Quantitative analysis of chromatin compaction in living cells using FLIM–FRET

We present a quantitative Förster resonance energy transfer (FRET)–based assay using multiphoton fluorescence lifetime imaging microscopy (FLIM) to measure chromatin compaction at the scale of nucleosomal arrays in live cells. The assay uses a human cell line coexpressing histone H2B tagged to either enhanced green fluorescent protein (FP) or mCherry FPs (HeLa^H2B-2FP). FRET occurs between FP-tagged histones on separate nucleosomes and is increased when chromatin compacts. Interphase cells consistently show three populations of chromatin with low, medium, or high FRET efficiency, reflecting spatially distinct regions with different levels of chromatin compaction. Treatment with inhibitors that either increase chromatin compaction (i.e., depletion of adenosine triphosphate) or decrease chromosome compaction (trichostatin A) results in a parallel increase or decrease in the FLIM–FRET signal. In mitosis, the assay showed variation in compaction level, as reflected by different FRET efficiency populations, throughout the length of all chromosomes, increasing to a maximum in late anaphase. These data are consistent with extensive higher order folding of chromatin fibers taking place during anaphase.     

A new polyaniline–catalase–glutaraldehyde-modified biosensor for hydrogen peroxide detection

A new biosensor based on catalase enzyme immobilized on electrochemically constructed polyaniline (PANI) film modified with glutaraldehyde has been developed for the determination of hydrogen peroxide (H₂O₂) in milk samples. Assembly processes of polyaniline and immobilization of the enzyme were monitored with the help of electrochemical impedance spectroscopy. Amperometric measurements have been performed at cathodic peak (?0.3?V vs. Ag/AgCI) which was attributed to reduction of PANI. Hydrogen peroxide was determined by using amperometric method at ?0.3?V. The biosensor responses were correlated linearly with the hydrogen peroxide concentrations between 5.0?×?10^?6 and 1.0?×?10^?4?M by amperometric method. Detection limit of the biosensor is 2.18?×?10^?6?M for H₂O₂. In the optimization studies of the biosensor, some parameters such as optimum pH, temperature, concentration of aniline, amount of enzyme, and number of scans during electropolymerization were investigated.     

Involvement of caspase activation and mitochondrial stress in taxol-induced apoptosis of Epstein–Barr virus-infected Akata cells

Taxol (paclitaxel) is one of the most potent antimicrotubule agents currently used in cancer chemoprevention and treatment. However, the effects of taxol on the induction of apoptosis in Epstein–Barr virus (EBV)-infected cells are unknown. This study investigated the mechanisms of taxol on cell cycle arrest and apoptosis induction using the EBV-infected cell line, Akata. Taxol treatment sensitively and dose-independently induced growth inhibition, cytotoxicity, and apoptosis in the cells, which was demonstrated by the decreased level of tritium incorporation and cell viability, the increased number of positively stained cells in the trypan blue staining and TUNEL assay, the increased population of cells in the sub-G₀/G₁ phase in flow cytometric analysis, and ladder formation of the genomic DNA. Treatment with z-VAD-fmk almost completely protected the cells from taxol-induced apoptosis indicating that the taxol-induced apoptosis of Akata cells is caspase-dependent. In addition, taxol-induced apoptosis is proposed to be associated with a lower mitochondrial membrane potential and G₂/M arrest. However, the tubulin expression level doses not appear to be a direct mediator of taxol-induced apoptosis in cells. The presence of EBV in these cells was not related to the sensitivity of the cells to the induction of apoptosis by taxol. Overall, these results demonstrate that taxol induces apoptosis in EBV-infected Akata cells in a dose-independent manner, and that caspase activation and mitochondrial stress are involved in the induction.     

