Quantitative analysis of spontaneous mitochondrial depolarizations

Spontaneous transient depolarizations in mitochondrial membrane potential (DeltaPsi(m)), mitochondrial flickers, have been observed in isolated mitochondria and intact cells using the fluorescent probe, tetramethylrhodamine ethyl ester (TMRE). In theory, the ratio of [TMRE] in cytosol and mitochondrion allows DeltaPsi(m) to be calculated with the Nernst equation, but this has proven difficult in practice due to fluorescence quenching and binding of dye to mitochondrial membranes. We developed a new method to determine the amplitude of flickers in terms of millivolts of depolarization. TMRE fluorescence was monitored using high-speed, high-sensitivity three-dimensional imaging to track individual mitochondria in freshly dissociated smooth muscle cells. Resting mitochondrial fluorescence, an exponential function of resting DeltaPsi(m), varied among mitochondria and was approximately normally distributed. Spontaneous changes in mitochondrial fluorescence, indicating depolarizations and repolarizations in DeltaPsi(m), were observed. The depolarizations were reversible and did not result in permanent depolarization of the mitochondria. The magnitude of the flickers ranged from <10 mV to >100 mV with a mean of 17.6 +/- 1.0 mV (n = 360) and a distribution skewed to smaller values. Nearly all mitochondria flickered, and they did so independently of one another, indicating that mitochondria function as independent units in the myocytes employed here.     

Mitochondrial and nonmitochondrial reduction of MTT: interaction of MTT with TMRE, JC-1, and NAO mitochondrial fluorescent probes

BACKGROUND: Bioreduction of water-soluble tetrazolium salts (e.g., MTS, XTT, and MTT) to their respective formazans is generally regarded as an indicator of cell "redox activity." The reaction is attributed mainly to mitochondrial enzymes and electron carriers. However, MTT reduction may also be catalyzed by a number of other nonmitochondrial enzymes. The goal of this work was to establish the sites of MTT reduction in intact HepG2 human hepatoma cells in culture. METHODS: In order to establish the subcellular localization of the sites of reduction of MTT, we imaged the formation of MTT-formazan deposits using backscattered light confocal microscopy. Mitochondria were visualized in viable cells using fluorescent dyes that bind in a manner dependent (JC-1 and TMRE) or independent (NAO) of mitochondrial electric potential. RESULTS: Only 25-45% of MTT-formazan was associated with mitochondria after 25 min of incubation. No more than 25% of the mitochondrial area on images was occupied by MTT-formazan. Mitochondrial fluorescence of TMRE, NAO, and the monomeric form of JC-1 decreased rapidly in cells incubated with MTT. However, the intensity of fluorescence of JC-1 aggregates dropped by less than 30% at the onset of incubation and remained constant as reduction of MTT proceeded further. CONCLUSIONS: (1) Most of MTT-formazan deposits are not coincident with mitochondria. (2) Monomeric JC-1, as well as TMRE and NAO, accumulating in mitochondria may be displaced by MTT. Thus, the presence of positively charged organic compounds (like MTT) may distort measurements of mitochondrial transmembrane electric potential, which are based on accumulation of fluorescent dyes.     

Phagocytic Activity of FRTL-5 Rat Thyroid Follicular Cells as Measured by Ingestion of Fluorescent Latex Beads

A rat thyroid cell line (FRTL-5) was used to study the phagocytic activity of thyroid follicular cells using fluorescent latex beads and flow cytometric analysis. Morphologic studies demonstrated that latex beads were engulfed and located within cytoplasmic vacuoles of thyrocytes. Flow cytometric evaluation of cell suspensions revealed high levels of fluorescence in cells engulfing latex beads. Using thyrotropin (TSH) as a stimulator of thyroid function and human interleukin-1β as an inhibitor, protocols were established for measuring the effects of these substances on either basal or TSH-induced phagocytosis. Cells exposed to latex beads over time in basal (0H) or TSH-containing medium had an increase in time-dependent phagocytic activity which was maximal after 24 or 8 h, respectively. Treatment of FRTL-5 cells with either a stimulator or an inhibitor revealed maximal change in phagocytic activity after 72 h as measured by the percentage of phagocytic cells as well as the mean fluorescence intensity. Phagocytic activity and iodide trapping by FRTL-5 cells were qualitatively similar in both sensitivity and magnitude of change in the assays used in this study. Phagocytosis of fluorescent latex beads represents a sensitive nonradioactive assay of thyrocyte function whose regulation is similar to iodide trapping.     

