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Sample records for subcellular distribution

  1. THE SUBCELLULAR DISTRIBUTION OF ANTIGEN IN MACROPHAGES

    PubMed Central

    Kölsch, E.; Mitchison, N. A.

    1968-01-01

    The intracellular fate of phagocytosed antigens in cells from peritoneal exudate in CBA mice has been studied by using 126I and 131I labeled antigens. After uptake of labeled antigen, cells were homogenized and the subcellular fractions were analyzed by isopycnic centrifugation in a sucrose gradient. The uptake of heat-denatured BSA (c BSA) by these cells in vivo is 3.5 µg/mg c BSA injected/108 cells. The uptake by cells in animals which were exposed 2 days earlier to 900 r whole body irradiation is slightly lower but does not differ significantly. 90% of the phagocytosed material is degraded within 2–3 hr, the residual 10% is retained at least over an 8 hr periods. Using a pulse and chase technique, with 125I and 131I c BSA in vitro and in vivo it was shown that newly phagocytosed antigen is found mainly in a lysosomal turnover compartment of a density 1.19 g cm–3. Antigen which has been in the cells for longer was found in a denser fraction (1.26 g cm–3). In a comparison of nhrmal and X-irradiated cells it can be shown that after irradiation with 900 r less c BSA is found in this storage compartment. Binding of the antigen to the subcellular fractions, and its behavior towards several detergents has been studied. Subcellular fractions do not have the increased immunogenic capacity of antigen enclosed in living macrophages. Two synthetic polypeptide antigens, poly(D-Tyr, D-Glu, D-Ala) and poly-(L-Tyr, L-Glu) have a different subcellular distribution from c BSA, BSA, or bovine gamma globulin. Apart from also being found in the 1.26 storage compartment the polypeptide antigens are mainly located in a 1.15 compartment and only to a small extent in the 1.19 compartment. The half-life of these antigens in the cells is much longer than the half-life of the protein antigens studied. The finding of several subcellular compartments is discussed in connection with the functions possibly performed by macrophages. PMID:5682940

  2. Subcellular distribution of potassium in striated muscles

    SciTech Connect

    Edelmann, L.

    1984-01-01

    Microanalytical experiments have been performed to answer the question whether the main cellular cation, K+, follows the water distribution in the striated muscle cell or whether K+ follows the distribution of negative fixed charges (beta- and gamma-carboxyl groups of aspartic and glutamic acid residues). Subcellular localization of K and/or of the K surrogates Rb, Cs, and Tl has been investigated by the following methods: Chemical precipitation of K with tetraphenylborate. Autoradiography of alkali-metals and Tl in air-dried and frozen-hydrated preparations. TEM visualization of electron dense Cs and Tl in sections of freeze-dried and plastic embedded muscle. X-ray microanalysis of air-dried myofibrils and muscle cryosections. The experiments consistently show that K, Rb, Cs, and Tl do not follow the water distribution but are mainly accumulated in the A band, especially in the marginal regions, and at Z lines. The same sites preferentially accumulate Cs or uranyl cations when sections of freeze-dried, embedded muscle are exposed to these electron microscopic stains. It is concluded that the detected uneven distribution of K, Rb, Cs, and Tl in muscle is neither a freeze-drying artifact nor an embedding artifact and may result from a weak ion binding to the beta- and gamma-carboxyl groups of cellular proteins.

  3. Subcellular distribution of phospholipids in different types of skeletal muscle.

    PubMed

    Domonkos, J; Heiner, L; Vargha, M

    1975-01-01

    Subcellular distribution of choline and non-choline phosphatides has been studied in tetanic (fast-twitch) and tonic (slow-twitch) muscles of rabbits. The choline phosphatide content of the subcellular fraction including the sarcolemma was greater in the tetanic than in the tonic muscle. On the other hand, the choline phosphatide content of the mitochondria-free sarcoplasmic fraction was greater in the tonic than in the tetanic muscle. A greater amount of non-choline phosphatide was found in each subcellular fraction of the tonic muscle as compared with those of the tetanic one. There was more fatty aldehyde in the non-choline phosphatides of each subcellular fraction of the tetanic muscle, than in those of the tonic one, of this type being much smaller in the tetanic muscle. There is not such an expressed difference in the fatty aldehyde contents of choline phosphatides of the subcellular fractions between the two kinds of muscle.

  4. Functional platform for controlled subcellular distribution of carbon nanotubes.

    PubMed

    Serag, Maged F; Kaji, Noritada; Venturelli, Enrica; Okamoto, Yukihiro; Terasaka, Kazuyoshi; Tokeshi, Manabu; Mizukami, Hajime; Braeckmans, Kevin; Bianco, Alberto; Baba, Yoshinobu

    2011-11-22

    As nanoparticles can cross different cellular barriers and access different tissues, control of their uptake and cellular fate presents a functional approach that will be broadly applicable to nanoscale technologies in cell biology. Here we show that the trafficking of single-walled carbon nanotubes (SWCNTs) through various subcellular membranes of the plant cell is facilitated or inhibited by attaching a suitable functional tag and controlling medium components. This enables a unique control over the uptake and the subcellular distribution of SWCNTs and provides a key strategy to promote their cellular elimination to minimize toxicity. Our results also demonstrate that SWCNTs are involved in a carrier-mediated transport (CMT) inside cells; this is a phenomenon that scientists could use to obtain novel molecular insights into CMT, with the potential translation to advances in subcellular nanobiology.

  5. Imaging trace element distributions in single organelles and subcellular features

    PubMed Central

    Kashiv, Yoav; Austin, Jotham R.; Lai, Barry; Rose, Volker; Vogt, Stefan; El-Muayed, Malek

    2016-01-01

    The distributions of chemical elements within cells are of prime importance in a wide range of basic and applied biochemical research. An example is the role of the subcellular Zn distribution in Zn homeostasis in insulin producing pancreatic beta cells and the development of type 2 diabetes mellitus. We combined transmission electron microscopy with micro- and nano-synchrotron X-ray fluorescence to image unequivocally for the first time, to the best of our knowledge, the natural elemental distributions, including those of trace elements, in single organelles and other subcellular features. Detected elements include Cl, K, Ca, Co, Ni, Cu, Zn and Cd (which some cells were supplemented with). Cell samples were prepared by a technique that minimally affects the natural elemental concentrations and distributions, and without using fluorescent indicators. It could likely be applied to all cell types and provide new biochemical insights at the single organelle level not available from organelle population level studies. PMID:26911251

  6. Imaging trace element distributions in single organelles and subcellular features

    NASA Astrophysics Data System (ADS)

    Kashiv, Yoav; Austin, Jotham R.; Lai, Barry; Rose, Volker; Vogt, Stefan; El-Muayed, Malek

    2016-02-01

    The distributions of chemical elements within cells are of prime importance in a wide range of basic and applied biochemical research. An example is the role of the subcellular Zn distribution in Zn homeostasis in insulin producing pancreatic beta cells and the development of type 2 diabetes mellitus. We combined transmission electron microscopy with micro- and nano-synchrotron X-ray fluorescence to image unequivocally for the first time, to the best of our knowledge, the natural elemental distributions, including those of trace elements, in single organelles and other subcellular features. Detected elements include Cl, K, Ca, Co, Ni, Cu, Zn and Cd (which some cells were supplemented with). Cell samples were prepared by a technique that minimally affects the natural elemental concentrations and distributions, and without using fluorescent indicators. It could likely be applied to all cell types and provide new biochemical insights at the single organelle level not available from organelle population level studies.

  7. Tissue and subcellular distribution of CLIC1

    PubMed Central

    Ulmasov, Barbara; Bruno, Jonathan; Woost, Philip G; Edwards, John C

    2007-01-01

    Background CLIC1 is a chloride channel whose cellular role remains uncertain. The distribution of CLIC1 in normal tissues is largely unknown and conflicting data have been reported regarding the cellular membrane fraction in which CLIC1 resides. Results New antisera to CLIC1 were generated and were found to be sensitive and specific for detecting this protein. These antisera were used to investigate the distribution of CLIC1 in mouse tissue sections and three cultured cell lines. We find CLIC1 is expressed in the apical domains of several simple columnar epithelia including glandular stomach, small intestine, colon, bile ducts, pancreatic ducts, airway, and the tail of the epididymis, in addition to the previously reported renal proximal tubule. CLIC1 is expressed in a non-polarized distribution in the basal epithelial cell layer of the stratified squamous epithelium of the upper gastrointesitinal tract and the basal cells of the epididymis, and is present diffusely in skeletal muscle. Distribution of CLIC1 was examined in Panc1 cells, a relatively undifferentiated, non-polarized human cell line derived from pancreatic cancer, and T84 cells, a human colon cancer cell line which can form a polarized epithelium that is capable of regulated chloride transport. Digitonin extraction was used to distinguish membrane-inserted CLIC1 from the soluble cytoplasmic form of the protein. We find that digitonin-resistant CLIC1 is primarily present in the plasma membrane of Panc1 cells. In T84 cells, we find digitonin-resistant CLIC1 is present in an intracellular compartment which is concentrated immediately below the apical plasma membrane and the extent of apical polarization is enhanced with forskolin, which activates transepithelial chloride transport and apical membrane traffic in these cells. The sub-apical CLIC1 compartment was further characterized in a well-differentiated mouse renal proximal tubule cell line. The distribution of CLIC1 was found to overlap that of

  8. Subcellular distribution of hydrolases in Naegleria fowleri.

    PubMed

    Lowrey, D M; McLaughlin, J

    1985-11-01

    The presence and particle association of various hydrolytic enzymes in Naegleria fowleri has been studied in whole cell extracts of trophozoites in an effort to establish authentic markers for surface membrane and lysosomal components. Evidence from the experiments reported here indicates that in N. fowleri a) acid proteinase, N-acetylglucosaminidase, and acid phosphatase are associated with cytoplasmic granules closely resembling lysosomes; b) 5'-nucleotidase is associated with the surface membrane, probably on the external surface; c) aspartate aminotransferase is associated with mitochondria; d) alpha-D-glucosidase and an aminopeptidase have bimodal distributions, activity being associated with both the surface membrane and lysosomal particles.

  9. Imaging trace element distributions in single organelles and subcellular features

    DOE PAGES

    Kashiv, Yoav; Austin, Jotham R.; Lai, Barry; Rose, Volker; Vogt, Stefan; El-Muayed, Malek

    2016-02-25

    The distributions of chemical elements within cells are of prime importance in a wide range of basic and applied biochemical research. An example is the role of the subcellular Zn distribution in Zn homeostasis in insulin producing pancreatic beta cells and the development of type 2 diabetes mellitus. We combined transmission electron microscopy with micro- and nano-synchrotron X-ray fluorescence to image unequivocally for the first time, to the best of our knowledge, the natural elemental distributions, including those of trace elements, in single organelles and other subcellular features. Detected elements include Cl, K, Ca, Co, Ni, Cu, Zn and Cdmore » (which some cells were supplemented with). Cell samples were prepared by a technique that minimally affects the natural elemental concentrations and distributions, and without using fluorescent indicators.We find it could likely be applied to all cell types and provide new biochemical insights at the single organelle level not available from organelle population level studies.« less

  10. Hepatic subcellular distribution of (tritium)T-2 toxin

    SciTech Connect

    Pace, J.G.; Watts, M.R.

    1989-01-01

    Hepatic subcellular distribution of ({sup 3}H)T-2 toxin. The subcellular distribution of T-2 mycotoxin and its metabolites was studied in isolated rat livers perfused with ({sup 3}H)T-2 toxin. After a 120-min perfusion, the distribution of radiolabel was to bile 53%, perfusate 38% and liver 7%. Livers were fractionated into mitochondria, endoplasmic reticulum (smooth and rough), plasma membrane and nuclei. Plasma membrane fractions contained 38% of the radiolabel within 5 min, decreasing to <1% at the end of the 120-min perfusion. Smooth endoplasmic reticulum contained 27% of the radiolabel by 5 min and increased to 43% over the 120-min perfusion. The mitochondrial fraction contained 3% of the radiolabel by 30 min and increased to 10% after 120-min perfusion. Label in the nuclear fraction remained constant at 7% from 30 to 120 min. By 15 min, only the parent toxin was detected in the mitochondrial fraction. In the other fractions, radiolabel was associated with HT-2, 4-deacetylneosolaniol, T-2 tetraol, and glucuronide conjugates. Glucuronide conjugates accounted for radiolabel eliminated via the bile. The time course for distribution of radiolabel in liver suggested an immediate association of ({sup 3}H)T-2 with plasma membranes and a subsequent association of toxin and metabolites with endoplasmic reticulum, mitochondria and nuclei, the known sites of action of this toxin.

  11. Subcellular distribution and chemical form of cadmium in bean plants

    SciTech Connect

    Weigel, H.J.; Jaeger, H.J.

    1980-03-01

    The subcellular distribution and chemical form of Cd in bean plants grown in nutrient solutions containing Cd were investigated. Cd was accumulated mainly in roots and to a minor extent in leaves. Subcellular fractionation of Cd-containing tissues (pH 7.5) showed that more than 70% of the element was localized in the cytoplasmic fraction in roots as well as in leaves. Little Cd (8 to 14%) was bound either to the cell wall fraction or to the organelles. Gel filtration of the soluble fraction showed Cd to be associated mainly with 5000 to 10,000 molecular weight components in roots, and 700 to 5000 molecular weight components in leaves. Small amounts of Cd were found in the high molecular weight proteins (molecular weight 150,000). Only traces of Cd could be detected as a free ion. Chemical characterization of the low molecular weight components resulted in the identification of nine amino acids which were identical in roots and leaves. Cd in bean plants is assumed to be bound to peptides and/or proteins of low molecular weight.

  12. Biochemical and subcellular distribution of arachidonic acid in rat myocardium

    SciTech Connect

    Miyazaki, Y.; Gross, R.W.; Sobel, B.E.; Saffitz, J.E. )

    1987-12-01

    Selective release of arachidonic acid from prelabeled phospholipid pools has been observed following exposure of neonatal rat cardiac myocytes to metabolic inhibitors in vitro and has been correlated temporally with the development of irreversible sarcolemmal damage. Hydrolysis of phospholipids with release of arachidonic acid may be an important mechanism in the pathogenesis of sarcolemmal damage induced by ischemia. To elucidate potential subcellular loci of arachidonic acid release in ischemic myocardium, the authors characterized the phospholipid composition of adult rat myocardial sarcolemma and delineated the biochemical and subcellular distribution of radiolabeled arachidonic acid in neonatal rat myocytes incubated with ({sup 3}H)-arachidonic acid for selected intervals. Radioactivity was located almost exclusively in mitochondria and internal cytoplasmic membranes (primarily sarcoplasmic reticulum), which collectively contained 90% of myocyte radioactivity. These results indicate that radiolabeled arachidonic acid released from prelabeled phospholipid pools on exposure of neonatal rat myocytes to oxidative inhibitors is derived from mitochondria and internal cell membranes. The diminutive labeling of the sarcolemma suggests that turnover of arachidonoyl phospholipids is slower in the sarcolemma than in other membranous organelles.

  13. Subcellular distribution and translocation of radionuclides in plants

    SciTech Connect

    Gouthu, S.; Weginwar, R.; Arie, Tsutomu; Ambe, Shizuko; Ozaki, Takuo; Enomoto, Shuichi; Ambe, Fumitoshi; Yamaguchi, Isamu

    1999-09-01

    The subcellular distribution of radionuclides in Glycine max Merr. (soybean) and Cucumis sativus L. (cucumber) and translocation of plant absorbed radionuclides with growth in soybean were studied. More than 60% of cellular incorporated Rb{sup {minus}83}, Sr{sup {minus}85}, Mn{sup {minus}54}, Nb{sup {minus}95}, and Se{sup {minus}75} remained in the supernatant fraction; 55% and 20% of Cr{sup {minus}51} was bound to soybean and cucumber cell wall fractions, respectively; 70% or more of Be{sup {minus}7}, Y{sup {minus}88}, and Fe{sup {minus}59} was fixed in the chloroplast fraction; and approx. 10% of Sc{sup {minus}46}, Fe{sup {minus}59}, V{sup {minus}48}, and As were fixed in the mitochondrial fraction. Translocation of nuclides within the soybean plant at different stages of growth has been determined. Vanadium, Y{sup {minus}88}, Be{sup {minus}7}, Se{sup {minus}75}, Nb{sup {minus}95}, Sc{sup {minus}46}, Cr{sup {minus}51}, and Zr{sup {minus}88} were predominantly accumulated in the root. Although the total percentage of plant uptake of Sc{sup {minus}46}, Zr{sup {minus}88}, Nb{sup {minus}95}, Sc{sup {minus}46}, and Cr{sup {minus}51} was high, because of low mobility and translocation to shoot, their accumulation in the fruit fraction was negligible. The translocation of mobile nuclides in plants was demonstrated clearly by Rb{sup {minus}83}, Zn{sup {minus}65}, and Fe{sup {minus}59}. Data on the nuclide fraction mobilized from vegetative parts into edible parts was used to assess the percentage of accumulated radionuclides in plants that may reach humans through beans.

  14. Polyprenols of Aspergillus niger. Their characterization, biosynthesis and subcellular distribution.

    PubMed

    Barr, R M; Hemming, F W

    1972-03-01

    A polyprenol complex of Aspergillus niger was shown, by using spectrometric methods, to consist of a family of exo-methylene-hexahydroprenols that contain between 18 and 24 isoprene residues per molecule. Each prenol contains two trans residues, three saturated residues (alpha, omega and psi) and an exo-methylene substituent on the carbon atom beta to the isopropyl group in each omega-residue. The ubiquinone complex consisted of 90% ubiquinone-9, 9% ubiquinone-8 and 1% ubiquinone-10. The amount of polyprenol complex present reached a maximum of 1.7mg/culture bottle after 9-10 days of growth, coincident with the maximum weight of mycelium. The amount of ergosterol (10mg/culture bottle) and ubiquinone (1mg/culture bottle) reached a peak at 8 days. By the 13th day of growth the yield of ergosterol had fallen by 20% and that of ubiquinone by 85%. A study of the incorporation of [2-(14)C]mevalonate over different time-intervals confirmed that there was a slow turnover of prenol, a more rapid turnover of ergosterol and a very rapid turnover of ubiquinone. At any one time each member of the prenol complex had essentially the same specific radioactivity as other members of the complex. A similar conclusion was made about the ubiquinone mixture. Just over half of the polyprenol present was esterified to fatty acids. Subcellular fractionation studies indicated that the unesterified prenol is associated primarily with a mitochondrial fraction, whereas the ester is more widely distributed.

  15. The Subcellular Distribution of Small Molecules: from Pharmacokinetics to Synthetic Biology

    PubMed Central

    Zheng, Nan; Tsai, Hobart Ng; Zhang, Xinyuan; Rosania, Gus R.

    2011-01-01

    The systemic pharmacokinetics and pharmacodynamics of small molecules are determined by subcellular transport phenomena. Although approaches used to study the subcellular distribution of small molecules have gradually evolved over the past several decades, experimental analysis and prediction of cellular pharmacokinetics remains a challenge. In this article, we surveyed the progress of subcellular distribution research since the 1960s, with a focus on the advantages, disadvantages and limitations of the various experimental techniques. Critical review of the existing body of knowledge pointed to many opportunities to advance the rational design of organelle-targeted chemical agents. These opportunities include: 1) development of quantitative, nonfluorescence-based, whole cell methods and techniques to measure the subcellular distribution of chemical agents in multiple compartments; 2) exploratory experimentation with nonspecific transport probes that have not been enriched with putative, organelle-targeting features; 3) elaboration of hypothesis-driven, mechanistic and modeling-based approaches to guide experiments aimed at elucidating subcellular distribution and transport; and 4) introduction of revolutionary conceptual approaches borrowed from the field of synthetic biology combined with cutting edge experimental strategies. In our laboratory, state-of-the-art subcellular transport studies are now being aimed at understanding the formation of new intracellular membrane structures in response to drug therapy, exploring the function of drug-membrane complexes as intracellular drug depots, and synthesizing new organelles with extraordinary physical and chemical properties. PMID:21805990

  16. Relationship between subcellular cadmium distribution in prey and cadmium trophic transfer to a predator

    USGS Publications Warehouse

    Wallace, W.G.; Lopez, G.R.

    1996-01-01

    We tested the hypothesis that exposure-related alterations in the subcellular Cd distribution in prey relate to changes in Cd absorption by a predator. Oligochaete worms, Limnodrilus hoffmeisteri were exposed for 1 wk or 6 wk to 0.5 ??g Cd l-1, 47 ??g l-1, or 140 ??g Cd l-1 (including 109Cd as a tracer) and relationship between oligochaete subcellular Cd distribution and Cd absorption by a predator, the grass shrimp (Palaemonetes pugio), were determined. Concentration and duration of Cd exposure had direct effects on oligochaete subcellular Cd distribution. Changes in oligochaete subcellular Cd distribution were characterized by increases in both the amount and proportion of Cd bound to the cytosolic fraction. The induction of Cd-binding proteins (e.g., metallothioneins) were suspected to be responsible for these changes. We found 1:1 relationships between the amount and percentage of Cd in oligochaete cytosol and the amount and percentage of Cd absorbed by shrimp. These results demonstrate that only metal bound to the soluble fraction of prey is available to higher trophic levels, and that factors influencing subcellular metal distribution in prey will directly alter metal trophic transfer to predators.

  17. Characteristic subcellular distribution, in brain, heart and lung, of biperiden, trihexyphenidyl, and (-)-quinuclidinyl benzylate in rats.

    PubMed

    Ishizaki, J; Yokogawa, K; Nakashima, E; Ohkuma, S; Ichimura, F

    1998-01-01

    The subcellular distribution of biperiden (BP), trihexyphenidyl (TP) and (-)-quinuclidinyl benzylate (QNB) in brain, heart and lung following high dose (3.2 mg/kg) i.v. administration was investigated in rats. The subcellular distribution of BP or TP used clinically conformed with that of QNB, a typical potent central muscarinic antagonist. The concentration-time courses of the brain subcellular fractions for these drugs were of two types which decreased slowly and in parallel to the plasma concentration. The subcellular distribution in the brain and heart was dependent on the protein amount of each fraction. The percent post-nuclear fraction (P2) of the total concentration in the lung was characteristically about 3-5 times larger than that in the heart. It was elucidated that the distribution in the lung differs from that in the brain and heart, with high affinity which is not dependent on the protein amount in the P2 fraction containing lysosomes. On the other hand, at a low dose (650 ng/kg) of 3H-QNB, each fraction as a percentage of the total concentration in the brain increased in synaptic membrane and synaptic vesicles and decreased in nuclei and cytosol as compared with the high dose. These results show that although the tissue concentration-time courses of anticholinergic drugs appear to decrease simply in parallel to plasma concentration, the subcellular distribution exhibits a variety of patterns among various tissues.

  18. Quantitative Analysis of Subcellular Distribution of the SUMO Conjugation System by Confocal Microscopy Imaging.

    PubMed

    Mas, Abraham; Amenós, Montse; Lois, L Maria

    2016-01-01

    Different studies point to an enrichment in SUMO conjugation in the cell nucleus, although non-nuclear SUMO targets also exist. In general, the study of subcellular localization of proteins is essential for understanding their function within a cell. Fluorescence microscopy is a powerful tool for studying subcellular protein partitioning in living cells, since fluorescent proteins can be fused to proteins of interest to determine their localization. Subcellular distribution of proteins can be influenced by binding to other biomolecules and by posttranslational modifications. Sometimes these changes affect only a portion of the protein pool or have a partial effect, and a quantitative evaluation of fluorescence images is required to identify protein redistribution among subcellular compartments. In order to obtain accurate data about the relative subcellular distribution of SUMO conjugation machinery members, and to identify the molecular determinants involved in their localization, we have applied quantitative confocal microscopy imaging. In this chapter, we will describe the fluorescent protein fusions used in these experiments, and how to measure, evaluate, and compare average fluorescence intensities in cellular compartments by image-based analysis. We show the distribution of some components of the Arabidopsis SUMOylation machinery in epidermal onion cells and how they change their distribution in the presence of interacting partners or even when its activity is affected. PMID:27424751

  19. How to unveil self-quenched fluorophores and subsequently map the subcellular distribution of exogenous peptides

    PubMed Central

    Swiecicki, Jean-Marie; Thiebaut, Frédéric; Di Pisa, Margherita; Gourdin -Bertin, Simon; Tailhades, Julien; Mansuy, Christelle; Burlina, Fabienne; Chwetzoff, Serge; Trugnan, Germain; Chassaing, Gérard; Lavielle, Solange

    2016-01-01

    Confocal laser scanning microscopy (CLSM) is the most popular technique for mapping the subcellular distribution of a fluorescent molecule and is widely used to investigate the penetration properties of exogenous macromolecules, such as cell-penetrating peptides (CPPs), within cells. Despite the membrane-association propensity of all these CPPs, the signal of the fluorescently labeled CPPs did not colocalize with the plasma membrane. We studied the origin of this fluorescence extinction and the overall consequence on the interpretation of intracellular localizations from CLSM pictures. We demonstrated that this discrepancy originated from fluorescence self-quenching. The fluorescence was unveiled by a “dilution” protocol, i.e. by varying the ratio fluorescent/non-fluorescent CPP. This strategy allowed us to rank with confidence the subcellular distribution of several CPPs, contributing to the elucidation of the penetration mechanism. More generally, this study proposes a broadly applicable and reliable method to study the subcellular distribution of any fluorescently labeled molecules. PMID:26839211

  20. Canine submandibular-gland hyaluronidase. Identification and subcellular distribution.

    PubMed

    Tan, Y H; Bowness, J M

    1968-11-01

    1. Submandibular glands from four species of mammal have been shown to contain a hyaluronidase active at acid pH; glands from dog and cat had a much higher content of this enzyme than has been found in other sources. 2. Product formation from hyaluronate after 24hr. incubation was almost the same as with testicular hyaluronidase, indicating that the enzyme is an endo-poly-beta-hexosaminidase. 3. When submandibular-gland homogenates were fractionated by the scheme developed for liver by de Duve, Pressman, Gianetto, Wattiaux & Appelmans (1955), all the enzymes assayed, except cytochrome c oxidase, were found to occur partly in the soluble fraction and partly in the particulate fractions. Among the particular fractions, the highest specific activity was found in the heavy-mitochondrial fraction for cytochrome c oxidase, in the microsomal fraction for alkaline phosphatase and in the light-mitochondrial fraction for acid phosphatase, beta-N-acetylhexosaminidase and acid-active hyaluronidase. 4. Release of the enzyme activity from the sedimentable fractions occurred in 0.1% Triton X-100 or after high-speed homogenization. 5. Stimulation of dogs by pilocarpine was found to decrease the hyaluronidase content of the submandibular gland by 5% and to cause the occurrence of a corresponding amount of acid-active hyaluronidase in the submandibular saliva. 6. The results are discussed in relation to the subcellular localization of hyaluronidase. PMID:4301907

  1. Toxicity and subcellular distribution of cadmium in wheat as affected by dissolved organic acids.

    PubMed

    Li, Dandan; Zhou, Dongmei

    2012-01-01

    We aim to investigate the effects of humic acid (HA) and citric acid (CA) on the toxicity and subcellular distribution of Cd in wheat. Results show that the toxicity and uptake of Cd decreased with increasing HA. The EC50 values of Cd increased from 3.36 micromol/L to 4.96 and 7.33 micromol/L at 50 and 250 mg/L HA, respectively, but decreased to 1.39 micromol/L in the presence of CA based on free ion activity model (FIAM). HA decreased the relative subcellular distribution of Cd in the heat-denatured proteins (decreased from 54% to 33%) but increased Cd in the heat-stable proteins in root (from 25% to 50%) at 7.61 micromol/L {Cd2+} (free Cd activity), which resulted in decreasing Cd toxicity. However, CA increased Cd toxicity due to the increased internalization of Cd although the relative subcellular distributions of Cd exhibited a decrease in the heat-denatured proteins and increase in the granule fraction compared to the control at high-level Cd. The FIAM could not predict the toxicity of Cd in the presence of organic acids. Alternatively, the internal Cd accumulation and subcellular Cd concentration were better to describe the toxicity of Cd to wheat.

  2. The distribution, abundance and subcellular localization of kinesin

    PubMed Central

    1989-01-01

    An antiserum which binds kinesin specifically on Western blots was used to determine the distribution and abundance of chicken kinesin by correlated immunoblotting and immunolocalization. Quantitative immunoblotting showed that the abundance of kinesin varied widely in different cell and tissue types, from 0.039% of total protein in epidermal fibroblasts to 0.309% in sympathetic neurons; of the types examined, only red blood cells lacked detectable kinesin. The molar ratio of tubulin/kinesin varied over a narrower range. To analyze the intracellular distribution of kinesin, cultured fibroblasts were fractionated by sequential extraction with saponin-, Triton X-100-, and SDS-containing buffer. Quantitative blotting of the resulting cell fractions indicated that 68% of fibroblast kinesin is in soluble form, 32% is membrane- or organelle-associated, and none is detectable in cytoskeletal fractions. To visualize this distribution, cells treated by the same extraction protocol were immunofluorescently stained with antikinesin and antitubulin. Without extraction, kinesin staining was located throughout cultured neurons and fibroblasts. However, when fibroblasts were extracted with saponin or Brij 58 before fixation, subsequent staining revealed that the remaining kinesin fraction was colocalized with interphase microtubules, but not with mitotic spindles. Prefixation extraction with Triton abolished antikinesin staining. These data suggest that kinesin may play a role in tubovesicular movement but provide no evidence for a role in mitosis. PMID:2525563

  3. Calcium: Some aspects of subcellular accumulation and distribution in milk

    SciTech Connect

    Shappell, N.W.

    1989-01-01

    Distribution and bioavailability of {sup 47}calcium in milk labeled by extrinsic and intrinsic methods was investigated. Milk from Sprague Dawley rats was labeled by both methods, and milk from a cow was labeled by the extrinsic method. Retention of {sup 47}Ca from milks administered to young male Sprague Dawley rats was determined through whole body counting for 6 days after administration of milk. Percent of {sup 47}Ca dose retained was 72% for extrinsically labeled cow milk, 62% for extrinsically labeled rat milk, and 55% for intrinsically labeled rat milk. Samples were fractionated by ultracentrifugation and by gel exclusion chromatography. {sup 47}Calcium distributions in rat milk labeled intrinsically or extrinsically were similar. The majority of {sup 47}Ca was found in a particulate, >30,000 molecular weight fraction. The amount of milk calcium retained by rats appeared to be related to the amount of noncasein micelle-associated calcium. When administered by intraperitoneal injection into rats, {sup 45}Ca specific activity of milk peaked in 60 to 90 minutes. In vitro {sup 45}Ca accumulation was compared in Golgi apparatus and endoplasmic reticulum from liver and mammary gland of lactating Dunkin Hartley guinea pigs. In the presence of ATP, highest accumulation per unit total fraction protein was found in Golgi apparatus (mammary gland 28% of available {sup 45}Ca, liver 11%) while 8% was accumulated by endoplasmic reticulum fractions.

  4. Subcellular distribution of jacalin in Artocarpus integrifolia seed.

    PubMed

    Majumder, M; Chatterjee, B P

    1996-01-01

    Seeds of Artocarpus integrifolia (jack fruit) contain large amounts of the anti-T lectin, jacalin. The mature seeds of jack fruit were homogenized in 0.25 M sucrose and separated by differential centrifugation into four fractions, viz wall, intermediate, and microsomal pellets and soluble supernatant. The lectin activity was associated with the wall pellet collected at low speed centrifugation. The other three fractions obtained by centrifugation at gradually higher speeds contained a similar lectin but of very low specific activity. The distribution pattern of jacalin remained unchanged in the presence of EDTA and/or Triton X-100 indicating that the lectin was not membrane bound. Immunofluorescent staining of jack fruit seeds showed that jacalin was localized in the cell wall in the intracellular space, which corroborated the results of fractionation studies. The possible relevance of these results to the function of lectin in the plant cell is discussed.

  5. Immunohistochemical distribution and electron microscopic subcellular localization of the proteasome in the rat CNS.

    PubMed

    Mengual, E; Arizti, P; Rodrigo, J; Giménez-Amaya, J M; Castaño, J G

    1996-10-15

    The proteasome multicatalytic proteinase (MCP) is a 20S complex that plays a major role in nonlysosomal pathways of intracellular protein degradation. A polyclonal antibody against rat liver MCP was used to investigate the distribution of MCP in the CNS of the rat and its subcellular localization within the neurons. As expected, MCP immunoreactivity (MCP-IR) was distributed ubiquitously in the rat CNS but not homogeneously. The most intensely stained neurons were the pyramidal cortical neurons of layer 5 and the motor neurons of the ventral horn in the spinal cord, which show an intense nuclear and cytoplasmatic MCP-IR and clearly stained processes. Additionally, some populations of large neurons in the mesencephalon and brainstem also displayed a moderate MCP-IR in their perikarya. The vast majority of neurons in the remaining structures did not show a strong cytoplasmatic MCP-IR, but their nuclei displayed an intense MCP-IR. The subcellular localization also was studied by immunoelectron microscopy. MCP-IR was intense in the neuronal nuclei, and significant staining also was found in the cytoplasm, dendritic, and axonic processes (including some myelinated axons) and in synaptic boutons, as illustrated in the cerebellar cortex. The distribution of MCP in the rat CNS and its subcellular localization are discussed in relation to (1) the distribution of calpain, the other major nonlysosomal cellular protease, and (2) the possible role of MCP in the degradation of regulatory proteins and key transcription factors that are essential in many neuronal responses.

  6. Kandelia obovata (S., L.) Yong tolerance mechanisms to Cadmium: subcellular distribution, chemical forms and thiol pools.

    PubMed

    Weng, Bosen; Xie, Xiangyu; Weiss, Dominik J; Liu, Jingchun; Lu, Haoliang; Yan, Chongling

    2012-11-01

    In order to explore the detoxification mechanisms adopted by mangrove under cadmium (Cd) stress, we investigated the subcellular distribution and chemical forms of Cd, in addition to the change of the thiol pools in Kandelia obovata (S., L.) Yong, which were cultivated in sandy culture medium treated with sequential Cd solution. We found that Cd addition caused a proportional increase of Cd in the organs of K. obovata. The investigation of subcellular distribution verified that most of the Cd was localized in the cell wall, and the lowest was in the membrane. Results showed sodium chloride and acetic acid extractable Cd fractions were dominant. The contents of non-protein thiol compounds, Glutathione and phytochelatins in K. obovata were enhanced by the increasing strength of Cd treatment. Therefore, K. obovata can be defined as Cd tolerant plant, which base on cell wall compartmentalization, as well as protein and organic acids combination.

  7. Subcellular distribution of zinc in Daphnia magna and implication for toxicity.

    PubMed

    Wang, Wen-Xiong; Guan, Rui

    2010-08-01

    We examined the subcellular partitioning of zinc (Zn) in Daphnia magna both under acute and chronic exposures. In the acute Zn toxicity tests, the daphnids were exposed to different Zn concentrations for 48 h or to one lethal concentration (1,000 microg/L) for different durations (time to death for up to 47 h). Significant mortality of daphnids was observed when the newly accumulated Zn concentration reached a threshold level of approximately 40 microg/g wet weight (or 320 microg/g dry wt), approximately 3.5 times higher than the background tissue concentration (92 microg/g dry wt). Chronic exposure (14 d) to Zn resulted in nonobservable effect on survivorship and growth at newly accumulated tissue concentration of over 40 microg/g wet weight. With increasing Zn acute exposure, more Zn was partitioned into the cellular debris fraction, indicating that this fraction was presumably the first targeted site of binding for Zn upon entering the animals. The importance of other subcellular fractions either decreased accordingly or remained comparable. We found that the metal-sensitive fraction (Zn distribution in the organelles and heat-denatured proteins) did not predict the acute Zn toxicity in Daphnia. During chronic exposure, however, no major change of the subcellular partitioning of Zn with increasing Zn exposure was documented. Zinc was mainly found in the organelles and heat-stable protein fractions during chronic exposure, suggesting that any subcellular repartitioning occurred primarily during acute exposure. Metallothioneins were induced upon chronic Zn exposure, but its induction evidently lagged behind the Zn accumulation. Our present study showed that the subcellular fractionation approach could not be readily used to predict the acute and chronic toxicities of Zn in Daphnia. A tissue-based Zn accumulation approach with a threshold Zn tissue concentration was better in predicting acute Zn toxicity. PMID:20821640

  8. High Accumulation and Subcellular Distribution of Thallium in Green Cabbage (Brassica Oleracea L. Var. Capitata L.).

    PubMed

    Ning, Zengping; He, Libin; Xiao, Tangfu; Márton, László

    2015-01-01

    The accumulation of thallium (Tl) in brassicaceous crops is widely known, but both the uptake extents of Tl by the individual cultivars of green cabbage and the distribution of Tl in the tissues of green cabbage are not well understood. Five commonly available cultivars of green cabbage grown in the Tl-spiked pot-culture trials were studied for the uptake extent and subcellular distribution of Tl. The results showed that all the trial cultivars mainly concentrated Tl in the leaves (101∼192 mg/kg, DW) rather than in the roots or stems, with no significant differences among cultivars (p = 0.455). Tl accumulation in the leaves revealed obvious subcellular fractionation: cell cytosol and vacuole > cell wall > cell organelles. The majority (∼ 88%) of leaf-Tl was found to be in the fraction of cytosol and vacuole, which also served as the major storage site for other major elements such as Ca and Mg. This specific subcellular fractionation of Tl appeared to enable green cabbage to avoid Tl damage to its vital organelles and to help green cabbage tolerate and detoxify Tl. This study demonstrated that all the five green cabbage cultivars show a good application potential in the phytoremediation of Tl-contaminated soils.

  9. Phosphoenolpyruvate carboxykinase of kidney. Subcellular distribution and response to acid–base changes

    PubMed Central

    Flores, H.; Alleyne, G. A. O.

    1971-01-01

    1. A method for the assay of phosphoenolpyruvate carboxykinase is presented, based on the enzymic determination of the phosphoenolpyruvate produced by the enzyme reaction. 2. The subcellular distribution of phosphoenolpyruvate carboxykinase in the kidney of several animal species resembled the distribution in the liver. 3. The rise in enzyme activity in the kidney cortex of rats made acidotic by feeding with ammonium chloride was not prevented by administration of ethionine or actinomycin. 4. The possibility is suggested that in the kidney acidosis causes activation of an inactive form of the enzyme already present. PMID:5128664

  10. Subcellular distribution of azithromycin and clarithromycin in rat alveolar macrophages (NR8383) in vitro.

    PubMed

    Togami, Kohei; Chono, Sumio; Morimoto, Kazuhiro

    2013-01-01

    Azithromycin (AZM), a 15-membered ring macrolide antimicrobial agent, has an antibacterial spectrum that includes intracellular parasitic pathogens that survive or intracellularly multiply in alveolar macrophages (AMs). The subcellular distribution of AZM in AMs was evaluated in vitro in comparison with clarithromycin (CAM). AZM and CAM (50 µM) were applied to the NR8383 cells, used as an in vitro model of AMs, followed by incubation at 37°C or 4°C. The total amount of AZM in cells and subcellular distribution (cell fractionation) was determined after incubation. High level of AZM accumulation was observed in the NR8383 cells at 37°C, and the equilibrium intracellular to extracellular concentration ratio (I/E ratio) was approximately 680, which was remarkably higher than that of CAM (equilibrium I/E ratio=28). The intracellular accumulation of AZM and CAM was temperature dependent. In addition, AZM distributed to the granules fraction including organelles and soluble fraction including cytosol in the NR8383 cells, whereas CAM mainly distributed in soluble fraction. The amount of AZM in the granules fraction was markedly reduced in the presence of ammonium chloride for increase in intracellular pH. These results indicate that AZM is distributed in acidic compartment in AMs. This study suggests that high AZM accumulation in the NR8383 cells is due to the trapping and/or binding in acidic organelles, such as lysosomes.

  11. Subcellular distribution and chemical forms of cadmium in a dark septate endophyte (DSE), Exophiala pisciphila.

    PubMed

    Zhan, Fangdong; He, Yongmei; Li, Yuan; Li, Tao; Yang, Yun-Ya; Toor, Gurpal S; Zhao, Zhiwei

    2015-11-01

    Our objective was to understand the cadmium (Cd) tolerance mechanisms by investigating the subcellular distribution, chemical forms of Cd and adsorptive groups in the mycelia of Exophiala pisciphila. We grew E. pisciphila in the liquid media with increasing Cd concentrations (0, 25, 50, 100, 200, and 400 mg L(-1)). Increased Cd in the media caused a proportional increase in the Cd uptake by E. pisciphila. Subcellular distribution indicated that 81 to 97% of Cd was associated with the cell walls. The largest amount and proportion (45-86%) of Cd was extracted with 2% acetic acid, and a concentration-dependent extraction was observed, both of which suggest that Cd-phosphate complexes were the major chemical form in E. pisciphila. A large distribution of phosphate and Cd on the mycelia surface was observed by scanning electron microscopy-energy dispersive spectrometer (SEM-EDS). The precipitates associated with the mycelia were observed to contain Cd by transmission electron microscopy-energy dispersive X-ray spectroscopy (TEM-EDX). Fourier transform infrared (FTIR) identified that hydroxyl, amine, carboxyl, and phosphate groups were responsible for binding Cd. We conclude that Cd associated with cell walls and integrated with phosphate might be responsible for the tolerance of E. pisciphila to Cd. PMID:26165995

  12. Bioaccumulation, subcellular distribution, and acute effects of chromium in Japanese medaka (Oryzias latipes).

    PubMed

    Li, Lixia; Chen, Hongxing; Bi, Ran; Xie, Lingtian

    2015-11-01

    Chromium (Cr) is an essential element but is toxic to aquatic organisms at elevated concentrations. In the present study, adult Japanese medaka (Oryzias latipes) were exposed to a sublethal hexavalent chromium (Cr(VI)) concentration via dissolved and dietary exposures for 6 d. Various measurements of Cr were made: bioaccumulation in different tissues, subcellular distribution in the liver, effects on antioxidants and acetylcholinesterase (AChE), and Cr-induced lipid peroxidation. The results showed that bioaccumulation increased dramatically in all tested tissues from dissolved exposure but only significantly in the intestine from dietary treatment, implying that dissolved exposure may be predominant for Cr accumulation in medaka. Subcellular distribution revealed that Cr accumulated in the liver was mainly (46%) associated with the heat-stable protein fraction. Among the antioxidants examined, catalase (CAT) responded to dissolved Cr exposure in most tissues whereas superoxide dismutase (SOD) was less responsive. Malondialdehyde concentrations were significantly elevated in most tissues examined in the dissolved Cr-exposed fish, but were only elevated in the liver and intestine in the dietary Cr-exposed fish. The AChE activity in the brain was stimulated by 49% in the dissolved Cr-exposed fish. Reductions in condition factor and gonadosomatic index were also observed. These data help in an understanding of Cr tissue distribution and the acute effects of Cr in Japanese medaka.

  13. Do arbuscular mycorrhizal fungi affect cadmium uptake kinetics, subcellular distribution and chemical forms in rice?

    PubMed

    Li, Hui; Luo, Na; Zhang, Li Jun; Zhao, Hai Ming; Li, Yan Wen; Cai, Quan Ying; Wong, Ming Hung; Mo, Ce Hui

    2016-11-15

    Rice (Oryza sativa L.) plants were inoculated with two species of arbuscular mycorrhizal fungi (AMF) - Rhizophagus intraradices (RI) and Funneliformis mosseae (FM) and grown for 60days to ensure strong colonization. Subsequently, a short-term hydroponic experiment was carried out to investigate the effects of AMF on cadmium (Cd) uptake kinetics, subcellular distribution and chemical forms in rice exposed to six Cd levels (0, 0.005, 0.01, 0.025, 0.05, 0.1mM) for three days. The results showed that the uptake kinetics of Cd fitted the Michaelis-Menten model well (R(2)>0.89). AMF significantly decreased the Cd concentrations both in shoots and roots in Cd solutions. Furthermore, the decrement of Cd concentrations by FM was significantly higher than RI treatment in roots. AMF reduced the Cd concentrations markedly in the cell wall fractions at high Cd substrate (≥0.025mM). The main subcellular fraction contributed to Cd detoxification was cell wall at low Cd substrate (<0.05mM), while vacuoles at high Cd substrate (≥0.05mM). Moreover, the concentrations and proportions of Cd in inorganic and water-soluble form also reduced by AMF colonization at high Cd substrate (≥0.05mM), both in shoots and roots. This suggested that AMF could convert Cd into inactive forms which were less toxic. Therefore, AMF could enhance rice resistance to Cd through altering subcellular distribution and chemical forms of Cd in rice. PMID:27450963

  14. Comparison of subcellular distribution and chemical forms of cadmium among four soybean cultivars at young seedlings.

    PubMed

    Wang, Peng; Deng, Xiaojuan; Huang, Yian; Fang, Xiaolong; Zhang, Jie; Wan, Haibo; Yang, Cunyi

    2015-12-01

    The hydroponic experiment was carried out to investigate the Cd subcellular distribution and chemical forms in roots and shoots among four soybean seedling cultivars with two Cd treatments. HX3 and GC8, two tolerant and low-grain-Cd-accumulating cultivars, had the lowest Cd concentration in roots and high Cd concentration in shoots, while BX10 and ZH24, two sensitive and high-grain-Cd-accumulating cultivars, had the highest Cd concentration in roots and the lowest Cd concentration in shoots at young seedling stage. Furthermore, the sequence of Cd subcellular distribution in roots at two Cd levels was cell wall (53.4-75.5 %) > soluble fraction (15.8-40.4 %) > organelle fraction (2.0-14.7 %), but in shoots, was soluble fraction (39.3-74.8 %) > cell wall (16.0-52.0 %) > organelle (4.8-19.5 %). BX10 and ZH24 had higher Cd concentration in all subcellular fractions in roots, but HX3 and GC8 had higher Cd concentration of soluble fraction in shoots. The sequence of Cd chemical forms in roots was FNacl (64.1-79.5 %) > FHAC (3.4-21.5 %) > Fd-H2O (3.6-13.0 %) > Fethanol (1.4-21.8) > FHCl (0.3-1.6 %) > Fother (0.2-1.4 %) at two Cd levels but, in shoots, was FNacl (19.7-51.4 %) ≥ FHAC (10.2-31.4 %) ≥ Fd-H2O (8.8-28.2 %) ≥ Fethanol (8.9-38.6 %) > FHCl (0.2-9.6 %) > Fother (2.5-11.2 %). BX10 and ZH24 had higher Cd concentrations in each extracted solutions from roots, but from shoots for GC8 and HX3. Taken together, the results uncover that root cell walls and leaf vacuoles might play important roles in Cd detoxification and limiting the symplastic movement of Cd. PMID:26272289

  15. Do arbuscular mycorrhizal fungi affect cadmium uptake kinetics, subcellular distribution and chemical forms in rice?

    PubMed

    Li, Hui; Luo, Na; Zhang, Li Jun; Zhao, Hai Ming; Li, Yan Wen; Cai, Quan Ying; Wong, Ming Hung; Mo, Ce Hui

    2016-11-15

    Rice (Oryza sativa L.) plants were inoculated with two species of arbuscular mycorrhizal fungi (AMF) - Rhizophagus intraradices (RI) and Funneliformis mosseae (FM) and grown for 60days to ensure strong colonization. Subsequently, a short-term hydroponic experiment was carried out to investigate the effects of AMF on cadmium (Cd) uptake kinetics, subcellular distribution and chemical forms in rice exposed to six Cd levels (0, 0.005, 0.01, 0.025, 0.05, 0.1mM) for three days. The results showed that the uptake kinetics of Cd fitted the Michaelis-Menten model well (R(2)>0.89). AMF significantly decreased the Cd concentrations both in shoots and roots in Cd solutions. Furthermore, the decrement of Cd concentrations by FM was significantly higher than RI treatment in roots. AMF reduced the Cd concentrations markedly in the cell wall fractions at high Cd substrate (≥0.025mM). The main subcellular fraction contributed to Cd detoxification was cell wall at low Cd substrate (<0.05mM), while vacuoles at high Cd substrate (≥0.05mM). Moreover, the concentrations and proportions of Cd in inorganic and water-soluble form also reduced by AMF colonization at high Cd substrate (≥0.05mM), both in shoots and roots. This suggested that AMF could convert Cd into inactive forms which were less toxic. Therefore, AMF could enhance rice resistance to Cd through altering subcellular distribution and chemical forms of Cd in rice.

  16. Differential subcellular distribution of rat brain dopamine receptors and subtype-specific redistribution induced by cocaine

    PubMed Central

    Voulalas, Pamela J.; Schetz, John; Undieh, Ashiwel S.

    2011-01-01

    We investigated the subcellular distribution of dopamine D1, D2 and D5 receptor subtypes in rat frontal cortex, and examined whether psychostimulant-induced elevation of synaptic dopamine could alter the receptor distribution. Differential detergent solubilization and density gradient centrifugation were used to separate various subcellular fractions, followed by semi-quantitative determination of the relative abundance of specific receptor proteins in each fraction. D1 receptors were predominantly localized to detergent-resistant membranes, and a portion of these receptors also floated on sucrose gradients. These properties are characteristic of proteins found in lipid rafts and caveolae. D2 receptors exhibited variable distribution between cytoplasmic, detergent-soluble and detergent-resistant membrane fractions, yet were not present in buoyant membranes. Most D5 receptor immunoreactivity was distributed into the cytoplasmic fraction, failing to sediment at forces up to 300,000g, while the remainder was localized to detergent-soluble membranes in cortex. D5 receptors were undetectable in detergent-resistant fractions or raft-like subdomains. Following daily cocaine administration for seven days, a significant portion of D1 receptors translocated from detergent-resistant membranes to detergent-soluble membranes and the cytoplasmic fraction. The distributions of D5 and D2 receptor subtypes were not significantly altered by cocaine treatment. These data imply that D5 receptors are predominantly cytoplasmic, D2 receptors are diffusely distributed within the cell, whereas D1 receptors are mostly localized to lipid rafts within the rat frontal cortex. Dopamine receptor subtype localization is susceptible to modulation by pharmacological manipulations that elevate synaptic dopamine, however the functional implications of such drug-induced receptor warrant further investigation. PMID:21236347

  17. Subcellular distribution of mutant movement proteins of Cucumber mosaic virus fused to green fluorescent proteins.

    PubMed

    Canto, Tomas; Palukaitis, Peter

    2005-04-01

    The subcellular distribution of the movement proteins (MPs) of nine alanine-scanning mutants of Cucumber mosaic virus (CMV), fused to the green fluorescent protein (GFP) and expressed from CMV, was determined by confocal microscopy of infected epidermal cells of Nicotiana tabacum and Nicotiana benthamiana, as well as infected N. benthamiana protoplasts. Only those mutant MPs that were functional for movement in all host species tested localized to plasmodesmata of infected epidermal cells and to tubules extending from the surface of infected protoplasts, as for wild-type CMV 3a MP. Various mutant MPs that were either conditionally functional for movement or dysfunctional for movement did not localize to plasmodesmata and did not form tubules on the surface of infected protoplasts. Rather, they showed distribution to different extents throughout the infected cells, including the cytoplasm, nucleus or the plasma membrane. The CMV 3a MP also did not associate with microtubules.

  18. Mating changes the subcellular distribution and the functionality of estrogen receptors in the rat oviduct

    PubMed Central

    2009-01-01

    Background Mating changes the mode of action of 17beta-estradiol (E2) to accelerate oviductal egg transport from a nongenomic to a genomic mode, although in both pathways estrogen receptors (ER) are required. This change was designated as intracellular path shifting (IPS). Methods Herein, we examined the subcellular distribution of ESR1 and ESR2 (formerly known as ER-alpha and ER-beta) in oviductal epithelial cells of rats on day 1 of cycle (C1) or pregnancy (P1) using immunoelectron microscopy for ESR1 and ESR2. The effect of mating on intraoviductal ESR1 or ESR2 signaling was then explored comparing the expression of E2-target genes c-fos, brain creatine kinase (Ckb) and calbindin 9 kDa (s100g) in rats on C1 or P1 treated with selective agonists for ESR1 (PPT) or ESR2 (DPN). The effect of ER agonists on egg transport was also evaluated on C1 or P1 rats. Results Receptor immunoreactivity was associated with the nucleus, cytoplasm and plasma membrane of the epithelial cells. Mating affected the subcellular distribution of both receptors as well as the response to E2. In C1 and P1 rats, PPT increased Ckb while both agonists increased c-fos. DPN increased Ckb and s100g only in C1 and P1 rats, respectively. PPT accelerated egg transport in both groups and DPN accelerated egg transport only in C1 rats. Conclusion Estrogen receptors present a subcellular distribution compatible with E2 genomic and nongenomic signaling in the oviductal epithelial cells of C1 and P1 although IPS occurs independently of changes in the distribution of ESR1 and ESR2 in the oviductal epithelial cells. Mating affected intraoviductal ER-signaling and induced loss of functional involvement of ESR2 on E2-induced accelerated egg transport. These findings reveal a profound influence on the ER signaling pathways exerted by mating in the oviduct. PMID:19948032

  19. Subcellular distribution and chemical forms of cadmium in Phytolacca americana L.

    PubMed

    Fu, Xiaoping; Dou, Changming; Chen, Yingxu; Chen, Xincai; Shi, Jiyan; Yu, Mingge; Xu, Jie

    2011-02-15

    Phytolacca americana L. (pokeweed) is a promising species for Cd phytoextraction with large biomass and fast growth rate. To further understand the mechanisms involved in Cd tolerance and detoxification, the present study investigated subcellular distribution and chemical forms of Cd in pokeweed. Subcellular fractionation of Cd-containing tissues indicated that both in root and leaves, the majority of the element was located in soluble fraction and cell walls. Meanwhile, Cd taken up by pokeweed existed in different chemical forms. Results showed that the greatest amount of Cd was found in the extraction of 80% ethanol in roots, followed by 1 M NaCl, d-H(2)O and 2% HAc, while in leaves and stems, most of the Cd was extracted by 1 M NaCl, and the subdominant amount of Cd was extracted by 80% ethanol. It could be suggested that Cd compartmentation with organo-ligands in vacuole or integrated with pectates and proteins in cell wall might be responsible for the adaptation of pokeweed to Cd stress.

  20. Unveiling the mechanism of uptake and sub-cellular distribution of cerium oxide nanoparticles†

    PubMed Central

    Singh, Sanjay; Kumar, Amit; Karakoti, Ajay; Seal, Sudipta; Self, William T.

    2011-01-01

    Cerium oxide nanoparticles (CNPs) have been recently studied for their potent superoxide scavenging properties in both cell and animal model systems. Data from these model systems have shown that exposure of cells to CNPs results in the protection against reactive oxygen species (ROS). Despite these exciting findings, very little is known regarding the uptake or subcellular distribution of these nanomaterials inside cells. In this study we utilized fluorophore (carboxyfluorescein) conjugated cerium oxide NPs (CCNPs) to study the mechanism of uptake and to elucidate the subcellular localization of CNPs using a keratinocyte model system. We observed rapid uptake (within 3 h) of CCNPs that was governed by energy-dependent, clathrin-mediated and caveolae-mediated endocytic pathways. We found CCNPs co-localized with mitochondria, lysosomes and endoplasmic reticulum as well as being abundant in the cytoplasm and the nucleus. Given the radical scavenging properties of cerium oxide and the widespread cellular disposition we observed, CNPs likely act as cellular antioxidants in multiple compartments of the cell imparting protection against a variety of oxidant injuries. PMID:20697616

  1. Apolipoprotein D subcellular distribution pattern in neuronal cells during oxidative stress.

    PubMed

    Martínez-Pinilla, Eva; Navarro, Ana; Ordóñez, Cristina; del Valle, Eva; Tolivia, Jorge

    2015-07-01

    Apolipoprotein D (Apo D) is a secreted glycoprotein, member of the lipocalin superfamily, with a related beneficial role in metabolism and lipid transport due to the presence of a binding pocket that allows its interaction with several lipids. Nowadays, it has been clearly demonstrated that Apo D expression is induced and its subcellular location undergoes modifications in stressful and pathological conditions that characterize aging processes and neurodegenerative diseases. The aim of the present work was to study in detail the effect of H2O2 on the subcellular location of Apo D, in the hippocampal cell line HT22, by structural, ultrastructural, immunocytochemical, and molecular techniques in order to characterize the Apo D distribution pattern in neurons during oxidative stress. Our results indicate that Apo D is located in the cytoplasm under physiological conditions but treatment with H2O2 induces apoptosis and causes a displacement of Apo D location to the nucleus, coinciding with DNA fragmentation. In addition, we demonstrated that Apo D tends to accumulate around the nuclear envelope in neurons and glial cells of different brain areas in some neurodegenerative diseases and during human aging, but never inside the nucleus. These data suggest that the presence of Apo D in the nucleus, which some authors related with a specific transport, is a consequence of structural and functional alterations during oxidative stress and not the result of a specific role in the regulation of nuclear processes.

  2. Cadmium sensitivity, uptake, subcellular distribution and thiol induction in a marine diatom: exposure to cadmium.

    PubMed

    Wang, Meng-Jiao; Wang, Wen-Xiong

    2011-01-25

    The aims of this study were to (1) evaluate the changes in the Cd tolerance of a marine diatom after exposure under different Cd concentrations for various durations and (2) to explore the potential subcellular and biochemical mechanisms underlying these changes. The 72-h toxicity, short-term Cd uptake, subcellular Cd distribution, as well as the synthesis of phytochelatins (PCs) were measured in a marine diatom Thalassiosira nordenskioeldii after exposure to a range of free Cd ion concentrations ([Cd(2+)], 0.01-84nM) for 1-15 days. Surprisingly, the diatoms did not acquire higher resistance to Cd after exposure; instead their sensitivity to Cd increased with a higher exposed [Cd(2+)] and a longer exposure period. The underlying mechanisms could be traced to the responses of Cd cellular accumulation and the intrinsic detoxification ability of the preconditioned diatoms. Generally, exposure to a higher [Cd(2+)] and for a longer period increased the Cd uptake rate, cellular accumulation, as well as the Cd concentration in metal-sensitive fraction (MSF) in these diatoms. In contrast, although PCs were induced by the environmental Cd stress (with PC(2) being the most affected), the increased intracellular Cd to PC-SH ratio implied that the PCs' detoxification ability had reduced after Cd exposure. All these responses resulted in an elevated Cd sensitivity as exposed [Cd(2+)] and duration increased. This study shows that the physiological/biochemical and kinetic responses of phytoplankton upon metal exposure deserve further investigation.

  3. Changes in subcellular elemental distributions accompanying the acrosome reaction in sea urchin sperm

    SciTech Connect

    Cantino, M.E.; Schackmann, R.W.; Johnson, D.E.

    1983-05-01

    Energy-dispersive x-ray microanalysis was used to analyze changes in the subcellular distributions of Na, Mg, P, S, Cl, K, and Ca associated with the acrosome reaction of sea urchin sperm. Within 5 sec after induction of the acrosome reaction, nuclear Na and mitochondrial Ca increased and nuclear and mitochondrial K decreased. Uptake of mitochondrial P was detected after several minutes, and increases in nuclear Mg were detected only after 5-10 min of incubation following induction of the reaction. The results suggest that sudden permeability changes in the sperm plasma membrane are associated with the acrosome reaction, but that complete breakdown of membrane and cell function does not occur for several minutes.

  4. Subcellular distribution of technetium-99m-N-NOEt in rat myocardium

    SciTech Connect

    Uccelli, L.; Giganti, M.; Duatti, A. ||

    1995-11-01

    The aim of this study was to determine the subcellular distribution of bis(N-ethoxy N-ethyl)dithiocarbamato, nitrido technetium(V) ({sup 99m}TcN-NOEt) in rat heart by differential centrifugation techniques. Extraction of the activity from homogenized rat heart tissue was also performed to assess whether myocardial retention might induce changes in the chemical identity of the complex. Anesthetized rats were intravenously injected with {sup 99m}TcN-NOEt, the heart tissue was extracted and homogenized and tissue fractions were obtained by differential centrifugation. The efficiency of organelle separation was determined by assay of each centrifugal fraction using enzyme markers. Lactate dehydrogenase (LDH), acid phosphatase (ACP), alkaline phosphatase (ALP) and 5{prime}-nucleotidase (5{prime}ND) activities were assayed using standard spectrophotometric methods. Succinic dehydrogenase (SDH) activity was determined using a p-iodo-nitrotetrazolium-linked assay. Severe cell membrane and organelle disruption were induced by prolonging the homogenization time and their effect on the subcellular distribution of {sup 99m}TcN-NOEt was studied. The activity from homogenized heart tissue was extracted using the Folch technique and analyzed by TLC and HPLC. Most of the {sup 99}TcN-NOEt activity was found to be associated with the hydrophobic components of the cell. No evidence of specific association of activity with the cytosolic and mitochondrial components was observed. Organelle and membrane cleavage did not cause release of activity into the cytosol. Approximately 90% of {sup 99m}TcN-NOEt activity was extracted from ventricular tissue and the chemical nature of {sup 99m}TcN-NOEt was not altered by uptake by myocardium. Cell membranes are the most apparent site of localization of {sup 99m}TcN-NOEt in heart tissue. 21 refs., 4 figs., 1 tab.

  5. CHARACTERIZATION OF AMINOPEPTIDASE IN THE FREE-LIVING NEMATODE PANAGRELLUS REDIVIVUS: SUBCELLULAR DISTRIBUTION AND POSSIBLE ROLE IN NEUROPEPTIDE METABOLISM

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aminopeptidase was detected in homogenates of the free-living nematode Panagrellus redivivus with the aminoacyl substrate L-alanine-4-nitroanilide (Ala-4-NA). Subcellular distribution of the enzyme was unequal, with approximately 80 percent of total aminopeptidase in the soluble fraction and the rem...

  6. Subcellular distribution of folate and folate binding protein in renal proximal tubules

    SciTech Connect

    Sharkey, C.; Hjelle, J.T.; Selhub, J.

    1986-03-01

    High affinity folate binding protein (FBP) found in brush border membranes derived from renal cortices is thought to be involved in the renal conservation of folate. To examine the mechanisms of folate recovery, the subcellular distribution of FBP and /sup 3/H-folate in rabbit renal proximal tubules (PT) was examined using analytical cell fractionation techniques. Tubules contain 3.41 +/- 0.32 picomoles FBP/mg protein (X +/- S.D.; n = 5). Postnuclear supernates (PNS) of PT were layered atop Percoll-sucrose gradients, centrifuged, fractions collected and assayed for various marker enzymes and FBP. Pooled fractions from such gradients were subsequently treated with digitonin and centrifuged in a stoichiometric manner with the activity of the microvillar enzyme, alanylaminopeptidase (AAP); excess FBP distributed with more buoyant particles. Infusion of /sup 3/H-folate into rabbit kidneys followed by tubule isolation and fractionation revealed a time dependent shift in distribution of radiolabel from the AAP-rich gradient fractions to a region containing more buoyant particles; radiolevel was not associated with lysosomal markers. EM-radioautography revealed grains over intracellular vesicles. These results are consistent with the hypothesis that folate is recovered by a process involving receptor-mediated endocytosis or transcytosis.