High-efficiency recovery of target cells using improved yeast display system for detection of protein–protein interactions

We constructed a high-throughput screening (HTS) system for target cells based on the detection of protein–protein interactions by flow cytometric sorting due to the improvement in the yeast cell surface display system. Interaction model proteins, which are the ZZ domain derived from Staphylococcus aureus and the Fc part of human immunoglobulin G (IgG), were displayed on the yeast cell surface. We achieved a rapid and enhanced expression of these proteins as a result of adopting an appropriate yeast strain and a suitable promoter. The displayed ZZ domain had an ability to bind to rabbit IgG and the displayed Fc part to protein A. These were confirmed by flow cytometry and fluorescence microscopy. Furthermore, the cells displaying the ZZ domain or Fc part were isolated from the model libraries constructed by mixing the control yeast cells with the target yeast cells. The ratio of the target cells was increased from 0.0001% to more than 70% by two cycles of cell sorting. These results indicate that we can achieve a rapid and highly efficient isolation method for the target cells with FACSCalibur and that this method will further extend the application of flow cytometric sorting to library selections.     

Differences in the products of mitochondrial protein synthesis in vivo in human and mouse cells and their potential use as markers for the mitochondrial genome in human–mouse cell hybrids

The proteins synthesized in the mitochondria of mouse and human cells grown in tissue culture were examined by electrophoresis in polyacrylamide gels. The proteins were labelled by incubating the cells in the presence of [(35)S]methionine and an inhibitor of cytoplasmic protein synthesis (emetine or cycloheximide). A detailed comparison between the labelled products of mouse and human mitochondrial protein synthesis was made possible by developing radioautograms after exposure to slab-electrophoresis gels. Patterns obtained for different cell types of the same species were extremely similar, whereas reproducible differences were observed on comparison of the profiles obtained for mouse and human cells. Four human-mouse somatic cell hybrids were examined, and in each one only components corresponding to mouse mitochondrially synthesized proteins were detected.     

Constructing glucagon like peptide-1 receptor fused with derivatives of GFP for visualizing protein–protein interaction in living cells

The glucagon-like peptide-1 receptor (GLP-1 receptor) mediates important antidiabetogenic effects on peripheral tissues. It appears to be one of the most promising therapeutic targets for treatment of diabetes mellitus type 2. Surprisingly, very little is known about the cellular mechanisms that regulate receptor function in living cells. One of the approaches how to study receptor dynamics is by using tagged fluorescent proteins. In this study, YFP-tagged GLP-1 (YFP-GLP-1) receptor and CFP-tagged GLP-1 (CFP-GLP-1) receptor for visualizing protein–protein interaction in living cells were constructed and localized in CHO cells. Cells expressing YFP-GLP-1 and CFP-GLP-1 receptor showed characteristic GLP-1 mediated increase in cAMP, similar to cells expressing a wild type GLP-1 receptor. This means that both types of receptors are functional and localized in plasma membrane.     

Molecular dynamics simulation of diffusion of hydrogen in binary hydrogen–tetrahydrofuran hydrate

The binary structure II hydrogen–tetrahydrofuran (THF) hydrate was studied with molecular dynamics simulation. The simulations were carried out at 300, 310 K and 10.1 MPa, and with various contents of hydrogen and THF. The migrations of hydrogen molecules from cage to cage were observed. The migration process of hydrogen was also analysed, and the diffusion coefficients of hydrogen in the hydrate were calculated. The calculated diffusion coefficients qualitatively agreed with the experimental data. Double and quintet occupancies of hydrogen molecules were observed in the small and large cages, respectively, without changing the hydrate structure.     

Dynamics of RhoA and ROKα translocation in single living cells

The RhoA-binding kinase (ROK) is one of the target kinases of RhoA and is known to play a critical role in regulating cytoskeletal rearrangement in cells. ROK translocates to the plasma membrane fraction; however, the mechanism of the translocation of ROK still remains obscure. To clarify the molecular mechanisms of the translocation of ROK, we co-transfected MDCK cells wity cyan fluorescent protein-tagged RhoA and yellow fluorescent protein-tagged ROKα, or their variants, and monitored the localization and translocation of the two different fluorescent tagged-molecules in single living cells during epithelial growth factor (EGF) stimulation. Both RhoA (wild-type) and ROKα (wild-type) translocated to ruffling membrane with EGF stimulation in several minutes. A ROKα mutant, in which Rho-binding ability is disrupted, is unable to translocate to the membrane with RhoA. However, RhoA mutant Q63L/C190R, an active form lacking membrane localization activity, abolished the translocation of wild-type ROKα, suggesting that the translocation of RhoA is critical for ROK translocation to the membrane. Another mutant lacking the pleckstrin homology domain failed in translocation as well. On the other hand, it was surprising that the kinase dead mutant succeeded in translocation to the membrane after EGF stimulation. Based on these results, we propose the following ROKα translocation mechanism. ROKα binds to RhoA in cytosol and translocates to the membrane based on the membrane-targeting ability of active RhoA. After ROKα associates with the membrane, the pleckstrin homology domain provides the stability of ROKα on the membrane. The activation of enzymatic activity or adenosine triphosphate binding, however, is not directly related to the translocation mechanism, although we found that the membrane association is critical for the activation of the kinase activity.     

