MitoTracker Green FM Research Articles

Strategies for regulation of hemolymph pH in acidic and alkaline water by the larval mosquitoAedes aegypti(L.) (Diptera; Culicidae)

The responses of larval Aedes aegypti to media of pH 4, 7 and 11 provide evidence for pH regulatory strategies. Drinking rates in pH 4 media were elevated 3- to 5-fold above those observed in pH 7 or 11. Total body water was elevated during acute exposure to acidic media. During chronic exposure, total body water was decreased and Malpighian tubule mitochondrial luminosity, quantified using Mitotracker Green FM, increased. Malpighian tubule secretion rates and energy demands thus appear to increase dramatically during acid exposure. In alkaline media, drinking rates were quite low. Larvae in pH 11 media excreted net acid (0.12 nequiv H(+) g(-1) h(-1)) and the pH indicators azolitmin and bromothymol blue revealed that the rectal lumen is acidic in vivo at all ambient pH values. The anal papillae (AP) were found to be highly permeant to acid-base equivalents. Ambient pH influenced the length, and the mass-specific length, of the AP in the presence of NaCl (59.9 mmol l(-1)). In contrast, the length and mass-specific length of AP were not influenced by ambient pH in low NaCl conditions. Mitochondrial luminosity was reduced in AP of larvae reared in acidic media, and was not elevated in alkaline media, relative to that of larvae reared in neutral media. These data suggest that the AP may compromise acid-base balance in acidic media, and may also be an important site of trade-offs between H(+) homeostasis and NaCl uptake in dilute, acidic media.

Practical Techniques for Bovine Sperm Simultaneous Fluorimetric Assessment of Plasma, Acrosomal and Mitochondrial Membranes

This experiment was performed to develop and validate practical techniques for simultaneous evaluation of the integrity of plasma and acrosomal membranes, as well as mitochondrial function in bovine spermatozoa using associations of fluorescent probes. Four protocols of fluorescent probes association were defined: protocol 1: propidium iodide (PI), fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and rhodamine 123; protocol 2: PI, FITC-PSA and MitoTracker Green FM (MITO); protocol 3: PI, Hoechst 33342 (H342), FITC-PSA and CMXRos; and protocol 4: PI, H342, FITC-PSA and JC-1. Three ejaculates from each of the four bulls (n = 12) were utilized, showing sperm motility >/=80% and abnormal morphology </=10%. The semen was diluted in Modified Tyrode's medium (TALP) (25 x 10(6) spermatozoa/ml) and split into two aliquots, one sample was flash-frozen in liquid nitrogen and thawed. Samples for three treatments were prepared with the following ratio of fresh semen : flash-frozen semen: 100 : 0, 50 : 50 and 0 : 100. Samples were stained in all four protocols and evaluated by epifluorescence microscopy. Protocol 1 did not result in a satisfactory stain, so it could not be validated. Protocols 2, 3 and 4 were validated and showed high determination coefficient to plasma membrane integrity (R(2) = 0.95, 0.93 and 0.92, respectively), acrosome integrity (R(2) = 0.95, 0.92 and 0.91, respectively) and mitochondrial function (R(2) = 0.84, 0.93 and R(2) = 0.93, respectively). These techniques are efficient for the simultaneous integrity evaluation of plasma and acrosomal membranes and mitochondrial function in bovine spermatozoa. However, JC-1 has an advantage over MITO and CMXRos, as it separates two cell populations with high and low mitochondrial membrane potential.

A Role for Mitochondrial Dysfunction in Perpetuating Radiation-Induced Genomic Instability