  7. Silicon modifies root anatomy, and uptake and subcellular distribution of cadmium in young maize plants

    PubMed Central

    Vaculík, Marek; Landberg, Tommy; Greger, Maria; Luxová, Miroslava; Stoláriková, Miroslava; Lux, Alexander

    2012-01-01

    Background and Aims Silicon (Si) has been shown to ameliorate the negative influence of cadmium (Cd) on plant growth and development. However, the mechanism of this phenomenon is not fully understood. Here we describe the effect of Si on growth, and uptake and subcellular distribution of Cd in maize plants in relation to the development of root tissues. Methods Young maize plants (Zea mays) were cultivated for 10 d hydroponically with 5 or 50 µm Cd and/or 5 mm Si. Growth parameters and the concentrations of Cd and Si were determined in root and shoot by atomic absorption spectrometry or inductively coupled plasma mass spectroscopy. The development of apoplasmic barriers (Casparian bands and suberin lamellae) and vascular tissues in roots were analysed, and the influence of Si on apoplasmic and symplasmic distribution of 109Cd applied at 34 nm was investigated between root and shoot. Key Results Si stimulated the growth of young maize plants exposed to Cd and influenced the development of Casparian bands and suberin lamellae as well as vascular tissues in root. Si did not affect the distribution of apoplasmic and symplasmic Cd in maize roots, but considerably decreased symplasmic and increased apoplasmic concentration of Cd in maize shoots. Conclusions Differences in Cd uptake of roots and shoots are probably related to the development of apoplasmic barriers and maturation of vascular tissues in roots. Alleviation of Cd toxicity by Si might be attributed to enhanced binding of Cd to the apoplasmic fraction in maize shoots. PMID:22455991

  8. Subcellular distribution and chemical forms of thorium in Brassica juncea var. foliosa.

    PubMed

    Zhou, Sai; Kai, Hailu; Zha, Zhongyong; Fang, Zhendong; Wang, Dingna; Du, Liang; Zhang, Dong; Feng, Xiaojie; Jin, Yongdong; Xia, Chuanqin

    2016-06-01

    Brassica juncea var. foliosa (B. juncea var. foliosa) is a promising species for thorium (Th) phytoextraction due to its large biomass, fast growth rate and high tolerance toward Th. To further understand the mechanisms of Th tolerance, the present study investigated the subcellular distribution and chemical forms of Th found in B. juncea var. foliosa Our results indicated that in both roots and leaves, Th contents in different parts of the cells follow the order of cell wall > membranes and soluble fraction > organelles. In particular, Transmission Electron Microscope (TEM) analysis showed that Th was abundantly located in cell walls of the roots. Additionally, when plants were exposed to different concentrations of Th, we have found that Th existed in B. juncea var. foliosa with different chemical forms. Much of the Th extracted by 2% acetic acid (HAc), 1 M NaCl and HCl in roots with the percentage distribution varied from 47.2% to 62.5%, while in leaves, most of the Th was in the form of residue and the subdominant amount of Th was extracted by HCl, followed by 2% HAc. This suggested that Th compartmentation in cytosol and integration with phosphate or proteins in cell wall might be responsible for the tolerance of B. juncea var. foliosa to the stress of Th. PMID:27010411

  9. Subcellular potassium and sodium distribution in Saccharomyces cerevisiae wild-type and vacuolar mutants.

    PubMed

    Herrera, Rito; Álvarez, María C; Gelis, Samuel; Ramos, José

    2013-09-15

    Living cells accumulate potassium (K⁺) to fulfil multiple functions. It is well documented that the model yeast Saccharomyces cerevisiae grows at very different concentrations of external alkali cations and keeps high and low intracellular concentrations of K⁺ and sodium (Na⁺) respectively. However less attention has been paid to the study of the intracellular distribution of these cations. The most widely used experimental approach, plasma membrane permeabilization, produces incomplete results, since it usually considers only cytoplasm and vacuoles as compartments where the cations are present in significant amounts. By isolating and analysing the main yeast organelles, we have determined the subcellular location of K⁺ and Na⁺ in S. cerevisiae. We show that while vacuoles accumulate most of the intracellular K⁺ and Na⁺, the cytosol contains relatively low amounts, which is especially relevant in the case of Na⁺. However K⁺ concentrations in the cytosol are kept rather constant during the K⁺-starvation process and we conclude that, for that purpose, vacuolar K⁺ has to be rapidly mobilized. We also show that this intracellular distribution is altered in four different mutants with impaired vacuolar physiology. Finally, we show that both in wild-type and vacuolar mutants, nuclei contain and keep a relatively constant and important percentage of total intracellular K⁺ and Na⁺, which most probably is involved in the neutralization of negative charges.

  10. MRI investigation of subcellular water compartmentalization and gas distribution in apples.

    PubMed

    Winisdorffer, Guillaume; Musse, Maja; Quellec, Stéphane; Devaux, Marie-Françoise; Lahaye, Marc; Mariette, François

    2015-06-01

    Water status and distribution at subcellular level in whole apple fruit were evaluated by Magnetic Resonance Imaging (MRI) measurement of the multi-exponential transverse (T2) relaxation of water protons. Apparent microporosity, also estimated by MRI, provided mapping of gas distribution in fruit tissues. Measuring for the first time the multi-exponential relaxation of water and apparent tissue microporosity in whole fruit and combining these with histological measurements provided a more reliable interpretation of the origins of variations in the transverse relaxation time (T2) and better characterization of the fruit tissue. Measurements were performed on 54 fruits from 3 different cultivars. Fruits of different sizes were selected for each cultivar to provide tissues with cells of different dimensions. Macrovision measurements were carried out on parenchymal tissue from all fruits to investigate the impact of cell size on T2 value. The results showed that the MRI transverse relaxation signal is well fitted by a tri-exponential decay curve that reflects cell compartmentalization. Variations in cell size partially explained the different T2 observed. This study highlighted the heterogeneity of apple tissues in terms of relaxation parameters, apparent microporosity and cell morphology and in relation to specific variations between fruit of different cultivars.

  11. Cadmium uptake, chemical forms, subcellular distribution, and accumulation in Echinodorus osiris Rataj.

    PubMed

    Zhang, Chaolan; Zhang, Peng; Mo, Chuangrong; Yang, Weiwei; Li, Qinfeng; Pan, Liping; Lee, D K

    2013-07-01

    Phytoremediation is a technology for extracting or inactivating pollutants in soil. Echinodorus osiris (E. osiris) is a fast growing perennial wetland plant that is common in tropical and subtropical areas and has a high tolerance to cadmium (Cd). However, the absorption dynamics, subcellular distribution and accumulation of Cd by E. osiris had not been investigated. In this paper, hydroponic experiments with different levels of Cd(2+) (0, 5.0, 15.0 mg L(-1)) were carried out to determine these characteristics of E. osiris. The results indicated that the Cd absorption rate of Echinodorus osiris decreased over time, and the absorption rate within 0.5-1.0 h was faster than after 1.0 h. In a 6.0 hour time period, the rate of Cd uptake fit a quadratic polynomial curve when E. osiris was grown under the 5 mg L(-1) Cd treatment. However, the rate of Cd uptake by E. osiris fit a cubic polynomial model with the 15 mg L(-1) Cd treatment. In the roots, the ethanol-extractable Cd, water-extractable Cd, and NaCl-extractable Cd were the largest proportions of the total Cd. The HAc-extractable Cd, HCl-extractable Cd, and residual-Cd represented a larger proportion of the total Cd in the leaves which was combined with phosphate including CdHPO4, Cd3 (PO4)2, and oxalic acid. When analyzing the subcellular distribution of Cd in the plant, the soluble fraction containing Cd accounted for the largest part (69.49-88.39%) followed by the Cd bound to the cell wall (8.44-25.62%). Both the lower and the higher Cd treatments demonstrated that compartmentation by the vacuole and cell wall binding were two effective defense mechanisms of the plant. However, the vacuole became the main site for Cd accumulation in the leaves under the 15 mg L(-1) Cd treatment. E. osiris was able to accumulate high concentrations of Cd in both the roots and the leaves. The Cd concentration reached 502.97 mg kg(-1) and 2742.95 mg kg(-1) in the shoots and roots, respectively, after 27 days of cultivation. It was

  12. Intracellular pH governs the subcellular distribution of hexokinase in a glioma cell line.

    PubMed Central

    Miccoli, L; Oudard, S; Sureau, F; Poirson, F; Dutrillaux, B; Poupon, M F

    1996-01-01

    Hexokinase plays a key role in regulating cell energy metabolism. Hexokinase is mainly particulate, bound to the mitochondrial outer membrane in brain and tumour cells. We hypothesized that the intracellular pH (pH1) controls the intracellular distribution of hexokinase. Using the SNB-19 glioma cell line, pH1 variations were imposed by incubating cells in a high-K+ medium at different pH values containing specific ionophores (nigericin and valinomycin), without affecting cell viability. Subcellular fractions of cell homogenates were analysed for hexokinase activity. Imposed pH1 changes were verified microspectrofluorimetrically by using the pH1-sensitive probe SNARF-1-AM (seminaphtho-rhodafluor-1-acetoxymethyl ester). Imposition of an acidic pH1 for 30 min strongly decreased the particulate/total hexokinase ratio, from 63% in the control sample to 31%. Conversely, when a basic pH1, was imposed, the particulate/total hexokinase ratio increased to 80%. The glycolytic parameters, namely lactate/pyruvate ratio, glucose 6-phosphate and ATP levels, were measured concomitantly. Lactate/pyruvate ratio and ATP level were both markedly decreased by acidic pH1 and increased by basic pH1. Conversely, the glucose 6-phosphate level was increased by acidic pH1 and decreased by basic pH1. To demonstrate that the change of hexokinase distribution was not due to altered metabolite levels of glycolysis, a pH1 was imposed for a 5 min incubation time. Modification of the hexokinase distribution was similar to that noted after a 30 min incubation, whereas metabolite levels of glycolysis were not affected. These results provide evidence that the intracellular distribution of hexokinase is highly sensitive to variations of the pH1, and regulates hexokinase activity. PMID:8611181

  13. Acid phosphatases of the rat epididymis. II. Biochemical characteristics, subcellular distribution and histochemical localization.

    PubMed

    Nikkanen, V; Vanha-Perttula, T

    1977-01-01

    After separation of three epididymal acid phosphatases their biochemical properties were differently studied. With appropriate substrate and inhibitor selection the distribution of the enzymes in different segments as well as the subcellular fractions of the rat epididymis was also demonstrated. The same biochemical differences were also utilized in the histochemical localization of the enzymes. It was found that Enzyme I had a pH-optimum at 5.0, a molecular weight of 97 000 and Km-constant of 0.901 mM. It was highly sensitive to tartrate and fluoride and it was localized in lysosomes as well as in the epididymal spermatozoa. Enzyme II had an optimum at pH 5.7, a molecular weight of 67 000 and Km-constant of 0.806 mM. It was also inhibited by fluoride but more resistant to tartrate. Its subcellular site was also particulate, but it was also found in the epididymal fluid. Enzyme III had an optimum at pH 5.2, a molecular weight of 135 000 and Km-constant of 0.685 m. It was resistant to low concentrations of fluoride and tartrate but sensitive to heavy metal ions. The enzyme was soluble and it behaved incoherently in thermal inactivation. All enzymes revealed the highest activity in the thin middle segments of the epididymis. Histochemical naphthol substrates gave a diffuse reaction in the epididymal epithelial cells. With the lead salt methods glycerophosphates and p-nitrophenylphosphate gave somewhat different results depending on their specificity as substrates for the epididymal enzymes. Both substrates gave a strong reaction supranuclearly in the Golgi area of the chief cells. This activity was inhibited by tartrate and was most probably due to Enzyme I. The epididymal corpus and cauda showed additionally a very strong apical activity in the chief cells with p-nitrophenylphosphate. This activity was resitant to tartrate but sensitive to fluoride. It was concluded that this enzyme represents Enzyme II activity. Similar activity was also found in the dissolving

  14. Subcellular distribution and chemical forms of cadmium in the edible seaweed, Porphyra yezoensis.

    PubMed

    Zhao, Yanfang; Wu, Jifa; Shang, Derong; Ning, Jinsong; Zhai, Yuxiu; Sheng, Xiaofeng; Ding, Haiyan

    2015-02-01

    The subcellular distribution and chemical forms of Cd were investigated in the edible seaweed, Porphyra yezoensis. The seaweed was exposed to different Cd concentrations (0.01, 0.05, 0.1, 0.5, 1.0 and 5.0mgl(-1)) for up to 96h. In both the controls (no Cd added) and treatment groups, 41.2-79.2% of Cd was localised in the cell wall, and the proportion of Cd in the cell wall increased with increasing concentrations of Cd and exposure time. In the control groups, 74.8% of Cd was extracted by 1M NaCl, followed by 2% acetic acid, HAC (18.9%). In the treatment groups, most Cd was extracted by 2% HAC. The proportion of Cd extracted by 2% HAC increased with exposure to increasing concentrations of Cd and over time. Cell wall deposition and forming of precipitates with phosphate may be a key strategy to reduce Cd toxicity in P. yezoensis.

  15. Functional domains and sub-cellular distribution of the Hedgehog transducing protein Smoothened in Drosophila.

    PubMed

    Nakano, Y; Nystedt, S; Shivdasani, A A; Strutt, H; Thomas, C; Ingham, P W

    2004-06-01

    The Hedgehog signalling pathway is deployed repeatedly during normal animal development and its inappropriate activity is associated with various tumours in human. The serpentine protein Smoothened (Smo) is essential for cells to respond to the Hedeghog (Hh) signal; oncogenic forms of Smo have been isolated from human basal cell carcinomas. Despite similarities with ligand binding G-protein coupled receptors, the molecular basis of Smo activity and its regulation remains unclear. In non-responding cells, Smo is suppressed by the activity of another multipass membrane spanning protein Ptc, which acts as the Hh receptor. In Drosophila, binding of Hh to Ptc has been shown to cause an accumulation of phosphorylated Smo protein and a concomitant stabilisation of the activated form of the Ci transcription factor. Here, we identify domains essential for Smo activity and investigate the sub-cellular distribution of the wild type protein in vivo. We find that deletion of the amino terminus and the juxtamembrane region of the carboxy terminus of the protein result in the loss of normal Smo activity. Using Green Fluorescent Protein (GFP) and horseradish peroxidase fusion proteins we show that Smo accumulates in the plasma membrane of cells in which Ptc activity is abrogated by Hh but is targeted to the degradative pathway in cells where Ptc is active. We further demonstrate that Smo accumulation is likely to be a cause, rather than a consequence, of Hh signal transduction.

  16. Intracellular accumulation, subcellular distribution, and efflux of tilmicosin in bovine mammary, blood, and lung cells.

    PubMed

    Scorneaux, B; Shryock, T R

    1999-06-01

    Tilmicosin is a semisynthetic macrolide antibiotic currently approved for veterinary use in cattle and swine to combat respiratory disease. Because the concentrations of tilmicosin are generally low in bovine serum, the interaction of tilmicosin with three types of bovine phagocytes (monocyte-macrophages, macrophages, and neutrophils from blood, lungs, and mammary gland, respectively) and mammary gland epithelial cells was evaluated to provide an understanding of potential clinical efficacy. After incubation with radiolabeled tilmicosin, uptake was determined and expressed as the ratio of the intracellular to the extracellular drug concentration. Accumulation of tilmicosin at 4 h of incubation by the alveolar macrophages (Cc/Ce 193) was 4 to 13 times more than that observed in monocyte-macrophages (Cc/Ce 43), neutrophils, (Cc/Ce 13), or mammary epithelial cells (Cc/Ce 20). Subcellular distribution showed that 70 to 80% of tilmicosin was localized in the lysosomes. Uptake in mammary gland cells was dependent on cell viability, temperature, and pH, but was not influenced by metabolic inhibitors or anaerobiosis. However, lipopolysaccharide exposure increased tilmicosin uptake by the bovine mammary macrophages and epithelial cells. When neutrophils and epithelial cells were incubated in the presence of tilmicosin and extracellular tilmicosin was then removed, 40% of the intracellular tilmicosin remained cell associated after 4 h of incubation (i.e., 60% effluxed), but only 25% remained in macrophages. These in vitro interactions of tilmicosin with bovine phagocytes and epithelial cells suggest an integral role in effecting clinical efficacy.

  17. Subcellular distribution of uranium in the roots of Spirodela punctata and surface interactions

    NASA Astrophysics Data System (ADS)

    Nie, Xiaoqin; Dong, Faqin; Liu, Ning; Liu, Mingxue; Zhang, Dong; Kang, Wu; Sun, Shiyong; Zhang, Wei; Yang, Jie

    2015-08-01

    The subcellular distribution of uranium in roots of Spirodela punctata (duckweed) and the process of surface interaction were studied upon exposure to U (0, 5-200 mg/L) at pH 5. The concentration of uranium in each subcelluar fraction increased significantly with increasing solution U level, after 200 mg/L uranium solution treatment 120 h, the proportion of uranium concentration approximate as 8:2:1 in the cell wall organelle and cytosol fractions of roots of S. punctata. OM SEM and EDS showed after 5-200 mg/L U treatment 4-24 h, some intracellular fluid released from the root cells, after 100 mg/L U treatment 48 h, the particles including 35% Fe (wt%) and other organic matters such as EPS released from the cells, most of the uranium bound onto the root surface and contacted with phosphorus ligands and formed as nano-scales U-P lamellar crystal, similar crystal has been found in the cell wall and organelle fractions after 50 mg/L U treatment 120 h. FTIR and XPS analyses result indicates the uranium changed the band position and shapes of phosphate group, and the region of characteristic peak belongs to U(VI) and U(IV) were also observed.

  18. Changing pattern of the subcellular distribution of erythroblast macrophage protein (Emp) during macrophage differentiation.

    PubMed

    Soni, Shivani; Bala, Shashi; Kumar, Ajay; Hanspal, Manjit

    2007-01-01

    Erythroblast macrophage protein (Emp) mediates the attachment of erythroid cells to macrophages and is required for normal differentiation of both cell lineages. In erythroid cells, Emp is believed to be involved in nuclear extrusion, however, its role in macrophage differentiation is unknown. Information on the changes in the expression level and subcellular distribution of Emp in differentiating macrophages is essential for understanding the function of Emp. Macrophages of varying maturity were examined by immunofluorescence microscopy and biochemical methods. Our data show that Emp is expressed in all stages of maturation, but its localization pattern changes dramatically during maturation: in immature macrophages, a substantial fraction of Emp is associated with the nuclear matrix, whereas in more mature cells, Emp is expressed largely at cell surface. Pulse-chase experiments show that nascent Emp migrates intracellularly from the cytoplasm to the plasma membrane more efficiently in mature macrophages than in immature cells. Incubation of erythroid cells with macrophages in culture shows that erythroid cells attach to mature macrophages but not to immature macrophage precursors. Together, our data show that the temporal and spatial expression of Emp correlates with its role in erythroblastic island formation and suggest that Emp may be involved in multiple cellular functions.

  19. Subcellular distribution and chemical forms of cadmium in Impatiens walleriana in relation to its phytoextraction potential.

    PubMed

    Lai, Hung-Yu

    2015-11-01

    Impatiens (Impatiens walleriana) has been shown to be a potential cadmium (Cd) hyperaccumulator, but its mechanisms in accumulation and detoxification have not been reported. Rooted cuttings of Impatiens were planted in artificially Cd-contaminated soils for 50 days with total target concentrations of 0, 10, 20, 40, 80, and 120 mg/kg. The subcellular distribution and chemical forms of Cd in the different organs were analyzed after the pot experiment. Compared with the control group, various Cd treatments affected the growth exhibitions of Impatiens, but most of them were not statistically significant. The Cd accumulation of different organs increased with an increase in the soil Cd concentrations for most of the treatments, and it was in the decreasing order of root>stem>leaf. In the roots of Impatiens, Cd was mainly compartmentalized in the soluble fraction (Fs), which has a high migration capacity and will further translocate to the shoot. The Cd was mainly compartmentalized in the cell wall fraction (Fcw) in the shoots as a mechanism of tolerance. Most of the Cd in the various organs of Impatiens was mainly in the forms of pectate and protein-integrated (FNaCl), whereas a minor portion was a water soluble fraction (FW). The experimental results show that the Cd in the Fs, FW, and FNaCl in the roots of Impatiens had a high mobility and will further translocate to the shoot. They could be used to estimate the Cd accumulated in the shoots of Impatiens.

  20. Localization and subcellular distribution of N-copine in mouse brain.

    PubMed

    Nakayama, T; Yaoi, T; Kuwajima, G

    1999-01-01

    N-Copine is a novel protein with two C2 domains. Its expression is brain specific and up-regulated by neuronal activity such as kainate stimulation and tetanus stimulation evoking hippocampal CA1 long-term potentiation. We examined the localization and subcellular distribution of N-copine in mouse brain. In situ hybridization analysis showed that N-copine mRNA was expressed exclusively in neurons of the hippocampus and in the main and accessory olfactory bulb, where various forms of synaptic plasticity and memory formation are known to occur. In immunohistochemical analyses, N-copine was detected mainly in the cell bodies and dendrites in the neurons, whereas presynaptic proteins such as synaptotagmin I and rab3A were detected in the regions where axons pass through. In fractionation experiments of brain homogenate, N-copine was associated with the membrane fraction in the presence of Ca2+ but not in its absence. As a GST-fusion protein with the second C2 domain of N-copine showed Ca2+-dependent binding to phosphatidylserine, this domain was considered to be responsible for the Ca2+-dependent association of N-copine with the membrane. Thus, N-copine may have a role as a Ca2+ sensor in postsynaptic events, in contrast to the known roles of "double C2 domain-containing proteins," including synaptotagmin I, in presynaptic events. PMID:9886090

  1. Btf and TRAP150 have distinct roles in regulating subcellular mRNA distribution

    PubMed Central

    Varia, Sapna; Potabathula, Divya; Deng, Zhihui; Bubulya, Athanasios; Bubulya, Paula A.

    2013-01-01

    Transcription of protein-coding genes in mammalian cells is coordinated with pre-mRNA processing as well as the assembly and nuclear export of mRNPs. Btf (BCLAF1) and TRAP150 (THRAP3) were previously reported to associate with in vitro spliced mRNPs and also as a part of the spliceosome, suggesting they are involved in pre-mRNA processing. Btf and TRAP150 are serine-arginine-rich (SR) proteins with significant sequence similarity, but the extent of their functional overlap is not yet clear. We show that both Btf and TRAP150 localize at a constitutively active β-tropomyosin (BTM) reporter minigene locus in mammalian cells. Both proteins also localize at a U2OS 2–6-3 reporter gene locus in a RNA polymerase II (RNAPII) transcription-dependent manner. While Btf and TRAP150 showed some overlap with reporter RNA and other pre-mRNA processing factors at transcription loci, they showed the most precise overlap with the exon junction complex (EJC) protein Magoh. Since EJC components have roles in nuclear export, we examined nuclear/cytoplasmic mRNA distribution after Btf or TRAP150 knockdown. Btf depletion caused an increase of β-tropomyosin minigene reporter transcripts in the cytoplasm as well as global increase of endogenous polyadenylated RNA in the cytoplasm, while TRAP150 depletion did not. We provide evidence that Btf has functions distinct from TRAP150 in regulating the subcellular distribution of mRNAs in human cells. PMID:23778535

  2. Diverse and pervasive subcellular distributions for both coding and long noncoding RNAs

    PubMed Central

    Wilk, Ronit; Hu, Jack; Blotsky, Dmitry; Krause, Henry M.

    2016-01-01

    In a previous analysis of 2300 mRNAs via whole-mount fluorescent in situ hybridization in cellularizing Drosophila embryos, we found that 70% of the transcripts exhibited some form of subcellular localization. To see whether this prevalence is unique to early Drosophila embryos, we examined ∼8000 transcripts over the full course of embryogenesis and ∼800 transcripts in late third instar larval tissues. The numbers and varieties of new subcellular localization patterns are both striking and revealing. In the much larger cells of the third instar larva, virtually all transcripts observed showed subcellular localization in at least one tissue. We also examined the prevalence and variety of localization mechanisms for >100 long noncoding RNAs. All of these were also found to be expressed and subcellularly localized. Thus, subcellular RNA localization appears to be the norm rather than the exception for both coding and noncoding RNAs. These results, which have been annotated and made available on a recompiled database, provide a rich and unique resource for functional gene analyses, some examples of which are provided. PMID:26944682

  3. DNA Damage Dependence on the Subcellular Distribution of Low-Energy Beta Emitters

    NASA Astrophysics Data System (ADS)

    Cutaia, Claudia; Alloni, Daniele; Mariotti, Luca; Friedland, Werner; Ottolenghi, Andrea

    risk, (particularly for Tritium intake), due to the distribution of the low energy emitters at subcellular levels.

  4. Measurement of rat heart fatty acid binding protein by ELISA. Tissue distribution, developmental changes and subcellular distribution.

    PubMed

    Crisman, T S; Claffey, K P; Saouaf, R; Hanspal, J; Brecher, P

    1987-05-01

    A class of soluble, low molecular weight proteins collectively called fatty acid binding proteins (FABP) are thought to function in the intracellular movement of fatty acids. To understand more clearly the role of FABP in cardiac metabolism, we used ELISA and immunoblotting techniques to study the distribution of heart FABP in several rat tissues, compare male and female rat heart content, quantitate developmental changes, and determine its subcellular distribution. Immunoreactive protein was found in appreciable amounts in rat heart, red skeletal muscle and kidney. Adult rat heart contained about 1.5 mg FABP/g tissue wet weight with the atrial content being approximately 50% of the ventricular concentration. No significant difference was detected between the sexes. The amount of FABP increased progressively during development from fetal to adult animals, and measureable amounts were found in 17-day-old fetal tissue. Comparisons between myoglobin and FABP showed that FABP appeared earlier than myoglobin in development, but myoglobin was more abundant than FABP at birth. Using immunoblots it was determined that rat heart FABP was localized in the cytosol with no detectable intramitochondrial material. PMID:3625779

  5. Drosophila melanogaster lipins are tissue-regulated and developmentally regulated and present specific subcellular distributions.

    PubMed

    Valente, Valeria; Maia, Rafaela Martins; Vianna, Murilo Carlos Bizam; Paçó-Larson, Maria Luisa

    2010-11-01

    Lipins constitute a novel family of Mg(2+)-dependent phosphatidate phosphatases that catalyze the dephosphorylation of phosphatidic acid to yield diacylglycerol, an important intermediate in lipid metabolism and cell signaling. Whereas a single lipin is detected in less complex organisms, in mammals there are distinct lipin isoforms and paralogs that are differentially expressed among tissues. Compatible with organism tissue complexity, we show that the single Drosophila Lpin1 ortholog (CG8709, here named DmLpin) expresses at least three isoforms (DmLpinA, DmLpinK and DmLpinJ) in a temporal and spatially regulated manner. The highest levels of lipin in the fat body, where DmLpinA and DmLpinK are expressed, correlate with the highest levels of triacylglycerol (TAG) measured in this tissue. DmLpinK is the most abundant isoform in the central nervous system, where TAG levels are significantly lower than in the fat body. In the testis, where TAG levels are even lower, DmLpinJ is the predominant isoform. Together, these data suggest that DmLpinA might be the isoform that is mainly involved in TAG production, and that DmLpinK and DmLpinJ could perform other cellular functions. In addition, we demonstrate by immunofluorescence that lipins are most strongly labeled in the perinuclear region of the fat body and ventral ganglion cells. In visceral muscles of the larval midgut and adult testis, lipins present a sarcomeric distribution. In the ovary chamber, the lipin signal is concentrated in the internal rim of the ring canal. These specific subcellular localizations of the Drosophila lipins provide the basis for future investigations on putative novel cellular functions of this protein family. PMID:20977671

  6. Cadmium sensitivity, uptake, subcellular distribution and thiol induction in a marine diatom: Recovery from cadmium exposure.

    PubMed

    Wang, Meng-Jiao; Wang, Wen-Xiong

    2011-01-25

    Studies in the recovery from metal stress and the tolerance development to metal exposure of aquatic organisms are important for the understanding of epidemic pollution. In this study, the responses of a marine diatom, Thalassiosira nordenskioeldii, following recovery from environmental cadmium (Cd) stress were investigated. The diatoms were exposed to different concentrations of Cd for 7 days, and were then allowed different periods of time to recover. The Cd sensitivity increased after recovery from Cd stress, followed by a gradual restoration. The extent of restoration depended on both the recovery time and the environmental Cd stress during the exposure period. A complete restoration of Cd tolerance proved to be impossible for cells pre-exposed to High-Cd. The Cd cellular burden and subcellular Cd concentration decreased to the control level within the first day of recovery, indicating that the elevated sensitivity may have been due to the accumulation of functional damage caused by Cd exposure instead of a result of physical Cd accumulation. The rapid change in phytochelatins (PC) to both the increase in and the withdrawal of environmental Cd stress made it a good quantitative bioindicator of environmental Cd contamination. However, the relationships between Cd distribution in the metal sensitive fraction (MSF-Cd) or intracellular Cd to thiol ratio (intra-Cd/PC-SH) and the relative change in the median inhibition [Cd(2+)] ([Cd(2+)]-based-IC(50), i.e., Cd sensitivity) differed for the various exposure and recovery periods tested. Our study suggests that more attention should be given to the recovery of aquatic organisms from episodic metal exposure.

  7. Subcellular distribution of peptides associated with gastric mucosal healing and neoplasia.

    PubMed

    Sarraf, C E; Alison, M R; Ansari, T W; Wright, N A

    1995-06-15

    The trefoil peptides pS2 and human spasmolytic peptide are putative growth factors, particularly associated with mucus-producing cells of the gastrointestinal tract including those of the stomach. The receptor for transforming growth factor alpha (TGF alpha) takes its name from one of its alternative ligands, epidermal growth factor, and is called the epidermal growth factor receptor. Although there is immunoreactive epidermal growth factor in the stomach, it is TGF alpha and the epidermal growth factor receptor that are abundant. Immunolabelling at electron microscope level allows for subcellular localisation of antigens; pS2 and human spasmolytic peptide co-localise to cytomembranes, including the Golgi apparatus, and thecae of surface/pit mucous cells. TGF alpha is abundant on the membranes of tubulovesicles of parietal cells and is also present in chief cells: in mucous producing cells it can be detected but not in association with mucous. The distribution of the epidermal growth factor receptor mimics that of TGF alpha but with preferential clustering on the basolateral membranes of gastric cells. The trefoil peptides are associated with healing and probably act, together with mucus, to protect the gastric mucosa and maintain a viable environment. TGF alpha, transduced via the epidermal growth factor receptor, inhibits gastric acid secretion, thus aids the trefoils in the maintenance of a gastric microenvironment conducive to healing after damage. TGF alpha, however, is also a potent mitogen; while this property plays a vital part in repairing mucosal defects, if this peptide or indeed its receptor are overexpressed, the result can be neoplasia.

  8. Dosimetric characterization of radionuclides for systemic tumor therapy: Influence of particle range, photon emission, and subcellular distribution

    SciTech Connect

    Uusijaervi, Helena; Bernhardt, Peter; Ericsson, Thomas; Forssell-Aronsson, Eva

    2006-09-15

    Various radionuclides have been proposed for systemic tumor therapy. However, in most dosimetric analysis of proposed radionuclides the charged particles are taken into consideration while the potential photons are ignored. The photons will cause undesirable irradiation of normal tissue, and increase the probability of toxicity in, e.g., the bone marrow. The aim of this study was to investigate the dosimetric properties according to particle range, photon emission, and subcellular radionuclide distribution, of a selection of radionuclides used or proposed for radionuclide therapy, and to investigate the possibility of dividing radionuclides into groups according to their dosimetric properties. The absorbed dose rate to the tumors divided by the absorbed dose rate to the normal tissue (TND) was estimated for different tumor sizes in a mathematical model of the human body. The body was simulated as a 70-kg ellipsoid and the tumors as spheres of different sizes (1 ng-100 g). The radionuclides were either assumed to be uniformly distributed throughout the entire tumor and normal tissue, or located in the nucleus or the cytoplasm of the tumor cells and on the cell membrane of the normal cells. Fifty-nine radionuclides were studied together with monoenergetic electrons, positrons, and alpha particles. The tumor and normal tissue were assumed to be of water density. The activity concentration ratio between the tumor and normal tissue was assumed to be 25. The radionuclides emitting low-energy electrons combined with a low photon contribution, and the alpha emitters showed high TND values for most tumor sizes. Electrons with higher energy gave reduced TND values for small tumors, while a higher photon contribution reduced the TND values for large tumors. Radionuclides with high photon contributions showed low TND value for all tumor sizes studied. The radionuclides studied could be divided into four main groups according to their TND values: beta emitters, Auger electron

  9. Subcellular distribution of trace elements and liver histology of landlocked Arctic char (Salvelinus alpinus) sampled along a mercury contamination gradient.

    PubMed

    Barst, Benjamin D; Rosabal, Maikel; Campbell, Peter G C; Muir, Derek G C; Wang, Xioawa; Köck, Günter; Drevnick, Paul E

    2016-05-01

    We sampled landlocked Arctic char (Salvelinus alpinus) from four lakes (Small, 9-Mile, North, Amituk) in the Canadian High Arctic that span a gradient of mercury contamination. Metals (Hg, Se, Tl, and Fe) were measured in char tissues to determine their relationships with health indices (relative condition factor and hepatosomatic index), stable nitrogen isotope ratios, and liver histology. A subcellular partitioning procedure was employed to determine how metals were distributed between potentially sensitive and detoxified compartments of Arctic char livers from a low- and high-mercury lake (Small Lake and Amituk Lake, respectively). Differences in health indices and metal concentrations among char populations were likely related to differences in feeding ecology. Concentrations of Hg, Se, and Tl were highest in the livers of Amituk char, whereas concentrations of Fe were highest in Small and 9-Mile char. At the subcellular level we found that although Amituk char had higher concentrations of Tl in whole liver than Small Lake char, they maintained a greater proportion of this metal in detoxified fractions, suggesting an attempt at detoxification. Mercury was found mainly in potentially sensitive fractions of both Small and Amituk Lake char, indicating that Arctic char are not effectively detoxifying this metal. Histological changes in char livers, mainly in the form of melano-macrophage aggregates and hepatic fibrosis, could be linked to the concentrations and subcellular distributions of essential or non-essential metals.

  10. Effects of dietary salt on renal Na+ transporter subcellular distribution, abundance, and phosphorylation status.

    PubMed

    Yang, Li E; Sandberg, Monica B; Can, Argun D; Pihakaski-Maunsbach, Kaarina; McDonough, Alicia A

    2008-10-01

    During high-salt (HS) diet the kidney increases urinary Na+ and volume excretion to match intake. We recently reported that HS provokes a redistribution of distal convoluted tubule Na+-Cl- cotransporter (NCC) from apical to subapical vesicles and decreases NCC abundance. This study aimed to test the hypothesis that the other renal Na+ transporters' abundance and or subcellular distribution is decreased by HS diet. Six-week-old Sprague-Dawley rats were fed a normal (NS) 0.4% NaCl diet or a HS 4% NaCl diet for 3 wk or overnight. Kidneys excised from anesthetized rats were fractionated on density gradients or analyzed by microscopy; transporters and associated regulators were detected with specific antibodies. Three-week HS doubled Na+/H+ exchanger (NHE)3 phosphorylation at serine 552 and provoked a redistribution of NHE3, dipeptidyl peptidase IV (DPPIV), myosin VI, Na+-Pi cotransporter (NaPi)-2, ANG II type 2 receptor (AT2R), aminopeptidase N (APN), Na+-K+-2Cl- cotransporter (NKCC2), epithelial Na+ channel (ENaC) beta-subunit, and Na+-K+-ATPase (NKA) alpha1- and beta1-subunits from low-density plasma membrane-enriched fractions to higher-density intracellular membrane-enriched fractions. NHE3, myosin VI, and AT2R retraction to the base of the microvilli (MV) during HS was evident by confocal microscopy. HS did not change abundance of NHE3, NKCC, or NKA alpha1- or beta1-subunits but increased ENaC-beta in high-density intracellular enriched membranes. Responses to HS were fully apparent after just 18 h. We propose that retraction of NHE3 to the base of the MV, driven by myosin VI and NHE3 phosphorylation and accompanied by redistribution of the NHE3 regulator DPPIV, contributes to a decrease in proximal tubule Na+ reabsorption during HS and that redistribution of transporters out of low-density plasma membrane-enriched fractions in the thick ascending limb of the loop of Henle and distal nephron may also contribute to the homeostatic natriuretic response to HS diet

  11. Role of structural and functional elements of mouse methionine-S-sulfoxide reductase in its subcellular distribution.

    PubMed

    Kim, Hwa-Young; Gladyshev, Vadim N

    2005-06-01

    Oxidized forms of methionine residues in proteins can be repaired by methionine-S-sulfoxide reductase (MsrA) and methionine-R-sulfoxide reductase (MsrB). In mammals, three MsrBs are present, which are targeted to various subcellular compartments. In contrast, only a single mammalian MsrA gene is known whose products have been detected in both cytosol and mitochondria. Factors that determine the location of the protein in these compartments are not known. Here, we found that MsrA was present in cytosol, nucleus, and mitochondria in mouse cells and tissues and that the major enzyme forms detected in various compartments were generated from a single-translation product rather than by alternative translation initiation. Both cytosolic and mitochondrial forms were processed with respect to the N-terminal signal peptide, and the distribution of the protein occurred post-translationally. Deletion of amino acids 69-108, 69-83, 84-108, or 217-233, which contained elements important for MsrA structure and function, led to exclusive mitochondrial location of MsrA, whereas a region that affected substrate binding but was not part of the overall fold had no influence on the subcellular distribution. The data suggested that proper structure-function organization of MsrA played a role in subcellular distribution of this protein in mouse cells. These findings were recapitulated by expressing various forms of mouse MsrA in Saccharomyces cerevisiae, suggesting conservation of the mechanisms responsible for distribution of the mammalian enzyme among different cellular compartments. PMID:15924425

  12. Subcellular boron and fluorine distributions with SIMS ion microscopy in BNCT and cancer research

    SciTech Connect

    Subhash Chandra

    2008-05-30

    The development of a secondary ion mass spectrometry (SIMS) based technique of Ion Microscopy in boron neutron capture therapy (BNCT) was the main goal of this project, so that one can study the subcellular location of boron-10 atoms and their partitioning between the normal and cancerous tissue. This information is fundamental for the screening of boronated drugs appropriate for neutron capture therapy of cancer. Our studies at Cornell concentrated mainly on studies of glioblastoma multiforme (GBM). The early years of the grant were dedicated to the development of cryogenic methods and correlative microscopic approaches so that a reliable subcellular analysis of boron-10 atoms can be made with SIMS. In later years SIMS was applied to animal models and human tissues of GBM for studying the efficacy of potential boronated agents in BNCT. Under this grant the SIMS program at Cornell attained a new level of excellence and collaborative SIMS studies were published with leading BNCT researchers in the U.S.

  13. Current Gaps in the Understanding of the Subcellular Distribution of Exogenous and Endogenous Protein TorsinA

    PubMed Central

    Harata, N. Charles

    2014-01-01

    Background An in-frame deletion leading to the loss of a single glutamic acid residue in the protein torsinA (ΔE-torsinA) results in an inherited movement disorder, DYT1 dystonia. This autosomal dominant disease affects the function of the brain without causing neurodegeneration, by a mechanism that remains unknown. Methods We evaluated the literature regarding the subcellular localization of torsinA. Results Efforts to elucidate the pathophysiological basis of DYT1 dystonia have relied partly on examining the subcellular distribution of the wild-type and mutated proteins. A typical approach is to introduce the human torsinA gene (TOR1A) into host cells and overexpress the protein therein. In both neurons and non-neuronal cells, exogenous wild-type torsinA introduced in this manner has been found to localize mainly to the endoplasmic reticulum, whereas exogenous ΔE-torsinA is predominantly in the nuclear envelope or cytoplasmic inclusions. Although these outcomes are relatively consistent, findings for the localization of endogenous torsinA have been variable, leaving its physiological distribution a matter of debate. Discussion As patients’ cells do not overexpress torsinA proteins, it is important to understand why the reported distributions of the endogenous proteins are inconsistent. We propose that careful optimization of experimental methods will be critical in addressing the causes of the differences among the distributions of endogenous (non-overexpressed) vs. exogenously introduced (overexpressed) proteins. PMID:25279252

  14. Characterizations of bio-accumulations, subcellular distribution and chemical forms of cesium in Brassica juncea, and Vicia faba.

    PubMed

    Fu, Qian; Lai, Jin-long; Tao, Zong-ya; Han, Na; Wu, Guo

    2016-04-01

    We aim to investigate the tolerance and enrichment mechanism of cesium (Cs) in hyperaccumulation plants. In this study, Brassica juncea and Vicia faba were subjected to varying doses of Cs for 21 days to investigate the differences in bio-accumulations, subcellular distribution and chemical forms of Cs in two cultivars by differential centrifugation, and extraction of Cs in different chemical forms, respectively. The results showed that 49.87%-61.08% of the Cs were in the leaf of B. juncea, while in V. faba, 1.58%-79.29% of the Cs was in the root. The translocation factor (TF) arrived 2.79 to 3.71 in B. juncea, while it only reached 0.26 to 0.62 in V. faba. Cs subcellular distribution of the two plants was in sequence as follows: soluble fraction > cell wall > organelles. Cs was more easily distributed to metal-sensitive fractions of V. faba. The inorganic Cs (F-ethanol), and water-soluble Cs (F-dH2O) are the main existing types of Cs in the two plants. In B. juncea, the relative content of inorganic Cs, and organic acids/CsH2PO4 (F-dH2O) were higher than that of V. faba in the stem. This suggests that Cs may induce related transporter gene expression (such as phosphate transporter, organic cation, high affinity nitrate transporter, amino acid permease, etc.) to help the transport of Cs between root to shoot. PMID:26854554

  15. 2-([sup 125]I) iodomelatonin binding sites in rat adrenals: Pharmacological characteristics and subcellular distribution

    SciTech Connect

    Persengiev, S.P. )

    1992-01-01

    Specific binding sites for 2-[[sup 125]I] iodomelatonin, a selective radiolabeled melatonin receptor ligand, were detected and characterized in rat adrenal membranes. Saturation studies demonstrated that 2-[[sup 125]I]iodomelatonin binds to a single class of sites with an affinity constant (Kd) of 541 pM and a total binding capacity (Bmax) of 3.23 fmol/mg protein. Competition experiments revealed that the relative order of potency of compounds tested was as follows: 6-chloromelatonin > 2-iodomelatonin > melatonin > 5-methoxytryptamine > 5-methoxytryptophol. The highest density of binding sites was found in membranes from nuclear and mitochondrial subcellular fractions.

  16. Effects of extended growth periods on subcellular distribution, chemical forms, and the translocation of cadmium in Impatiens walleriana.

    PubMed

    Lai, Hung-Yu; Cai, Ming-Cyuan

    2016-01-01

    Impatiens walleriana plants accumulate sufficiently high concentrations of cadmium (Cd) for this species to be considered a potential Cd hyperaccumulator. Rooted cuttings were grown hydroponically for 25 and 50 days in solutions spiked with various Cd concentrations. The subcellular distribution and chemical forms of Cd in different organs were analyzed, and its upward translocation was also assessed. The plants accumulated large amounts of Cd; the Cd concentration in the roots and shoots reached 120-1900 and 60-1600 mg/kg, respectively. Regardless of the growth period, the Cd accumulated in the roots was primarily compartmentalized in the soluble fraction or ethanol and deionized water extractable chemical forms with high migration abilities. Translocation to the shoots was followed by an association of Cd mainly in the cell wall or with pectate and protein. The roots' Cd showed a high migration capacity for predicting the shoots' Cd concentrations. Different exposure periods significantly affected the subcellular distribution of Cd in the stems, and thus the upward translocation.

  17. Subcellular distribution of the transmissible agent in Creutzfeldt-Jakob disease mouse brain.

    PubMed

    Tamai, Y; Kojima, H; Ohtani, Y; Uchida, K; Taguchi, F; Kawaguchi, T; Miura, S; Tateishi, J

    1989-01-01

    To determine the intracellular localization of the Creutzfeldt-Jakob disease (CJD) agent in mouse brain, cerebrum tissue of the mouse brain affected with the Fukuoka-1 strain was separated into six subcellular fractions (microsome, nerve ending, myelin, mitochondria, nucleus, and soluble fractions) by differential sucrose density gradient, and then the CJD infectivity of these fractions was examined. Serially diluted samples of each subfraction were inoculated intracerebrally into groups of BALB/c mice, and the infectivity was determined as to end point titration value, incubation period, and number of affected mice. On the basis of the protein content, the highest CJD infectivity was observed in the microsomal fraction. The nerve ending (synaptic plasma membrane) and myelin fractions were also infective. The mitochondria and nucleus fractions showed the lower infectivity. The infectivity of the soluble fraction was the lowest among the six subcellular fractions. From the findings obtained in this study two possibilities as to the intracellular localization of CJD agent were suggested: 1) the transmissible agent of CJD is closely associated with surface membranes of neuronal and/or glial cells, including their processes; 2) the CJD agent is diffusely present intracellularly, including in the surface membranes, but for manifestation of infectivity the agent needs membrane components as prerequisite factors.

  18. Non-random subcellular distribution of variant EKLF in erythroid cells

    PubMed Central

    Quadrini, Karen J.; Gruzglin, Eugenia; Bieker, James J.

    2008-01-01

    EKLF protein plays a prominent role during erythroid development as a nuclear transcription factor. Not surprisingly, exogenous EKLF quickly localizes to the nucleus. However, using two different assays we have unexpectedly found that a substantial proportion of endogenous EKLF resides in the cytoplasm at steady state in all erythroid cells examined. While EKLF localization does not appear to change during either erythroid development or terminal differentiation, we find that the protein displays subtle yet distinct biochemical and functional differences depending on which subcellular compartment it is isolated from, with PEST sequences possibly playing a role in these differences. Localization is unaffected by inhibition of CRM1 activity and the two populations are not differentiated by stability. Heterokaryon assays demonstrate that EKLF is able to shuttle out of the nucleus although its nuclear re-entry is rapid. These studies suggest there is an unexplored role for EKLF in the cytoplasm that is separate from its well-characterized nuclear function. PMID:18329016

  19. Non-random subcellular distribution of variant EKLF in erythroid cells

    SciTech Connect

    Quadrini, Karen J.; Gruzglin, Eugenia; Bieker, James J.

    2008-04-15

    EKLF protein plays a prominent role during erythroid development as a nuclear transcription factor. Not surprisingly, exogenous EKLF quickly localizes to the nucleus. However, using two different assays we have unexpectedly found that a substantial proportion of endogenous EKLF resides in the cytoplasm at steady state in all erythroid cells examined. While EKLF localization does not appear to change during either erythroid development or terminal differentiation, we find that the protein displays subtle yet distinct biochemical and functional differences depending on which subcellular compartment it is isolated from, with PEST sequences possibly playing a role in these differences. Localization is unaffected by inhibition of CRM1 activity and the two populations are not differentiated by stability. Heterokaryon assays demonstrate that EKLF is able to shuttle out of the nucleus although its nuclear re-entry is rapid. These studies suggest there is an unexplored role for EKLF in the cytoplasm that is separate from its well-characterized nuclear function.

  20. The subcellular distribution and properties of hexokinases in the guinea-pig cerebral cortex.

    PubMed

    Bachelard, H S

    1967-07-01

    1. Hexokinase activities were estimated in primary subcellular fractions from guinea-pig cerebral cortex and in sucrose-density-gradient subfractions of the mitochondrial and microsomal fractions. 2. Appreciable activities were observed in mitochondrial, microsomal and soluble fractions. The activity in the mitochondrial fraction was associated with the mitochondria rather than with myelin or nerve endings and that in the microsomal fraction was associated with membrane fragments. 3. Most of the mitochondrial activity was extracted in soluble form by osmotic ;shock'. The activity of the mitochondrial extract differed from the soluble activity in kinetic properties and in electrophoretic behaviour. 4. No evidence was obtained for the presence of a high-K(m) glucokinase in the brain. 5. The results are discussed in terms of relevance to considerations of glucose utilization by the brain.

  1. Subcellular distribution of 65,000 calmodulin-binding protein (p65) and synaptophysin (p38) in adrenal medulla.

    PubMed

    Fournier, S; Novas, M L; Trifaró, J M

    1989-10-01

    Both neuronal and endocrine cells contain secretory vesicles that store and release neurotransmitters and peptides. Neuronal cells release their secretory material from both small synaptic vesicles and large dense-core vesicles (LDCVs), whereas endocrine cells release secretory products from LDCVs. Neuronal small synaptic vesicles are known to express three integral membrane proteins: 65,000 calmodulin-binding protein (65-CMBP) (p65), synaptophysin (p38), and SV2. A controversial question surrounding these three proteins is whether they are present in LDCV membranes of endocrine and neuronal cells. Sucrose density centrifugation of adrenal medulla was performed to study and compare the subcellular distribution of two of these small synaptic vesicle proteins (65-CMBP and synaptophysin). Subsequent immunoblotting and 125I-Protein A binding experiments performed on the fractions obtained from sucrose gradients showed that 65-CMBP was present in fractions corresponding to granule membranes and intact chromaffin granules. Similar immunoblotting and 125I-Protein A binding experiments with synaptophysin antibodies showed that this protein was also present in intact granules and granule membrane fractions. However, an additional membrane component, equilibrating near the upper portion of the sucrose gradient, also showed strong immunoreactivity with anti-synaptophysin and high 125I-Protein A binding activity. In addition, immunoblotting experiments on purified plasma and granule membranes demonstrated that 65-CMBP was a component of both membranes, whereas synaptophysin was only present in granule membranes. Thus, there appears to be a different subcellular localization between 65-CMBP and synaptophysin in the chromaffin cell.

  2. Fine and distributed subcellular retinotopy of excitatory inputs to the dendritic tree of a collision-detecting neuron.

    PubMed

    Zhu, Ying; Gabbiani, Fabrizio

    2016-06-01

    Individual neurons in several sensory systems receive synaptic inputs organized according to subcellular topographic maps, yet the fine structure of this topographic organization and its relation to dendritic morphology have not been studied in detail. Subcellular topography is expected to play a role in dendritic integration, particularly when dendrites are extended and active. The lobula giant movement detector (LGMD) neuron in the locust visual system is known to receive topographic excitatory inputs on part of its dendritic tree. The LGMD responds preferentially to objects approaching on a collision course and is thought to implement several interesting dendritic computations. To study the fine retinotopic mapping of visual inputs onto the excitatory dendrites of the LGMD, we designed a custom microscope allowing visual stimulation at the native sampling resolution of the locust compound eye while simultaneously performing two-photon calcium imaging on excitatory dendrites. We show that the LGMD receives a distributed, fine retinotopic projection from the eye facets and that adjacent facets activate overlapping portions of the same dendritic branches. We also demonstrate that adjacent retinal inputs most likely make independent synapses on the excitatory dendrites of the LGMD. Finally, we show that the fine topographic mapping can be studied using dynamic visual stimuli. Our results reveal the detailed structure of the dendritic input originating from individual facets on the eye and their relation to that of adjacent facets. The mapping of visual space onto the LGMD's dendrites is expected to have implications for dendritic computation. PMID:27009157

  3. Global Identification and Differential Distribution Analysis of Glycans in Subcellular Fractions of Bladder Cells

    PubMed Central

    Yang, Ganglong; Huang, Luyu; Zhang, Jiaxu; Yu, Hanjie; Li, Zheng; Guan, Feng

    2016-01-01

    Compartmentalization of cellular components and their associated biological processes is crucial for cellular function. Protein glycosylation provides a basis for diversity of protein functions. Diversity of glycan composition in animal cells remains poorly understood. We used differential centrifugation techniques to isolate four subcellular protein fractions from homogenate of metastatic bladder YTS1 cells, low grade nonmuscle invasive bladder cancer KK47 cells and normal bladder epithelia HCV29 cells: microsomal (Mic), mitochondrial (Mito), nuclear (Nuc), and cytosolic (Cyto). An integrated strategy combining lectin microarray and mass spectrometry (MS) analysis was then applied to evaluate protein glycosylation of the four fractions. Lectin microarray analysis revealed significant differences among the four fractions in terms of glycan binding to the lectins LCA, AAL, MPL, WGA and PWM in YTS1 cell, STL, Jacalin, VVA, LCA and WGA in KK47, and ConA, GNA, VVA and ACA in HCV29 cell. Among a total of 40, 32 and 15 N-glycans in four fractions of three cells detected by MS analysis, high-mannose and fucosylated structures were predominant, 10 N-glycans in YTS1, 5 N-glycans in KK47 and 7 N-glycans in HCV29 were present in all four fractions; and 10 N-glycans in YTS1, 16 N-glycans in KK47, and 3 N-glycans in HCV29 were present in only one fraction. Glycans in the latter category are considered potential markers for the corresponding organelles. The integrated strategy described here allows detailed examination of glycomes subcellular fraction with high resolution and sensitivity, and will be useful for elucidation of the functional roles of glycans and corresponding glycosylated proteins in distinct organelles. PMID:27313494

  4. Subcellular distribution of okadaic acid in the digestive gland of Mytilus galloprovincialis: first evidences of lipoprotein binding to okadaic acid.

    PubMed

    Rossignoli, Araceli E; Blanco, Juan

    2010-01-01

    The subcellular distribution of okadaic acid, the main diarrhetic shellfish poisoning (DSP) toxin, in the cells of the digestive gland of the mussel Mytilus galloprovincialis was studied. By means of differential centrifugation, ultrafiltration and extraction with methanol, it was found that most okadaic acid was stored in the cytosol. Notwithstanding only a small proportion of the total toxin was found to be in free form, being most of it bound to a soluble cellular compound with a molecular mass which ranged from 30 to 300 kDa. A series of fractionations of samples digested with a protease, a lipase, and amylase suggested that the component to which okadaic acid is bound is a high density lipoprotein. A new fractionation after digestion with a protein lipase additionally supports the previous conclusion.

  5. Microfilaments and microtubules control the shape, motility, and subcellular distribution of cortical mitochondria in characean internodal cells.

    PubMed

    Foissner, I

    2004-12-01

    The shape, motility, and subcellular distribution of mitochondria in characean internodal cells were studied by visualizing fluorescent dyes with confocal laser scanning microscopy and conducting drug-inhibitor experiments. Shape, size, number, and distribution of mitochondria varied according to the growth status and the metabolic activity within the cell. Vermiform (sausage-shaped), disc-, or amoeba-like mitochondria were present in elongating internodes, whereas very young cells and older cells that had completed growth contained short, rodlike organelles only. Mitochondria were evenly distributed and passively transported in the streaming endoplasm. In the cortex, mitochondria were sandwiched between the plasma membrane and the stationary chloroplast files and distributed in relation to the pattern of pH banding. Highest mitochondrial densities were found at the acid, photosynthetically more active regions, whereas the alkaline sites contained fewer and smaller mitochondria. In the cortex of elongating cells, small mitochondria moved slowly along microtubules or actin filaments. The shape and motility of giant mitochondria depended on the simultaneous interaction with both cytoskeletal systems. There was no microtubule-dependent motility in the cortex of nonelongating mature cells and mitochondria only occasionally travelled along actin filaments. These observations suggest that mitochondria of characean internodes possess motor proteins for microtubules and actin filaments, both of which can be used either as tracks for migration or for immobilization. The cortical cytoskeleton probably controls the spatiotemporal distribution of mitochondria within the cell and promotes their association with chloroplasts, which is necessary for exchange of metabolites during photosynthesis and detoxification.

  6. Subcellular distribution of apolipoprotein E along the lipoprotein synthetic pathway of rat liver

    SciTech Connect

    Cole, T.G.; Stockhausen, D.C.

    1986-03-01

    Apolipoprotein E (apoE) is synthesized by the liver and is secreted as a component of VLDL. To define the intracellular locations of apoE, liver from 10 nonfasted male rats were removed and subcellular organelles prepared by differential pelleting through sucrose gradients. Mass of apoE was measured by radioimmunoassay. Approximately 10% of total hepatic apoE was recovered in rough endoplasmic reticulum (RER), smooth endoplasmic reticulum (SER) and Golgi fractions. Concentrations of apoE (ng/mg protein) were: homogenate, 302 +/- 59; RER, 653 +/- 251; SER, 1250 +/- 471; Golgi, 11,044 +/- 4291. Total apoE content of each reaction (..mu..g/organelle) was: homogenate (whole liver), 517 +/- 103; RER, 15 +/- 3; SER, 9 +/- 3; Golgi, 28 +/- 8. These data indicate that along the putative pathway of lipoprotein synthesis (RER->SER->Golgi), apoE concentration increases in each successive organelle and that flux of apoE is apparently most rapid through SER. Furthermore, the majority of apoE in the rat liver is apparently not directly associated with the lipoprotein synthetic pathway and may be associated with internalized lipoproteins or may be involved in non-lipoprotein related functions.

  7. Uptake and subcellular distributions of cadmium and selenium in transplanted aquatic insect larvae.

    PubMed

    Rosabal, Maikel; Ponton, Dominic E; Campbell, Peter G C; Hare, Landis

    2014-11-01

    We transplanted larvae of the phantom midge Chaoborus punctipennis from a lake having lower concentrations of Cd and Se (Lake Dasserat) to a more contaminated lake (Lake Dufault) located near a metal smelter in Rouyn-Noranda, Quebec. Transplanted individuals were held in mesh mesocosms for up to 16 days where they were fed with indigenous contaminated zooplankton. Larval Cd and Se burdens increased over time, and came to equal those measured in indigenous C. punctipennis from contaminated Lake Dufault. Larval Se burdens increased steadily, whereas those of Cd showed an initial lag phase that we explain by a change in the efficiency with which this insect assimilated Cd from its prey. We measured Cd and Se in subcellular fractions and found that larvae sequestered the majority (60%) of the incoming Cd in a detoxified fraction containing metal-binding proteins, whereas a minority of this nonessential metal was in sensitive fractions (20%). In contrast, a much higher proportion of the essential element Se (40%) was apportioned to metabolically active sensitive fractions. Larvae took up equimolar quantities of these elements over the course of the experiment. Likewise, Cd and Se concentrations in wild larvae were equimolar, which suggests that they are exposed to equimolar bioavailable concentrations of these elements in our study lakes. PMID:25268462

  8. Changes in subcellular distribution of ependymins in goldfish brain induced by learning.

    PubMed

    Schmidt, R

    1987-06-01

    Goldfish were trained for 4 h to swim with an attached polystyrene foam float and tested for retention 3 days later. Intracerebroventricular injection of anti-ependymin antisera was shown to prevent long-term memory formation of this vestibulomotor learning task, as reported previously. In further experiments, fish were killed 4-14 h after the start of training. The brains were dissected, incubated in an isoosmolar solution for collection of proteins of the brain extracellular fluid (ECF), homogenized, and fractionated by differential centrifugation. The ECF, a supernatant fraction enriched in cytoplasmic constituents (S3), and various particulate subcellular fractions were analyzed for their ependymin contents by radioimmunoassay. No statistically significant changes that might be induced by the learning were revealed in any of the particulate fractions. Steady-state concentrations of ependymins in the cytoplasm, however, increased temporarily by 39% in fish that had mastered the training task as compared with nonlearning animals (passive and active controls). In the ECF, the specific concentration of ependymins first decreased to 88% of control levels (4-5 h after the start of training), but later on, it increased to 138% (8-14 h). Apparently, ependymins present in the ECF are used during biochemical reactions of memory consolidation. The resulting decrease in extracellular ependymin concentrations might trigger their resynthesis in the cytoplasm and lead to an increased release of these glycoproteins into the ECF.

  9. Expression and Subcellular Distribution of GFP-Tagged Human Tetraspanin Proteins in Saccharomyces cerevisiae.

    PubMed

    Skaar, Karin; Korza, Henryk J; Tarry, Michael; Sekyrova, Petra; Högbom, Martin

    2015-01-01

    Tetraspanins are integral membrane proteins that function as organizers of multimolecular complexes and modulate function of associated proteins. Mammalian genomes encode approximately 30 different members of this family and remotely related eukaryotic species also contain conserved tetraspanin homologs. Tetraspanins are involved in a number of fundamental processes such as regulation of cell migration, fusion, immunity and signaling. Moreover, they are implied in numerous pathological states including mental disorders, infectious diseases or cancer. Despite the great interest in tetraspanins, the structural and biochemical basis of their activity is still largely unknown. A major bottleneck lies in the difficulty of obtaining stable and homogeneous protein samples in large quantities. Here we report expression screening of 15 members of the human tetraspanin superfamily and successful protocols for the production in S. cerevisiae of a subset of tetraspanins involved in human cancer development. We have demonstrated the subcellular localization of overexpressed tetraspanin-green fluorescent protein fusion proteins in S. cerevisiae and found that despite being mislocalized, the fusion proteins are not degraded. The recombinantly produced tetraspanins are dispersed within the endoplasmic reticulum membranes or localized in granule-like structures in yeast cells. The recombinantly produced tetraspanins can be extracted from the membrane fraction and purified with detergents or the poly (styrene-co-maleic acid) polymer technique for use in further biochemical or biophysical studies. PMID:26218426

  10. Arbuscular Mycorrhizal Colonization Alters Subcellular Distribution and Chemical Forms of Cadmium in Medicago sativa L. and Resists Cadmium Toxicity

    PubMed Central

    Gao, Yanzheng

    2012-01-01

    Some plants can tolerate and even detoxify soils contaminated with heavy metals. This detoxification ability may depend on what chemical forms of metals are taken up by plants and how the plants distribute the toxins in their tissues. This, in turn, may have an important impact on phytoremediation. We investigated the impact of arbuscular mycorrhizal (AM) fungus, Glomus intraradices, on the subcellular distribution and chemical forms of cadmium (Cd) in alfalfa (Medicago sativa L.) that were grown in Cd-added soils. The fungus significantly colonized alfalfa roots by day 25 after planting. Colonization of alfalfa by G. intraradices in soils contaminated with Cd ranged from 17% to 69% after 25–60 days and then decreased to 43%. The biomass of plant shoots with AM fungi showed significant 1.7-fold increases compared to no AM fungi addition under the treatment of 20 mg·kg−1 Cd. Concentrations of Cd in the shoots of alfalfa under 0.5, 5, and 20 mg·kg−1 Cd without AM fungal inoculation are 1.87, 2.92, and 2.38 times higher, respectively, than those of fungi-inoculated plants. Fungal inoculation increased Cd (37.2–80.5%) in the cell walls of roots and shoots and decreased in membranes after 80 days of incubation compared to untreated plants. The proportion of the inactive forms of Cd in roots was higher in fungi-treated plants than in controls. Furthermore, although fungi-treated plants had less overall Cd in subcellular fragments in shoots, they had more inactive Cd in shoots than did control plants. These results provide a basis for further research on plant-microbe symbioses in soils contaminated with heavy metals, which may potentially help us develop management regimes for phytoremediation. PMID:23139811

  11. Nano-hydroxyapatite and nano-titanium dioxide exhibit different subcellular distribution and apoptotic profile in human oral epithelium.

    PubMed

    Tay, Chor Yong; Fang, Wanru; Setyawati, Magdiel Inggrid; Chia, Sing Ling; Tan, Kai Soo; Hong, Catherine Hsu Ling; Leong, David Tai

    2014-05-14

    Nanomaterials (NMs) such as titanium dioxide (nano-TiO2) and hydroxyapatite (nano-HA) are widely used in food, personal care, and many household products. Due to their extensive usage, the risk of human exposure is increased and may trigger NMs specific biological outcomes as the NMs interface with the cells. However, the interaction of nano-TiO2 and nano-HA with cells, their uptake and subcellular distribution, and the cytotoxic effects are poorly understood. Herein, we characterized and examined the cellular internalization, inflammatory response and cytotoxic effects of nano-TiO2 and nano-HA using TR146 human oral buccal epithelial cells as an in vitro model. We showed both types of NMs were able to bind to the cellular membrane and passage into the cells in a dose dependent manner. Strikingly, both types of NMs exhibited distinct subcellular distribution profile with nano-HA displaying a higher preference to accumulate near the cell membrane compared to nano-TiO2. Exposure to both types of NMs caused an elevated reactive oxygen species (ROS) level and expression of inflammatory transcripts with increasing NMs concentration. Although cells treated with nano-HA induces minimal apoptosis, nano-TiO2 treated samples displayed approximately 28% early apoptosis after 24 h of NMs exposure. We further showed that nano-TiO2 mediated cell death is independent of the classical p53-Bax apoptosis pathway. Our findings provided insights into the potential cellular fates of human oral epithelial cells as they interface with industrial grade nano-HA and nano-TiO2.

  12. Nano-hydroxyapatite and nano-titanium dioxide exhibit different subcellular distribution and apoptotic profile in human oral epithelium.

