Single Mitochondria Research Articles

Cryptosporidium parvum mitochondrial-type HSP70 targets homologous and heterologous mitochondria.

A mitochondrial HSP70 gene (Cp-mtHSP70) is described for the apicomplexan Cryptosporidium parvum, an agent of diarrhea in humans and animals. Mitochondrial HSP70 is known to have been acquired from the proto-mitochondrial endosymbiont. The amino acid sequence of Cp-mtHSP70 shares common domains with mitochondrial and proteobacterial homologues, including 34 amino acids of an NH2-terminal mitochondrion-like targeting presequence. Phylogenetic reconstruction places Cp-mtHSP70 within the mitochondrial clade of HSP70 homologues. Using reverse transcription-PCR, Cp-mtHSP70 mRNA was observed in C. parvum intracellular stages cultured in HCT-8 cells. Polyclonal antibodies to Cp-mtHSP70 recognize a approximately 70-kDa protein in Western blot analysis of sporozoite extracts. Both fluorescein- and immunogold-labeled anti-Cp-mtHSP70 localize to a single mitochondrial compartment in close apposition to the nucleus. Furthermore, the NH2-terminal presequence of Cp-mtHSP70 can correctly target green fluorescent protein to the single mitochondrion of the apicomplexan Toxoplasma gondii and the mitochondrial network of the yeast Saccharomyces cerevisiae. When this presequence was truncated, the predicted amphiphilic alpha-helix was shown to be essential for import into the yeast mitochondrion. These data further support the presence of a secondarily reduced relict mitochondrion in C. parvum.

Role of mitochondrial inner membrane permeabilization in necrotic cell death, apoptosis, and autophagy.

Inhibition of mitochondrial oxidative phosphorylation progresses to uncoupling when opening of cyclosporin A-sensitive permeability transition pores increases permeability of the mitochondrial inner membrane to small solutes. Involvement of the mitochondrial permeability transition (MPT) in necrotic and apoptotic cell death is implicated by demonstrations of protection by cyclosporin A against oxidative stress, ischemia/reperfusion, tumor necrosis factor-alpha exposure, Fas ligation, calcium overload, and a variety of toxic chemicals. Confocal microscopy directly visualizes the MPT in single mitochondria within living cells from the translocation of impermeant fluorophores, such as calcein, across the inner membrane. Simultaneously, mitochondria release potential-indicating fluorophores. Subsequently, mitochondria swell, causing outer membrane rupture and release of cytochrome c and other proapoptotic proteins from the intermembrane space. In situ a sequence of decreased NAD(P)H, increased free calcium, and increased reactive oxygen species formation within mitochondria promotes the MPT and subsequent cell death. Necrotic and apoptotic cell death after the MPT depends, in part, on ATP levels. If ATP levels fall profoundly, glycine-sensitive plasma membrane permeabilization and rupture ensue. If ATP levels are partially maintained, apoptosis follows the MPT. The MPT also signals mitochondrial autophagy, a process that may be important in removing damaged mitochondria. Cellular features of necrosis, apoptosis, and autophagy frequently occur together after death signals and toxic stresses. A new term, necrapoptosis, describes such death processes that begin with a common stress or death signal, progress by shared pathways, but culminate in either cell lysis (necrosis) or programmed cellular resorption (apoptosis), depending on modifying factors such as ATP.

