An impedimetric assay for the identification of abnormal mitochondrial dynamics in living cells

Mitochondrial dynamics (fission and fusion) plays an important role in cell functions. Disruption in mitochondrial dynamics has been associated with diseases such as neurobiological disorders and cardiovascular diseases. Analysis of mitochondrial fission/fusion has been mostly achieved through direct visualization of the fission/fusion events in live-cell imaging of fluorescently labeled mitochondria. In this study, we demonstrated a label-free, non-invasive Electrical Impedance Spectroscopy (EIS) approach to analyze mitochondrial dynamics in a genetically modified human neuroblastoma SH-SY5Y cell line with no huntingtin protein expression. Huntingtin protein has been shown to regulate mitochondria dynamics. We performed EIS studies on normal SH-SY5Y cells and two independent clones of huntingtin-null cells. The impedance data was used to determine the suspension conductivity and further cytoplasmic conductivity and relate to the abnormal mitochondrial dynamics. For instance, the cytoplasm conductivity value was increased by 11% from huntingtin-null cells to normal cells. Results of this study demonstrated that EIS is sensitive to characterize the abnormal mitochondrial dynamics that can be difficult to quantify by the conventional microscopic method.     

Detection of carbonyl-modified proteins in interfibrillar rat mitochondria using N'-aminooxymethylcarbonylhydrazino-D-biotin as an aldehyde/keto-reactive probe in combination with Western blot analysis and tandem mass spectrometry

There is now a large body of supporting data available that links oxidative modifications of proteins to a large number of diseases, degenerative disorders and aging. However, the detailed analysis of oxidative protein modifications remains challenging. Here, we report a new efficient method for identification of oxidatively modified proteins in complex biological samples which is based on the use of an aldehyde-reactive probe, N'-aminooxymethylcarbonylhydrazino-D-biotin (ARP), in combination with Western-type analyses and MS. The biotinylated hydroxylamine derivative forms a chemically stable oxime derivative with the aldehyde/keto group found in carbonyl-modified proteins. The biotin tag is detected by avidin affinity staining. ARP-positive proteins are subsequently subjected to in-gel trypsinization and MS/MS for protein identification. We demonstrate the usefulness of the method for the analysis of protein extracts obtained from interfibrillar heart mitochondria (IFM) from young and old rats. In this study, we identified as putative major protein targets of oxidative modifications the mitochondrial matrix protein, aconitase, the inner mitochondrial membrane protein, ADP/ATP translocase, and constituents of the electron transport chain complexes IV and V. An age-related increase of carbonyl levels was found for aconitase and ATP synthase.     

Effect of La on heat production by mitochondria isolated from hybrid rice

The effect of lanthanum on mitochondria isolated from hybrid rice Fengyou 559 (Oryza sativa L.) was investigated. Through in vivo culture, low-dose La³⁺ promoted, but higher dose La³⁺, restrained mitochondrial heat production. However, through in vitro incubation, La³⁺ manifested only inhibitory action on mitochondrial energy turnover, the concentration required for 50% and 100% inhibition being 50.9 and 230.2 μM (57.6 nmol/mg protein), respectively. In addition, La³⁺, like Ca²⁺, induced rice mitochondrial swelling and decreased membrane potential (Δψ), which was inhibited by the specific permeability transition inhibitor cyclosporine A (CsA). The induction approached a constant limitation while mitochondrial metabolism was completely prevented by La³⁺, and microscopy observation showed a high disruption of inner mitochondrial membrane in this state. These results demonstrated that lanthanum influenced rice mitochondria in vivo and in vitro via different action pathways, and the latter involved the opening of rice mitochondrial permeability.     