    PubMed

    Tay, Chor Yong; Fang, Wanru; Setyawati, Magdiel Inggrid; Chia, Sing Ling; Tan, Kai Soo; Hong, Catherine Hsu Ling; Leong, David Tai

    2014-05-14

    Nanomaterials (NMs) such as titanium dioxide (nano-TiO2) and hydroxyapatite (nano-HA) are widely used in food, personal care, and many household products. Due to their extensive usage, the risk of human exposure is increased and may trigger NMs specific biological outcomes as the NMs interface with the cells. However, the interaction of nano-TiO2 and nano-HA with cells, their uptake and subcellular distribution, and the cytotoxic effects are poorly understood. Herein, we characterized and examined the cellular internalization, inflammatory response and cytotoxic effects of nano-TiO2 and nano-HA using TR146 human oral buccal epithelial cells as an in vitro model. We showed both types of NMs were able to bind to the cellular membrane and passage into the cells in a dose dependent manner. Strikingly, both types of NMs exhibited distinct subcellular distribution profile with nano-HA displaying a higher preference to accumulate near the cell membrane compared to nano-TiO2. Exposure to both types of NMs caused an elevated reactive oxygen species (ROS) level and expression of inflammatory transcripts with increasing NMs concentration. Although cells treated with nano-HA induces minimal apoptosis, nano-TiO2 treated samples displayed approximately 28% early apoptosis after 24 h of NMs exposure. We further showed that nano-TiO2 mediated cell death is independent of the classical p53-Bax apoptosis pathway. Our findings provided insights into the potential cellular fates of human oral epithelial cells as they interface with industrial grade nano-HA and nano-TiO2. PMID:24734929

  13. Lead tolerance mechanism in Conyza canadensis: subcellular distribution, ultrastructure, antioxidative defense system, and phytochelatins.

    PubMed

    Li, Ying; Zhou, Chuifan; Huang, Meiying; Luo, Jiewen; Hou, Xiaolong; Wu, Pengfei; Ma, Xiangqing

    2016-03-01

    We used hydroponic experiments to examine the effects of different concentrations of lead (Pb) on the performance of the Pb-tolerable plant Conyza canadensis. In these experiments, most of the Pb was accumulated in the roots; there was very little Pb accumulated in stems and leaves. C. canadensis is able to take up significant amounts of Pb whilst greatly restricting its transportation to specific parts of the aboveground biomass. High Pb concentrations inhibited plant growth, increased membrane permeability, elevated antioxidant enzyme activity in roots, and caused a significant increase in root H2O2 and malondialdehyde content. Analysis of Pb content at the subcellular level showed that most Pb was associated with the cell wall fraction, followed by the nucleus-rich fraction, and with a minority present in the mitochondrial and soluble fractions. Furthermore, transmission electron microscopy and energy dispersive X-ray analysis of root cells revealed that the cell wall and intercellular space in C. canadensis roots are the main locations of Pb accumulation. Additionally, high Pb concentrations adversely affected the cellular structure of C. canadensis roots. The increased enzyme activity suggests that the antioxidant system may play an important role in eliminating or alleviating Pb toxicity in C. canadensis roots. However, the levels of non-protein sulfhydryl compounds, glutathione, and phytochelatin did not significantly change in either the roots or leaves under Pb-contaminated treatments. Our results provide strong evidence that cell walls restrict Pb uptake into the root and act as an important barrier protecting root cells, while demonstrating that antioxidant enzyme levels are correlated with Pb exposure. These findings demonstrate the roles played by these detoxification mechanisms in supporting Pb tolerance in C. canadensis. PMID:26733305

  14. Lead tolerance mechanism in Conyza canadensis: subcellular distribution, ultrastructure, antioxidative defense system, and phytochelatins.

    PubMed

    Li, Ying; Zhou, Chuifan; Huang, Meiying; Luo, Jiewen; Hou, Xiaolong; Wu, Pengfei; Ma, Xiangqing

    2016-03-01

    We used hydroponic experiments to examine the effects of different concentrations of lead (Pb) on the performance of the Pb-tolerable plant Conyza canadensis. In these experiments, most of the Pb was accumulated in the roots; there was very little Pb accumulated in stems and leaves. C. canadensis is able to take up significant amounts of Pb whilst greatly restricting its transportation to specific parts of the aboveground biomass. High Pb concentrations inhibited plant growth, increased membrane permeability, elevated antioxidant enzyme activity in roots, and caused a significant increase in root H2O2 and malondialdehyde content. Analysis of Pb content at the subcellular level showed that most Pb was associated with the cell wall fraction, followed by the nucleus-rich fraction, and with a minority present in the mitochondrial and soluble fractions. Furthermore, transmission electron microscopy and energy dispersive X-ray analysis of root cells revealed that the cell wall and intercellular space in C. canadensis roots are the main locations of Pb accumulation. Additionally, high Pb concentrations adversely affected the cellular structure of C. canadensis roots. The increased enzyme activity suggests that the antioxidant system may play an important role in eliminating or alleviating Pb toxicity in C. canadensis roots. However, the levels of non-protein sulfhydryl compounds, glutathione, and phytochelatin did not significantly change in either the roots or leaves under Pb-contaminated treatments. Our results provide strong evidence that cell walls restrict Pb uptake into the root and act as an important barrier protecting root cells, while demonstrating that antioxidant enzyme levels are correlated with Pb exposure. These findings demonstrate the roles played by these detoxification mechanisms in supporting Pb tolerance in C. canadensis.

  15. The effect of selenium on the subcellular distribution of antimony to regulate the toxicity of antimony in paddy rice.

    PubMed

    Ding, Yongzhen; Wang, Ruigang; Guo, Junkang; Wu, Fengchang; Xu, Yingming; Feng, Renwei

    2015-04-01

    Selenium (Se) can alleviate the toxicity of antimony (Sb) in plants; however, the associated mechanisms have not been fully clarified. In this study, we hypothesize that Se can affect the subcellular distribution of Sb to regulate Sb toxicity. To test our hypothesis, two nested hydroponic experiments were performed by using paddy rice (Fengmeizhan). The results showed that Sb exerted toxic effects on the growth of paddy rice, and Se caused beneficial effects that were limited to the shoot growth. In general, Se and Sb mutually showed antagonistic effects on their uptake and concentrations in different subcellular fractions. However, in some cases, the stimulation effects of Sb on the Se concentration in chlorophyll (Chl) and cytosol (Cy) fractions or of Se on the Sb concentration in the cell wall fraction (Cw) were also observed in the shoots, which might suggest that Sb detoxification by Se is also related to the migration of both Se and Sb in cells. Selenium and Sb were primarily concentrated in the Cw and Cy, suggesting the important roles of these two fractions in detoxifying Se and Sb. When paddy rice was subjected to increasing Sb concentrations and a fixed Se concentration, most of the Se in the shoots was sequestered in the Cy (59.81-79.51% of total Se) and more Se was transferred into the inner cell from Cw; however, in the roots, Se was primarily concentrated in the Cw (53.28-72.10%). When paddy rice was exposed to increasing Se concentrations with a fixed Sb concentration, the Cw in both the shoots and roots might play an important role in binding Se, especially in the roots where up to 78.92% of the total Se was sequestered in the Cw.

  16. Changes in chemical forms, subcellular distribution, and thiol compounds involved in Pb accumulation and detoxification in Athyrium wardii (Hook.).

    PubMed

    Zhao, Li; Li, Tingxuan; Yu, Haiying; Chen, Guangdeng; Zhang, Xizhou; Zheng, Zicheng; Li, Jinxing

    2015-08-01

    Athyrium wardii is one of the dominant plant species flourishing on the Pb-Zn mine tailings in Sichuan Province, China. A greenhouse pot experiment was conducted to evaluate the chemical forms, subcellular distribution, and thiol compounds in A. wardii under different Pb treatments. The results showed that plants of the mining ecotype (ME) of A. wardii were more tolerant to Pb than those of the non-mining ecotype (NME) in spite of accumulation of higher Pb concentrations. The Pb concentrations in shoots and roots of the ME were 3.2∼8.6 times and 3.0∼24.6 times higher than those of the NME, respectively. The ME was more efficient in Pb uptake than the NME. Moreover, 27.8∼39.0% of the total Pb in ME was sodium chloride (NaCl) extractable and 38.0∼48.5% was acetic acid (HAc) extractable, whereas only a minority of total Pb was in ethanol and H2O extractable. In subcellular level, 77.4∼88.8% of total Pb was stored in the cell walls of ME and 9.0∼18.9% in soluble fractions. Increasing Pb concentrations enhanced sequestration of Pb into the cell walls and soluble fractions of ME tissues to protect organelles against Pb. Synthesis of non-protein thiols (NP-SH) and phytochelatins (PCs) in roots of ME significantly enhanced in response to Pb stress, and significant increases in glutathione (GSH) were observed in shoots of ME. Higher levels of NP-SH, GSH, and PCs were observed in roots of the ME comparing with NME, especially under high Pb treatments. The results indicated that Pb was localized mainly in cell wall and soluble fraction of ME plants with low biological activity by cell wall deposition and vacuolar compartmentalization, which might be the important adapted Pb detoxification mechanisms of ME. PMID:25913310

  17. Quantitative evaluation of berberine subcellular distribution and cellular accumulation in non-small cell lung cancer cells by UPLC-MS/MS.

    PubMed

    Yuan, Zhong-Wen; Leung, Elaine Lai-Han; Fan, Xing-Xing; Zhou, Hua; Ma, Wen-Zhe; Liu, Liang; Xie, Ying

    2015-11-01

    Berberine, an isoquinoline alkaloid, has been demonstrated to be a safe anti-cancer agent with multiple effects on mitochondria. Intracellular concentration and distribution around the targeting sites are determinants of efficacy, but subcellular distribution of berberine has not been fully elucidated yet, which relies on the sensitive and robustness assay. In this study, a sensitive and robust UPLC-MS/MS method has been developed and validated with optimized extraction solvents and detection conditions. Key factors such as the purity and integrity of isolated organelle fractions, and the effects of isolation procedures on the subcellular concentration of berberine were systemically evaluated. With the developed assay, we found that the intracellular accumulations of berberine in two gefitinib resistant NSCLC cell lines H1650 and H1975 were 2-3 folds higher than that of normal epithelial cells BEAS-2B. Moreover, significantly different subcellular distribution profiles in NSCLC cancer cells from that of BEAS-2B cells with a striking increase in content in most organelles may contribute to its selective cytotoxicity to cancer cells. Furthermore, a predominant accumulation of berberine was observed for the first time in microsomal fraction for all three cell lines. Therefore, this method could be used for quantitative evaluation of subcellular distribution and cellular accumulation of berberine and for further evaluation of the concentration-effects relationship.

  18. Cellular and subcellular distributions of delta opioid receptor activation sites in the ventral oral pontine tegmentum of the cat.

    PubMed

    Alvira-Botero, Maria Ximena; Garzón, Miguel

    2006-12-01

    The ventral division of the reticular oral pontine nucleus (vRPO) is a pontine tegmentum region critically involved in REM sleep generation. Previous reports of morphine microinjections in the cat pontine tegmentum have shown that opioid receptor activation in this region modulates REM sleep. Even though opiate administration has marked effects on sleep-wake cycle architecture, the distribution of opioid receptors in vRPO has only been partially described. Using an antiserum directed against delta opioid receptor (DOR), to which morphine binds, in the present study, we use (1) light microscopy to determine DOR cellular distribution in the rostral pontine tegmentum and (2) electron microscopy to determine DOR subcellular distribution in the cat vRPO. In the dorsal pons, DOR immunoreactivity was evenly distributed throughout the neuropil of the reticular formation and was particularly intense in the parabrachial nuclei and locus coeruleus; the ventral and central areas of the RPO and locus coeruleus complex were especially rich in DOR-labeled somata. Within the vRPO, DOR was localized mainly in the cytoplasm and on plasma membranes of medium to large dendrites (47.8% of DOR-labeled profiles), which received both symmetric and asymmetric synaptic contacts mainly from non-labeled (82% of total inputs) axon terminals. Less frequently, DOR was distributed presynaptically in axon terminals (19% of DOR-labeled profiles). Our results suggest that DOR activation in vRPO regulates REM sleep occurrence by modulating postsynaptic responses to both excitatory and inhibitory afferents. DOR activation in vRPO could have, however, an additional role in direct modulation of neurotransmitter release from axon terminals.

  19. Arsenic species uptake and subcellular distribution in Vallisneria natans (Lour.) Hara as influenced by aquatic pH.

    PubMed

    Chen, Guoliang; Liu, Xingmei; Xu, Jianming; Brookes, Philip C; Wu, Jianjun

    2014-04-01

    Vallisneria natans (Lour.) Hara, a widely distributed submerged aquatic plant, is a promising species for arsenic (As) removal from contaminated water. We investigated the effects of pH on the accumulation, subcellular distribution and detoxification of As in V. natans. The results showed that the optimum pH for submerged V. natans growth is close to 7.0. The accumulation of As in the plant increased with the increase of pH (p < 0.05). This may have been due to arsenic/phosphate transporters with a higher affinity for the more highly electronegative AsO4 (3-) than for HAsO4 (2-) and H2AsO4 (-). After As(V) was accumulated by plants, more than 80 % was reduced to As(III), but As reduction decreased with increased pH. The majority of accumulated As and reduced As(III) (47 %-66 %) was found in the vacuoles. Higher As concentrations in vacuoles could be considered as an important mechanism for As detoxification in submerged plants. PMID:24420344

  20. Subcellular distribution of small interfering RNA: directed delivery through RNA polymerase III expression cassettes and localization by in situ hybridization.

    PubMed

    Paul, Cynthia P

    2005-01-01

    Reduction in the expression of specific genes through small interfering RNAs (siRNAs) is dependent on the colocalization of siRNAs with other components of the RNA interference (RNAi) pathways within the cell. The expression of siRNAs within cells from cassettes that are derived from genes transcribed by RNA polymerase III (pol III) and provide for selective subcellular distribution of their products can be used to direct siRNAs to the cellular pathways. Expression from the human U6 promoter, resulting in siRNA accumulation in the nucleus, is effective in reducing gene expression, whereas cytoplasmic and nucleolar localization of the siRNA when expressed from the 5S or 7 SL promoters is not effective. The distribution of siRNA within the cell is determined by fluorescence in situ hybridization. Although the long uninterrupted duplex of siRNA makes it difficult to detect with DNA oligonucleotide probes, labeled oligonucleotide probes with 2'-O-methyl RNA backbones provide the stability needed for a strong signal. These methods contribute to studies of the interconnected cellular RNAi pathways and are useful in adapting RNAi as a tool to determine gene function and develop RNA-based therapeutics. PMID:15644179

  1. Subcellular distribution and uptake mechanism of di-n-butyl phthalate in roots of pumpkin (Cucurbita moschata) seedlings.

    PubMed

    Lin, Qingqi; Yang, Xiuhong; Huang, Xiongfei; Wang, Shizhong; Chao, Yuanqing; Qiu, Rongliang

    2016-01-01

    Phthalate acid esters (PAEs) are of particular concern due to their potential environmental risk to human and nonhuman organisms. Although uptake of PAEs by plants has been reported by several researchers, information about the intracellular distribution and uptake mechanisms of PAEs is still lacking. In this study, a series of hydroponic experiments using intact pumpkin (Cucurbita moschata) seedlings was conducted to investigate how di-n-butyl phthalate (DnBP), one of the most frequently identified PAEs in the environment, enters and is distributed in roots. DnBP was transported into subcellular tissues rapidly in the initial uptake period (<12 h). More than 80% of DnBP was detected in the cell walls and organelles, which suggests that DnBP is primarily accumulated in these two fractions due to their high affinity to DnBP. The kinetics of DnBP uptake were fitted well with the Michaelis-Menten equation, suggesting that a carrier-mediated process was involved. The application of 2,4-dinitrophenol and sodium vanadate reduced the uptake of DnBP by 37 and 26%, respectively, while aquaporin inhibitors, silver and glycerol, had no effect on DnBP uptake. These data demonstrated that the uptake of DnBP included a carrier-mediated and energy-dependent process without the participation of aquaporins.

  2. Deposition pattern and subcellular distribution of disease-associated prion protein in cerebellar organotypic slice cultures infected with scrapie

    PubMed Central

    Wolf, Hanna; Hossinger, André; Fehlinger, Andrea; Büttner, Sven; Sim, Valerie; McKenzie, Debbie; Vorberg, Ina M.

    2015-01-01

    Organotypic cerebellar slices represent a suitable model for characterizing and manipulating prion replication in complex cell environments. Organotypic slices recapitulate prion pathology and are amenable to drug testing in the absence of a blood-brain-barrier. So far, the cellular and subcellular distribution of disease-specific prion protein in organotypic slices is unclear. Here we report the simultaneous detection of disease-specific prion protein and central nervous system markers in wild-type mouse cerebellar slices infected with mouse-adapted prion strain 22L. The disease-specific prion protein distribution profile in slices closely resembles that in vivo, demonstrating granular spot like deposition predominately in the molecular and Purkinje cell layers. Double immunostaining identified abnormal prion protein in the neuropil and associated with neurons, astrocytes and microglia, but absence in Purkinje cells. The established protocol for the simultaneous immunohistochemical detection of disease-specific prion protein and cellular markers enables detailed analysis of prion replication and drug efficacy in an ex vivo model of the central nervous system. PMID:26581229

  3. Interaction of cadmium and zinc on accumulation and sub-cellular distribution in leaves of hyperaccumulator Potentilla griffithii.

    PubMed

    Qiu, Rong-Liang; Thangavel, Palaniswamy; Hu, Peng-Jie; Senthilkumar, Palaninaicker; Ying, Rong-Rong; Tang, Ye-Tao

    2011-02-28

    Potentilla griffithii Hook is a newly found hyperaccumulator plant capable of high tolerance and accumulation of Zn and Cd. We investigated the interactive effects between Cd and Zn on accumulation and vacuolar sequestration in P. griffithii. Stimulatory effect of growth was noted at 0.2 mM Cd and 1.25 and 2.5 mM Zn tested. Accumulation of Zn and Cd in roots, petioles and leaves were increased significantly with addition of these metals individually. However, the Zn supplement decreased root Cd accumulation but increased the concentration of Cd in petioles and leaves. The results from sub-cellular distribution showed that up to 94% and 70% of the total Zn and Cd in the leaves were present in the protoplasts, and more than 90% Cd and Zn in the protoplasts were localized in the vacuoles. Nearly, 88% and 85% of total Cd and Zn were extracted in the cell sap of the leaves suggesting that most of the Cd and Zn in the leaves were available in soluble form. The present results indicate that Zn supplement significantly enhanced the petiole accumulation of Cd and further vacuolar sequestration plays an important role in tolerance, detoxification and hyperaccumulation of these metals in P. griffithii. PMID:21211902

  4. Cellular Distribution and Subcellular Localization of Molecular Components of Vesicular Transmitter Release in Horizontal Cells of Rabbit Retina

    PubMed Central

    HIRANO, ARLENE A.; BRANDSTÄTTER, JOHANN H.; BRECHA, NICHOLAS C.

    2010-01-01

    The mechanism underlying transmitter release from retinal horizontal cells is poorly understood. We investigated the possibility of vesicular transmitter release from mammalian horizontal cells by examining the expression of synaptic proteins that participate in vesicular transmitter release at chemical synapses. Using immunocytochemistry, we evaluated the cellular and subcellular distribution of complexin I/II, syntaxin-1, and synapsin I in rabbit retina. Strong labeling for complexin I/II, proteins that regulate a late step in vesicular transmitter release, was found in both synaptic layers of the retina, and in somata of A- and B-type horizontal cells, of γ-aminobutyric acid (GABA)- and glycinergic amacrine cells, and of ganglion cells. Immunoelectron microscopy demonstrated the presence of complexin I/II in horizontal cell processes postsynaptic to rod and cone ribbon synapses. Syntaxin-1, a core protein of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) complex known to bind to complexin, and synapsin I, a synaptic vesicle-associated protein involved in the Ca2+-dependent recruitment of synaptic vesicles for transmitter release, were also present in the horizontal cells and their processes at photoreceptor synapses. Photoreceptors and bipolar cells did not express any of these proteins at their axon terminals. The presence of complexin I/II, syntaxin-1, and synapsin I in rabbit horizontal cell processes and tips suggests that a vesicular mechanism may underlie transmitter release from mammalian horizontal cells. PMID:15912504

  5. Subcellular Distribution of Heavy Metals in Organs of Bivalve Modiolus Modiolus Living Along a Metal Contamination Gradient

    NASA Astrophysics Data System (ADS)

    Podgurskaya, Olga V.; Kavun, Victor Ya.

    2006-03-01

    Concentration and distribution of Fe, Zn, Cu, Cd, Mn, Pb, Ni among subcellular fractions (cellular membrane structures and cytosol) and Zn, Cu, Cd among cytoplasmic proteins in the kidney and digestive gland of mussel Modiolus modiolus living along a polymetallic concentration gradient were studied. It was found in the kidney of M. modiolus from contaminated sites that the Fe percent increased in the “membrane” fraction, whereas Zn, Pb, Ni and Mn percent increased in the cytosol compared to the kidney of the control mussel. Note kidney cytosol of M. modiolus from clean and contaminated sites sequestered major parts of Cu and Cd. In the digestive gland of M. modiolus from contaminated sites Fe, Zn, Cd, Mn, Ni percent increased in the “membrane” fraction, whereas Cu, Pb percent increased in the cytosol compared to digestive gland of control mussel. Gel-filtration chromatography shows kidney of M. modiolus contains increased metallothionein-like protein levels irrespective of ambient dissolved metal concentrations. It was shown that the metal detoxification system in the kidney and digestive gland of M. modiolus was efficient under extremely high ambient metal levels. However, under complex environmental contamination in the kidney of M. modiolus, the metal detoxification capacity of metallothionein-like proteins was damaged.

  6. Influence of metal exposure history on the bioaccumulation and subcellular distribution of aqueous cadmium in the insect Hydropsyche californica

    USGS Publications Warehouse

    Cain, D.J.; Buchwalter, D.B.; Luoma, S.N.

    2006-01-01

    The influence of metal exposure history on rates of aqueous Cd accumulation, elimination, and subcellular distribution was examined in the aquatic insect Hydropsyche californica. Specimens were obtained from a reference site and a metal-contaminated site and returned to the laboratory where they were continuously exposed to aqueous Cd (518 ng/L, nominal) for 6 d, followed by 9 d of depuration. Rates of Cd accumulation and elimination were similar in insects from the two sites. Efflux rate constants, ke, ranged from 0.20 to 0.24/d (t1/2 ??? 3 d). Immediately following exposure, the cytosol accounted for 40% of the body burden in insects from both sites; however, 89 ?? 2% of the cytosolic Cd was associated with metallothionein-like proteins (MTLP) in insects from the contaminated site, compared to 60 ?? 0% in insects from the reference site. The concentration of Cd bound to non-MTLPs (representing potentially Cd-sensitive proteins) was significantly greater in the insects from the reference site (134 ?? 7 ng/g) than in those from the contaminated site (42 ?? 2 ng/g). At the end of the depuration period, 90% of the accumulated Cd body burden had been eliminated, and Cd concentrations in MTLPs and non-MTLPs were similar between the sites. Results suggested that differences in exposure history had no influence on the bioaccumulation of Cd, but did affect the concentrations of Cd bound to MTLP during Cd exposure in these insects. ?? 2006 SETAC.

  7. Transmembrane topology, subcellular distribution and turnover of the gamma-aminobutyric acid/benzodizaepine receptor in chick brain cell cultures

    SciTech Connect

    Czajkowski, C.M.

    1987-01-01

    Experiments were performed utilizing trypsinization of the GABA/BZD-R in intact cells to determine (1) the subcellular distribution of membrane-associated GABA/BZD-Rs and (2) aspects of the transmembrane topology of the BZD-R. Additionally, R07-0213, a positively charged benzodiazepine, was used to distinguish between cell surface and intracellular BZD-Rs. Following trypsin treatment of intact cells a cleaved receptor fragment of M{sub r} = 24,000 (xRF24) is generated. It remains anchored in the plasma membrane and not only retains the ability to bind ({sup 3}H)flunitrazepan reversibly and irreversibly but also retains the ability to be modulated by GABA. xRF24 is not observed following trypsinization of saponin-treated cells or cell homogenates, indicating that it has a cytoplasmic domain as well as a cell surface domain, as expected for a transmembrane fragment of the BZD-R. By utilizing ({sup 3}H)flunitrazepam as an irreversible photoaffinity label, BZD-R turnover was also investigated.

  8. Immunocytochemical analysis of the subcellular distribution of ferritin in Imperata cylindrica (L.) Raeuschel, an iron hyperaccumulator plant.

    PubMed

    de la Fuente, Vicenta; Rodríguez, Nuria; Amils, Ricardo

    2012-05-01

    Ferritin is of interest at the structural and functional level not only as storage for iron, a critical element, but also as a means to prevent cell damage produced by oxidative stress. The main objective of this work was to confirm by immunocytochemistry the presence and the subcellular distribution of the ferritin detected by Mösbauer spectroscopy in Imperata cylindrica, a plant which accumulates large amounts of iron. The localization of ferritin was performed in epidermal, parenchymal and vascular tissues of shoots and leaves of I. cylindrica. The highest density of immunolabeling in shoots appeared in the intracellular space of cell tissues, near the cell walls and in the cytoplasm. In leaves, ferritin was detected in the proximity of the dense network of the middle lamella of cell walls, following a similar path to that observed in shoots. Immunolabeling was also localized in chloroplasts. The abundance of immunogold labelling in mitochondria for I. cylindrica was rather low, probably because the study dealt with tissues from old plants. These results further expand the localization of ferritin in cell components other than chloroplasts and mitochondria in plants. PMID:21764425

  9. Subcellular cadmium distribution and antioxidant enzymatic activities in the leaves of two castor (Ricinus communis L.) cultivars exhibit differences in Cd accumulation.

    PubMed

    Zhang, Hanzhi; Guo, Qingjun; Yang, Junxing; Shen, Jianxiu; Chen, Tongbin; Zhu, Guangxu; Chen, Hui; Shao, Chunyan

    2015-10-01

    The aims of this study were: (1) the study of cadmium (Cd) accumulation and toxicity in different castor cultivars (Ricinus communis L.); (2) to investigate changes in antioxidant enzymatic activities and the subcellular distribution of Cd in young and old leaves from two different castor cultivars, after exposure to two different Cd concentrations, and explore the underlying mechanism of Cd detoxification focusing on antioxidant enzymes and subcellular compartmentalization. The Cd concentration, toxicity, and subcellular distribution, as well as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activities were measured in Zibo-3 and Zibo-9 cultivars after exposure to two different concentrations of Cd (2mg/L and 5mg/L) for 10 days. This research revealed Cd accumulation characteristics in castor are root>stem>young leaf>old leaf. Castor tolerance was Cd dose exposure and the cultivars themselves dependent. Investigation of subcellular Cd partitioning showed that Cd accumulated mainly in the heat stable protein (HSP) and cellular debris fractions, followed by the Cd rich granule (MRG), heat denatured protein (HDP), and organelle fractions. With increasing Cd concentration in nutrient solution, the decreased detoxified fractions (BDM) and the increased Cd-sensitive fractions (MSF) in young leaves may indicate the increased Cd toxicity in castor cultivars. The BDM-Cd fractions or MSF-Cd in old leaves may be linked with Cd tolerance of different cultivars of castor. The antioxidant enzymes that govern Cd detoxification were not found to be active in leaves. Taken together, these results indicate Cd tolerance and toxicity in castor can be explained by subcellular partitioning.

  10. Intracellular uptake, trafficking and subcellular distribution of folate conjugated single walled carbon nanotubes within living cells

    NASA Astrophysics Data System (ADS)

    Kang, Bin; Yu, De-cai; Chang, Shu-quan; Chen, Da; Dai, Yao-dong; Ding, Yitao

    2008-09-01

    Herein we studied the uptake, trafficking and distribution of folate conjugated single walled carbon nanotubes (SWNTs) within living cells. SWNTs were noncovalently functionalized with chitosan and then linked with folate acid and fluorescence dye Alexa Fluor 488 (denoted FA-SWNTs). Hep G2 cells were cultured in vitro and incubated with FA-SWNTs at different levels. The FA-SWNTs exhibited a concentration-dependent uptake within Hep G2 cells, and Hep G2 cells were able to internalize FA-SWNTs via a folate receptor-mediated pathway. The distribution of nanotubes inside cells demonstrated that the FA-SWNTs only locate in the cytoplasm and not in nuclei, indicating the failure of transporting through the nuclear envelope. Transmission electron microscope (TEM) results showed the presence of FA-SWNTs in lysosomes and the discharge to extracellular space after incubation with nanotubes for 5 h. No obvious cellular death rate was observed when the concentration of nanotubes was below 50 µg ml-1. However, cells with FA-SWNT uptake showed a concentration-dependent apoptosis. These discoveries might be helpful for understanding the interaction of SWNTs and living cells.

  11. The subcellular distribution and properties of aldehyde dehydrogenases in rat liver

    PubMed Central

    Tottmar, S. O. C.; Pettersson, H.; Kiessling, K.-H.

    1973-01-01

    1. Kinetic experiments suggested the possible existence of at least two different NAD+-dependent aldehyde dehydrogenases in rat liver. Distribution studies showed that one enzyme, designated enzyme I, was exclusively localized in the mitochondria and that another enzyme, designated enzyme II, was localized in both the mitochondria and the microsomal fraction. 2. A NADP+-dependent enzyme was also found in the mitochondria and the microsomal fraction and it is suggested that this enzyme is identical with enzyme II. 3. The Km for acetaldehyde was apparently less than 10μm for enzyme I and 0.9–1.7mm for enzyme II. The Km for NAD+ was similar for both enzymes (20–30μm). The Km for NADP+ was 2–3mm and for acetaldehyde 0.5–0.7mm for the NADP+-dependent activity. 4. The NAD+-dependent enzymes show pH optima between 9 and 10. The highest activity was found in pyrophosphate buffer for both enzymes. In phosphate buffer there was a striking difference in activity between the two enzymes. Compared with the activity in pyrophosphate buffer, the activity of enzyme II was uninfluenced, whereas the activity of enzyme I was very low. 5. The results are compared with those of earlier investigations on the distribution of aldehyde dehydrogenase and with the results from purified enzymes from different sources. PMID:4149764

  12. Intracellular Trafficking and Subcellular Distribution of a Large Array of HPMA Copolymers

    PubMed Central

    Callahan, Jon; Kopečková, Pavla; Kopeček, Jindřich

    2011-01-01

    The basic physicochemical properties that determine the distribution and fate of synthetic macromolecules in living cells were characterized using fluorescently labeled HPMA (N-(2-hydroxypropyl)methacrylamide) copolymers. Twelve different classes of water-soluble copolymers were created by incorporating eight different functionalized comonomers. These comonomers possessed functional groups with positive or negative charges or contained short hydrophobic peptides. The copolymers were fractionated to create parallel “ladders” consisting of 10 fractions of narrow polydispersity with molecular weights ranging from 10 to 200 kDa. The intracellular distributions were characterized for copolymer solutions microinjected into the cytoplasm of cultured ovarian carcinoma cells. Even the highest molecular weight HPMA copolymers were shown to quickly and evenly diffuse throughout the cytoplasm and remain excluded from membrane-bound organelles, regardless of composition. The exceptions were the strongly cationic copolymers, which demonstrated a pronounced localization to microtubules. For all copolymers, nuclear entry was consistent with passive transport through the nuclear pore complex (NPC). Nuclear uptake was shown to be largely dictated by the molecular weight of the copolymers, however, detailed kinetic analyses showed that nuclear import rates were moderately, but significantly, affected by differences in comonomer composition. HPMA copolymers containing amide-terminated phenylalanine-glycine (FG) sequences, analogous to those found in the NPC channel protein, demonstrated a potential to regulate import to the nuclear compartment. Kinetic analyses showed that 15 kDa copolymers containing GGFG, but not those containing GGLFG, peptide pendant groups altered the size-exclusion characteristics of NPC-mediated nuclear import. PMID:21197960

  13. Intracellular trafficking and subcellular distribution of a large array of HPMA copolymers.

    PubMed

    Callahan, Jon; Kopečkov, Pavla; Kopeček, Jindřich

    2009-07-13

    The basic physicochemical properties that determine the distribution and fate of synthetic macromolecules in living cells were characterized using fluorescently labeled HPMA (N-(2-hydroxypropyl)methacrylamide) copolymers. Twelve different classes of water-soluble copolymers were created by incorporating eight different functionalized comonomers. These comonomers possessed functional groups with positive or negative charges or contained short hydrophobic peptides. The copolymers were fractionated to create parallel "ladders" consisting of 10 fractions of narrow polydispersity with molecular weights ranging from 10 to 200 kDa. The intracellular distributions were characterized for copolymer solutions microinjected into the cytoplasm of cultured ovarian carcinoma cells. Even the highest molecular weight HPMA copolymers were shown to quickly and evenly diffuse throughout the cytoplasm and remain excluded from membrane-bound organelles, regardless of composition. The exceptions were the strongly cationic copolymers, which demonstrated a pronounced localization to microtubules. For all copolymers, nuclear entry was consistent with passive transport through the nuclear pore complex (NPC). Nuclear uptake was shown to be largely dictated by the molecular weight of the copolymers, however, detailed kinetic analyses showed that nuclear import rates were moderately, but significantly, affected by differences in comonomer composition. HPMA copolymers containing amide-terminated phenylalanine-glycine (FG) sequences, analogous to those found in the NPC channel protein, demonstrated a potential to regulate import to the nuclear compartment. Kinetic analyses showed that 15 kDa copolymers containing GGFG, but not those containing GGLFG, peptide pendant groups altered the size-exclusion characteristics of NPC-mediated nuclear import. PMID:21197960

  14. Asymmetric subcellular mRNA distribution correlates with carbonic anhydrase activity in Acetabularia acetabulum.

    PubMed

    Serikawa, K A; Porterfield, D M; Mandoli, D F

    2001-02-01

    The unicellular green macroalga Acetabularia acetabulum L. Silva is an excellent system for studying regional differentiation within a single cell. In late adults, physiologically mediated extracellular alkalinity varies along the long axis of the alga with extracellular pH more alkaline along the apical and middle regions of the stalk than at and near the rhizoid. Respiration also varies with greater respiration at and near the rhizoid than along the stalk. We hypothesized that the apical and middle regions of the stalk require greater carbonic anhydrase (CA) activity to facilitate inorganic carbon uptake for photosynthesis. Treatment of algae with the CA inhibitors acetazolamide and ethoxyzolamide decreased photosynthetic oxygen evolution along the stalk but not at the rhizoid, indicating that CA facilitates inorganic carbon uptake in the apical portions of the alga. To examine the distribution of enzymatic activity within the alga, individuals were dissected into apical, middle, and basal tissue pools and assayed for both total and external CA activity. CA activity was greatest in the apical portions. We cloned two CA genes (AaCA1 and AaCA2). Northern analysis demonstrated that both genes are expressed throughout much of the life cycle of A. acetabulum. AaCA1 mRNA first appears in early adults. AaCA2 mRNA appears in juveniles. The AaCA1 and AaCA2 mRNAs are distributed asymmetrically in late adults with highest levels of each in the apical portion of the alga. mRNA localization and enzyme activity patterns correlate for AaCA1 and AaCA2, indicating that mRNA localization is one mechanism underlying regional differentiation in A. acetabulum.

  15. Subcellular distribution of small GTP binding proteins in pancreas: Identification of small GTP binding proteins in the rough endoplasmic reticulum

    SciTech Connect

    Nigam, S.K. )

    1990-02-01

    Subfractionation of a canine pancreatic homogenate was performed by several differential centrifugation steps, which gave rise to fractions with distinct marker profiles. Specific binding of guanosine 5{prime}-({gamma}-({sup 35}S)thio)triphosphate (GTP({gamma}-{sup 35}S)) was assayed in each fraction. Enrichment of GTP({gamma}-{sup 35}S) binding was greatest in the interfacial smooth microsomal fraction, expected to contain Golgi and other smooth vesicles. There was also marked enrichment in the rough microsomal fraction. Electron microscopy and marker protein analysis revealed the rough microsomes (RMs) to be highly purified rough endoplasmic reticulum (RER). The distribution of small (low molecular weight) GTP binding proteins was examined by a ({alpha}-{sup 32}P)GTP blot-overlay assay. Several apparent GTP binding proteins of molecular masses 22-25 kDa were detected in various subcellular fractions. In particular, at least two such proteins were found in the Golgi-enriched and RM fractions, suggesting that these small GTP binding proteins were localized to the Golgi and RER. To more precisely localize these proteins to the RER, native RMs and RMs stripped of ribosomes by puromycin/high salt were subjected to isopycnic centrifugation. The total GTP({gamma}-{sup 35}S) binding, as well as the small GTP binding proteins detected by the ({alpha}-{sup 32}P)GTP blot overlay, distributed into fractions of high sucrose density, as did the RER marker ribophorin I. Consistent with a RER localization, when the RMS were stripped of ribosomes and subjected to isopycnic centrifugation, the total GTP({gamma}-{sup 35}S) binding and the small GTP binding proteins detected in the blot-overlay assay shifted to fractions of lighter sucrose density along with the RER marker.

  16. Quantitative immunofluorescence microscopy of subcellular GLUT4 distribution in human skeletal muscle: effects of endurance and sprint interval training

    PubMed Central

    Bradley, Helen; Shaw, Christopher S.; Worthington, Philip L.; Shepherd, Sam O.; Cocks, Matthew; Wagenmakers, Anton J. M.

    2014-01-01

    Abstract Increases in insulin‐mediated glucose uptake following endurance training (ET) and sprint interval training (SIT) have in part been attributed to concomitant increases in glucose transporter 4 (GLUT4) protein content in skeletal muscle. This study used an immunofluorescence microscopy method to investigate changes in subcellular GLUT4 distribution and content following ET and SIT. Percutaneous muscle biopsy samples were taken from the m. vastus lateralis of 16 sedentary males in the overnight fasted state before and after 6 weeks of ET and SIT. An antibody was fully validated and used to show large (> 1 μm) and smaller (<1 μm) GLUT4‐containing clusters. The large clusters likely represent trans‐Golgi network stores and the smaller clusters endosomal stores and GLUT4 storage vesicles (GSVs). Density of GLUT4 clusters was higher at the fibre periphery especially in perinuclear regions. A less dense punctate distribution was seen in the rest of the muscle fibre. Total GLUT4 fluorescence intensity increased in type I and type II fibres following both ET and SIT. Large GLUT4 clusters increased in number and size in both type I and type II fibres, while the smaller clusters increased in size. The greatest increases in GLUT4 fluorescence intensity occurred within the 1 μm layer immediately adjacent to the PM. The increase in peripheral localisation and protein content of GLUT4 following ET and SIT is likely to contribute to the improvements in glucose homeostasis observed after both training modes. PMID:25052490

  17. Cdk5 promotes synaptogenesis by regulating the subcellular distribution of the MAGUK family member CASK

    PubMed Central

    Samuels, Benjamin Adam; Hsueh, Yi-Ping; Shu, Tianzhi; Liang, Haoya; Tseng, Huang-Chun; Hong, Chen-Jei; Su, Susan C; Volker, Janet; Neve, Rachael L; Yue, David T; Tsai, Li-Huei

    2007-01-01

    Synaptogenesis is a highly regulated process that underlies formation of neural circuitry. Considerable work has demonstrated the capability of some adhesion molecules, such as SynCAM and Neurexins/Neuroligins, to induce synapse formation in vitro. Furthermore, Cdk5 gain-of-function results in an increased number of synapses in vivo. To gain a better understanding of how Cdk5 might promote synaptogenesis, we investigated potential crosstalk between Cdk5 and the cascade of events mediated by synapse-inducing proteins. One protein recruited to developing terminals by SynCAM and Neurexins/Neuroligins is the MAGUK family member CASK. We found that Cdk5 phosphorylates and regulates CASK distribution to membranes. In the absence of Cdk5-dependent phosphorylation, CASK is not recruited to developing synapses and thus fails to interact with essential presynaptic components. Functional consequences include alterations in calcium influx. Mechanistically, Cdk5 regulates the interaction between CASK and liprin-α. These results provide a molecular explanation of how Cdk5 can promote synaptogenesis. PMID:18054859

  18. Tissue and subcellular distribution of glucokinase in rat liver and their changes during fasting-refeeding.

    PubMed

    Toyoda, Y; Miwa, I; Kamiya, M; Ogiso, S; Nonogaki, T; Aoki, S; Okuda, J

    1995-01-01

    The distribution of glucokinase in rat liver under both normal feeding and fasting-refeeding conditions was investigated immunohistochemically. Under normal feeding conditions, glucokinase immunoreactivity was observed in both nuclei and cytoplasm of parenchymal cells. The nuclei were stained intensely and evenly, whereas the cytoplasm showed weak immunoreactivity of different degrees of staining intensity depending on the location of the cells. The cytoplasm of perivenous hepatocytes was stained more intensely, though not so much more, than that of periportal hepatocytes. The cytoplasm of hepatocytes surrounding the terminal hepatic venule (THV), of hepatocytes surrounding the portal triad, and of some other hepatocytes showed a stronger immunoreactivity than that of residual hepatocytes. The nuclear immunoreactivity in hepatocytes surrounding the portal triad and in some other hepatocytes was weak or absent, and positive immunoreactivity was detected at the plasma membrane of some of these cells. After 72 h of fasting, glucokinase immunoreactivity was markedly decreased in all hepatocytes. After the start of refeeding, the cytoplasmic immunoreactivity began to increase first in the parenchymal cells surrounding the THV and extended to those in the intermediate zone followed by those in the periportal zone. In contrast, the increase in nuclear immunoreactivity started in hepatocytes situated in the intermediate zone adjacent to the perivenous zone and then extended to those in the perivenous zone followed by those in the periportal zone. Hepatocytes surrounding either THV or portal triad showed a distinctive change in immunoreactivity during the refeeding period. After 10 h of refeeding, strong immunoreactivity was observed in both the cytoplasm and the nuclei of all hepatocytes, and appreciable glucokinase immunoreactivity was detected at the plasma membrane of some hepatocytes. These findings are discussed from the standpoint of a functional role of glucokinase

  19. Subcellular Size

    PubMed Central

    Marshall, Wallace F.

    2016-01-01

    All of the same conceptual questions about size in organisms apply equally at the level of single cells. What determines the size, not only of the whole cell, but of all its parts? What ensures that subcellular components are properly proportioned relative to the whole cell? How does alteration in organelle size affect biochemical function? Answering such fundamental questions requires us to understand how the size of individual organelles and other cellular structures is determined. Knowledge of organelle biogenesis and dynamics has advanced rapidly in recent years. Does this knowledge give us enough information to formulate reasonable models for organelle size control, or are we still missing something? PMID:25957302

  20. The UL24 protein of herpes simplex virus 1 affects the sub-cellular distribution of viral glycoproteins involved in fusion

    SciTech Connect

    Ben Abdeljelil, Nawel; Rochette, Pierre-Alexandre; Pearson, Angela

    2013-09-15

    Mutations in UL24 of herpes simplex virus type 1 can lead to a syncytial phenotype. We hypothesized that UL24 affects the sub-cellular distribution of viral glycoproteins involved in fusion. In non-immortalized human foreskin fibroblasts (HFFs) we detected viral glycoproteins B (gB), gD, gH and gL present in extended blotches throughout the cytoplasm with limited nuclear membrane staining; however, in HFFs infected with a UL24-deficient virus (UL24X), staining for the viral glycoproteins appeared as long, thin streaks running across the cell. Interestingly, there was a decrease in co-localized staining of gB and gD with F-actin at late times in UL24X-infected HFFs. Treatment with chemical agents that perturbed the actin cytoskeleton hindered the formation of UL24X-induced syncytia in these cells. These data support a model whereby the UL24 syncytial phenotype results from a mislocalization of viral glycoproteins late in infection. - Highlights: • UL24 affects the sub-cellular distribution of viral glycoproteins required for fusion. • Sub-cellular distribution of viral glycoproteins varies in cell-type dependent manner. • Drugs targeting actin microfilaments affect formation of UL24-related syncytia in HFFs.

  1. Analysis of sublethal arsenic toxicity to Ceratophyllum demersum: subcellular distribution of arsenic and inhibition of chlorophyll biosynthesis

    PubMed Central

    Mishra, Seema; Alfeld, Matthias; Sobotka, Roman; Andresen, Elisa; Falkenberg, Gerald; Küpper, Hendrik

    2016-01-01

    Arsenic (As) pollution is a serious concern worldwide. Recent studies under environmentally relevant conditions revealed that, in the aquatic plant Ceratophyllum demersum, pigments are the first observable target of toxicity, prior to any effect on photosynthetic parameters or to oxidative stress. Lethal toxicity was initiated by a change of As species and their distribution pattern in various tissues. Here, the localization of As was investigated at the subcellular level through X-ray fluorescence using a submicron beam and a Maia detector. Further, it was possible to obtain useful tissue structural information from the ratio of the tomogram of photon flux behind the sample to the tomogram of Compton scattering. The micro-X-ray fluorescence tomograms showed that As predominantly accumulated in the nucleus of the epidermal cells in young mature leaves exposed to sublethal 1 µM As. This suggests that As may exert toxic effects in the nucleus, for example, by interfering with nucleic acid synthesis by replacing phosphorous with As. At higher cellular concentrations, As was mainly stored in the vacuole, particularly in mature leaves. An analysis of precursors of chlorophyll and degradation metabolites revealed that the observed decrease in chlorophyll concentration was associated with hindered biosynthesis, and was not due to degradation. Coproporphyrinogen III could not be detected after exposure to only 0.5 µM As. Levels of subsequent precursors, for example, protoporphyrin IX, Mg-protoporphyrin, Mg-protoporphyrin methyl ester, and divinyl protochlorophyllide, were significantly decreased at this concentration as well, indicating that the pathway was blocked upstream of tetrapyrrole synthesis. PMID:27340233

  2. Imaging P2X4 receptor subcellular distribution, trafficking, and regulation using P2X4-pHluorin

    PubMed Central

    Xu, Ji; Chai, Hua; Ehinger, Konstantin; Egan, Terrance M.; Srinivasan, Rahul; Frick, Manfred

    2014-01-01

    P2X4 receptors are adenosine triphosphate (ATP)-gated cation channels present on the plasma membrane (PM) and also within intracellular compartments such as vesicles, vacuoles, lamellar bodies (LBs), and lysosomes. P2X4 receptors in microglia are up-regulated in epilepsy and in neuropathic pain; that is to say, their total and/or PM expression levels increase. However, the mechanisms underlying up-regulation of microglial P2X4 receptors remain unclear, in part because it has not been possible to image P2X4 receptor distribution within, or trafficking between, cellular compartments. Here, we report the generation of pH-sensitive fluorescently tagged P2X4 receptors that permit evaluations of cell surface and total receptor pools. Capitalizing on information gained from zebrafish P2X4.1 crystal structures, we designed a series of mouse P2X4 constructs in which a pH-sensitive green fluorescent protein, superecliptic pHluorin (pHluorin), was inserted into nonconserved regions located within flexible loops of the P2X4 receptor extracellular domain. One of these constructs, in which pHluorin was inserted after lysine 122 (P2X4-pHluorin123), functioned like wild-type P2X4 in terms of its peak ATP-evoked responses, macroscopic kinetics, calcium flux, current–voltage relationship, and sensitivity to ATP. P2X4-pHluorin123 also showed pH-dependent fluorescence changes, and was robustly expressed on the membrane and within intracellular compartments. P2X4-pHluorin123 identified cell surface and intracellular fractions of receptors in HEK-293 cells, hippocampal neurons, C8-B4 microglia, and alveolar type II (ATII) cells. Furthermore, it showed that the subcellular fractions of P2X4-pHluorin123 receptors were cell and compartment specific, for example, being larger in hippocampal neuron somata than in C8-B4 cell somata, and larger in C8-B4 microglial processes than in their somata. In ATII cells, P2X4-pHluorin123 showed that P2X4 receptors were secreted onto the PM when LBs

  3. Molecular cloning and subcellular distribution of the novel PDE4B4 cAMP-specific phosphodiesterase isoform.

    PubMed Central

    Shepherd, Malcolm; McSorley, Theresa; Olsen, Aileen E; Johnston, Lee Ann; Thomson, Neil C; Baillie, George S; Houslay, Miles D; Bolger, Graeme B

    2003-01-01

    We have isolated cDNAs encoding PDE4B4, a new cAMP-specific phosphodiesterase (PDE4) isoform with novel properties. The amino acid sequence of PDE4B4 demonstrates that it is encoded by the PDE4B gene, but that it differs from the previously isolated PDE4B1, PDE4B2 and PDE4B3 isoforms by the presence of a novel N-terminal region of 17 amino acids. PDE4B4 contains both of the upstream conserved region 1 (UCR1) and UCR2 regulatory units that are characteristic of 'long' PDE4 isoforms. RNase protection demonstrated that PDE4B4 mRNA is expressed preferentially in liver, skeletal muscle and various regions of the brain, which differs from the pattern of tissue distribution of the other known PDE4B long forms, PDE4B1 and PDE4B3. Expression of PDE4B4 cDNA in COS7 cells produced a protein of 85 kDa under denaturing conditions. Subcellular fractionation of recombinant, COS7-cell expressed PDE4B4 showed that the protein was localized within the cytosol, which was confirmed by confocal microscopic analysis of living COS7 cells transfected with a green fluorescent protein-PDE4B4 chimaera. PDE4B4 exhibited a K(m) for cAMP of 5.4 microM and a V(max), relative to that of the long PDE4B1 isoform, of 2.1. PDE4B4 was inhibited by the prototypical PDE4 inhibitor rolipram [4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidinone] with an IC(50) of 83 nM. Treatment of COS7 cells with forskolin, to elevate cAMP levels, produced activation of PDE4B4, which was associated with the phosphorylation of PDE4B4 on Ser-56 within UCR1. The unique tissue distribution and intracellular targeting of PDE4B4 suggests that this isoform may have a distinct functional role in regulating cAMP levels in specific cell types. PMID:12441002

  4. N-Glycan Branching Affects the Subcellular Distribution of and Inhibition of Matriptase by HAI-2/Placental Bikunin

    PubMed Central

    Lai, Hongyu; Xu, Yuan; Shiao, Frank; Huang, Nanxi; Li, Linpei; Lee, Ming-Shyue; Johnson, Michael D.; Wang, Jehng-Kang; Lin, Chen-Yong

    2015-01-01

    The gene product of SPINT 2, that encodes a transmembrane, Kunitz-type serine protease inhibitor independently designated as HAI-2 or placenta bikunin (PB), is involved in regulation of sodium absorption in human gastrointestinal track. Here, we show that SPINT 2 is expressed as two species of different size (30-40- versus 25-kDa) due to different N-glycans on Asn-57. The N-glycan on 25-kDa HAI-2 appears to be of the oligomannose type and that on 30-40-kDa HAI-2 to be of complex type with extensive terminal N-acetylglucosamine branching. The two different types of N-glycan differentially mask two epitopes on HAI-2 polypeptide, recognized by two different HAI-2 mAbs. The 30-40-kDa form may be mature HAI-2, and is primarily localized in vesicles/granules. The 25-kDa form is likely immature HAI-2, that remains in the endoplasmic reticulum (ER) in the perinuclear regions of mammary epithelial cells. The two different N-glycans could, therefore, represent different maturation stages of N-glycosylation with the 25-kDa likely a precursor of the 30-40-kDa HAI-2, with the ratio of their levels roughly similar among a variety of cells. In breast cancer cells, a significant amount of the 30-40-kDa HAI-2 can translocate to and inhibit matriptase on the cell surface, followed by shedding of the matriptase-HAI-2 complex. The 25-kDa HAI-2 appears to have also exited the ER/Golgi, being localized at the cytoplasmic face of the plasma membrane of breast cancer cells. While the 25-kDa HAI-2 was also detected at the extracellular face of plasma membrane at very low levels it appears to have no role in matriptase inhibition probably due to its paucity on the cell surface. Our study reveals that N-glycan branching regulates HAI-2 through different subcellular distribution and subsequently access to different target proteases. PMID:26171609

  5. The influence of metal speciation on the bioavailability and sub-cellular distribution of cadmium to the terrestrial isopod, Porcellio dilatatus.

    PubMed

    Calhôa, Carla Filipa; Monteiro, Marta S; Soares, Amadeu M V M; Mann, Reinier M

    2011-04-01

    Cadmium is a non-essential toxic metal that is able to bioaccumulate in both flora fauna and has the potential to biomagnify in some food chains. However, the form in which cadmium is presented to consumers can alter the bioavailability and possibly the internal distribution of assimilated Cd. Previous studies in our laboratory highlighted differences in Cd assimilation among isopods when they were provided with a plant-based food with either Cd biologically incorporated into plant tissue or superficially amended with ionic Cd(2+). Cd is known for its high affinity for sulphur ligands in cysteine residues which form the basis for metal-binding proteins such as metallothionein. This study compares Cd assimilation efficiency (AE) in Porcellio dilatatus fed with food amended with either cadmium cysteinate or cadmium nitrate in an examination of the influence of Cd speciation on metal bioavailability followed by an examination of the sub-cellular distribution using a centrifugal fractionation protocol. As hypothesized the AE of Cd among isopods fed with Cd(NO(3))(2) (64%, SE=5%) was higher than AE for isopods fed with Cd(Cys)(2) (20%, SE=3%). The sub-cellular distribution also depended on the Cd species provided. Those isopods fed Cd(Cys)(2) allocated significantly more Cd to the cell debris and organelles fractions at the expense of allocation to metal-rich granules (MRG). The significance of the difference in sub-cellular distribution with regard to toxicity is discussed. This paper demonstrates that the assimilation and internal detoxification of Cd is dependent on the chemical form of Cd presented to the isopod.

  6. MitoTimer probe reveals the impact of autophagy, fusion, and motility on subcellular distribution of young and old mitochondrial protein and on relative mitochondrial protein age.

    PubMed

    Ferree, Andrew W; Trudeau, Kyle; Zik, Eden; Benador, Ilan Y; Twig, Gilad; Gottlieb, Roberta A; Shirihai, Orian S

    2013-11-01

    To study mitochondrial protein age dynamics, we targeted a time-sensitive fluorescent protein, MitoTimer, to the mitochondrial matrix. Mitochondrial age was revealed by the integrated portions of young (green) and old (red) MitoTimer protein. Mitochondrial protein age was dependent on turnover rates as pulsed synthesis, decreased import, or autophagic inhibition all increased the proportion of aged MitoTimer protein. Mitochondrial fusion promotes the distribution of young mitochondrial protein across the mitochondrial network as cells lacking essential fusion genes Mfn1 and Mfn2 displayed increased heterogeneity in mitochondrial protein age. Experiments in hippocampal neurons illustrate that the distribution of older and younger mitochondrial protein within the cell is determined by subcellular spatial organization and compartmentalization of mitochondria into neurites and soma. This effect was altered by overexpression of mitochondrial transport protein, RHOT1/MIRO1. Collectively our data show that distribution of young and old protein in the mitochondrial network is dependent on turnover, fusion, and transport.

  7. Interaction of HSP20 with a viral RdRp changes its sub-cellular localization and distribution pattern in plants

    PubMed Central

    Li, Jing; Xiang, Cong-Ying; Yang, Jian; Chen, Jian-Ping; Zhang, Heng-Mu

    2015-01-01

    Small heat shock proteins (sHSPs) perform a fundamental role in protecting cells against a wide array of stresses but their biological function during viral infection remains unknown. Rice stripe virus (RSV) causes a severe disease of rice in Eastern Asia. OsHSP20 and its homologue (NbHSP20) were used as baits in yeast two-hybrid (YTH) assays to screen an RSV cDNA library and were found to interact with the viral RNA-dependent RNA polymerase (RdRp) of RSV. Interactions were confirmed by pull-down and BiFC assays. Further analysis showed that the N-terminus (residues 1–296) of the RdRp was crucial for the interaction between the HSP20s and viral RdRp and responsible for the alteration of the sub-cellular localization and distribution pattern of HSP20s in protoplasts of rice and epidermal cells of Nicotiana benthamiana. This is the first report that a plant virus or a viral protein alters the expression pattern or sub-cellular distribution of sHSPs. PMID:26359114

  8. High-Resolution Secondary Ion Mass Spectrometry Reveals the Contrasting Subcellular Distribution of Arsenic and Silicon in Rice Roots1[C][W][OA

    PubMed Central

    Moore, Katie L.; Schröder, Markus; Wu, Zhongchang; Martin, Barry G.H.; Hawes, Chris R.; McGrath, Steve P.; Hawkesford, Malcolm J.; Feng Ma, Jian; Zhao, Fang-Jie; Grovenor, Chris R.M.

    2011-01-01

    Rice (Oryza sativa) takes up arsenite mainly through the silicic acid transport pathway. Understanding the uptake and sequestration of arsenic (As) into the rice plant is important for developing strategies to reduce As concentration in rice grain. In this study, the cellular and subcellular distributions of As and silicon (Si) in rice roots were investigated using high-pressure freezing, high-resolution secondary ion mass spectrometry, and transmission electron microscopy. Rice plants, both the lsi2 mutant lacking the Si/arsenite efflux transporter Lsi2 and its wild-type cultivar, with or without an iron plaque, were treated with arsenate or arsenite. The formation of iron plaque on the root surface resulted in strong accumulation of As and phosphorous on the epidermis. The lsi2 mutant showed stronger As accumulation in the endodermal vacuoles, where the Lsi2 transporter is located in the plasma membranes, than the wild-type line. As also accumulated in the vacuoles of some xylem parenchyma cells and in some pericycle cells, particularly in the wild-type mature root zone. Vacuolar accumulation of As is associated with sulfur, suggesting that As may be stored as arsenite-phytochelatin complexes. Si was localized in the cell walls of the endodermal cells with little apparent effect of the Lsi2 mutation on its distribution. This study reveals the vacuolar sequestration of As in rice roots and contrasting patterns of As and Si subcellular localization, despite both being transported across the plasma membranes by the same transporters. PMID:21490163

  9. Regulation of the Regulators: Post-Translational Modifications, Subcellular, and Spatiotemporal Distribution of Plant 14-3-3 Proteins

    PubMed Central

    Wilson, Rashaun S.; Swatek, Kirby N.; Thelen, Jay J.

    2016-01-01

    14-3-3 proteins bind to and modulate the activity of phosphorylated proteins that regulate a variety of metabolic processes in eukaryotes. Multiple 14-3-3 isoforms are expressed in most organisms and display redundancy in both sequence and function. Plants contain the largest number of 14-3-3 isoforms. For example, Arabidopsis thaliana contains thirteen 14-3-3 genes, each of which is expressed. Interest in the plant 14-3-3 field has swelled over the past decade, largely due to the vast number of possibilities for 14-3-3 metabolic regulation. As the field progresses, it is essential to understand these proteins' activities at both the spatiotemporal and subcellular levels. This review summarizes current knowledge of 14-3-3 proteins in plants, including 14-3-3 interactions, regulatory functions, isoform specificity, and post-translational modifications. We begin with a historical overview and structural analysis of 14-3-3 proteins, which describes the basic principles of 14-3-3 function, and then discuss interactions and regulatory effects of plant 14-3-3 proteins in specific tissues and subcellular compartments. We conclude with a summary of 14-3-3 phosphorylation and current knowledge of the functional effects of this modification in plants. PMID:27242818

  10. Subcellular distribution of calcium-binding proteins and a calcium- ATPase in canine pancreas [published erratum appears in J Cell Biol 1990 Oct;111(4):1726

    PubMed Central

    1990-01-01

    Using a 45Ca blot-overlay assay, we monitored the subcellular fractionation pattern of several Ca binding proteins of apparent molecular masses 94, 61, and 59 kD. These proteins also appeared to stain blue with "Stains-All." Additionally, using a monoclonal antiserum raised against canine cardiac sarcoplasmic reticulum Ca- ATPase, we examined the subcellular distribution of a canine pancreatic 110-kD protein recognized by this antiserum. This protein had the same electrophoretic mobility as the cardiac protein against which the antiserum was raised. The three Ca binding proteins and the Ca-ATPase cofractionated into the rough microsomal fraction (RM), previously shown to consist of highly purified RER, in a pattern highly similar to that of the RER marker, ribophorin I. To provide further evidence for an RER localization, native RM were subjected to isopycnic flotation in sucrose gradients. The Ca binding proteins and the Ca-ATPase were found in dense fractions, along with ribophorin I. When RM were stripped of ribosomes with puromycin/high salt, the Ca binding proteins and the Ca- ATPase exhibited a shift to less dense fractions, as did ribophorin I. We conclude that, in pancreas, the Ca binding proteins and Ca-ATPase we detect are localized to the RER (conceivably a subcompartment of the RER) or, possibly, a structure intimately associated with the RER. PMID:2142161

  11. A Celiac Cellular Phenotype, with Altered LPP Sub-Cellular Distribution, Is Inducible in Controls by the Toxic Gliadin Peptide P31-43

    PubMed Central

    Nanayakkara, Merlin; Kosova, Roberta; Lania, Giuliana; Sarno, Marco; Gaito, Alessandra; Galatola, Martina; Greco, Luigi; Cuomo, Marialaura; Troncone, Riccardo; Auricchio, Salvatore

    2013-01-01

    Celiac disease (CD) is a frequent inflammatory intestinal disease, with a genetic background, caused by gliadin-containing food. Undigested gliadin peptides P31-43 and P57-68 induce innate and adaptive T cell-mediated immune responses, respectively. Alterations in the cell shape and actin cytoskeleton are present in celiac enterocytes, and gliadin peptides induce actin rearrangements in both the CD mucosa and cell lines. Cell shape is maintained by the actin cytoskeleton and focal adhesions, sites of membrane attachment to the extracellular matrix. The locus of the human Lipoma Preferred Partner (LPP) gene was identified as strongly associated with CD using genome-wide association studies (GWAS). The LPP protein plays an important role in focal adhesion architecture and acts as a transcription factor in the nucleus. In this study, we examined the hypothesis that a constitutive alteration of the cell shape and the cytoskeleton, involving LPP, occurs in a cell compartment far from the main inflammation site in CD fibroblasts from skin explants. We analyzed the cell shape, actin organization, focal adhesion number, focal adhesion proteins, LPP sub-cellular distribution and adhesion to fibronectin of fibroblasts obtained from CD patients on a Gluten-Free Diet (GFD) and controls, without and with treatment with A-gliadin peptide P31-43. We observed a “CD cellular phenotype” in these fibroblasts, characterized by an altered cell shape and actin organization, increased number of focal adhesions, and altered intracellular LPP protein distribution. The treatment of controls fibroblasts with gliadin peptide P31-43 mimics the CD cellular phenotype regarding the cell shape, adhesion capacity, focal adhesion number and LPP sub-cellular distribution, suggesting a close association between these alterations and CD pathogenesis. PMID:24278174

  12. Glutamine synthetase stability and subcellular distribution in astrocytes are regulated by γ-aminobutyric type B receptors.

    PubMed

    Huyghe, Deborah; Nakamura, Yasuko; Terunuma, Miho; Faideau, Mathilde; Haydon, Philip; Pangalos, Menelas N; Moss, Stephen J

    2014-10-17

    Emerging evidence suggests that functional γ-aminobutyric acid B receptors (GABABRs) are expressed by astrocytes within the mammalian brain. GABABRs are heterodimeric G-protein-coupled receptors that are composed of R1/R2 subunits. To date, they have been characterized in neurons as the principal mediators of sustained inhibitory signaling; however their roles in astrocytic physiology have been ill defined. Here we reveal that the cytoplasmic tail of the GABABR2 subunit binds directly to the astrocytic protein glutamine synthetase (GS) and that this interaction determines the subcellular localization of GS. We further demonstrate that the binding of GS to GABABR2 increases the steady state expression levels of GS in heterologous cells and in mouse primary astrocyte culture. Mechanistically this increased stability of GS in the presence of GABABR2 occurs via reduced proteasomal degradation. Collectively, our results suggest a novel role for GABABRs as regulators of GS stability. Given the critical role that GS plays in the glutamine-glutamate cycle, astrocytic GABABRs may play a critical role in supporting both inhibitory and excitatory neurotransmission.

  13. Subcellular distribution of ( sup 3 H)-dexamethasone mesylate binding sites in Leydig cells using electron microscope radioautography

    SciTech Connect

    Stalker, A.; Hermo, L.; Antakly, T. )

    1991-01-01

    The present view is that glucocorticoid hormones bind to their cytoplasmic receptors before reaching their nuclear target sites, which include specific DNA sequences. Although it is believed that cytoplasmic sequestration of steroid receptors and other transcription factors (such as NFKB) may regulate the overall activity of these factors, there is little information on the exact subcellular sites of steroid receptors or even of any other transcription factors. Tritiated (3H)-dexamethasone 21-mesylate (DM) is an affinity label that binds covalently to the glucocorticoid receptor (GR), thereby allowing morphological localization of the receptor at the light and electron microscope levels as well as for quantitative radioautographic (RAG) analysis. After injection of 3H-DM into the testis, a specific radioautographic signal was observed in Leydig cells, which correlated with a high level of immunocytochemically demonstrable GR in these cells at the light-microscope level. To localize the 3H-DM binding sites at the electron microscope (EM) level, the testes of 5 experimental and 3 control adrenalectomized rats were injected directly with 20 microCi 3H-DM; control rats received simultaneously a 25-fold excess of unlabeled dexamethasone; 15 min later, rats were fixed with glutaraldehyde and the tissue was processed for EM RAG analysis combined with quantitative morphometry. The radioautographs showed that the cytosol, nucleus, smooth endoplasmic reticulum (sER), and mitochondria were labeled. Since the cytosol was always adjacent to tubules of the sER, the term sER-rich cytosol was used to represent label over sER networks, which may also represent cytosol labeling due to the limited resolution of the radioautographic technique. Labeling was highest in sER-rich cytosol and mitochondria, at 53% and 31% of the total, respectively.