Radiation-induced genomic instability (RIGI) manifests as a heritable increased rate of genetic alterations in the progeny of irradiated cells generations after the initial insult. The progeny can show an increased frequency of chromosomal translocations, deletions, mutations, micronuclei, and decreased plating efficiency. What perpetuates RIGI is unclear; however, persistently increased levels of reactive oxygen species (ROS) are frequently associated with genomically unstable clones. Furthermore, addition of free radical scavengers (e.g., DMSO, glycerol, and cationic thiol cysteamine) reduces the incidence of instability after irradiation, implicating a ROS-mediated role in RIGI induction. Because mitochondria are a major natural cellular source of ROS, we tested the hypothesis that mitochondrial dysfunction has a role in maintaining the elevated ROS levels in our irradiated, genetically unstable GM10115 Chinese hamster ovary cells. Amplex Red fluorometry measurements indicate that the relative contribution of uncoupler-sensitive mitochondrial hydrogen peroxide production to total cellular hydrogen peroxide generation is greater in unstable cells. Measurements of mitochondrial DNA levels and cell cytometric fluorescent measurements of Mitotracker Green FM indicate that differences in mitochondrial ROS production are not due to varying mitochondrial levels. However, mitochondrial respiration measured in digitonin-permeabilized cells is impaired in unstable clones. In addition, manganese superoxide dismutase, a major mitochondrial antioxidant enzyme, exhibits increased immunoreactivity but decreased enzyme activity in unstable clones, which along with decreased respiration rates may explain the increased levels of cellular ROS. These studies show that mitochondria from unstable cells are abnormal and likely contribute to the persistent oxidative stress in the unstable clones.

Application of fluorescent probes to study structural changes in Aspergillus fumigatus exposed to amphotericin B, itraconazole, and voriconazole

The broad objective of this study was to document patterns of structural changes following antifungal treatment, and to determine any relationship with minimum inhibitory concentration (MIC) of an antifungal. Three clinical isolates of Aspergillus fumigatus, with high, intermediate, and low amphotericin B (AB), itraconazole (IZ), and voriconazole (VZ) MICs were studied in 24-well plates with cover slips. The fluorescent probes used were Calcofluor White (cell wall), propidium iodide (nucleus), and MitoTracker Green FM (mitochondria). Fluorescent microscopy as early as 3-h after exposure revealed that AB treated hyphae had intact cell wall with deformed mitochondria and nuclei while IZ and VZ treated hyphae revealed no intact cell wall, and deformation of mitochondria and nuclei. At 48 h, AB treated cells revealed rupture of hyphae and disintegration of mitochondria, and nuclei, IZ treated hyphae were swollen with disintegration of mitochondria, and nuclei while VZ treated hyphae showed rupture and disintegration of mitochondria and nuclei. The structural changes for the three strains studied were similar in fluorescent microscopy as long as the incubation time and their respective MICs were used. Thus, AB, IZ, and VZ induced gross organelle defects in A. fumigatus nuclei, mitochondria, and cell wall, which were consistent with respective MICs of antifungals used.

Insights into the interstitium of ventricular myocardium: interstitial Cajal-like cells (ICLC)

We have previously described interstitial Cajal-like cells (ICLC) in human atrial myocardium. Several complementary approaches were used to verify the existence of ICLC in the interstitium of rat or human ventricular myocardium: primary cell cultures, vital stainings (e.g.: methylene blue), traditional stainings (including silver impregnation), phase contrast and non-conventional light microscopy (Epon-embedded semithin sections), transmission electron microscopy (TEM) (serial ultrathin sections), stereology, immunohistochemistry (IHC) and immunofluorescence (IF) with molecular probes. Cardiomyocytes occupy about 75% of rat ventricular myocardium volume. ICLC represent ∼32% of the number of interstitial cells and the ratio cardiomyocytes/ICLC is about 70/1. In the interstitium, ICLC establish close contacts with nerve fibers, myocytes, blood capillaries and with immunoreactive cells (stromal synapses). ICLC show characteristic cytoplasmic processes, frequently two or three, which are very long (tens up to hundreds of μm), very thin (0.1-0.5μm thick), with uneven caliber, having dilations, resulting in a moniliform aspect. Gap junctions between such processes can be found. Usually, the dilations are occupied by mitochondria (as revealed by Janus green B and Mito Tracker Green FM) and elements of endoplasmic reticulum. Characteristically, some prolongations are flat, with a veil-like appearance, forming a labyrinthic system. ICLC display caveolae (about 1 caveola/1μm cell membrane length, or 2-4% of the relative cytoplasmic volume, Mitochondria and endoplasmic reticulum (rough and smooth) occupy 5-10% and 1-2% of cytoplasmic volume, respectively. IHC revealed positive staining for CD34, EGFR and vimentin and, only in a few cases for CD117. IHC was negative for: desmin, CD57, tau, chymase, tryptase and CD13. IF showed that ventricular ICLC expressed connexin 43. We may speculate that possible ICLC roles might be: intercellular signaling (neurons, myocytes, capillaries etc.) and/or chemomechanical sensors. For pathology, it seems attractive to think that ICLC might participate in the process of cardiac repair/remodeling, arrhythmogenesis and, eventually, sudden death.

