Videomicroscopic Studies of the Movement of Mitochondria

A stable cell line CV-1 was obtained for vital observation of the transport of mitochondria in animals cells, which express a fragment of the resident protein of mitochondria marked by yellow fluorescent protein. The parameters and conditions of movement of the mitochondria in living cells were established using fluorescence videomicroscopy. Under the normal conditions, only a small part of mitochondria (ca. 7%) was transported over significant distances, while others were in the state of relative rest. The effective transport of mitochondria strictly depended on the dynamic properties of microtubules. Incubation of cell in a serum-free medium suppressed active transport of mitochondria, thus suggesting its dependence on certain, not yet determined environmental factors.     

Chronic high glucose induced INS‐1β cell mitochondrial dysfunction: A comparative mitochondrial proteome with SILAC

As glucose‐stimulated insulin secretion of pancreatic β cell is triggered and promoted by the metabolic messengers derived from mitochondria, mitochondria take a central stage in the normal function of β cells. β cells in diabetics were chronically exposed to hyperglycemia stimulation, which have been reported to exert deleterious effects on β‐cell mitochondria. However, the mechanism of the toxic effects of hyperglycemia on β‐cell mitochondria was not clear. In this study, we characterized the biological functional changes of rat INS‐1β cells and their mitochondria with chronic exposure to hyperglycemia and created a research model of chronic hyperglycemia‐induced dysfunctional β cells with damaged mitochondria. Then, SILAC‐based quantitative proteomic approach was used to compare the mitochondrial protein expression from high glucose treated INS‐1β cells and control cells. The expression of some mitochondrial proteins was found with significant changes. Functional classification revealed most of these proteins were related with oxidative phosphorylation, mitochondrial protein biosynthesis, substances metabolism, transport, and cell death. These results presented some useful information about the effect of glucotoxicity on the β‐cell mitochondria.     

Involvement of p32 and microtubules in alteration of mitochondrial functions by rubella virus

The interaction of the rubella virus (RV) capsid (C) protein and the mitochondrial p32 protein is believed to participate in virus replication. In this study, the physiological significance of the association of RV with mitochondria was investigated by silencing p32 through RNA interference. It was demonstrated that downregulation of p32 interferes with microtubule-directed redistribution of mitochondria in RV-infected cells. However, the association of the viral C protein with mitochondria was not affected. When cell lines either pretreated with respiratory chain inhibitors or cultivated under (mild) hypoxic conditions were infected with RV, viral replication was reduced in a time-dependent fashion. Additionally, RV infection induces increased activity of mitochondrial electron transport chain complex III, which was associated with an increase in the mitochondrial membrane potential. These effects are outstanding among the examples of mitochondrial alterations caused by viruses. In contrast to the preferential localization of p32 to the mitochondrial matrix in most cell lines, RV-permissive cell lines were characterized by an almost exclusive membrane association of p32. Conceivably, this contributes to p32 function(s) during RV replication. The data presented suggest that p32 fulfills an essential function for RV replication in directing trafficking of mitochondria near sites of viral replication to meet the energy demands of the virus.     

Mrs3p, Mrs4p, and frataxin provide iron for Fe-S cluster synthesis in mitochondria

Yeast Mrs3p and Mrs4p are evolutionarily conserved mitochondrial carrier proteins that transport iron into mitochondria under some conditions. Yeast frataxin (Yfh1p), the homolog of the human protein implicated in Friedreich ataxia, is involved in iron homeostasis. However, its precise functions are controversial. Anaerobically grown triple mutant cells (Deltamrs3/4/Deltayfh1) displayed a severe growth defect corrected by in vivo iron supplementation. Because anaerobically grown cells do not synthesize heme, and they do not experience oxidative stress, this growth defect was most likely due to Fe-S cluster deficiency. Fe-S cluster formation was assessed in anaerobically grown cells shifted to air for a brief period. In isolated mitochondria, Fe-S clusters were detected on newly imported yeast ferredoxin precursor and on endogenous aconitase by means of [35S]cysteine labeling and native gel separation. New cluster formation was dependent on iron addition to mitochondria, and the iron concentration dependence was shifted dramatically upward in the Deltamrs3/4 mutant, indicating a role of Mrs3/4p in iron transport. The frataxin mutant strain lacked protein import capacity because of low mitochondrial membrane potential, although this was partially restored by growth in the presence of high iron. Under these conditions, a kinetic defect in new Fe-S cluster formation was still noted. Import of frataxin into frataxin-minus isolated mitochondria promptly corrected the Fe-S cluster assembly defect without further iron addition. These findings show that Mrs3/4p transports iron into mitochondria, whereas frataxin makes iron already within mitochondria available for Fe-S cluster synthesis.     