  14. [Effects of Different Modifier Concentrations on Lead-Zinc Tolerance, Subcellular Distribution and Chemical Forms for Four Kinds of Woody Plants].

    PubMed

    Chen, Yong-hua; Zhang, Fu-yun; Wu, Xiao-fu; Liang, Xi; Yuan, Si-wen

    2015-10-01

    Four kinds of lead-zinc tolerant woody plants: Nerium oleander, Koelreuteria paniculata, Paulownia and Boehmeria were used as materials to estimate their enrichment and transferable capacity of lead (Pb) and zinc (Zn) and analyze the subcellular distribution and chemical speciation of Zn and Ph in different parts of plants, under different modifier concentrations (CK group: 100% lead-zinc slag plus a small amount of phosphate fertilizer, improved one: 85% of lead-zinc slag ± 10% peat ± 5% bacterial manure plus a small amount of phosphate fertilizer, improved two: 75% lead-zinc slag ± 20% peat ± 5% bacterial manure ± a small amount of phosphate). Results showed that: (1) The content of Pb, Zn in matrix after planting four kinds of plants was lower than before, no significant difference between improved one and improved two of Nerium oleander and Boehmeria was found, but improved two was better than improved one of Paulownia, while improved one was better than improved two of Koelreuteria paniculata; Four plants had relatively low aboveground enrichment coefficient of Pb and Zn, but had a high transfer coefficient, showed that the appropriate modifier concentration was able to improve the Pb and Zn enrichment and transfer ability of plants. (2) In subcellular distribution, most of Pb and Zn were distributed in plant cell wall components and soluble components while the distribution in cell organelles such as mitochondria, chloroplasts and nucleus component were less. Compared with CK group, two improved group made soluble components of the cell walls of Pb fixation and retention of zinc role in the enhancement. (3) As for the chemical forms of Pb and Zn in plants, the main chemical forms of Pb were hydrochloric acid, sodium chloride and ethanol extractable forms, while other chemical form contents were few, the main chemical forms of Zn were different based on plant type. Compared with CK group, the proportion of the active Pb chemical form in different plant

  15. [Effects of Different Modifier Concentrations on Lead-Zinc Tolerance, Subcellular Distribution and Chemical Forms for Four Kinds of Woody Plants].

    PubMed

    Chen, Yong-hua; Zhang, Fu-yun; Wu, Xiao-fu; Liang, Xi; Yuan, Si-wen

    2015-10-01

    Four kinds of lead-zinc tolerant woody plants: Nerium oleander, Koelreuteria paniculata, Paulownia and Boehmeria were used as materials to estimate their enrichment and transferable capacity of lead (Pb) and zinc (Zn) and analyze the subcellular distribution and chemical speciation of Zn and Ph in different parts of plants, under different modifier concentrations (CK group: 100% lead-zinc slag plus a small amount of phosphate fertilizer, improved one: 85% of lead-zinc slag ± 10% peat ± 5% bacterial manure plus a small amount of phosphate fertilizer, improved two: 75% lead-zinc slag ± 20% peat ± 5% bacterial manure ± a small amount of phosphate). Results showed that: (1) The content of Pb, Zn in matrix after planting four kinds of plants was lower than before, no significant difference between improved one and improved two of Nerium oleander and Boehmeria was found, but improved two was better than improved one of Paulownia, while improved one was better than improved two of Koelreuteria paniculata; Four plants had relatively low aboveground enrichment coefficient of Pb and Zn, but had a high transfer coefficient, showed that the appropriate modifier concentration was able to improve the Pb and Zn enrichment and transfer ability of plants. (2) In subcellular distribution, most of Pb and Zn were distributed in plant cell wall components and soluble components while the distribution in cell organelles such as mitochondria, chloroplasts and nucleus component were less. Compared with CK group, two improved group made soluble components of the cell walls of Pb fixation and retention of zinc role in the enhancement. (3) As for the chemical forms of Pb and Zn in plants, the main chemical forms of Pb were hydrochloric acid, sodium chloride and ethanol extractable forms, while other chemical form contents were few, the main chemical forms of Zn were different based on plant type. Compared with CK group, the proportion of the active Pb chemical form in different plant

  16. MitoTimer probe reveals the impact of autophagy, fusion, and motility on subcellular distribution of young and old mitochondrial protein and on relative mitochondrial protein age

    PubMed Central

    Ferree, Andrew W; Trudeau, Kyle; Zik, Eden; Benador, Ilan Y; Twig, Gilad; Gottlieb, Roberta A; Shirihai, Orian S

    2013-01-01

    To study mitochondrial protein age dynamics, we targeted a time-sensitive fluorescent protein, MitoTimer, to the mitochondrial matrix. Mitochondrial age was revealed by the integrated portions of young (green) and old (red) MitoTimer protein. Mitochondrial protein age was dependent on turnover rates as pulsed synthesis, decreased import, or autophagic inhibition all increased the proportion of aged MitoTimer protein. Mitochondrial fusion promotes the distribution of young mitochondrial protein across the mitochondrial network as cells lacking essential fusion genes Mfn1 and Mfn2 displayed increased heterogeneity in mitochondrial protein age. Experiments in hippocampal neurons illustrate that the distribution of older and younger mitochondrial protein within the cell is determined by subcellular spatial organization and compartmentalization of mitochondria into neurites and soma. This effect was altered by overexpression of mitochondrial transport protein, RHOT1/MIRO1. Collectively our data show that distribution of young and old protein in the mitochondrial network is dependent on turnover, fusion, and transport. PMID:24149000

  17. Biochemical properties and subcellular distribution of the BI and rbA isoforms of alpha 1A subunits of brain calcium channels

    PubMed Central

    1996-01-01

    Biochemical properties and subcellular distribution of the class A calcium channel alpha 1 subunits (alpha 1A) from rat and rabbit brain were examined using site-directed anti-peptide antibodies specific for rat rbA (anti-CNA3) and for rabbit BI (anti-NBI-1 and anti-NBI-2) isoforms of alpha 1A. In immunoblotting experiments, anti-CNA3 specifically identifies multiple alpha 1A polypeptides with apparent molecular masses of 210, 190, and 160 kD, and anti-NBI-1 and anti-NBI-2 specifically recognize 190-kD alpha 1A polypeptides in rat brain membrane. In rabbit brain, anti-NBI-1 or anti-NBI-2 specifically detect alpha 1A polypeptides with apparent molecular masses of 220, 200, and 190 kD, while anti-CNA3 specifically recognizes 190-kD alpha 1A polypeptides. These polypeptides evidently represent multiple isoforms of alpha 1A present in both rat and rabbit brain. Anti-CNA3 specifically immunoprecipitates high affinity receptor sites for omega- conotoxin MVIIC (Kd approximately 100 pM), whereas anti-NBI-2 immunoprecipitates two distinct affinity receptor sites for omega- conotoxin MVIIC (Kd approximately 100 pM and approximately 1 microM). Coimmunoprecipitation experiments indicate that alpha 1A subunits recognized by anti-CNA3 and anti-NBI-2 are associated with syntaxin in a stable, SDS-resistant complex and with synaptotagmin. Immunofluorescence studies reveal that calcium channels recognized by anti-NBI-2 are localized predominantly in dendrites and nerve terminals forming synapses on them, while calcium channels recognized by anti- CNA3 are localized more prominently in cell bodies and in nerve terminals. The mossy fiber terminals in hippocampus and the terminals of climbing and parallel fibers in cerebellum are differentially stained by these isoform-specific antibodies. These results indicate that both rbA and BI isoforms of alpha 1A are expressed in rat and rabbit brain and form calcium channels having alpha 1A subunits with distinct molecular mass, pharmacology

  18. Subcellular distribution of heavy metals in liver and kidney of a narwhal whale (Monodon monoceros): an evaluation for the presence of metallothionein.

    PubMed

    Wagemann, R; Hunt, R; Klaverkamp, J F

    1984-01-01

    The subcellular distribution of Zn, Cd, Cu and Hg in liver and kidney from a narwhal was determined by ultracentrifugation and gel filtration. Most of the total mercury in the liver and kidney was bound by the cellular pellet (88 and 73%, respectively). Of the total mercury, 7 and 11% was in the form of methylmercury in the liver and kidney, respectively. More than half (74%) of the total Zn and Cu in the kidney was in the cytosol and somewhat less than this was in the cytosol of the liver. Almost all of the cadmium in liver and kidney (88 and 92%, respectively) was in cytosol. Cytosolic fractions from liver and kidney were evaluated for the presence of metallothionein by analysing for Zn, Cd, Hg, Cu, Fe and--SH groups, by molecular weight estimation and by u.v. absorption spectra. Metallothionein was found in these organs in estimated concentrations similar to those present in terrestrial and other marine mammals. PMID:6149069

  19. Analysis of subcellular [57Co] cobalamin distribution in SH-SY5Y neurons and brain tissue.

    PubMed

    Zhao, Hua; Ruberu, Kalani; Li, Hongyun; Garner, Brett

    2013-07-15

    Cobalamin (Cbl) utilization as a cofactor for methionine synthase and methylmalonyl-CoA mutase is dependent on the transport of Cbl through lysosomes and its subsequent delivery to the cytosol and mitochondria. We speculated that neuropathological conditions that impair lysosomal function (e.g., age-related lipofuscinosis and specific neurodegenerative diseases) might impair lysosomal Cbl transport. To address this question, an appropriate method to quantify intracellular Cbl transport in neuronal cell types and brain tissue is required. Thus, we developed methods to measure [57Co] Cbl levels in lysosomes, mitochondria and cytosol obtained from in vitro and in vivo sources. Human SH-SY5Y neurons or HT1080 fibroblasts were labeled with [57Co] Cbl and homogenized using a ball-bearing homogenizer, and the lysates were separated into 10 fractions using ultracentrifugation in an OptiPrep density gradient. Lysosomes were recovered from the top of the gradient (fractions 1-5), which were clearly separated from mitochondria (fractions 7-9) on the basis of the expression of the marker proteins, LAMP2 and VDAC1. The isolated lysosomes were intact based on their colocalization with acid phosphatase activity. The lysosomal and mitochondrial fractions were free of the cytosolic markers beta-actin and methionine synthase. The relative distribution of [57Co] Cbl in both neurons and fibroblasts was as follows: 6% in the lysosomes, 14% in the mitochondria and 80% in the cytosol. This technique was also used to fractionate organelles from mouse brain, where marker proteins were detected in the gradient at positions similar to those observed for the cell lines, and the relative distribution of [57Co] Cbl was as follows: 12% in the lysosomes, 15% in the mitochondria and 73% in the cytosol. These methods provide a useful tool for the investigation of intracellular Cbl trafficking in a neurobiological setting. PMID:23608310

  20. High Resolution Imaging of Temporal and Spatial Changes of Subcellular Ascorbate, Glutathione and H2O2 Distribution during Botrytis cinerea Infection in Arabidopsis

    PubMed Central

    Simon, Uwe K.; Polanschütz, Lisa M.; Koffler, Barbara E.; Zechmann, Bernd

    2013-01-01

    In order to study the mechanisms behind the infection process of the necrotrophic fungus Botrytis cinerea, the subcellular distribution of hydrogen peroxide (H2O2) was monitored over a time frame of 96 h post inoculation (hpi) in Arabidopsis thaliana Col-0 leaves at the inoculation site (IS) and the area around the IS which was defined as area adjacent to the inoculation site (AIS). H2O2 accumulation was correlated with changes in the compartment-specific distribution of ascorbate and glutathione and chloroplast fine structure. This study revealed that the severe breakdown of the antioxidative system, indicated by a drop in ascorbate and glutathione contents at the IS at later stages of infection correlated with an accumulation of H2O2 in chloroplasts, mitochondria, cell walls, nuclei and the cytosol which resulted in the development of chlorosis and cell death, eventually visible as tissue necrosis. A steady increase of glutathione contents in most cell compartments within infected tissues (up to 600% in chloroplasts at 96 hpi) correlated with an accumulation of H2O2 in chloroplasts, mitochondria and cell walls at the AIS indicating that high glutathione levels could not prevent the accumulation of reactive oxygen species (ROS) which resulted in chlorosis. Summing up, this study reveals the intracellular sequence of events during Botrytis cinerea infection and shows that the breakdown of the antioxidative system correlated with the accumulation of H2O2 in the host cells. This resulted in the degeneration of the leaf indicated by severe changes in the number and ultrastructure of chloroplasts (e.g. decrease of chloroplast number, decrease of starch and thylakoid contents, increase of plastoglobuli size), chlorosis and necrosis of the leaves. PMID:23755284

  1. Ultrastructure and subcellular distribution of Cr in Iris pseudacorus L. using TEM and X-ray microanalysis.

    PubMed

    Caldelas, Cristina; Bort, Jordi; Febrero, Anna

    2012-02-01

    Chromium pollution of freshwater is hazardous for humans and other organisms, and places a limitation on the use of polluted water sources. Phytoremediation, the use of plants to remove pollutants from the environment, is a cost-effective, environmentally friendly approach for water decontamination. To improve the efficiency of the process, it is essential to increase the current knowledge about Cr accumulation in macrophytes. Plants of Iris pseudacorus L. were treated with Cr(III) at 0.75 mM for 5 weeks to investigate Cr localization by means of transmission electron microscopy and energy dispersive X-ray analysis. Chromium induced severe ultrastructural alterations in the rhizodermis (cell wall disorganisation, thickening, plasmolysis, and electron-dense inclusions) and rhizome parenchyma (reduced cell size, cell wall detachment, vacuolation, and opaque granules). The highest Cr contents were found in the cell walls of the cortex in the roots and in the cytoplasm and intercellular spaces of the rhizome. The Cr concentration in root tissues was in the order cortex>rhizodermis>stele, whereas in the rhizome, Cr was evenly distributed. It is proposed that root and rhizome have distinct functions in the response of I. pseudacorus to Cr. The rhizodermis limits Cr uptake by means of Si deposition and cell wall thickening. The rhizome cortex generates vacuoles and granules where Cr co-occurs with S, indicating Cr sequestration by metal-binding proteins. PMID:22009188

  2. Subcellular localization and mechanisms of nucleocytoplasmic distribution of p202, an interferon-inducible candidate for lupus susceptibility.

    PubMed

    Choubey, Divaker; Pramanik, Rocky; Xin, Hong

    2003-10-23

    Increased expression of p202 (52 kDa), an interferon (IFN)-inducible murine protein, in splenic cells (B- and T-cells) derived from female mice of the lupus-prone strains is correlated with increased susceptibility to develop systemic lupus erythematosus. However, the molecular mechanisms remain unclear. Our previous studies have indicated that, in IFN-treated fibroblasts, p202 is detected both in the cytoplasm and in the nucleus. Moreover, in the cytoplasm, a fraction of p202 associates with a membranous organelle. Here we report that, in the cytoplasm, a fraction of p202 associated with mitochondria. Additionally, we found that the constitutive p202 is primarily detected in the cytoplasm. Remarkably, the IFN treatment of cells potentiated nuclear accumulation of p202. Our observations are consistent with the possibility that IFN signaling regulates p202 levels as well as its nucleocytoplasmic distribution. These observations will serve as a basis to elucidate the molecular mechanisms by which p202 contributes to lupus susceptibility.

  3. Subcellular Distribution of M2-muscarinic Receptors in Relation to Dopaminergic Neurons of the Rat Ventral Tegmental Area

    PubMed Central

    Garzón, Miguel; Pickel, Virginia M.

    2008-01-01

    Acetylcholine can affect cognitive functions and reward, in part, through activation of muscarinic receptors in the ventral tegmental area (VTA) to evoke changes in mesocorticolimbic dopaminergic transmission. Of the known muscarinic receptor subtypes present in the VTA, the M2 receptor (M2R) is most implicated in autoregulation, and also may play a heteroreceptor role in regulation of the output of the dopaminergic neurons. We sought to determine the functionally relevant sites for M2R activation in relation to VTA dopaminergic neurons by examining the electron microscopic immunolabeling of M2R and the dopamine transporter (DAT) in the VTA of rat brain. The M2R was localized to endomembranes in DAT-containing somatodendritic profiles, but showed a more prominent, size-dependent plasmalemmal location in non-dopaminergic dendrites. M2R also was located on the plasma membrane of morphologically heterogenous axon terminals contacting unlabeled as well as M2R or DAT-labeled dendrites. Some of these terminals formed asymmetric synapses resembling those of cholinergic terminals in the VTA. The majority, however, formed symmetric, inhibitory-type synapses, or were apposed without recognized junctions. Our results provide the first ultrastructural evidence that the M2R is expressed, but largely not available for local activation, on the plasma membrane of VTA dopaminergic neurons. Instead, the M2R in this region has a distribution suggesting more indirect regulation of mesocorticolimbic transmission through autoregulation of acetylcholine release and changes in the physiological activity or release of other, largely inhibitory transmitters. These findings could have implications for understanding the muscarinic control of cognitive and goal-directed behaviors within the VTA. PMID:16927256

  4. Concentration and subcellular distribution of trace elements in liver of small cetaceans incidentally caught along the Brazilian coast.

    PubMed

    Kunito, Takashi; Nakamura, Shinji; Ikemoto, Tokutaka; Anan, Yasumi; Kubota, Reiji; Tanabe, Shinsuke; Rosas, Fernando C W; Fillmann, Gilberto; Readman, James W

    2004-10-01

    Concentrations of trace elements (V, Cr, Mn, Fe, Co, Cu, Zn, Ga, As, Se, Rb, Sr, Mo, Ag, Cd, Sb, Cs, Ba, T-Hg, Org-Hg, Tl and Pb) were determined in liver samples of estuarine dolphin (Sotalia guianensis; n = 20), Franciscana dolphin (Pontoporia blainvillei; n = 23), Atlantic spotted dolphin (Stenella frontalis; n = 2), common dolphin (Delphinus capensis; n = 1) and striped dolphin (Stenella coeruleoalba; n = 1) incidentally caught along the coast of Sao Paulo State and Parana State, Brazil, from 1997 to 1999. The hepatic concentrations of trace elements in the Brazilian cetaceans were comparable to the data available in literature on marine mammals from Northern Hemisphere. Concentrations of V, Se, Mo, Cd, T-Hg and Org-Hg increased with increasing age in liver of both estuarine and Franciscana dolphins. Very high concentrations of Cu (range, 262-1970 microg/g dry wt.) and Zn (range, 242-369 microg/g dry wt.) were observed in liver of sucklings of estuarine dolphin. Hepatic concentrations of V, Se, T-Hg, Org-Hg and Pb were significantly higher in estuarine dolphin, whereas Franciscana dolphin showed higher concentrations of Mn, Co, As and Rb. Ratio of Org-Hg to T-Hg in liver was significantly higher in Franciscana dolphin than estuarine dolphin, suggesting that demethylation ability of methyl Hg might be lower in liver of Franciscana than estuarine dolphins. High hepatic concentrations of Ag were found in some specimens of Franciscana dolphin (maximum, 20 microg/g dry wt.), and 17% of Franciscana showed higher concentrations of Ag than Hg. These samples with high Ag concentration also exhibited elevated hepatic Se concentration, implying that Ag might be detoxified by Se in the liver. Higher correlation coefficient between (Hg+0.5 Ag) and Se than between Hg and Se and the large distribution of Ag in non-soluble fraction in nuclear and mitochondrial fraction of the liver also suggests that Ag might be detoxified by Se via formation of Ag2Se in the liver of

  5. Subcellular Localization and Clues for the Function of the HetN Factor Influencing Heterocyst Distribution in Anabaena sp. Strain PCC 7120

    PubMed Central

    Corrales-Guerrero, Laura; Mariscal, Vicente; Nürnberg, Dennis J.; Elhai, Jeff; Mullineaux, Conrad W.; Flores, Enrique

    2014-01-01

    In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, heterocysts are formed in the absence of combined nitrogen, following a specific distribution pattern along the filament. The PatS and HetN factors contribute to the heterocyst pattern by inhibiting the formation of consecutive heterocysts. Thus, inactivation of any of these factors produces the multiple contiguous heterocyst (Mch) phenotype. Upon N stepdown, a HetN protein with its C terminus fused to a superfolder version of green fluorescent protein (sf-GFP) or to GFP-mut2 was observed, localized first throughout the whole area of differentiating cells and later specifically on the peripheries and in the polar regions of mature heterocysts, coinciding with the location of the thylakoids. Polar localization required an N-terminal stretch comprising residues 2 to 27 that may represent an unconventional signal peptide. Anabaena strains expressing a version of HetN lacking this fragment from a mutant gene placed at the native hetN locus exhibited a mild Mch phenotype. In agreement with previous results, deletion of an internal ERGSGR sequence, which is identical to the C-terminal sequence of PatS, also led to the Mch phenotype. The subcellular localization in heterocysts of fluorescence resulting from the fusion of GFP to the C terminus of HetN suggests that a full HetN protein is present in these cells. Furthermore, the full HetN protein is more conserved among cyanobacteria than the internal ERGSGR sequence. These observations suggest that HetN anchored to thylakoid membranes in heterocysts may serve a function besides that of generating a regulatory (ERGSGR) peptide. PMID:25049089

  6. Incremental genetic perturbations to MCM2-7 expression and subcellular distribution reveal exquisite sensitivity of mice to DNA replication stress.

    PubMed

    Chuang, Chen-Hua; Wallace, Marsha D; Abratte, Christian; Southard, Teresa; Schimenti, John C

    2010-09-01

    Mutations causing replication stress can lead to genomic instability (GIN). In vitro studies have shown that drastic depletion of the MCM2-7 DNA replication licensing factors, which form the replicative helicase, can cause GIN and cell proliferation defects that are exacerbated under conditions of replication stress. To explore the effects of incrementally attenuated replication licensing in whole animals, we generated and analyzed the phenotypes of mice that were hemizygous for Mcm2, 3, 4, 6, and 7 null alleles, combinations thereof, and also in conjunction with the hypomorphic Mcm4(Chaos3) cancer susceptibility allele. Mcm4(Chaos3/Chaos3) embryonic fibroblasts have ∼40% reduction in all MCM proteins, coincident with reduced Mcm2-7 mRNA. Further genetic reductions of Mcm2, 6, or 7 in this background caused various phenotypes including synthetic lethality, growth retardation, decreased cellular proliferation, GIN, and early onset cancer. Remarkably, heterozygosity for Mcm3 rescued many of these defects. Consistent with a role in MCM nuclear export possessed by the yeast Mcm3 ortholog, the phenotypic rescues correlated with increased chromatin-bound MCMs, and also higher levels of nuclear MCM2 during S phase. The genetic, molecular and phenotypic data demonstrate that relatively minor quantitative alterations of MCM expression, homeostasis or subcellular distribution can have diverse and serious consequences upon development and confer cancer susceptibility. The results support the notion that the normally high levels of MCMs in cells are needed not only for activating the basal set of replication origins, but also "backup" origins that are recruited in times of replication stress to ensure complete replication of the genome. PMID:20838603

  7. Subcellular Distribution and Dynamics of Active Proteasome Complexes Unraveled by a Workflow Combining in Vivo Complex Cross-Linking and Quantitative Proteomics*

    PubMed Central

    Fabre, Bertrand; Lambour, Thomas; Delobel, Julien; Amalric, François; Monsarrat, Bernard; Burlet-Schiltz, Odile; Bousquet-Dubouch, Marie-Pierre

    2013-01-01

    Through protein degradation, the proteasome plays fundamental roles in different cell compartments. Although the composition of the 20S catalytic core particle (CP) has been well documented, little is known about the composition and dynamics of the regulatory complexes that play a crucial role in its activity, or about how they associate with the CP in different cell compartments, different cell lines, and in response to external stimuli. Because of difficulties performing acceptable cell fractionation while maintaining complex integrity, it has been challenging to characterize proteasome complexes by proteomic approaches. Here, we report an integrated protocol, combining a cross-linking procedure on intact cells with cell fractionation, proteasome immuno-purification, and robust label-free quantitative proteomic analysis by mass spectrometry to determine the distribution and dynamics of cellular proteasome complexes in leukemic cells. Activity profiles of proteasomes were correlated fully with the composition of protein complexes and stoichiometry. Moreover, our results suggest that, at the subcellular level, proteasome function is regulated by dynamic interactions between the 20S CP and its regulatory proteins—which modulate proteasome activity, stability, localization, or substrate uptake—rather than by profound changes in 20S CP composition. Proteasome plasticity was observed both in the 20S CP and in its network of interactions following IFNγ stimulation. The fractionation protocol also revealed specific proteolytic activities and structural features of low-abundance microsomal proteasomes from U937 and KG1a cells. These could be linked to their important roles in the endoplasmic reticulum associated degradation pathway in leukemic cells. PMID:23242550

  8. Subcellular localization and clues for the function of the HetN factor influencing heterocyst distribution in Anabaena sp. strain PCC 7120.

    PubMed

    Corrales-Guerrero, Laura; Mariscal, Vicente; Nürnberg, Dennis J; Elhai, Jeff; Mullineaux, Conrad W; Flores, Enrique; Herrero, Antonia

    2014-10-01

    In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, heterocysts are formed in the absence of combined nitrogen, following a specific distribution pattern along the filament. The PatS and HetN factors contribute to the heterocyst pattern by inhibiting the formation of consecutive heterocysts. Thus, inactivation of any of these factors produces the multiple contiguous heterocyst (Mch) phenotype. Upon N stepdown, a HetN protein with its C terminus fused to a superfolder version of green fluorescent protein (sf-GFP) or to GFP-mut2 was observed, localized first throughout the whole area of differentiating cells and later specifically on the peripheries and in the polar regions of mature heterocysts, coinciding with the location of the thylakoids. Polar localization required an N-terminal stretch comprising residues 2 to 27 that may represent an unconventional signal peptide. Anabaena strains expressing a version of HetN lacking this fragment from a mutant gene placed at the native hetN locus exhibited a mild Mch phenotype. In agreement with previous results, deletion of an internal ERGSGR sequence, which is identical to the C-terminal sequence of PatS, also led to the Mch phenotype. The subcellular localization in heterocysts of fluorescence resulting from the fusion of GFP to the C terminus of HetN suggests that a full HetN protein is present in these cells. Furthermore, the full HetN protein is more conserved among cyanobacteria than the internal ERGSGR sequence. These observations suggest that HetN anchored to thylakoid membranes in heterocysts may serve a function besides that of generating a regulatory (ERGSGR) peptide.

  9. Sterol liganding of OSBP-related proteins (ORPs) regulates the subcellular distribution of ORP-VAPA complexes and their impacts on organelle structure.

    PubMed

    Kentala, Henriikka; Pfisterer, Simon G; Olkkonen, Vesa M; Weber-Boyvat, Marion

    2015-07-01

    Oxysterol-binding protein (OSBP) and its homologues (ORPs) are lipid-binding/transfer proteins with affinity for oxysterols, cholesterol and glycerophospholipids. In addition to a ligand-binding domain, a majority of the ORPs carry a pleckstrin homology domain that targets organelle membranes via phosphoinositides, and a motif targeting the endoplasmic reticulum (ER) via VAMP-associated proteins (VAPs). We employed here Bimolecular Fluorescence Complementation (BiFC) to systematically assess the effects of sterol manipulation of HuH7 cells on complexes of established sterol-binding ORPs with their ER receptor, VAMP-associated protein A (VAPA). Depletion of cellular cholesterol with lipoprotein-deficient medium and Mevastatin caused concentration of OSBP-VAPA complexes and Golgi complex markers at a juxtanuclear position, an effect reversed by low-density lipoprotein treatment. A similar redistribution of OSBP-VAPA but not of sterol-binding deficient mutant OSBP(ΔELSK)-VAPA, occurred upon treatment with the high-affinity ligand, 25-hydroxycholesterol (25OHC), which reduced total and free cholesterol. ORP2-VAPA complexes, which localize in untreated cells at blob-like ER structures with associated lipid droplets, were redistributed upon treatment with the ORP2 ligand 22(R)OHC to a diffuse cytoplasmic/ER pattern and the plasma membrane. Analogously, distribution of ORP4L-VAPA complexes between the plasma membrane and vimentin intermediate filament associated compartments was modified by statin or 25OHC treatment. The treatments resulted in loss of vimentin co-localization, and sterol-binding deficient ORP4L(ΔELSR)-VAPA localized predominantly to the plasma membrane. In conclusion, treatment with statin or oxysterol ligands modify the subcellular targeting of ORP-VAPA complexes, consistent with the notion that this machinery controls lipid homeostasis and signaling at organelle interfaces.

  10. The role of subcellular distribution of cadmium and phytochelatins in the generation of distinct phenotypes of AtPCS1- and CePCS3-expressing tobacco.

    PubMed

    Wojas, Sylwia; Ruszczyńska, Anna; Bulska, Ewa; Clemens, Stephan; Antosiewicz, Danuta Maria

    2010-08-15

    Exposure to Cd2+ leads to activation of phytochelatin synthase (PCS) and the formation of phytochelatins (PCs) in the cytosol. Binding of Cd by PCs and the subsequent transport of PC-Cd complexes to the vacuole are essential for Cd tolerance. Attempts to improve Cd detoxification by PCS overexpression have resulted in contrasting plant phenotypes, ranging from enhanced Cd tolerance to Cd hypersensitivity. In the present paper, changes in the subcellular phytochelatin, glutathione, gamma-glutamylcysteine and cadmium vacuolar and cytosolic distribution underlying these phenotypes were examined. Cadmium and PCs levels were determined in protoplasts and vacuoles isolated from leaves of Nicotiana tabacum expressing either of two phytochelatin synthase genes, AtPCS1 and CePCS (differing in their level of Cd tolerance; being Cd hypersensitive or more Cd-tolerant as compared to wild-type plants, respectively). We showed that Cd hypersensitivity of AtPCS1-expressing tobacco results from a significant decrease in both the cytosolic and vacuolar pool of PCs, indicating a decreased cadmium detoxification capacity. By contrast, enhanced Cd tolerance of CePCS plants was accompanied by an increased cytosolic and vacuolar SH of PC/Cd ratio, suggesting more efficient Cd detoxification. Surprisingly, the substantially reduced level of PCs did not influence Cd accumulation in vacuoles of AtPCS1-transformed tobacco (relative to the wild-type), which suggests the important role of mechanisms other than PC-Cd transport in Cd translocation to the vacuole. Our data suggest that the key role of the PCs in Cd tolerance is temporary binding of Cd2+ in the cytosol, and contrary to the current view, their contribution to cadmium sequestration seems to be less important.

  11. Daytime restricted feeding modifies 24 h rhythmicity and subcellular distribution of liver glucocorticoid receptor and the urea cycle in rat liver.

    PubMed

    Luna-Moreno, Dalia; García-Ayala, Braulio; Díaz-Muñoz, Mauricio

    2012-12-14

    The timing system in mammals is formed by a set of peripheral biological clocks coordinated by a light-entrainable pacemaker located in the suprachiasmatic nucleus. Daytime restricted feeding (DRF) modifies the circadian control and uncouples the light-dependent physiological rhythmicity, food access becoming the principal external time cue. In these conditions, an alternative biological clock is expressed, the food-entrainable oscillator (FEO). Glucocorticoid hormones are an important part of the humoral mechanisms in the daily synchronisation of the metabolic response of peripheral oscillators by the timing system. A peak of circulating corticosterone has been reported before food access in DRF protocols. In the present study we explored in the liver the 24 h variations of: (1) the subcellular distribution of glucocorticoid receptor (GCR), (2) the activities of the corticosterone-forming and NADPH-generating enzymes (11β-hydroxysteroid dehydrogenase type 1 (11β-HSD-1) and hexose-6-phosphate dehydrogenase (H6PDH)), and, (3) parameters related with the urea cycle (circulating urea and activities of carbamoyl phosphate synthetase and ornithine transcarbamylase) elicited by DRF. The results showed that DRF promoted an increase of more than two times of the hepatic GCR, but exclusively in the cytosolic compartment, since the GCR in the nuclear fraction showed a reduction. No changes were observed in the activities of 11β-HSD-1 and H6PDH, but the rhythmicity of all of the urea cycle-related parameters was modified. It is concluded that liver glucocorticoid signalling and the urea cycle are responsive to feeding-restricted schedules and could be part of the FEO.

  12. Subcellular localization and clues for the function of the HetN factor influencing heterocyst distribution in Anabaena sp. strain PCC 7120.

    PubMed

    Corrales-Guerrero, Laura; Mariscal, Vicente; Nürnberg, Dennis J; Elhai, Jeff; Mullineaux, Conrad W; Flores, Enrique; Herrero, Antonia

    2014-10-01

    In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, heterocysts are formed in the absence of combined nitrogen, following a specific distribution pattern along the filament. The PatS and HetN factors contribute to the heterocyst pattern by inhibiting the formation of consecutive heterocysts. Thus, inactivation of any of these factors produces the multiple contiguous heterocyst (Mch) phenotype. Upon N stepdown, a HetN protein with its C terminus fused to a superfolder version of green fluorescent protein (sf-GFP) or to GFP-mut2 was observed, localized first throughout the whole area of differentiating cells and later specifically on the peripheries and in the polar regions of mature heterocysts, coinciding with the location of the thylakoids. Polar localization required an N-terminal stretch comprising residues 2 to 27 that may represent an unconventional signal peptide. Anabaena strains expressing a version of HetN lacking this fragment from a mutant gene placed at the native hetN locus exhibited a mild Mch phenotype. In agreement with previous results, deletion of an internal ERGSGR sequence, which is identical to the C-terminal sequence of PatS, also led to the Mch phenotype. The subcellular localization in heterocysts of fluorescence resulting from the fusion of GFP to the C terminus of HetN suggests that a full HetN protein is present in these cells. Furthermore, the full HetN protein is more conserved among cyanobacteria than the internal ERGSGR sequence. These observations suggest that HetN anchored to thylakoid membranes in heterocysts may serve a function besides that of generating a regulatory (ERGSGR) peptide. PMID:25049089

  13. Regulation of gene expression and subcellular protein distribution in MLO-Y4 osteocytic cells by lysophosphatidic acid: Relevance to dendrite outgrowth.

    SciTech Connect

    Waters, Katrina M.; Jacobs, Jon M.; Gritsenko, Marina A.; Karin, Norman J.

    2011-02-26

    Osteoblastic and osteocytic cells are highly responsive to the lipid growth factor lysophosphatidic acid (LPA) but the mechanisms by which LPA alters bone cell functions are largely unknown. A major effect of LPA on osteocytic cells is the stimulation of dendrite membrane outgrowth, a process that we predicted to require changes in gene expression and protein distribution. We employed DNA microarrays for global transcriptional profiling of MLO-Y4 osteocytic cells grown for 6 and 24h in the presence or absence of LPA. We identified 932 transcripts that displayed statistically significant changes in abundance of at least 1.25-fold in response to LPA treatment. Gene ontology (GO) analysis revealed that the regulated gene products were linked to diverse cellular processes, including DNA repair, response to unfolded protein, ossification, protein-RNA complex assembly, and amine biosynthesis. Gene products associated with the regulation of actin microfilament dynamics displayed the most robust expression changes, and LPA-induced dendritogenesis in vitro was blocked by the stress fiber inhibitor cytochalasin D. Mass spectrometry-based proteomic analysis of MLO-Y4 cells revealed significant LPA-induced changes in the abundance of 284 proteins at 6h and 844 proteins at 24h. GO analysis of the proteomic data linked the effects of LPA to cell processes that control of protein distribution and membrane outgrowth, including protein localization, protein complex assembly, Golgi vesicle transport, cytoskeleton-dependent transport, and membrane invagination/endocytosis. Dendrites were isolated from LPA-treated MLO-Y4 cells and subjected to proteomic analysis to quantitatively assess the subcellular distribution of proteins. Sets of 129 and 36 proteins were enriched in the dendrite fraction as compared to whole cells after 6h and 24h of LPA exposure, respectively. Protein markers indicated that membranous organelles were largely excluded from the dendrites. Highly represented among

  14. Variation in cadmium accumulation among 30 cultivars and cadmium subcellular distribution in 2 selected cultivars of water spinach (Ipomoea aquatica Forsk.).

    PubMed

    Wang, Junli; Yuan, Jiangang; Yang, Zhongyi; Huang, Baifei; Zhou, Yihui; Xin, Junliang; Gong, Yulian; Yu, Hui

    2009-10-14

    To reduce the influx of cadmium (Cd), a toxic heavy metal, into the human food chain through vegetable intake, a pot experiment for the selection of a pollution-safe cultivar (PSC) of water spinach (Ipomoea aquatica Forsk.) was carried out. The experiment with 30 tested cultivars revealed that the maximum differences in Cd concentration between the cultivars containing the highest and the lowest Cd were 3.0-3.9-fold under low-Cd treatment (soil Cd = 0.593 mg kg(-1)), 2.7-3.5-fold under middle-Cd treatment (soil Cd = 1.091 mg kg(-1)), and 2.6-2.7-fold under high-Cd treatment (soil Cd = 1.824 mg kg(-1)), large enough to define the Cd-PSCs. Concentrations of Cd in edible parts of six cultivars, cv. Daxingbaigu, Huifengqing, Qiangkunbaigu, Qiangkunqinggu, Shenniuliuye, and Xingtianqinggu, were lower than 0.2 mg kg(-1), the maximum level (ML) of Cd allowed by the Codex Alimentarius Commission (CAC) standard, even under middle-Cd treatment. Accordingly, these cultivars were treated as typical Cd-PSCs. Four cultivars, cv. Jieyangbaigeng, Xianggangdaye, Sannongbaigeng, and Taiwan 308, contained Cd in edible parts exceeding the ML even under low-Cd treatment, and they were defined as typical non-Cd-PSCs. The correlations of the Cd concentrations among the tested cultivars between the three treatments were significant at the p < 0.05 level. A conspicuous difference in Cd subcellular distribution in hydroponic plant tissues between cv. Qiangkunqinggu (a typical Cd-PSC) and cv. Taiwan 308 (a typical non-Cd-PSC) were observed. Cd absorbed by cv. Qiangkunqinggu seemed to be well-compartmentalized in root and in cell wall fragment, which may be one of the mechanisms leading to its low Cd accumulating property. The results indicated that water spinach, a leafy vegetable, could be easily polluted by soils contaminated with Cd, as 80% of the tested cultivars had exceeded the ML of Cd according to the CAC standard even under the middle-Cd treatment. Much of the evidence obtained from

  15. Evolution of alanine:glyoxylate aminotransferase 1 peroxisomal and mitochondrial targeting. A survey of its subcellular distribution in the livers of various representatives of the classes Mammalia, Aves and Amphibia.

    PubMed

    Danpure, C J; Fryer, P; Jennings, P R; Allsop, J; Griffiths, S; Cunningham, A

    1994-08-01

    As part of a wider study on the molecular evolution of alanine:glyoxylate aminotransferase 1 (AGT1) intracellular compartmentalization, we have determined the subcellular distribution of immunoreactive AGT1, using postembedding protein A-gold immunoelectron microscopy, in the livers of various members of the classes Mammalia, Aves, and Amphibia. As far as organellar distribution is concerned, three categories could be distinguished. In members of the first category (type I), all, or nearly all, of the immunoreactive AGT1 was concentrated within the peroxisomes. In the second category (type II), AGT1 was found more evenly distributed in both peroxisomes and mitochondria. In the third category (type III), AGT1 was localized mainly within the mitochondria with much lower, but widely variable, amounts in the peroxisomes. Type I animals include the human, two great apes (gorilla, orangutan), two Old World monkeys (anubis baboon, Japanese macaque), a New World monkey (white-faced Saki monkey), a lago, morph (European rabbit), a bat (Seba's short-tailed fruit bat), two caviomorph rodents (guinea pig, orange-rumped agouti), and two Australian marsupials (koala, Bennett's wallaby). Type II animals include two New World monkeys (common marmoset, cotton-top tamarin), three prosimians (brown lemur, fat-tailed dwarf lemur, pygmy slow loris), five rodents (a hybrid crested porcupine, Colombian ground squirrel, laboratory rat, laboratory mouse, golden hamster), an American marsupial (grey short-tailed opossum), and a bird (raven). Type III animals include the large tree shrew, three insectivores (common Eurasian mole, European hedgehog, house shrew), four carnivores (domestic cat, ocelot, domestic dog, polecat ferret), and an amphibian (common frog). In addition to these categories, some animals (e.g. guinea pig, common frog) possessed significant amounts of cytosolic AGT1. Whereas the subcellular distribution of AGT1 in some orders (e.g. Insectivora and Carnivora) did not appear

  16. Unique subcellular distribution of phosphorylated Plk1 (Ser137 and Thr210) in mouse oocytes during meiotic division and pPlk1(Ser137) involvement in spindle formation and REC8 cleavage.

    PubMed

    Du, Juan; Cao, Yan; Wang, Qian; Zhang, Nana; Liu, Xiaoyu; Chen, Dandan; Liu, Xiaoyun; Xu, Qunyuan; Ma, Wei

    2015-01-01

    Polo-like kinase 1 (Plk1) is pivotal for proper mitotic progression, its targeting activity is regulated by precise subcellular positioning and phosphorylation. Here we assessed the protein expression, subcellular localization and possible functions of phosphorylated Plk1 (pPlk1(Ser137) and pPlk1(Thr210)) in mouse oocytes during meiotic division. Western blot analysis revealed a peptide of pPlk1(Ser137) with high and stable expression from germinal vesicle (GV) until metaphase II (MII), while pPlk1(Thr210) was detected as one large single band at GV stage and 2 small bands after germinal vesicle breakdown (GVBD), which maintained stable up to MII. Immunofluorescence analysis showed pPlk1(Ser137) was colocalized with microtubule organizing center (MTOC) proteins, γ-tubulin and pericentrin, on spindle poles, concomitantly with persistent concentration at centromeres and dynamic aggregation between chromosome arms. Differently, pPlk1(Thr210) was persistently distributed across the whole body of chromosomes after meiotic resumption. The specific Plk1 inhibitor, BI2536, repressed pPlk1(Ser137) accumulation at MTOCs and between chromosome arms, consequently disturbed γ-tubulin and pericentrin recruiting to MTOCs, destroyed meiotic spindle formation, and delayed REC8 cleavage, therefore arresting oocytes at metaphase I (MI) with chromosome misalignment. BI2536 completely reversed the premature degradation of REC8 and precocious segregation of chromosomes induced with okadaic acid (OA), an inhibitor to protein phosphatase 2A. Additionally, the protein levels of pPlk1(Ser137) and pPlk1(Thr210), as well as the subcellular distribution of pPlk1(Thr210), were not affected by BI2536. Taken together, our results demonstrate that Plk1 activity is required for meiotic spindle assembly and REC8 cleavage, with pPlk1(Ser137) is the action executor, in mouse oocytes during meiotic division. PMID:26654596

  17. Metabolism of cerebroside sulfate and subcellular distribution of its metabolites in cultured skin fibroblasts from controls, metachromatic leukodystrophy, and globoid cell leukodystrophy.

    PubMed

    Inui, K; Furukawa, M; Okada, S; Yabuuchi, H

    1988-02-01

    With pulse-chase study of 1-[14C]stearic acid-labeled cerebroside sulfate (14C-CS) and subsequent subcellular fractionation by Percoll gradient, the metabolism of CS and translocation of its metabolites in human skin fibroblasts from controls, metachromatic leukodystrophy (MLD), and globoid cell leukodystrophy (GLD) were studied. In control skin fibroblasts, CS was transported to lysosome and metabolized there to galactosylceramide (GalCer) and ceramide (Cer) within 1 h. During the chase period, radioactivity was increased at plasma membrane plus Golgi as phospholipids and no accumulation of GalCer or Cer was found in lysosome. In MLD fibroblasts, 95% of 14C-CS taken up was unhydrolyzed at 24 h-chase and accumulated at not only lysosome but also plasma membrane. In GLD fibroblasts, GalCer was accumulated throughout the subcellular fractions and more accumulated mainly at plasma membrane plus Golgi with longer pulse. This translocation of lipid from lysosome seems to have considerable function, even in lipidosis, which may result in an imbalance of the sphingolipid pattern on the cell surface and these changes might be one of causes of neuronal dysfunction in sphingolipidosis.

  18. Determination of elemental distribution in green micro-algae using synchrotron radiation nano X-ray fluorescence (SR-nXRF) and electron microscopy techniques--subcellular localization and quantitative imaging of silver and cobalt uptake by Coccomyxa actinabiotis.

    PubMed

    Leonardo, T; Farhi, E; Boisson, A-M; Vial, J; Cloetens, P; Bohic, S; Rivasseau, C

    2014-02-01

    The newly discovered unicellular micro-alga Coccomyxa actinabiotis proves to be highly radio-tolerant and strongly concentrates radionuclides, as well as large amounts of toxic metals. This study helps in the understanding of the mechanisms involved in the accumulation and detoxification of silver and cobalt. Elemental distribution inside Coccomyxa actinabiotis cells was determined using synchrotron nano X-ray fluorescence spectroscopy at the ID22 nano fluorescence imaging beamline of the European Synchrotron Radiation Facility. The high resolution and high sensitivity of this technique enabled the assessment of elemental associations and exclusions in subcellular micro-algae compartments. A quantitative treatment of the scans was implemented to yield absolute concentrations of each endogenous and exogenous element with a spatial resolution of 100 nm and compared to the macroscopic content in cobalt and silver determined using inductively coupled plasma-mass spectrometry. The nano X-ray fluorescence imaging was complemented by transmission electron microscopy coupled to X-ray microanalysis (TEM-EDS), yielding differential silver distribution in the cell wall, cytosol, nucleus, chloroplast and mitochondria with unique resolution. The analysis of endogenous elements in control cells revealed that iron had a unique distribution; zinc, potassium, manganese, molybdenum, and phosphate had their maxima co-localized in the same area; and sulfur, copper and chlorine were almost homogeneously distributed among the whole cell. The subcellular distribution and quantification of cobalt and silver in micro-alga, assessed after controlled exposure to various concentrations, revealed that exogenous metals were mainly sequestered inside the cell rather than on mucilage or the cell wall, with preferential compartmentalization. Cobalt was homogeneously distributed outside of the chloroplast. Silver was localized in the cytosol at low concentration and in the whole cell excluding the

  19. Determination of elemental distribution in green micro-algae using synchrotron radiation nano X-ray fluorescence (SR-nXRF) and electron microscopy techniques--subcellular localization and quantitative imaging of silver and cobalt uptake by Coccomyxa actinabiotis.

    PubMed

    Leonardo, T; Farhi, E; Boisson, A-M; Vial, J; Cloetens, P; Bohic, S; Rivasseau, C

    2014-02-01

    The newly discovered unicellular micro-alga Coccomyxa actinabiotis proves to be highly radio-tolerant and strongly concentrates radionuclides, as well as large amounts of toxic metals. This study helps in the understanding of the mechanisms involved in the accumulation and detoxification of silver and cobalt. Elemental distribution inside Coccomyxa actinabiotis cells was determined using synchrotron nano X-ray fluorescence spectroscopy at the ID22 nano fluorescence imaging beamline of the European Synchrotron Radiation Facility. The high resolution and high sensitivity of this technique enabled the assessment of elemental associations and exclusions in subcellular micro-algae compartments. A quantitative treatment of the scans was implemented to yield absolute concentrations of each endogenous and exogenous element with a spatial resolution of 100 nm and compared to the macroscopic content in cobalt and silver determined using inductively coupled plasma-mass spectrometry. The nano X-ray fluorescence imaging was complemented by transmission electron microscopy coupled to X-ray microanalysis (TEM-EDS), yielding differential silver distribution in the cell wall, cytosol, nucleus, chloroplast and mitochondria with unique resolution. The analysis of endogenous elements in control cells revealed that iron had a unique distribution; zinc, potassium, manganese, molybdenum, and phosphate had their maxima co-localized in the same area; and sulfur, copper and chlorine were almost homogeneously distributed among the whole cell. The subcellular distribution and quantification of cobalt and silver in micro-alga, assessed after controlled exposure to various concentrations, revealed that exogenous metals were mainly sequestered inside the cell rather than on mucilage or the cell wall, with preferential compartmentalization. Cobalt was homogeneously distributed outside of the chloroplast. Silver was localized in the cytosol at low concentration and in the whole cell excluding the

  20. Metal-induced stress in bivalves living along a gradient of Cd contamination: relating sub-cellular metal distribution to population-level responses.

    PubMed

    Perceval, Olivier; Couillard, Yves; Pinel-Alloul, Bernadette; Giguère, Anik; Campbell, Peter G C

    2004-09-20

    The use of biomarkers to assess the impacts of contaminants on aquatic ecosystems has noticeably increased over the past few years. Few of these studies, however, have contributed to the prediction of ecologically significant effects (i.e., at the population or community levels). The present field study was designed to evaluate the potential of metallothionein (MT) and sub-cellular metal partitioning measurements for predicting toxic effects at higher levels of the biological organization in freshwater bivalves (Pyganodon grandis) chronically exposed to Cd. For that purpose, we quantitatively sampled P. grandis populations in the littoral zone of nine lakes on the Precambrian Canadian Shield during two consecutive summers (1998 and 1999); lakes were characterized by contrasting Cd levels but similar trophic status. We tested relationships between the population status of P. grandis (i.e., growth parameters, density, biomass, secondary production, turnover ratio and cumulative fecundity) and (i) ambient Cd concentrations, (ii) sub-organismal responses (MT concentrations in the gill cytosol of individuals and Cd concentrations in three metal-ligand pools identified as M-HMW, the high molecular weight pool, M-MT, the metallothionein-like pool and M-LMW, the low molecular weight pool) and (iii) ecological confounding factors (food resources, presence of host fishes for the obligatory parasitic larval stage of P. grandis). Our results show that littoral density, live weight, dry viscera biomass, production and cumulative fecundity decreased with increasing concentrations of the free-cadmium ion in the environment (Pearson's r ranging from -0.63 to -0.78). On the other hand, theoretical maximum shell lengths (L( infinity )) in our populations were related to both the dissolved Ca concentration and food quality (sestonic C and N concentrations). Overall, Cd concentrations in the gill cytosolic HMW pool of the individual molluscs were the biomarker response that was most

  1. Metal-induced stress in bivalves living along a gradient of Cd contamination: relating sub-cellular metal distribution to population-level responses.

    PubMed

    Perceval, Olivier; Couillard, Yves; Pinel-Alloul, Bernadette; Giguère, Anik; Campbell, Peter G C

    2004-09-20

    The use of biomarkers to assess the impacts of contaminants on aquatic ecosystems has noticeably increased over the past few years. Few of these studies, however, have contributed to the prediction of ecologically significant effects (i.e., at the population or community levels). The present field study was designed to evaluate the potential of metallothionein (MT) and sub-cellular metal partitioning measurements for predicting toxic effects at higher levels of the biological organization in freshwater bivalves (Pyganodon grandis) chronically exposed to Cd. For that purpose, we quantitatively sampled P. grandis populations in the littoral zone of nine lakes on the Precambrian Canadian Shield during two consecutive summers (1998 and 1999); lakes were characterized by contrasting Cd levels but similar trophic status. We tested relationships between the population status of P. grandis (i.e., growth parameters, density, biomass, secondary production, turnover ratio and cumulative fecundity) and (i) ambient Cd concentrations, (ii) sub-organismal responses (MT concentrations in the gill cytosol of individuals and Cd concentrations in three metal-ligand pools identified as M-HMW, the high molecular weight pool, M-MT, the metallothionein-like pool and M-LMW, the low molecular weight pool) and (iii) ecological confounding factors (food resources, presence of host fishes for the obligatory parasitic larval stage of P. grandis). Our results show that littoral density, live weight, dry viscera biomass, production and cumulative fecundity decreased with increasing concentrations of the free-cadmium ion in the environment (Pearson's r ranging from -0.63 to -0.78). On the other hand, theoretical maximum shell lengths (L( infinity )) in our populations were related to both the dissolved Ca concentration and food quality (sestonic C and N concentrations). Overall, Cd concentrations in the gill cytosolic HMW pool of the individual molluscs were the biomarker response that was most

  2. Fractionation of Subcellular Organelles.

    PubMed

    Graham, John M

    2015-01-01

    This unit provides both a theoretical and a practical background to all the techniques associated with the application of differential and density gradient centrifugation for the analysis of subcellular membranes. The density gradient information focuses on the use of the modern gradient solute iodixanol, chosen for its ease of use, versatility, and compatibility with biological particles. Its use in both pre-formed discontinuous and continuous gradients and in self-generated gradients is discussed. Considerable emphasis is given to selection of the appropriate centrifuge rotors and tubes and their influence on the methods used for creation, fractionation, and analysis of density gradients. Without proper consideration of these critical ancillary procedures, the resolving power of the gradient can be easily compromised. PMID:26621372

  3. Fractionation of Subcellular Organelles.

    PubMed

    Graham, John M

    2015-12-01

    This unit provides both a theoretical and a practical background to all the techniques associated with the application of differential and density gradient centrifugation for the analysis of subcellular membranes. The density gradient information focuses on the use of the modern gradient solute iodixanol, chosen for its ease of use, versatility, and compatibility with biological particles. Its use in both pre-formed discontinuous and continuous gradients and in self-generated gradients is discussed. Considerable emphasis is given to selection of the appropriate centrifuge rotors and tubes and their influence on the methods used for creation, fractionation, and analysis of density gradients. Without proper consideration of these critical ancillary procedures, the resolving power of the gradient can be easily compromised.

  4. Expression of an abscisic acid-binding single-chain antibody influences the subcellular distribution of abscisic acid and leads to developmental changes in transgenic potato plants.

    PubMed

    Strauss, M; Kauder, F; Peisker, M; Sonnewald, U; Conrad, U; Heineke, D

    2001-07-01

    Potato (Solanum tuberosum L. cv. Désirée) plants were transformed to express a single-chain variable-fragment antibody against abscisic acid (ABA), and present in the endoplasmic reticulum at to up to 0.24% of the soluble leaf protein. The resulting transgenic plants were only able to grow normally at 95% humidity and moderate light. Four-week-old plants accumulated ABA to high extent, were retarded in growth and their leaves were smaller than those of control plants. Leaf stomatal conductivity was increased due to larger stomates. The subcellular concentrations of ABA in the chloroplast, cytoplasm and vacuole, and the apoplastic space of leaves were determined. In the 4-week-old transgenic plants the concentration of ABA not bound to the antibody was identical to that of control plants and the stomates were able to close in response to lower humidity of the atmosphere. A detailed analysis of age-dependent changes in plant metabolism showed that leaves of young transformed plants developed in ABA deficiency and leaves of older plants in ABA excess. Phenotypic changes developed in ABA deficiency partly disappeared in older plants.

  5. Human Gyrovirus Apoptin shows a similar subcellular distribution pattern and apoptosis induction as the chicken anaemia virus derived VP3/Apoptin

    PubMed Central

    Bullenkamp, J; Cole, D; Malik, F; Alkhatabi, H; Kulasekararaj, A; Odell, E W; Farzaneh, F; Gäken, J; Tavassoli, M

    2012-01-01

    The chicken anaemia virus-derived protein Apoptin/VP3 (CAV-Apoptin) has the important ability to induce tumour-selective apoptosis in a variety of human cancer cells. Recently the first human Gyrovirus (HGyV) was isolated from a human skin swab. It shows significant structural and organisational resemblance to CAV and encodes a homologue of CAV-Apoptin/VP3. Using overlapping primers we constructed a synthetic human Gyrovirus Apoptin (HGyV-Apoptin) fused to green fluorescent protein in order to compare its apoptotic function in various human cancer cell lines to CAV-Apoptin. HGyV-Apoptin displayed a similar subcellular expression pattern as observed for CAV-Apoptin, marked by translocation to the nucleus of cancer cells, although it is predominantly located in the cytosol of normal human cells. Furthermore, expression of either HGyV-Apoptin or CAV-Apoptin in several cancer cell lines triggered apoptosis at comparable levels. These findings indicate a potential anti-cancer role for HGyV-Apoptin. PMID:22495351

  6. Immunodetection and subcellular distribution of imidazoline receptor proteins with three antibodies in mouse and human brains: Effects of treatments with I1- and I2-imidazoline drugs.

    PubMed

    Keller, Benjamin; García-Sevilla, Jesús A

    2015-09-01

    Various imidazoline receptor (IR) proteins have been proposed to mediate the effects of selective I1- and I2-IR drugs. However, the association of these IR-binding proteins with classic I1- and I2-radioligand binding sites remains somewhat controversial. In this study, three IR antibodies (anti-NISCH and anti-nischarin for I1-IRs; and anti-IRBP for I1/I2-IRs) were used to immunodetect, characterize and compare IR protein patterns in brain (mouse and human; total homogenate, subcellular fractionation, grey and white matter) and some cell systems (neurones, astrocytes, human platelets). Various immunoreactive IRs (specific molecular weight bands coincidently detected with the different antibodies) were related to I1-IR (167 kDa, 105/115 kDa and 85 kDa proteins) or I2-IR (66 kDa, 45 kDa and 30 kDa proteins) types. The biochemical characterization of cortical 167 kDa protein, localized in the membrane/cytosol but not in the nucleus, indicated that this I1-IR also forms part of higher order nischarin-related complexes. The contents of I1-IR (167 kDa, 105/115 kDa, and 85 kDa) proteins in mouse brain cortex were upregulated by treatment with I1-drugs (moxonidine, efaroxan) but not with I2-drugs (BU-224, LSL 61122). Conversely, the contents of I2-IR (66 kDa, 45 kDa and 30 kDa) proteins in mouse brain cortex were modulated by treatment with I2-drugs (decreases after BU-224 and LSL 61122, and increases after idazoxan) but not with I1-drugs (with the exception of moxonidine). These findings further indicate that brain immunoreactive IR proteins exist in multiple forms that can be grouped in the already known I1- and I2-IR types, which are expressed both in neurones and astrocytes.

  7. Immunodetection and subcellular distribution of imidazoline receptor proteins with three antibodies in mouse and human brains: Effects of treatments with I1- and I2-imidazoline drugs.

    PubMed

    Keller, Benjamin; García-Sevilla, Jesús A

    2015-09-01

    Various imidazoline receptor (IR) proteins have been proposed to mediate the effects of selective I1- and I2-IR drugs. However, the association of these IR-binding proteins with classic I1- and I2-radioligand binding sites remains somewhat controversial. In this study, three IR antibodies (anti-NISCH and anti-nischarin for I1-IRs; and anti-IRBP for I1/I2-IRs) were used to immunodetect, characterize and compare IR protein patterns in brain (mouse and human; total homogenate, subcellular fractionation, grey and white matter) and some cell systems (neurones, astrocytes, human platelets). Various immunoreactive IRs (specific molecular weight bands coincidently detected with the different antibodies) were related to I1-IR (167 kDa, 105/115 kDa and 85 kDa proteins) or I2-IR (66 kDa, 45 kDa and 30 kDa proteins) types. The biochemical characterization of cortical 167 kDa protein, localized in the membrane/cytosol but not in the nucleus, indicated that this I1-IR also forms part of higher order nischarin-related complexes. The contents of I1-IR (167 kDa, 105/115 kDa, and 85 kDa) proteins in mouse brain cortex were upregulated by treatment with I1-drugs (moxonidine, efaroxan) but not with I2-drugs (BU-224, LSL 61122). Conversely, the contents of I2-IR (66 kDa, 45 kDa and 30 kDa) proteins in mouse brain cortex were modulated by treatment with I2-drugs (decreases after BU-224 and LSL 61122, and increases after idazoxan) but not with I1-drugs (with the exception of moxonidine). These findings further indicate that brain immunoreactive IR proteins exist in multiple forms that can be grouped in the already known I1- and I2-IR types, which are expressed both in neurones and astrocytes. PMID:26038110

  8. Reversible Oxidation of a Conserved Methionine in the Nuclear Export Sequence Determines Subcellular Distribution and Activity of the Fungal Nitrate Regulator NirA

    PubMed Central

    Schinko, Thorsten; Gesslbauer, Bernd; Hortschansky, Peter; Dattenböck, Christoph; Muro-Pastor, María Isabel; Kungl, Andreas; Brakhage, Axel A.; Scazzocchio, Claudio; Strauss, Joseph

    2015-01-01

    The assimilation of nitrate, a most important soil nitrogen source, is tightly regulated in microorganisms and plants. In Aspergillus nidulans, during the transcriptional activation process of nitrate assimilatory genes, the interaction between the pathway-specific transcription factor NirA and the exportin KapK/CRM1 is disrupted, and this leads to rapid nuclear accumulation and transcriptional activity of NirA. In this work by mass spectrometry, we found that in the absence of nitrate, when NirA is inactive and predominantly cytosolic, methionine 169 in the nuclear export sequence (NES) is oxidized to methionine sulfoxide (Metox169). This oxidation depends on FmoB, a flavin-containing monooxygenase which in vitro uses methionine and cysteine, but not glutathione, as oxidation substrates. The function of FmoB cannot be replaced by alternative Fmo proteins present in A. nidulans. Exposure of A. nidulans cells to nitrate led to rapid reduction of NirA-Metox169 to Met169; this reduction being independent from thioredoxin and classical methionine sulfoxide reductases. Replacement of Met169 by isoleucine, a sterically similar but not oxidizable residue, led to partial loss of NirA activity and insensitivity to FmoB-mediated nuclear export. In contrast, replacement of Met169 by alanine transformed the protein into a permanently nuclear and active transcription factor. Co-immunoprecipitation analysis of NirA-KapK interactions and subcellular localization studies of NirA mutants lacking different parts of the protein provided evidence that Met169 oxidation leads to a change in NirA conformation. Based on these results we propose that in the presence of nitrate the activation domain is exposed, but the NES is masked by a central portion of the protein (termed nitrate responsive domain, NiRD), thus restricting active NirA molecules to the nucleus. In the absence of nitrate, Met169 in the NES is oxidized by an FmoB-dependent process leading to loss of protection by the Ni

  9. Reversible Oxidation of a Conserved Methionine in the Nuclear Export Sequence Determines Subcellular Distribution and Activity of the Fungal Nitrate Regulator NirA.

    PubMed

    Gallmetzer, Andreas; Silvestrini, Lucia; Schinko, Thorsten; Gesslbauer, Bernd; Hortschansky, Peter; Dattenböck, Christoph; Muro-Pastor, María Isabel; Kungl, Andreas; Brakhage, Axel A; Scazzocchio, Claudio; Strauss, Joseph

    2015-07-01

    The assimilation of nitrate, a most important soil nitrogen source, is tightly regulated in microorganisms and plants. In Aspergillus nidulans, during the transcriptional activation process of nitrate assimilatory genes, the interaction between the pathway-specific transcription factor NirA and the exportin KapK/CRM1 is disrupted, and this leads to rapid nuclear accumulation and transcriptional activity of NirA. In this work by mass spectrometry, we found that in the absence of nitrate, when NirA is inactive and predominantly cytosolic, methionine 169 in the nuclear export sequence (NES) is oxidized to methionine sulfoxide (Metox169). This oxidation depends on FmoB, a flavin-containing monooxygenase which in vitro uses methionine and cysteine, but not glutathione, as oxidation substrates. The function of FmoB cannot be replaced by alternative Fmo proteins present in A. nidulans. Exposure of A. nidulans cells to nitrate led to rapid reduction of NirA-Metox169 to Met169; this reduction being independent from thioredoxin and classical methionine sulfoxide reductases. Replacement of Met169 by isoleucine, a sterically similar but not oxidizable residue, led to partial loss of NirA activity and insensitivity to FmoB-mediated nuclear export. In contrast, replacement of Met169 by alanine transformed the protein into a permanently nuclear and active transcription factor. Co-immunoprecipitation analysis of NirA-KapK interactions and subcellular localization studies of NirA mutants lacking different parts of the protein provided evidence that Met169 oxidation leads to a change in NirA conformation. Based on these results we propose that in the presence of nitrate the activation domain is exposed, but the NES is masked by a central portion of the protein (termed nitrate responsive domain, NiRD), thus restricting active NirA molecules to the nucleus. In the absence of nitrate, Met169 in the NES is oxidized by an FmoB-dependent process leading to loss of protection by the Ni

  10. Reversible Oxidation of a Conserved Methionine in the Nuclear Export Sequence Determines Subcellular Distribution and Activity of the Fungal Nitrate Regulator NirA.