ER-associated CTRP1 regulates mitochondrial fission via interaction with DRP1

C1q/TNF-related protein 1 (CTRP1) is a CTRP family member that has collagenous and globular C1q-like domains. The secreted form of CTRP1 is known to be associated with cardiovascular and metabolic diseases, but its cellular roles have not yet been elucidated. Here, we showed that cytosolic CTRP1 localizes to the endoplasmic reticulum (ER) membrane and that knockout or depletion of CTRP1 leads to mitochondrial fission defects, as demonstrated by mitochondrial elongation. Mitochondrial fission events are known to occur through an interaction between mitochondria and the ER, but we do not know whether the ER and/or its associated proteins participate directly in the entire mitochondrial fission event. Interestingly, we herein showed that ablation of CTRP1 suppresses the recruitment of DRP1 to mitochondria and provided evidence suggesting that the ER–mitochondrion interaction is required for the proper regulation of mitochondrial morphology. We further report that CTRP1 inactivation-induced mitochondrial fission defects induce apoptotic resistance and neuronal degeneration, which are also associated with ablation of DRP1. These results demonstrate for the first time that cytosolic CTRP1 is an ER transmembrane protein that acts as a key regulator of mitochondrial fission, providing new insight into the etiology of metabolic and neurodegenerative disorders.Subject terms: Endoplasmic reticulum, Mitochondria     

Possible involvement of mitochondrial calcium transport in causing cell injury in experimental hepatic chronic iron overload

The functional state of isolated mitochondria and, specifically, the integrity of the inner membrane were investigated in the livers of rats made siderotic by dietary supplementation with carbonyl iron. The concentration of iron in the mitochondrial fraction increased progressively up to nearly 40 days and it reached a steady-state level. When the iron content reached a threshold value (higher than 30 nmol/mg protein) the occurrence of in vivo lipid peroxidation in the mitochondrial membrane was detected. This process did not result in gross alterations in the mitochondrial membrane, as indicated by phosphorylative capability and membrane potential measurements. On the contrary, the induction of the lipoperoxidative reaction appeared to be associated with the activation of Ca²⁺ release from mitochondria. This was shown to occur as a consequence of rather subtle modifications in the inner membrane structure via a specific efflux route, which appeared to be linked to the oxidation level of mitochondrial pyridine nucleotides. The induction of this Ca²⁺ release from iron treated mitochondria resulted in enhancement of Ca²⁺ cycling, a process which dissipates energy to reaccumulate the releaased Ca²⁺ into mitochondria. The perturbation in mitochondrial Ca²⁺ homeostasis reported here may be a factor in the onset of cell damage in this experimental model of hepatic chronic iron overload.     

Eine gentherapeutische Strategie zur Behandlung von Energiestoffwechsel-Erkrankungen

Genetic variations of the mitochondrial genome lead to severe neuromuscular diseases in man. A treatment of these utilizing a somatic gene therapy approach is invariably linked to a mitochondrial transfection system. A novel technique of targeting nucleic acids to mitochondria has been developed that takes advantage of the protein import pathway. The system is based on chimerical molecules that are composed of a DNA and a protein moiety, harboring the information for mitochondrial targeting. Upon recognition of these molecules by a receptor on the outer mitochondrial membrane, the molecules are able to cross the membrane system and are released into the matrix of the organelle. The further development of this technique will give to rise to strategies for the treatment of mitochondrial DNA diseases by a somatic gene therapy approach.     

Wafer-scale mitochondrial membrane potential assays

It has been reported that mitochondrial metabolic and biophysical parameters are associated with degenerative diseases and the aging process. To evaluate these biochemical parameters, current technology requires several hundred milligrams of isolated mitochondria for functional assays. Here, we demonstrate manufacturable wafer-scale mitochondrial functional assay lab-on-a-chip devices, which require mitochondrial protein quantities three orders of magnitude less than current assays, integrated onto 4' standard silicon wafer with new fabrication processes and materials. Membrane potential changes of isolated mitochondria from various well-established cell lines such as human HeLa cell line (Heb7A), human osteosarcoma cell line (143b) and mouse skeletal muscle tissue were investigated and compared. This second generation integrated lab-on-a-chip system developed here shows enhanced structural durability and reproducibility while increasing the sensitivity to changes in mitochondrial membrane potential by an order of magnitude as compared to first generation technologies. We envision this system to be a great candidate to substitute current mitochondrial assay systems.     