Development of an Optical Tweezers Methodology to Separate Single Cells and Single Mitochondria to Determine the Location of Heteroplasmy in Mitochondrial DNA

Mitochondrial DNA heteroplasmies are well documented at the multi-cell level. Some have been linked to various degrees of chronic symptoms of mitochondria-based diseases. However, the mechanism producing heteroplasmy is undetermined. The presence of heteroplasmy may differ depending on the tissue examined (e.g., somatic cells may be heteroplasmic, while germ cells can be homoplasmic). The presence and degree of heteroplasmy is important in both the forensic and medical communities. The question of whether heteroplasmy is present in the single cell and in the single mitochondrion remains. We developed an effective protocol to isolate single human leukocyte cells and single mitochondria from those cells. The cells from an HL-60 cell culture were labeled with Mitotracker Green FM (tested and shown not to interfere with PCR), and individually separated using optical tweezers (5 watt, CW, Ytterbium (Yb) fiber laser) and an Axiovert 100 M fluorescent microscope. Once it was determined that the individual cells were heteroplasmic (PCR and sequencing showed a 50/50 C/T heteroplasmy at nucleotide position 12071), individual dyed mitochondria were separated by the optical tweezers method and tested in the same manner. Preliminary results suggest that single mitochondria also contain the heteroplasmy.

Bilirubin exhibits a novel anti-cancer effect on human adenocarcinoma

Anti-oxidants are essential for intracellular free radical scavenging, as free radicals are one of the causes for tumorigenesis. Our objective was to use bilirubin and investigate its action on human carcinoma cell lines. Bilirubin manifested as a prooxidant showing its cytopathic effect on TMK-1, showing growth inhibition close to 50%. Cell cycle analysis showed an arrest at G0/G1. Flow cytometry investigations with Red CC-1 showed an increase by more than 2 times suggesting a prooxidative role of bilirubin. To check the effect of radicals on DNA, a Comet Assay displayed a typical comet’s tail with bilirubin treated slides, only. Further, staining with DAPI showed apoptotic action of bilirubin. Decreased mitochondrial function by bilirubin was observed with Mitotracker Green FM staining. These unexpected data have led us to conclude that bilirubin has anti-cancer activity as a prooxidant and may have a more vital role in the human body than realized.

Effect of new photosensitizer CDHS801-mediated photodynamic therapy on bladder cancer: an experimental study

To investigate the effects of dynamic photodynamic therapy (PDT) on bladder cancer. Human bladder cancer cells of the line T24 were co-cultured with CDHS801, a photosensitizer, and MitoTracker RED CMXRose and MitoTracker GREEN FM, mitochondria specific fluorescence probe dyes. Laser scanning confocal fluorescence microimaging system was applied to collect the fluorescence of the photosensitizer and the probes. T24 cells were cultured and divided into 4 groups: Group 1 as blank control group, Group 2 undergoing laser irradiation, Group 3, added with CDHS801 for 6h, and Group 4 (PDT group), added with CDHS801 and undergoing laser irradiation. The survival of the cells was examined by MTT colorimetric assay. The morphological changes and apoptosis of the photo-activated T24 cells were investigated by transmission electron microscopy, confocal laser scan microscopy, and flow cytometry. The fluorescence of the photosensitizer and that of the probes were detected in the cytoplasm and in the peri-nuclear region, mainly in the mitochondria, of the T24 cells. 2. The inhibitory rates of PDT on T24 cells were 0%, 7.3%, 10.8%, and 71.4% in the control group, Group 1, Group 2, and Group 3 respectively. T24 cell photo-activated with CDHS801 showed cell size shrinkage, condensed chromatin and formation of apoptotic body. Flow cytometry showed that apoptosis was seen in 55.31% of the photo-activated cells and peaked in the sub-G1 phase. However, no such changes were seen in the control group. CDHS801-based PDT can kill bladder cancer T24 cells. CDHS801 is localized in the cytoplasm and peri-nuclear region, mainly mitochondria, of tumor cell. CDHS801 based PDT maybe eliminate the T24 cell by the induction of apoptosis.