    PubMed

    Gallmetzer, Andreas; Silvestrini, Lucia; Schinko, Thorsten; Gesslbauer, Bernd; Hortschansky, Peter; Dattenböck, Christoph; Muro-Pastor, María Isabel; Kungl, Andreas; Brakhage, Axel A; Scazzocchio, Claudio; Strauss, Joseph

    2015-07-01

    The assimilation of nitrate, a most important soil nitrogen source, is tightly regulated in microorganisms and plants. In Aspergillus nidulans, during the transcriptional activation process of nitrate assimilatory genes, the interaction between the pathway-specific transcription factor NirA and the exportin KapK/CRM1 is disrupted, and this leads to rapid nuclear accumulation and transcriptional activity of NirA. In this work by mass spectrometry, we found that in the absence of nitrate, when NirA is inactive and predominantly cytosolic, methionine 169 in the nuclear export sequence (NES) is oxidized to methionine sulfoxide (Metox169). This oxidation depends on FmoB, a flavin-containing monooxygenase which in vitro uses methionine and cysteine, but not glutathione, as oxidation substrates. The function of FmoB cannot be replaced by alternative Fmo proteins present in A. nidulans. Exposure of A. nidulans cells to nitrate led to rapid reduction of NirA-Metox169 to Met169; this reduction being independent from thioredoxin and classical methionine sulfoxide reductases. Replacement of Met169 by isoleucine, a sterically similar but not oxidizable residue, led to partial loss of NirA activity and insensitivity to FmoB-mediated nuclear export. In contrast, replacement of Met169 by alanine transformed the protein into a permanently nuclear and active transcription factor. Co-immunoprecipitation analysis of NirA-KapK interactions and subcellular localization studies of NirA mutants lacking different parts of the protein provided evidence that Met169 oxidation leads to a change in NirA conformation. Based on these results we propose that in the presence of nitrate the activation domain is exposed, but the NES is masked by a central portion of the protein (termed nitrate responsive domain, NiRD), thus restricting active NirA molecules to the nucleus. In the absence of nitrate, Met169 in the NES is oxidized by an FmoB-dependent process leading to loss of protection by the Ni

  11. Comparison of GLUT1, GLUT3, and GLUT4 mRNA and the subcellular distribution of their proteins in normal human muscle

    NASA Technical Reports Server (NTRS)

    Stuart, C. A.; Wen, G.; Gustafson, W. C.; Thompson, E. A.

    2000-01-01

    Basal, "insulin-independent" glucose uptake into skeletal muscle is provided by glucose transporters positioned at the plasma membrane. The relative amount of the three glucose transporters expressed in muscle has not been previously quantified. Using a combination of qualitative and quantitative ribonuclease protection assay (RPA) methods, we found in normal human muscle that GLUT1, GLUT3, and GLUT4 mRNA were expressed at 90 +/- 10, 46 +/- 4, and 156 +/- 12 copies/ng RNA, respectively. Muscle was fractionated by DNase digestion and differential sedimentation into membrane fractions enriched in plasma membranes (PM) or low-density microsomes (LDM). GLUT1 and GLUT4 proteins were distributed 57% to 67% in LDM, whereas GLUT3 protein was at least 88% in the PM-enriched fractions. These data suggest that basal glucose uptake into resting human muscle could be provided in part by each of these three isoforms.

  12. Subcellular distribution of swine vesicular disease virus proteins and alterations induced in infected cells: A comparative study with foot-and-mouth disease virus and vesicular stomatitis virus

    SciTech Connect

    Martin-Acebes, Miguel A.; Gonzalez-Magaldi, Monica; Rosas, Maria F.; Borrego, Belen; Brocchi, Emiliana; Armas-Portela, Rosario; Sobrino, Francisco

    2008-05-10

    The intracellular distribution of swine vesicular disease virus (SVDV) proteins and the induced reorganization of endomembranes in IBRS-2 cells were analyzed. Fluorescence to new SVDV capsids appeared first upon infection, concentrated in perinuclear circular structures and colocalized to dsRNA. As in foot-and-mouth disease virus (FMDV)-infected cells, a vesicular pattern was predominantly found in later stages of SVDV capsid morphogenesis that colocalized with those of non-structural proteins 2C, 2BC and 3A. These results suggest that assembly of capsid proteins is associated to the replication complex. Confocal microscopy showed a decreased fluorescence to ER markers (calreticulin and protein disulfide isomerase), and disorganization of cis-Golgi gp74 and trans-Golgi caveolin-1 markers in SVDV- and FMDV-, but not in vesicular stomatitis virus (VSV)-infected cells. Electron microscopy of SVDV-infected cells at an early stage of infection revealed fragmented ER cisternae with expanded lumen and accumulation of large Golgi vesicles, suggesting alterations of vesicle traffic through Golgi compartments. At this early stage, FMDV induced different patterns of ER fragmentation and Golgi alterations. At later stages of SVDV cytopathology, cells showed a completely vacuolated cytoplasm containing vesicles of different sizes. Cell treatment with brefeldin A, which disrupts the Golgi complex, reduced SVDV ({approx} 5 log) and VSV ({approx} 4 log) titers, but did not affect FMDV growth. Thus, three viruses, which share target tissues and clinical signs in natural hosts, induce different intracellular effects in cultured cells.

  13. Subcellular distribution of the calcium-storing inositol 1,4,5-trisphosphate-sensitive organelle in rat liver. Possible linkage to the plasma membrane through the actin microfilaments.

    PubMed Central

    Rossier, M F; Bird, G S; Putney, J W

    1991-01-01

    The role of Ins(1,4,5)P3 in the mobilization of Ca2+ from intracellular stores of non-muscle cells has been extensively demonstrated; however, the nature of the organelle releasing the Ca2+ is still poorly understood. The distributions of the Ins(1,4,5)P3-binding sites and of the Ins(1,4,5)P3-sensitive Ca2+ pool were investigated in subcellular fractions obtained from rat liver and compared with those of other markers. The Ins(1,4,5)P3-binding vesicles appeared to be completely distinct from the endoplasmic-reticulum-derived microsomes and were enriched in the same fractions which were enriched in alkaline phosphodiesterase I activity. This co-purification of the plasma-membrane marker with the Ins(1,4,5)P3-binding sites was dramatically altered after freezing or after treatment of the homogenate with the microfilament-disruptive drug cytochalasin B, suggesting that the Ins(1,4,5)P3-sensitive organelle may be linked to the plasma membrane through the actin microfilaments. No correlation was observed between the Ins(1,4,5)P3-binding capacity and the portion of the Ca2+ pool that was released by Ins(1,4,5)P3. This may result from the disruption of the native organelle during homogenization, leading to the formation of vesicles containing the Ins(1,4,5)P3 receptor, but lacking the Ca2+ pump. These results are consistent with the idea of a specialized Ins(1,4,5)P3-regulated organelle distinct from the endoplasmic reticulum, and we propose a model of the structural organization of this organelle, in which the anchorage to the cytoskeleton as well as the spatial separation of the Ca2+ pump from the Ins(1,4,5)P3 receptor have important functional significance. PMID:1849402

  14. Bioavailability of purified subcellular metals to a marine fish.

    PubMed

    Guo, Feng; Yao, Jie; Wang, Wen-Xiong

    2013-09-01

    In the present study, the authors used a supply of naturally contaminated oysters to investigate how the subcellular metal distribution and the metal burden in prey affected the transfer of metals to a marine fish, the grunt Terapon jarbua. The oysters, Crassostrea hongkongensis, each with different contamination histories, were collected and separated into 3 subcellular fractions: 1) metal-rich granules, 2) cellular debris, and 3) a combined fraction of organelles, heat-denatured proteins, and metallothionein-like proteins, defined as the trophically available metal (TAM). These purified fractions showed a wide range of metal concentrations and were fed to the fish for a period of 7 d at a daily comparable feeding rate of 3% of fish body weight. After 7 d exposure, the newly absorbed metals were mainly distributed in the intestine and liver, indicating a significant tissue-specific trophic transfer, especially for Cd and Cu. The trophic transfer factors (TTFs) showed a sequence of cellular debris >TAM > metal-rich granules, suggesting the impact of subcellular distribution in prey on metal bioavailability. However, significant inverse relationships between the TTFs and the metal concentrations in diets were also found in the present study, especially for Cd and Zn. The subcellular metal compartmentalization might be less important than the metal concentration in prey influencing the trophic transfer. The authors' results have important implications for bioavailability and environmental assessment of dietary metals.

  15. Regulating Subcellular Metal Homeostasis: The Key to Crop Improvement.

    PubMed

    Bashir, Khurram; Rasheed, Sultana; Kobayashi, Takanori; Seki, Motoaki; Nishizawa, Naoko K

    2016-01-01

    Iron (Fe), zinc (Zn), manganese (Mn), and copper (Cu) are essential micronutrient mineral elements for living organisms, as they regulate essential cellular processes, such as chlorophyll synthesis and photosynthesis (Fe, Cu, and Mn), respiration (Fe and Cu), and transcription (Zn). The storage and distribution of these minerals in various cellular organelles is strictly regulated to ensure optimal metabolic rates. Alteration of the balance in uptake, distribution, and/or storage of these minerals severely impairs cellular metabolism and significantly affects plant growth and development. Thus, any change in the metal profile of a cellular compartment significantly affects metabolism. Different subcellular compartments are suggested to be linked through complex retrograde signaling networks to regulate cellular metal homeostasis. Various genes regulating cellular and subcellular metal distribution have been identified and characterized. Understanding the role of these transporters is extremely important to elaborate the signaling between various subcellular compartments. Moreover, modulation of the proteins involved in cellular metal homeostasis may help in the regulation of metabolism, adaptability to a diverse range of environmental conditions, and biofortification. Here, we review progress in the understanding of different subcellular metal transport components in plants and discuss the prospects of regulating cellular metabolism and strategies to develop biofortified crop plants. PMID:27547212

  16. Regulating Subcellular Metal Homeostasis: The Key to Crop Improvement

    PubMed Central

    Bashir, Khurram; Rasheed, Sultana; Kobayashi, Takanori; Seki, Motoaki; Nishizawa, Naoko K.

    2016-01-01

    Iron (Fe), zinc (Zn), manganese (Mn), and copper (Cu) are essential micronutrient mineral elements for living organisms, as they regulate essential cellular processes, such as chlorophyll synthesis and photosynthesis (Fe, Cu, and Mn), respiration (Fe and Cu), and transcription (Zn). The storage and distribution of these minerals in various cellular organelles is strictly regulated to ensure optimal metabolic rates. Alteration of the balance in uptake, distribution, and/or storage of these minerals severely impairs cellular metabolism and significantly affects plant growth and development. Thus, any change in the metal profile of a cellular compartment significantly affects metabolism. Different subcellular compartments are suggested to be linked through complex retrograde signaling networks to regulate cellular metal homeostasis. Various genes regulating cellular and subcellular metal distribution have been identified and characterized. Understanding the role of these transporters is extremely important to elaborate the signaling between various subcellular compartments. Moreover, modulation of the proteins involved in cellular metal homeostasis may help in the regulation of metabolism, adaptability to a diverse range of environmental conditions, and biofortification. Here, we review progress in the understanding of different subcellular metal transport components in plants and discuss the prospects of regulating cellular metabolism and strategies to develop biofortified crop plants. PMID:27547212

  17. Subcellular trace element distribution in Geosiphon pyriforme

    NASA Astrophysics Data System (ADS)

    Maetz, Mischa; Schüßler, Arthur; Wallianos, Alexandros; Traxel, Kurt

    1999-04-01

    Geosiphon pyriforme is a unique endosymbiotic consortium consisting of a soil dwelling fungus and the cyanobacterium Nostoc punctiforme. At present this symbiosis becomes very interesting because of its phylogenetic relationship to the arbuscular mycorrhizal (AM) fungi. Geosiphon pyriforme could be an important model system for these obligate symbiotic fungi, which supply 80-90% of all land plant species with nutrients, in particular phosphorous and trace elements. Combined PIXE and STIM analyses of the various compartments of Geosiphon give hints for the matter exchange between the symbiotic partners and their environment and the kind of nutrient storage and acquisition, in particular related to nitrogen fixation and metabolism. To determine the quality of our PIXE results we analysed several geological and biological standards over a time period of three years. This led to an overall precision of about 6% and an accuracy of 5-10% for nearly all detectable elements. In combination with the correction model for the occurring mass loss during the analyses this holds true even for biological targets.

  18. Plasma effects on subcellular structures

    SciTech Connect

    Gweon, Bomi; Kim, Dan Bee; Jung, Heesoo; Choe, Wonho; Kim, Daeyeon; Shin, Jennifer H.

    2010-03-08

    Atmospheric pressure helium plasma treated human hepatocytes exhibit distinctive zones of necrotic and live cells separated by a void. We propose that plasma induced necrosis is attributed to plasma species such as oxygen radicals, charged particles, metastables and/or severe disruption of charged cytoskeletal proteins. Interestingly, uncharged cytoskeletal intermediate filaments are only minimally disturbed by plasma, elucidating the possibility of plasma induced electrostatic effects selectively destroying charged proteins. These bona fide plasma effects, which inflict alterations in specific subcellular structures leading to necrosis and cellular detachment, were not observed by application of helium flow or electric field alone.

  19. Cholesterol Depletion Alters Cardiomyocyte Subcellular Signaling and Increases Contractility

    PubMed Central

    McIntosh, Victoria J.; Abou Samra, Abdul B.; Mohammad, Ramzi M.; Lasley, Robert D.

    2016-01-01

    Membrane cholesterol levels play an important factor in regulating cell function. Sarcolemmal cholesterol is concentrated in lipid rafts and caveolae, which are flask-shaped invaginations of the plasma membrane. The scaffolding protein caveolin permits the enrichment of cholesterol in caveolae, and caveolin interactions with numerous proteins regulate their function. The purpose of this study was to determine whether acute reductions in cardiomyocyte cholesterol levels alter subcellular protein kinase activation, intracellular Ca2+ and contractility. Methods: Ventricular myocytes, isolated from adult Sprague Dawley rats, were treated with the cholesterol reducing agent methyl-β-cyclodextrin (MβCD, 5 mM, 1 hr, room temperature). Total cellular cholesterol levels, caveolin-3 localization, subcellular, ERK and p38 mitogen activated protein kinase (MAPK) signaling, contractility, and [Ca2+]i were assessed. Results: Treatment with MβCD reduced cholesterol levels by ~45 and shifted caveolin-3 from cytoskeleton and triton-insoluble fractions to the triton-soluble fraction, and increased ERK isoform phosphorylation in cytoskeletal, cytosolic, triton-soluble and triton-insoluble membrane fractions without altering their subcellular distributions. In contrast the primary effect of MβCD was on p38 subcellular distribution of p38α with little effect on p38 phosphorylation. Cholesterol depletion increased cardiomyocyte twitch amplitude and the rates of shortening and relaxation in conjunction with increased diastolic and systolic [Ca2+]i. Conclusions: These results indicate that acute reductions in membrane cholesterol levels differentially modulate basal cardiomyocyte subcellular MAPK signaling, as well as increasing [Ca2+]i and contractility. PMID:27441649

  20. Sequence conserved for subcellular localization

    PubMed Central

    Nair, Rajesh; Rost, Burkhard

    2002-01-01

    The more proteins diverged in sequence, the more difficult it becomes for bioinformatics to infer similarities of protein function and structure from sequence. The precise thresholds used in automated genome annotations depend on the particular aspect of protein function transferred by homology. Here, we presented the first large-scale analysis of the relation between sequence similarity and identity in subcellular localization. Three results stood out: (1) The subcellular compartment is generally more conserved than what might have been expected given that short sequence motifs like nuclear localization signals can alter the native compartment; (2) the sequence conservation of localization is similar between different compartments; and (3) it is similar to the conservation of structure and enzymatic activity. In particular, we found the transition between the regions of conserved and nonconserved localization to be very sharp, although the thresholds for conservation were less well defined than for structure and enzymatic activity. We found that a simple measure for sequence similarity accounting for pairwise sequence identity and alignment length, the HSSP distance, distinguished accurately between protein pairs of identical and different localizations. In fact, BLAST expectation values outperformed the HSSP distance only for alignments in the subtwilight zone. We succeeded in slightly improving the accuracy of inferring localization through homology by fine tuning the thresholds. Finally, we applied our results to the entire SWISS-PROT database and five entirely sequenced eukaryotes. PMID:12441382

  1. Subcellular localization of pituitary enzymes

    NASA Technical Reports Server (NTRS)

    Smith, R. E.

    1970-01-01

    A cytochemical procedure is reported for identifying subcellular sites of enzymes hydrolyzing beta-naphthylamine substrates, and to study the sites of reaction product localization in cells of various tissues. Investigations using the substrate Leu 4-methoxy-8-naphthylamine, a capture with hexonium pararosaniline, and the final chelation of osmium have identified the hydrolyzing enzyme of rat liver cells; this enzyme localized on cell membranes with intense deposition in the areas of the parcanaliculi. The study of cells in the anterior pituitary of the rat showed the deposition of reaction product on cell membrane; and on the membranes of secretion granules contained within the cell. The deposition of reaction product on the cell membrane however showed no increase or decrease with changes in the physiological state of the gland and release of secretion granules from specific cells.

  2. Modeling biosilicification at subcellular scales.

    PubMed

    Javaheri, Narjes; Cronemberger, Carolina M; Kaandorp, Jaap A

    2013-01-01

    Biosilicification occurs in many organisms. Sponges and diatoms are major examples of them. In this chapter, we introduce a modeling approach that describes several biological mechanisms controlling silicification. Modeling biosilicification is a typical multiscale problem where processes at very different temporal and spatial scales need to be coupled: processes at the molecular level, physiological processes at the subcellular and cellular level, etc. In biosilicification morphology plays a fundamental role, and a spatiotemporal model is required. In the case of sponges, a particle simulation based on diffusion-limited aggregation is presented here. This model can describe fractal properties of silica aggregates in first steps of deposition on an organic template. In the case of diatoms, a reaction-diffusion model is introduced which can describe the concentrations of chemical components and has the possibility to include polymerization chain of reactions. PMID:24420712

  3. Effects of alpha-linolenic acid vs. docosahexaenoic acid supply on the distribution of fatty acids among the rat cardiac subcellular membranes after a short- or long-term dietary exposure

    PubMed Central

    Brochot, Amandine; Guinot, Marine; Auchere, Daniel; Macaire, Jean-Paul; Weill, Pierre; Grynberg, Alain; Rousseau-Ralliard, Delphine

    2009-01-01

    Background Previous work showed that the functional cardiac effect of dietary alpha-linolenic acid (ALA) in rats requires a long feeding period (6 months), although a docosahexaenoic (DHA) acid-supply affects cardiac adrenergic response after 2 months. However, the total cardiac membrane n-3 polyunsaturated fatty acid (PUFA) composition remained unchanged after 2 months. This delay could be due to a specific reorganization of the different subcellular membrane PUFA profiles. This study was designed to investigate the evolution between 2 and 6 months of diet duration of the fatty acid profile in sarcolemmal (SL), mitochondrial (MI), nuclear (NU) and sarcoplasmic reticulum (SR) membrane fractions. Methods Male Wistar rats were randomly assigned to 3 dietary groups (n = 10/diet/period), either n-3 PUFA-free diet (CTL), or ALA or DHA-rich diets. After 2 or 6 months, the subcellular cardiac membrane fractions were separated by differential centrifugations and sucrose gradients. Each membrane profile was analysed by gas chromatography (GC) after lipid extraction. Results As expected the n-3 PUFA-rich diets incorporated n-3 PUFA instead of n-6 PUFA in all the subcellular fractions, which also exhibited individual specificities. The diet duration increased SFA and decreased PUFA in SL, whereas NU remained constant. The SR and MI enriched in n-3 PUFA exhibited a decreased DHA level with ageing in the DHA and CTL groups. Conversely, the n-3 PUFA level remained unchanged in the ALA group, due to a significant increase in docosapentaenoic acid (DPA). N-3 PUFA rich diets lead to a better PUFA profile in all the fractions and significantly prevent the profile modifications induced by ageing. Conclusion With the ALA diet the n-3 PUFA content, particularly in SR and SL kept increasing between 2 and 6 months, which may partly account for the delay to achieve the modification of adrenergic response. PMID:19320987

  4. Intracellular And Subcellular Partitioning Of Nickel In Aureococcus Anophagefferens

    NASA Astrophysics Data System (ADS)

    Wang, B.; Axe, L.; Wei, L.; Bagheri, S.; Michalopoulou, Z.

    2008-12-01

    Brown tides are caused by Aureococcus anophagefferens, a species of Pelagophyceae, and have been observed in NY/NJ waterways effecting ecosystems by attenuating light, changing water color, reducing eelgrass beds, decreasing shellfisheries, and further impacting the food web by reducing phytoplankton. Although the impact of macronutrients and iron on A. anophagefferens has been well studied, contaminants, and specifically trace metals have not. In long-term experiments designed to investigate the growth and toxicity, Cd, Cu, Ni, and Zn exposure was evaluated over 10-13 to 10-7 M for the free metal ion. While growth was inhibited or terminated from exposure to Cd and Cu, nickel addition ([Ni2+]: 10-11.23 to 10-10.23 M) promoted A. anophagefferens growth. Short-term experiments are being conducted to better understand mechanistically nickel speciation and distribution. Both total intracellular and subcellular metal concentrations are being assessed with radio-labeled 63Ni. Subcellular fractions are defined as metal-sensitive fractions (MSF) constituting organelles, cell debris, and heat-denatured protein [HDP] and biologically detoxified metal comprising heat-stabilized protein [HSP] and metal-rich granules [MRG]. Based on subcellular distribution, aqueous [Ni2+] concentrations, and A. anophagefferens growth rates, potential reaction pathways promoting A. anophagefferens growth can be addressed.

  5. Subcellular Dynamics of Multifunctional Protein Regulation: Mechanisms of GAPDH Intracellular Translocation

    PubMed Central

    Sirover, Michael A.

    2012-01-01

    Multidimensional proteins such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) exhibit distinct activities unrelated to their originally identified functions. Apart from glycolysis, GAPDH participates in iron metabolism, membrane trafficking, histone biosynthesis, the maintenance of DNA integrity and receptor mediated cell signaling. Further, multifunctional proteins exhibit distinct changes in their subcellular localization reflecting their new activities. As such, GAPDH is not only a cytosolic protein but is localized in the membrane, the nucleus, polysomes, the ER and the Golgi. In addition, although the initial subcellular localizations of multifunctional proteins may be of significance, dynamic changes in intracellular distribution may occur as a consequence of those new activities. As such, regulatory mechanisms may exist through which cells control multifunctional protein expression as a function of their subcellular localization. The temporal sequence through which subcellular translocation and the acquisition of new GAPDH functions is considered as well as post-translational modification as a basis for its intracellular transport. PMID:22388977

  6. Intracellular mannose binding lectin mediates subcellular trafficking of HIV-1 gp120 in neurons.

    PubMed

    Teodorof, C; Divakar, S; Soontornniyomkij, B; Achim, C L; Kaul, M; Singh, K K

    2014-09-01

    Human immunodeficiency virus-1 (HIV-1) enters the brain early during infection and leads to severe neuronal damage and central nervous system impairment. HIV-1 envelope glycoprotein 120 (gp120), a neurotoxin, undergoes intracellular trafficking and transport across neurons; however mechanisms of gp120 trafficking in neurons are unclear. Our results show that mannose binding lectin (MBL) that binds to the N-linked mannose residues on gp120, participates in intravesicular packaging of gp120 in neuronal subcellular organelles and also in subcellular trafficking of these vesicles in neuronal cells. Perinuclear MBL:gp120 vesicular complexes were observed and MBL facilitated the subcellular trafficking of gp120 via the endoplasmic reticulum (ER) and Golgi vesicles. The functional carbohydrate recognition domain of MBL was required for perinuclear organization, distribution and subcellular trafficking of MBL:gp120 vesicular complexes. Nocodazole, an agent that depolymerizes the microtubule network, abolished the trafficking of MBL:gp120 vesicles, suggesting that these vesicular complexes were transported along the microtubule network. Live cell imaging confirmed the association of the MBL:gp120 complexes with dynamic subcellular vesicles that underwent trafficking in neuronal soma and along the neurites. Thus, our findings suggest that intracellular MBL mediates subcellular trafficking and transport of viral glycoproteins in a microtubule-dependent mechanism in the neurons.

  7. Analysis of Subcellular Prefoldin 1 Redistribution During Rabies Virus Infection

    PubMed Central

    Zhang, Jinyang; Han, Qinqin; Song, Yuzhu; Chen, Qiang; Xia, Xueshan

    2015-01-01

    Background: Rabies virus (RABV) is one of the old deadly zoonotic viruses. It attacks the central nervous system and causes acute encephalitis in humans and animals. Host factors are known to be essential for virus infection and replication in cells. The identification of the key host factors required for RABV infection may provide important information on RABV replication and may provide new potential targets for RABV drug discovery. Objectives: This study aimed to investigate the change in the subcellular distribution and expression of the host protein Prefoldin subunit 1 (PFDN1) in RABV-infected cells and the viral expression of plasmids in the transfected cells. Materials and Methods: Mouse Neuro-2a (N2a) cells were infected by RABV or transfected with the plasmids of the nucleoprotein (N) and/or phosphoprotein (P) gene of RABV. The subcellular distribution of PFDN1 was analyzed by confocal microscopy, and the transcription levels of PFDN1 in the N and/or P gene of the RABV-transfected or RABV-infected N2a cells were assessed via real-time quantitative polymerase chain reaction. Results: Confocal microscopy showed that PFDN1 was colocalized with the N protein of RABV in the infected N2a cells and was mainly recruited to the characteristic Negri-Body-Like (NBL) structures in the cytoplasm, as well as the cotransfection of the N and P genes of RABV. The transcription of PFDN1 in the RABV-infected N2a cells was upregulated, whereas the transfection of the N and/or P genes did not result in the upregulation of PFDN1. Conclusions: The results of this work demonstrated that the subcellular distribution of PFDN1 was altered in the RABV-infected N2a cells and colocalized with the N protein of RABV in the NBL structures. PMID:26421138

  8. Self-calibrating viscosity probes: Design and subcellular localization

    PubMed Central

    Dakanali, Marianna; Do, Thai H.; Horn, Austin; Chongchivivat, Akaraphon; Jarusreni, Tuptim; Lichlyter, Darcy; Guizzunti, Gianni; Haidekker, Mark A.; Theodorakis, Emmanuel A.

    2012-01-01

    We describe the design, synthesis and fluorescence profiles of new self-calibrating viscosity dyes in which a coumarin (reference fluorophore) has been covalently linked with a molecular rotor (viscosity sensor). Characterization of their fluorescence properties was made with separate excitation of the units and through Resonance Energy Transfer from the reference to the sensor dye. We have modified the linker and the substitution of the rotor in order to change the hydrophilicity of these probes thereby altering their subcellular localization. For instance, hydrophilic dye 12 shows a homogeneous distribution inside the cell and represents a suitable probe for viscosity measurements in the cytoplasm. 2012 Elsevier Ltd. All rights reserved. PMID:22698784

  9. CHARACTERIZATION OF RAT LIVER SUBCELLULAR MEMBRANES

    PubMed Central

    DeHeer, David H.; Olson, Merle S.; Pinckard, R. Neal

    1974-01-01

    The induction of acute hepatocellular necrosis in rats resulted in the production of complement fixing, IgM autoantibodies directed toward inner and outer mitochondrial membranes, microsomal membrane, lysosomal membrane, nuclear membrane, cytosol, but not to plasma membrane. Utilizing selective absorption procedures it was demonstrated that each subcellular membrane fraction possessed unique autoantigenic activity with little or no cross-reactivity between the various membrane fractions. It is proposed that the development of membrane-specific autoantibodies may provide an immunological marker useful in the differential characterization of various subcellular membranes. PMID:4813214

  10. Sequestration of total and methyl mercury in different subcellular pools in marine caged fish.

    PubMed

    Onsanit, Sarayut; Wang, Wen-Xiong

    2011-12-30

    Mercury contamination is an important issue in marine fish, and can cause toxicity to human by fish consumption. Many studies have measured the mercury concentrations in fish and estimated the threshold levels of its risk to human, but the mercury sequestration in different subcellular pools of fish is unclear. In this study, we investigated the concentration and distribution of total mercury (THg) and methylmercury (MeHg) in different subcellular fractions in the farmed red seabream, red drum, and black seabream from Fujian marine fish farms, China. We found that both THg and MeHg were dominantly bound with the cellular debris, followed by metallothionein-like protein>metal-rich granule>heat-denatured protein>organelles pools. In general, Hg bound with the metal-sensitive fraction was small, indicating that Hg may have little toxicity to the fish (muscle). For the first time we showed that MTLP fraction had the highest % of total Hg as MeHg (88-91%) among all the subcellular fractions. Furthermore, the mercury concentration and subcellular distribution in the black seabream were both dependent on the fish size. Subcellular study may shed light on the detoxification of marine fish to Hg exposure and the potential bioavailability to humans due to fish consumption. PMID:22056886

  11. Subcellular localization of ammonium transporters in Dictyostelium discoideum

    PubMed Central

    Kirsten, Janet H; Xiong, Yanhua; Davis, Carter T; Singleton, Charles K

    2008-01-01

    Background With the exception of vertebrates, most organisms have plasma membrane associated ammonium transporters which primarily serve to import a source of nitrogen for nutritional purposes. Dictyostelium discoideum has three ammonium transporters, Amts A, B and C. Our present work used fluorescent fusion proteins to determine the cellular localization of the Amts and tested the hypothesis that the transporters mediate removal of ammonia generated endogenously from the elevated protein catabolism common to many protists. Results Using RFP and YFP fusion constructs driven by the actin 15 promoter, we found that the three ammonium transporters were localized on the plasma membrane and on the membranes of subcellular organelles. AmtA and AmtB were localized on the membranes of endolysosomes and phagosomes, with AmtB further localized on the membranes of contractile vacuoles. AmtC also was localized on subcellular organelles when it was stabilized by coexpression with either the AmtA or AmtB fusion transporter. The three ammonium transporters exported ammonia linearly with regard to time during the first 18 hours of the developmental program as revealed by reduced export in the null strains. The fluorescently tagged transporters rescued export when expressed in the null strains, and thus they were functional transporters. Conclusion Unlike ammonium transporters in most organisms, which import NH3/NH4+ as a nitrogen source, those of Dictyostelium export ammonia/ammonium as a waste product from extensive catabolism of exogenously derived and endogenous proteins. Localization on proteolytic organelles and on the neutral contractile vacuole suggests that Dictyostelium ammonium transporters may have unique subcellular functions and play a role in the maintenance of intracellular ammonium distribution. A lack of correlation between the null strain phenotypes and ammonia excretion properties of the ammonium transporters suggests that it is not the excretion function that

  12. Subcellular partitioning of metals in Aporrectodea caliginosa along a gradient of metal exposure in 31 field-contaminated soils.

    PubMed

    Beaumelle, Léa; Gimbert, Frédéric; Hedde, Mickaël; Guérin, Annie; Lamy, Isabelle

    2015-07-01

    Subcellular fractionation of metals in organisms was proposed as a better way to characterize metal bioaccumulation. Here we report the impact of a laboratory exposure to a wide range of field-metal contaminated soils on the subcellular partitioning of metals in the earthworm Aporrectodea caliginosa. Soils moderately contaminated were chosen to create a gradient of soil metal availability; covering ranges of both soil metal contents and of several soil parameters. Following exposure, Cd, Pb and Zn concentrations were determined both in total earthworm body and in three subcellular compartments: cytosolic, granular and debris fractions. Three distinct proxies of soil metal availability were investigated: CaCl2-extractable content dissolved content predicted by a semi-mechanistic model and free ion concentration predicted by a geochemical speciation model. Subcellular partitionings of Cd and Pb were modified along the gradient of metal exposure, while stable Zn partitioning reflected regulation processes. Cd subcellular distribution responded more strongly to increasing soil Cd concentration than the total internal content, when Pb subcellular distribution and total internal content were similarly affected. Free ion concentrations were better descriptors of Cd and Pb subcellular distribution than CaCl2 extractable and dissolved metal concentrations. However, free ion concentrations and soil total metal contents were equivalent descriptors of the subcellular partitioning of Cd and Pb because they were highly correlated. Considering lowly contaminated soils, our results raise the question of the added value of three proxies of metal availability compared to soil total metal content in the assessment of metal bioavailability to earthworm.

  13. Recent advances in imaging subcellular processes

    PubMed Central

    Myers, Kenneth A.; Janetopoulos, Christopher

    2016-01-01

    Cell biology came about with the ability to first visualize cells. As microscopy techniques advanced, the early microscopists became the first cell biologists to observe the inner workings and subcellular structures that control life. This ability to see organelles within a cell provided scientists with the first understanding of how cells function. The visualization of the dynamic architecture of subcellular structures now often drives questions as researchers seek to understand the intricacies of the cell. With the advent of fluorescent labeling techniques, better and new optical techniques, and more sensitive and faster cameras, a whole array of questions can now be asked. There has been an explosion of new light microscopic techniques, and the race is on to build better and more powerful imaging systems so that we can further our understanding of the spatial and temporal mechanisms controlling molecular cell biology. PMID:27408708

  14. Recent advances in imaging subcellular processes.

    PubMed

    Myers, Kenneth A; Janetopoulos, Christopher

    2016-01-01

    Cell biology came about with the ability to first visualize cells. As microscopy techniques advanced, the early microscopists became the first cell biologists to observe the inner workings and subcellular structures that control life. This ability to see organelles within a cell provided scientists with the first understanding of how cells function. The visualization of the dynamic architecture of subcellular structures now often drives questions as researchers seek to understand the intricacies of the cell. With the advent of fluorescent labeling techniques, better and new optical techniques, and more sensitive and faster cameras, a whole array of questions can now be asked. There has been an explosion of new light microscopic techniques, and the race is on to build better and more powerful imaging systems so that we can further our understanding of the spatial and temporal mechanisms controlling molecular cell biology. PMID:27408708

  15. Subcellular SIMS imaging of gadolinium isotopes in human glioblastoma cells treated with a gadolinium containing MRI agent

    NASA Astrophysics Data System (ADS)

    Smith, Duane R.; Lorey, Daniel R.; Chandra, Subhash

    2004-06-01

    Neutron capture therapy is an experimental binary radiotherapeutic modality for the treatment of brain tumors such as glioblastoma multiforme. Recently, neutron capture therapy with gadolinium-157 has gained attention, and techniques for studying the subcellular distribution of gadolinium-157 are needed. In this preliminary study, we have been able to image the subcellular distribution of gadolinium-157, as well as the other six naturally abundant isotopes of gadolinium, with SIMS ion microscopy. T98G human glioblastoma cells were treated for 24 h with 25 mg/ml of the metal ion complex diethylenetriaminepentaacetic acid Gd(III) dihydrogen salt hydrate (Gd-DTPA). Gd-DTPA is a contrast enhancing agent used for MRI of brain tumors, blood-brain barrier impairment, diseases of the central nervous system, etc. A highly heterogeneous subcellular distribution was observed for gadolinium-157. The nuclei in each cell were distinctly lower in gadolinium-157 than in the cytoplasm. Even within the cytoplasm the gadolinium-157 was heterogeneously distributed. The other six naturally abundant isotopes of gadolinium were imaged from the same cells and exhibited a subcellular distribution consistent with that observed for gadolinium-157. These observations indicate that SIMS ion microscopy may be a viable approach for subcellular studies of gadolinium containing neutron capture therapy drugs and may even play a major role in the development and validation of new gadolinium contrast enhancing agents for diagnostic MRI applications.

  16. Cellular and subcellular localization of Marlin-1 in the brain

    PubMed Central

    Vidal, René L; Valenzuela, José I; Luján, Rafael; Couve, Andrés

    2009-01-01

    Background Marlin-1 is a microtubule binding protein that associates specifically with the GABAB1 subunit in neurons and with members of the Janus kinase family in lymphoid cells. In addition, it binds the molecular motor kinesin-I and nucleic acids, preferentially single stranded RNA. Marlin-1 is expressed mainly in the central nervous system but little is known regarding its cellular and subcellular distribution in the brain. Results Here we have studied the localization of Marlin-1 in the rodent brain and cultured neurons combining immunohistochemistry, immunofluorescence and pre-embedding electron microscopy. We demonstrate that Marlin-1 is enriched in restricted areas of the brain including olfactory bulb, cerebral cortex, hippocampus and cerebellum. Marlin-1 is abundant in dendrites and axons of GABAergic and non-GABAergic hippocampal neurons. At the ultrastructural level, Marlin-1 is present in the cytoplasm and the nucleus of CA1 neurons in the hippocampus. In the cytoplasm it associates to microtubules in the dendritic shaft and occasionally with the Golgi apparatus, the endoplasmic reticulum (ER) and dendritic spines. In the nucleus, clusters of Marlin-1 associate to euchromatin. Conclusion Our results demonstrate that Marlin-1 is expressed in discrete areas of the brain. They also confirm the microtubule association at the ultrastructural level in neurons. Together with the abundance of the protein in dendrites and axons they are consistent with the emerging role of Marlin-1 as an intracellular protein linking the cytoskeleton and transport. Our study constitutes the first detailed description of the cellular and subcellular distribution of Marlin-1 in the brain. As such, it will set the basis for future studies on the functional implications of Marlin-1 in protein trafficking. PMID:19386132

  17. Subcellular Localization of Class I Histone Deacetylases in the Developing Xenopus tectum

    PubMed Central

    Guo, Xia; Ruan, Hangze; Li, Xia; Qin, Liming; Tao, Yi; Qi, Xianjie; Gao, Juanmei; Gan, Lin; Duan, Shumin; Shen, Wanhua

    2016-01-01

    Histone deacetylases (HDACs) are thought to localize in the nucleus to regulate gene transcription and play pivotal roles in neurogenesis, apoptosis, and plasticity. However, the subcellular distribution of class I HDACs in the developing brain remains unclear. Here, we show that HDAC1 and HDAC2 are located in both the mitochondria and the nucleus in the Xenopus laevis stage 34 tectum and are mainly restricted to the nucleus following further brain development. HDAC3 is widely present in the mitochondria, nucleus, and cytoplasm during early tectal development and is mainly distributed in the nucleus in stage 45 tectum. In contrast, HDAC8 is broadly located in the mitochondria, nucleus, and cytoplasm during tectal development. These data demonstrate that HDAC1, HDAC2, and HDAC3 are transiently localized in the mitochondria and that the subcellular distribution of class I HDACs in the Xenopus tectum is heterogeneous. Furthermore, we observed that spherical mitochondria accumulate in the cytoplasm at earlier stages, whereas elongated mitochondria are evenly distributed in the tectum at later stages. The activity of histone acetylation (H4K12) remains low in mitochondria during tectal development. Pharmacological blockades of HDACs using a broad spectrum HDAC inhibitor of Trichostatin A (TSA) or specific class I HDAC inhibitors of MS-275 and MGCD0103 decrease the number of mitochondria in the tectum at stage 34. These findings highlight a link between the subcellular distribution of class I HDACs and mitochondrial dynamics in the developing optic tectum of Xenopus laevis. PMID:26793062

  18. An image analysis method to quantify CFTR subcellular localization.

    PubMed

    Pizzo, Lucilla; Fariello, María Inés; Lepanto, Paola; Aguilar, Pablo S; Kierbel, Arlinet

    2014-08-01

    Aberrant protein subcellular localization caused by mutation is a prominent feature of many human diseases. In Cystic Fibrosis (CF), a recessive lethal disorder that results from dysfunction of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), the most common mutation is a deletion of phenylalanine-508 (pF508del). Such mutation produces a misfolded protein that fails to reach the cell surface. To date, over 1900 mutations have been identified in CFTR gene, but only a minority has been analyzed at the protein level. To establish if a particular CFTR variant alters its subcellular distribution, it is necessary to quantitatively determine protein localization in the appropriate cellular context. To date, most quantitative studies on CFTR localization have been based on immunoprecipitation and western blot. In this work, we developed and validated a confocal microscopy-image analysis method to quantitatively examine CFTR at the apical membrane of epithelial cells. Polarized MDCK cells transiently transfected with EGFP-CFTR constructs and stained for an apical marker were used. EGFP-CFTR fluorescence intensity in a region defined by the apical marker was normalized to EGFP-CFTR whole cell fluorescence intensity, rendering "apical CFTR ratio". We obtained an apical CFTR ratio of 0.67 ± 0.05 for wtCFTR and 0.11 ± 0.02 for pF508del. In addition, this image analysis method was able to discriminate intermediate phenotypes: partial rescue of the pF508del by incubation at 27 °C rendered an apical CFTR ratio value of 0.23 ± 0.01. We concluded the method has a good sensitivity and accurately detects milder phenotypes. Improving axial resolution through deconvolution further increased the sensitivity of the system as rendered an apical CFTR ratio of 0.76 ± 0.03 for wild type and 0.05 ± 0.02 for pF508del. The presented procedure is faster and simpler when compared with other available methods and it is therefore suitable as a screening method to identify

  19. Dissecting the Subcellular Compartmentation of Proteins and Metabolites in Arabidopsis Leaves Using Non-aqueous Fractionation *

    PubMed Central

    Arrivault, Stéphanie; Guenther, Manuela; Florian, Alexandra; Encke, Beatrice; Feil, Regina; Vosloh, Daniel; Lunn, John E.; Sulpice, Ronan; Fernie, Alisdair R.; Stitt, Mark; Schulze, Waltraud X.

    2014-01-01

    Non-aqueous fractionation is a technique for the enrichment of different subcellular compartments derived from lyophilized material. It was developed to study the subcellular distribution of metabolites. Here we analyzed the distribution of about 1,000 proteins and 70 metabolites, including 22 phosphorylated intermediates in wild-type Arabidopsis rosette leaves, using non-aqueous gradients divided into 12 fractions. Good separation of plastidial, cytosolic, and vacuolar metabolites and proteins was achieved, but cytosolic, mitochondrial, and peroxisomal proteins clustered together. There was considerable heterogeneity in the fractional distribution of transcription factors, ribosomal proteins, and subunits of the vacuolar-ATPase, indicating diverse compartmental location. Within the plastid, sub-organellar separation of thylakoids and stromal proteins was observed. Metabolites from the Calvin–Benson cycle, photorespiration, starch and sucrose synthesis, glycolysis, and the tricarboxylic acid cycle grouped with their associated proteins of the respective compartment. Non-aqueous fractionation thus proved to be a powerful method for the study of the organellar, and in some cases sub-organellar, distribution of proteins and their association with metabolites. It remains the technique of choice for the assignment of subcellular location to metabolites in intact plant tissues, and thus the technique of choice for doing combined metabolite–protein analysis on a single tissue sample. PMID:24866124

  20. A Computational Modeling and Simulation Approach to Investigate Mechanisms of Subcellular cAMP Compartmentation.

    PubMed

    Yang, Pei-Chi; Boras, Britton W; Jeng, Mao-Tsuen; Docken, Steffen S; Lewis, Timothy J; McCulloch, Andrew D; Harvey, Robert D; Clancy, Colleen E

    2016-07-01

    distribution of cAMP at the subcellular level could be important for developing new strategies for the prevention or treatment of unfavorable responses associated with different disease states. PMID:27409243

  1. A Computational Modeling and Simulation Approach to Investigate Mechanisms of Subcellular cAMP Compartmentation

    PubMed Central

    Yang, Pei-Chi; Boras, Britton W.; Jeng, Mao-Tsuen; Lewis, Timothy J.; McCulloch, Andrew D.; Harvey, Robert D.; Clancy, Colleen E.

    2016-01-01

    distribution of cAMP at the subcellular level could be important for developing new strategies for the prevention or treatment of unfavorable responses associated with different disease states. PMID:27409243

  2. Tuning the Catalytic Activity of Subcellular Nanoreactors.

    PubMed

    Jakobson, Christopher M; Chen, Yiqun; Slininger, Marilyn F; Valdivia, Elias; Kim, Edward Y; Tullman-Ercek, Danielle

    2016-07-31

    Bacterial microcompartments are naturally occurring subcellular organelles of bacteria and serve as a promising scaffold for the organization of heterologous biosynthetic pathways. A critical element in the design of custom biosynthetic organelles is quantitative control over the loading of heterologous enzymes to the interior of the organelles. We demonstrate that the loading of heterologous proteins to the 1,2-propanediol utilization microcompartment of Salmonella enterica can be controlled using two strategies: by modulating the transcriptional activation of the microcompartment container and by coordinating the expression of the microcompartment container and the heterologous cargo. These strategies allow general control over the loading of heterologous proteins localized by two different N-terminal targeting peptides and represent an important step toward tuning the catalytic activity of bacterial microcompartments for increased biosynthetic productivity.

  3. Controlling subcellular delivery to optimize therapeutic effect

    PubMed Central

    Mossalam, Mohanad; Dixon, Andrew S; Lim, Carol S

    2010-01-01

    This article focuses on drug targeting to specific cellular organelles for therapeutic purposes. Drugs can be delivered to all major organelles of the cell (cytosol, endosome/lysosome, nucleus, nucleolus, mitochondria, endoplasmic reticulum, Golgi apparatus, peroxisomes and proteasomes) where they exert specific effects in those particular subcellular compartments. Delivery can be achieved by chemical (e.g., polymeric) or biological (e.g., signal sequences) means. Unidirectional targeting to individual organelles has proven to be immensely successful for drug therapy. Newer technologies that accommodate multiple signals (e.g., protein switch and virus-like delivery systems) mimic nature and allow for a more sophisticated approach to drug delivery. Harnessing different methods of targeting multiple organelles in a cell will lead to better drug delivery and improvements in disease therapy. PMID:21113240

  4. Subcellular localization of calcium deposits during zebrafish (Danio rerio) oogenesis.

    PubMed

    Golpour, Amin; Pšenička, Martin; Niksirat, Hamid

    2016-01-01

    Calcium plays prominent roles in regulating a broad range of physiological events in reproduction. The aim of this study was to describe the subcellular distribution of calcium deposits during stages of oogenesis in zebrafish using a combined oxalate-pyroantimonate technique. The oocyte development of zebrafish was categorized into four stages: primary growth, cortical-alveolus, vitellogenic, and maturation, based on morphological criteria. Calcium deposits in the primary growth stage were detected in the cytoplasm, mitochondria, nucleus, and follicular cells. At the cortical-alveolus stage, calcium particles were transported from follicular cells and deposited in the cortical alveoli. In the vitellogenic stage, some cortical alveoli were compacted and transformed from flocculent electron-lucent to electron-dense objects with the progression of the stage. Calcium deposits were transformed from larger to smaller particles, coinciding with compaction of cortical alveoli. In the maturation stage, calcium deposits in all oocyte compartments decreased, with the exception of those in mitochondria. The proportion of area covered by calcium deposits in the mitochondria and cortical alveoli of oocytes at different stages of development was significantly different (p<0.05). The extent of calcium deposits in the cortical alveoli of mature oocytes was substantially lower than in earlier stages. Basic information about calcium distribution during zebrafish oogenesis may contribute to better understanding of its role in oogenesis.

  5. Subcellular localization of the heparin-neutralizing factor in blood platelets.

    PubMed Central

    Da Prada, M; Jakábová, M; Lüscher, E F; Pletscher, A; Richards, J G

    1976-01-01

    1. The distribution of the heparin-neutralizing factor (platelet factor 4, PF4) in subcellular organelles of blood platelets of rabbits and man was investigated. 2. In both species the organelles storing 5-hydroxytryptamine (5-HT storage organelles) contained only trivial amounts of PF4. 3. In contrast, the content of PF4 was highest in the subcellular fractions rich in alpha-granules. 4. In conclusion, PF4 is probably localized in the alpha-granules and therefore the platelets contain at least two types of organelles (5-HT organelles and alpha-granules) capable of releasing their contents in response to the same stimuli, such as exposure to collagen, thrombin, etc. Images Plate 1 Plate 2 PMID:950602

  6. Spatial variation and subcellular binding of metals in oysters from a large estuary in China.

    PubMed

    Yu, Xiu-Juan; Pan, Ke; Liu, Fengjie; Yan, Yan; Wang, Wen-Xiong

    2013-05-15

    Pearl River Estuary (PRE) is the largest estuary in Southern China and there has been an increasing concern of metal pollution due to regional industrialization. In this study, we investigated the spatial variation of metal pollution (Ag, As, Cd, Cu, Pb, and Zn) as well as their subcellular handling in the oyster Crassostrea hongkongensis. Hot spots of metal contamination in the oysters were found in different sites, suggesting that there were different sources of metals in the estuary associated with industrial activity. Metals differed in their subcellular bindings in the oysters from different locations. Metal distribution in the biologically detoxified fraction decreased for Cu but increased for Zn with increasing contamination in the oysters. For Zn, there was a significant difference in its two detoxification pools (metal-rich granules and metallothionein-like proteins) in response to Zn contamination. The high Cd concentrations in oysters may carry a high Cd hazard to the consumers.

  7. Subcellular Localization of Hexokinases I and II Directs the Metabolic Fate of Glucose

    PubMed Central

    John, Scott; Weiss, James N.; Ribalet, Bernard

    2011-01-01

    Background The first step in glucose metabolism is conversion of glucose to glucose 6-phosphate (G-6-P) by hexokinases (HKs), a family with 4 isoforms. The two most common isoforms, HKI and HKII, have overlapping tissue expression, but different subcellular distributions, with HKI associated mainly with mitochondria and HKII associated with both mitochondrial and cytoplasmic compartments. Here we tested the hypothesis that these different subcellular distributions are associated with different metabolic roles, with mitochondrially-bound HK's channeling G-6-P towards glycolysis (catabolic use), and cytoplasmic HKII regulating glycogen formation (anabolic use). Methodology/Principal Findings To study subcellular translocation of HKs in living cells, we expressed HKI and HKII linked to YFP in CHO cells. We concomitantly recorded the effects on glucose handling using the FRET based intracellular glucose biosensor, FLIPglu-600 mM, and glycogen formation using a glycogen-associated protein, PTG, tagged with GFP. Our results demonstrate that HKI remains strongly bound to mitochondria, whereas HKII translocates between mitochondria and the cytosol in response to glucose, G-6-P and Akt, but not ATP. Metabolic measurements suggest that HKI exclusively promotes glycolysis, whereas HKII has a more complex role, promoting glycolysis when bound to mitochondria and glycogen synthesis when located in the cytosol. Glycogen breakdown upon glucose removal leads to HKII inhibition and dissociation from mitochondria, probably mediated by increases in glycogen-derived G-6-P. Conclusions/Significance These findings show that the catabolic versus anabolic fate of glucose is dynamically regulated by extracellular glucose via signaling molecules such as intracellular glucose, G-6-P and Akt through regulation and subcellular translocation of HKII. In contrast, HKI, which activity and regulation is much less sensitive to these factors, is mainly committed to glycolysis. This may be an

  8. Tau regulates the subcellular localization of calmodulin

    SciTech Connect

    Barreda, Elena Gomez de

    2011-05-13

    Highlights: {yields} In this work we have tried to explain how a cytoplasmic protein could regulate a cell nuclear function. We have tested the role of a cytoplasmic protein (tau) in regulating the expression of calbindin gene. We found that calmodulin, a tau-binding protein with nuclear and cytoplasmic localization, increases its nuclear localization in the absence of tau. Since nuclear calmodulin regulates calbindin expression, a decrease in nuclear calmodulin, due to the presence of tau that retains it at the cytoplasm, results in a change in calbindin expression. -- Abstract: Lack of tau expression in neuronal cells results in a change in the expression of few genes. However, little is known about how tau regulates gene expression. Here we show that the presence of tau could alter the subcellular localization of calmodulin, a protein that could be located at the cytoplasm or in the nucleus. Nuclear calmodulin binds to co-transcription factors, regulating the expression of genes like calbindin. In this work, we have found that in neurons containing tau, a higher proportion of calmodulin is present in the cytoplasm compared with neurons lacking tau and that an increase in cytoplasmic calmodulin correlates with a higher expression of calbindin.

  9. Subcellular storage compartments of bacteriopheophorbide sensitizers

    NASA Astrophysics Data System (ADS)

    Moser, Joerg G.; Dembeck, U.; Hubert, M.; Spengler, Bernhard; Bayer, Rainer; Wagner, Birgit

    1994-03-01

    Fluorescence colocalization with the Golgi specific stain, NBD-ceramide, and the mitochondrial localizing stain, Rhodamine 123, confirmed the earlier assumption that the Golgi apparatus is one of the prominent storage compartments for bacteriopheophorbide esters in OAT 75 SCLC cells and several amelanotic melanoma cell lines (A375, Melur SP18, SkAMel 25). Furthermore, a diffuse staining of mitochondria, of non-structured cytoplasm, and an additional storage in melanine vesicles of the amelanotic melanoma cells suggests further storage compartments with quantitatively different contributions to the phototoxicity of bacteriochlorophyll-derived photosensitizers. Independent observations of early phototoxic effects on microfilamentous networks, enzymatic activities (succinate dehydrogenase, lactate dehydrogenase), and redistribution phenomena following primary uptake of the sensitizers let us assume that only a part of the 108 molecules taken up by a cell contribute directly to phototoxicity. Thus it may be asked if a proper subcellular positioning of only a few sensitizer molecules may have similar phototoxic effects as the huge amounts stored at apparently ineffective sites.

  10. Subcellular proteomics of Trypanosoma cruzi reservosomes

    PubMed Central

    Sant’Anna, Celso; Nakayasu, Ernesto S.; Pereira, Miria G.; Lourenço, Daniela; de Souza, Wanderley; Almeida, Igor C.; Cunha-e-Silva, Narcisa L.

    2009-01-01

    Reservosomes are the endpoint of the endocytic pathway in Trypanosoma cruzi epimastigotes. These organelles have the particular ability to concentrate proteins and lipids obtained from medium together with the main proteolytic enzymes originated from the secretory pathway, being at the same time a storage organelle and the main site of protein degradation. Subcellular proteomics have been extensively used for profiling organelles in different cell types. Here, we combine cell fractionation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to identify reservosome-resident proteins. Starting from a purified reservosome fraction, we established a protocol to isolate reservosome membranes. Transmission electron microscopy was applied to confirm the purity of the fractions. To achieve a better coverage of identified proteins we analyzed the fractions separately and combined the results. LC-MS/MS analysis identified in total 709 T. cruzi-specific proteins; of these, 456 had predicted function and 253 were classified as hypothetical proteins. We could confirm the presence of most of the proteins validated by previous work and identify new proteins from different classes such as enzymes, proton pumps, transport proteins and others. The definition of the reservosome protein profile is a good tool to assess their molecular signature, identify molecular markers, and understand their relationship with different organelles. PMID:19288526

  11. Stargazing: Monitoring subcellular dynamics of brain astrocytes.

    PubMed

    Benjamin Kacerovsky, J; Murai, K K

    2016-05-26

    Astrocytes are major non-neuronal cell types in the central nervous system that regulate a variety of processes in the brain including synaptic transmission, neurometabolism, and cerebrovasculature tone. Recent discoveries have revealed that astrocytes perform very specialized and heterogeneous roles in brain homeostasis and function. Exactly how astrocytes fulfill such diverse roles in the brain remains to be fully understood and is an active area of research. In this review, we focus on the complex subcellular anatomical features of protoplasmic gray matter astrocytes in the mature, healthy brain that likely empower these cells with the ability to detect and respond to changes in neuronal and synaptic activity. In particular, we discuss how intricate processes on astrocytes allow these cells to communicate with neurons and their synapses and strategically deliver specific cellular organelles such as mitochondria and ribosomes to active compartments within the neuropil. Understanding the properties of these structural elements will lead to a better understanding of how astrocytes function in the healthy and diseased brain. PMID:26162237

  12. Myeloperoxidase in human peripheral blood lymphocytes: Production and subcellular localization.

    PubMed

    Okada, Sabrina Sayori; de Oliveira, Edson Mendes; de Araújo, Tomaz Henrique; Rodrigues, Maria Rita; Albuquerque, Renata Chaves; Mortara, Renato Arruda; Taniwaki, Noemi Nosomi; Nakaya, Helder Imoto; Campa, Ana; Moreno, Ana Carolina Ramos

    2016-02-01

    Myeloperoxidase (MPO) is an important enzyme in the front-line protection against microorganisms. In peripheral blood, it is accepted that MPO is only produced by myeloid-lineage cells. Thus, MPO presence is unexpected in lymphocytes. We showed recently that B1-lymphocytes from mice have MPO. Here, we showed that subsets of human peripheral B, CD4(+) and CD8(+) T lymphocytes express MPO. The content of MPO in lymphocytes was very low compared to neutrophils/monocytes with a preferential distribution in the nucleus and perinuclear region. Also, we performed a MPO mRNA expression analysis from human blood cells derived from microarray raw data publicly available, showing that MPO is modulated in infectious disease. MPO was increased in CD4(+) T lymphocytes from HIV chronic infection and in CD8(+) T lymphocytes from HCV-positive patients. Our study points out MPO as a multifunctional protein due to its subcellular localization and expression modulation in lymphocytes indicating alternative unknown functions for MPO in lymphocytes. PMID:26632272

  13. Subcellular analysis by laser ablation electrospray ionization mass spectrometry

    DOEpatents

    Vertes, Akos; Stolee, Jessica A; Shrestha, Bindesh

    2014-12-02

    In various embodiments, a method of laser ablation electrospray ionization mass spectrometry (LAESI-MS) may generally comprise micro-dissecting a cell comprising at least one of a cell wall and a cell membrane to expose at least one subcellular component therein, ablating the at least one subcellular component by an infrared laser pulse to form an ablation plume, intercepting the ablation plume by an electrospray plume to form ions, and detecting the ions by mass spectrometry.

  14. Proteome-wide Subcellular Topologies of E. coli Polypeptides Database (STEPdb)*

    PubMed Central

    Orfanoudaki, Georgia; Economou, Anastassios

    2014-01-01

    Cell compartmentalization serves both the isolation and the specialization of cell functions. After synthesis in the cytoplasm, over a third of all proteins are targeted to other subcellular compartments. Knowing how proteins are distributed within the cell and how they interact is a prerequisite for understanding it as a whole. Surface and secreted proteins are important pathogenicity determinants. Here we present the STEP database (STEPdb) that contains a comprehensive characterization of subcellular localization and topology of the complete proteome of Escherichia coli. Two widely used E. coli proteomes (K-12 and BL21) are presented organized into thirteen subcellular classes. STEPdb exploits the wealth of genetic, proteomic, biochemical, and functional information on protein localization, secretion, and targeting in E. coli, one of the best understood model organisms. Subcellular annotations were derived from a combination of bioinformatics prediction, proteomic, biochemical, functional, topological data and extensive literature re-examination that were refined through manual curation. Strong experimental support for the location of 1553 out of 4303 proteins was based on 426 articles and some experimental indications for another 526. Annotations were provided for another 320 proteins based on firm bioinformatic predictions. STEPdb is the first database that contains an extensive set of peripheral IM proteins (PIM proteins) and includes their graphical visualization into complexes, cellular functions, and interactions. It also summarizes all currently known protein export machineries of E. coli K-12 and pairs them, where available, with the secretory proteins that use them. It catalogs the Sec- and TAT-utilizing secretomes and summarizes their topological features such as signal peptides and transmembrane regions, transmembrane topologies and orientations. It also catalogs physicochemical and structural features that influence topology such as abundance

  15. Subcellular Partitioning and Analysis of Gd3+-Loaded Ultrashort Single-Walled Carbon Nanotubes.

    PubMed

    Holt, Brian D; Law, Justin J; Boyer, Patrick D; Wilson, Lon J; Dahl, Kris Noel; Islam, Mohammad F

    2015-07-15

    Magnetic resonance imaging (MRI) is of vast clinical utility, with tens of millions of scans performed annually. Chemical contrast agents (CAs) can greatly enhance the diagnostic potential of MRI, and ∼50% of MRI scans use CAs. However, CAs have significant limitations such as low contrast enhancement, lack of specificity, and potential toxicity. Recently developed, Gd3+-loaded ultrashort single-walled carbon nanotubes, also referred to as gadonanotubes or GNTs, exhibit ∼40 times the relaxivities of clinical CAs, representing a potential major advance in clinically relevant MRI CA materials. Although initial cytotoxicity and MRI studies have suggested great promise for GNTs, relatively little is known regarding their subcellular interactions, which are crucial for further, safe development of GNTs as CAs. In this work, we administered GNTs to a well-established human cell line (HeLa) and to murine macrophage-like cells (J774A.1). GNTs were not acutely cytotoxic and did not reduce proliferation, except for the highest exposure concentration of 27 μg/mL for J774A.1 macrophages, yet bulk uptake of GNTs occurred in minutes at picogram quantities, or millions of GNTs per cell. J774A.1 macrophages internalized substantially more GNTs than HeLa cells in a dose-dependent manner, and Raman imaging of the subcellular distribution of GNTs revealed perinuclear localization. Fluorescence intensity and lifetime imaging demonstrated that GNTs did not grossly alter subcellular compartments, including filamentous-actin structures. Together, these results provide subcellular evidence necessary to establish GNTs as a new MRI CA material.

  16. Proteome-wide subcellular topologies of E. coli polypeptides database (STEPdb).

    PubMed

    Orfanoudaki, Georgia; Economou, Anastassios

    2014-12-01

    Cell compartmentalization serves both the isolation and the specialization of cell functions. After synthesis in the cytoplasm, over a third of all proteins are targeted to other subcellular compartments. Knowing how proteins are distributed within the cell and how they interact is a prerequisite for understanding it as a whole. Surface and secreted proteins are important pathogenicity determinants. Here we present the STEP database (STEPdb) that contains a comprehensive characterization of subcellular localization and topology of the complete proteome of Escherichia coli. Two widely used E. coli proteomes (K-12 and BL21) are presented organized into thirteen subcellular classes. STEPdb exploits the wealth of genetic, proteomic, biochemical, and functional information on protein localization, secretion, and targeting in E. coli, one of the best understood model organisms. Subcellular annotations were derived from a combination of bioinformatics prediction, proteomic, biochemical, functional, topological data and extensive literature re-examination that were refined through manual curation. Strong experimental support for the location of 1553 out of 4303 proteins was based on 426 articles and some experimental indications for another 526. Annotations were provided for another 320 proteins based on firm bioinformatic predictions. STEPdb is the first database that contains an extensive set of peripheral IM proteins (PIM proteins) and includes their graphical visualization into complexes, cellular functions, and interactions. It also summarizes all currently known protein export machineries of E. coli K-12 and pairs them, where available, with the secretory proteins that use them. It catalogs the Sec- and TAT-utilizing secretomes and summarizes their topological features such as signal peptides and transmembrane regions, transmembrane topologies and orientations. It also catalogs physicochemical and structural features that influence topology such as abundance

  17. Subcellular Characterization of Porcine Oocytes with Different Glucose-6-phosphate Dehydrogenase Activities

    PubMed Central

    Fu, Bo; Ren, Liang; Liu, Di; Ma, Jian-Zhang; An, Tie-Zhu; Yang, Xiu-Qin; Ma, Hong; Zhang, Dong-Jie; Guo, Zhen-Hua; Guo, Yun-Yun; Zhu, Meng; Bai, Jing

    2015-01-01

    The in vitro maturation (IVM) efficiency of porcine embryos is still low because of poor oocyte quality. Although brilliant cresyl blue positive (BCB+) oocytes with low glucose-6-phosphate dehydrogenase (G6PDH) activity have shown superior quality than BCB negative (−) oocytes with high G6PDH activity, the use of a BCB staining test before IVM is still controversial. This study aimed to shed more light on the subcellular characteristics of porcine oocytes after selection using BCB staining. We assessed germinal vesicle chromatin configuration, cortical granule (CG) migration, mitochondrial distribution, the levels of acetylated lysine 9 of histone H3 (AcH3K9) and nuclear apoptosis features to investigate the correlation between G6PDH activity and these developmentally related features. A pattern of chromatin surrounding the nucleoli was seen in 53.0% of BCB+ oocytes and 77.6% of BCB+ oocytes showed peripherally distributed CGs. After IVM, 48.7% of BCB+ oocytes had a diffused mitochondrial distribution pattern. However, there were no significant differences in the levels of AcH3K9 in the nuclei of blastocysts derived from BCB+ and BCB− oocytes; at the same time, we observed a similar incidence of apoptosis in the BCB+ and control groups. Although this study indicated that G6PDH activity in porcine oocytes was correlated with several subcellular characteristics such as germinal vesicle chromatin configuration, CG migration and mitochondrial distribution, other features such as AcH3K9 level and nuclear apoptotic features were not associated with G6PDH activity and did not validate the BCB staining test. In using this test for selecting porcine oocytes, subcellular characteristics such as the AcH3K9 level and apoptotic nuclear features should also be considered. Adding histone deacetylase inhibitors or apoptosis inhibitors into the culture medium used might improve the efficiency of IVM of BCB+ oocytes. PMID:26580437

  18. Investigation of the functional properties and subcellular localization of alpha human and rainbow trout estrogen receptors within a unique yeast cellular context.

