MitoTracker Deep Red Research Articles

TRIM34 facilitates the formation of multinucleated giant cells by enhancing cell fusion and phagocytosis in epithelial cells

Persistent microbial infection promotes the fusion of several kinds of somatic cells, such as macrophages and endothelial cells, leading to the formation of multinucleated giant cells (MGCs). However, the molecular mechanisms of MGCs formation are still poorly understood. By laser confocal microscope, we discovered that TRIM34 increased the efficiency of cell fusion in Human Embryonic Kidney cells (HEK293T). By means of DiD cell membrane probes, LysoTracker Deep Red or MitoTracker Deep Red staining, we also demonstrated that TRIM34 stimulated cell fusion in paraformaldehyde fixed or living HEK293T cells. Moreover, we discovered that the nuclei shapes of MGCs induced by TRIM34 were diversiform, such as horseshoe shape, ring like shape etc. Through 3D reconstruction of confocal z-stacks images, we found that TRIM34-EGFP proteins could form macromolecule aggregates in the central area of MGCs, while the nuclei were arranged in ring like shape and distributed around the plasma membrane. Cell fusion assay showed that cocultured TRIM34-EGFP+ cells and TRIM34-DsRed1+ cells could fuse to form MGCs. We speculate that the formation of MGCs can be divided into two phase: primary multinucleated cells (PMCs) and secondary multinucleated cells (SMCs). Firstly, TRIM34 induced fusion of multiple adjacent cells resulting in PMCs formation, and then PMCs were endowed with the capacity of phagocytosis and turned into SMCs. Collectively, these results suggest that TRIM34 proteins contribute to the formation of MGCs by promoting cell fusion and phagocytosis in epithelial cells.

Abstract 545: A Multifunctional Genetic Probe for Investigating Mitochondrial Calcium Dynamics in Cardiac Cells

Introduction: Calcium cycling plays a critical role in regulating function of cardiomyocytes (CMs) under both physiological and pathological conditions. Among their many important functions, mitochondria are known to involve in calcium handling. However, little is known about the calcium dynamics of human stem cell-derived cardiomyocytes (hiPSC-CM) and what little is known shows much greater variability than what is seen in adult cardiomyocytes. Methods: We produced adenovirus encoding the red Ca 2+ indicator RCaMP prepended with a mitochondrial targeting sequence fused to the green Ca 2+ indicator GCaMP with a cleavable linker. We then used this virus to induce expression of this calcium probe in CMs & recorded calcium activity with a confocal microscope. Results & Discussion: Mitochondrial targeting was confirmed by confocal imaging showing GCaMP (green), mRCaMP (red), and costaining with Mitotracker Deep Red (Magenta) (Figure 1A, 1B). Probe functionality was validated by observing beat-by-beat calcium dynamics in paced neonatal rat CMs with and without blocking mitochondrial calcium uptake with the mitochondrial calcium uniport inhibitor Ru360. Recording GCaMP and mRCaMP dynamics in adult CMs (Figure 1C) showed synchronous beat-by-beat calcium activity in the cytosol and mitochondria. However, iPSC-CMs (Figure 1D) showed variable mitochondrial activity, with some synchronous mitochondria and some showing inverse activity. Conclusions: hiPSC-CMs and adult CMs show differences in beat-by-beat mitochondrial calcium dynamics, offering a potential investigatory target for improving the therapeutic effect of stem cell-derived heart therapies.

MoNa \u2013 A Cost-Efficient, Portable System for the Nanoinjection of Living Cells

Injection techniques to deliver macromolecules to cells such as microinjection have been around for decades with applications ranging from probing whole organisms to the injection of fluorescent molecules into single cells. A similar technique that has raised recent interest is nanoinjection. The pipettes used here are much smaller and allow for the precise deposition of molecules into single cells via electrokinetics with minimal influence on the cells’ health. Unfortunately, the equipment utilized for nanoinjection originates from scanning ion conductance microscopy (SICM) and is therefore expensive and not portable, but usually fixed to a specific microscope setup. The level of precision that these systems achieve is much higher than what is needed for the more robust nanoinjection process. We present Mobile Nanoinjection (MoNa), a portable, cost-efficient and easy to build system for the injection of single cells. Sacrificing unnecessary sub-nanometer accuracy and low ion current noise levels, we were able to inject single living cells with high accuracy. We determined the noise of the MoNa system and investigated the injection conditions for 16 prominent fluorescent labels and fluorophores. Further, we performed proof of concepts by injection of ATTO655-Phalloidin and MitoTracker Deep Red to living human osteosarcoma (U2OS) cells and of living adult human inferior turbinate stem cells (ITSC’s) following neuronal differentiation with the MoNa system. We achieved significant cost reductions of the nanoinjection technology and gained full portability and compatibility to most optical microscopes.