Tagging of functional ribosomes in living cells by HaloTag? technology

Ribosomal proteins and ribosomal associated proteins are complicated subjects to target and study because of their high conservation through evolution which led to highly structured and regulated proteins. Tagging of ribosomal proteins may allow following of protein synthesis in vivo and isolating translated mRNAs. HaloTag? is a new technology which allows detection in living cells, biochemical purification, and localization studies. In the present work, we tested HaloTag?-based ribosomal tagging. We focused on eIF6 (eukaryotic Initiation Factor 6 free 60S ribosomal marker), RACK1 (Receptor for Activated C Kinase 1; 40S and polysomes, not nuclear), and rpS9 (40S ribosomes, both in the nucleus and in the cytoplasm). Experiments performed on HEK293 cells included ribosomal profiles and Western blot on the fractions, purification of HaloTag? proteins, and fluorescence with time-lapse microscopy. We show that tagged proteins can be incorporated on ribosomes and followed by time-lapse microscopy. eIF6 properly accumulates in the nucleolus, and it is redistributed upon actinomycin D treatment. RACK1 shows a specific cytoplasmic localization, whereas rpS9 is both nucleolar and cytoplasmic. However, efficiency of purification varies due to steric hindrances. In addition, the level of overexpression and degradation may vary upon different constructs. In summary, HaloTag? technology is highly suitable to ribosome tagging, but requires prior characterization for each construct.     

Regulation of β-catenin trafficking to the membrane in living cells

β-catenin is a key mediator of the Wnt signaling process and accumulates in the nucleus and at the membrane in response to Wnt-mediated inhibition of GSK-3β. In this study we used live cell photobleaching experiments to determine the dynamics and rate of recruitment of β-catenin at membrane adherens junctions (cell adhesion) and membrane ruffles (cell migration). First, we confirmed the nuclear-cytoplasmic shuttling of GFP-tagged β-catenin, and found that a small mobile pool of β-catenin can move from the nucleus to membrane ruffles in NIH 3T3 fibroblasts with a t_0.5 of ~ 30 s. Thus, β-catenin can shuttle between the nucleus and plasma membrane. The localized recruitment of β-catenin-GFP to membrane ruffles was more rapid, and the strong recovery observed after bleaching (mobile fraction 53%, t_0.5 ~5 s) is indicative of high turnover and transient association. In contrast, β-catenin-GFP displayed poor recovery at adherens junctions in MDCK epithelial cells (mobile fraction 10%, t_0.5 ~8 s), indicating stable retention at these membrane structures. We previously identified IQGAP1 as an upstream regulator of β-catenin at the membrane, and this is supported by photobleaching assays which now reveal IQGAP1 to be more stably anchored at membrane ruffles than β-catenin. Further analysis showed that LiCl-mediated inactivation of the kinase GSK-3β increased β-catenin membrane ruffle staining; this correlated with a faster rate of recruitment and not increased membrane retention of β-catenin. In summary, β-catenin displays a high turnover rate at membrane ruffles consistent with its dynamic internalization and recycling at these sites by macropinocytosis.     

Analysis of sitosteryl oleate esters in phytosterols esters enriched foods by HPLC–ESI-MS2

Phytosteryl esters (PE)-enriched spreads are marketed for eating and cooking purposes. Temperature and also light exposure are the major factors leading to the formation of PE oxides in food matrix. In this study a high-speed HPLC–MS² method was developed to analyze the major PE present in PE-enriched spreads: sitosteryl oleate (SO) and its oxidation products, by using synthesized compounds as standards. This analytical method was used to quantify seven SO oxides formed in PE-enriched spreads after heating at different temperatures for varying time periods and after prolonged exposure to sunlight. Quantification of remaining native SO was also performed after these different treatments. It was found that under specific heating conditions the decrease of the SO amount was much more important compared to the formation of SO oxides showing that many other products are formed. In contrast to heating, sunlight radiation did not result in the degradation of SO and very few oxides were formed.     