Safranine O as a fluorescent probe for mitochondrial membrane potential studied on the single particle level and in suspension

The permeant cationic dye safranine O is often used to measure mitochondrial membrane potential due to the dependence of both its absorption and fluorescence on mitochondrial energization, which causes its oligomerization inside mitochondria. In the present study we have used fluorescent correlation spectroscopy (FCS) to record the fluorescence changes on a micro level, i.e. under conditions permitting resolution of contributions from single particles (molecules of the dye and stained mitochondria). We have shown that the decrease in fluorescence signal from a suspension of energized mitochondria stained with a high safranine concentration (10 μM) is explained by the decrease in dye concentration in the medium in parallel with the accumulation of the dye inside the mitochondria, which results in fluorescence quenching. With 1 μM safranine O, the fluorescence rise after energization is caused by the accumulation of the dye up to a level not sufficient for full fluorescence quenching and also by the higher intensity of mitochondrial fluorescence on immersion of the dye in the hydrophobic milieu. Besides the estimation of the inner mitochondrial membrane potential, this approach also assesses the concentration of fluorescent particles. The non-monotonic dependence of the FCS parameter 1/G(τ→0) on the concentration of mitochondrial protein suggests heterogeneity of the system with respect to fluorescence of particles. An important advantage of the described method is its high sensitivity, which allows measurements with low concentrations and quantities of mitochondrial protein in samples (less than 10 μg).     

High throughput particle analysis: combining dielectrophoretic particle focussing with confocal optical detection

A micro flow cytometer has been fabricated that detects and counts fluorescent particles flowing through a microchannel at high speed based upon their fluorescence emission intensity. Dielectrophoresis is used to continuously focus particles within the flowing fluid stream into the centre of the device, which is 40 microm high and 250 microm wide. The method ensures that all the particles pass through an interrogation region approximately 5 microm in diameter, which is created by focusing a beam of light into a spot. The functioning of the device was demonstrated by detecting and counting fluorescent latex particles at a rate of up to 250 particles/s. A mixture of three different populations of latex particle was used, each sub-population with a distinct level of fluorescent intensity. The device was evaluated by comparison with a conventional fluorescent activated cell sorter (FACS) and numerical simulation demonstrated that for 6 microm beads, and for this design of chip the theoretical throughput is of the order of 1000 particles/s (corresponding to a particle velocity of 10 mm s(-1)).     

Effect of Farnesyltransferase Inhibitor R115777 on Mitochondria of Plasmodium falciparum

The parasite Plasmodium falciparum causes severe malaria and is the most dangerous to humans. However, it exhibits resistance to their drugs. Farnesyltransferase has been identified in pathogenic protozoa of the genera Plasmodium and the target of farnesyltransferase includes Ras family. Therefore, the inhibition of farnesyltransferase has been suggested as a new strategy for the treatment of malaria. However, the exact functional mechanism of this agent is still unknown. In addition, the effect of farnesyltransferase inhibitor (FTIs) on mitochondrial level of malaria parasites is not fully understood. In this study, therefore, the effect of a FTI R115777 on the function of mitochondria of P. falciparum was investigated experimentally. As a result, FTI R115777 was found to suppress the infection rate of malaria parasites under in vitro condition. It also reduces the copy number of mtDNA-encoded cytochrome c oxidase III. In addition, the mitochondrial membrane potential (ΔΨm) and the green fluorescence intensity of MitoTracker were decreased by FTI R115777. Chloroquine and atovaquone were measured by the mtDNA copy number as mitochondrial non-specific or specific inhibitor, respectively. Chloroquine did not affect the copy number of mtDNA-encoded cytochrome c oxidase III, while atovaquone induced to change the mtDNA copy number. These results suggest that FTI R115777 has strong influence on the mitochondrial function of P. falciparum. It may have therapeutic potential for malaria by targeting the mitochondria of parasites.     