Effects of alginate encapsulation on mitochondrial activity

The long-term objective of our research is to study the biochemical consequences of primary genetic defects of the Pyruvate Dehydrogenase Complex, a key mitochondrial enzyme complex, by NMR spectroscopy. An established method to obtain energetic and metabolic information from intact cells involves the use of 31P and 13C NMR spectroscopic techniques. NMR spectra from live and fully functional cells can be obtained from cells encapsulated within alginate beads and maintained in a perfusion bioreactor throughout the NMR experiment. However, before spectroscopic studies can commence, the effects of alginate encapsulation on the general metabolism and mitochondrial activity of fibroblasts need to be determined. in this study we report glucose consumption and flow cytometry measurements (with the fluorescent markers MitoTracker GreenFM and Nonyl-acridine Orange to determine the mitochondrial status and mass) of healthy human fibroblasts encapsulated in a mannuronic acid-rich alginate matrix. The results show that alginate encapsulation of fibroblasts does not affect the glucose consumption, the mitochondrial integrity, or the mitochondrial mass during 21 days of in vitro culture.

Novel type of interstitial cell (Cajal-like) in human fallopian tube.

We describe here--presumably for the first time--a Cajal-like type of tubal interstitial cells (t-ICC), resembling the archetypal enteric ICC. t-ICC were demonstrated in situ and in vitro on fresh preparations (tissue cryosections and primary cell cultures) using methylene-blue, crystal-violet, Janus-Green B or MitoTracker-Green FM Probe vital stainings. Also, t-ICC were identified in fixed specimens by light microscopy (methylene-blue, Giemsa, trichrome stainings, Gomori silver-impregnation) or transmission electron microscopy (TEM). The positive diagnosis of t-ICC was strengthened by immunohistochemistry (IHC; CD117/c-kit+ and other 14 antigens) and immunofluorescence (IF; CD117/c-kit+ and other 7 antigens). The spatial density of t-ICC (ampullar-segment cryosections) was 100-150 cells/mm2. Non-conventional light microscopy (NCLM) of Epon semithin-sections revealed a network-like distribution of t-ICC in lamina propria and smooth muscle meshwork. t-ICC appeared located beneath of epithelium, in a 10-15 microm thick 'belt', where 18+/-2% of cells were t-ICC. In the whole lamina propria, t-ICC were about 9%, and in muscularis approximately 7%. In toto, t-ICC represent ~8% of subepithelial cells, as counted by NCLM. In vitro, t-ICC were 9.9+/-0.9% of total cell population. TEM showed that the diagnostic 'gold standard' (Huizinga et al., 1997) is fulfilled by 'our' t-ICC. However, we suggest a 'platinum standard', adding a new defining criterion- characteristic cytoplasmic processes (number: 1-5; length: tens of microm; thickness: < or =0.5 microm; aspect: moniliform; branching: dichotomous; organization: network, labyrinthic-system). Quantitatively, the ultrastructural architecture of t-ICC is: nucleus, 23.6+/-3.2% of cell volume, with heterochromatin 49.1+/-3.8%; mitochondria, 4.8+/-1.7%; rough and smooth endoplasmic-reticulum (1.1+/-0.6%, 1.0+/-0.2%, respectively); caveolae, 3.4+/-0.5%. We found more caveolae on the surface of cell processes versus cell body, as confirmed by IF for caveolins. Occasionally, the so-called 'Ca2+-release units' (subplasmalemmal close associations of caveolae+endoplasmic reticulum+mitochondria) were detected in the dilations of cell processes. Electrophysiological single unit recordings of t-ICC in primary cultures indicated sustained spontaneous electrical activity (amplitude of membrane potentials: 57.26+/-6.56 mV). Besides the CD117/c-kit marker, t-ICC expressed variously CD34, caveolins 1&2, alpha-SMA, S-100, vimentin, nestin, desmin, NK-1. t-ICC were negative for: CD68, CD1a, CD62P, NSE, GFAP, chromogranin-A, PGP9.5, but IHC showed the possible existence of (neuro)endocrine cells in tubal interstitium. We call them 'JF cells'. In conclusion, the identification of t-ICC might open the door for understanding some tubal functions, e.g. pace-making/peristaltism, secretion (auto-, juxta- and/or paracrine), regulation of neurotransmission (nitrergic/purinergic) and intercellular signaling, via the very long processes. Furthermore, t-ICC might even be uncommitted bipotential progenitor cells.