The accessory subunit of mitochondrial DNA polymerase γ determines the DNA content of mitochondrial nucleoids in human cultured cells

The accessory subunit of mitochondrial DNA polymerase γ, POLGβ, functions as a processivity factor in vitro. Here we show POLGβ has additional roles in mitochondrial DNA metabolism. Mitochondrial DNA is arranged in nucleoprotein complexes, or nucleoids, which often contain multiple copies of the mitochondrial genome. Gene-silencing of POLGβ increased nucleoid numbers, whereas over-expression of POLGβ reduced the number and increased the size of mitochondrial nucleoids. Both increased and decreased expression of POLGβ altered nucleoid structure and precipitated a marked decrease in 7S DNA molecules, which form short displacement-loops on mitochondrial DNA. Recombinant POLGβ preferentially bound to plasmids with a short displacement-loop, in contrast to POLGα. These findings support the view that the mitochondrial D-loop acts as a protein recruitment centre, and suggest POLGβ is a key factor in the organization of mitochondrial DNA in multigenomic nucleoprotein complexes.     

α-Synuclein modifies huntingtin aggregation in living cells

Several neurodegenerative disorders are characterized by the accumulation of proteinaceous inclusions in the central nervous system. These inclusions are frequently composed of a mixture of aggregation-prone proteins. Here, we used a bimolecular fluorescence complementation assay to study the initial steps of the co-aggregation of huntingtin (Htt) and α-synuclein (α-syn), two aggregation-prone proteins involved in Huntington's disease (HD) and Parkinson's disease (PD), respectively. We found that Htt (exon 1) oligomerized with α-syn and sequestered it in the cytosol. In turn, α-syn increased the number of cells displaying aggregates, decreased the number of aggregates per cell and increased the average size of the aggregates. Our results support the idea that co-aggregation of aggregation-prone proteins can contribute to the histopathology of neurodegenerative disorders.     

NMR detection of side chain–side chain hydrogen bonding interactions in 13C/15N-labeled proteins

We describe the direct observation of side chain–side chain hydrogen bonding interactions in proteins with sensitivity-enhanced NMR spectroscopy. Specifically, the remote correlation between the guanidinium nitrogen ¹⁵N of arginine 71, which serves as the hydrogen donor, and the acceptor carboxylate carbon ¹³CO₂ of aspartate 100 in a 12 kDa protein, human FKBP12, is detected via the trans-hydrogen bond ^3h J _{N CO2} coupling by employing a novel HNCO-type experiment, soft CPD-HNCO. The ^3h J _{N CO2} coupling constant appears to be even smaller than the average value of backbone ^3h J _NC couplings, consistent with more extensive local dynamics in protein side chains. The identification of trans-hydrogen bond J-couplings between protein side chains should provide useful markers for monitoring hydrogen bonding interactions that contribute to the stability of protein folds, to alignments within enzyme active sites and to recognition events at macromolecular interfaces.     

A new Rhodamine B-based ‘on–off’ chemical sensor with high selectivity and sensitivity toward Fe3+ and its imaging in living cells

A new fluorescent chemosensor based on a Rhodamine B and pyrrole conjugate (RBPY) has been designed and synthesized. UV–vis absorption and fluorescence spectroscopic studies show that RBPY exhibits a high selectivity and sensitivity toward Fe³⁺ among many other metal cations in a MeOH/H₂O solution (3:2, v/v, pH 7.10, HEPES buffer, 0.1 mM) by forming a 1:1 complex with Fe³⁺. Furthermore, results reveal that the formation of the RBPY–Fe³⁺ complex is fully reversible in the presence of sulfide anions and could also be used as an efficient sensor for S²⁻. Importantly, fluorescence microscopy experiments further demonstrated that RBPY can be utilized as a fluorescent probe for the detection of Fe³⁺ in human liver (L-02) cells.