Mmm1p, a mitochondrial outer membrane protein, is connected to mitochondrial DNA (mtDNA) nucleoids and required for mtDNA stability

In the yeast Saccharomyces cerevisiae, mitochondria form a branched, tubular reticulum in the periphery of the cell. Mmm1p is required to maintain normal mitochondrial shape and in mmm1 mutants mitochondria form large, spherical organelles. To further explore Mmm1p function, we examined the localization of a Mmm1p-green fluorescent protein (GFP) fusion in living cells. We found that Mmm1p-GFP is located in small, punctate structures on the mitochondrial outer membrane, adjacent to a subset of matrix-localized mitochondrial DNA nucleoids. We also found that the temperature-sensitive mmm1-1 mutant was defective in transmission of mitochondrial DNA to daughter cells immediately after the shift to restrictive temperature. Normal mitochondrial nucleoid structure also collapsed at the nonpermissive temperature with similar kinetics. Moreover, we found that mitochondrial inner membrane structure is dramatically disorganized in mmm1 disruption strains. We propose that Mmm1p is part of a connection between the mitochondrial outer and inner membranes, anchoring mitochondrial DNA nucleoids in the matrix.     

Electrostatic interaction between merocyanine 540 and liposomal and mitochondrial membranes

Summary The fluorescence of merocyanine 540 (MC) in liposomal and mitochondrial suspensions was measured under various conditions. Under a given condition, both the amount of dye bound to the membrane and the zeta potential were determined simultaneously. It was found that the fluorescence intensity was proportional to the amount of bound dye and correlated with the zeta potential of particles. The fluorescence intensity was represented quantitatively in terms of the Langmuir adsorption isotherm, when the electrostatic interaction acting between MC and membrane surface was properly taken into account. It was concluded that the changes in MC fluorescence in the liposomal and mitochondrial suspensions are mainly attributed to the changes in the surface potential of the membranes.     

Three-dimensional analysis of the association of viral particles with mitochondria during the replication of Rice gall dwarf virus

Examination of cultured insect vector cells that had been infected with Rice gall dwarf virus (RGDV), using transmission electron microscopy and confocal microscopy, revealed the presence of clusters of virus-coated mitochondria around viroplasms in which replication and assembly of RGDV occurred, suggesting a role for mitochondria in supplying the energy required for viral morphogenetic processes. Electron tomography revealed that RGDV particles on the surface of mitochondria are arrayed in an orderly but loose manner, unlike tightly packaged particles in vesicular compartments, suggesting the presence of counterpart molecules on the surface of mitochondria. The viral particles in close proximity to mitochondria were aligned along intermediate filaments, which might serve as scaffolds for the anchorage of these particles. RGDV has a putative mitochondrion-targeting sequence on the outer surface of the outer-capsid protein P8. The arrangement of RGDV particles around mitochondria suggests that the region of the P8 protein containing the mitochondrion-targeting sequence might attach to a molecule like a receptor on the outer mitochondrial membrane. Our analysis demonstrates the three-dimensional arrangement and molecular basis for the mitochondrial proximity of RGDV particles during viral replication.     

Phagocytosis and membrane fluidity: application to the evaluation of opsonizing properties of fibronectin

The uptake of particles by phagocytic cells involves an increase in the membrane fluidity determined by steady-state fluorescence polarization. Binding and endocytosis of target particles is in vivo enhanced by humoral factors called opsonins. In this work, fluorescence polarization was used to detect in vitro the opsonic activity of a plasma protein: fibronectin. The assay is based on the analysis of membrane fluidity variations following the uptake of gelatinized latex beads by phagocytic cells in the presence or in the absence of fibronectin. Using TMA-DPH as fluorescent probe, it was observed that the increase in membrane fluidity was enhanced in presence of fibronectin and depended upon the enhanced in presence of fibronectin and depended upon the opsonic activity was related to the integrity of the molecule. Using this method, the opsonic activity of various plasmas could be also determined.     

A possible pivotal role of mitochondrial free calcium in neurotoxicity mediated by N-methyl-d-aspartate receptors in cultured rat hippocampal neurons

We have previously shown that mitochondrial membrane potential disruption is involved in mechanisms underlying differential vulnerabilities to the excitotoxicity mediated by N-methyl-d-aspartate (NMDA) receptors between primary cultured neurons prepared from rat cortex and hippocampus. To further elucidate the role of mitochondria in the excitotoxicity after activation of NMDA receptors, neurons were loaded with the fluorescent dye calcein diffusible in the cytoplasm and organelles for determination of the activity of mitochondrial permeability transition pore (mPTP) responsible for the leakage of different mitochondrial molecules. The addition of CoCl₂ similarly quenched the intracellular fluorescence except mitochondria in both cultured neurons, while further addition of NMDA led to a leakage of the dye into the cytoplasm in hippocampal neurons only. An mPTP inhibitor prevented the NMDA-induced loss of viability in hippocampal neurons, while an activator of mPTP induced a similarly potent loss of viability in cortical and hippocampal neurons. Although NMDA was more effective in increasing rhodamine-2 fluorescence as a mitochondrial calcium indicator in hippocampal than cortical neurons, a mitochondrial calcium uniporter inhibitor significantly prevented the NMDA-induced loss of viability in hippocampal neurons. Expression of mRNA was significantly higher for the putative uniporter uncoupling protein-2 in hippocampal than cortical neurons. These results suggest that mitochondrial calcium uniporter would be at least in part responsible for the NMDA neurotoxicity through a mechanism relevant to promotion of mPTP orchestration in hippocampal neurons.     