Vimentin Protects Cells Against Doxorubicin and Vincristine

Vimentin is a class III IF protein that among other functions in the cells interacts with mitochondria causing the elevation of their membrane potential. This interaction may be very important although they are still poorly understood. In this study vimentin-null cells and derivative cell lines that express different mutant forms of human vimentin were used as a model to investigate the ability of this protein to provide the resistance of cells against antitumor drugs and its dependence on the interaction with mitochondria. The half maximal inhibitory concentration (IC₅₀) of vincristine and doxorubicin, two commonly used anticancer drugs, demonstrated that drug resistance of the cells increases only with the vimentin forms that can bind mitochondria. Mutant forms of vimentin lacking the ability to bind mitochondria had no effect on drug resistance. The effect of vimentin on cell viability was observed in the presence of verapamil, the inhibitor of P-glycoprotein, product of Multi-Drug Resistance (MDR) gene. We propose that the interaction of mitochondria with vimentin protects and stabilizes these organelles in the stress conditions.     

In search of an effective cell disruption method to isolate intact mitochondria from Chinese hamster ovary cells

An efficient isolation of mitochondria from cells under physiological conditions is crucial for many studies in life sciences but still challenging in many cases such as in metabolic characterization of mitochondria. In this work, four methods for the disruption of Chinese hamster ovary cells were evaluated regarding their influence on mitochondrial integrity and yield. After cell disruption, mitochondria released from cells were separated from the remaining cell homogenate by differential centrifugation. Sonication was shown to be a rapid and sensitive isolation method. Yields of 14.0 ± 0.3 mg raw mitochondrial protein per 10⁸ cells were obtained. The mitochondria were morphologically intact, with membrane integrities of 67% (outer membrane) to 94% (inner membrane). Compared with the methods using Dounce homogenization, digitonin permeabilization, or electroporation for cell disruption the ultrasound method provided the highest yield of isolated mitochondria. Furthermore, this method is rapid (≈ 45 s for disruption), more robust than Dounce homogenization regarding their influence on mitochondrial integrity and especially suitable for preparing a relatively large amount of mitochondria. The results of this work can be helpful for quantitative and dynamic studies of molecular processes related to mitochondria under physiological conditions for many questions in both biomedicine and biotechnology.     

Mitochondrial dynamics in plant male gametophyte visualized by fluorescent live imaging

Visualization of organelles in living cells is a powerful method for studying their dynamic behavior. Here we attempted to visualize mitochondria in angiosperm male gametophyte (pollen grain from Arabidopsis thaliana) that are composed of one vegetative cell (VC) and two sperm cells (SCs). Combination of mitochondria-targeted fluorescent proteins with VC- or SC-specific expression allowed us to observe the precise number and dynamic behavior of mitochondria in the respective cell types. Furthermore, live imaging of SC mitochondria during double fertilization confirmed previous observations, demonstrated by electron microscopy in other species, that sperm mitochondria enter into the egg and central cells. We also attempted to visualize mutant mitochondria that were elongated due to a defect in mitochondrial division. This mutant phenotype was indeed detectable in VC mitochondria of a heterozygous F(1) plant, suggesting active mitochondrial division in male gametophyte. Finally, we performed mutant screening and isolated a putative mitochondrial protein transport mutant whose phenotype was detectable only in haploid cells. The transgenic materials presented in this work are useful not only for live imaging but also for studying mitochondrial functions by mutant analysis.     