Hemarthrosis alters mitochondrial dynamics in situ after traumatic injury

Purpose: Hemarthrosis occurs after a variety of joint injuries and contributes to rapid progression of posttraumatic osteoarthritis (PTOA). Similarly, mitochondrial responses to traumatic injury appear to modulate rate of PTOA progression. In vitro studies have shown that chondrocyte mitochondria are impaired by a variety of signaling molecules present in whole blood and that pro-inflammatory activity is responsive to manipulations of mitochondrial function. The combination of oxidizing mitochondria and oxidizing, labile iron derived from blood already associated with trauma may cause more severe mitochondrial damage in situ than previously characterized in agarose models. This study tested whether the addition of whole blood after injury would exacerbate losses in mitochondrial content observed after impact in a chelation-inhibitable fashion. Methods: Fresh, skeletally mature, bovine stifle (knee) joints were obtained from a local abattoir (Bud’s Custom Meats, Riverside, IA) and 1 cm diameter cylindrical osteochondral explants from the loaded articular surface of the femoral condyle were harvested. Explants were equilibrated for 1 day in 45% F-12/45% Dulbecco’s modified Eagle medium/10% fetal bovine serum at 37ºC with 5% O2 and 5% CO2. Explants were impacted with a 2 J impact via drop tower to the articular surface using a flat impermeable platen (or sham impact for controls) and placed into one of three solutions: normal media, normal media + 10% whole blood from the same animal, or normal media + 10% blood + the iron chelator desferrioxamine mesylate (DFO) (100 μM, Sigma-Aldrich). Samples were returned to the incubator in these solutions and 2 h later stained in serum free media with 1 μM calcein AM, a viability dye, and 200 nM MitoTracker Deep Red, a stain for mitochondria (both ThermoFisher Scientific). Cells were visualized with an Olympus FV1000 confocal microscope with a 20X objective immersed in phenol red-free media, as well as with a 100X immersion objective placed in contact with the impacted site. Images were analyzed using a custom-written two-part analysis routine developed in Matlab. Viable cells were automatically identified from the calcein AM-stained images using intensity thresholding and area-based shape measures, and resultant cell counts were expressed as viable cell density (cells/mm2). A mask image of only viable cells was applied to the MitoTracker images to extract mitochondrial staining intensity from viable cells. Data reported are the average results from explants obtained from 4 separate animals represented in all groups. Results: Impact resulted in a ∼50% reduction in viable cell density in the zone of impact across all groups and conditions. Total viable cell mitochondrial staining decreased after impact (p < 0.05) and this was not altered with whole blood addition after impact, Figure 1. A trend towards recovery of mitochondrial content was observed with whole blood and DFO after impact; however this has not reached statistical significance at n = 4 (p = 0.2 for reversing the effect of impact). A clear difference in mitochondrial network morphology was observed comparing samples with and without whole blood, Figure 2, with samples exposed to whole blood demonstrating a much more punctate, fragmented morphology. No significant differences in cell viability were observed after including blood in our studies, suggesting that impact itself was largely determinate of cell death at sites of impact. The mitochondrial phenotype after impact appears very different in the presence of hemarthrosis, with brighter and more punctate mitochondria than impact alone. Conclusions: Iron chelation may be effective in preventing loss of overall mitochondrial intensity, i.e. mitochondrial mass, but greater numbers of replicates will be needed to reach statistical significance. While DFO provided some recovery of intensity, it did not appear to rescue the morphology changes associated with exposure to blood. This study contributes to ongoing efforts in the field to improve the physiological relevance of in vitro models of cartilage injury by demonstrating a clear effect of whole blood on intracellular responses to mechanical injury.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Inhibition of lysosomal function enhances inflammatory gene expression, induce oxidative stress and apoptosis in human chondrocytes

