppressed mitochondrial swelling, depolarization, and a release of cytochrome c induced by a single Ca2+ bolus. Li+ robustly protected individual brain mitochondria loaded with rhodamine 123 against Ca2+-induced depolarization. In the experiments with slow calcium infusion, replacement of KCl by LiCl in the incubation medium increased resilience of synaptic and nonsynaptic brain mitochondria as well as resilience of liver and heart mitochondria to the deleterious effect of Ca2+. In LiCl medium, mitochondria accumulated larger amounts of Ca2+ before they lost the ability to sequester Ca2+. However, lithium appeared to be ineffective if mitochondria were challenged by Sr2+ instead of Ca2+. Cyclosporin A, sanglifehrin A, and Mg2+, inhibitors of the mitochondrial permeability transition (mPT), increased mitochondrial Ca2+ capacity in KCl medium but failed to do so in LiCl medium. This suggests that the mPT might be a common target for Li+ and mPT inhibitors. In addition, lithium protected mitochondria against high Ca2+ in the presence of ATP, where cyclosporin A was reported to be ineffective. SB216763 and SB415286, inhibitors of glycogen synthase kinase-3beta, which is implicated in regulating reactive oxygen species-induced mPT in cardiac mitochondria, did not increase Ca2+ capacity of brain mitochondria. Altogether, these findings suggest that Li+ desensitizes mitochondria to elevated Ca2+ and diminishes cytochrome c release from brain mitochondria by antagonizing the Ca2+-induced mPT.     

Comparative study of functional characteristics of mitochondrial preparations from cell cultures

A comparative study was performed of coupled oxidative phosphorylation in mitochondria and the composition of mitochondria from some cell cultures. The mitochondria from VNK-21 and Vero cells had a high degree of coupled oxidative phosphorylation, which was estimated by the respiratory control coefficient. The cell cultures can be regarded as a promising source of mitochondria to be used for toxicological control of the products of the microbiological synthesis.     

The bioenergetics of brown fat mitochondria from UCP1-ablated mice. Ucp1 is not involved in fatty acid-induced de-energization ("uncoupling").

The bioenergetics of brown fat mitochondria isolated from UCP1-ablated mice were investigated. The mitochondria had lost the high GDP-binding capacity normally found in brown fat mitochondria, and they were innately in an energized state, in contrast to wild-type mitochondria. GDP, which led to energization of wild-type mitochondria, was without effect on the brown fat mitochondria from UCP1-ablated mice. The absence of thermogenic function did not result in reintroduction of high ATP synthase activity. Remarkably and unexpectedly, the mitochondria from UCP1-ablated mice were as sensitive to the de-energizing ("uncoupling") effect of free fatty acids as were UCP1-containing mitochondria. Therefore, the de-energizing effect of free fatty acids does not appear to be mediated via UCP1, and free fatty acids would not seem to be the intracellular physiological activator involved in mediation of the thermogenic signal from the adrenergic receptor to UCP1. In the UCP1-ablated mice, Ucp2 mRNA levels in brown adipose tissue were 14-fold higher and Ucp3 mRNA levels were marginally lower than in wild-type. The Ucp2 and Ucp3 mRNA levels were therefore among the highest found in any tissue. These high mRNA levels did not confer on the isolated mitochondria any properties associated with de-energization. Thus, the mere observation of a high level of Ucp2 or Ucp3 mRNA in a tissue cannot be taken as an indication that mitochondria isolated from that tissue will display innate de-energization or thermogenesis.     

Developmental changes of the sensitivity of cardiac and liver mitochondrial permeability transition pore to calcium load and oxidative stress

Opening of the mitochondrial membrane permeability transition pore (MPTP) is an important factor in the activation of apoptotic and necrotic processes in mammalian cells. In a previous paper we have shown that cardiac mitochondria from neonatal rats are more resistant to calcium load than mitochondria from adult animals. In this study we have analyzed the ontogenetic development of this parameter both in heart and in liver mitochondria. We found that the high resistance of heart mitochondria decreases from day 14 to adulthood. On the other hand, we did not observe a similar age-dependent sensitivity in liver mitochondria, particularly in the neonatal period. Some significant but relatively smaller increase could be observed only after day 30. When compared with liver mitochondria cardiac mitochondria were more resistant also to the peroxide activating effect on calcium-induced mitochondrial swelling. These data thus indicate that the MPTP of heart mitochondria is better protected against damaging effects of the calcium load and oxidative stress. We can only speculate that the lower sensitivity to calcium-induced swelling may be related to the higher ischemic tolerance of the neonatal heart.     