    PubMed

    Le Grand, Adélaïde; Bouter, Anthony; Couturier, Anne; Mulner-Lorillon, Odile; Le Goff, Xavier; Chesnel, Franck; Sire, Olivier; Le Tilly, Véronique

    2015-05-01

    Estrogens are steroid hormones that play a pivotal role in growth, differentiation and function of reproductive and non-reproductive tissues, mediated through estrogen receptors (ERs). Estrogens are involved in different genomic and non-genomic cell signaling pathways which involve well-defined subcellular ER localizations. Thus, ER activity results from complex interplays between intrinsic binding properties and specific subcellular localization. Since these two factors are deeply intricate, we carried out, in a unique yeast cell context, a comparative study to better understand structure/function/subcellular distribution relationships. This was carried out by comparing two ERs: the human ER α subtype (hERα) and the short form of the α isoform of the rainbow trout ER (rtERαS). Their distinct binding properties to agonist and antagonist ligands and subcellular localizations were characterized in Saccharomyces cerevisiae yeast cells. An unexpected partial agonistic effect of ICI 182-780 was observed for rtERαS. Concomitant to distinct binding properties, distinct subcellular localizations were observed before and after ligand stimulation. Due to the unique cell context, the link between ERs intrinsic binding properties and subcellular localizations is partly unveiled and issues are hypothesized based on the role of cytoplasmic transient complexes which play a role in the ER cytoplasmic/nuclear partition, which in turn is critical for the recruitment of co-regulators in the nucleus.

  19. Correlation of N-myc downstream-regulated gene 1 subcellular localization and lymph node metastases of colorectal neoplasms

    SciTech Connect

    Song, Yan; Lv, Liyang; Du, Juan; Yue, Longtao; Cao, Lili

    2013-09-20

    Highlights: •We clarified NDRG1 subcellular location in colorectal cancer. •We found the changes of NDRG1 distribution during colorectal cancer progression. •We clarified the correlation between NDRG1 distribution and lymph node metastasis. •It is possible that NDRG1 subcellular localization may determine its function. •Maybe NDRG1 is valuable early diagnostic markers for metastasis. -- Abstract: In colorectal neoplasms, N-myc downstream-regulated gene 1 (NDRG1) is a primarily cytoplasmic protein, but it is also expressed on the cell membrane and in the nucleus. NDRG1 is involved in various stages of tumor development in colorectal cancer, and it is possible that the different subcellular localizations may determine the function of NDRG1 protein. Here, we attempt to clarify the characteristics of NDRG1 protein subcellular localization during the progression of colorectal cancer. We examined NDRG1 expression in 49 colorectal cancer patients in cancerous, non-cancerous, and corresponding lymph node tissues. Cytoplasmic and membrane NDRG1 expression was higher in the lymph nodes with metastases than in those without metastases (P < 0.01). Nuclear NDRG1 expression in colorectal neoplasms was significantly higher than in the normal colorectal mucosa, and yet the normal colorectal mucosa showed no nuclear expression. Furthermore, our results showed higher cytoplasmic NDRG1 expression was better for differentiation, and higher membrane NDRG1 expression resulted in a greater possibility of lymph node metastasis. These data indicate that a certain relationship between the cytoplasmic and membrane expression of NDRG1 in lymph nodes exists with lymph node metastasis. NDRG1 expression may translocate from the membrane of the colorectal cancer cells to the nucleus, where it is involved in lymph node metastasis. Combination analysis of NDRG1 subcellular expression and clinical variables will help predict the incidence of lymph node metastasis.

  20. Subcellular and intranuclear localization of neptunium-237 (V) in rat liver.

    PubMed

    Paquet, F; Verry, M; Grillon, G; Landesman, C; Masse, R; Taylor, D M

    1995-08-01

    The present investigation was aimed at establishing the distribution of 237Np within the different structures of hepatocytes. Rats were contaminated experimentally by intravenous injection of 237Np (V) and the subcellular structures of the liver were separated by ultracentrifugation. Twenty-four hours after contamination, the nuclear and cytosolic fractions bound 54 and 32%, respectively, of the total radionuclide. Purification of the nuclei followed by dissociation of the protein components in medium of increasing ionic strength showed a specific binding of neptunium to the structural proteins of the nuclear matrix.

  1. Subcellular Localized Chemical Imaging of Benthic Algal Nutritional Content via HgCdTe Array FT-IR

    SciTech Connect

    Wetzel, D.; Murdock, J; Dodds, W

    2008-01-01

    Algae respond rapidly and uniquely to changes in nutrient availability by adjusting pigment, storage product, and organelle content and quality. Cellular and subcellular variability of the relative abundance of macromolecular pools (e.g. protein, lipid, carbohydrate, and phosphodiesters) within the benthic (bottom dwelling) alga Cladophora glomerata (a common nuisance species in fresh and saline waters) was revealed by FT-IR microspectroscopic imaging. Nutrient heterogeneity was compared at the filament, cellular, and subcellular level, and localized nutrient uptake kinetics were studied by detecting the gradual incorporation of isotopically labeled nitrogen (N) (as K15NO3) from surrounding water into cellular proteins. Nutritional content differed substantially among filament cells, with differences driven by protein and lipid abundance. Whole cell imaging showed high subcellular macromolecular variability in all cells, including adjacent cells on a filament that developed clonally. N uptake was also very heterogeneous, both within and among cells, and did not appear to coincide with subcellular protein distribution. Despite high intercellular variability, some patterns emerged. Cells acquired more 15N the further they were away from the filament attachment point, and 15N incorporation was more closely correlated with phosphodiester content than protein, lipid, or carbohydrate content. Benthic algae are subject to substantial environmental heterogeneity induced by microscale hydrodynamic factors and spatial variability in nutrient availability. Species specific responses to nutrient heterogeneity are central to understanding this key component of aquatic ecosystems. FT-IR microspectroscopy, modified for benthic algae, allows determination of algal physiological responses at scales not available using current techniques.

  2. Classification of protein motifs based on subcellular localization uncovers evolutionary relationships at both sequence and functional levels

    PubMed Central

    2013-01-01

    Background Most proteins have evolved in specific cellular compartments that limit their functions and potential interactions. On the other hand, motifs define amino acid arrangements conserved between protein family members and represent powerful tools for assigning function to protein sequences. The ideal motif would identify all members of a protein family but in practice many motifs identify both family members and unrelated proteins, referred to as True Positive (TP) and False Positive (FP) sequences, respectively. Results To address the relationship between protein motifs, protein function and cellular localization, we systematically assigned subcellular localization data to motif sequences from the comprehensive PROSITE sequence motif database. Using this data we analyzed relationships between localization and function. We find that TPs and FPs have a strong tendency to localize in different compartments. When multiple localizations are considered, TPs are usually distributed between related cellular compartments. We also identified cases where FPs are concentrated in particular subcellular regions, indicating possible functional or evolutionary relationships with TP sequences of the same motif. Conclusions Our findings suggest that the systematic examination of subcellular localization has the potential to uncover evolutionary and functional relationships between motif-containing sequences. We believe that this type of analysis complements existing motif annotations and could aid in their interpretation. Our results shed light on the evolution of cellular organelles and potentially establish the basis for new subcellular localization and function prediction algorithms. PMID:23865897

  3. Subcellular localization and compartmentation of thiamine derivatives in rat brain.

    PubMed

    Bettendorff, L; Wins, P; Lesourd, M

    1994-05-26

    The subcellular distribution of thiamine derivatives in rat brain was studied. Thiamine diphosphate content was highest in the mitochondrial and synaptosomal fractions, and lowest in microsomal, myelin and cytosolic fractions. Only 3-5% of total thiamine diphosphate was bound to transketolase, a cytosolic enzyme. Thiamine triphosphate was barely detectable in the microsomal and cytosolic fraction, but synaptosomes were slightly enriched in this compound compared to the crude homogenate. Both myelin and mitochondrial fractions contained significant amounts of thiamine triphosphate. In order to estimate the relative turnover rates of these compounds, the animals received an intraperitoneal injection of either [14C]thiamine or [14C]sulbutiamine (isobutyrylthiamine disulfide) 1 h before decapitation. The specific radioactivities of thiamine compounds found in the brain decreased in the order: thiamine > thiamine triphosphate > thiamine monophosphate > thiamine diphosphate. Incorporation of radioactivity into thiamine triphosphate was more marked with [14C]sulbutiamine than with [14C]thiamine. The highest specific radioactivity of thiamine diphosphate was found in the cytosolic fraction of the brain, though this pool represents less than 10% of total thiamine diphosphate. Cytosolic thiamine diphosphate had a twice higher specific radioactivity when [14C]sulbutiamine was used as precursor compared with thiamine though no significant differences were found in the other cellular compartments. Our results suggest the existence of two thiamine diphosphate pools: the bound cofactor pool is essentially mitochondrial and has a low turnover; a much smaller cytosolic pool (6-7% of total TDP) of high turnover is the likely precursor of thiamine triphosphate. PMID:8186256

  4. Subcellular optogenetics – controlling signaling and single-cell behavior

    PubMed Central

    Karunarathne, W. K. Ajith; O'Neill, Patrick R.; Gautam, Narasimhan

    2015-01-01

    ABSTRACT Variation in signaling activity across a cell plays a crucial role in processes such as cell migration. Signaling activity specific to organelles within a cell also likely plays a key role in regulating cellular functions. To understand how such spatially confined signaling within a cell regulates cell behavior, tools that exert experimental control over subcellular signaling activity are required. Here, we discuss the advantages of using optogenetic approaches to achieve this control. We focus on a set of optical triggers that allow subcellular control over signaling through the activation of G-protein-coupled receptors (GPCRs), receptor tyrosine kinases and downstream signaling proteins, as well as those that inhibit endogenous signaling proteins. We also discuss the specific insights with regard to signaling and cell behavior that these subcellular optogenetic approaches can provide. PMID:25433038

  5. Protein subcellular localization assays using split fluorescent proteins

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2009-09-08

    The invention provides protein subcellular localization assays using split fluorescent protein systems. The assays are conducted in living cells, do not require fixation and washing steps inherent in existing immunostaining and related techniques, and permit rapid, non-invasive, direct visualization of protein localization in living cells. The split fluorescent protein systems used in the practice of the invention generally comprise two or more self-complementing fragments of a fluorescent protein, such as GFP, wherein one or more of the fragments correspond to one or more beta-strand microdomains and are used to "tag" proteins of interest, and a complementary "assay" fragment of the fluorescent protein. Either or both of the fragments may be functionalized with a subcellular targeting sequence enabling it to be expressed in or directed to a particular subcellular compartment (i.e., the nucleus).

  6. Tissue and subcellular localizations of 3H-cyclosporine A in mice.

    PubMed

    Bäckman, L; Brandt, I; Appelkvist, E L; Dallner, G

    1988-02-01

    The tissue and subcellular localizations of 3H-cyclosporine A after administration to mice were determined with whole-body autoradiography and scintillation counting of lipid extracts of tissues and subcellular fractions. The radioactivity was widely distributed in the body and the pattern of distribution after oral or parenteral administration was the same, except that tissue levels were generally lower after oral administration. Pretreatment of the animals with a diet containing cyclosporine A for 30 days before the injection of radioactive cyclosporine A did not change the pattern of distribution substantially. No significant radioactivity was found in the central nervous system, except for the choroidal plexus and the area postrema region of the brain. In pregnant mice no passage of radioactivity from the placentas to fetuses was observed after a single injection. 3H-cyclosporine A and/or its metabolites showed a high affinity for the lympho-myeloid tissues, with a marked long-term retention in bone marrow and lymph nodes. There was massive excretion in the intestinal tract after parenteral administration, and the liver, bile, pancreas and salivary glands contained high levels of radioactivity. In the kidney radioactivity was confined to the outer zone of the outer kidney medulla. In liver homogenates no quantitatively significant binding of 3H-cyclosporine A and/or its metabolites to cellular molecules such as proteins, DNA, phospho- or neutral lipids was found. After lipid extraction with organic solvents, almost all radioactivity was recovered in the organic phase.

  7. Cellular and Subcellular Immunohistochemical Localization and Quantification of Cadmium Ions in Wheat (Triticum aestivum)

    PubMed Central

    Gao, Wei; Nan, Tiegui; Tan, Guiyu; Zhao, Hongwei; Tan, Weiming; Meng, Fanyun; Li, Zhaohu; Li, Qing X.; Wang, Baomin

    2015-01-01

    The distribution of metallic ions in plant tissues is associated with their toxicity and is important for understanding mechanisms of toxicity tolerance. A quantitative histochemical method can help advance knowledge of cellular and subcellular localization and distribution of heavy metals in plant tissues. An immunohistochemical (IHC) imaging method for cadmium ions (Cd2+) was developed for the first time for the wheat Triticum aestivum grown in Cd2+-fortified soils. Also, 1-(4-Isothiocyanobenzyl)-ethylenediamine-N,N,N,N-tetraacetic acid (ITCB-EDTA) was used to chelate the mobile Cd2+. The ITCB-EDTA/Cd2+ complex was fixed with proteins in situ via the isothiocyano group. A new Cd2+-EDTA specific monoclonal antibody, 4F3B6D9A1, was used to locate the Cd2+-EDTA protein complex. After staining, the fluorescence intensities of sections of Cd2+-positive roots were compared with those of Cd2+-negative roots under a laser confocal scanning microscope, and the location of colloidal gold particles was determined with a transmission electron microscope. The results enable quantification of the Cd2+ content in plant tissues and illustrate Cd2+ translocation and cellular and subcellular responses of T. aestivum to Cd2+ stress. Compared to the conventional metal-S coprecipitation histochemical method, this new IHC method is quantitative, more specific and has less background interference. The subcellular location of Cd2+ was also confirmed with energy-dispersive X-ray microanalysis. The IHC method is suitable for locating and quantifying Cd2+ in plant tissues and can be extended to other heavy metallic ions. PMID:25941807

  8. Cellular and Subcellular Immunohistochemical Localization and Quantification of Cadmium Ions in Wheat (Triticum aestivum).

    PubMed

    Gao, Wei; Nan, Tiegui; Tan, Guiyu; Zhao, Hongwei; Tan, Weiming; Meng, Fanyun; Li, Zhaohu; Li, Qing X; Wang, Baomin

    2015-01-01

    The distribution of metallic ions in plant tissues is associated with their toxicity and is important for understanding mechanisms of toxicity tolerance. A quantitative histochemical method can help advance knowledge of cellular and subcellular localization and distribution of heavy metals in plant tissues. An immunohistochemical (IHC) imaging method for cadmium ions (Cd2+) was developed for the first time for the wheat Triticum aestivum grown in Cd2+-fortified soils. Also, 1-(4-Isothiocyanobenzyl)-ethylenediamine-N,N,N,N-tetraacetic acid (ITCB-EDTA) was used to chelate the mobile Cd2+. The ITCB-EDTA/Cd2+ complex was fixed with proteins in situ via the isothiocyano group. A new Cd2+-EDTA specific monoclonal antibody, 4F3B6D9A1, was used to locate the Cd2+-EDTA protein complex. After staining, the fluorescence intensities of sections of Cd2+-positive roots were compared with those of Cd2+-negative roots under a laser confocal scanning microscope, and the location of colloidal gold particles was determined with a transmission electron microscope. The results enable quantification of the Cd2+ content in plant tissues and illustrate Cd2+ translocation and cellular and subcellular responses of T. aestivum to Cd2+ stress. Compared to the conventional metal-S coprecipitation histochemical method, this new IHC method is quantitative, more specific and has less background interference. The subcellular location of Cd2+ was also confirmed with energy-dispersive X-ray microanalysis. The IHC method is suitable for locating and quantifying Cd2+ in plant tissues and can be extended to other heavy metallic ions.

  9. The subcellular localization of Otx2 is cell-type specific and developmentally regulated in the mouse retina.

    PubMed

    Baas, D; Bumsted, K M; Martinez, J A; Vaccarino, F M; Wikler, K C; Barnstable, C J

    2000-05-31

    Recent evidence implicates homeodomain-containing proteins in the specification of cell fates in the central nervous system. Here we report that in the embryonic mouse eye Otx2, a paired homeodomain transcription factor, was found in retinal pigment epithelial cells and a restricted subset of retinal neurons, including ganglion cells. In the postnatal and adult eye, however, both the cellular and subcellular distribution of the Otx2 protein were cell type-specific. Otx2 was detected only in the nuclei of retinal pigment epithelial and bipolar cells, but was present in the cytoplasm of rod photoreceptors. Immunohistochemical studies of retinal explants and transfected cell lines both suggested that the retention of Otx2 in the cytoplasm of immature rods is a developmentally regulated process. The differential distribution of Otx2 in the cytoplasm of rods and the nucleus of other cell types, suggests that subcellular localization of this transcription factor may participate cell fate determination during specific phases of retinal development.

  10. Rapid subcellular fractionation of the rat liver endocytic compartments involved in transcytosis of polymeric immunoglobulin A and endocytosis of asialofetuin.

    PubMed Central

    Branch, W J; Mullock, B M; Luzio, J P

    1987-01-01

    The distributions of two endocytosed radiolabelled ligands (polymeric immunoglobulin A and asialofetuin) in rat liver endocytic compartments were investigated by using rapid subcellular fractionation of post-mitochondrial supernatants on vertical density gradients of Ficoll or Nycodenz. Two endocytic compartments were identified, both ligands being initially associated with a light endocytic-vesicle fraction on Ficoll gradients, asialofetuin then accumulating in denser endosomes before transfer to lysosomes for degradation. PMID:2444213

  11. Predicted Protein Subcellular Localization in Dominant Surface Ocean Bacterioplankton

    PubMed Central

    2012-01-01

    Bacteria consume dissolved organic matter (DOM) through hydrolysis, transport and intracellular metabolism, and these activities occur in distinct subcellular localizations. Bacterial protein subcellular localizations for several major marine bacterial groups were predicted using genomic, metagenomic and metatranscriptomic data sets following modification of MetaP software for use with partial gene sequences. The most distinct pattern of subcellular localization was found for Bacteroidetes, whose genomes were substantially enriched with outer membrane and extracellular proteins but depleted of inner membrane proteins compared with five other taxa (SAR11, Roseobacter, Synechococcus, Prochlorococcus, oligotrophic marine Gammaproteobacteria). When subcellular localization patterns were compared between genes and transcripts, three taxa had expression biased toward proteins localized to cell locations outside of the cytosol (SAR11, Roseobacter, and Synechococcus), as expected based on the importance of carbon and nutrient acquisition in an oligotrophic ocean, but two taxa did not (oligotrophic marine Gammaproteobacteria and Bacteroidetes). Diel variations in the fraction and putative gene functions of transcripts encoding inner membrane and periplasmic proteins compared to cytoplasmic proteins suggest a close coupling of photosynthetic extracellular release and bacterial consumption, providing insights into interactions between phytoplankton, bacteria, and DOM. PMID:22773648

  12. SUBCELLULAR PHARMACOKINETICS AND ITS POTENTIAL FOR LIBRARY FOCUSING (R826652)

    EPA Science Inventory

    Abstract

    Subcellular pharmacokinetics (SP) optimizes biology-related factors in the design of libraries for high throughput screening by defining comparatively narrow ranges of properties (lipophilicity, amphiphilicity, acidity, reactivity, 3D-structural features) of t...

  13. Regulation of intracellular heme trafficking revealed by subcellular reporters.

    PubMed

    Yuan, Xiaojing; Rietzschel, Nicole; Kwon, Hanna; Walter Nuno, Ana Beatriz; Hanna, David A; Phillips, John D; Raven, Emma L; Reddi, Amit R; Hamza, Iqbal

    2016-08-30

    Heme is an essential prosthetic group in proteins that reside in virtually every subcellular compartment performing diverse biological functions. Irrespective of whether heme is synthesized in the mitochondria or imported from the environment, this hydrophobic and potentially toxic metalloporphyrin has to be trafficked across membrane barriers, a concept heretofore poorly understood. Here we show, using subcellular-targeted, genetically encoded hemoprotein peroxidase reporters, that both extracellular and endogenous heme contribute to cellular labile heme and that extracellular heme can be transported and used in toto by hemoproteins in all six subcellular compartments examined. The reporters are robust, show large signal-to-background ratio, and provide sufficient range to detect changes in intracellular labile heme. Restoration of reporter activity by heme is organelle-specific, with the Golgi and endoplasmic reticulum being important sites for both exogenous and endogenous heme trafficking. Expression of peroxidase reporters in Caenorhabditis elegans shows that environmental heme influences labile heme in a tissue-dependent manner; reporter activity in the intestine shows a linear increase compared with muscle or hypodermis, with the lowest heme threshold in neurons. Our results demonstrate that the trafficking pathways for exogenous and endogenous heme are distinct, with intrinsic preference for specific subcellular compartments. We anticipate our results will serve as a heuristic paradigm for more sophisticated studies on heme trafficking in cellular and whole-animal models.

  14. Regulation of intracellular heme trafficking revealed by subcellular reporters.

    PubMed

    Yuan, Xiaojing; Rietzschel, Nicole; Kwon, Hanna; Walter Nuno, Ana Beatriz; Hanna, David A; Phillips, John D; Raven, Emma L; Reddi, Amit R; Hamza, Iqbal

    2016-08-30

    Heme is an essential prosthetic group in proteins that reside in virtually every subcellular compartment performing diverse biological functions. Irrespective of whether heme is synthesized in the mitochondria or imported from the environment, this hydrophobic and potentially toxic metalloporphyrin has to be trafficked across membrane barriers, a concept heretofore poorly understood. Here we show, using subcellular-targeted, genetically encoded hemoprotein peroxidase reporters, that both extracellular and endogenous heme contribute to cellular labile heme and that extracellular heme can be transported and used in toto by hemoproteins in all six subcellular compartments examined. The reporters are robust, show large signal-to-background ratio, and provide sufficient range to detect changes in intracellular labile heme. Restoration of reporter activity by heme is organelle-specific, with the Golgi and endoplasmic reticulum being important sites for both exogenous and endogenous heme trafficking. Expression of peroxidase reporters in Caenorhabditis elegans shows that environmental heme influences labile heme in a tissue-dependent manner; reporter activity in the intestine shows a linear increase compared with muscle or hypodermis, with the lowest heme threshold in neurons. Our results demonstrate that the trafficking pathways for exogenous and endogenous heme are distinct, with intrinsic preference for specific subcellular compartments. We anticipate our results will serve as a heuristic paradigm for more sophisticated studies on heme trafficking in cellular and whole-animal models. PMID:27528661

  15. Imaging Subcellular Structures in the Living Zebrafish Embryo.

    PubMed

    Engerer, Peter; Plucinska, Gabriela; Thong, Rachel; Trovò, Laura; Paquet, Dominik; Godinho, Leanne

    2016-04-02

    In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

  16. Endo-N-acetyl-beta-D-glucosaminidase activity in rat liver. Studies on substrate specificity, enzyme inhibition, subcellular localization and partial purification.

    PubMed

    Lisman, J J; van der Wal, C J; Overdijk, B

    1985-07-15

    Endo-N-acetyl-beta-D-glucosaminidase (EC 3.2.1.96, endoglucosaminidase) has been partially purified (520-fold with respect to the cytoplasmic activity) by using concanavalin A-Sepharose, CM-Sephadex and Bio-Gel P-150 chromatography. From the influence of exogenous glycopeptides on the endoglucosaminidase activity it can be concluded that this activity consists of one enzyme hydrolysing both N-acetyl-lactosaminic-type and oligomannosidic-type substrates. Glycoproteins present in the homogenate inhibit the endoglucosaminidase activity. On re-examination of the subcellular distribution of endoglucosaminidase (after removal of inhibiting glycoproteins from the respective subcellular fractions), its cytoplasmic localization was confirmed. PMID:3929770

  17. Endo-N-acetyl-beta-D-glucosaminidase activity in rat liver. Studies on substrate specificity, enzyme inhibition, subcellular localization and partial purification.

    PubMed Central

    Lisman, J J; van der Wal, C J; Overdijk, B

    1985-01-01

    Endo-N-acetyl-beta-D-glucosaminidase (EC 3.2.1.96, endoglucosaminidase) has been partially purified (520-fold with respect to the cytoplasmic activity) by using concanavalin A-Sepharose, CM-Sephadex and Bio-Gel P-150 chromatography. From the influence of exogenous glycopeptides on the endoglucosaminidase activity it can be concluded that this activity consists of one enzyme hydrolysing both N-acetyl-lactosaminic-type and oligomannosidic-type substrates. Glycoproteins present in the homogenate inhibit the endoglucosaminidase activity. On re-examination of the subcellular distribution of endoglucosaminidase (after removal of inhibiting glycoproteins from the respective subcellular fractions), its cytoplasmic localization was confirmed. PMID:3929770

  18. Mapping the subcellular localization of Fe3O4@TiO2 nanoparticles by X-ray Fluorescence Microscopy

    NASA Astrophysics Data System (ADS)

    Yuan, Y.; Chen, S.; Gleber, S. C.; Lai, B.; Brister, K.; Flachenecker, C.; Wanzer, B.; Paunesku, T.; Vogt, S.; Woloschak, G. E.

    2013-10-01

    The targeted delivery of Fe3O4@TiO2 nanoparticles to cancer cells is an important step in their development as nanomedicines. We have synthesized nanoparticles that can bind the Epidermal Growth Factor Receptor, a cell surface protein that is overexpressed in many epithelial type cancers. In order to study the subcellular distribution of these nanoparticles, we have utilized the sub-micron resolution of X-ray Fluorescence Microscopy to map the location of Fe3O4@TiO2 NPs and other trace metal elements within HeLa cervical cancer cells. Here we demonstrate how the higher resolution of the newly installed Bionanoprobe at the Advanced Photon Source at Argonne National Laboratory can greatly improve our ability to distinguish intracellular nanoparticles and their spatial relationship with subcellular compartments.

  19. Mapping the subcellular localization of Fe3O4@TiO2 nanoparticles by X-ray Fluorescence Microscopy

    PubMed Central

    Yuan, Y; Chen, S; Gleber, SC; Lai, B; Brister, K; Flachenecker, C; Wanzer, B; Paunesku, T; Vogt, S; Woloschak, GE

    2013-01-01

    The targeted delivery of Fe3O4@TiO2 nanoparticles to cancer cells is an important step in their development as nanomedicines. We have synthesized nanoparticles that can bind the Epidermal Growth Factor Receptor, a cell surface protein that is overexpressed in many epithelial type cancers. In order to study the subcellular distribution of these nanoparticles, we have utilized the sub-micron resolution of X-ray Fluorescence Microscopy to map the locationof Fe3O4@TiO2 NPs and other trace metal elements within HeLa cervical cancer cells. Here we demonstrate how the higher resolution of the newly installed Bionanoprobe at the Advanced Photon Source at Argonne National Laboratory can greatly improve our ability to distinguish intracellular nanoparticles and their spatial relationship with subcellular compartments. PMID:26413134

  20. Immunostaining: detection of signaling protein location in tissues, cells and subcellular compartments.

    PubMed

    Maity, Biswanath; Sheff, David; Fisher, Rory A

    2013-01-01

    The purpose of this protocol is to describe various methodologies used to detect the distribution and localization of specific proteins within individual cells or tissues using immunostaining, defined as the use of specific antibodies to detect a single target protein. Detection of antigens in cultured cells is referred to as immunocytochemistry, whereas their detection in tissues is generally referred to as immunohistochemistry. Both methods involve exposure of fixed cells or tissues to primary antibodies directed against one or more proteins of interest. Bound antibodies are then detected using commercially available secondary antibodies directed against the invariant portion of the primary antibody. Two primary methodologies exist to visualize antigen-antibody complexes: immunofluorescence using fluorophore-conjugated antibodies or chemiluminescence using antibodies coupled to horse-radish peroxidase. This protocol details the steps involved and appropriate use of both methodologies. Immunostaining is used in cell biology to study differential protein expression, localization and distribution at the tissue, cellular, and subcellular level. PMID:23317899

  1. Hepatic Subcellular Compartmentation of Cytoplasmic Phosphoenolpyruvate Carboxykinase Determined by Immunogold Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Gao, Kuixiong; Cardell, Emma Lou; Morris, Randal E.; Giffin, Bruce F.; Cardell, Robert R.

    1995-08-01

    Phosphoenolpyruvate carboxykinase (PEPCK) is the rate-limiting gluconeogenic enzyme and in liver occurs in a lobular gradient from periportal to pericentral regions. The subcellular distribution of cytoplasmic PEPCK molecules within hepatocytes and its relationship to organelles have not been determined previously. In this study, we have used immunogold electron microscopy to evaluate the subcellar distribution of the enzyme, in addition to brightfield and epipolarized light microscopy. Cryosections (10 [mu]m) of perfusion-fixed rat liver were collected on silanated slides and immunostained using goat anti-rat PEPCK followed by 5-nm gold-labeled secondary and tertiary antibodies. Additionally, free-floating vibratome sections (25, 50, and 100 [mu]m) of perfusion-immersion-fixed rat liver were immunogold stained using goat anti-rat PEPCK and 5-nm gold-labeled secondary antibody, with and without silver enhancement. The immunogold labeled sections from both procedures were embedded in epoxy resin for the preparation of thin sections for electron microscopy. The results showed that the gold-labeled antibodies penetrated the entire thickness of cryosections, resulting in a high signal for PEPCK, but membranes in general, the smooth endoplasmic reticulum in particular, were not identifiable as electron dense unit membranes. On the other hand, the vibratome sections of well-fixed tissue allowed good visualization of the ultrastructure of cellular organelles, with the smooth endoplasmic reticulum appearing as vesicles and tubules with electron dense unit membranes; however, the penetration of the gold-labeled antibody was limited to cells at the surface of the vibratome sections. In both procedures, PEPCK, as indicated by gold particles, is predominantly in the glycogen areas of the cytosome and not in mitochondria, nuclei, Golgi apparatus, or other cell organelles. Hepatocytes in periportal regions have a compact subcellular distribution of PEPCK shown by gold particles

  2. Subcellular SIMS imaging of isotopically labeled amino acids in cryogenically prepared cells

    NASA Astrophysics Data System (ADS)

    Chandra, Subhash

    2004-06-01

    Ion microscopy is a potentially powerful technique for localization of isotopically labeled molecules. In this study, L-arginine and phenylalanine amino acids labeled with stable isotopes 13C and 15N were localized in cultured cells with the ion microscope at 500 nm spatial resolution. Cells were exposed to the labeled amino acids and cryogenically prepared. SIMS analyses were made in fractured freeze-dried cells. A dynamic distribution was observed from labeled arginine-treated LLC-PK 1 kidney cells at mass 28 ( 13C15N) in negative secondaries, revealing cell-to-cell heterogeneity and preferential accumulation of the amino acid (or its metabolite) in the nucleus and nucleolus of some cells. The smaller nucleolus inside the nucleus was clearly resolved in SIMS images and confirmed by correlative light microscopy. The distribution of labeled phenylalanine contrasted with arginine as it was rather homogeneously distributed in T98G human glioblastoma cells. Images of 39K, 23Na and 40Ca were also recorded to confirm the reliability of sample preparation and authenticity of the observed amino acid distributions. These observations indicate that SIMS techniques can provide a valuable technology for subcellular localization of nitrogen-containing molecules in proteomics since nitrogen does not have a radionuclide tracer isotope. Amino acids labeled with stable isotopes can be used as tracers for studying their transport and metabolism in distinct subcellular compartments with SIMS. Further studies of phenylalanine uptake in human glioblastoma cells may have special significance in boron neutron capture therapy (BNCT) as a boron analogue of phenylalanine, boronophenylalanine is a clinically approved compound for the treatment of brain tumors.

  3. Classification of Subcellular Phenotype Images by Decision Templates for Classifier Ensemble

    NASA Astrophysics Data System (ADS)

    Zhang, Bailing

    2010-01-01

    Subcellular localization is a key functional characteristic of proteins. An automatic, reliable and efficient prediction system for protein subcellular localization is needed for large-scale genome analysis. The automated cell phenotype image classification problem is an interesting "bioimage informatics" application. It can be used for establishing knowledge of the spatial distribution of proteins within living cells and permits to screen systems for drug discovery or for early diagnosis of a disease. In this paper, three well-known texture feature extraction methods including local binary patterns (LBP), Gabor filtering and Gray Level Coocurrence Matrix (GLCM) have been applied to cell phenotype images and the multiple layer perceptron (MLP) method has been used to classify cell phenotype image. After classification of the extracted features, decision-templates ensemble algorithm (DT) is used to combine base classifiers built on the different feature sets. Different texture feature sets can provide sufficient diversity among base classifiers, which is known as a necessary condition for improvement in ensemble performance. For the HeLa cells, the human classification error rate on this task is of 17% as reported in previous publications. We obtain with our method an error rate of 4.8%.

  4. Subcellular targeting of nine calcium-dependent protein kinase isoforms from Arabidopsis

    NASA Technical Reports Server (NTRS)

    Dammann, Christian; Ichida, Audrey; Hong, Bimei; Romanowsky, Shawn M.; Hrabak, Estelle M.; Harmon, Alice C.; Pickard, Barbara G.; Harper, Jeffrey F.; Evans, M. L. (Principal Investigator)

    2003-01-01

    Calcium-dependent protein kinases (CDPKs) are specific to plants and some protists. Their activation by calcium makes them important switches for the transduction of intracellular calcium signals. Here, we identify the subcellular targeting potentials for nine CDPK isoforms from Arabidopsis, as determined by expression of green fluorescent protein (GFP) fusions in transgenic plants. Subcellular locations were determined by fluorescence microscopy in cells near the root tip. Isoforms AtCPK3-GFP and AtCPK4-GFP showed a nuclear and cytosolic distribution similar to that of free GFP. Membrane fractionation experiments confirmed that these isoforms were primarily soluble. A membrane association was observed for AtCPKs 1, 7, 8, 9, 16, 21, and 28, based on imaging and membrane fractionation experiments. This correlates with the presence of potential N-terminal acylation sites, consistent with acylation as an important factor in membrane association. All but one of the membrane-associated isoforms targeted exclusively to the plasma membrane. The exception was AtCPK1-GFP, which targeted to peroxisomes, as determined by covisualization with a peroxisome marker. Peroxisome targeting of AtCPK1-GFP was disrupted by a deletion of two potential N-terminal acylation sites. The observation of a peroxisome-located CDPK suggests a mechanism for calcium regulation of peroxisomal functions involved in oxidative stress and lipid metabolism.

  5. Analysis of the subcellular targeting of the smaller replicase protein of Pelargonium flower break virus.

    PubMed

    Martínez-Turiño, Sandra; Hernández, Carmen

    2012-02-01

    Replication of all positive RNA viruses occurs in association with intracellular membranes. In many cases, the mechanism of membrane targeting is unknown and there appears to be no correlation between virus phylogeny and the membrane systems recruited for replication. Pelargonium flower break virus (PFBV, genus Carmovirus, family Tombusviridae) encodes two proteins, p27 and its read-through product p86 (the viral RNA dependent-RNA polymerase), that are essential for replication. Recent reports with other members of the family Tombusviridae have shown that the smaller replicase protein is targeted to specific intracellular membranes and it is assumed to determine the subcellular localization of the replication complex. Using in vivo expression of green fluorescent protein (GFP) fusions in plant and yeast cells, we show here that PFBV p27 localizes in mitochondria. The same localization pattern was found for p86 that contains the p27 sequence at its N-terminus. Cellular fractionation of p27GFP-expressing cells confirmed the confocal microscopy observations and biochemical treatments suggested a tight association of the protein to membranes. Analysis of deletion mutants allowed identification of two regions required for targeting of p27 to mitochondria. These regions mapped toward the N- and C-terminus of the protein, respectively, and could function independently though with distinct efficiency. In an attempt to search for putative cellular factors involved in p27 localization, the subcellular distribution of the protein was checked in a selected series of knockout yeast strains and the outcome of this approach is discussed.

  6. Protein expression and subcellular localization of the general purine transporter UapC from Aspergillus nidulans.

    PubMed

    Valdez-Taubas, J; Diallinas, G; Scazzocchio, C; Rosa, A L

    2000-07-01

    The uapC gene of Aspergillus nidulans belongs to a family of nucleobase-specific transporters conserved in prokaryotic and eucaryotic organisms. We report the use of immunological and green fluorescent protein based strategies to study protein expression and subcellular distribution of UapC. A chimeric protein containing a plant-adapted green fluorescent protein (sGFP) fused to the C-terminus of UapC was shown to be functional in vivo, as it complements a triple mutant (i.e., uapC(-) uapA(-) azgA(-)) unable to grow on uric acid as the sole nitrogen source. UapC-GFP is located in the plasma membrane and, secondarily, in internal structures observed as fluorescent dots. A strong correlation was found between cellular levels of UapC-GFP fluorescence and known patterns of uapC gene expression. This work represents the first in vivo study of protein expression and subcellular localization of a filamentous fungal nucleobase transporter.

  7. Subcellular targeting of nine calcium-dependent protein kinase isoforms from Arabidopsis.

    PubMed

    Dammann, Christian; Ichida, Audrey; Hong, Bimei; Romanowsky, Shawn M; Hrabak, Estelle M; Harmon, Alice C; Pickard, Barbara G; Harper, Jeffrey F

    2003-08-01

    Calcium-dependent protein kinases (CDPKs) are specific to plants and some protists. Their activation by calcium makes them important switches for the transduction of intracellular calcium signals. Here, we identify the subcellular targeting potentials for nine CDPK isoforms from Arabidopsis, as determined by expression of green fluorescent protein (GFP) fusions in transgenic plants. Subcellular locations were determined by fluorescence microscopy in cells near the root tip. Isoforms AtCPK3-GFP and AtCPK4-GFP showed a nuclear and cytosolic distribution similar to that of free GFP. Membrane fractionation experiments confirmed that these isoforms were primarily soluble. A membrane association was observed for AtCPKs 1, 7, 8, 9, 16, 21, and 28, based on imaging and membrane fractionation experiments. This correlates with the presence of potential N-terminal acylation sites, consistent with acylation as an important factor in membrane association. All but one of the membrane-associated isoforms targeted exclusively to the plasma membrane. The exception was AtCPK1-GFP, which targeted to peroxisomes, as determined by covisualization with a peroxisome marker. Peroxisome targeting of AtCPK1-GFP was disrupted by a deletion of two potential N-terminal acylation sites. The observation of a peroxisome-located CDPK suggests a mechanism for calcium regulation of peroxisomal functions involved in oxidative stress and lipid metabolism.

  8. Analysis of potato virus X replicase and TGBp3 subcellular locations

    SciTech Connect

    Bamunusinghe, Devinka; Hemenway, Cynthia L.; Nelson, Richard S.; Sanderfoot, Anton A.; Ye, Chang M.; Silva, Muniwarage A.T.; Payton, M.; Verchot-Lubicz, Jeanmarie

    2009-10-25

    Potato virus X (PVX) infection leads to certain cytopathological modifications of the host endomembrane system. The subcellular location of the PVX replicase was previously unknown while the PVX TGBp3 protein was previously reported to reside in the ER. Using PVX infectious clones expressing the green fluorescent protein reporter, and antisera detecting the PVX replicase and host membrane markers, we examined the subcellular distribution of the PVX replicase in relation to the TGBp3. Confocal and electron microscopic observations revealed that the replicase localizes in membrane bound structures that derive from the ER. A subset of TGBp3 resides in the ER at the same location as the replicase. Sucrose gradient fractionation showed that the PVX replicase and TGBp3 proteins co-fractionate with ER marker proteins. This localization represents a region where both proteins may be synthesized and/or function. There is no evidence to indicate that either PVX protein moves into the Golgi apparatus. Cerulenin, a drug that inhibits de novo membrane synthesis, also inhibited PVX replication. These combined data indicate that PVX replication relies on ER-derived membrane recruitment and membrane proliferation.

  9. Different subcellular localization of muscarinic and serotonin (S2) receptors in human, dog, and rat brain.

    PubMed

    Luabeya, M K; Maloteaux, J M; De Roe, C; Trouet, A; Laduron, P M

    1986-02-01

    Cortex from rat, dog, and human brain was submitted to subcellular fractionation using an analytical approach consisting of a two-step procedure. First, fractions were obtained by differential centrifugation and were analyzed for their content of serotonin S2 and muscarinic receptors, serotonin uptake, and marker enzymes. Second, the cytoplasmic extracts were subfractionated by equilibration in sucrose density gradient. In human brain, serotonin and muscarinic receptors were found associated mostly with mitochondrial fractions which contain synaptosomes, whereas in rat brain they were concentrated mainly in the microsomal fractions. Density gradient centrifugation confirmed a more marked synaptosomal localization of receptors in human than in rat brain, the dog displaying an intermediate profile. In human brain, indeed, more receptor sites were found to be associated with the second peak characterized in electron microscopy by the largest number of nerve terminals. In addition, synaptosomes from human brain are denser than those from rat brain and some marker enzymes reveal different subcellular distribution in the three species. These data indicate that more receptors are of synaptosomal nature in human brain than in other species and this finding is compatible with a larger amount of synaptic contacts in human brain. PMID:2934515

  10. Fluorescence lifetime imaging to quantify sub-cellular oxygen measurements in live macrophage during bacterial invasion

    NASA Astrophysics Data System (ADS)

    Dragavon, Joe; Amiri, Megdouda; Marteyn, Benoit; Sansonetti, Philipe; Shorte, Spencer

    2011-03-01

    Fluorophore concentration, the surrounding microenvironment, and photobleaching greatly influence the fluorescence intensity of a fluorophore, increasing the difficulty to directly observe micro-environmental factors such as pH and oxygen. However, the fluorescence lifetime of a fluorophore is essentially independent of both the fluorophore concentration and photobleaching, providing a viable alternative to intensity measurements. The development of fluorescence lifetime imaging (FLI) allows for the direct measurement of the microenvironment surrounding a fluorophore. Pt-porphyrin is a fluorophore whose optical properties include a very stable triplet excited state. This energy level overlaps strongly with the ground triplet state of oxygen, making the phosphorescent lifetime directly proportional to the surrounding oxygen concentration. Initial experiments using this fluorophore involved the use of individual microwells coated with the porphyrin. Cells were allowed to enter the micro-wells before being sealed to create a diffusionally isolated volume. The decrease in the extracellular oxygen concentration was observed using FLI. However, this isolation technique provides only the consumption rate but cannot indicate the subcellular oxygen distribution. To improve upon this, live macrophages are loaded with the porphyrin and the fluorescence lifetime determined using a Lambert Instruments Lifa-X FLI system. Initial results indicate that an increase in subcellular oxygen is observed upon initial exposure to invasive bacteria. A substantial decrease in oxygen is observed after about 1 hour of exposure. The cells remain in this deoxygenated state until the bacteria are removed or cell death occurs.

  11. Subcellular organization of ureide biogenesis from glycolytic intermediates and ammonium in nitrogen-fixing soybean nodules.

    PubMed

    Boland, M J; Hanks, J F; Reynolds, P H; Blevins, D G; Tolbert, N E; Schubert, K R

    1982-06-01

    Subcellular organelle fractionation of nitrogen-fixing nodules of soybean (Glycine max (L.) Merr.) indicates that a number of enzymes involved in the assimilation of ammonia into amino acids and purines are located in the proplastids. These include asparagine synthetase (EC 6.3.1.1), phosphoribosyl amidotransferase (EC 2.4.2.14), phosphoglycerate dehydrogenase (EC 1.1.1.95), serine hydroxymethylase (EC 2.1.2.1), and methylene-tetrahydrofolate dehydrogenase (EC 1.5.1.5). Of the two isoenzymes of asparate aminotransferase (EC 2.6.1.1) in the nodule, only one was located in the proplastid fraction. Both glutamate synthase (EC 1.4.1.14) and triosephosphate isomerase (EC 5.3.1.1) were associated at least in part with the proplastids. Glutamine synthetase (EC 6.3.1.2) and xanthine dehydrogenase (EC 1.2.1.37) were found in significant quantities only in the soluble fraction. Phosphoribosylpyrophosphate synthetase (EC 2.7.6.1) was found mostly in the soluble fraction, although small amounts of it were detected in other organelle fractions. These results together with recent organelle fractionation and electron microscopic studies form the basis for a model of the subcellular distribution of ammonium assimilation, amide synthesis and uredie biogenesis in the nodule.

  12. Mechanosensitive subcellular rheostasis drives emergent single-cell mechanical homeostasis

    NASA Astrophysics Data System (ADS)

    Weng, Shinuo; Shao, Yue; Chen, Weiqiang; Fu, Jianping

    2016-09-01

    Mechanical homeostasis--a fundamental process by which cells maintain stable states under environmental perturbations--is regulated by two subcellular mechanotransducers: cytoskeleton tension and integrin-mediated focal adhesions (FAs). Here, we show that single-cell mechanical homeostasis is collectively driven by the distinct, graduated dynamics (rheostasis) of subcellular cytoskeleton tension and FAs. Such rheostasis involves a mechanosensitive pattern wherein ground states of cytoskeleton tension and FA determine their distinct reactive paths through either relaxation or reinforcement. Pharmacological perturbations of the cytoskeleton and molecularly modulated integrin catch-slip bonds biased the rheostasis and induced non-homeostasis of FAs, but not of cytoskeleton tension, suggesting a unique sensitivity of FAs in regulating homeostasis. Theoretical modelling revealed myosin-mediated cytoskeleton contractility and catch-slip-bond-like behaviours in FAs and the cytoskeleton as sufficient and necessary mechanisms for quantitatively recapitulating mechanosensitive rheostasis. Our findings highlight the previously underappreciated physical nature of the mechanical homeostasis of cells.

  13. Subcellular localization of legionella Dot/Icm effectors.

    PubMed

    Vogrin, Adam J; Mousnier, Aurelie; Frankel, Gad; Hartland, Elizabeth L

    2013-01-01

    The translocation of effector proteins by the Dot/Icm type IV secretion system is central to the ability of Legionella pneumophila to persist and replicate within eukaryotic cells. The subcellular localization of translocated Dot/Icm proteins in host cells provides insight into their function. Through co-staining with host cell markers, effector proteins may be localized to specific subcellular compartments and membranes, which frequently reflects their host cell target and mechanism of action. In this chapter, we describe protocols to (1) localize effector proteins within cells by ectopic expression using green fluorescent protein fusions and (2) localize effector proteins within infected cells using epitope-tagged effector proteins and immuno-fluorescence microscopy.

  14. Genetically targeted fluorogenic macromolecules for subcellular imaging and cellular perturbation.

    PubMed

    Magenau, Andrew J D; Saurabh, Saumya; Andreko, Susan K; Telmer, Cheryl A; Schmidt, Brigitte F; Waggoner, Alan S; Bruchez, Marcel P

    2015-10-01

    The alteration of cellular functions by anchoring macromolecules to specified organelles may reveal a new area of therapeutic potential and clinical treatment. In this work, a unique phenotype was evoked by influencing cellular behavior through the modification of subcellular structures with genetically targetable macromolecules. These fluorogen-functionalized polymers, prepared via controlled radical polymerization, were capable of exclusively decorating actin, cytoplasmic, or nuclear compartments of living cells expressing localized fluorgen-activating proteins. The macromolecular fluorogens were optimized by establishing critical polymer architecture-biophysical property relationships which impacted binding rates, binding affinities, and the level of internalization. Specific labeling of subcellular structures was realized at nanomolar concentrations of polymer, in the absence of membrane permeabilization or transduction domains, and fluorogen-modified polymers were found to bind to protein intact after delivery to the cytosol. Cellular motility was found to be dependent on binding of macromolecular fluorogens to actin structures causing rapid cellular ruffling without migration.

  15. Genetically Targeted Fluorogenic Macromolecules for Subcellular Imaging and Cellular Perturbation

    PubMed Central

    Magenau, Andrew J. D.; Saurabh, Saumya; Andreko, Susan K.; Telmer, Cheryl A.; Schmidt, Brigitte F.; Waggoner, Alan S.; Bruchez, Marcel P.

    2015-01-01

    The alteration of cellular functions by anchoring macromolecules to specified organelles may reveal a new area of therapeutic potential and clinical treatment. In this work, a unique phenotype was evoked by influencing cellular behavior through the modification of subcellular structures with genetically targetable macromolecules. These fluorogen-functionalized polymers, prepared via controlled radical polymerization, were capable of exclusively decorating actin, cytoplasmic, or nuclear compartments of living cells expressing localized fluorgen-activating proteins. The macromolecular fluorogens were optimized by establishing critical polymer architecture-biophysical property relationships which impacted binding rates, binding affinities, and the level of internalization. Specific labeling of subcellular structures was realized at nanomolar concentrations of polymer, in the absence of membrane permeabilization or transduction domains, and fluorogen-modified polymers were found to bind to protein intact after delivery to the cytosol. Cellular motility was found to be dependent on binding of macromolecular fluorogens to actin structures causing rapid cellular ruffling without migration. PMID:26183934

  16. Subcellular patterning: axonal domains with specialized structure and function

    PubMed Central

    Normand, Elizabeth A.; Rasband, Matthew N.

    2015-01-01

    Myelinated axons are patterned into discrete and often repeating domains responsible for the efficient and rapid transmission of electrical signals. These domains include nodes of Ranvier and axon initial segments. Disruption of axonal patterning leads to nervous system dysfunction. In this review we introduce the concept of subcellular patterning as applied to axons and discuss how these patterning events depend on both intrinsic, cytoskeletal mechanisms, and extrinsic, myelinating-glia dependent mechanisms. PMID:25710532

  17. Geometric modeling of subcellular structures, organelles, and multiprotein complexes

    PubMed Central

    Feng, Xin; Xia, Kelin; Tong, Yiying; Wei, Guo-Wei

    2013-01-01

    SUMMARY Recently, the structure, function, stability, and dynamics of subcellular structures, organelles, and multi-protein complexes have emerged as a leading interest in structural biology. Geometric modeling not only provides visualizations of shapes for large biomolecular complexes but also fills the gap between structural information and theoretical modeling, and enables the understanding of function, stability, and dynamics. This paper introduces a suite of computational tools for volumetric data processing, information extraction, surface mesh rendering, geometric measurement, and curvature estimation of biomolecular complexes. Particular emphasis is given to the modeling of cryo-electron microscopy data. Lagrangian-triangle meshes are employed for the surface presentation. On the basis of this representation, algorithms are developed for surface area and surface-enclosed volume calculation, and curvature estimation. Methods for volumetric meshing have also been presented. Because the technological development in computer science and mathematics has led to multiple choices at each stage of the geometric modeling, we discuss the rationales in the design and selection of various algorithms. Analytical models are designed to test the computational accuracy and convergence of proposed algorithms. Finally, we select a set of six cryo-electron microscopy data representing typical subcellular complexes to demonstrate the efficacy of the proposed algorithms in handling biomolecular surfaces and explore their capability of geometric characterization of binding targets. This paper offers a comprehensive protocol for the geometric modeling of subcellular structures, organelles, and multiprotein complexes. PMID:23212797

  18. PSCL: predicting protein subcellular localization based on optimal functional domains.

    PubMed

    Wang, Kai; Hu, Le-Le; Shi, Xiao-He; Dong, Ying-Song; Li, Hai-Peng; Wen, Tie-Qiao

    2012-01-01

    It is well known that protein subcellular localizations are closely related to their functions. Although many computational methods and tools are available from Internet, it is still necessary to develop new algorithms in this filed to gain a better understanding of the complex mechanism of plant subcellular localization. Here, we provide a new web server named PSCL for plant protein subcellular localization prediction by employing optimized functional domains. After feature optimization, 848 optimal functional domains from InterPro were obtained to represent each protein. By calculating the distances to each of the seven categories, PSCL showing the possibilities of a protein located into each of those categories in ascending order. Toward our dataset, PSCL achieved a first-order predicted accuracy of 75.7% by jackknife test. Gene Ontology enrichment analysis showing that catalytic activity, cellular process and metabolic process are strongly correlated with the localization of plant proteins. Finally, PSCL, a Linux Operate System based web interface for the predictor was designed and is accessible for public use at http://pscl.biosino.org/.

  19. Geometric modeling of subcellular structures, organelles, and multiprotein complexes.

    PubMed

    Feng, Xin; Xia, Kelin; Tong, Yiying; Wei, Guo-Wei

    2012-12-01

    Recently, the structure, function, stability, and dynamics of subcellular structures, organelles, and multiprotein complexes have emerged as a leading interest in structural biology. Geometric modeling not only provides visualizations of shapes for large biomolecular complexes but also fills the gap between structural information and theoretical modeling, and enables the understanding of function, stability, and dynamics. This paper introduces a suite of computational tools for volumetric data processing, information extraction, surface mesh rendering, geometric measurement, and curvature estimation of biomolecular complexes. Particular emphasis is given to the modeling of cryo-electron microscopy data. Lagrangian-triangle meshes are employed for the surface presentation. On the basis of this representation, algorithms are developed for surface area and surface-enclosed volume calculation, and curvature estimation. Methods for volumetric meshing have also been presented. Because the technological development in computer science and mathematics has led to multiple choices at each stage of the geometric modeling, we discuss the rationales in the design and selection of various algorithms. Analytical models are designed to test the computational accuracy and convergence of proposed algorithms. Finally, we select a set of six cryo-electron microscopy data representing typical subcellular complexes to demonstrate the efficacy of the proposed algorithms in handling biomolecular surfaces and explore their capability of geometric characterization of binding targets. This paper offers a comprehensive protocol for the geometric modeling of subcellular structures, organelles, and multiprotein complexes.

  20. High sequence variability, diverse subcellular localizations, and ecological implications of alkaline phosphatase in dinoflagellates and other eukaryotic phytoplankton.

    PubMed

    Lin, Xin; Zhang, Huan; Cui, Yudong; Lin, Senjie

    2012-01-01

    Alkaline phosphatase (AP) is a key enzyme for phytoplankton to utilize dissolved organic phosphorus (DOP) when dissolved inorganic phosphorus is limited. While three major types of AP and their correspondingly diverse subcellular localization have been recognized in bacteria, little is known about AP in eukaryotic phytoplankton such as dinoflagellates. Here, we isolated a full-length AP cDNA from a latest-diverging dinoflagellate genus Alexandrium, and conducted comparative analyses with homologs from a relatively basal (Amphidinium carterae) and late-diverging (Karenia brevis) lineage of dinoflagellates as well as other eukaryotic algae. New data and previous studies indicate that AP is common in dinoflagellates and most other major eukaryotic groups of phytoplankton. AP sequences are more variable than many other genes studied in dinoflagellates, and are divergent among different eukaryotic phytoplankton lineages. Sequence comparison to the other characterized APs suggests that dinoflagellates and some other eukaryotic phytoplankton possess the putative AP as phoA type, but some other eukaryotic phytoplankton seem to have other types. Phylogenetic analyses based on AP amino acid sequences indicated that the "red-type" eukaryotic lineages formed a monophyletic group, suggesting a common origin of their APs. As different amino acid sequences have been found to predictably determine different spatial distribution in the cells, which may facilitate access to different pools of DOP, existing computational models were adopted to predict the subcellular localizations of putative AP in the three dinoflagellates and other eukaryotic phytoplankton. Results showed different subcellular localizations of APs in different dinoflagellates and other lineages. The linkage between AP sequence divergence, subcellular localization, and ecological niche differentiation requires rigorous experimental verification, and this study now provides a framework for such a future effort.

  1. High Sequence Variability, Diverse Subcellular Localizations, and Ecological Implications of Alkaline Phosphatase in Dinoflagellates and Other Eukaryotic Phytoplankton

    PubMed Central

    Lin, Xin; Zhang, Huan; Cui, Yudong; Lin, Senjie

    2012-01-01

    Alkaline phosphatase (AP) is a key enzyme for phytoplankton to utilize dissolved organic phosphorus (DOP) when dissolved inorganic phosphorus is limited. While three major types of AP and their correspondingly diverse subcellular localization have been recognized in bacteria, little is known about AP in eukaryotic phytoplankton such as dinoflagellates. Here, we isolated a full-length AP cDNA from a latest-diverging dinoflagellate genus Alexandrium, and conducted comparative analyses with homologs from a relatively basal (Amphidinium carterae) and late-diverging (Karenia brevis) lineage of dinoflagellates as well as other eukaryotic algae. New data and previous studies indicate that AP is common in dinoflagellates and most other major eukaryotic groups of phytoplankton. AP sequences are more variable than many other genes studied in dinoflagellates, and are divergent among different eukaryotic phytoplankton lineages. Sequence comparison to the other characterized APs suggests that dinoflagellates and some other eukaryotic phytoplankton possess the putative AP as phoA type, but some other eukaryotic phytoplankton seem to have other types. Phylogenetic analyses based on AP amino acid sequences indicated that the “red-type” eukaryotic lineages formed a monophyletic group, suggesting a common origin of their APs. As different amino acid sequences have been found to predictably determine different spatial distribution in the cells, which may facilitate access to different pools of DOP, existing computational models were adopted to predict the subcellular localizations of putative AP in the three dinoflagellates and other eukaryotic phytoplankton. Results showed different subcellular localizations of APs in different dinoflagellates and other lineages. The linkage between AP sequence divergence, subcellular localization, and ecological niche differentiation requires rigorous experimental verification, and this study now provides a framework for such a future effort

  2. Analysis of the influence of subcellular localization of the HIV Rev protein on Rev-dependent gene expression by multi-fluorescence live-cell imaging

    SciTech Connect

    Wolff, Horst; Hadian, Kamyar; Ziegler, Manja; Weierich, Claudia; Kramer-Hammerle, Susanne; Kleinschmidt, Andrea; Erfle, Volker; Brack-Werner, Ruth . E-mail: brack@gsf.de

    2006-02-15

    The human immunodeficiency virus Rev protein is a post-transcriptional activator of HIV gene expression. Rev is a nucleocytoplasmic shuttle protein that displays characteristic nuclear/nucleolar subcellular localization in various cell lines. Cytoplasmic localization of Rev occurs under various conditions disrupting Rev function. The goal of this study was to investigate the relationship between localization of Rev and its functional activity in living cells. A triple-fluorescent imaging assay, called AQ-FIND, was established for automatic quantitative evaluation of nucleocytoplasmic distribution of fluorescently tagged proteins. This assay was used to screen 500 rev genes generated by error-prone PCR for Rev mutants with different localization phenotypes. Activities of the Rev mutants were determined with a second quantitative, dual-fluorescent reporter assay. In HeLa cells, the majority of nuclear Rev mutants had activities similar to wild-type Rev. The activities of Rev mutants with abnormal cytoplasmic localization ranged from moderately impaired to nonfunctional. There was no linear correlation between subcellular distribution and levels of Rev activity. In astrocytes, nuclear Rev mutants showed similar impaired activities as the cytoplasmic wild-type Rev. Our data suggest that steady-state subcellular localization is not a primary regulator of Rev activity but may change as a secondary consequence of altered Rev function. The methodologies described here have potential for studying the significance of subcellular localization for functions of other regulatory factors.

  3. Subcellular localization of alkaline phosphatase in Bacillus licheniformis 749/C by immunoelectron microscopy with colloidal gold

    SciTech Connect

    Tinglu, G.; Ghosh, A.; Ghosh, B.K.

    1984-08-01

    Subcellular distribution of the alkaline phosphatase of Bacillus licheniformis 749/C was determined by an immunoelectron microscopy method. Anti-alkaline phosphatase antibody labeled with 15- to 18-nm colloidal gold particles (gold-immunoglobulin G (IgG) complex) were used for the study. Both the plasma membrane and cytoplasmic material were labeled with the gold-IgG particles. These particles formed clusters in association with the plasma membrane; in contrast, in the cytoplasm the particles were largely dispersed, and only a few clusters were found. The gold-IgG binding was quantitatively estimated by stereological analysis of labeled, frozen thin sections. This estimation of a variety of control samples showed that the labeling was specific for the alkaline phosphatase. Cluster formation of the gold -IgG particles in association with the plasma membrane suggests that existence of specific alkaline phosphatase binding sites (receptors) in the plasma membrane of B. licheniformis 749/C. 27 references, 6 figures, 1 table.

  4. Subcellular localization and antiviral activity of carminic acid/poly r(A-U) combinations.

    PubMed

    Krabill, K; Jamison, J M; Gilloteaux, J; Summers, J L

    1993-10-01

    Carminic acid (CAR) enhances the antiviral activity of poly r(A-U) twelve-fold without increasing interferon induction, inactivating the vesicular stomatitis virus or inducing host cell cytotoxicity. Phase contrast photomicrographs of human foreskin fibroblasts (HSF) incubated with CAR alone, poly r(A-U) alone or with a CAR/poly r(A-U) combination illustrate that the CAR/poly r(A-U) combinations display altered subcellular distribution with the CAR being localized in the nucleoli and chromatin. Phase contrast and fluorescence photomicrographs of adriamycin (ADR)-treated and ADR/poly r(A-U)-treated HSF cells corroborate these findings. These results suggest that modulation of one or more nucleolar processes may be responsible for the enhanced antiviral activity. PMID:8287022

  5. In vivo imaging of specific drug target binding at subcellular resolution

    PubMed Central

    Dubach, J.M.; Vinegoni, C.; Mazitschek, R.; Fumene Feruglio, P.; Cameron, L.A.; Weissleder, R.

    2015-01-01

    The possibility to measure binding of small molecule drugs to desired targets in live cells could provide a better understanding of drug action. However, current approaches mostly yield static data, require lysis or rely on indirect assays and thus often provide an incomplete understanding of drug action. Here, we present a multiphoton fluorescence anisotropy microscopy live cell imaging technique to measure and map drug-target interaction in real time at subcellular resolution. This approach is generally applicable using any fluorescently labeled drug and enables high resolution spatial and temporal mapping of bound and unbound drug distribution. To illustrate our approach we measure intracellular target engagement of the chemotherapeutic Olaparib, a poly(ADP-ribose) polymerase inhibitor, in live cells and within a tumor in vivo. These results are the first generalizable approach to directly measure drug-target binding in vivo and present a promising tool to enhance understanding of drug activity. PMID:24867710

  6. Iodination by stimulated human neutrophils. Studies on its stoichiometry, subcellular localization and relevance to microbial killing.

    PubMed Central

    Segal, A W; Garcia, R C; Harper, A M; Banga, J P

    1983-01-01

    Myeloperoxidase of phagocytic leucocytes is thought to utilize H2O2 to oxidize halides, which then react with and kill ingested microbes. This hypothesis was based largely on the incorporation of radiolabelled iodide into cells that had phagocytosed bacteria. The present studies investigated the stoichiometry of these reactions and the subcellular localization and electrophoretic pattern of the cellular components that became iodinated. 1. The stoichiometry of the reactions are such that only a small proportion (less than 0.3%) of the total oxygen consumed is utilized for iodination. Iodination after stimulation with the soluble stimulus phorbol myristate acetate (PMA), which is not known to involve the azurophil granules and their contained myeloperoxidase, was comparable with that occurring after bacterial ingestion. 2. Analytical subcellular fractionation of cells that had phagocytosed bacteria localized about 25% of the radioactivity to the membranes, and most of the residual radioactivity distributed with the bacteria and dense granules. In cells stimulated with PMA, more of the radioactivity was associated with the membranes, but about half was still associated with the dense granules. 3. Autoradiographs after dodecyl sulphate/polyacrylamide-gel electrophoresis of cells stimulated with opsonized bacteria gave a similar distribution of iodinated components to that obtained with cells that had been stimulated with PMA or iodinated with Iodogen. These patterns of iodination were very different from those obtained when bacteria alone were iodinated with Iodogen or myeloperoxidase and H2O2. Preparations in which bacteria had been phagocytosed did not show evidence of iodination of bacterial proteins or coating opsonins. Thus positive evidence for the iodination of bacteria has not been produced, and the role of iodination in the microbicidal process of neutrophils remains to be established. Images Fig. 6. Fig. 7. PMID:6303312

  7. Motion Compensation for in vivo Sub-Cellular Optical Microscopy

    PubMed Central

    Lucotte, Bertrand; Balaban, Robert S.