Purpose: Lysosomes are the major catabolic organelle of the cells and are essential for the clearance of autophagosomes. Loss of lysosomal function with age has been associated with mitochondrial dysfunction, oxidative stress and apoptosis of the cells. However, the role of lysosomes in chondrocytes homeostasis under pathological conditions has not been explored. Here, we used Chloroquine (CQ) and Bafilomycin A1 (Baf) to inhibit lysosomal function and investigated its consequences on chondrocyte homeostasis and viability in vitro. Methods: Human articular chondrocytes and cartilage explants were prepared from OA knee cartilage obtained from donors undergoing knee arthroplasty at the SUMMA Health Barberton Hospital. Human normal cartilage was obtained from NDRI. The expression of lysosomal associated membrane protein 1 (LAMP1) in normal and OA cartilage was determined by immunohistochemistry. Primary human OA chondrocytes or cartilage explants were maintained in complete DMEM/F12 and treated with CQ or Baf for 24 h or indicated time in DMEM/F12 medium without serum. Induction of apoptosis in chondrocytes treated with CQ or Baf was determined by measuring the activity of LDH released in the culture supernatant, caspase 3/7-luciferase reporter assay and TUNEL staining. The level of IL-1β and IL-6 was determined by TaqMan assays. The level of reactive oxygen species (ROS) was determined by DHR123 staining followed by flow cytometry and MitoSOX Red staining followed by confocal microscopy. The loss of mitochondrial membrane potential was determined by JC-1 dye staining and ATP levels were determined using a kit. The accumulation of dysfunctional mitochondria was determined by staining with MitoTracker Deep Red followed by flow cytometry. The signaling pathway activation was investigated by immunoblot analysis of NFκB and MAPK pathways and its involvement was confirmed by using small molecule inhibitors of the induced pathway. The role of ROS in CQ and Baf induced expression of IL-1β and IL-6 was determined by using anti-oxidants, MitoTempo and Trolox. Results: The expression of LAMP1 was highly downregulated in OA cartilage compared to normal cartilage. CQ and Baf significantly induced apoptosis of primary human OA chondrocytes as determined by LDH activity assay, caspase3/7 luciferase reporter assay and TUNEL staining. Similar results were obtained with mouse chondrocytes. Human OA cartilage explants as well as mouse femoral head cartilage explant culture treated with CQ or Baf showed significant increase in LDH release and TUNEL staining. The CQ and Baf induced inhibition of lysosomal function resulted in the loss of mitochondrial membrane potential, excessive levels of ROS, lower levels of ATP and the accumulation of dysfunctional mitochondria in the treated chondrocytes. Treatment of primary human OA chondrocytes with CQ or Baf significantly increased the gene and protein expression of IL-1β and IL-6. Investigation of the signaling pathways showed no role of NFκB signaling in the CQ and Baf induced upregulation of IL-1β and IL-6 expression. However, the JNK-MAPK, but not ERK- or p38-MAPK pathway, was activated in response to Baf treatment. Inhibition of JNK activation by using a specific inhibitor abrogated the upregulation of IL-1β and IL-6 expression in Baf treated human chondrocytes. The use of anti-oxidants, MitoTempo and Trolox also suppressed the CQ and Baf induced expression of IL-1β and IL-6. JNK inhibition and treatment with MitoTempo or Trolox failed to suppress apoptosis suggesting that this was independent of ROS levels and JNK pathway in chondrocytes with inhibited lysosomal function. Conclusions: We demonstrate here that inhibition of lysosomal function promotes inflammation, oxidative stress and chondrocyte apoptosis through JNK activation. Our data suggest that protecting lysosomal function may be a therapeutic target for OA management.

ABRO1 promotes NLRP3 inflammasome activation through regulation of NLRP3 deubiquitination.

Deubiquitination of NLRP3 has been suggested to contribute to inflammasome activation, but the roles and molecular mechanisms are still unclear. We here demonstrate that ABRO1, a subunit of the BRISC deubiquitinase complex, is necessary for optimal NLRP3-ASC complex formation, ASC oligomerization, caspase-1 activation, and IL-1β and IL-18 production upon treatment with NLRP3 ligands after the priming step, indicating that efficient NLRP3 activation requires ABRO1. Moreover, we report that ABRO1 deficiency results in a remarkable attenuation in the syndrome severity of NLRP3-associated inflammatory diseases, including MSU- and Alum-induced peritonitis and LPS-induced sepsis in mice. Mechanistic studies reveal that LPS priming induces ABRO1 binding to NLRP3 in an S194 phosphorylation-dependent manner, subsequently recruiting the BRISC to remove K63-linked ubiquitin chains of NLRP3 upon stimulation with activators. Furthermore, deficiency of BRCC3, the catalytically active component of BRISC, displays similar phenotypes to ABRO1 knockout mice. Our findings reveal an ABRO1-mediated regulatory signaling system that controls activation of the NLRP3 inflammasome and provide novel potential targets for treating NLRP3-associated inflammatory diseases.