S2J1 はじめに : 単一ミトコンドリア膜電位のリアルタイムイメージング(オルガネラ:そのダイナミクスから見た機能発現戦略,シンポジウム,日本生物物理学会第40回年会)

S2J1 はじめに : 単一ミトコンドリア膜電位のリアルタイムイメージング(オルガネラ:そのダイナミクスから見た機能発現戦略,シンポジウム,日本生物物理学会第40回年会)

3P227単一ミトコンドリアにおける膜透過性変化の検出とその機構の研究

3P227単一ミトコンドリアにおける膜透過性変化の検出とその機構の研究

Asymmetries in the Maternal and Paternal Genetic Histories of Colombian Populations

The mitochondrial genome was among the earliest molecules to be surveyed for variation at the DNA level in humans. Some of the advantages offered by mtDNA in the pre-PCR era were purely practical: it is abundant in most cells, and its circularity and small size allowed it to be purified from nuclear DNA by centrifugation in cesium chloride gradients. Even without cloning or PCR amplification, sufficient DNA could be obtained for surveys of restriction fragment–length polymorphisms. In addition to these features, which provide for its easy manipulation in the laboratory, the molecule possesses further advantages to the population geneticist. The most exploited feature has been its apparent inability to undergo genetic recombination; despite the large number of mitochondrial genomes in most cells (and even within single mitochondria) there is still no convincing evidence that portions of the genome are exchanged in a recombinational process between molecules (Jorde and Bamshad 2000; Kivisild and Villems 2000; Kumar et al. 2000; Parsons and Irwin 2000). This failure to recombine means that all modern mtDNA sequences descend from a single ancestral molecule at some point in the past (a feature known as “coalescence”) and that a modern mtDNA sequence will differ from an ancestral molecule only by the accumulation of mutations—a process that appears to be fairly regular when viewed over appreciable evolutionary periods. Thus, it has been a relatively straightforward task to assess the extent of genetic variation in living humans and to estimate the time required to generate it. In practice, this is done by counting the number of mutations that have occurred between a reconstructed ancestral DNA sequence and a sample of modern sequences and then multiplying this by the mutation rate of mtDNA. Allan Wilson and his associates were among the first to exploit this feature of mtDNA and to recognize that our origins as a species should not antedate the coalescence time of mtDNA (barring a very complicated history of natural selection and migration) (Cann et al. 1987; Vigilant et al. 1991).

2L1700 単一ミトコンドリアを対象とした蛍光色素による膜電位計測方法の開発

2L1700 単一ミトコンドリアを対象とした蛍光色素による膜電位計測方法の開発

A transient decrease of electrochemical gradient stabilizes DNA structural change in single mitochondria of living cells

The effect of controlled and reversible perturbation of the electrochemical gradient on the structural changes of mitochondrial DNA has been studied in living cells by fluorescence microscopy. Electrochemical gradient perturbations were induced by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone and quantified by measuring the mitochondrial membrane potential using tetramethyl rhodamine methyl ester. Under our experimental conditions, we have shown that ethidium fluorescence was mainly due to ethidium molecules intercalated in mtDNA. Ethidium fluorescence variations have been used to probe DNA structural changes. This showed that: i) electrochemical gradient perturbations induced mtDNA structural change; ii) this change was readily reversible following a total but short collapse of the electrochemical gradient; iii) in contrast, a short and weak perturbation of the electrochemical gradient stabilized the mtDNA structural change; and iv) the degree of weak depolarization varied from cell to cell, showing the necessity of studying the effect of energetic perturbations at the level of an individual cell.

A transient decrease of electrochemical gradient stabilizes DNA structural change in single mitochondria of living cells

The effect of controlled and reversible perturbation of the electrochemical gradient on the structural changes of mitochondrial DNA has been studied in living cells by fluorescence microscopy. Electrochemical gradient perturbations were induced by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone and quantified by measuring the mitochondrial membrane potential using tetramethyl rhodamine methyl ester. Under our experimental conditions, we have shown that ethidium fluorescence was mainly due to ethidium molecules intercalated in mtDNA. Ethidium fluorescence variations have been used to probe DNA structural changes. This showed that: i) electrochemical gradient perturbations induced mtDNA structural change; ii) this change was readily reversible following a total but short collapse of the electrochemical gradient; iii) in contrast, a short and weak perturbation of the electrochemical gradient stabilized the mtDNA structural change; and iv) the degree of weak depolarization varied from cell to cell, showing the necessity of studying the effect of energetic perturbations at the level of an individual cell.