Studies on the Active Transport of Calcium in Human Red Cells

The Ca⁺⁺ transport mechanism in the red cell membrane was studied in resealed ghost cells. It was found that the red cell membrane can transport Ca⁺⁺ from inside the cell into the medium against great concentration gradient ratios. Tracing the movement of ⁴⁵Ca infused inside red cells indicated that over 95% of all Ca⁺⁺ in the cells was transported into media in 20 min incubation under the optimum experimental conditions. The influence of temperature on the rate constant of transport indicated an activation energy of 13,500 cal per mole. The optimum pH range of media for the transport was between 7.5 and 8.5. As energy sources, ATP¹, CTP, and UTP were about equally effective, GTP somewhat less effective, and ITP least effective among the nucleotides tested. The Ca⁺⁺ transport does not appear to involve exchange of Ca⁺⁺ with any monovalent or divalent cations. Also, it is not influenced by oligomycin, sodium azide, or ouabain in high concentrations, which inhibit the Ca⁺⁺ transport in mitochondria or in sarcoplasmic reticulum. In these respects, the Ca⁺⁺ transport mechanism in the red cell membrane is different from those of mitochondria and the sarcoplasmic reticulum.     

Regulation of palmitoyl-CoA inhibition of mitochondrial adenine nucleotide transport by cytosolic fatty acid binding protein.

Defatted liver fatty acid binding protein (FABP) reverses the inhibitory effect of palmitoyl-CoA on adenine nucleotide transport in rat liver mitochondria; addition of titrating amounts of FABP to mitochondria pretreated with palmitoyl-CoA stimulates nucleotide transport and that activation parallels the removal of the inhibitor from mitochondria. This effect is specific only for FABP; all other cytosolic proteins which do not bind fatty acids do not influence nucleotide transport activity. Addition of free fatty acids (which can compete for ligand binding sites on FABP) to mitochondria pretreated with palmitoyl-CoA interferes with the reversal activity of FABP. Adding FABP alone to freshly isolated mitochondria also activates nucleotide transport activity suggesting that the originally submaximal activity is probably due to the presence of endogenous long-chain acyl-CoA esters in the mitochondrial preparation. Because FABP is present in relatively high concentration in most mammalian cells, these observations offer a likely explanation of why the potent inhibitory effects of long-chain acyl-CoA esters on adenine nucleotide transport in isolated mitochondria are not seen in the intact cell.     

A yeast mitochondrial leader peptide functions in vivo as a dual targeting signal for both chloroplasts and mitochondria.

A fusion protein was expressed in transgenic tobacco and yeast cells to examine the functional conservation of mechanisms for importing precursor proteins from the cytosol into mitochondria and chloroplasts. The test protein consisted of the mitochondrial leader peptide from the yeast precursor to cytochrome oxidase subunit Va (prC5) fused to the reporter protein chloramphenicol acetyltransferase. This protein, denoted prC5/CAT, was transported into the mitochondrial interior in yeast and tobacco cells. In both organisms, the mitochondrial form of prC5/CAT was smaller than the primary translation product, suggesting that proteolytic processing occurred during the transport process. prC5/CAT also was translocated into chloroplasts in vivo, accumulating to approximately the same levels as in plant mitochondria. However, accumulation of prC5/CAT in chloroplasts relative to mitochondria varied with the conditions under which plants were grown. The chloroplast form of prC5/CAT also appeared to have been proteolytically processed, yielding a mature protein of the same apparent size as that seen in mitochondria of either tobacco or yeast. Chloramphenicol acetyltransferase lacking a mitochondrial targeting peptide did not associate with either chloroplasts or mitochondria. The results demonstrated that in plant cells a single leader peptide can interact functionally with the protein translocation systems of both chloroplasts and mitochondria, and raised the possibility that certain native proteins might be shared between these two organelles.     