SU27 - MOOD STABILIZERS IMPROVE CELL VIABILITY AND MITOCHONDRIAL MEMBRANE POTENTIAL IN VITRO

Background Lithium and valproate are the first line of drugs to treat Bipolar Affective Disorder (BPAD). The role of mitochondria in BPAD pathophysiology has often been investigated. The current study aims to explore the effects of lithium and valproate on cell death and mitochondrial membrane potential using Lymphoblastoid Cell Lines (LCL) model. Methods The patients were recruited from outpatient services of NIMHANS, Bangalore. The blood sample for LCL generation using Epstein Barr Virus transformation was obtained from BPAD patients (N=25) and healthy controls (N=11) after obtaining informed consent. The healthy controls were ethnically matched and selected randomly. Lithium response was assessed for the BPAD subjects using “Retrospective criteria of long term treatment response in research subjects with bipolar disorder” for whom NIMH life charts were available. Score ≥ 7 were considered as responders (N=14) and score For the experiment, 5 million cells were treated with medium containing lithium (1 mM) or valproate (0.7 mM) for 7 days. On the 8th day, one million cells were taken for flow cytometric analysis after incubation with the indicator dyes: Sytox Green (an indicator of dead cells) and Mitotracker Deep Red (an indicator of mitochondrial membrane potential; MMP). The experiments were performed in triplicates. FlowJo software was used for flow cytometry analysis and appropriate statistical analyses were performed. Results At baseline, the population of dead cells was significantly higher in BPAD LCLs (p The Mitochondrial Membrane Potential (MMP) was significantly lower in BPAD LCLs (p Analyses were performed at baseline and after mood stabilizer treatment between lithium response groups. Neither dead cell population nor high MMP population shown any significant difference between the responders and the non-responders group. Discussion BPAD LCL populations showed significantly higher cell death and lower mitochondrial membrane potential. On treatment with the mood stabilizer, it was found the dead cell population was decreased in LCLs. However, the MMP was found to be increased only in BPAD LCLs when treated. In conclusion, the effect of treatment was more pronounced in BPAD than in controls. We believe it would be interesting to look into the expression of genes involved in the pathways regulating apoptosis and mitochondrial activity.

Sulfuretin protects hepatic cells through regulation of ROS levels and autophagic flux.

Palmitate (PA) exposure induces stress conditions featuring ROS accumulation and upregulation of p62 expression, resulting in autophagic flux blockage and cell apoptosis. Sulfuretin (Sul) is a natural product isolated from Rhus verniciflua Stokes; the cytoprotective effect of Sul on human hepatic L02 cells and mouse primary hepatocytes under PA-induced stress conditions was investigated in this study. Sul induced mitophagy by activation of p-TBK1 and LC3 and produced a concomitant decline in p62 expression. Autophagosome formation and mitophagy were assessed by the sensitive dual fluorescence reporter mCherry-EGFP-LC3B, and mitochondrial fragmentation was analyzed using MitoTracker Deep Red FM. A preliminary structure-activity relationship (SAR) for Sul was also investigated, and the phenolic hydroxyl group was found to be pivotal for maintaining the cytoprotective bioactivity of Sul. Furthermore, experiments using flow cytometry and western blots revealed that Sul reversed the cytotoxic effect stimulated by the autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CQ), and its cytoprotective effect was almost eliminated when the autophagy-related 5 (Atg5) gene was knocked down. These studies suggest that, in addition to its antioxidative effects, Sul stimulates mitophagy and restores impaired autophagic flux, thus protecting hepatic cells from apoptosis, and that Sul has potential future medical applications for hepatoprotection.

Pioglitazone, a PPARγ agonist, reduces cisplatin-evoked neuropathic pain by protecting against oxidative stress.

Painful peripheral neuropathy is a dose-limiting side effect of cisplatin treatment. Using a murine model of cisplatin-induced hyperalgesia, we determined whether a PPARγ synthetic agonist, pioglitazone, attenuated the development of neuropathic pain and identified underlying mechanisms. Cisplatin produced mechanical and cold hyperalgesia and decreased electrical thresholds of Aδ and C fibers, which were attenuated by coadministration of pioglitazone (10 mg/kg, intraperitoneally [i.p.]) with cisplatin. Antihyperalgesic effects of pioglitazone were blocked by the PPARγ antagonist T0070907 (10 mg/kg, i.p.). We hypothesized that the ability of pioglitazone to reduce the accumulation of reactive oxygen species (ROS) in dorsal root ganglion (DRG) neurons contributed to its antihyperalgesic activity. Effects of cisplatin and pioglitazone on somatosensory neurons were studied on dissociated mouse DRG neurons after 24 hours in vitro. Incubation of DRG neurons with cisplatin (13 µM) for 24 hours increased the occurrence of depolarization-evoked calcium transients, and these were normalized by coincubation with pioglitazone (10 µM). Oxidative stress in DRG neurons was considered a significant contributor to cisplatin-evoked hyperalgesia because a ROS scavenger attenuated hyperalgesia and normalized the evoked calcium responses when cotreated with cisplatin. Pioglitazone increased the expression and activity of ROS-reducing enzymes in DRG and normalized cisplatin-evoked changes in oxidative stress and labeling of mitochondria with the dye MitoTracker Deep Red, indicating that the antihyperalgesic effects of pioglitazone were attributed to its antioxidant properties in DRG neurons. These data demonstrate clear benefits of broadening the use of the antidiabetic drug pioglitazone, or other PPARγ agonists, to minimize the development of cisplatin-induced painful neuropathy.