Presynaptic mitochondria and the temporal pattern of neurotransmitter release.

Mitochondria are critical for the function of nerve terminals as the cycling of synaptic vesicle membrane requires an efficient supply of ATP. In addition, the presynaptic mitochondria take part in functions such as Ca2+ buffering and neurotransmitter synthesis. To learn more about presynaptic mitochondria, we have examined their organization in two types of synapse in the lamprey, both of which are glutamatergic but are adapted to different temporal patterns of activity. The first is the giant lamprey reticulospinal synapse, which is specialized to transmit phasic signals (i.e. bursts of impulses). The second is the synapse established by sensory dorsal column axons, which is adapted to tonic activity. In both cases, the presynaptic axons were found to contain two distinct types of mitochondria; small 'synaptic' mitochondria, located near release sites, and larger mitochondria located in more central parts of the axon. The size of the synapse-associated mitochondria was similar in both types of synapse. However, their number differed considerably. Whereas the reticulospinal synapses contained only single mitochondria within 1 micron distance from the edge of the active zone (on average 1.2 per active zone, range of 1-3), the tonic dorsal column synapses were surrounded by clusters of mitochondria (4.5 per active zone, range of 3-6), with individual mitochondria sometimes apparently connected by intermitochondrial contacts. In conjunction with studies of crustacean neuromuscular junctions, these observations indicate that the temporal pattern of transmitter release is an important determinant of the organization of presynaptic mitochondria.

3PD045 蛍光画像解析による単一心筋ミトコンドリアの活性測定

3PD045 蛍光画像解析による単一心筋ミトコンドリアの活性測定

Do single mitochondria contain zones with different membrane potential?

Do single mitochondria contain zones with different membrane potential?

Integrating cytosolic calcium signals into mitochondrial metabolic responses.

Stimulation of hepatocytes with vasopressin evokes increases in cytosolic free Ca2+ ([Ca2+]c) that are relayed into the mitochondria, where the resulting mitochondrial Ca2+ ([Ca2+]m) increase regulates intramitochondrial Ca2+-sensitive targets. To understand how mitochondria integrate the [Ca2+]c signals into a final metabolic response, we stimulated hepatocytes with high vasopressin doses that generate a sustained increase in [Ca2+]c. This elicited a synchronous, single spike of [Ca2+]m and consequent NAD(P)H formation, which could be related to changes in the activity state of pyruvate dehydrogenase (PDH) measured in parallel. The vasopressin-induced [Ca2+]m spike evoked a transient increase in NAD(P)H that persisted longer than the [Ca2+]m increase. In contrast, PDH activity increased biphasically, with an initial rapid phase accompanying the rise in [Ca2+]m, followed by a sustained secondary activation phase associated with a decline in cellular ATP. The decline of NAD(P)H in the face of elevated PDH activity occurred as a result of respiratory chain activation, which was also manifest in a calcium-dependent increase in the membrane potential and pH gradient components of the proton motive force (PMF). This is the first direct demonstration that Ca2+-mobilizing hormones increase the PMF in intact cells. Thus, Ca2+ plays an important role in signal transduction from cytosol to mitochondria, with a single [Ca2+]m spike evoking a complex series of changes to activate mitochondrial oxidative metabolism.