Pontentiometric cyanine dyes are sensitive probes for mitochondria in intact plant cells : kinetin enhances mitochondrial fluorescence

Selected fluorescent dyes were tested for uptake by mitochrondria in intact cells of barley, maize, and onion. The cationic cyanine dye 3,3′-diheptyloxacarbocyanine iodide [DiOC₇(3)] accumulated in mitochondria within 15 to 30 minutes without appreciable staining of other protoplasmic constituents. The number, shape, and movement of the fluorescent mitochondria could be seen readily, and the fluorescence intensity of the mitochondria could be monitored with a microscope photometer. Fluorescence was eliminated in 1 to 5 minutes by the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) indicating that maintenance of dye concentration was dependent on the inside-negative transmembrane potential maintained by functional mitochondria. Fluorescence of prestained mitochondria was enhanced within 5 to 10 minutes after addition of 0.1 millimolar kinetin to cells. The fluorescence in kinetintreated cells was dissipated by CCCP. These results suggest that kinetin interacted with respiratory processes resulting in higher potential across the mitochondrial membrane.     

Lipidomics of intact mitochondria by MALDI-TOF/MS

A simple and fast method of lipid analysis of isolated intact mitochondria by means of MALDI-TOF mass spectrometry is described. Mitochondria isolated from bovine heart and yeast have been employed to set up and validate the new method of lipid analysis. The mitochondrial suspension is directly applied over the target and, after drying, covered by a thin layer of the 9-aminoacridine matrix solution. The lipid profiles acquired with this procedure contain all peaks previously obtained by analyzing the lipid extracts of isolated mitochondria by TLC and/or mass spectrometry. The novel procedure allows the quick, simple, precise, and accurate analysis of membrane lipids, utilizing only a tiny amount of isolated organelle; it has also been tested with intact membranes of the bacterium Paracoccus denitrificans for its evolutionary link to present-day mitochondria. The method is of general validity for the lipid analysis of other cell fractions and isolated organelles.     

A cell-penetrating peptide derived from mammalian cell uptake protein of Mycobacterium tuberculosis

A Mycobacterium tuberculosis membrane protein called Mycobacterium cell entry protein (Mce1A) was previously shown to mediate the uptake of nonpathogenic Escherichia coli and latex beads by nonphagocytic mammalian cells. Here we characterize further the in vitro invasive activity of Mce1A using colloidal gold nanoparticles and fluorescent latex microspheres. Mce1A-coated colloidal gold particles induced plasma membrane invagination and entered membrane-bound compartments inside HeLa cells. Few of the protein-coated particles were also found in the cytosol compartment. Cytochalasin D and nocodazole inhibited the uptake by HeLa cells, indicating that rearrangement of both microtubules and microfilaments was necessary for the uptake. The functional domain of Mce1A for invasion was narrowed to a highly basic 22-amino acid sequence termed Inv3. A synthetic Inv3 peptide stimulated uptake of colloidal gold particles as well as latex microspheres by HeLa cells. A chimeric protein composed of Inv3 sequence at the N terminus of beta-galactosidase appeared to stain the nuclear membrane, suggesting that it entered the HeLa cell cytoplasm. These observations suggest that the cell uptake activity of Mce1A is confined to a small peptide domain located in the core region of the protein. Inv3 could be used to ferry any protein in fusion with it into mammalian cells and may serve as a potent nonviral delivery system.     

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.     