Glucose promotes the formation of tunneling nanotubes between bladder cancer cells by up-regulating Fyn/Rho-associated kinase signaling pathway

Objective To investigate the effects of glucose in formation of tunneling nanotubes (TNTs) between bladder cancer cells and the potential mechanism. Methods The highly invasive bladder cancer cells T24 were labeled with Mito-Tracker Deep Red, and then co-cultured with the less invasive bladder cancer cells RT4 for 24 hours. Actin-Tracker Green was used to label the cytoskeletal protein to detect the TNTs between T24 and RT4 cells. T24 and RT4 cells were cultured separately for 12 hours and 24 hours respectively, and the medium was collected and analyzed by liquid chromatography and mass spectrometry to screened out the difference factors between the micro-environment. T24 cells were cultured with the factors to confirm whether they could induce and enhance TNTs formation. The expression level of Fyn, Rho-associated kinase (ROCK) and p-paxillin proteins were detected and compared by Western blotting analysis. Results The formation of TNTs was found between T24 and RT4 cells. And the main component of TNTs was F-actin. Metabolomics analysis revealed that glucose was a differential factor between the micro-environments of T24 and RT4 cells, which could induce the formation of TNTs between T24 and RT4 cells (P=0.005). Concentration gradient of glucose between T24 and RT4 cells can up-regulate the expression of Fyn/ROCK and p-paxillin proteins (P=0.001, P=0.004, P=0.010). Conclusion Concentration gradient of glucose in the micro-environment of bladder cancer cells can promote the formation of TNTs between highly invasive and less invasive bladder cancer cells. The potential mechanism of the formation of TNTs induced by glucose might be explained by concentration gradient of glucose in the micro-environment of bladder cancer cells which can up-regulate the activity of Fyn/ROCK signaling pathway. Key words: Tunneling nanotubes; Bladder cancer; Cell conjunction; Micro-environment

Effects of biphasic and monophasic electrical stimulation on mitochondrial dynamics, cell apoptosis, and cell proliferation.

Currently, electrical stimulation (ES) is used to induce changes in various tissues and cellular processes, but its effects on mitochondrial dynamics and mechanisms are unknown. The aim of this study was to compare the effects of monophasic and biphasic, anodal, and cathodal ES on apoptosis, proliferation, and mitochondrial dynamics in neuroblastoma SH-SY5Y cells. Cells were cultured and treated with ES. Alamar blue assay was performed to measure cell proliferation. The proteins expression of apoptotic-related proteins Bcl-2 associated X (Bax), B cell lymphoma 2 (Bcl-2), optic-atrophy-1 (OPA1), mitofusin2 (Mfn2), phosphorylated dynamin-related protein 1 at serine 616 (p-DRP1), and total dynamin-related protein 1 (Total-DRP1) were also determined. The results showed that monophasic anodal and biphasic anodal/cathodal (Bi Anod) ES for 1 hr at 125 pulses per minute (2.0 Hz) produced the most significant increase in cell proliferation. In addition, monophasic anodal and Bi Anod ES treated cells displayed a significant increase in the levels of anti-apoptotic protein Bcl-2, whereas the Bax levels were not changed. Moreover, the levels of Mfn2 were increased in the cells treated by Bi Anod, and OPA1 was increased by monophasic anodal and Bi Anod ES, indicating increased mitochondrial fusion in these ES-treated cells. However, the levels of mitochondrial fission indicated by DRP1 remained unchanged compared with non-stimulated cells. These findings were confirmed through visualization of mitochondria using Mitotracker Deep Red, demonstrating that monophasic anodal and Bi Anod ES could induce pro-survival effects in SH-SY5Y cells through increasing cell proliferation and mitochondrial fusion. Future research is needed to validate these findings for the clinical application of monophasic anodal and Bi Anod ES.

Identification of mitochondrial hormone receptors in avian muscle cells.

The major objective of this study was to assess the expression of mitochondrial hormone receptors for progesterone (PR), estrogen (ER), glucocorticoid (GR), thyroid (TR), and insulin (IR) in avian muscle cells (quail muscle 7, QM7) and in breast muscle of quail and broilers. Visualization of receptor location in QM7 cells was accomplished by immunofluorescence. QM7 cells were stained with Mito Tracker Deep Red CMX, fixed in methanol, immune stained with anti-PR, -GR, -TR, -IR, and -ER primary antibodies overnight at 4°C, and visualized with Alexa Fluor 488-conjugated secondary antibody. After staining the nucleus with 4',6-diamidino-2-phenylindole, images were obtained by immunofluorescence microscopy. Merged images revealed the presence of all 5 hormone receptors on mitochondria in QM7 cells. Western blot analysis identified; (a) the β-isoform of the PR, (b) the α-isoform of GR, (c) the α-receptor of TR, (d) the β-subunit of IR, and (e) the α-isoform of the ER on mitochondria isolated from broiler breast muscle. Similar results were obtained in quail breast muscle mitochondria with the exception that the α-isoform of the GR was not detected. To our knowledge, this is the first report of hormone receptors (PR, TR, GR, IR, and ER) on mitochondria in avian cells. We hypothesize that these receptors could play important roles in regulating mitochondrial function in avian muscle cells.