    2014-01-01

    In this review we focus on the impact of tissue motion on attempting to conduct sub-cellular resolution optical microscopy, in vivo. Our position is that tissue motion is one of the major barriers to conducting these studies along with light induced damage, optical probe loading as well as absorbance and scattering effects on the excitation point spread function and collection of emitted light. Recent developments in the speed of image acquisition have reached the limit, in most cases, where the signal from a sub-cellular voxel limits the speed and not the scanning rate of the microscope. Different schemes for compensating for tissue displacements due to rigid body and deformation are presented from tissue restriction, gating, adaptive gating and active tissue tracking. We argue that methods that minimally impact the natural physiological motion of the tissue are desirable since the major reason to perform in vivo studies is to evaluate normal physiological functions. Towards this goal, the methods for active tracking using the optical imaging data itself to monitor tissue displacement and actively move the FOV of the microscope to match the tissue deformation in near real time. Critical for this development was the implementation of near real time image processing in conjunction with the control of the microscope imaging parameters. Clearly the continuing development of methods of motion compensation as well as significant technological solutions to the other barriers to tissue sub-cellular optical imaging in vivo, including optical aberrations and overall signal to noise, will make major contributions to the understanding of cell biology within the body. PMID:24673143

  8. Exploitation of Eukaryotic Subcellular Targeting Mechanisms by Bacterial Effectors

    PubMed Central

    Hicks, Stuart W.; Galán, Jorge E.

    2013-01-01

    Several bacteria have evolved specialized secretion systems to deliver bacterial effector proteins into eukaryotic cells with the capacity to modulate cellular pathways to promote bacterial survival and replication. The spatial and temporal context in which effectors exert their biochemical activities is critical for their function. Understanding the mechanisms that lead to their precise subcellular localization following delivery into host cells is essential for understanding effector function in the context of infection. Recent studies have shown that bacterial effectors exploit host cellular machinery to accurately target their biochemical activities within the host cell. PMID:23588250

  9. Cellular and subcellular localization of hexokinase, glutamate dehydrogenase, and alanine aminotransferase in the honeybee drone retina.

    PubMed

    Veuthey, A L; Tsacopoulos, M; Millan de Ruiz, L; Perrottet, P

    1994-05-01

    Subcellular localization of hexokinase in the honeybee drone retina was examined following fractionation of cell homogenate using differential centrifugation. Nearly all hexokinase activity was found in the cytosolic fraction, following a similar distribution as the cytosolic enzymatic marker, phosphoglycerate kinase. The distribution of enzymatic markers of mitochondria (succinate dehydrogenase, rotenone-insensitive cytochrome c reductase, and adenylate kinase) indicated that the outer mitochondrial membrane was partly damaged, but their distributions were different from that of hexokinase. The activity of hexokinase in purified suspensions of cells was fivefold higher in glial cells than in photoreceptors. This result is consistent with the hypothesis based on quantitative 2-deoxy[3H]glucose autoradiography that only glial cells phosphorylate significant amounts of glucose to glucose-6-phosphate. The activities of alanine aminotransferase and to a lesser extent of glutamate dehydrogenase were higher in the cytosolic than in the mitochondrial fraction. This important cytosolic activity of glutamate dehydrogenase was consistent with the higher activity found in mitochondria-poor glial cells. In conclusion, this distribution of enzymes is consistent with the model of metabolic interactions between glial and photoreceptor cells in the intact bee retina. PMID:8158142

  10. Cellular and subcellular localization of hexokinase, glutamate dehydrogenase, and alanine aminotransferase in the honeybee drone retina.

    PubMed

    Veuthey, A L; Tsacopoulos, M; Millan de Ruiz, L; Perrottet, P

    1994-05-01

    Subcellular localization of hexokinase in the honeybee drone retina was examined following fractionation of cell homogenate using differential centrifugation. Nearly all hexokinase activity was found in the cytosolic fraction, following a similar distribution as the cytosolic enzymatic marker, phosphoglycerate kinase. The distribution of enzymatic markers of mitochondria (succinate dehydrogenase, rotenone-insensitive cytochrome c reductase, and adenylate kinase) indicated that the outer mitochondrial membrane was partly damaged, but their distributions were different from that of hexokinase. The activity of hexokinase in purified suspensions of cells was fivefold higher in glial cells than in photoreceptors. This result is consistent with the hypothesis based on quantitative 2-deoxy[3H]glucose autoradiography that only glial cells phosphorylate significant amounts of glucose to glucose-6-phosphate. The activities of alanine aminotransferase and to a lesser extent of glutamate dehydrogenase were higher in the cytosolic than in the mitochondrial fraction. This important cytosolic activity of glutamate dehydrogenase was consistent with the higher activity found in mitochondria-poor glial cells. In conclusion, this distribution of enzymes is consistent with the model of metabolic interactions between glial and photoreceptor cells in the intact bee retina.

  11. Alternative splicing affects the subcellular localization of Drosha.

    PubMed

    Link, Steffen; Grund, Stefanie E; Diederichs, Sven

    2016-06-20

    The RNase III enzyme Drosha is a key factor in microRNA (miRNA) biogenesis and as such indispensable for cellular homeostasis and developmental processes. Together with its co-factor DGCR8, it converts the primary transcript (pri-miRNA) into the precursor hairpin (pre-miRNA) in the nucleus. While the middle and the C-terminal domain are crucial for pri-miRNA processing and DGCR8 binding, the function of the N-terminus remains cryptic. Different studies have linked this region to the subcellular localization of Drosha, stabilization and response to stress. In this study, we identify alternatively spliced Drosha transcripts that are devoid of a part of the arginine/serine-rich (RS-rich) domain and expressed in a large set of human cells. In contrast to their expected habitation, we find two isoforms also present in the cytoplasm, while the other two isoforms reside exclusively in the nucleus. Their processing activity for pri-miRNAs and the binding to co-factors remains unaltered. In multiple cell lines, the endogenous mRNA expression of the Drosha isoforms correlates with the localization of endogenous Drosha proteins. The pri-miRNA processing efficiency is not significantly different between groups of cells with or without cytoplasmic Drosha expression. In summary, we discovered novel isoforms of Drosha with differential subcellular localization pointing toward additional layers of complexity in the regulation of its activity. PMID:27185895

  12. Endothelial Caveolar Subcellular Domain Regulation of Endothelial Nitric Oxide Synthase

    PubMed Central

    Ramadoss, Jayanth; Pastore, Mayra B.; Magness, Ronald R.

    2015-01-01

    SUMMARY Complex regulatory processes alter the activity of endothelial nitric oxide synthase (eNOS) leading to nitric oxide (NO) production by endothelial cells under various physiological states. These complex processes require specific sub-cellular eNOS partitioning between plasma membrane caveolar domains and non-caveolar compartments.eNOS translocation from the plasma membrane to intracellular compartments is important for eNOS activation and subsequent NO biosynthesis. We present data reviewing and interpreting information: 1) the coupling of endothelial plasma membrane receptor systems in the caveolar structure relative to eNOS trafficking; 2) how eNOS trafficking relates to specific protein-protein interaction for inactivation and activation of eNOS; and 3) how these complex mechanisms confer specific subcellular location relative to eNOS multi-site phosphorylation and signaling.Dysfunction in regulation of eNOS activation may contribute to several disease states; in particular gestational endothelial abnormalities (preeclampsia, gestational diabetes, etc) that have life-long deleterious health consequences that predispose the offspring to develop hypertensive disease, type II diabetes and adiposity.1 PMID:23745825

  13. 3D subcellular SIMS imaging in cryogenically prepared single cells

    NASA Astrophysics Data System (ADS)

    Chandra, Subhash

    2004-06-01

    The analysis of a cell with dynamic SIMS ion microscopy depends on the gradual erosion (sputtering) of the cell surface for obtaining spatially resolved chemical information in the X-, Y-, and Z-dimensions. This ideal feature of ion microscopy is rarely explored in probing microfeatures hidden beneath the cell surface. In this study, this capability is explored for the analysis of cells undergoing cell division. The mitotic cells required 3D SIMS imaging in order to study the chemical composition of specialized subcellular regions, like the mitotic spindle, hidden beneath the cell surface. Human glioblastoma T98G cells were grown on silicon chips and cryogenically prepared with a sandwich freeze-fracture method. The fractured freeze-dried cells were used for SIMS analysis with the microscope mode of the CAMECA IMS-3f, which is capable of producing 500 nm lateral image resolution. SIMS analysis of calcium in the spindle region of metaphase cells required sequential recording of as many as 10 images. The T98G human glioblastoma tumor cells revealed an unusual depletion/lack of calcium store in the metaphase spindle, which is in contrast to the accumulation of calcium stores generally observed in normal cells. This study shows the feasibility of the microscope mode imaging in resolving subcellular microfeatures in 3D and opens new avenues of research in spatially resolved chemical analysis of dividing cells.

  14. Nanodiamond landmarks for subcellular multimodal optical and electron imaging.

    PubMed

    Zurbuchen, Mark A; Lake, Michael P; Kohan, Sirus A; Leung, Belinda; Bouchard, Louis-S

    2013-01-01

    There is a growing need for biolabels that can be used in both optical and electron microscopies, are non-cytotoxic, and do not photobleach. Such biolabels could enable targeted nanoscale imaging of sub-cellular structures, and help to establish correlations between conjugation-delivered biomolecules and function. Here we demonstrate a sub-cellular multi-modal imaging methodology that enables localization of inert particulate probes, consisting of nanodiamonds having fluorescent nitrogen-vacancy centers. These are functionalized to target specific structures, and are observable by both optical and electron microscopies. Nanodiamonds targeted to the nuclear pore complex are rapidly localized in electron-microscopy diffraction mode to enable "zooming-in" to regions of interest for detailed structural investigations. Optical microscopies reveal nanodiamonds for in-vitro tracking or uptake-confirmation. The approach is general, works down to the single nanodiamond level, and can leverage the unique capabilities of nanodiamonds, such as biocompatibility, sensitive magnetometry, and gene and drug delivery. PMID:24036840

  15. Protein Subcellular Relocalization Increases the Retention of Eukaryotic Duplicate Genes

    PubMed Central

    Byun, S. Ashley; Singh, Sarabdeep

    2013-01-01

    Gene duplication is widely accepted as a key evolutionary process, leading to new genes and novel protein functions. By providing the raw genetic material necessary for functional expansion, the mechanisms that involve the retention and functional diversification of duplicate genes are one of the central topics in evolutionary and comparative genomics. One proposed source of retention and functional diversification is protein subcellular relocalization (PSR). PSR postulates that changes in the subcellular location of eukaryotic duplicate proteins can positively modify function and therefore be beneficial to the organism. As such, PSR would promote retention of those relocalized duplicates and result in significantly lower death rates compared with death rates of nonrelocalized duplicate pairs. We surveyed both relocalized and nonrelocalized duplicate proteins from the available genomes and proteomes of 59 eukaryotic species and compared their relative death rates over a Ks range between 0 and 1. Using the Cox proportional hazard model, we observed that the death rates of relocalized duplicate pairs were significantly lower than the death rates of the duplicates without relocalization in most eukaryotic species examined in this study. These observations suggest that PSR significantly increases retention of duplicate genes and that it plays an important, but currently underappreciated, role in the evolution of eukaryotic genomes. PMID:24265504

  16. Subcellular Neural Probes from Single-Crystal Gold Nanowires

    PubMed Central

    2014-01-01

    Size reduction of neural electrodes is essential for improving the functionality of neuroprosthetic devices, developing potent therapies for neurological and neurodegenerative diseases, and long-term brain–computer interfaces. Typical neural electrodes are micromanufactured devices with dimensions ranging from tens to hundreds of micrometers. Their further miniaturization is necessary to reduce local tissue damage and chronic immunological reactions of the brain. Here we report the neural electrode with subcellular dimensions based on single-crystalline gold nanowires (NWs) with a diameter of ∼100 nm. Unique mechanical and electrical properties of defect-free gold NWs enabled their implantation and recording of single neuron-activities in a live mouse brain despite a ∼50× reduction of the size compared to the closest analogues. Reduction of electrode dimensions enabled recording of neural activity with improved spatial resolution and differentiation of brain activity in response to different social situations for mice. The successful localization of the epileptic seizure center was also achieved using a multielectrode probe as a demonstration of the diagnostics potential of NW electrodes. This study demonstrated the realism of single-neuron recording using subcellular-sized electrodes that may be considered a pivotal point for use in diverse studies of chronic brain diseases. PMID:25112683

  17. Understanding metal homeostasis in primary cultured neurons. Studies using single neuron subcellular and quantitative metallomics.

    PubMed

    Colvin, Robert A; Lai, Barry; Holmes, William R; Lee, Daewoo

    2015-07-01

    The purpose of this study was to demonstrate how single cell quantitative and subcellular metallomics inform us about both the spatial distribution and cellular mechanisms of metal buffering and homeostasis in primary cultured neurons from embryonic rat brain, which are often used as models of human disease involving metal dyshomeostasis. The present studies utilized synchrotron radiation X-ray fluorescence (SRXRF) and focused primarily on zinc and iron, two abundant metals in neurons that have been implicated in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Total single cell contents for calcium, iron, zinc, copper, manganese, and nickel were determined. Resting steady state zinc showed a diffuse distribution in both soma and processes, best defined by the mass profile of the neuron with an enrichment in the nucleus compared with the cytoplasm. Zinc buffering and homeostasis was studied using two modes of cellular zinc loading - transporter and ionophore (pyrithione) mediated. Single neuron zinc contents were shown to statistically significantly increase by either loading method - ionophore: 160 million to 7 billion; transporter 160 million to 280 million atoms per neuronal soma. The newly acquired and buffered zinc still showed a diffuse distribution. Soma and processes have about equal abilities to take up zinc via transporter mediated pathways. Copper levels are distributed diffusely as well, but are relatively higher in the processes relative to zinc levels. Prior studies have observed iron puncta in certain cell types, but others have not. In the present study, iron puncta were characterized in several primary neuronal types. The results show that iron puncta could be found in all neuronal types studied and can account for up to 50% of the total steady state content of iron in neuronal soma. Although other metals can be present in iron puncta, they are predominantly iron containing and do not appear to be

  18. Understanding metal homeostasis in primary cultured neurons. Studies using single neuron subcellular and quantitative metallomics.

    PubMed

    Colvin, Robert A; Lai, Barry; Holmes, William R; Lee, Daewoo

    2015-07-01

    The purpose of this study was to demonstrate how single cell quantitative and subcellular metallomics inform us about both the spatial distribution and cellular mechanisms of metal buffering and homeostasis in primary cultured neurons from embryonic rat brain, which are often used as models of human disease involving metal dyshomeostasis. The present studies utilized synchrotron radiation X-ray fluorescence (SRXRF) and focused primarily on zinc and iron, two abundant metals in neurons that have been implicated in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Total single cell contents for calcium, iron, zinc, copper, manganese, and nickel were determined. Resting steady state zinc showed a diffuse distribution in both soma and processes, best defined by the mass profile of the neuron with an enrichment in the nucleus compared with the cytoplasm. Zinc buffering and homeostasis was studied using two modes of cellular zinc loading - transporter and ionophore (pyrithione) mediated. Single neuron zinc contents were shown to statistically significantly increase by either loading method - ionophore: 160 million to 7 billion; transporter 160 million to 280 million atoms per neuronal soma. The newly acquired and buffered zinc still showed a diffuse distribution. Soma and processes have about equal abilities to take up zinc via transporter mediated pathways. Copper levels are distributed diffusely as well, but are relatively higher in the processes relative to zinc levels. Prior studies have observed iron puncta in certain cell types, but others have not. In the present study, iron puncta were characterized in several primary neuronal types. The results show that iron puncta could be found in all neuronal types studied and can account for up to 50% of the total steady state content of iron in neuronal soma. Although other metals can be present in iron puncta, they are predominantly iron containing and do not appear to be

  19. Subcellular Localization of Proteins Responding to Mitoxantrone-Induced DNA Damage in Leukaemic Cells.

    PubMed

    Ćmielová, J; Lesná, M; Řezáčová, M

    2015-01-01

    The aim of the present study was to investigate the subcellular localization of proteins participating in the double-strand break response pathway - p53, Mdm2, p21 and Chk2. MOLT-4 cells were pre-treated with mitoxantrone in concentrations 1 nmol/l and 5 nmol/l. The trypan blue technique was used to determine cell viability and proliferation. Western blotting was used to evaluate changes in p53, Mdm2 and Chk2 protein expression and sandwich ELISA was used to evaluate changes in the p21 protein amount. After 1 nmol/l mitoxantrone cells did not die, but their ability to proliferate was decreased. The p53 protein was activated and phosphorylated at serines 15 and 392 and accumulated in the nucleus after 24 and 48 h. The Mdm2 protein was present in the cytoplasm with its maximal level after 8 and 16 h. The p21 protein was detected in the nucleus after 24 and 48 h. Increased levels of phosphorylated Chk2 at threonine 68 were observed in the cytoplasmic fraction after 24 and 48 h of mitoxantrone treatment. We used mitoxantrone as an inducer of double-strand breaks to bring new data about the subcellular distribution of proteins responding to DNA damage. In MOLT-4 cells, the p53 protein was activated. p53 was phosphorylated at serines 15 and 392 and accumulated in the nucleus. The Mdm2 protein was activated in advance to p53 and occurred in the cytoplasm. The p21 protein was present in the nucleus. Chk2 kinase was activated by the phosphorylation at threonine 68 and we observed increased levels of this protein in the cytoplasmic fraction.

  20. Active nuclear import and export pathways regulate E2F-5 subcellular localization.

    PubMed

    Apostolova, Margarita D; Ivanova, Iordanka A; Dagnino, Carla; D'Souza, Sudhir J A; Dagnino, Lina

    2002-09-13

    Epidermal keratinocyte differentiation is accompanied by differential regulation of E2F genes, including up-regulation of E2F-5 and its concomitant association with the retinoblastoma family protein p130. This complex appears to play a role in irreversible withdrawal from the cell cycle in differentiating keratinocytes. We now report that keratinocyte differentiation is also accompanied by changes in E2F-5 subcellular localization, from the cytoplasm to the nucleus. To define the molecular determinants of E2F-5 nuclear import, we tested its ability to enter the nucleus in import assays in vitro using digitonin-permeabilized cells. We found that E2F-5 enters the nucleus through mediated transport processes that involve formation of nuclear pore complexes. It has been proposed that E2F-4 and E2F-5, which lack defined nuclear localization signal (NLS) consensus sequences, enter the nucleus in association with NLS-containing DP-2 or pRB family proteins. However, we show that nuclear import of E2F-5 only requires the first N-terminal 56 amino acid residues and is not dependent on interaction with DP or pRB family proteins. Because E2F-5 is predominantly cytoplasmic in undifferentiated keratinocytes and in other intact cells, we also examined whether this protein is subjected to active nuclear export. Indeed, E2F-5 is exported from the nucleus through leptomycin B-sensitive, CRM1-mediated transport, through a region corresponding to amino acid residues 130-154. This region excludes the DNA- and the p130-binding domains. Thus, the subcellular distribution of E2F-5 is tightly regulated in intact cells, through multiple functional domains that direct nucleocytoplasmic shuttling of this protein.

  1. Human skeletal muscle glycogen utilization in exhaustive exercise: role of subcellular localization and fibre type

    PubMed Central

    Nielsen, Joachim; Holmberg, Hans-Christer; Schrøder, Henrik D; Saltin, Bengt; Ørtenblad, Niels

    2011-01-01

    Abstract Although glycogen is known to be heterogeneously distributed within skeletal muscle cells, there is presently little information available about the role of fibre types, utilization and resynthesis during and after exercise with respect to glycogen localization. Here, we tested the hypothesis that utilization of glycogen with different subcellular localizations during exhaustive arm and leg exercise differs and examined the influence of fibre type and carbohydrate availability on its subsequent resynthesis. When 10 elite endurance athletes (22 ± 1 years, = 68 ± 5 ml kg−1 min−1, mean ± SD) performed one hour of exhaustive arm and leg exercise, transmission electron microscopy revealed more pronounced depletion of intramyofibrillar than of intermyofibrillar and subsarcolemmal glycogen. This phenomenon was the same for type I and II fibres, although at rest prior to exercise, the former contained more intramyofibrillar and subsarcolemmal glycogen than the latter. In highly glycogen-depleted fibres, the remaining small intermyofibrillar and subsarcolemmal glycogen particles were often found to cluster in groupings. In the recovery period, when the athletes received either a carbohydrate-rich meal or only water the impaired resynthesis of glycogen with water alone was associated primarily with intramyofibrillar glycogen. In conclusion, after prolonged high-intensity exercise the depletion of glycogen is dependent on subcellular localization. In addition, the localization of glycogen appears to be influenced by fibre type prior to exercise, as well as carbohydrate availability during the subsequent period of recovery. These findings provide insight into the significance of fibre type-specific compartmentalization of glycogen metabolism in skeletal muscle during exercise and subsequent recovery. PMID:21486810

  2. Stochastic Subcellular Organization of Dense-Core Vesicles Revealed by Point Pattern Analysis.

    PubMed

    Robinson, Benjamin J; Stanisavljevic, Bogdan; Silverman, Michael A; Scalettar, Bethe A

    2016-08-23

    Dense-core vesicles (DCVs) are regulated secretory organelles found in many types of neurons. In neurons of the hippocampus, their cargo includes proteins that mediate several pivotal processes, including differentiation and synaptic plasticity. Motivated by interest in DCV distribution and its impact on cargo action, we have used fluorescence microscopy and statistical analysis to develop a quantitative model of the subcellular organization of DCVs in hippocampal neurons that are spontaneously active (their most prevalent state). We also have tested the functionally motivated hypothesis that these organelles are synaptically enriched. Variance-to-mean ratio, frequency distribution, and Moran's autocorrelation analyses reveal that DCV distribution along shafts, and within synapses, follows Poisson statistics, establishing that stochastically dictated organization sustains cargo function. Occupancy in boutons exceeds that at nearby extrasynaptic axonal sites by approximately threefold, revealing significant local presynaptic enrichment. Widespread stochastic organization is consistent with the emerging functional importance of synaptically and extrasynaptically localized DCVs. Presynaptic enrichment is consistent with the established importance of protecting presynaptic sites from depletion of DCV cargo. These results enhance understanding of the link between DCV organization and mechanisms of cargo action, and they reinforce the emerging theme that randomness is a prevalent aspect of synaptic organization and composition. PMID:27558728

  3. Genetically Anchored Fluorescent Probes for Subcellular Specific Imaging of Hydrogen Sulfide

    PubMed Central

    Jiang, Xiqian; Sizovs, Antons; Wang, Meng C.; Provost, Christopher R.; Huang, Jia

    2016-01-01

    Imaging hydrogen sulfide (H2S) at the subcellular resolution will greatly improve the understanding of functions of this signaling molecule. Taking advantage of the protein labeling technologies, we report a general strategy for the development of organelle specific H2S probes, which enables sub-cellular H2S imaging essentially in any organelles of interest. PMID:26806071

  4. Microscopy with spatial filtering for monitoring subcellular morphology

    NASA Astrophysics Data System (ADS)

    Zheng, Jing-Yi

    Dynamic alteration in organelle morphology is an important indicator of cellular function and many efforts have been made to monitor the subcellular morphology. Optical scatter imaging (OSI), which combines light scattering spectroscopy with microscopic imaging, was developed to non-invasively track real-time changes in particle morphology in situ. Using a variable diameter iris as a Fourier spatial filter, the technique consisted of collecting images that encoded the intensity ratio of wide-to-narrow angle scatter (OSIR, optical scatter imaging ratio) at each pixel in the full field of view. For spherical particles, the OSIR was shown to decrease monotonically with diameter. In living cells, we reported this technique is able to detect mitochondrial morphological alterations, which were mediated by the Bcl- xL transmembrane domain, but could not be observed by fluorescence or DIC images1. However, the initial design was based on Mie theory of scattering by spheres, and hence only adequate for measuring spherical particles. This limits the applicability of OSI to cellular functional studies involving organelles, which are naturally non-spherical. In this project, we aim to enhance the current capability of the existing optical scatter microscope to assess size and shape information for both spherical and non-spherical particles, and eventually apply this technique for monitoring and quantifying subcellular morphology within living cells. To reach this goal, we developed an improved system, in which the variable diameter iris is replaced with a digital micromirror device and adopted the concept of Gabor filtering to extend our assessment of morphology to the characterization of particle shape and orientation. Using bacteria and polystyrene spheres, we show how this system can be used to assess particle aspect ratio even when imaged at low resolution. We also show the feasibility of detecting alterations in organelle aspect ratio in situ within living cells. This

  5. Dynamic subcellular localization of a respiratory complex controls bacterial respiration.

    PubMed

    Alberge, François; Espinosa, Leon; Seduk, Farida; Sylvi, Léa; Toci, René; Walburger, Anne; Magalon, Axel

    2015-01-01

    Respiration, an essential process for most organisms, has to optimally respond to changes in the metabolic demand or the environmental conditions. The branched character of their respiratory chains allows bacteria to do so by providing a great metabolic and regulatory flexibility. Here, we show that the native localization of the nitrate reductase, a major respiratory complex under anaerobiosis in Escherichia coli, is submitted to tight spatiotemporal regulation in response to metabolic conditions via a mechanism using the transmembrane proton gradient as a cue for polar localization. These dynamics are critical for controlling the activity of nitrate reductase, as the formation of polar assemblies potentiates the electron flux through the complex. Thus, dynamic subcellular localization emerges as a critical factor in the control of respiration in bacteria.

  6. Analysis of the Phosphoinositide Composition of Subcellular Membrane Fractions.

    PubMed

    Sarkes, Deborah A; Rameh, Lucia E

    2016-01-01

    Phosphoinositides play critical roles in the transduction of extracellular signals through the plasma membrane and also in endomembrane events important for vesicle trafficking and organelle function (Di Paolo and De Camilli, Nature 443(7112):651-657, 2006). The response triggered by these lipids is heavily dependent on the microenvironment in which they are found. HPLC analysis of labeled phosphoinositides allows quantification of the levels of each phosphoinositide species relative to their precursor, phosphatidylinositol. When combined with subcellular fractionation techniques, this strategy allows measurement of the relative phosphoinositide composition of each membrane fraction or organelle and determination of the microenvironment in which each species is enriched. Here, we describe the steps to separate and quantify total or localized phosphoinositides from cultured cells. PMID:26552687

  7. Dynamic subcellular localization of a respiratory complex controls bacterial respiration

    PubMed Central

    Alberge, François; Espinosa, Leon; Seduk, Farida; Sylvi, Léa; Toci, René; Walburger, Anne; Magalon, Axel

    2015-01-01

    Respiration, an essential process for most organisms, has to optimally respond to changes in the metabolic demand or the environmental conditions. The branched character of their respiratory chains allows bacteria to do so by providing a great metabolic and regulatory flexibility. Here, we show that the native localization of the nitrate reductase, a major respiratory complex under anaerobiosis in Escherichia coli, is submitted to tight spatiotemporal regulation in response to metabolic conditions via a mechanism using the transmembrane proton gradient as a cue for polar localization. These dynamics are critical for controlling the activity of nitrate reductase, as the formation of polar assemblies potentiates the electron flux through the complex. Thus, dynamic subcellular localization emerges as a critical factor in the control of respiration in bacteria. DOI: http://dx.doi.org/10.7554/eLife.05357.001 PMID:26077726

  8. Subcellular Localization of Carotenoid Biosynthesis in Synechocystis sp. PCC 6803

    PubMed Central

    Selstam, Eva; Norling, Birgitta

    2015-01-01

    The biosynthesis pathway of carotenoids in cyanobacteria is partly described. However, the subcellular localization of individual steps is so far unknown. Carotenoid analysis of different membrane subfractions in Synechocystis sp. PCC6803 shows that “light” plasma membranes have a high carotenoid/protein ratio, when compared to “heavier” plasma membranes or thylakoids. The localization of CrtQ and CrtO, two well-defined carotenoid synthesis pathway enzymes in Synechocystis, was studied by epitope tagging and western blots. Both enzymes are locally more abundant in plasma membranes than in thylakoids, implying that the plasma membrane has higher synthesis rates of β-carotene precursor molecules and echinenone. PMID:26083372

  9. Vacuoles in mammals: a subcellular structure indispensable for early embryogenesis.

    PubMed

    Wada, Yoh

    2013-01-01

    A vacuole is a membrane-bound subcellular structure involved in intracellular digestion. Instead of the large "vacuolar" organelles that are found in plants and fungi, animal cells possess lysosomes that are smaller in size and are enriched with hydrolytic enzymes similar to those found in the vacuoles. Large vacuolar structures are often observed in highly differentiated mammalian tissues such as embryonic visceral endoderm and absorbing epithelium. Vacuoles/lysosomes share a conserved mechanism of biogenesis, and they are at the terminal of the endocytic pathways, Recent genetic studies of the mammalian orthologs of Vam/Vps genes, which have essential functions for vacuole assembly, revealed that the dynamics of vacuoles/lysosomes are important for tissue differentiation and patterning through regulation of various molecular signaling events in mammals.

  10. Laserspritzer: A Simple Method for Optogenetic Investigation with Subcellular Resolutions

    PubMed Central

    Sun, Qian-Quan; Wang, Xinjun; Yang, Weiguo

    2014-01-01

    To build a detailed circuit diagram in the brain, one needs to measure functional synaptic connections between specific types of neurons. A high-resolution circuit diagram should provide detailed information at subcellular levels such as soma, distal and basal dendrites. However, a limitation lies in the difficulty of studying long-range connections between brain areas separated by millimeters. Brain slice preparations have been widely used to help understand circuit wiring within specific brain regions. The challenge exists because long-range connections are likely to be cut in a brain slice. The optogenetic approach overcomes these limitations, as channelrhodopsin 2 (ChR2) is efficiently transported to axon terminals that can be stimulated in brain slices. Here, we developed a novel fiber optic based simple method of optogenetic stimulation: the laserspritzer approach. This method facilitates the study of both long-range and local circuits within brain slice preparations. This is a convenient and low cost approach that can be easily integrated with a slice electrophysiology setup, and repeatedly used upon initial validation. Our data with direct ChR2 mediated-current recordings demonstrates that the spatial resolution of the laserspritzer is correlated with the size of the laserspritzer, and the resolution lies within the 30 µm range for the 5 micrometer laserspritzer. Using olfactory cortical slices, we demonstrated that the laserspritzer approach can be applied to selectively activate monosynaptic perisomatic GABAergic basket synapses, or long-range intracortical glutamatergic inputs formed on different subcellular domains within the same cell (e.g. distal and proximal dendrites). We discuss significant advantages of the laserspritzer approach over the widely used collimated LED whole-field illumination method in brain slice electrophysiological research. PMID:24992677

  11. Quantitative Dose-Response Curves from Subcellular Lipid Multilayer Microarrays

    PubMed Central

    Kusi-Appiah, A. E.; Lowry, T. W.; Darrow, E. M.; Wilson, K.; Chadwick, B. P.; Davidson, M. W.; Lenhert, S.

    2015-01-01

    The dose-dependent bioactivity of small molecules on cells is a crucial factor in drug discovery and personalized medicine. Although small-molecule microarrays are a promising platform for miniaturized screening, it has been a challenge to use them to obtain quantitative dose-response curves in vitro, especially for lipophilic compounds. Here we establish a small-molecule microarray assay capable of controlling the dosage of small lipophilic molecules delivered to cells by varying the sub-cellular volumes of surface supported lipid micro- and nanostructure arrays fabricated with nanointaglio. Features with sub-cellular lateral dimensions were found necessary to obtain normal cell adhesion with HeLa cells. The volumes of the lipophilic drug-containing nanostructures were determined using a fluorescence microscope calibrated by atomic-force microscopy. We used the surface supported lipid volume information to obtain EC-50 values for the response of HeLa cells to three FDA-approved lipophilic anticancer drugs, docetaxel, imiquimod and triethylenemelamine, which were found to be significantly different from neat lipid controls. No significant toxicity was observed on the control cells surrounding the drug/lipid patterns, indicating lack of interference or leakage from the arrays. Comparison of the microarray data to dose-response curves for the same drugs delivered liposomally from solution revealed quantitative differences in the efficacy values, which we explain in terms of cell-adhesion playing a more important role in the surface-based assay. The assay should be scalable to a density of at least 10,000 dose response curves on the area of a standard microtiter plate. PMID:26167949

  12. Enhanced Glycogen Storage of a Subcellular Hot Spot in Human Skeletal Muscle during Early Recovery from Eccentric Contractions.

    PubMed

    Nielsen, Joachim; Farup, Jean; Rahbek, Stine Klejs; de Paoli, Frank Vincenzo; Vissing, Kristian

    2015-01-01

    Unaccustomed eccentric exercise is accompanied by muscle damage and impaired glucose uptake and glycogen synthesis during subsequent recovery. Recently, it was shown that the role and regulation of glycogen in skeletal muscle are dependent on its subcellular localization, and that glycogen synthesis, as described by the product of glycogen particle size and number, is dependent on the time course of recovery after exercise and carbohydrate availability. In the present study, we investigated the subcellular distribution of glycogen in fibers with high (type I) and low (type II) mitochondrial content during post-exercise recovery from eccentric contractions. Analysis was completed on five male subjects performing an exercise bout consisting of 15 x 10 maximal eccentric contractions. Carbohydrate-rich drinks were subsequently ingested throughout a 48 h recovery period and muscle biopsies for analysis included time points 3, 24 and 48 h post exercise from the exercising leg, whereas biopsies corresponding to prior to and at 48 h after the exercise bout were collected from the non-exercising, control leg. Quantitative imaging by transmission electron microscopy revealed an early (post 3 and 24 h) enhanced storage of intramyofibrillar glycogen (defined as glycogen particles located within the myofibrils) of type I fibers, which was associated with an increase in the number of particles. In contrast, late in recovery (post 48 h), intermyofibrillar, intramyofibrillar and subsarcolemmal glycogen in both type I and II fibers were lower in the exercise leg compared with the control leg, and this was associated with a smaller size of the glycogen particles. We conclude that in the carbohydrate-supplemented state, the effect of eccentric contractions on glycogen metabolism depends on the subcellular localization, muscle fiber's oxidative capacity, and the time course of recovery. The early enhanced storage of intramyofibrillar glycogen after the eccentric contractions may

  13. Enhanced Glycogen Storage of a Subcellular Hot Spot in Human Skeletal Muscle during Early Recovery from Eccentric Contractions.

    PubMed

    Nielsen, Joachim; Farup, Jean; Rahbek, Stine Klejs; de Paoli, Frank Vincenzo; Vissing, Kristian

    2015-01-01

    Unaccustomed eccentric exercise is accompanied by muscle damage and impaired glucose uptake and glycogen synthesis during subsequent recovery. Recently, it was shown that the role and regulation of glycogen in skeletal muscle are dependent on its subcellular localization, and that glycogen synthesis, as described by the product of glycogen particle size and number, is dependent on the time course of recovery after exercise and carbohydrate availability. In the present study, we investigated the subcellular distribution of glycogen in fibers with high (type I) and low (type II) mitochondrial content during post-exercise recovery from eccentric contractions. Analysis was completed on five male subjects performing an exercise bout consisting of 15 x 10 maximal eccentric contractions. Carbohydrate-rich drinks were subsequently ingested throughout a 48 h recovery period and muscle biopsies for analysis included time points 3, 24 and 48 h post exercise from the exercising leg, whereas biopsies corresponding to prior to and at 48 h after the exercise bout were collected from the non-exercising, control leg. Quantitative imaging by transmission electron microscopy revealed an early (post 3 and 24 h) enhanced storage of intramyofibrillar glycogen (defined as glycogen particles located within the myofibrils) of type I fibers, which was associated with an increase in the number of particles. In contrast, late in recovery (post 48 h), intermyofibrillar, intramyofibrillar and subsarcolemmal glycogen in both type I and II fibers were lower in the exercise leg compared with the control leg, and this was associated with a smaller size of the glycogen particles. We conclude that in the carbohydrate-supplemented state, the effect of eccentric contractions on glycogen metabolism depends on the subcellular localization, muscle fiber's oxidative capacity, and the time course of recovery. The early enhanced storage of intramyofibrillar glycogen after the eccentric contractions may

  14. Subcellular partitioning profiles and metallothionein levels in indigenous clams Moerella iridescens from a metal-impacted coastal bay.

    PubMed

    Wang, Zaosheng; Feng, Chenglian; Ye, Chun; Wang, Youshao; Yan, Changzhou; Li, Rui; Yan, Yijun; Chi, Qiaoqiao

    2016-07-01

    In this study, the effect of environmental metal exposure on the accumulation and subcellular distribution of metals in the digestive gland of clams with special emphasis on metallothioneins (MTs) was investigated. Specimens of indigenous Moerella iridescens were collected from different natural habitats in Maluan Bay (China), characterized by varying levels of metal contamination. The digestive glands were excised, homogenized and six subcellular fractions were separated by differential centrifugation procedures and analyzed for their Cu, Zn, Cd and Pb contents. MTs were quantified independently by spectrophotometric measurements of thiols. Site-specific differences were observed in total metal concentrations in the tissues, correlating well with variable environmental metal concentrations and reflecting the gradient trends in metal contamination. Concentrations of the non-essential Cd and Pb were more responsive to environmental exposure gradients than were tissue concentrations of the essential metals, Cu and Zn. Subcellular partitioning profiles for Cu, Zn and Cd were relatively similar, with the heat-stable protein (HSP) fraction as the dominant metal-binding compartment, whereas for Pb this fraction was much less important. The variations in proportions and concentrations of metals in this fraction along with the metal bioaccumulation gradients suggested that the induced MTs play an important role in metal homeostasis and detoxification for M. iridescens in the metal-contaminated bay. Nevertheless, progressive accumulation of non-essential metals (Cd, and especially Pb) resulting from "spillover" was observed in putative metal- sensitive (e.g., mitochondria and heat-denaturable protein (HDP)) or lysosome/microsome fractions, demonstrating that metal detoxification was incomplete and increased the toxicological risk to M. iridescens inhabiting the metal-impacted environments. Through multiple stepwise regression analysis, the induction of MTs was statistically

  15. Cell segmentation in time-lapse fluorescence microscopy with temporally varying sub-cellular fusion protein patterns.

    PubMed

    Bunyak, Filiz; Palaniappan, Kannappan; Chagin, Vadim; Cardoso, M

    2009-01-01

    Fluorescently tagged proteins such as GFP-PCNA produce rich dynamically varying textural patterns of foci distributed in the nucleus. This enables the behavioral study of sub-cellular structures during different phases of the cell cycle. The varying punctuate patterns of fluorescence, drastic changes in SNR, shape and position during mitosis and abundance of touching cells, however, require more sophisticated algorithms for reliable automatic cell segmentation and lineage analysis. Since the cell nuclei are non-uniform in appearance, a distribution-based modeling of foreground classes is essential. The recently proposed graph partitioning active contours (GPAC) algorithm supports region descriptors and flexible distance metrics. We extend GPAC for fluorescence-based cell segmentation using regional density functions and dramatically improve its efficiency for segmentation from O(N(4)) to O(N(2)), for an image with N(2) pixels, making it practical and scalable for high throughput microscopy imaging studies.

  16. Sub-cellular localisation of a 15N-labelled peptide vector using NanoSIMS imaging

    NASA Astrophysics Data System (ADS)

    Römer, Winfried; Wu, Ting-Di; Duchambon, Patricia; Amessou, Mohamed; Carrez, Danièle; Johannes, Ludger; Guerquin-Kern, Jean-Luc

    2006-07-01

    Dynamic SIMS imaging is proposed to map sub-cellular distributions of isotopically labelled, exogenous compounds. NanoSIMS imaging allows the characterisation of the intracellular transport pathways of exogenous molecules, including peptide vectors employed in innovative therapies, using stable isotopes as molecular markers to detect the compound of interest. Shiga toxin B-subunit (STxB) was chosen as a representative peptide vector. The recombinant protein ( 15N-STxB) was synthesised in Escherichia coli using 15NH 4Cl as sole nitrogen source resulting in 15N enrichment in the molecule. Using the NanoSIMS 50 ion microprobe (Cameca), different ion species ( 12C 14N -, 12C 15N -, 31P -) originating from the same sputtered micro volume were simultaneously detected. High mass resolving power enabled the discrimination of 12C 15N - from its polyatomic isobars of mass 27. We imaged the membrane binding and internalisation of 15N-STxB in HeLa cells at spatial resolutions of less than 100 nm. Thus, the use of rare stable isotopes like 15N with dynamic SIMS imaging permits sub-cellular detection of isotopically labelled, exogenous molecules and imaging of their transport pathways at high mass and spatial resolution. Application of stable isotopes as markers can replace the large and chemically complex tags used for fluorescence microscopy, without altering the chemical and physical properties of the molecule.

  17. Bioaccumulation, subcellular, and molecular localization and damage to physiology and ultrastructure in Nymphoides peltata (Gmel.) O. Kuntze exposed to yttrium.

    PubMed

    Fu, Yongyang; Li, Feifei; Xu, Ting; Cai, Sanjuan; Chu, Weiyue; Qiu, Han; Sha, Sha; Cheng, Guangyu; Xu, Qinsong

    2014-02-01

    Bioaccumulation, subcellular distribution, and acute toxicity of yttrium (Y) were evaluated in Nymphoides peltata. The effects of Y concentrations of 1-5 mg L(-1) applied for 4 days were assessed by measuring changes in photosynthetic pigments, nutrient contents, enzymatic and non-enzymatic antioxidants, and ultrastructure. The accumulation of Y in subcellular fractions decreased in the order of cell wall > organelle > soluble fraction. Much more Y was located in cellulose and pectin than in other biomacromolecules. The content of some mineral elements (Mg, Ca, Fe, Mn, and Mo) increased in N. peltata, but there was an opposite effect for P and K. Meanwhile, ascorbate, and catalase activity decreased significantly for all Y concentrations. In contrast, peroxidase activity was induced, while initial rises in superoxide dismutase activity and glutathione content were followed by subsequent declines. Morphological symptoms of senescence, such as chlorosis and damage to chloroplasts and mitochondria, were observed even at the lowest Y concentration. Pigment content decreased as the Y concentration rose and the calculated EC50 and MPC of Y for N. peltata were 2 and 0.2 mg L(-1) after 4 days of exposure, respectively. The results showed that exogenous Y was highly available in water and that its high concentration in water bodies might produce harmful effects on aquatic organisms. N. peltata is proposed as a biomonitor for the assessment of metal pollution in aquatic ecosystems.

  18. Developmental changes in the subcellular localization of calretinin.

    PubMed

    Hack, N J; Wride, M C; Charters, K M; Kater, S B; Parks, T N

    2000-04-01

    Brainstem auditory neurons in the chick nucleus magnocellularis (NM) express high levels of the neuron-specific calcium-binding protein calretinin (CR). CR has heretofore been considered a diffusible calcium buffer that is dispersed uniformly throughout the cytosol. Using high-resolution confocal microscopy and complementary biochemical analyses, we have found that during the development of NM neurons, CR changes from being expressed diffusely at low concentrations to being highly concentrated beneath the plasma membrane. This shift in CR localization occurs at the same time as the onset of spontaneous activity, synaptic transmission, and synapse refinement in NM. In the chick brainstem auditory pathway, this subcellular localization appears to occur only in NM neurons and only with respect to CR, because calmodulin remains diffusely expressed in NM. Biochemical analyses show the association of calretinin with the membrane is detergent-soluble and calcium-independent. Because these are highly active neurons with a large number of Ca2+-permeable synaptic AMPA receptors, we hypothesize that localization of CR beneath the plasma membrane is an adaptation to spatially restrict the calcium influxes.

  19. PSORTdb: a protein subcellular localization database for bacteria

    PubMed Central

    Rey, Sébastien; Acab, Michael; Gardy, Jennifer L.; Laird, Matthew R.; deFays, Katalin; Lambert, Christophe; Brinkman, Fiona S. L.

    2005-01-01

    Information about bacterial subcellular localization (SCL) is important for protein function prediction and identification of suitable drug/vaccine/diagnostic targets. PSORTdb (http://db.psort.org/) is a web-accessible database of SCL for bacteria that contains both information determined through laboratory experimentation and computational predictions. The dataset of experimentally verified information (∼2000 proteins) was manually curated by us and represents the largest dataset of its kind. Earlier versions have been used for training SCL predictors, and its incorporation now into this new PSORTdb resource, with its associated additional annotation information and dataset version control, should aid researchers in future development of improved SCL predictors. The second component of this database contains computational analyses of proteins deduced from the most recent NCBI dataset of completely sequenced genomes. Analyses are currently calculated using PSORTb, the most precise automated SCL predictor for bacterial proteins. Both datasets can be accessed through the web using a very flexible text search engine, a data browser, or using BLAST, and the entire database or search results may be downloaded in various formats. Features such as GO ontologies and multiple accession numbers are incorporated to facilitate integration with other bioinformatics resources. PSORTdb is freely available under GNU General Public License. PMID:15608169

  20. Subcellular localization of transiently expressed fluorescent fusion proteins.

    PubMed

    Collings, David A

    2013-01-01

    The recent and massive expansion in plant genomics data has generated a large number of gene sequences for which two seemingly simple questions need to be answered: where do the proteins encoded by these genes localize in cells, and what do they do? One widespread approach to answering the localization question has been to use particle bombardment to transiently express unknown proteins tagged with green fluorescent protein (GFP) or its numerous derivatives. Confocal fluorescence microscopy is then used to monitor the localization of the fluorescent protein as it hitches a ride through the cell. The subcellular localization of the fusion protein, if not immediately apparent, can then be determined by comparison to localizations generated by fluorescent protein fusions to known signalling sequences and proteins, or by direct comparison with fluorescent dyes. This review aims to be a tour guide for researchers wanting to travel this hitch-hiker's path, and for reviewers and readers who wish to understand their travel reports. It will describe some of the technology available for visualizing protein localizations, and some of the experimental approaches for optimizing and confirming localizations generated by particle bombardment in onion epidermal cells, the most commonly used experimental system. As the non-conservation of signal sequences in heterologous expression systems such as onion, and consequent mis-targeting of fusion proteins, is always a potential problem, the epidermal cells of the Argenteum mutant of pea are proposed as a model system.

  1. Automatic Identification of Subcellular Phenotypes on Human Cell Arrays

    PubMed Central

    Conrad, Christian; Erfle, Holger; Warnat, Patrick; Daigle, Nathalie; Lörch, Thomas; Ellenberg, Jan; Pepperkok, Rainer; Eils, Roland

    2004-01-01

    Light microscopic analysis of cell morphology provides a high-content readout of cell function and protein localization. Cell arrays and microwell transfection assays on cultured cells have made cell phenotype analysis accessible to high-throughput experiments. Both the localization of each protein in the proteome and the effect of RNAi knock-down of individual genes on cell morphology can be assayed by manual inspection of microscopic images. However, the use of morphological readouts for functional genomics requires fast and automatic identification of complex cellular phenotypes. Here, we present a fully automated platform for high-throughput cell phenotype screening combining human live cell arrays, screening microscopy, and machine-learning-based classification methods. Efficiency of this platform is demonstrated by classification of eleven subcellular patterns marked by GFP-tagged proteins. Our classification method can be adapted to virtually any microscopic assay based on cell morphology, opening a wide range of applications including large-scale RNAi screening in human cells. PMID:15173118

  2. Subcellular localization of hepatitis E virus (HEV) replicase

    SciTech Connect

    Rehman, Shagufta; Kapur, Neeraj; Durgapal, Hemlata; Panda, Subrat Kumar

    2008-01-05

    Hepatitis E virus (HEV) is a hepatotropic virus with a single sense-strand RNA genome of {approx} 7.2 kb in length. Details of the intracellular site of HEV replication can pave further understanding of HEV biology. In-frame fusion construct of functionally active replicase-enhanced green fluorescent protein (EGFP) gene was made in eukaryotic expression vector. The functionality of replicase-EGFP fusion protein was established by its ability to synthesize negative-strand viral RNA in vivo, by strand-specific anchored RT-PCR and molecular beacon binding. Subcellular co-localization was carried out using organelle specific fluorophores and by immuno-electron microscopy. Fluorescence Resonance Energy Transfer (FRET) demonstrated the interaction of this protein with the 3' end of HEV genome. The results show localization of replicase on the endoplasmic reticulum membranes. The protein regions responsible for membrane localization was predicted and identified by use of deletion mutants. Endoplasmic reticulum was identified as the site of replicase localization and possible site of replication.

  3. Expression and subcellular localization of ORC1 in Leishmania major

    SciTech Connect

    Kumar, Diwakar; Mukherji, Agnideep; Saha, Swati

    2008-10-10

    The mechanism of DNA replication is highly conserved in eukaryotes, with the process being preceded by the ordered assembly of pre-replication complexes (pre-RCs). Pre-RC formation is triggered by the association of the origin replication complex (ORC) with chromatin. Leishmania major appears to have only one ORC ortholog, ORC1. ORC1 in other eukaryotes is the largest of the ORC subunits and is believed to play a significant role in modulating replication initiation. Here we report for the first time, the cloning of ORC1 from L. major, and the analysis of its expression in L. major promastigotes. In human cells ORC1 levels have been found to be upregulated in G1 and subsequently degraded, thus playing a role in controlling replication initiation. We examine the subcellular localization of L. major ORC1 in relation to the different stages of the cell cycle. Our results show that, unlike what is widely believed to be the case with ORC1 in human cells, ORC1 in L. major is nuclear at all stages of the cell cycle.

  4. Tetraspan vesicle membrane proteins: synthesis, subcellular localization, and functional properties.

    PubMed

    Hübner, Kirsten; Windoffer, Reinhard; Hutter, Harald; Leube, Rudolf E

    2002-01-01

    Tetraspan vesicle membrane proteins (TVPs) are characterized by four transmembrane regions and cytoplasmically located end domains. They are ubiquitous and abundant components of vesicles in most, if not all, cells of multicellular organisms. TVP-containing vesicles shuttle between various membranous compartments and are localized in biosynthetic and endocytotic pathways. Based on gene organization and amino acid sequence similarities TVPs can be grouped into three distinct families that are referred to as physins, gyrins, and secretory carrier-associated membrane proteins (SCAMPs). In mammals synaptophysin, synaptoporin, pantophysin, and mitsugumin29 constitute the physins, synaptogyrin 1-4 the gyrins, and SCAMP1-5 the SCAMPs. Members of each family are cell-type-specifically synthesized resulting in unique patterns of TVP coexpression and subcellular colocalization. TVP orthologs have been identified in most multicellular organisms, including diverse animal and plant species, but have not been detected in unicellular organisms. They are subject to protein modification, most notably to phosphorylation, and are part of multimeric complexes. Experimental evidence is reviewed showing that TVPs contribute to vesicle trafficking and membrane morphogenesis. PMID:11893164

  5. Subcellular and in-vivo Nano-Endoscopy

    PubMed Central

    Cheemalapati, Surya Venkatasekhar; Winskas, John; Wang, Hao; Konnaiyan, Karthik; Zhdanov, Arseny; Roth, Alison; Adapa, Swamy Rakesh; Deonarine, Andrew; Noble, Mark; Das, Tuhin; Gatenby, Robert; Westerheide, Sandy D.; Jiang, Rays H. Y.; Pyayt, Anna

    2016-01-01

    Analysis of individual cells at the subcellular level is important for understanding diseases and accelerating drug discovery. Nanoscale endoscopes allow minimally invasive probing of individual cell interiors. Several such instruments have been presented previously, but they are either too complex to fabricate or require sophisticated external detectors because of low signal collection efficiency. Here we present a nanoendoscope that can locally excite fluorescence in labelled cell organelles and collect the emitted signal for spectral analysis. Finite Difference Time Domain (FDTD) simulations have shown that with an optimized nanoendoscope taper profile, the light emission and collection was localized within ~100 nm. This allows signal detection to be used for nano-photonic sensing of the proximity of fluorophores. Upon insertion into the individual organelles of living cells, the nanoendoscope was fabricated and resultant fluorescent signals collected. This included the signal collection from the nucleus of Acridine orange labelled human fibroblast cells, the nucleus of Hoechst stained live liver cells and the mitochondria of MitoTracker Red labelled MDA-MB-231 cells. The endoscope was also inserted into a live organism, the yellow fluorescent protein producing nematode Caenorhabditis elegans, and a fluorescent signal was collected. To our knowledge this is the first demonstration of in vivo, local fluorescence signal collection on the sub-organelle level. PMID:27694854

  6. Neurovascular Events After Subarachnoid Hemorrhage: Focusing on Subcellular Organelles

    PubMed Central

    Chen, Sheng; Wu, Haijian; Tang, Jiping; Zhang, Jianmin; Zhang, John H.

    2015-01-01

    Subarachnoid hemorrhage (SAH) is a devastating condition with high morbidity and mortality rates due to the lack of effective therapy. Early brain injury (EBI) and cerebral vasospasm (CVS) are the two most important pathophysiological mechanisms for brain injury and poor outcomes for patients with SAH. CVS has traditionally been considered the sole cause of delayed ischemic neurological deficits after SAH. However, the failure of antivasospastic therapy in patients with SAH supported changing the research target from CVS to other mechanisms. Currently, more attention has been focused on global brain injury within 3 days after ictus, designated as EBI. The dysfunction of subcellular organelles, such as endoplasmic reticulum stress, mitochondrial failure, and autophagy–lysosomal system activation, has developed during EBI and delayed brain injury after SAH. To our knowledge, there is a lack of review articles addressing the direction of organelle dysfunction after SAH. In this review, we discuss the roles of organelle dysfunction in the pathogenesis of SAH and present the opportunity to develop novel therapeutic strategies of SAH via modulating the functions of organelles. PMID:25366597

  7. Novel subcellular localization for α-synuclein: possible functional consequences

    PubMed Central

    Guardia-Laguarta, Cristina; Area-Gomez, Estela; Schon, Eric A.; Przedborski, Serge

    2015-01-01

    α-synuclein (α-syn) is one of the genes that when mutated or overexpressed causes Parkinson’s Disease (PD). Initially, it was described as a synaptic terminal protein and later was found to be localized at mitochondria. Mitochondria-associated membranes (MAM) have emerged as a central endoplasmic reticulum (ER) subcellular compartments where key functions of the cell occur. These domains, enriched in cholesterol and anionic phospholipids, are where calcium homeostasis, lipid transfer, and cholesterol metabolism are regulated. Some proteins, related to mitochondrial dynamics and function, are also localized to this area. Several neurodegenerative diseases have shown alterations in MAM functions and resident proteins, including Charcot Marie-Tooth and Alzheimer’s disease (AD). We have recently reported that MAM function is downregulated in cell and mouse models of PD expressing pathogenic mutations of α-syn. This review focuses on the possible role of α-syn in these cellular domains and the early pathogenic features of PD that could be explained by α-syn-MAM disturbances. PMID:25755636

  8. Subcellular localization of rickettsial invasion protein, InvA.

    PubMed

    Gaywee, Jariyanart; Sacci, John B; Radulovic, Suzana; Beier, Magda S; Azad, Abdu F

    2003-01-01

    To understand further the molecular basis of rickettsial host cell invasion, Rickettsia prowazekii invasion gene homolog (invA) has been characterized. Our previous experiments have shown that InvA is an Ap5A pyrophosphatase, a member of the Nudix hydrolase family, which is up-regulated during the internalization, early growth phase, and exit steps during rickettsial mammalian cell infection. In addition to the molecular characterization, subcellular localization of InvA was investigated. InvA-specific antibodies were raised in mice and used for immunoelectron microscopy. The generated antibodies were shown to recognize InvA and by immunogold labeling showed InvA in the cytoplasm of rickettsiae. A cytoplasmic location for InvA would allow for a rapid response to any internal substance and efficient functioning in hydrolysis of toxic metabolic by-products that are accumulated in the rickettsial cytoplasm during host cell invasion. Protecting bacteria from a hazardous environment could enhance their viability and allow them to remain metabolically active, which is a necessary step for the rickettsial obligate intracellular lifestyle.

  9. Global, quantitative and dynamic mapping of protein subcellular localization

    PubMed Central

    Itzhak, Daniel N; Tyanova, Stefka; Cox, Jürgen; Borner, Georg HH

    2016-01-01

    Subcellular localization critically influences protein function, and cells control protein localization to regulate biological processes. We have developed and applied Dynamic Organellar Maps, a proteomic method that allows global mapping of protein translocation events. We initially used maps statically to generate a database with localization and absolute copy number information for over 8700 proteins from HeLa cells, approaching comprehensive coverage. All major organelles were resolved, with exceptional prediction accuracy (estimated at >92%). Combining spatial and abundance information yielded an unprecedented quantitative view of HeLa cell anatomy and organellar composition, at the protein level. We subsequently demonstrated the dynamic capabilities of the approach by capturing translocation events following EGF stimulation, which we integrated into a quantitative model. Dynamic Organellar Maps enable the proteome-wide analysis of physiological protein movements, without requiring any reagents specific to the investigated process, and will thus be widely applicable in cell biology. DOI: http://dx.doi.org/10.7554/eLife.16950.001 PMID:27278775

  10. Reconstruction of Danio rerio Metabolic Model Accounting for Subcellular Compartmentalisation

    PubMed Central

    Bekaert, Michaël

    2012-01-01

    Plant and microbial metabolic engineering is commonly used in the production of functional foods and quality trait improvement. Computational model-based approaches have been used in this important endeavour. However, to date, fish metabolic models have only been scarcely and partially developed, in marked contrast to their prominent success in metabolic engineering. In this study we present the reconstruction of fully compartmentalised models of the Danio rerio (zebrafish) on a global scale. This reconstruction involves extraction of known biochemical reactions in D. rerio for both primary and secondary metabolism and the implementation of methods for determining subcellular localisation and assignment of enzymes. The reconstructed model (ZebraGEM) is amenable for constraint-based modelling analysis, and accounts for 4,988 genes coding for 2,406 gene-associated reactions and only 418 non-gene-associated reactions. A set of computational validations (i.e., simulations of known metabolic functionalities and experimental data) strongly testifies to the predictive ability of the model. Overall, the reconstructed model is expected to lay down the foundations for computational-based rational design of fish metabolic engineering in aquaculture. PMID:23166792

  11. Subcellular localization of hepatitis E virus (HEV) replicase.

    PubMed

    Rehman, Shagufta; Kapur, Neeraj; Durgapal, Hemlata; Panda, Subrat Kumar

    2008-01-01

    Hepatitis E virus (HEV) is a hepatotropic virus with a single sense-strand RNA genome of approximately 7.2 kb in length. Details of the intracellular site of HEV replication can pave further understanding of HEV biology. In-frame fusion construct of functionally active replicase-enhanced green fluorescent protein (EGFP) gene was made in eukaryotic expression vector. The functionality of replicase-EGFP fusion protein was established by its ability to synthesize negative-strand viral RNA in vivo, by strand-specific anchored RT-PCR and molecular beacon binding. Subcellular co-localization was carried out using organelle specific fluorophores and by immuno-electron microscopy. Fluorescence Resonance Energy Transfer (FRET) demonstrated the interaction of this protein with the 3' end of HEV genome. The results show localization of replicase on the endoplasmic reticulum membranes. The protein regions responsible for membrane localization was predicted and identified by use of deletion mutants. Endoplasmic reticulum was identified as the site of replicase localization and possible site of replication.

  12. Subcellular localization of transiently expressed fluorescent fusion proteins.

    PubMed

    Collings, David A

    2013-01-01

    The recent and massive expansion in plant genomics data has generated a large number of gene sequences for which two seemingly simple questions need to be answered: where do the proteins encoded by these genes localize in cells, and what do they do? One widespread approach to answering the localization question has been to use particle bombardment to transiently express unknown proteins tagged with green fluorescent protein (GFP) or its numerous derivatives. Confocal fluorescence microscopy is then used to monitor the localization of the fluorescent protein as it hitches a ride through the cell. The subcellular localization of the fusion protein, if not immediately apparent, can then be determined by comparison to localizations generated by fluorescent protein fusions to known signalling sequences and proteins, or by direct comparison with fluorescent dyes. This review aims to be a tour guide for researchers wanting to travel this hitch-hiker's path, and for reviewers and readers who wish to understand their travel reports. It will describe some of the technology available for visualizing protein localizations, and some of the experimental approaches for optimizing and confirming localizations generated by particle bombardment in onion epidermal cells, the most commonly used experimental system. As the non-conservation of signal sequences in heterologous expression systems such as onion, and consequent mis-targeting of fusion proteins, is always a potential problem, the epidermal cells of the Argenteum mutant of pea are proposed as a model system. PMID:23996319

  13. A Graphical Model to Determine the Subcellular Protein Location in Artificial Tissues

    PubMed Central

    Glory-Afshar, Estelle; Osuna-Highley, Elvira; Granger, Brian; Murphy, Robert F.

    2010-01-01

    Location proteomics is concerned with the systematic analysis of the subcellular location of proteins. In order to perform comprehensive analysis of all protein location patterns, automated methods are needed. With the goal of extending automated subcellular location pattern analysis methods to high resolution images of tissues, 3D confocal microscope images of polarized CaCo2 cells immunostained for various proteins were collected. A three-color staining protocol was developed that permits parallel imaging of proteins of interest as well as DNA and the actin cytoskeleton. The collection is composed of 11 to 21 images for each of the 9 proteins that depict major subcellular patterns. A classifier was trained to recognize the subcellular location pattern of segmented cells with an accuracy of 89.2%. Using the Prior Updating method allowed improvement of this accuracy to 99.6%. This study demonstrates the benefit of using a graphical model approach for improving the pattern classification in tissue images. PMID:21625289

  14. Correction: Understanding metal homeostasis in primary cultured neurons. Studies using single neuron subcellular and quantitative metallomics.

    PubMed

    Colvin, Robert A; Lai, Barry; Holmes, William R; Lee, Daewoo

    2015-09-01

    Correction for 'Understanding metal homeostasis in primary cultured neurons. Studies using single neuron subcellular and quantitative metallomics' by Robert A. Colvin et al., Metallomics, 2015, 7, 1111-1123.

  15. Sequential fractionation and isolation of subcellular proteins from tissue or cultured cells.

    PubMed

    Baghirova, Sabina; Hughes, Bryan G; Hendzel, Michael J; Schulz, Richard

    2015-01-01

    Many types of studies require the localization of a protein to, or isolation of enriched protein from a specific cellular compartment. Many protocols in the literature and from commercially available kits claim to yield pure cellular fractions. However, in our hands, the former often do not work effectively and the latter may be prohibitively expensive if a large number of fractionations are required. Furthermore, the largely proprietary composition of reagents in commercial kits means that the user is not able to make adjustments if, for example, a particular component affects the activity of a protein of interest. The method described here allows the isolation of purified proteins from three cellular fractions: the cytosol, membrane-bound organelles, and the nucleus. It uses gentle buffers with increasing detergent strength that sequentially lyse the cell membrane, organelle membranes and finally the nuclear membrane.•Quick, simple to replicate or adjust; this method does not require expensive reagents or use of commercial kits•The protocol can be applied to tissue samples or cultured cells without changing buffer components•Yields purified fractions of cytosolic, membrane bound and nuclear proteins, with the proper distribution of the appropriate subcellular markers: GAPDH, VDAC, SERCA2 and lamin A/C. PMID:26740924

  16. Lesion profiling and subcellular prion localization of cervid chronic wasting disease in domestic cats.

    PubMed

    Seelig, D M; Nalls, A V; Flasik, M; Frank, V; Eaton, S; Mathiason, C K; Hoover, E A

    2015-01-01

    Chronic wasting disease (CWD) is an efficiently transmitted, fatal, and progressive prion disease of cervids with an as yet to be fully clarified host range. While outbred domestic cats (Felis catus) have recently been shown to be susceptible to experimental CWD infection, the neuropathologic features of the infection are lacking. Such information is vital to provide diagnostic power in the event of natural interspecies transmission and insights into host and strain interactions in interspecies prion infection. Using light microscopy and immunohistochemistry, we detail the topographic pattern of neural spongiosis (the "lesion profile") and the distribution of misfolded prion protein in the primary and secondary passage of feline CWD (Fel(CWD)). We also evaluated cellular and subcellular associations between misfolded prion protein (PrP(D)) and central nervous system neurons and glial cell populations. From these studies, we (1) describe the novel neuropathologic profile of Fel(CWD), which is distinct from either cervid CWD or feline spongiform encephalopathy (FSE), and (2) provide evidence of serial passage-associated interspecies prion adaptation. In addition, we demonstrate through confocal analysis the successful co-localization of PrP(D) with neurons, astrocytes, microglia, lysosomes, and synaptophysin, which, in part, implicates each of these in the neuropathology of Fel(CWD). In conclusion, this work illustrates the simultaneous role of both host and strain in the development of a unique Fel(CWD) neuropathologic profile and that such a profile can be used to discriminate between Fel(CWD) and FSE.