Further studies on the effect of phosphoenolpyruvate on respiration-dependent calcium transport by rat heart mitochondria.

Phosphoenolpyruvate was found to depress extra oxygen consumption associated with Ca2+ -induced respiratory jump by rat heart mitochondria. Addition of phosphoenolpyruvate to mitochondria which have accumulated Ca2+ in the presence of glutamate and inorganic phosphate causes the release of Ca2+ from mitochondria. The phosphoenolpyruvate-stimulated Ca2+ efflux can be observed with mitochondria loaded with low initial Ca2+ concentration (0.12 mM) in the incubation medium. Measurements of mitochondrial H+ translocation produced by addition of Ca2+ to respiring mitochondria show that phosphoenolpyruvate depresses H+ ejection and enhances H+ uptake by mitochondria. The Ca2+ -releasing effect of phosphoenolpyruvate was found to be significantly stronger than that produced by rotenone when added to mitochondria loaded with Ca2+ in the presence of glutamate and inorganic phosphate. Dithiothreitol cannot overcome the effect of phosphoenolpyruvate on mitochondrial Ca2+ transport.     

The effect of irradiation by a He-Ne laser and phytohemagglutinin on lymphocyte mitochondria

Electron-microscopic morphometry has been applied to study mitochondria on ultrathin sections of lymphocytes from human peripheral blood. It has been shown that the stimulation of lymphocytes by the mitogen phytohemagglutinin (PHA) 1 h causes increases in the quantity of mitochondria per cellular section (17%) as well as in the total area of mitochondria per cell section (35%), i.e. an increase in mitochondrial mass. Taking into account known facts about growth and division of mitochondria in late phases of cellular cycle, one can suppose that described above changes in mitochondria during G0----G1 transition under action of PHA belong to an early phase of biogenesis of mitochondria. In the contrary, irradiation of lymphocytes with He-Ne-laser (lambda = 632.8 nm) in dose 56 J/m2 which does not cause the G0----C1 transition, results in the increase in the number of mitochondria per cellular section (20%) but not increase in the total area of mitochondria per cell section. The last finding indicates to some modification of space configuration of the mitochondria without any changes in their mass. The increase in the quantity of mitochondria per cellular section after the irradiation could be related with the increase in electrochemical proton gradient and in phosphorylating activity of mitochondria. He-Ne-laser radiation as well as mitogen PHA cause some deaggregation of mitochondria (this is more pronounced in case of PHA) which may be related to their functional activation.     

Mieap与线粒体的质量控制

线粒体在细胞能量代谢和细胞凋亡中起着至关重要的作用.质量控制是线粒体在细胞中维持正常状态的关键机制.2011年Miyamoto等发现Mieap参与线粒体质量控制的两个新机制.Mieap诱导的溶酶体样细胞器,进入线粒体内,并在线粒体积累,能通过特异性的清除氧化的线粒体蛋白来修复异常线粒体,使得线粒体维持在正常状态.Mieap诱导的通过细胞膜内吞机制形成的囊泡,识别异常线粒体,并对其特异性的清除.Mieap诱导的这两个过程参与了线粒体质量控制,并决定线粒体的命运.     

A retention mechanism for distribution of mitochondria during cell division in budding yeast.

Mitochondria are indispensable for normal eukaryotic cell function. As they cannot be synthesized de novo and are self-replicating, mitochondria must be transferred from mother to daughter cells. Studies in the budding yeast Saccharomyces cerevisiae indicate that mitochondria enter the bud immediately after bud emergence, interact with the actin cytoskeleton for linear, polarized movement of mitochondria from mother to bud, but are equally distributed among mother and daughter cells [1] [2] [3]. It is not clear how the mother cell maintains its own supply of mitochondria. Here, we found that mother cells retain mitochondria by immobilization of some mitochondria in the 'retention zone', the base of the mother cell distal to the bud. Retention requires the actin cytoskeleton as mitochondria colocalized with actin cables in the retention zone, and mutations that perturb actin dynamics or actin-mitochondrial interactions produced retention defects. Our results support the model that equal distribution of mitochondria during cell division is a consequence of two actin-dependent processes: movement of some mitochondria into the daughter bud and immobilization of others in the mother cell.