Evidence of Oxidative Phosphorylation in Zebrafish Photoreceptor Outer Segments at Different Larval Stages.

Previous studies on purified bovine rod outer segments (OS) disks pointed to Oxidative Phosphorylation (OXPHOS) as being the most likely mechanism involved in ATP production, as yet not fully understood, to support the first phototransduction steps. Bovine and murine rod OS disks, devoid of mitochondria, would house respiratory chain complexes I to IV and ATP synthase, similar to mitochondria. Zebrafish ( Danio rerio) is a well-suited animal model to study vertebrate embryogenesis as well as the retina, morphologically and functionally similar to its human counterpart. The present article reports fluorescence and Transmission Electron Microscopy colocalization analyses of respiratory complexes I and IV and ATP synthase with zpr3, the rod OS marker, in adult and larval zebrafish retinas. MitoTracker Deep Red 633 staining and assays of complexes I and III-IV activity suggest that those proteins are active in OS. Results show that an extramitochondrial aerobic metabolism is active in the zebrafish OS at 4 and 10 days of larval development, as well as in adults, suggesting that it is probably maintained during embryogenesis. Data support the hypothesis of an extramitochondrial aerobic metabolism in the OS of zebrafish.

Apoptotic Effects of Temozolomide and Naturopathic Agents upon Glioblastoma Cells

Temozolomide (TMZ), thymoquinone (TMQ), epigallocatechin gallate (EPIGAL) and staurosporine (STAURO) were used as apoptotic inducing agents acting upon the U87-MG (ATCC, HTB15), LN18 (ATCC, CRL2610) and U118-MG (ATCC, HTB14) glioblastoma multiforme (GBM) cell lines. TMZ is the current drug of choice for primary treatment and adjuvant therapy for recurrent GBM. TMQ and EPIGAL are naturopathic agents, while STAURO is a well-studied apoptotic-inducing agent. The degree and time course of apoptosis were measured by flow cytometry techniques capable of detecting changes in mitochondrial function using the fluorescent dye MitoTracker Deep Red. Phosphatidylserine exposure and plasma membrane permeability were detected simultaneously using violet fluorescent reactive dye (VFRD) in combination with AnnexinV-488. The apoptotic effectiveness of the inducing agents TMZ, TMQ, EPIGAL and STAURO were compared with their ability to inhibit invasiveness and degrade Class I major histocompatibility complex (MHC) determinants. Invasiveness was measured in vitro by the 3D matrigel spheroid invasion assay. The density of the class 1 MHC determinants was measured by flow cytometry. Although TMZ is widely used for the chemotherapeutic treatment of GBM, it was determined that the time course for TMZ induced apoptosis was slower than those of TMQ, EPIGAL and STAURO. Unexpectedly, TMZ was ineffective in its ability to inhibit in vitro invasiveness and did not degrade the class I MHC determinants as effectively as the other apoptotic inducing agents. The findings raise the question of whether in vitro assays of apoptosis and invasiveness are the best measures of the effectiveness of chemotherapeutic agents for the primary treatment and adjuvant therapy of recurrent GBM. The findings also point to the in vivo complexity of the efficacy of chemotherapeutic agents whereby preserving the components of natural and acquired immune mechanisms may be more important than the rapid apoptotic effects of the chemotherapeutic agent.

A nontoxic, photostable and high signal-to-noise ratio mitochondrial probe with mitochondrial membrane potential and viscosity detectivity

Herein, we reported a yellow emission probe 1-methyl-4-(6-morpholino-1, 3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl) pyridin-1-ium iodide which could specifically stain mitochondria in living immortalized and normal cells. In comparison to the common mitochondria tracker (Mitotracker Deep Red, MTDR), this probe was nontoxic, photostable and ultrahigh signal-to-noise ratio, which could real-time monitor mitochondria for a long time. Moreover, this probe also showed high sensitivity towards mitochondrial membrane potential and intramitochondrial viscosity change. Consequently, this probe was used for imaging mitochondria, detecting changes in mitochondrial membrane potential and intramitochondrial viscosity in physiological and pathological processes.