Development of a protocol for multiple staining with fluorochromes to assess the functional status of boar spermatozoa

The aim of this study was to design a simple and reliable method for the simultaneous evaluation of the nucleus, the acrosome, and the mitochondrial sheath of boar spermatozoa. Sperm samples coming from healthy and sexually mature Pietrain boars were incubated with two nuclear fluorochromes--bis-benzamide specific for viable cells, and propidium iodide specific for nonviable cells--the fluorochrome Mitotracker Green FM specific for functional mitochondria, and the lectin Trypsin inhibitor from Soybean (SBTI) conjugated with the fluorochrome Alexa Fluor 488 specific for proacrosin. The results obtained from assessing the functional status of the spermatozoa using fluorochromes were compared with the conventional sperm parameters of sperm vitality using the eosin exclusion test (EE test), and sperm motility and morphology using the computer-assisted semen analyzer SCA 2002Producció. Applying the multiple staining test, it was found that the frequency of viable spermatozoa with intact acrosome and intact mitochondria was not different from the frequency of viable spermatozoa obtained with the EE test, and also correlated positively with the frequency of motile spermatozoa and the frequency of mature spermatozoa. Therefore, this technique is useful to characterize the status of boar spermatozoa by assessing the nuclear, acrosomal, and mitochondrial integrity. Moreover, it provides reliable diagnostic information about the fertility potential of boars.

64 OOPLASMIC TRANSFER AFTER INTERSPECIES NUCLEAR TRANSFER: PRESENCE OF FOREIGN MITOCHONDRIA, PATTERN OF MIGRATION, AND EFFECT ON EMBRYO DEVELOPMENT

Interspecies somatic cell nuclear transfer (iSCNT) using the bovine cytoplast as universal recipient has potential applications in the conservation of exotic species. However, an in vitro developmental block has been observed using this approach. It has been suggested that mitochondrial mismatch between donor cell and recipient oocyte could be a cause for the embryonic developmental arrest. A series of experiments were conducted to investigate the effect of mixed mitochondrial populations (heteroplasmy) on early development of cloned embryos. In Experiment 1, we examined the effect of combining the technique of ooplasmic transfer (OT) with somatic cell nuclear transfer (SCNT) in the bovine model. In addition, presence and pattern of migration of foreign mitochondria after OT were examined by MitoTracker® (Molecular Probes, Inc., Eugene, OR, USA) staining. In Experiment 2, we examined the effect of transferring caprine ooplasm into bovine enucleated oocytes (iOT) used as recipients for goat iSCNT. Ooplasm from donor oocytes was aspirated until the oolema was ruptured and filled about 200 μm of the micropipette. Aspirated ooplasm was injected into recipient oocyte; the oolema of the recipient oocyte was also ruptured by partial aspiration into the micropipette to ensure mixing. Mean cleavage rates and embryo development were compared by chi-square analysis. Percentages (except for parthenogenic controls) were calculated from number of fused couplets. In Experiment 1, there was no significant effect of the sequence of events (OT-SCNT or SCNT-OT) on the number of fused, cleaved, blastocyst (BLST), or hatched blastocyst (HCHD) embryos (Table 1). MitoTracker Green FM staining of donor oocytes used for OT revealed foreign mitochondria were introduced by the procedure. Their pattern of distribution remained in a distinct cluster after 12, 74 and 144 h of in vitro culture. However, when goat ooplasm was injected into bovine enucleated oocytes used for iSCNT, there was a significant reduction in fusion (52 vs. 82%) and cleavage rates (55 vs. 78%) (P &lt; 0.05). In addition, the procedure of iOT prior to iSCNT was not effective in overcoming the 8- to 16-cell in vitro developmental block and only parthenogenic cow and goat controls reached blastocyst (36 and 32%) and hatched blastocyst (25 and 12%) stages, respectively (Table 1). This study demonstrates that foreign mitochondria are introduced at the time of OT and these mitochondria remain in a cluster without relocation after a few mitotic divisions. Although the bovine cytoplast appears capable of supporting mitotic divisions after iOT-iSCNT, heteroplasmy or mitochondrial incompatibilities may affect nuclear-ooplasmic events occurring at genomic activation. To our knowledge, this is the first scientific report of iOT used in combination with iSCNT in an attempt to overcome the in vitro developmental block. Further research is needed to determine characteristics of foreign mitochondrial dynamics as well as replication of foreign mitochondria introduced into NT embryos. Table 1. Intraspecies (cow) and interspecies (goat-cow) ooplasmic transfer and nuclear transfer