Identification of novel anti cancer agents by applying insilico methods for inhibition of TSPO protein

Cancer is a genomic disease characterised as impaired cellular energy metabolism. Cancer cells derive most of their energy from oxidative phosphorylation unlike normal ones during cell progression TSPO protein present in external mitochondrial membrane, is involved in various cellular functions like Cell proliferation, mitochondrial respiration, synthesis of steroids and also participates in import of cholesterol into the inner mitochondrial membrane from outside of the membrane of mitochondria.The 3D model of TSPO protein is built using comparative homology modelling techniques and validated by proSA, Ramachandran plot and ERRAT in the present work. Active site prediction is carried out using SiteMap and literature, which allows the prediction of the important binding pockets for the identification of putative active site. New molecular entities as TSPO inhibitors were obtained from Virtual screening using MS Spectrum databank in Schrodinger suite and were prioritised based on Glide Score. Docking was performed using Autodock to identify molecules with different scaffolds and were prioritised based on binding energy and RMSD values. Qikprop is used to calculate pharmacokinetic properties of the screened molecules which are found to be in permissible range as possible novel inhibitors of TSPO protein to supress cell proliferation.     

Photophysical, photochemical, and tumor-selectivity properties of bromine derivatives of rhodamine-123

The conceptual basis for the development of mitochondrial targeting as a novel therapeutic strategy for both chemotherapy and photochemotherapy of neoplastic diseases rests on the observation that enhanced mitochondrial membrane potential is a common tumor cell phenotype. The potential of this strategy is highlighted by the fact that the toxic effects associated with a number of cationic dyes known to localize in energized cell mitochondria are much more pronounced in tumor cells than in normal cells. Here we evaluate the phototoxic properties of four bromine derivatives of rhodamine-123 toward human uterine sarcoma (MES-SA) and green monkey kidney (CV-1) cells and compare the degrees of tumor cell selectivity associated with these dyes with those associated with two model mitochondrial triarylmethanes (crystal violet and ethyl violet). Selective phototoxicity toward tumor cells was found to be highly dependent upon the lipophilic/hydrophilic character of the cationic photosensitizer. Our experimental data have indicated that the probability of success of mitochondrial targeting in (photo)chemotherapy of neoplastic diseases is higher when the octan-1-ol/water partition coefficient of the drug candidate falls within approximately two orders of magnitude from that of the prototypical mitochondria-specific dye rhodamine-123.     

用于检测生物体系中线粒体活性氧的荧光探针

In addition to being the energy powerhouse of the cell, mitochondria are an important source of reactive oxygen species (ROS) during the process of molecular oxygen metabolism. Mitochondrial ROS are closely associated with normal physiological functions as well as human diseases, and participate in cell signaling, nucleic acid and protein damage, and oxidative stress induction. However, the complicated interplay between mitochondrial ROS and the cellular pathological state has not been fully elucidated. It is expected that research on the mitochondrial ROS undertaking in the molecular pathogenesis of human diseases would benefit from development of efficient tools for the detection of these ROS. In recent years, an increasing number of fluorescent probes for mitochondrial ROS with high sensitivity and selectivity have been developed. Here, we present a review of the recent advances in small molecular fluorescent probes for selective detection of ROS inside the mitochondria. In this review, the design, synthesis, characteristics, and applications of the published fluorescent probes for mitochondrial ROS are discussed in detail.     

Action of propranolol on mitochondrial proton fluxes.

The action of propranolol, a Beta-blocking adrenergic agent, on mitochondrial proton fluxes in nonenergized and energized conditions has been studied. 1. The drug inhibited the actions of valinomycin, higericin and FCCP on the inner mitochondrial membrane. 2. It decreased the rate and extent of active energized proton expulsion and passive collapse of the proton gradient so formed. 3. Propranolol was able to increase the permeation of chloride ion through the inner mitochondrial membrane in nonenergized and energized conditions. 4. The drug inhibited mitochondrial contraction but stimulated swelling in various conditions. It is suggested that propranolol is is able to change the proton and chloride permeabilities of mitochondria by perturbing the structure of inner membrane phospholipids, thus enlarging the water-lipid interface.     