Cationic Cytosine Morpholino‐Based Transporters: Synthesis and Regulation of Intracellular Localization

AbstractCationic guanidinium and phosphonium functionalized cytosine morpholino tetramer (G3) and trimer (P3) have been reported for the first time. They show efficient cellular uptake properties in full growth medium, quantified by fluorescence activated cell sorting (FACS) and visualized by fluorescence microscopy. An interesting feature of these transporters is their localization in the nucleolus or the rest of nucleus, determined by the nature of cationic moieties attached with cytosine base. Co‐staining with MitoTracker Deep Red depicts their ability to penetrate mitochondrial membrane. Fluorescence imaging displays their uniform distribution throughout zebrafish larvae with no significant toxicity. FACS analysis at 4°C indicated that transfection of G3 transporter is energy dependent whereas P3 is energy independent. When covalently conjugated, G3 is capable of transporting a 25‐mer phosphorodiamidate morpholino oligomer (PMO) into the cells. It exhibits antisense effect against Gli1 gene in hedgehog pathway, confirmed in preliminary β‐glactosidase assay performed in Ptch1–/– cells. Further antisense effect was confirmed when G3‐conjugated zebrafish Gli1‐PMO was injected into embryo to obtain Gli1‐dependent phenotypes. To the best of our knowledge, herein, we first report that molecular transporters with a minimum number of three cationic groups are capable of efficiently passing through plasma, nuclear and mitochondrial membranes and show distribution in zebrafish larvae.

MP07-18 IMPACTS OF SMOKING ON THE GLYCOCALYX OF HUMAN SPERMATOZOA

You have accessJournal of UrologyInfertility: Basic Research & Pathophysiology I1 Apr 2017MP07-18 IMPACTS OF SMOKING ON THE GLYCOCALYX OF HUMAN SPERMATOZOA Rick Paschold, Susanne Bour, Armin Becker, Christian Stief, and Matthias Trottmann Rick PascholdRick Paschold More articles by this author , Susanne BourSusanne Bour More articles by this author , Armin BeckerArmin Becker More articles by this author , Christian StiefChristian Stief More articles by this author , and Matthias TrottmannMatthias Trottmann More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.284AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES About 10-15% of all infertility patients are diagnosed with idiopathic infertility. Particularly, long term smokers often suffer from a reduction of basic sperm parameters (Ramlau-Hansen, 2007). The effects of smoking on male fertility are still discussed controversially. It was shown, that the essential binding between sperm and oviduct is based on a lectine-carbohydrate interaction (Koelle, 2012). A functional reduction of fertility could occur due to a lack of binding capacity. Therefore, our group characterized the proteins on glycocalyx of human spermatozoa that are capable to bind sugar residues. Further it was evaluated, if smokers show a restricted sugar-binding ability compared to non-smokers. METHODS We separated two study populations (smokers, non-smokers) out of 78 fresh human ejaculate samples. A direct staining with Mitotracker DeepRed, NucBlue (DAPI) and FITC-conjugated sugar residues (sialic acid- (SA), mannose- (MA) and fucose- (FU)) was performed. We used confocal microscopy to examine the fluorescence-marked samples. The fluorescent cells were analysed quantitatively and qualitatively within the study populations.Additionally, we extracted the sperm's proteins from smokers and control group which we applied on SDS-Page (4-12.5 %). Western Blot was used to proof the presence of LMAN2 and CatSper1 proteins on the plasma membrane surface. RESULTS We located proteins at the middle part of the spermatozoa's head that are capable of binding sugar residues.The ratio of fluorescence-labelled cells to the total cell count, which correspond to the capacity of binding sugar residues, was measured. We showed a significant difference between the groups: For smokers, we counted a proportion of 0.07±0.006 (SA), 0.05±0.006 (MA), 0.05±0.005 (FU) compared to 0.15±0.01 (SA), 0.20±0.02 (MA), 0.19±0.02 (FU) for the non-smokers (p<0.05). Fluorescence intensity did not vary significantly between the groups.Protein candidates were found in Western Blot but first experiments did not show a significant difference in the amount between smokers and non-smokers. CONCLUSIONS Our results point out, that smoking could possibly lead to a reduction of sugar binding proteins on the human sperm glycocalyx. This could be a reason for a decreased binding capacity of sperms to the female reproductive tract which could lead to a reduced fertility potential of smokers. Further work is necessary to lighten the exact molecular interaction between spermatozoa and female reproductive tract. Based on these facts it might be possible to examine new diagnostic and therapeutic approach in the future. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e88-e89 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Rick Paschold More articles by this author Susanne Bour More articles by this author Armin Becker More articles by this author Christian Stief More articles by this author Matthias Trottmann More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

A New Method for Evaluating Stallion Sperm Viability and Mitochondrial Membrane Potential in Fixed Semen Samples.

Multiparametric assessment of stallion sperm quality using flow cytometry can be a useful adjunct in semen evaluation; however, the availability of flow cytometers in veterinary practice is limited. The ability to preserve and transport sperm samples for later flow cytometric analysis using fixable probes would potentially facilitate this process. In the current study, we validated the combination of live/dead Zombie Green® (a fixable dye used to assess live and dead sperm) and MitoTracker Deep Red® (used to assess mitochondrial membrane potential). The assay was validated against classic, non-fixable, membrane assays (SYBR-14/PI). Our results demonstrated the feasibility of the assay. In conclusion, stained and fixed semen samples stored for 72 h obtained equivalent results to the exam on the same day; this new protocol shall facilitate the wider use of flow cytometry in stallion andrology in the future. © 2017 International Clinical Cytometry Society.