Partial contribution of mitochondrial permeability transition to t-butyl hydroperoxide-induced cell death

Mitochondrial permeability transition (MPT) is thought to determine cell death under oxidative stress. However, MPT inhibitors only partially suppress oxidative stress-induced cell death. Here, we demonstrate that cells in which MPT is inhibited undergo cell death under oxidative stress. When C6 cells were exposed to 250 μM t-butyl hydroperoxide (t-BuOOH), the loss of a membrane potential-sensitive dye (tetramethylrhodamine ethyl ester, TMRE) from mitochondria was observed, indicating mitochondrial depolarization leading to cell death. The fluorescence of calcein entrapped in mitochondria prior to addition of t-BuOOH was significantly decreased to 70% after mitochondrial depolarization. Cyclosporin A suppressed the decrease in mitochondrial calcein fluorescence, but not mitochondrial depolarization. These results show that t-BuOOH induced cell death even when it did not induce MPT. Prior to MPT, lactate production and respiration were hampered. Taken together, these data indicate that the decreased turnover rate of glycolysis and mitochondrial respiration may be as vital as MPT for cell death induced under moderate oxidative stress.     

Anilinonaphthalenesulfonate fluorescence changes induced by non-enzymatic generation of membrane potential in mitochondria and submitochondrial particles

Changes in the fluorescence of 1-anilino-8-naphthalenesulfonate (ANS⁻) accompanying non-enzymatic generation of the membrane potential in mitochondria and sonicated submitochondrial particles have been demonstrated. Generation of the membrane potential was induced by addition of an ionophore (valinomycin for K⁺, or tetrachlorotri-fluoromethylbenzimidazole for H⁺) under conditions where there existed K⁺ (or H⁺) gradients across the mitochondrial membrane. The ANS⁻ fluorescence decreased when the mitochondrial (or particle) interior became more negative, and increased when it became more positive. Collapse of the membrane potential reversed the ANS⁻ responses. A hypothesis is put forward to explain the energy-dependent ANS⁻ responses in mitochondria and particles by the membrane potential-induced redistribution of ANS⁻ between the membrane and water phases.     

XIAP impairs Smac release from the mitochondria during apoptosis

X-linked inhibitor of apoptosis protein (XIAP) is a potent inhibitor of caspases 3, 7 and 9, and mitochondrial Smac (second mitochondria-derived activator of caspase) release during apoptosis inhibits the activity of XIAP. In this study we show that cytosolic XIAP also feeds back to mitochondria to impair Smac release. We constructed a fluorescent XIAP-fusion protein by labelling NH₂- and COOH-termini with Cerulean fluorescent protein (C-XIAP-C). Immunoprecipitation confirmed that C-XIAP-C retained the ability to interact with Smac and impaired extrinsically and intrinsically activated apoptosis in response to tumour necrosis factor-related apoptosis-inducing ligand/cycloheximide and staurosporine. In C-XIAP-C-expressing cells, cytochrome c release from mitochondria proceeded normally, whereas Smac release was significantly prolonged and incomplete. In addition, physiological expression of native XIAP prolonged or limited Smac release in HCT-116 colon cancer cells and primary mouse cortical neurons. The Smac-binding capacity of XIAP, but not caspase inhibition, was central for mitochondrial Smac retention, as evidenced in experiments using XIAP mutants that cannot bind to Smac or effector caspases. Similarly, the release of a Smac mutant that cannot bind to XIAP was not impaired by C-XIAP-C expression. Full Smac release could however be provoked by rapid cytosolic C-XIAP-C depletion upon digitonin-induced plasma membrane permeabilization. Our findings suggest that although mitochondria may already contain pores sufficient for cytochrome c release, elevated amounts of XIAP can selectively impair and limit the release of Smac.     

Membrane potentials in mitochondrial preparations as measured by means of a cyanine dye

Changes in the fluorescent intensity of the dye 3,3′-dipropylthiodicarbocyanine iodide were measured in suspensions of hamster liver mitochondria upon the development of a K⁺ diffusion potential by the addition of valinomycin and upon the development of the energized state by the addition of succinate or ATP. The changes (large decreases) seen with the addition of succinate or ATP (inhibitable by NaCN and oligomycin respectively) were comparable to those recorded upon the addition of valinomycin to mitochondria suspended in media containing low concentrations of K⁺. The change observed with succinate was partially reversed by the addition of either 2,4-dinitrophenol or ADP. Oligomycin prevented the reversal seen with ADP. Decreases in fluorescent intensity were also recorded when succinate was added to suspensions of inner membranes (prepared from rat liver mitochondria) containing the dye. With submitochondrial particles (also from rat liver mitochondria), however, increases in fluorescent intensity were seen upon the addition of succinate or ATP. These observations are consistent with the idea that a large negative (internal) potential develops across the inner membrane of the mitochondrion during energization and with other aspects of the chemiosmotic hypothesis.