Imaging the Permeability Pore Transition in Single Mitochondria

In mitochondria the opening of a large proteinaceous pore, the “mitochondrial permeability transition pore” (MTP), is known to occur under conditions of oxidative stress and matrix calcium overload. MTP opening and the resulting cellular energy deprivation have been implicated in processes such as hypoxic cell damage, apoptosis, and neuronal excitotoxicity. Membrane potential (ΔΨ m) in single isolated heart mitochondria was measured by confocal microscopy with a voltage-sensitive fluorescent dye. Measurements in mitochondrial populations revealed a gradual loss of ΔΨ m due to the light-induced generation of free radicals. In contrast, the depolarization in individual mitochondria was fast, sometimes causing marked oscillations of ΔΨ m. Rapid depolarizations were accompanied by an increased permeability of the inner mitochondrial membrane to matrix-entrapped calcein (≈620 Da), indicating the opening of a large membrane pore. The MTP inhibitor cyclosporin A significantly stabilized ΔΨ m in single mitochondria, thereby slowing the voltage decay in averaged recordings. We conclude that the spontaneous depolarizations were caused by repeated stochastic openings and closings of the transition pore. The data demonstrate a much more dynamic regulation of membrane permeability at the level of a single organelle than predicted from ensemble behavior of mitochondrial populations.

The protein phosphatase inhibitor okadaic acid induces defects in cytokinesis and organellar genome segregation in Trypanosoma brucei

Mitosis and cytokinesis are events that are highly coordinated in most eukaryotic cell cycles. African trypanosomes possess a single mitochondrion and must additionally coordinate the organellar division cycle. Here we report that okadaic acid, a potent and specific inhibitor of protein phosphatases PP1and PP2A, uncouples these cycles in living trypanosomes. Cell cycle analysis of treated cells revealed elevated DNA content. Microscopic examination indicated that okadaic acid treatment yielded multinucleate cells with a single mitochondrial network indicating these cells have undergone mitosis but failed to complete cytokinesis. Immunofluorescence analysis of 5-bromo-2-deoxyuridine incorporation demonstrated that the mitochondrial DNA was replicated but did not segregate. The dose response curve for inhibition of the normal cell cycle paralleled that for the in vitro inhibition of protein phosphatase activities with IC50s of approximately 20 nM okadaic acid. These results suggest the involvement of a PP1/PP2A-like activity in coordinating mitosis, mitochondrial DNA division and cytokinesis in trypanosomes.

Imaging in 5 dimensions: Quantitative measurement of membrane potential from single mitochondria in living cells

The energy released during the oxidation reactions in the mitochondrial respiratory chain is stored as an electrochemical potential consisting of a transmembrane electrical potential (Vmit) of about −180 mV and a proton gradient of about 1 pH unit; this drives the synthesis of the ATP required to fuel the cell’s energy-dependent processes. This key cellular system has been the subject of thousands of studies. However, data on how it is regulated by the physiological state of the cell has only been gathered via indirect studies on isolated mitochondrial suspensions; quantitative studies on individual mitochondria in situ have been precluded by their small size, their high motility, and the absence of appropriate methodologies.An approach toward a more quantitative assessment of Vmit within intact cells was based on the development of the potentiometric fluorescent dyes TMRE and TMRM, which rapidly equilibrate across membranes in accord with the Nernst equation.

Inner ear fine structures of the hamster in frozen sections used for immunohistochemical assays for inner ear diseases

Frozen sections of the inner ear of the hamster enable detailed investigations of the fine structures in immunofluorescence assays. At high magnification single mitochondria can be identified by their reactions with an antiserum containing antibodies against mitochondria. In the positive reaction with an antiserum against nuclei, the typical green fluorescence is restricted to the nuclei, which are mostly separated by the surrounding cytoplasm. The method of immunohistochemical assay using frozen sections from the non-decalcified inner ear is very time-consuming and cannot be recommended for the routine diagnosis of inner ear diseases, although it may be useful in research and for studying critical clinical cases.