Tudor protein is essential for the localization of mitochondrial RNAs in polar granules of Drosophila embryos

In Drosophila, polar plasm contains polar granules, which deposit the factors required for the formation of pole cells, germ line progenitors. Polar granules are tightly associated with mitochondria in early embryos, suggesting that mitochondria could contribute to pole cell formation. We have previously reported that mitochondrial large and small rRNAs (mtrRNAs) are transported from mitochondria to polar granules prior to pole cell formation and the large rRNA is essential for pole cell formation. Here we show that the localization of mtrRNAs is diminished in embryos laid by tudor mutant females, although the polar granules are maintained. We also found that Tud protein was colocalized with mtrRNAs at the boundaries between mitochondria and polar granules when the transport of mtrRNAs takes place. These observations suggest that Tud mediates the transport of mtrRNAs from mitochondria to polar granules.     

Axonal transport of mitochondria to synapses depends on milton,a novel Drosophila protein

A protein required to localize mitochondria to Drosophila nerve terminals has been identified genetically. Photoreceptors mutant for milton show aberrant synaptic transmission despite normal phototransduction. Without Milton, synaptic terminals and axons lack mitochondria, although mitochondria are numerous in neuronal cell bodies. In contrast, synaptic vesicles continue to be transported to and concentrated at synapses. Milton protein is associated with mitochondria and is present primarily in axons and synapses. A likely explanation of the apparent trafficking defect is offered by the coimmunoprecipitation of Milton and kinesin heavy chain. Transfected into HEK293T cells, Milton induces a redistribution of mitochondria within the cell. We propose that Milton is a mitochondria-associated protein required for kinesin-mediated transport of mitochondria to nerve terminals.     

Metabolic capacity regulates iron homeostasis in endothelial cells

The sensitivity of endothelial cells to oxidative stress and the high concentrations of iron in mitochondria led us to test the hypotheses that (1) changes in respiratory capacity alter iron homeostasis, and (2) lack of aerobic metabolism decreases labile iron stores and attenuates oxidative stress. Two respiration-deficient (rho(o)) endothelial cell lines with selective deletion of mitochondrial DNA (mtDNA) were created by exposing a parent endothelial cell line (EA) to ethidium bromide. Surviving cells were cloned and mtDNA-deficient cell lines were demonstrated to have diminished oxygen consumption. Total cellular and mitochondrial iron levels were measured, and iron uptake and compartmentalization were measured by inductively coupled plasma atomic emission spectroscopy. Iron transport and storage protein expression were analyzed by real-time polymerase chain reaction and Western blot or ELISA, and total and mitochondrial reactive oxygen species (ROS) generation was measured. Mitochondrial iron content was the same in all three cell lines, but both rho(o) lines had lower iron uptake and total cellular iron. Protein and mRNA expressions of major cytosolic iron transport constituents were down-regulated in rho(o) cells, including transferrin receptor, divalent metal transporter-1 (-IRE isoform), and ferritin. The mitochondrial iron-handling protein, frataxin, was also decreased in respiration-deficient cells. The rho(o) cell lines generated less mitochondrial ROS but released more extracellular H(2)O(2), and demonstrated significantly lower levels of lipid aldehyde formation than control cells. In summary, rho(o) cells with a minimal aerobic capacity had decreased iron uptake and storage. This work demonstrates that mitochondria regulate iron homeostasis in endothelial cells.     

Characteristics of Cultured Tobacco Cell Strains Producing High Levels of Ubiquinone-10 Selected by a Cell Cloning Technique

Characteristic differences were examined between tobacco cell strains producing high levels of ubiquinone (UQ) and the original tobacco cell line (Nicotiana tabacum L. cv BY-2). The growth rate of strains producing high levels of UQ was about half of that of the original cells. The maximum yield in cell dry weight was about two-thirds of that of original cells. The time-course of UQ formation by selected strains and the respiratory rates were similar to those in the original cells. The UQ contents were much higher than those in original cells, not only per g-dry weight but also per cell. Most UQ in the selected strains were also localized in mitochondria, as well as in the original cells. On protein basis, the yield of purified mitochondria from strains producing high levels of UQ was 4.3 times as much as that from original cells. UQ formation per mg of mitochondrial protein and the molar ratios of UQ to the other electron transport components in selected cells were similar to those in original cells. The ratio of the mitochondrial protein yield in strains producing high levels of UQ to the yield in original cells correlated closely with the ratio of UQ content per g-dry weight in UQ-producing strains to UQ content in original cells.     