Flow cytometric evaluation of sperm parameters in relation to fertility potential

Most laboratory methods used to evaluate semen quality have not correlated highly with fertilizing capacity. The discovery of a variety of fluorochromes and compounds conjugated to fluorescent probes has enabled a more widespread analysis of sperm attributes, and in conjunction with the flow cytometer, permit the evaluation of a large number of spermatozoa. A number of characteristics of sperm integrity, viability and function can be assessed by flow cytometry. The DNA status of spermatozoa has been determined using the metachromatic properties of acridine orange (AO). AO staining, when used in the sperm chromatin structure assay (SCSA ®), correlates with fertility in a number of species. DNA fragmentation can also be assessed using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, which identifies DNA strand breaks by labeling free 3′-OH termini with modified nucleotides. The status of the sperm acrosome can be determined using fluorescently labeled lectins and LysoTracker Green DND-26, a fluorescent acidotropic probe. Capacitation status has been observed through calcium-mediated changes using chlortetracycline (CTC) or by changes in membrane fluidity monitored by the binding of the fluorescent amphiphilic probe, Merocyanine 540. Fluorescently labeled annexin-V, C6NBD and Ro-09-0198 can also be used to detect changes in membrane phospholipid distribution. Cell viability can be determined using the propensity of propidium iodide (PI), ethidium homodimer-1 (EthD-1) or Yo-Pro-1 to permeate damaged membranes. These are generally more adaptable to clinical flow cytometry than the bisbenzimide membrane impermeable stain, Hoechst 33258, which excites in the ultraviolet range and requires UV laser equipment. Mitochondrial function can be determined using rhodamine 123 (R123) and MitoTracker Green FM (MITO) and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1). Flow cytometry is a tool that may be used in the future to monitor many new potential markers of sperm function.

Modulation of [Ca2+]i Signaling Dynamics and Metabolism by Perinuclear Mitochondria in Mouse Parotid Acinar Cells

Parotid acinar cells exhibit rapid cytosolic calcium signals ([Ca2+]i) that initiate in the apical region but rapidly become global in nature. These characteristic [Ca2+]i signals are important for effective fluid secretion, which critically depends on a synchronized activation of spatially separated ion fluxes. Apically restricted [Ca2+]i signals were never observed in parotid acinar cells. This is in marked contrast to the related pancreatic acinar cells, where the distribution of mitochondria has been suggested to contribute to restricting [Ca2+]i signals to the apical region. Therefore, the aim of this study was to determine the mitochondrial distribution and the role of mitochondrial Ca2+ uptake in shaping the spatial and temporal properties of [Ca2+]i signaling in parotid acinar cells. Confocal imaging of cells stained with MitoTracker dyes (MitoTracker Green FM or MitoTracker CMXRos) and SYTO dyes (SYTO-16 and SYTO-61) revealed that a majority of mitochondria is localized around the nucleus. Carbachol (CCh) and caged inositol 1,4,5-trisphosphate-evoked [Ca2+]i signals were delayed as they propagated through the nucleus. This delay in the CCh-evoked nuclear [Ca2+]i signal was abolished by inhibition of mitochondrial Ca2+ uptake with ruthenium red and Ru360. Likewise, simultaneous measurement of [Ca2+]i with mitochondrial [Ca2+] ([Ca2+]m), using fura-2 and rhod-FF, respectively, revealed that mitochondrial Ca2+ uptake was also inhibited by ruthenium red and Ru360. Finally, at concentrations of agonist that evoke[Ca2+]i oscillations, mitochondrial Ca2+ uptake, and a nuclear [Ca2+] delay, CCh also evoked a substantial increase in NADH autofluorescence. This autofluorescence exhibited a predominant perinuclear localization that was also sensitive to mitochondrial inhibitors. These data provide evidence that perinuclear mitochondria and mitochondrial Ca2+ uptake may differentially shape nuclear [Ca2+] signals but more importantly drive mitochondrial metabolism to generate ATP close to the nucleus. These effects may profoundly affect a variety of nuclear processes in parotid acinar cells while facilitating efficient fluid secretion.