The Quantitative Ultrastructure of the Heart Muscle of Japanese Quail by Hypergravitation, Hypodynamy and Combination

It is anticipated that Japanese quail /Coturnix coturnix japonica/ will provide animal proteins in long term space flights. Consequently this species of birds is of research interest of international space program INTERCOSMOS. In the year 1987 we reported on an experiment /2/ in which the effect of chronic acceleration of 2 G hypergravitation, the hypodynamy and the simultaneous effect of chronic acceleration and the location in the centre of the turntable of the centrifuge on the protein fractions in skeletal muscles was studied. The ultrastructure of the heart muscle was now in this experiments examined as well.Japanese quail cockerels, aged 48 days were exposed to 2 G hypergravitation /group IV/ in a 6,4 m diameter centrifuge, to hypodynamy /group III/ and their combination /group V/, respectively for 6 days / Fig.1/. The hypodynamy in group III was achieved by suspending the birds in jackets without contact the floor. The group II was located in the centre ofthe turntable of the centrifuge. The control group I. was kept under normal conditions. The quantitative ultrastructure of myocard was evaluated by the methods of Weibel/3/ - this enables to determine the number, relative size and volume of mitochondria volume of single mitochondria, defficiency of mitochondrial cristae and volume of myofibrils.

Calcium-shifts in anoxic cardiac myocytes. A cytochemical study

Cultivated heart muscle cells from adult rats were exposed to anoxia in a substrate-free Tyrode solution at constant pH. It has been shown previously that in this system anoxic changes of metabolism and morphology develop gradually during the first 60 min. In this study, intracellular Ca2+ localization was pursued cytochemically. Under aerobic control conditions, Ca2+-deposits are only detected along the sarcolemma and T-tubules. Under anoxia the pattern changes gradually. During the first 60 min, the number of sarcolemmal deposits is transiently increased and single deposits appear inside mitochondria. After 90 and 120 min of anoxia, an increasing number of cells have lost their ability of sarcolemmal Ca2+ binding, but exhibit clustered deposits in single mitochondria. These cells are hypercontracted, often contain condensed myofibrillar masses and are covered with large sarcolemmal protrusions, indicating that they are irreversible injured. Loss of sarcolemmal Ca2+ binding ability seems to be a crucial event on the edge of the development of irreversible injury. Since the sarcolemmal Ca2+-deposits in normal cells are believed to be causally related to a binding of Ca2+ to anionic phospholipids, a decrease of Ca2+ affinity of these phospholipids and a change in the sarcolemmal phospholipid composition may be considered as causes for the disappearance of sarcolemma-bound Ca2+.

Scanning electron-microscopic studies on the three-dimensional structure of mitochondria in the mammalian red, white and intermediate muscle fibers.

The three-dimensional structure and arrangement of mitochondria in the red, white and intermediate striated muscle fibers of the rat were examined under a field-emission type scanning electron microscope after removal of cytoplasmic matrices by means of the Osmium-DMSO-Osmium procedure. Beneath the sarcolemma, spherical or ovoid subsarcolemmal mitochondria show accumulations. The mitochondria are numerous and large in size in the red fibers, intermediate in the intermediate fibers, and few and small in the white fibers. Paired, slender I-band-limited mitochondria were located on both sides of the Z-line and partly embraced the myofibrils at the I-band level; they occurred in all three types of fibers. In the intermyofibrillar spaces, numerous mitochondria formed mitochondrial columns. These columns were classified into two types: 1) thick mitochondrial columns, formed by multiple mitochondria each with an intermyofibrillar space corresponding to one sarcomere in length, and 2) thin mitochondrial columns, established by single mitochondria corresponding to one sarcomere in length. In the red fibers mitochondrial columns were abundant and the ratio of the thick and thin columns was almost the same, while in the intermediate fibers most of the columns belonged to the thin type. The white fibers displayed rare, very thin columns.

Serial reconstruction of the mitochondrial reticulum in the antarctic flagellate,Pyramimonas gelidicola (Prasinophyceae, Chlorophyta)

A serial reconstruction ofPyramimonas gelidicolaMcFadden, Moestrup andWetherbee has revealed a large reticulated mitochondrion branching throughout the cell. The possibility of single mitochondria in other members of thePrasinophyceae and the uniformity of the morphology of this organelle within the class is discussed.