Identification of an N-Methyl-d-aspartate Receptor in Isolated Nervous System Mitochondria

NMDA ionotropic glutamate receptors gate the cytoplasmic influx of calcium, which may, depending on the intensity of the stimulus, subserve either normal synaptic communication or cell death. We demonstrate that when isolated mitochondria are exposed to calcium and NMDA agonists, there is a significant increase in mitochondrial calcium levels. The agonist/antagonist response studies on purified mitochondria suggest the presence of a receptor on mitochondria with features similar to plasma membrane NMDA receptors. Immunogold electron microscopy of hippocampal tissue sections revealed extensive localization of NR2a subunit immunoreactivity on mitochondria. Transient transfection of neuronal GT1-7 cells with an NR1-NR2a NMDA receptor subunit cassette specifically targeting mitochondria resulted in a significant increase in mitochondrial calcium and neuroprotection against glutamate-induced cell death. Mitochondria prepared from GT1-7 cells in which the NR1 subunit of NMDA receptors was silenced demonstrated a decrease in calcium uptake. Our findings are the first to demonstrate that mitochondria express a calcium transport protein that shares characteristics with the NMDA receptor and may play a neuroprotective role.     

Cytoskeleton proteins F-actin and tubulin distribution and interaction with mitochondria in the granulosa cells surrounding stage III zebrafish (Danio rerio) oocytes

The distributional arrangement of mitochondria in the granulosa cells surrounding stage III zebrafish oocyte has been reported as a contiguous aggregation of mitochondria at the margin of the each granulosa cell. The aim of the present study was to further investigate the mitochondrial distribution in the granulosa cell layer in stage III ovarian follicles and the interaction between mitochondria and cytoskeleton elements actin and tubulin. To determine mitochondrial distribution/transport, immunocytochemistry analysis of tubulin and mitochondrial COX-I was carried out along with phalloidin staining of polymerised F-actin. The follicles were also exposed to a range of conditions that are known to affect mitochondria and the cytoskeleton proteins actin and tubulin. The mitochondrial inhibitor FCCP, the anti-mitotic drug nocodazole, and actin polymerisation inhibitor cytochalasin B were used. Levels of ATP, mtDNA copy number, and viability assessed by Trypan blue were also studied after exposure to inhibitors in order to determine the relationship between mitochondrial distribution/activity and ATP production. F-actin showed a hexagonal-polygonal distribution surrounding the mitochondria in granulosa cells, with the F-actin network adjacent to the plasma membrane of each granulosa cell. Tubulin structure presented a less organised distribution than F-actin, it was sparse in the cytosol. Interaction between mitochondria and tubulin was found indicating that mitochondria and tubulin are colocalised in zebrafish ovarian follicles. The exposure of ovarian follicles to inhibitors induced the loss of mitochondrial structural integrity showing that mitochondria distribution in granulosa cells of stage III zebrafish ovarian follicles is determined by the microtubules network.     

ASTER-B regulates mitochondrial carotenoid transport and homeostasis

The scavenger receptor class B type 1 (SR-B1) facilitates uptake of cholesterol and carotenoids into the plasma membrane (PM) of mammalian cells. Downstream of SR-B1, ASTER-B protein mediates the nonvesicular transport of cholesterol to mitochondria for steroidogenesis. Mitochondria also are the place for the processing of carotenoids into diapocarotenoids by β-carotene oxygenase-2. However, the role of these lipid transport proteins in carotenoid metabolism has not yet been established. Herein, we showed that the recombinant StART-like lipid-binding domain of ASTER-A and B preferentially binds oxygenated carotenoids such as zeaxanthin. We established a novel carotenoid uptake assay and demonstrated that ASTER-B expressing A549 cells transport zeaxanthin to mitochondria. In contrast, the pure hydrocarbon β-carotene is not transported to the organelles, consistent with its metabolic processing to vitamin A in the cytosol by β-carotene oxygenase-1. Depletion of the PM from cholesterol by methyl-β-cyclodextrin treatment enhanced zeaxanthin but not β-carotene transport to mitochondria. Loss-of-function assays by siRNA in A549 cells and the absence of zeaxanthin accumulation in mitochondria of ARPE19 cells confirmed the pivotal role of ASTER-B in this process. Together, our study in human cell lines established ASTER-B protein as key player in nonvesicular transport of zeaxanthin to mitochondria and elucidated the molecular basis of compartmentalization of the metabolism of nonprovitamin A and provitamin A carotenoids in mammalian cells.     