可固定性荧光探针:线粒体成像的可靠工具

线粒体是一种具有双层膜结构的细胞器,参与细胞新陈代谢过程的能量循环以及离子平衡过程,在细胞生理过程中具有极其重要的意义。一些小分子荧光染料/探针结构中带有正电荷,因受到线粒体内膜负电势的牵引而标记在线粒体上,为研究线粒体的形态或功能提供了重要的可视化成像工具。然而,大多数线粒体染料/探针对线粒体的靶向标记稳定性仍不够理想,因为线粒体电势处于不断的动态变化中,当电势降低时,对染料的亲和力相应降低。尤其在病理条件下(比如细胞凋亡)细胞代谢受到阻滞时,线粒体膜电势显著降低,阳离子染料将扩散离开线粒体,造成非特异性荧光。最近,Kim团队和本人课题组提出可固定线粒体探针的新概念,用活性基团将荧光分子探针通过共价键固定在线粒体中,开发了稳定靶向线粒体中的定量探测微环境p H值、粘度、膜电势荧光探针。我们认为,对于追踪和探测具有高度动态变化特性的线粒体而言,开发可固定的线粒体荧光分子探针是必然趋势,因此本文进行评述和展望。     

Alterations of the mitochondrial proteome caused by the absence of mitochondrial DNA: A proteomic view

The proper functioning of mitochondria requires that both the mitochondrial and the nuclear genome are functional. To investigate the importance of the mitochondrial genome, which encodes only 13 subunits of the respiratory complexes, the mitochondrial rRNAs and a few tRNAs, we performed a comparative study on the 143B cell line and on its Rho-0 counterpart, i.e., devoid of mitochondrial DNA. Quantitative differences were found, of course in the respiratory complexes subunits, but also in the mitochondrial translation apparatus, mainly mitochondrial ribosomal proteins, and in the ion and protein import system, i.e., including membrane proteins. Various mitochondrial metabolic processes were also altered, especially electron transfer proteins and some dehydrogenases, but quite often on a few proteins for each pathway. This study also showed variations in some hypothetical or poorly characterized proteins, suggesting a mitochondrial localization for these proteins. Examples include a stomatin-like protein and a protein sharing homologies with bacterial proteins implicated in tyrosine catabolism. Proteins involved in apoptosis control are also found modulated in Rho-0 mitochondria.     

Die zitterfreie Thermogenese: Wärmeproduktion bei Kältestress

Non‐shivering thermogenesis is based on the increase of the permeability of the inner membrane of mitochondria to H⁺. An increase in the H⁺‐flux from the cytosolic to the matrix compartiment dissipates the electrochemical proton gradient and, therefore, activates the mitochondrial respiratory chain. Permeability of H⁺ is stimulated by artificial protonophores such as 2,4‐dinitrophenol or the interaction of non‐esterified fatty acids with certain proteins of the inner membrane of mitochondria, called uncoupling proteins (UCPs). UCPs are expressed in various tissues of mammalians, but also in riping plants. Despite of their role in thermogenesis, activated UCPs suppress the formation of the superoxid anion radical. Therefore, UCPs are also physiological tools to diminish oxidative stress within the cell.     

哮喘大鼠淋巴细胞蛋白质组的分析

通过建立卵蛋白(Ovalbumin, OVA) 致敏的Wistar大鼠哮喘动物模型, 利用二维凝胶电泳及MALDI-TOF-MS分析技术, 对哮喘大鼠与正常大鼠脾脏淋巴细胞差异表达蛋白质进行鉴定和分析. 结果显示, 在哮喘组中呈现高表达的蛋白质, 包括胶原蛋白调控蛋白质(hnRNP)以及细胞结构蛋白(Fibrinogen). 而与代谢相关的蛋白质(CoQ10)和线粒体内膜蛋白, 在哮喘组中呈低表达. 研究结果表明, 这些蛋白质与哮喘气道慢性炎症、气道高反应性和气道的重构有关.     

Mitochondria‐Targeted Cancer Therapy Using a Light‐Up Probe with Aggregation‐Induced‐Emission Characteristics

Subcellular organelle‐specific reagents for simultaneous tumor targeting, imaging, and treatment are of enormous interest in cancer therapy. Herein, we present a mitochondria‐targeting probe (AIE‐mito‐TPP) by conjugating a triphenylphosphine (TPP) with a fluorogen which can undergo aggregation‐induced emission (AIE). Owing to the more negative mitochondrial membrane potential of cancer cells than normal cells, the AIE‐mito‐TPP probe can selectively accumulate in cancer‐cell mitochondria and light up its fluorescence. More importantly, the probe exhibits selective cytotoxicity for studied cancer cells over normal cells. The high potency of AIE‐mito‐TPP correlates with its strong ability to aggregate in mitochondria, which can efficiently decrease the mitochondria membrane potential and increase the level of intracellular reactive oxygen species (ROS) in cancer cells. The mitochondrial light‐up probe provides a unique strategy for potential image‐guided therapy of cancer cells.     