Cytofluorometric Assessment of Mitophagic Flux in Mammalian Cells and Tissues.

Mitophagy is the selective autophagic degradation of mitochondria inside lysosomes and is the only mechanism capable of eliminating whole mitochondria that are either damaged or no longer required. Mitochondrial dysfunction is a common hallmark in many pathological conditions and has been implicated in cellular damage in aged tissues. The limited availability of mitophagy research methods underscores the need for more robust, quantitative, and objective tools in order to better understand this process. Here we describe a flow cytometry-based method using MitoTracker Deep Red for mitophagy assessment that can be applied to cells and tissues. Moreover, we demonstrate that when used in conjunction with lysosomal inhibitors, our method provides a novel means of assessing mitophagic flux, which is the most reliable indicator of whether mitochondria are truly delivered to lysosomes for degradation.

Endosome-Mitochondria Interface Controls Intracellular Iron Trafficking in Erythroid Cells

In erythroid cells, more than 90% of transferrin-derived iron enters mitochondria where ferrochelatase inserts Fe2+ into protoporphyrin IX. However, the path of iron from endosomes to mitochondrial ferrochelatase remains elusive. The prevailing opinion is that, after its export from endosomes, the redox-active metal spreads into the cytosol and mysteriously finds its way into mitochondria through passive diffusion. An opposing view is that the highly efficient transport of iron toward ferrochelatase in erythroid cells requires a direct interaction between transferrin-endosomes and mitochondria ("kiss-and-run" hypothesis; Ponka Blood 89:1, 1997). Despite the longevity of the prevailing opinion, experimental evidence (Zhang et al. Blood 105:368, 2005; Sheftel et al. Blood 110: 125, 2007) only supports the latter hypothesis.Using 3D live confocal imaging of reticulocytes following their incubation with MitoTracker Deep Red (MTDR) and Alexa Green Transferrin (AGTf), we have demonstrated transient endosome-mitochondria interactions. We have also documented these interactions by a novel method exploiting flow sub-cytometry to analyze reticulocyte lysates labeled with MTDR and AGTf. We have thusly identified a population of particles labeled with both fluorescent markers, representing endosomes interacting with mitochondria. FACS followed by 2D confocal microscopy confirmed the association of both organelles in the double-labeled population.In the current study, we examined whether reticulocyte mitochondria interact with transferrin (Tf) in a cell-free system. Lysates of reticulocytes previously labeled with MTDR were incubated with AGTf for various time intervals. Examination of lysates by 2D confocal microscopy revealed a time-dependent increase in the number of mitochondria in contact with fluorescent Tf. This can be prevented by the presence of excess, unlabeled Fe2-Tf, but not by albumin (Fig.1). Moreover, the addition of unlabeled Fe2-Tf to reticulocyte lysates removed AGTf from mitochondria, indicating that mitochondria from reticulocyte lysates are associated with TfR that can reversibly bind Tf.In addition, we demonstrate that endosomes containing mutated recombinant holotransferrin, which cannot release iron, remain associated with mitochondria, while endosomes containing mutated recombinant apotransferrin, which cannot bind iron, are not associated with mitochondria. Our findings indicate that endosomes containing holo-Tf promote their attachment to, and drive the detachment of apo-Tf-endosomes from, mitochondria, respectively.By co-immunoprecipitation assay (from murine eryhroleukemia [MEL] cells and reticulocytes lysates), we purified the voltage-dependent anion channel 2 (VDAC2), which is located at the outer membrane of the mitochondrion (Graham, et al. Curr Top Dev Biol. 59: 87, 2004) with DMT1. We confirmed the colocalization of VDAC2 and DMT1 in MEL cells and reticulocytes by both immunofluorescence and confocal microscopy. Moreover, we found a significant decrease in the number of mitochondria in contact with Tf-endosomes after depletion of VDAC2 in MEL cells or after treatment of reticulocyte lysates with the mitochondrial uncoupler CCCP, further supporting the concept of a physical interaction between endosomes and mitochondria.To examine a possible role of DMT1-VDAC2 interactions in iron trafficking, we depleted MEL cells of VDAC2 or inhibited VADC2 using erastin (a specific VDAC2 inhibitor that alters its gating) followed by the measurement of 59Fe incorporation from 59Fe-Tf into heme. Our finding of decreased 59Fe incorporation into heme of MEL cells with silenced or inhibited VDAC2 supports the idea that this outer-membrane mitochondrial protein is involved in the interaction of endosomes with mitochondria.We are currently continuing to delineate the molecular mechanisms involved in endosome-mitochondria interactions focusing on the "signal(s)" that direct iron-carrying endosomes towards mitochondria, the players involved in the docking of endosomes to mitochondria and the "signal(s)" that determine the detachment of iron-free endosomes from mitochondria. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.