Differential Segregation Patterns of Sperm Mitochondria in Embryos of the Blue Mussel (Mytilus edulis)

Abstract In Mytilus, females carry predominantly maternal mitochondrial DNA (mtDNA) but males carry maternal mtDNA in their somatic tissues and paternal mtDNA in their gonads. This phenomenon, known as doubly uniparental inheritance (DUI) of mtDNA, presents a major departure from the uniparental transmission of organelle genomes. Eggs of Mytilus edulis from females that produce exclusively daughters and from females that produce mostly sons were fertilized with sperm stained with MitoTracker Green FM, allowing observation of sperm mitochondria in the embryo by epifluorescent and confocal microscopy. In embryos from females that produce only daughters, sperm mitochondria are randomly dispersed among blastomeres. In embryos from females that produce mostly sons, sperm mitochondria tend to aggregate and end up in one blastomere in the two- and four-cell stages. We postulate that the aggregate eventually ends up in the first germ cells, thus accounting for the presence of paternal mtDNA in the male gonad. This is the first evidence for different behaviors of sperm mitochondria in developing embryos that may explain the tight linkage between gender and inheritance of paternal mitochondrial DNA in species with DUI.

Differential segregation patterns of sperm mitochondria in embryos of the blue mussel (Mytilus edulis).

In Mytilus, females carry predominantly maternal mitochondrial DNA (mtDNA) but males carry maternal mtDNA in their somatic tissues and paternal mtDNA in their gonads. This phenomenon, known as doubly uniparental inheritance (DUI) of mtDNA, presents a major departure from the uniparental transmission of organelle genomes. Eggs of Mytilus edulis from females that produce exclusively daughters and from females that produce mostly sons were fertilized with sperm stained with MitoTracker Green FM, allowing observation of sperm mitochondria in the embryo by epifluorescent and confocal microscopy. In embryos from females that produce only daughters, sperm mitochondria are randomly dispersed among blastomeres. In embryos from females that produce mostly sons, sperm mitochondria tend to aggregate and end up in one blastomere in the two- and four-cell stages. We postulate that the aggregate eventually ends up in the first germ cells, thus accounting for the presence of paternal mtDNA in the male gonad. This is the first evidence for different behaviors of sperm mitochondria in developing embryos that may explain the tight linkage between gender and inheritance of paternal mitochondrial DNA in species with DUI.

Discrimination of depolarized from polarized mitochondria by confocal fluorescence resonance energy transfer

Mitochondrial depolarization promotes apoptotic and necrotic cell death and possibly other cellular events. Polarized mitochondria take up cationic tetramethylrhodamine methylester (TMRM), which is released after depolarization. Thus, TMRM does not label depolarized mitochondria. To identify both polarized and depolarized mitochondria in living cells, cultured rat hepatocytes, and sinusoidal endothelial cells were co-loaded with green-fluorescing MitoTracker Green FM (MTG) and red-fluorescing TMRM for imaging by laser scanning confocal microscopy. Like TMRM, MTG is a cationic fluorophore that accumulates electrophoretically into polarized mitochondria. Unlike TMRM, MTG binds covalently to intramitochondrial protein thiols and remains bound after depolarization. In cells labeled only with MTG, excitation with blue (488 nm) light yielded green but almost no red fluorescence. After subsequent loading with TMRM, green MTG fluorescence became quenched. Instead, blue excitation yielded red fluorescence. Mitochondrial de-energization restored green fluorescence and abolished red fluorescence. Conversely, when MTG was added to TMRM-labeled cells, red fluorescence excited by blue light was enhanced, an effect again reversed by de-energization. These observations of reversible quenching of donor fluorescence and augmentation of acceptor fluorescence signify fluorescence resonance energy transfer (FRET). In undisturbed hepatocytes, spontaneous depolarization of a subfraction of mitochondria was an ongoing phenomenon. In conclusion, confocal FRET discriminates individual depolarized mitochondria against a background of hundreds of polarized mitochondria.

Suppression of mitochondria-dependent neutrophil apoptosis with thermal injury.