Changes in Mitochondrial Respiratory Chain Components of Petunia Cells during Culture in the Presence of Antimycin A

When petunia (Petunia hybrida Vilm, cv Rosy Morn) cells are cultured in the presence of 2 [mu]M antimycin A (AA), respiration proceeds mainly via the cyanide-resistant pathway. Cyanide-resistant respiratory rates were higher in mitochondria from AA cells than in control mitochondria. Compared with control cells, an increase in alternative oxidase protein was observed in AA cells, as well as an increase in ubiquinone (UQ) content. A change in the kinetics of succinate dehydrogenase was observed: there was a much higher activity at high UQ reduction in mitochondria from AA cells compared with control mitochondria. No changes were found for external NADH dehydrogenase kinetics. In AA cells in vivo, UQ reduction was only slightly higher than in control cells, indicating that increased electron transport via the alternative pathway can prevent high UQ reduction levels. Moreover, O2 consumption continues at a similar rate as in control cells, preventing O2 danger. These adaptations to stress conditions, in which the cytochrome pathway is restricted, apparently require, in addition to an increase in alternative oxidase protein, a new setup of the relative amounts and/or kinetic parameters of all of the separate components of the respiratory network.     

A specific role of the yeast mitochondrial carriers MRS3/4p in mitochondrial iron acquisition under iron-limiting conditions

The yeast genes MRS3 and MRS4 encode two members of the mitochondrial carrier family with high sequence similarity. To elucidate their function we utilized genome-wide expression profiling and found that both deletion and overexpression of MRS3/4 lead to up-regulation of several genes of the "iron regulon." We therefore analyzed the two major iron-utilizing processes, heme formation and Fe/S protein biosynthesis in vivo, in organello (intact mitochondria), and in vitro (mitochondrial extracts). Radiolabeling of yeast cells with 55Fe revealed a clear correlation between MRS3/4 expression levels and the efficiency of these biosynthetic reactions indicating a role of the carriers in utilization and/or transport of iron in vivo. Similar effects on both heme formation and Fe/S protein biosynthesis were seen in organello using mitochondria isolated from cells grown under iron-limiting conditions. The correlation between MRS3/4 expression levels and the efficiency of the two iron-utilizing processes was lost upon detergent lysis of mitochondria. As no significant changes in the mitochondrial membrane potential were observed upon overexpression or deletion of MRS3/4, our results suggest that Mrs3/4p carriers are directly involved in mitochondrial iron uptake. Mrs3/4p function in mitochondrial iron transport becomes evident under iron-limiting conditions only, indicating that the two carriers do not represent the sole system for mitochondrial iron acquisition.     

Specific features of changes in levels of endogenous respiration substrates in Saccharomyces cerevisiae cells at low temperature

The rate of endogenous respiration of Saccharomyces cerevisiae cells incubated at 0 degrees C under aerobic conditions in the absence of exogenous substrates decreased exponentially with a half-period of about 5 h when measured at 30 degrees C. This was associated with an indirectly shown decrease in the level of oxaloacetate in the mitochondria in situ. The initial concentration of oxaloacetate significantly decreased the activity of succinate dehydrogenase. The rate of cell respiration in the presence of acetate and other exogenous substrates producing acetyl-CoA in mitochondria also decreased, whereas the respiration rate on succinate increased. These changes were accompanied by an at least threefold increase in the L-malate concentration in the cells within 24 h. It is suggested that the increase in the L-malate level in the cells and the concurrent decrease in the oxaloacetate level in the mitochondria should be associated with a deceleration at 0 degrees C of the transport of endogenous respiration substrates from the cytosol into the mitochondria. This deceleration is likely to be caused by a high Arrhenius activation energy specific for transporters. The physiological significance of L-malate in regulation of the S. cerevisiae cell respiration is discussed.