Use of Highly Purified and Mixed Antibodies for Simultaneous Detection of Multiple Protein Species Released from Mitochondria upon Induction of the Permeability Transition

Concomitant with the induction of the mitochondrial permeability transition (PT), cytochrome c is released from mitochondria into the cytosol where it triggers subsequent steps of cellular apoptosis. Thus, inducers of the mitochondrial PT would become “seed compounds” of regulators of apoptosis. However, when we examine the actions of certain chemicals on the release of mitochondrial cytochrome c, the behaviors of not only cytochrome c but also multiple mitochondrial protein species must be carefully examined because the mitochondrial PT and release of proteins from mitochondria occur in diverse manners. In the present study, we examined whether it is possible to measure the behaviors of multiple protein species in a single experiment using purified and mixed antibodies. The results obtained clearly indicate that this procedure would be applicable for high-throughput screening of regulators of apoptosis. Further requirements necessary for the establishment of a useful screening system for apoptosis regulators are discussed.     

Mitochondria-penetrating peptides

Mitochondria are important targets for cancer chemotherapy and other disease treatments. Gaining access to this organelle can be difficult, as the inner membrane is a barrier limiting diffusive transport. A mitochondrial molecular carrier would be a boon to the development of organelle-specific therapeutics. Here, we report a significant advance in the development of mitochondrial transporters-synthetic cell-permeable peptides that are able to enter mitochondria. Efficient uptake of these mitochondria-penetrating peptides (MPPs) is observed in a variety of cell types, and organellar specificity is attained with sequences that possess specific chemical properties. The MPPs identified are cationic, but also lipophilic; this combination of characteristics facilitates permeation of the hydrophobic mitochondrial membrane. The examination of a panel of MPPs illustrates that mitochondrial localization can be rationally controlled and finely tuned by altering lipophilicity and charge.     

Opsin3 Downregulation Induces Apoptosis of Human Epidermal Melanocytes via Mitochondrial Pathway

G protein-coupled receptors (GPCRs) are core switches connecting excellular survival or death signals with cellular signaling pathways in a context-dependent manner. Opsin 3 (OPN3) belongs to the GPCR superfamily. However, whether OPN3 can control the survival or death of human melanocytes is not known. Here, we try to investigate the inherent function of OPN3 on the survival of melanocytes. Our results demonstrate that OPN3 knockdown by RNAi-OPN3 in human epidermal melanocytes leads to cell apoptosis. The downregulation of OPN3 markedly reduces intracellular calcium levels and decreases phosphorylation of BAD. Attenuated BAD phosphorylation and elevated BAD protein level alter mitochondria membrane permeability, which trigger activation of BAX and inhibition of BCL-2 and raf-1. Activated BAX results in the release of cytochrome c and the loss of mitochondrial membrane potential. Cytochrome c complexes associate with caspase 9, forming a postmitochondrial apoptosome that activate effector caspases including caspase 3 and caspase 7. The release of apoptotic molecules eventually promotes the occurrence of apoptosis. In conclusion, we hereby are the first to prove that OPN3 is a key signal responsible for cell survival through a calcium-dependent G protein-coupled signaling and mitochondrial pathway.     

Reversible Effects of Photodamage Directed Toward Mitochondria

When the initial effect of photodynamic therapy (PDT) involves mitochondrial photodamage, an early effect is loss of the mitochondrial membrane potential (ΔΨ_m). Using murine hepatoma 1c1c7 cells and a photosensitizing agent known to target mitochondria, we examined loss of ΔΨ_m, initiation of apoptosis and loss of viability as a function of time and light dose. There was a correlation between loss of viability and the rapid disappearance of ΔΨ_m, as detected by the potential‐sensitive probe Mitotracker Orange (MTO). Loss of ΔΨ_m was, however, reversible even with a substantial loss of viability. Unless there was a supralethal level of photodamage, 1c1c7 cells recovered their mitochondrial membrane potential, even if the cell population was on the pathway to apoptosis and cell death. These results indicate that when mitochondria are the initial PDT target, a qualitative estimate of photokilling can be provided by assessing the initial loss of ΔΨ_m.