Neutrophil apoptosis is delayed under trauma and/or sepsis conditions. The mechanism for the delay has remained unclear. We hypothesize that modulation of the mitochondrial pathway of apoptosis contributes to the delay in neutrophil apoptosis with burn injury. Rats were subjected to burn injury (30% of total body surface area, 98 degrees C for 10 s) and euthanatized 24 h postinjury. Blood neutrophils from sham and burn-injured rats were isolated by Ficoll gradient centrifugation and cultured for 2 or 8 h. Neutrophil apoptosis was determined by annexin V and propidium iodide (PI) labeling and flow cytometry. Neutrophil mitochondrial morphology was assessed via histochemical staining (MitoTracker GreenFM) and confocal microscopy. Neutrophils from rats with burn injury showed a decreased level of apoptosis compared with sham rat neutrophils at both 2 and 8 h of incubation. In incubated sham rat neutrophils, mitochondria showed a change from normal "tubular" to an "aggregated" morphology. In contrast, cultured neutrophils from burn rats did not exhibit this mitochondrial morphological transition until 8 h of incubation. Compared with sham rat neutrophils, neutrophils from burn rats showed decreased levels of active caspase-9 and -3. Whereas an upregulation of Bcl-xL and a downregulation of Bax seemed to contribute to decreased apoptosis in burn rat neutrophils at 2 h of incubation, the decreased apoptosis at 8 h appeared to be associated with a decrease in Bax and increased phosphorylated Bad. These data suggest that suppression of the mitochondrial pathway plays an essential role in the delay of polymorphonuclear neutrophil apoptosis with burn injury.

Asbestos induces mitochondrial DNA damage and dysfunction linked to the development of apoptosis.

To test the hypothesis that asbestos-mediated cell injury is mediated through an oxidant-dependent mitochondrial pathway, isolated mesothelial cells were examined for mitochondrial DNA damage as determined by quantitative PCR. Mitochondrial DNA damage occurred at fourfold lower concentrations of crocidolite asbestos compared with concentrations required for nuclear DNA damage. DNA damage by asbestos was preceded by oxidant stress as shown by confocal scanning laser microscopy using MitoTracker Green FM and the oxidant probe Redox Sensor Red CC-1. These events were associated with dose-related decreases in steady-state mRNA levels of cytochrome c oxidase, subunit 3 (COIII), and NADH dehydrogenase 5. Subsequently, dose-dependent decreases in formazan production, an indication of mitochondrial dysfunction, increased mRNA expression of pro- and antiapoptotic genes, and increased numbers of apoptotic cells were observed in asbestos-exposed mesothelial cells. The possible contribution of mitochondrial-derived pathways to asbestos-induced apoptosis was confirmed by its significant reduction after pretreatment of cells with a caspase-9 inhibitor. Apoptosis was decreased in the presence of catalase. Last, use of HeLa cells transfected with a mitochondrial transport sequence targeting the human DNA repair enzyme 8-oxoguanine DNA glycosylase to mitochondria demonstrated that asbestos-induced apoptosis was ameliorated with increased cell survival. Studies collectively indicate that mitochondria are initial targets of asbestos-induced DNA damage and apoptosis via an oxidant-related mechanism.

Visualizing Superoxide Production in Normal and Diabetic Rat Islets of Langerhans

Oxygen free radicals have been implicated in beta-cell dysfunction and apoptosis associated with type 1 and type 2 diabetes mellitus. The roles of free radicals in diabetes have thus far been defined indirectly by monitoring oxidative tissue damage and the effects of antioxidants, free radical scavengers, and overexpression of superoxide dismutase. We employed the superoxide-mediated oxidation of hydroethidine to ethidium to dynamically and directly assess the relative rates of mitochondrial superoxide anion generation in isolated islets in response to glucose stimulation. Superoxide content of isolated islets increased in response to glucose stimulation. We next compared the oxyradical levels in Zucker lean control and Zucker diabetic fatty rat islets by digital imaging microfluorometry. The superoxide content of Zucker diabetic fatty islets was significantly higher than Zucker lean control islets under resting conditions, relatively insensitive to elevated glucose concentrations, and correlated temporally with a decrease in glucose-induced hyperpolarization of the mitochondrial membrane. Importantly, superoxide levels were elevated in islets from young, pre-diabetic Zucker diabetic fatty animals. Overproduction of superoxide was associated with perturbed mitochondrial morphology and may contribute to abnormal glucose signaling found in the Zucker diabetic fatty model of type 2 diabetes mellitus.