  17. HSP60 interacts with YB-1 and affects its polysome association and subcellular localization

    SciTech Connect

    Ohashi, Sachiyo; Atsumi, Megumi; Kobayashi, Shunsuke

    2009-08-07

    YB-1 is a DNA/RNA-binding protein which, in the cytoplasm, associates with polysomes and regulates translation. However, YB-1 has a novel nuclear localization signal, and its nuclear accumulation is correlated with cancer induction. Here we designated the amino-acid sequence as YB-NLS and demonstrated that YB-NLS is necessary for the nuclear translocation of overexpressed YB-1 in NG108-15 cells. In addition, we found that a heat shock protein, HSP60, binds to YB-NLS in the cytoplasm. Interestingly, when HSP60 expression was repressed, an increase of polysome-associated YB-1 was observed in heavy-sedimenting fractions on a sucrose gradient. Overexpression of HSP60 resulted in a decrease of YB-1 in the heavy-sedimenting fractions and suppression of YB-NLS activity. Furthermore, the NLS-deleted YB-1 was apparently associated with the heavy-sedimenting polysomes. These results suggest that HSP60 interacts with YB-1 at the YB-NLS region and acts as a regulator of polysome association and the subcellular distribution of YB-1.

  18. Dual subcellular compartment delivery of doxorubicin to overcome drug resistant and enhance antitumor activity

    PubMed Central

    Song, Yan-feng; Liu, Dao-zhou; Cheng, Ying; Liu, Miao; Ye, Wei-liang; Zhang, Bang-le; Liu, Xin-you; Zhou, Si-yuan

    2015-01-01

    In order to overcome drug resistant and enhance antitumor activity of DOX, a new pH-sensitive micelle (DOX/DQA-DOX@DSPE-hyd-PEG-AA) was prepared to simultaneously deliver DOX to nucleus and mitochondria. Drug released from DOX/DQA-DOX@DSPE-hyd-PEG-AA showed a pH-dependent manner. DOX/DQA-DOX@DSPE-hyd-PEG-AA induced the depolarization of mitochondria and apoptosis in MDA-MB-231/ADR cells and A549 cells, which resulted in the high cytotoxicity of DOX/DQA-DOX@DSPE-hyd-PEG-AA against MDA-MB-231/ADR cells and A549 cells. Confocal microscopy confirmed that DOX/DQA-DOX@DSPE-hyd-PEG-AA simultaneously delivered DQA-DOX and DOX to the mitochondria and nucleus of tumor cell. After DOX/DQA-DOX@DSPE-hyd-PEG-AA was injected to the tumor-bearing nude mice by the tail vein, DOX was mainly found in tumor tissue. But DOX was widely distributed in the whole body after the administration of free DOX. Compared with free DOX, the same dose of DOX/DQA-DOX@DSPE-hyd-PEG-AA significantly inhibited the growth of DOX-resistant tumor in tumor-bearing mice without obvious systemic toxicity. Therefore, dual subcellular compartment delivery of DOX greatly enhanced the antitumor activity of DOX on DOX-resistant tumor. DOX/DQA-DOX@DSPE-hyd-PEG-AA has the potential in target therapy for DOX-resistant tumor. PMID:26530454

  19. Subcellular Evidence for Biogenesis of Autophagosomal Membrane during Spermiogenesis In vivo

    PubMed Central

    Huang, Yufei; Yang, Ping; Liu, Tengfei; Chen, Hong; Chu, Xiaoya; Ahmad, Nisar; Zhang, Qian; Li, Quanfu; Hu, Lisi; Liu, Yi; Chen, Qiusheng

    2016-01-01

    Although autophagosome formation has attracted substantial attention, the origin and the source of the autophagosomal membrane remains unresolved. The present study was designed to investigate in vivo subcellular evidence for the biogenesis of autophagosomal membrane during spermiogenesis using transmission-electron microscopy (TEM), Western blots and immunohistochemistry in samples from the Chinese soft-shelled turtle. The testis expressed LC3-II protein, which was located within spermatids at different stages of differentiation and indicated active autophagy. TEM showed that numerous autophagosomes were developed inside spermatids. Many endoplasmic reticulum (ER) were transferred into a special “Chrysanthemum flower center” (CFC) in which several double-layer isolation membranes (IM) were formed and extended. The elongated IM always engulfed some cytoplasm and various structures. Narrow tubules connected the ends of multiple ER and the CFC. The CFC was more developed in spermatids with compact nuclei than in spermatids with granular nuclei. An IM could also be transformed from a single ER. Sometimes an IM extended from a trans-Golgi network and wrapped different structures. The plasma membrane of the spermatid invaginated to form vesicles that were distributed among various endosomes around the CFC during spermiogenesis. All this cellular evidence suggests that, in vivo, IM was developed mainly by CFC produced from ER within differentiating spermatids during spermiogenesis. Vesicles from Golgi complexes, plasma membranes and endosomes might also be the sources of the autophagosome membrane. PMID:27803675

  20. Subcellular localization of PUMA regulates its pro-apoptotic activity in Burkitt's lymphoma B cells.

    PubMed

    Ambroise, Gorbatchev; Portier, Alain; Roders, Nathalie; Arnoult, Damien; Vazquez, Aimé

    2015-11-10

    The BH3-only protein PUMA (p53-upregulated modulator of apoptosis) is a major regulator of apoptosis. It belongs to the Bcl-2 family of proteins responsible for maintaining mitochondrial outer membrane integrity by controlling the intrinsic (mitochondrial) apoptotic pathway. We describe here a new pathway regulating PUMA activation through the control of its subcellular distribution. Surprisingly, neither PUMA upregulation in normal activated human B lymphocytes nor high levels of PUMA in Burkitt's lymphoma (BL) were associated with cell death. We show that PUMA is localized to the cytosol in these cells. By contrast, various apoptosis-triggering signals were found to promote the translocation of PUMA to the mitochondria in these cells, leading to their death by apoptosis. This apoptosis was associated with the binding of mitochondrial PUMA to anti-apoptotic members of the Bcl-2 family, such as Bcl-2 and Mcl-1. This translocation was caspase-independent but was prevented by inhibiting or knocking down the expression of the MAPK kinase p38. Our data suggest that the accumulation of PUMA in the cytosol may be important for the participation of this protein in apoptosis without the need for prior transcription. This regulatory pathway may be an important feature of differentiation and tumorigenic processes.

  1. Subcellular localization and properties of lipase activities in human polymorphonuclear leukocytes.

    PubMed

    Hack, N J; Smith, G P; Peters, T J

    1985-03-01

    A fluorimetric assay for lipase activity has been optimized for measurement of the enzyme in human neutrophils. Activity was maximal at acid (4.5) and alkaline (9.5) pH, although there was also a neutral peak of activity at pH 6.5. Neutrophils were homogenised in isotonic sucrose and subjected to analytical subcellular fractionation by sucrose density gradient centrifugation. The gradient fractions were assayed for acid, neutral and alkaline lipase activity and for the principal organelle marker enzymes. Neutral lipase showed a unimodal distribution with an equilibrium density of 1.19 g . cm-3, corresponding to the distribution of particulate leucine aminopeptidase. Acid and alkaline lipase activities showed very similar distribution profiles to each other with both soluble components and a broad peak of particulate activity. The broad modal density of 1.19-1.22 g . cm-3 suggests that acid and alkaline lipase activities could be localised to more than one population of cytoplasmic granule. Fractionation experiments with neutrophils homogenised in sucrose medium containing digitonin confirmed the localisation of neutral lipase and leucine aminopeptidase to the same cytoplasmic granule, and suggested that at least part of the acid lipase activity was localised to the specific granule. No lipase activity could be attributed to the alkaline phosphatase-containing granule. Neutrophils were isolated from control subjects, patients with chronic granulocytic leukaemia and women in the third trimester of pregnancy. The specific activity of acid, neutral and alkaline lipase, and leucine aminopeptidase, in contrast to that of alkaline phosphatase, were similar in the three patient groups.

  2. MetazSecKB: the human and animal secretome and subcellular proteome knowledgebase

    PubMed Central

    Meinken, John; Walker, Gary; Cooper, Chester R.; Min, Xiang Jia

    2015-01-01

    The subcellular location of a protein is a key factor in determining the molecular function of the protein in an organism. MetazSecKB is a secretome and subcellular proteome knowledgebase specifically designed for metazoan, i.e. human and animals. The protein sequence data, consisting of over 4 million entries with 121 species having a complete proteome, were retrieved from UniProtKB. Protein subcellular locations including secreted and 15 other subcellular locations were assigned based on either curated experimental evidence or prediction using seven computational tools. The protein or subcellular proteome data can be searched and downloaded using several different types of identifiers, gene name or keyword(s), and species. BLAST search and community annotation of subcellular locations are also supported. Our primary analysis revealed that the proteome sizes, secretome sizes and other subcellular proteome sizes vary tremendously in different animal species. The proportions of secretomes vary from 3 to 22% (average 8%) in metazoa species. The proportions of other major subcellular proteomes ranged approximately 21–43% (average 31%) in cytoplasm, 20–37% (average 30%) in nucleus, 3–19% (average 12%) as plasma membrane proteins and 3–9% (average 6%) in mitochondria. We also compared the protein families in secretomes of different primates. The Gene Ontology and protein family domain analysis of human secreted proteins revealed that these proteins play important roles in regulation of human structure development, signal transduction, immune systems and many other biological processes. Database URL: http://proteomics.ysu.edu/secretomes/animal/index.php PMID:26255309

  3. Intracellular lumen formation in Drosophila proceeds via a novel subcellular compartment.

    PubMed

    Nikolova, Linda S; Metzstein, Mark M

    2015-11-15

    Cellular tubes have diverse morphologies, including multicellular, unicellular and subcellular architectures. Subcellular tubes are found prominently within the vertebrate vasculature, the insect breathing system and the nematode excretory apparatus, but how such tubes form is poorly understood. To characterize the cellular mechanisms of subcellular tube formation, we have refined methods of high pressure freezing/freeze substitution to prepare Drosophila larvae for transmission electron microscopic (TEM) analysis. Using our methods, we have found that subcellular tube formation may proceed through a previously undescribed multimembrane intermediate composed of vesicles bound within a novel subcellular compartment. We have also developed correlative light/TEM procedures to identify labeled cells in TEM-fixed larval samples. Using this technique, we have found that Vacuolar ATPase (V-ATPase) and the V-ATPase regulator Rabconnectin-3 are required for subcellular tube formation, probably in a step resolving the intermediate compartment into a mature lumen. In general, our ultrastructural analysis methods could be useful for a wide range of cellular investigations in Drosophila larvae.

  4. Intracellular lumen formation in Drosophila proceeds via a novel subcellular compartment.

    PubMed

    Nikolova, Linda S; Metzstein, Mark M

    2015-11-15

    Cellular tubes have diverse morphologies, including multicellular, unicellular and subcellular architectures. Subcellular tubes are found prominently within the vertebrate vasculature, the insect breathing system and the nematode excretory apparatus, but how such tubes form is poorly understood. To characterize the cellular mechanisms of subcellular tube formation, we have refined methods of high pressure freezing/freeze substitution to prepare Drosophila larvae for transmission electron microscopic (TEM) analysis. Using our methods, we have found that subcellular tube formation may proceed through a previously undescribed multimembrane intermediate composed of vesicles bound within a novel subcellular compartment. We have also developed correlative light/TEM procedures to identify labeled cells in TEM-fixed larval samples. Using this technique, we have found that Vacuolar ATPase (V-ATPase) and the V-ATPase regulator Rabconnectin-3 are required for subcellular tube formation, probably in a step resolving the intermediate compartment into a mature lumen. In general, our ultrastructural analysis methods could be useful for a wide range of cellular investigations in Drosophila larvae. PMID:26428009

  5. Subcellular location and properties of bactericidal factors from human neutrophils.

    PubMed

    Gabay, J E; Heiple, J M; Cohn, Z A; Nathan, C F

    1986-11-01

    We examined the subcellular location of bactericidal factors (BF) in human neutrophils, using an efficient fractionation scheme. Nitrogen bomb cavitates of DIFP-treated PMN were centrifuged through discontinuous Percoll gradients, each fraction extracted with 0.05 M glycine, pH 2.0, and tested for the killing of Escherichia coli. greater than 90% of BF coisolated with the azurophil granules. After lysis of azurophils, 98% of azurophil-derived BF (ADBF) sedimented with the membrane. ADBF activity was solubilized from azurophil membrane with either acid or nonionic detergent (Triton X-100, Triton X-114). Bactericidal activity was linear with respect to protein concentration over the range 0.3-30 micrograms/ml. 0.1-0.3 microgram/ml ADBF killed 10(5) E. coli within 30 min at 37 degrees C. At 1.4 micrograms/ml, 50% of 2 X 10(5) bacteria were killed within 5 min. ADBF was effective between pH 5-8, with peak activity at pH 5.5. Glucose (20 mM), EDTA (1-25 mM), and physiologic concentrations of NaCl or KCl had little or no inhibitory effect on ADBF. ADBF killed both Gram-positive and Gram-negative virulent clinical isolates, including listeria, staphylococci, beta-hemolytic streptococci, and Pseudomonas aeruginosa. Thus, under these conditions of cell disruption, fractionation, extraction, and assay, almost all BF in human PMN appeared to be localized to the membrane of azurophilic granules as a highly potent, broad-spectrum, rapidly acting protein(s) effective in physiologic medium. Some of these properties appear to distinguish ADBF from previously described PMN bactericidal proteins. PMID:3772295

  6. A Sub-Cellular Viscoelastic Model for Cell Population Mechanics

    PubMed Central

    Jamali, Yousef; Azimi, Mohammad; Mofrad, Mohammad R. K.

    2010-01-01

    Understanding the biomechanical properties and the effect of biomechanical force on epithelial cells is key to understanding how epithelial cells form uniquely shaped structures in two or three-dimensional space. Nevertheless, with the limitations and challenges posed by biological experiments at this scale, it becomes advantageous to use mathematical and ‘in silico’ (computational) models as an alternate solution. This paper introduces a single-cell-based model representing the cross section of a typical tissue. Each cell in this model is an individual unit containing several sub-cellular elements, such as the elastic plasma membrane, enclosed viscoelastic elements that play the role of cytoskeleton, and the viscoelastic elements of the cell nucleus. The cell membrane is divided into segments where each segment (or point) incorporates the cell's interaction and communication with other cells and its environment. The model is capable of simulating how cells cooperate and contribute to the overall structure and function of a particular tissue; it mimics many aspects of cellular behavior such as cell growth, division, apoptosis and polarization. The model allows for investigation of the biomechanical properties of cells, cell-cell interactions, effect of environment on cellular clusters, and how individual cells work together and contribute to the structure and function of a particular tissue. To evaluate the current approach in modeling different topologies of growing tissues in distinct biochemical conditions of the surrounding media, we model several key cellular phenomena, namely monolayer cell culture, effects of adhesion intensity, growth of epithelial cell through interaction with extra-cellular matrix (ECM), effects of a gap in the ECM, tensegrity and tissue morphogenesis and formation of hollow epithelial acini. The proposed computational model enables one to isolate the effects of biomechanical properties of individual cells and the communication

  7. Oleosin of subcellular lipid droplets evolved in green algae.

    PubMed

    Huang, Nan-Lan; Huang, Ming-Der; Chen, Tung-Ling L; Huang, Anthony H C

    2013-04-01

    In primitive and higher plants, intracellular storage lipid droplets (LDs) of triacylglycerols are stabilized with a surface layer of phospholipids and oleosin. In chlorophytes (green algae), a protein termed major lipid-droplet protein (MLDP) rather than oleosin on LDs was recently reported. We explored whether MLDP was present directly on algal LDs and whether algae had oleosin genes and oleosins. Immunofluorescence microscopy revealed that MLDP in the chlorophyte Chlamydomonas reinhardtii was associated with endoplasmic reticulum subdomains adjacent to but not directly on LDs. In C. reinhardtii, low levels of a transcript encoding an oleosin-like protein (oleolike) in zygotes-tetrads and a transcript encoding oleosin in vegetative cells transferred to an acetate-enriched medium were found in transcriptomes and by reverse transcription-polymerase chain reaction. The C. reinhardtii LD fraction contained minimal proteins with no detectable oleolike or oleosin. Several charophytes (advanced green algae) possessed low levels of transcripts encoding oleosin but not oleolike. In the charophyte Spirogyra grevilleana, levels of oleosin transcripts increased greatly in cells undergoing conjugation for zygote formation, and the LD fraction from these cells contained minimal proteins, two of which were oleosins identified via proteomics. Because the minimal oleolike and oleosins in algae were difficult to detect, we tested their subcellular locations in Physcomitrella patens transformed with the respective algal genes tagged with a Green Fluorescent Protein gene and localized the algal proteins on P. patens LDs. Overall, oleosin genes having weak and cell/development-specific expression were present in green algae. We present a hypothesis for the evolution of oleosins from algae to plants. PMID:23391579

  8. Aberrant subcellular neuronal calcium regulation in aging and Alzheimer's disease.

    PubMed

    Camandola, Simonetta; Mattson, Mark P

    2011-05-01

    In this mini-review/opinion article we describe evidence that multiple cellular and molecular alterations in Alzheimer's disease (AD) pathogenesis involve perturbed cellular calcium regulation, and that alterations in synaptic calcium handling may be early and pivotal events in the disease process. With advancing age neurons encounter increased oxidative stress and impaired energy metabolism, which compromise the function of proteins that control membrane excitability and subcellular calcium dynamics. Altered proteolytic cleavage of the β-amyloid precursor protein (APP) in response to the aging process in combination with genetic and environmental factors results in the production and accumulation of neurotoxic forms of amyloid β-peptide (Aβ). Aβ undergoes a self-aggregation process and concomitantly generates reactive oxygen species that can trigger membrane-associated oxidative stress which, in turn, impairs the functions of ion-motive ATPases and glutamate and glucose transporters thereby rendering neurons vulnerable to excitotoxicity and apoptosis. Mutations in presenilin-1 that cause early-onset AD increase Aβ production, but also result in an abnormal increase in the size of endoplasmic reticulum calcium stores. Some of the events in the neurodegenerative cascade can be counteracted in animal models by manipulations that stabilize neuronal calcium homeostasis including dietary energy restriction, agonists of glucagon-like peptide 1 receptors and drugs that activate mitochondrial potassium channels. Emerging knowledge of the actions of calcium upstream and downstream of Aβ provides opportunities to develop novel preventative and therapeutic interventions for AD. This article is part of a Special Issue entitled: 11th European Symposium on Calcium. PMID:20950656

  9. Subcellular location and properties of bactericidal factors from human neutrophils.

    PubMed

    Gabay, J E; Heiple, J M; Cohn, Z A; Nathan, C F

    1986-11-01

    We examined the subcellular location of bactericidal factors (BF) in human neutrophils, using an efficient fractionation scheme. Nitrogen bomb cavitates of DIFP-treated PMN were centrifuged through discontinuous Percoll gradients, each fraction extracted with 0.05 M glycine, pH 2.0, and tested for the killing of Escherichia coli. greater than 90% of BF coisolated with the azurophil granules. After lysis of azurophils, 98% of azurophil-derived BF (ADBF) sedimented with the membrane. ADBF activity was solubilized from azurophil membrane with either acid or nonionic detergent (Triton X-100, Triton X-114). Bactericidal activity was linear with respect to protein concentration over the range 0.3-30 micrograms/ml. 0.1-0.3 microgram/ml ADBF killed 10(5) E. coli within 30 min at 37 degrees C. At 1.4 micrograms/ml, 50% of 2 X 10(5) bacteria were killed within 5 min. ADBF was effective between pH 5-8, with peak activity at pH 5.5. Glucose (20 mM), EDTA (1-25 mM), and physiologic concentrations of NaCl or KCl had little or no inhibitory effect on ADBF. ADBF killed both Gram-positive and Gram-negative virulent clinical isolates, including listeria, staphylococci, beta-hemolytic streptococci, and Pseudomonas aeruginosa. Thus, under these conditions of cell disruption, fractionation, extraction, and assay, almost all BF in human PMN appeared to be localized to the membrane of azurophilic granules as a highly potent, broad-spectrum, rapidly acting protein(s) effective in physiologic medium. Some of these properties appear to distinguish ADBF from previously described PMN bactericidal proteins.

  10. A sub-cellular viscoelastic model for cell population mechanics.

    PubMed

    Jamali, Yousef; Azimi, Mohammad; Mofrad, Mohammad R K

    2010-01-01

    Understanding the biomechanical properties and the effect of biomechanical force on epithelial cells is key to understanding how epithelial cells form uniquely shaped structures in two or three-dimensional space. Nevertheless, with the limitations and challenges posed by biological experiments at this scale, it becomes advantageous to use mathematical and 'in silico' (computational) models as an alternate solution. This paper introduces a single-cell-based model representing the cross section of a typical tissue. Each cell in this model is an individual unit containing several sub-cellular elements, such as the elastic plasma membrane, enclosed viscoelastic elements that play the role of cytoskeleton, and the viscoelastic elements of the cell nucleus. The cell membrane is divided into segments where each segment (or point) incorporates the cell's interaction and communication with other cells and its environment. The model is capable of simulating how cells cooperate and contribute to the overall structure and function of a particular tissue; it mimics many aspects of cellular behavior such as cell growth, division, apoptosis and polarization. The model allows for investigation of the biomechanical properties of cells, cell-cell interactions, effect of environment on cellular clusters, and how individual cells work together and contribute to the structure and function of a particular tissue. To evaluate the current approach in modeling different topologies of growing tissues in distinct biochemical conditions of the surrounding media, we model several key cellular phenomena, namely monolayer cell culture, effects of adhesion intensity, growth of epithelial cell through interaction with extra-cellular matrix (ECM), effects of a gap in the ECM, tensegrity and tissue morphogenesis and formation of hollow epithelial acini. The proposed computational model enables one to isolate the effects of biomechanical properties of individual cells and the communication between

  11. Oleosin of subcellular lipid droplets evolved in green algae.

    PubMed

    Huang, Nan-Lan; Huang, Ming-Der; Chen, Tung-Ling L; Huang, Anthony H C

    2013-04-01

    In primitive and higher plants, intracellular storage lipid droplets (LDs) of triacylglycerols are stabilized with a surface layer of phospholipids and oleosin. In chlorophytes (green algae), a protein termed major lipid-droplet protein (MLDP) rather than oleosin on LDs was recently reported. We explored whether MLDP was present directly on algal LDs and whether algae had oleosin genes and oleosins. Immunofluorescence microscopy revealed that MLDP in the chlorophyte Chlamydomonas reinhardtii was associated with endoplasmic reticulum subdomains adjacent to but not directly on LDs. In C. reinhardtii, low levels of a transcript encoding an oleosin-like protein (oleolike) in zygotes-tetrads and a transcript encoding oleosin in vegetative cells transferred to an acetate-enriched medium were found in transcriptomes and by reverse transcription-polymerase chain reaction. The C. reinhardtii LD fraction contained minimal proteins with no detectable oleolike or oleosin. Several charophytes (advanced green algae) possessed low levels of transcripts encoding oleosin but not oleolike. In the charophyte Spirogyra grevilleana, levels of oleosin transcripts increased greatly in cells undergoing conjugation for zygote formation, and the LD fraction from these cells contained minimal proteins, two of which were oleosins identified via proteomics. Because the minimal oleolike and oleosins in algae were difficult to detect, we tested their subcellular locations in Physcomitrella patens transformed with the respective algal genes tagged with a Green Fluorescent Protein gene and localized the algal proteins on P. patens LDs. Overall, oleosin genes having weak and cell/development-specific expression were present in green algae. We present a hypothesis for the evolution of oleosins from algae to plants.

  12. Chemical bioimaging for the subcellular localization of trace elements by high contrast TEM, TEM/X-EDS, and NanoSIMS.

    PubMed

    Penen, Florent; Malherbe, Julien; Isaure, Marie-Pierre; Dobritzsch, Dirk; Bertalan, Ivo; Gontier, Etienne; Le Coustumer, Philippe; Schaumlöffel, Dirk

    2016-09-01

    Chemical bioimaging offers an important contribution to the investigation of biochemical functions, biosorption and bioaccumulation processes of trace elements via their localization at the cellular and even at the subcellular level. This paper describes the combined use of high contrast transmission electron microscopy (HC-TEM), energy dispersive X-ray spectroscopy (X-EDS), and nano secondary ion mass spectrometry (NanoSIMS) applied to a model organism, the unicellular green algae Chlamydomonas reinhardtii. HC-TEM providing a lateral resolution of 1nm was used for imaging the ultrastructure of algae cells which have diameters of 5-10μm. TEM coupled to X-EDS (TEM/X-EDS) combined textural (morphology and size) analysis with detection of Ca, P, K, Mg, Fe, and Zn in selected subcellular granules using an X-EDS probe size of approx. 1μm. However, instrumental sensitivity was at the limit for trace element detection. NanoSIMS allowed chemical imaging of macro and trace elements with subcellular resolution (element mapping). Ca, Mg, and P as well as the trace elements Fe, Cu, and Zn present at basal levels were detected in pyrenoids, contractile vacuoles, and granules. Some metals were even localized in small vesicles of about 200nm size. Sensitive subcellular localization of trace metals was possible by the application of a recently developed RF plasma oxygen primary ion source on NanoSIMS which has shown good improvements in terms of lateral resolution (below 50nm), sensitivity, and stability. Furthermore correlative single cell imaging was developed combining the advantages of TEM and NanoSIMS. An advanced sample preparation protocol provided adjacent ultramicrotome sections for parallel TEM and NanoSIMS analyses of the same cell. Thus, the C. reinhardtii cellular ultrastructure could be directly related to the spatial distribution of metals in different cell organelles such as vacuoles and chloroplast. PMID:27288221

  13. Prot-Prop: J-tool to predict the subcellular location of proteins based on physiochemical characterization.

    PubMed

    Senthilkumar, Brindha; Sailo, Sangzuala; Guruswami, Gurusubramanian; Nachimuthu, Senthilkumar

    2012-12-01

    PROT-PROP is a computational tool to characterize 27 physicochemical properties of a protein along with its subcellular location (intra or extra) in a single-window application. Other significant features of this software include calculation of numerical values for hydrophobicity, hydrophilicity; composition of small and large amino acids; net hydrophobic content in terms of low/high; and Navie's algorithm to calculate theoretical pI. PROT-PROP is an easy-to-install platform independent implementation of JAVA under a user-friendly interface. It is a standalone version as a virtual appliance and source code for platforms supporting Java 1.5.0 and higher versions, and downloadable from the web http://www.mzu.edu.in/schools/biotechnology.html . PROT-PROP can run under Windows and Macintosh Operating Systems. PROT-PROP is distributed with its source code so that it may be adapted or customized, if desired. PMID:23354819

  14. Differential Site Accessibility Mechanistically Explains Subcellular-Specific N-Glycosylation Determinants

    PubMed Central

    Lee, Ling Yen; Lin, Chi-Hung; Fanayan, Susan; Packer, Nicolle H.; Thaysen-Andersen, Morten

    2014-01-01

    Glycoproteins perform extra- and intracellular functions in innate and adaptive immunity by lectin-based interactions to exposed glyco-determinants. Herein, we document and mechanistically explain the formation of subcellular-specific N-glycosylation determinants on glycoproteins trafficking through the shared biosynthetic machinery of human cells. LC-MS/MS-based quantitative glycomics showed that the secreted glycoproteins of eight human breast epithelial cells displaying diverse geno- and phenotypes consistently displayed more processed, primarily complex type, N-glycans than the high-mannose-rich microsomal glycoproteins. Detailed subcellular glycome profiling of proteins derived from three breast cell lines (MCF7/MDA468/MCF10A) demonstrated that secreted glycoproteins displayed significantly more α-sialylation and α1,6-fucosylation, but less α-mannosylation, than both the intermediately glycan-processed cell-surface glycoproteomes and the under-processed microsomal glycoproteomes. Subcellular proteomics and gene ontology revealed substantial presence of endoplasmic reticulum resident glycoproteins in the microsomes and confirmed significant enrichment of secreted and cell-surface glycoproteins in the respective subcellular fractions. The solvent accessibility of the glycosylation sites on maturely folded proteins of the 100 most abundant putative N-glycoproteins observed uniquely in the three subcellular glycoproteomes correlated with the glycan type processing thereby mechanistically explaining the formation of subcellular-specific N-glycosylation. In conclusion, human cells have developed mechanisms to simultaneously and reproducibly generate subcellular-specific N-glycosylation using a shared biosynthetic machinery. This aspect of protein-specific glycosylation is important for structural and functional glycobiology and discussed here in the context of the spatio-temporal interaction of glyco-determinants with lectins central to infection and immunity

  15. Metabolism of nitrofluoranthenes by rat lung subcellular fractions.

    PubMed

    Mitchell, C E; Bechtold, W E; Belinsky, S A

    1993-06-01

    The nitrofluoranthene (NF) family of compounds includes the potent pulmonary carcinogen 3,9-dinitrofluoranthene (3,9-DNF) and the weak carcinogen 3-nitrofluoranthene (3-NF). Although the specific molecular mechanisms involved in this difference in sensitivity for the induction of lung tumors in rats by 3,9-DNF and 3-NF have not been defined, these compounds most likely induce carcinogenesis by metabolic activation to electrophilic metabolites that bind DNA. The purpose of these investigations was to determine the activation pathways in the rat lung for the metabolism of the di-(3,9-DNF) and mono-nitroisomers (3-NF, 8-NF, 2-NF) of NFs. The metabolic rates of NFs were compared for lung subcellular fractions of pristine rats as well as rats previously treated with 3-methylcholanthrene (3-MC) or phenobarbital at levels that would induce cytochrome P450 enzymes. One major metabolite, the amino derivative, was detected by high pressure liquid chromatography following anaerobic incubation of rat lung cytosol with 3-NF, 8-NF, 2-NF or 3,9-DNF. 3,9-DNF was metabolized to its amino derivative, aminonitrofluoranthene, at a higher rate than 3-NF, 8-NF or 2-NF. Pretreatment of the rats with 3-MC or phenobarbital did not affect the metabolic rates of cytosolic reduction. Both 3-NF and 3,9-DNF were metabolized anaerobically to their amino derivatives by microsomal reductas(s). 3,9-DNF was metabolized twice as fast as 3-NF. The formation of the aminonitrofluoranthene metabolite was increased approximately 2 times with microsomes from 3-MC-induced rats, but was unaffected by microsomes from phenobarbital-treated rats. This suggests that the cytochrome P450 isozymes and reductase, which are induced by 3-MC, may be involved in the metabolism of 3-NF and 3,9-DNF. The metabolic products of 3-NF, formed aerobically, consisted of one major and three minor compounds. The major metabolite, tentatively identified as 3-NF-8-ol, was increased approximately 6 times using microsomes from 3-MC

  16. Regulation of Protein Levels in Subcellular Domains through mRNA Transport and Localized Translation*

    PubMed Central

    Willis, Dianna E.; Twiss, Jeffery L.

    2010-01-01

    Localized protein synthesis is increasingly recognized as a means for polarized cells to modulate protein levels in subcellular regions and the distal reaches of their cytoplasm. The axonal and dendritic processes of neurons represent functional domains of cytoplasm that can be separated from their cell body by vast distances. This separation provides a biological setting where the cell uses locally synthesized proteins to both autonomously respond to stimuli and to retrogradely signal the cell body of events occurring is this distal environment. Other cell types undoubtedly take advantage of this localized mechanism, but these have not proven as amenable for isolation of functional subcellular domains. Consequently, neurons have provided an appealing experimental platform for study of mRNA transport and localized protein synthesis. Molecular biology approaches have shown both the population of mRNAs that can localize into axons and dendrites and an unexpectedly complex regulation of their transport into these processes. Several lines of evidence point to similar complexities and specificity for regulation of mRNA translation at subcellular sites. Proteomics studies are beginning to provide a comprehensive view of the protein constituents of subcellular domains in neurons and other cell types. However, these have currently fallen short of dissecting temporal regulation of new protein synthesis in subcellular sites and mechanisms used to ferry mRNAs to these sites. PMID:20167945

  17. Subcellular location of serum- and glucocorticoid-induced kinase-1 in renal and mammary epithelial cells.

    PubMed

    Cordas, Emily; Náray-Fejes-Tóth, Anikó; Fejes-Tóth, Géza

    2007-05-01

    Serum- and glucocorticoid-induced kinase-1 (SGK1) is involved in aldosterone-induced Na(+) reabsorption by increasing epithelial Na(+) channel (ENaC) activity in cortical collecting duct (CCD) cells, but its exact mechanisms of action are unknown. Although several potential targets such as Nedd4-2 have been described in expression systems, endogenous substrates mediating SGK1's physiological effects remain to be identified. In addition, subcellular localization studies of SGK1 have provided controversial results. We determined the subcellular location of SGK1 using SGK1-autofluorescent protein (AFP) fusion proteins. Rabbit CCD (RCCT-28A) cells were transiently transfected with a construct encoding for SGK1-AFP and were stained or cotransfected with markers for various subcellular compartments. In live cells, transiently expressed SGK1-AFP clearly colocalized with the mitochondrial marker rhodamine 123. Similarly, SGK1-AFP colocalized with the mitochondrial marker MitoTracker when stably expressed using a retroviral system in either RCCT-28A cells or the mammary epithelial cell line MCF10A. To determine which region of SGK1 is responsible for this subcellular localization, we generated RCCT-28A cell lines stably expressing SGK1 mutants. The results indicate that the NH(2)-terminal 60-amino acid region of SGK1 is necessary and sufficient for its subcellular localization. Localization of SGK1 to the mitochondria raises the possibility that SGK1 may play a role in regulating energy metabolism.

  18. Chitin synthetases in Candida albicans: a review on their subcellular distribution and biological function.

    PubMed

    Martínez, J P; Gozalbo, D

    1994-09-01

    In the light of recent genetic advances, some results regarding chitin biosynthetic activities are reviewed in this paper. Genes coding for distinct enzymes displaying chitin synthetase activities have been characterized in Saccharomyces cerevisiae as well as in other fungal species including Candida albicans. Several activities seem to exist in the cells: (i) one zymogenic, located in cytoplasmic vesicles called chitosomes, although the presence of other types of vesicles with zymogenic activity cannot be completely discarded, and (ii) plasma membrane associated activities (the active enzyme and probably two distinct pools of zymogenic activity). Possible relationships between these activities, if any, remain to be determined. These multiplicity of enzymes is not surprising taking into account that chitin biosynthesis is required during very well defined temporal and spatial events of the cell cycle. A general repair function for one of the chitin biosynthetic activities is proposed as a possible salvage mechanism to warrant cell survival after wall damage has been caused, since chitin appears to be the most suitable polymer to carry out this function due to its particular physico-chemical properties.

  19. Trafficking regulates the subcellular distribution of voltage-gated sodium channels in primary sensory neurons.

    PubMed

    Bao, Lan

    2015-01-01

    Voltage-gated sodium channels (Navs) comprise at least nine pore-forming α subunits. Of these, Nav1.6, Nav1.7, Nav1.8 and Nav1.9 are the most frequently studied in primary sensory neurons located in the dorsal root ganglion and are mainly localized to the cytoplasm. A large pool of intracellular Navs raises the possibility that changes in Nav trafficking could alter channel function. The molecular mediators of Nav trafficking mainly consist of signals within the Navs themselves, interacting proteins and extracellular factors. The surface expression of Navs is achieved by escape from the endoplasmic reticulum and proteasome degradation, forward trafficking and plasma membrane anchoring, and it is also regulated by channel phosphorylation and ubiquitination in primary sensory neurons. Axonal transport and localization of Navs in afferent fibers involves the motor protein KIF5B and scaffold proteins, including contactin and PDZ domain containing 2. Localization of Nav1.6 to the nodes of Ranvier in myelinated fibers of primary sensory neurons requires node formation and the submembrane cytoskeletal protein complex. These findings inform our understanding of the molecular and cellular mechanisms underlying Nav trafficking in primary sensory neurons. PMID:26423360

  20. Interaction of insulin with the rat diaphragm. Subcellular distribution of insulin and its receptor

    SciTech Connect

    Brush, J.S.; Guzman-Diaz, A.; Celis, J.

    1987-05-01

    In studying the uptake and processing of A-14( SVI)monoiodoinsulin by isolated rat hemidiaphragms it was found that major amounts of the hormone are associated with the debris fraction. A method was developed for separating debris components by discontinuous sucrose density gradient centrifugation. Major amounts of radioactivity were associated with its myofibril component with much lower amounts in its sarcolemmal elements. Using a modified method of Marshall, et. al. insulin receptor was measurable in these fractions with greatest amounts in myofibril and microsomal fractions. Receptor was also detectable in the latter after gel electrophoresis and immunoblotting with receptor antiserum. Sarcolemmal marker enzyme (K -stimulated, ouabain-suppressible p-nitrophenylphosphate phosphatase) activity was insignificant in sarcolemmal and myofibril fractions, but was significant in the microsomal fraction. Sarcolemmal activity becomes significant after hemidiaphragm incubation with 1 M insulin for 90 sec. but does not change in the microsomal fraction. It is concluded that 1) a component bound to the myofibrils in muscle is important in insulin processing, and 2) the largest part of sarcolemmal insulin receptors are incorporated in the microsomal fraction of homogenized tissue.

  1. The accumulation and subcellular distribution of arsenic and antimony in four fern plants.

    PubMed

    Feng, R; Wang, X; Wei, C; Tu, S

    2015-01-01

    In the present study, Pteris cretica 'Albo-Lineata' (PC), Pteris fauriei (PF), Humata tyermanii Moore (HT), and Pteris ensiformis Burm (PE), were selected to explore additional plant materials for the phytoremediation of As and Sb co-contamination. To some extent, the addition of As and Sb enhanced the growth of HT, PE, and PF. Conversely, the addition of As and Sb negatively affected the growth of PC and was accompanied with the accumulation of high levels of As and Sb in the roots. The highest concentration of Sb was recorded as 6405 mg kg(-1) in the roots of PC, and that for As was 337 mg kg(-1) in the rhizome of PF. To some degree, As and Sb stimulated the uptake of each other in these ferns. Arsenic was mainly stored in the cytoplasmic supernatant (CS) fraction, followed by the cell wall (CW) fraction. In contrast, Sb was mainly found in the CW fraction and, to a lesser extent, in the CS fraction, suggesting that the cell wall and cytosol play different roles in As and Sb accumulation by fern plants. This study demonstrated that these fern plants show a good application potential in the phytoremediation of As and Sb co-contaminated environments.

  2. Eucalyptus tolerance mechanisms to lanthanum and cerium: subcellular distribution, antioxidant system and thiol pools.

    PubMed

    Shen, Yichang; Zhang, Shirong; Li, Sen; Xu, Xiaoxun; Jia, Yongxia; Gong, Guoshu

    2014-12-01

    Guanglin 9 (Eucalyptus grandis × Eucalyptus urophlla) and Eucalyptus grandis 5 are two eucalyptus species which have been found to grow normally in soils contaminated with lanthanum and cerium, but the tolerance mechanisms are not clear yet. In this study, a pot experiment was conducted to investigate the tolerance mechanisms of the eucalyptus to lanthanum and cerium. Cell walls stored 45.40-63.44% of the metals under lanthanum or cerium stress. Peroxidase and catalase activities enhanced with increasing soil La or Ce concentrations up to 200 mg kg(-1), while there were no obvious changes in glutathione and ascorbate concentrations. Non-protein thiols concentrations increased with increasing treatment levels up to 200 mg kg(-1), and then decreased. Phytochelatins concentrations continued to increase under La or Ce stress. Therefore, the two eucalyptus species are La and Ce tolerant plants, and the tolerance mechanisms include cell wall deposition, antioxidant system response, and thiol compound synthesis. PMID:25303462

  3. Lifelong maintenance of composition, function and cellular/subcellular distribution of proteasomes in human liver.

    PubMed

    Bellavista, Elena; Martucci, Morena; Vasuri, Francesco; Santoro, Aurelia; Mishto, Michele; Kloss, Alexander; Capizzi, Elisa; Degiovanni, Alessio; Lanzarini, Catia; Remondini, Daniel; Dazzi, Alessandro; Pellegrini, Sara; Cescon, Matteo; Capri, Miriam; Salvioli, Stefano; D'Errico-Grigioni, Antonia; Dahlmann, Burkhardt; Grazi, Gian Luca; Franceschi, Claudio

    2014-01-01

    Owing to organ shortage, livers from old donors are increasingly used for transplantation. The function and duration of such transplanted livers are apparently comparable to those from young donors, suggesting that, despite some morphological and structural age-related changes, no major functional changes do occur in liver with age. We tested this hypothesis by performing a comprehensive study on proteasomes, major cell organelles responsible for proteostasis, in liver biopsies from heart-beating donors. Oxidized and poly-ubiquitin conjugated proteins did not accumulate with age and the three major proteasome proteolytic activities were similar in livers from young and old donors. Analysis of proteasomes composition showed an age-related increased of β5i/α4 ratio, suggesting a shift toward proteasomes containing inducible subunits and a decreased content of PA28α subunit, mainly in the cytosol of hepatocytes. Thus our data suggest that, proteasomes activity is well preserved in livers from aged donors, concomitantly with subtle changes in proteasome subunit composition which might reflect the occurrence of a functional remodelling to maintain an efficient proteostasis. Gender differences are emerging and they deserve further investigations owing to the different aging trajectories between men and women. Finally, our data support the safe use of livers from old donors for transplantation.

  4. Phosphorylation and subcellular distribution of connexin43 in normal and stressed cells.

    PubMed

    Leykauf, Kerstin; Dürst, Matthias; Alonso, Angel

    2003-01-01

    We have developed polyclonal antibodies (SA226P) to a peptide of the human connexin43 (Cx43) protein between amino acids 271 and 288 containing phosphorylated S279 and S282. Antibodies specific for the phosphorylated form of the peptide were isolated by double immunoaffinity chromatography and were characterised using proteins of the cell line WB-F344, known to contain large amounts of Cx43. SA226P recognises specifically the slowest migrating Cx43 band in immunoblots of proteins isolated from untreated cells. In immunofluorescence experiments SA226P scarcely stains the plasma membrane in untreated cells in contrast to a commercial antibody recognising all isoforms of the Cx43 protein. EGF or stress treatment of the cells results in a rapid increase in the phosphorylated forms of Cx43 as revealed by immunoblotting. Immunofluorescence experiments reveal that both phosphorylated and non-phosphorylated Cx43 could be found at the plasma membrane. Whether phosphorylation of S279/S282 takes place before or after incorporation of Cx43 into the membranes is so far unknown. More interestingly, confocal microscopy using our antibodies and a commercial antibody recognising all isoforms of Cx43 shows the coexistence of differentially phosphorylated forms of the protein at the plasma membrane. Our results indicate that MAP kinases erk1/2 are mainly responsible for this phosphorylation, as already published. Nevertheless, treatment of the cells with anisomycin, known to activate stress kinase p38 but not erk1/2, also results in a weak but reproducible Cx43 phosphorylation. PMID:12483281

  5. Subcellular Profiling Reveals Distinct and Developmentally Regulated Repertoire of Growth Cone mRNAs

    PubMed Central

    Zivraj, Krishna H.; Tung, Yi Chun Loraine; Piper, Michael; Gumy, Laura; Fawcett, James W.; Yeo, Giles S. H.; Holt, Christine E.

    2013-01-01

    Cue-directed axon guidance depends partly on local translation in growth cones. Many mRNA transcripts are known to reside in developing axons, yet little is known about their subcellular distribution or, specifically, which transcripts are in growth cones. Here laser capture microdissection (LCM) was used to isolate the growth cones of retinal ganglion cell (RGC) axons of two vertebrate species, mouse and Xenopus, coupled with unbiased genomewide microarray profiling. An unexpectedly large pool of mRNAs defined predominant pathways in protein synthesis, oxidative phosphorylation, cancer, neurological disease, and signaling. Comparative profiling of “young” (pathfinding) versus “old” (target-arriving) Xenopus growth cones revealed that the number and complexity of transcripts increases dramatically with age. Many presynaptic protein mRNAs are present exclusively in old growth cones, suggesting that functionally related sets of mRNAs are targeted to growth cones in a developmentally regulated way. Remarkably, a subset of mRNAs was significantly enriched in the growth cone compared with the axon compartment, indicating that mechanisms exist to localize mRNAs selectively to the growth cone. Furthermore, some receptor transcripts (e.g., EphB4), present exclusively in old growth cones, were equally abundant in young and old cell bodies, indicating that RNA trafficking from the soma is developmentally regulated. Our findings show that the mRNA repertoire in growth cones is regulated dynamically with age and suggest that mRNA localization is tailored to match the functional demands of the growing axon tip as it transforms into the presynaptic terminal. PMID:21084603

  6. Stress-Responsive Expression, Subcellular Localization and Protein-Protein Interactions of the Rice Metacaspase Family.

    PubMed

    Huang, Lei; Zhang, Huijuan; Hong, Yongbo; Liu, Shixia; Li, Dayong; Song, Fengming

    2015-07-17

    Metacaspases, a class of cysteine-dependent proteases like caspases in animals, are important regulators of programmed cell death (PCD) during development and stress responses in plants. The present study was focused on comprehensive analyses of expression patterns of the rice metacaspase (OsMC) genes in response to abiotic and biotic stresses and stress-related hormones. Results indicate that members of the OsMC family displayed differential expression patterns in response to abiotic (e.g., drought, salt, cold, and heat) and biotic (e.g., infection by Magnaporthe oryzae, Xanthomonas oryzae pv. oryzae and Rhizoctonia solani) stresses and stress-related hormones such as abscisic acid, salicylic acid, jasmonic acid, and 1-amino cyclopropane-1-carboxylic acid (a precursor of ethylene), although the responsiveness to these stresses or hormones varies to some extent. Subcellular localization analyses revealed that OsMC1 was solely localized and OsMC2 was mainly localized in the nucleus. Whereas OsMC3, OsMC4, and OsMC7 were evenly distributed in the cells, OsMC5, OsMC6, and OsMC8 were localized in cytoplasm. OsMC1 interacted with OsLSD1 and OsLSD3 while OsMC3 only interacted with OsLSD1 and that the zinc finger domain in OsMC1 is responsible for the interaction activity. The systematic expression and biochemical analyses of the OsMC family provide valuable information for further functional studies on the biological roles of OsMCs in PCD that is related to abiotic and biotic stress responses.

  7. Regulation of the subcellular trafficking of CD36, a major determinant of cardiac fatty acid utilization.

    PubMed

    Glatz, Jan F C; Nabben, Miranda; Heather, Lisa C; Bonen, Arend; Luiken, Joost J F P

    2016-10-01

    Myocardial uptake of long-chain fatty acids largely occurs by facilitated diffusion, involving primarily the membrane-associated protein CD36. Other putative fatty acid transporters, such as FABPpm, FATP1 and FATP4, also play a role, but their quantitative contribution is much smaller or their involvement is rather permissive. Besides its sarcolemmal localization, CD36 is also present in intracellular compartments (endosomes). CD36 cycles between both pools via vesicle-mediated trafficking, and the relative distribution between endosomes versus sarcolemma determines the rate of cardiac fatty acid uptake. A net translocation of CD36 to the sarcolemma is induced by various stimuli, in particular hormones like insulin and myocyte contractions, so as to allow a proper coordination of the rate of fatty acid uptake with rapid fluctuations in myocardial energy needs. Furthermore, changes in cardiac fatty acid utilization that occur in both acute and chronic cardiac disease appear to be accompanied by concomitant changes in the sarcolemmal presence of CD36. Studies in various animal and cell models suggest that interventions aimed at modulating the sarcolemmal presence or functioning of CD36 hold promise as therapy to rectify aberrant rates of fatty acid uptake in order to fight cardiac metabolic remodeling and restore proper contractile function. In this review we discuss our current knowledge about the role of CD36 in cardiac fatty acid uptake and metabolism in health and disease with focus on the regulation of the subcellular trafficking of CD36 and its selective modulation as therapeutic approach for cardiac disease. This article is part of a Special Issue entitled: Heart Lipid Metabolism edited by G.D. Lopaschuk. PMID:27090938

  8. Function and subcellular localization of Gcn5, a histone acetyltransferase in Candida albicans.

    PubMed

    Chang, Peng; Fan, Xueyi; Chen, Jiangye

    2015-08-01

    Candida albicans is an opportunistic fungal pathogen commonly found in humans. It has the ability to switch reversibly between three growth forms: budding yeast, pseudohypha, and hypha. The transition between yeast and hyphal growth forms is critical for the pathogenesis of C. albicans. During the yeast-to-hypha morphologic transition, gene expression is regulated by transcriptional regulators including histone modifying complexes and chromatin remodeling complexes. We previously reported that Esa1, a catalytic subunit in the histone acetyltransferase complex NuA4, is essential for the hyphal development of C. albicans. In this study, we analyzed the functional roles of Gcn5, a catalytic subunit in the histone acetyltransferase complex SAGA, in C. albicans. Gcn5 is required for the invasive and filamentous growth of C. albicans. Deletion of GCN5 impaired hyphal elongation in sensing serum and attenuated the virulence of C. albicans in a mouse systemic infection model. The C. albicans gcn5/gcn5 mutant cells also exhibited sensitivity to cell wall stress. Functional analysis showed that the HAT domain and Bromodomain in Gcn5 play distinct roles in morphogenesis and cell wall stress response of C. albicans. Our results show that the conserved residue Glu188 is crucial for the Gcn5 HAT activity and for Gcn5 function during filamentous growth. In addition, the subcellular distribution of ectopically expressed GFP-Gcn5 correlates with the different growth states of C. albicans. In stationary phase, Gcn5 accumulated in the nucleus, while during vegetative growth it localized in the cytoplasm in a morpha-independent manner. Our results suggest that the nuclear localization of Gcn5 depends on the existence of its N-terminal NLS and HAT domains.

  9. Subcellular localization and trafficking of phytolongins (non-SNARE longins) in the plant secretory pathway

    PubMed Central

    de Marcos Lousa, Carine; Soubeyrand, Eric; Bolognese, Paolo; Wattelet-Boyer, Valerie; Bouyssou, Guillaume; Marais, Claireline; Boutté, Yohann; Filippini, Francesco; Moreau, Patrick

    2016-01-01

    SNARE proteins are central elements of the machinery involved in membrane fusion of eukaryotic cells. In animals and plants, SNAREs have diversified to sustain a variety of specific functions. In animals, R-SNARE proteins called brevins have diversified; in contrast, in plants, the R-SNARE proteins named longins have diversified. Recently, a new subfamily of four longins named ‘phytolongins’ (Phyl) was discovered. One intriguing aspect of Phyl proteins is the lack of the typical SNARE motif, which is replaced by another domain termed the ‘Phyl domain’. Phytolongins have a rather ubiquitous tissue expression in Arabidopsis but still await intracellular characterization. In this study, we found that the four phytolongins are distributed along the secretory pathway. While Phyl2.1 and Phyl2.2 are strictly located at the endoplasmic reticulum network, Phyl1.2 associates with the Golgi bodies, and Phyl1.1 locates mainly at the plasma membrane and partially in the Golgi bodies and post-Golgi compartments. Our results show that export of Phyl1.1 from the endoplasmic reticulum depends on the GTPase Sar1, the Sar1 guanine nucleotide exchange factor Sec12, and the SNAREs Sec22 and Memb11. In addition, we have identified the Y48F49 motif as being critical for the exit of Phyl1.1 from the endoplasmic reticulum. Our results provide the first characterization of the subcellular localization of the phytolongins, and we discuss their potential role in regulating the secretory pathway. PMID:26962210

  10. Subcellular localization of glycosidases and glycosyltransferases involved in the processing of N-linked oligosaccharides

    SciTech Connect

    Sturm, A.; Johnson, K.D.; Szumilo, T.; Elbein, A.D.; Chrispeels, M.J.

    1987-11-01

    Using isopycnic sucrose gradients, we have ascertained the subcellular location of several enzymes involved in the processing of the N-linked oligosaccharides of glycoproteins in developing cotyledons of the common bean, Phaseolus vulgaris. All are localized in the endoplasmic reticulum (ER) or Golgi complex as determined by co-sedimentation with the ER marker, NADH-cytochrome c reductase, or the Golgi marker, glucan synthase I. Glucosidase activity, which removes glucose residues from Glc/sub 3/Man/sub 9/(GlcNAc)/sub 2/, was found exclusively in the ER. All other processing enzymes, which act subsequent to the glucose trimming steps, are associates with Golgi. These include mannosidase I (removes 1-2 mannose residues from Man/sub 6-9/(GlcNAc)/sub 2/), mannosidase II (removes mannose residues from GlcNAcMan/sub 5/(GlcNAc)/sub 2/), and fucosyltransferase (transfers a fucose residue to the Asn-linked GlcNAc of appropriate glycans). The authors have previously reported the localization of two other glycan modifying enzymes (GlcNAc-transferase and xylosyltranferase activities) in the Golgi complex. Attempts at subfractionation of the Golgi fraction on shallow sucrose gradients yielded similar patterns of distribution for all the Golgi processing enzymes. Subfractionation on Percoll gradients resulted in two peaks of the Golgi marker enzyme inosine diphosphatase, whereas the glycan processing enzymes were all enriched in the peak of lower density. These results do not lend support to the hypothesis that N-linked oligosaccharide processing enzymes are associated with Golgi cisternae of different densities.

  11. Subcellular localization and displacement by diuretics of the peripheral benzodiazepine binding site (PBS) from rat kidney

    SciTech Connect

    Lukeman, S.; Fanestil, D.

    1986-03-05

    Although the PBS has been identified in many organs, its function and cellular location are speculative. Using rapid filtration, binding of (/sup 3/H)RO 5-4864 (*RO) (.75 nM) was assessed in four subcellular fractions (.3 mg/ml) derived from depapillated rat kidney by differential centrifugation: N (450g x 2 min), O (13,000 x 10), P (105,000 x 30), and S. The binding distribution was: N-18%, O-74%, P-6%, and S-2%. Marker enzyme analysis revealed that O was enriched in mitochondria (M), lysosomes (L), peroxisomes (P), and endoplasmic reticulum (ER), but not plasma membrane, and that N contained small amounts (10-15%) of markers for the above. Repeated washing of O removed ER enzymes but preserved *RO binding. O was further fractionated with centrifugation (57,000g x 4 hr) on a linear sucrose gradient (18-65%); *RO binding then comigrated with M but not P and L markers. Centrifugation of isolated M (5500 x 10 min) on another linear sucrose gradient (37-65%) gave low and high density bands, which contained 65% and 35% of *RO binding activity, resp. *RO binding in O was specific, saturable, reversible, and inhibited by diuretics. Inhibitors with the highest potency were indacrinone (K/sub d/ = 35 ..mu..M), hydrochlorothiazide (100 ..mu..M), and ethacrynic acid (325 ..mu..M). Low potency inhibitors (K/sub d/ greater than or equal to 1 mM) included amiloride, triamterene, furosemide, bumetanide, and ozolinone.

  12. Subcellular localization of multiple PREP2 isoforms is regulated by actin, tubulin, and nuclear export.

    PubMed

    Haller, Klaus; Rambaldi, Isabel; Daniels, Eugene; Featherstone, Mark

    2004-11-19

    The PREP, MEIS, and PBX families are mammalian members of the TALE (three amino acid loop extension) class of homeodomain-containing transcription factors. These factors have been implicated in cooperative DNA binding with the HOX class of homeoproteins, but PREP and MEIS interact with PBX in apparently non-HOX-dependent cooperative DNA binding as well. PREP, MEIS, and PBX have all been reported to reside in the cytoplasm in one or more tissues of the developing vertebrate embryo. In the case of PBX, cytoplasmic localization is due to the modulation of nuclear localization signals, nuclear export sequences, and interaction with a cytoplasmic anchoring factor, non-muscle myosin heavy chain II B. Here we report that murine PREP2 exists in multiple isoforms distinguished by interaction with affinity-purified antibodies raised to N- and C-terminal epitopes and by nuclear versus cytoplasmic localization. Alternative splicing gives rise to some of these PREP2 isoforms, including a 25-kDa variant lacking the C-terminal half of the protein and homeodomain and having the potential to act as dominant-negative. We further show that cytoplasmic localization is due to the concerted action of nuclear export, as evidenced by sensitivity to leptomycin B, and cytoplasmic retention by the actin and microtubule cytoskeletons. Cytoplasmic PREP2 colocalizes with both the actin and microtubule cytoskeletons and coimmunoprecipitates with actin and tubulin. Importantly, disruption of either cytoskeletal system redirects cytoplasmic PREP2 to the nucleus. We suggest that transcriptional regulation by PREP2 is modulated through the subcellular distribution of multiple isoforms and by interaction with two distinct cytoskeletal systems.

  13. Cadmium induced changes in subcellular glutathione contents within glandular trichomes of Cucurbita pepo L.

    PubMed

    Kolb, Dagmar; Müller, Maria; Zellnig, Günther; Zechmann, Bernd

    2010-07-01

    Plants cope with cadmium (Cd) stress by complexation with phytochelatins (Pc), metallothioneins and glutathione and sequestration within vacuoles. Especially glutathione was found to play a major role in Cd detoxification as Cd shows a high binding affinity towards thiols and as glutathione is a precursor for Pc synthesis. In the present study, we have used an immunohistochemical approach combined with computer-supported transmission electron microscopy in order to measure changes in the subcellular distribution of glutathione during Cd-stress in mesophyll cells and cells of different glandular trichomes (long and short stalked) of Cucurbita pepo L. subsp. pepo var. styriaca GREB: . Even though no ultrastructural alterations were observed in leaf and glandular trichome cells after the treatment of plants with 50 microM cadmium chloride (CdCl(2)) for 48 h, all cells showed a large decrease in glutathione contents. The strongest decrease was found in nuclei and the cytosol (up to 76%) in glandular trichomes which are considered as a major side of Cd accumulation in leaves. The ratio of glutathione between the cytosol and nuclei and the other cell compartments was strongly decreased only in glandular trichomes (more than 50%) indicating that glutathione in these two cell compartments is especially important for the detoxification of Cd in glandular trichomes. Additionally, these data indicate that large amounts of Cd are withdrawn from nuclei during Cd exposure. The present study gives a detailed insight into the compartment-specific importance of glutathione during Cd exposure in mesophyll cells and glandular trichomes of C. pepo L. plants.

  14. Aaknox1, a kn1-like homeobox gene in Acetabularia acetabulum, undergoes developmentally regulated subcellular localization.

    PubMed

    Serikawa, K A; Mandoli, D F

    1999-12-01

    Homeobox-containing genes play developmentally important roles in a wide variety of plants, animals and fungi. As a way of studying how development is controlled in the unicellular green macroalga Acetabularia acetabulum, we used degenerate PCR to clone a knotted1-like (kn1-like) homeobox gene, Aaknox1 (Acetabularia acetabulum kn1-like homeobox 1). Aaknorx1 is the first knotted1-like homeobox gene to be cloned from a non-vascular plant and shows strong conservation with kn1-like genes from the vascular plants (ca. 56% amino acid identity within the homeodomain). Sequencing of cDNA clones indicates that Aaknor1 possesses at least two distinct polyadenylation sites spaced ca. 600 bp apart. Southern analysis suggests that several other kn1-like homeobox genes exist in the Acetabularia genome. Northern analyses demonstrate that expression of Aaknox1 is developmentally regulated, with peak levels of expression during early reproductive phase. Northern analyses further demonstrate that Aaknox1 mRNA undergoes a change in its subcellular localization pattern during the progression from late vegetative to early reproductive phase. In late adult phase, Aaknox1 is distributed uniformly throughout the alga; in early reproductive phase, Aaknox1 is present in a gradient with the highest concentration of the mRNA at the base of the stalk, near the single nucleus. These data suggest that Aaknox1 may have a role during early reproductive development and that mRNA localization may be one mechanism by which A. acetabulum regulates gene expression posttranscriptionally.

  15. Analyzing subcellular structure with optical Fourier filtering based on Gabor filters

    NASA Astrophysics Data System (ADS)

    Boustany, Nada N.; Sierra, Heidy

    2013-02-01

    Label-free measurement of subcellular morphology can be used to track dynamically cellular function under various conditions and has important applications in cellular monitoring and in vitro cell assays. We show that optical filtering of scattered light by two-dimensional Gabor filters allows for direct and highly sensitive measurement of sample structure. The Gabor filters, which are defined by their spatial frequency, orientation and Gaussian envelope, can be used to track locally and in situ the characteristic size and orientation of structures within the sample. Our method consists of sequentially implementing a set of Gabor filters via a spatial light modulator placed in a conjugate Fourier plane during optical imaging and identifying the filters that yield maximum signal. Using this setup, we show that Gabor filtering of light forward-scattered by spheres yields an optical response which varies linearly with diameter between 100nm and 2000nm. The optical filtering sensitivity to changes in diameter is on the order of 20nm and can be achieved at low image resolution. We use numerical simulations to demonstrate that this linear response can be predicted from scatter theory and does not vary significantly with changes in refractive index ratio. By applying this Fourier filtering method in samples consisting of diatoms and cells, we generate false-color images of the object that encode at each pixel the size of the local structures within the object. The resolution of these encoded size maps in on the order of 0.36μm. The pixel histograms of these encoded images directly provide 20nm resolved "size spectra", depicting the size distribution of structures within the analyzed object. We use these size spectra to differentiate the morphology of apoptosis-competent and bax/bak null apoptosis-resistant cells during cell death. We also utilize the sensitivity of the Gabor filters to object orientation to track changes in organelle morphology, and detect mitochondrial

  16. Use of correspondence discriminant analysis to predict the subcellular location of bacterial proteins.

    PubMed

    Perrière, Guy; Thioulouse, Jean

    2003-02-01

    Correspondence discriminant analysis (CDA) is a multivariate statistical method derived from discriminant analysis which can be used on contingency tables. We have used CDA to separate Gram negative bacteria proteins according to their subcellular location. The high resolution of the discrimination obtained makes this method a good tool to predict subcellular location when this information is not known. The main advantage of this technique is its simplicity. Indeed, by computing two linear formulae on amino acid composition, it is possible to classify a protein into one of the three classes of subcellular location we have defined. The CDA itself can be computed with the ADE-4 software package that can be downloaded, as well as the data set used in this study, from the Pôle Bio-Informatique Lyonnais (PBIL) server at http://pbil.univ-lyon1.fr.

  17. Dual Function of NRP1 in Axon Guidance and Subcellular Target Recognition in Cerebellum.

    PubMed

    Telley, Ludovic; Cadilhac, Christelle; Cioni, Jean-Michel; Saywell, Veronique; Jahannault-Talignani, Céline; Huettl, Rosa E; Sarrailh-Faivre, Catherine; Dayer, Alexandre; Huber, Andrea B; Ango, Fabrice

    2016-09-21

    Subcellular target recognition in the CNS is the culmination of a multiple-step program including axon guidance, target recognition, and synaptogenesis. In cerebellum, basket cells (BCs) innervate the soma and axon initial segment (AIS) of Purkinje cells (PCs) to form the pinceau synapse, but the underlying mechanisms remain incompletely understood. Here, we demonstrate that neuropilin-1 (NRP1), a Semaphorin receptor expressed in BCs, controls both axonal guidance and subcellular target recognition. We show that loss of Semaphorin 3A function or specific deletion of NRP1 in BCs alters the stereotyped organization of BC axon and impairs pinceau synapse formation. Further, we identified NRP1 as a trans-synaptic binding partner of the cell adhesion molecule neurofascin-186 (NF186) expressed in the PC AIS during pinceau synapse formation. These findings identify a dual function of NRP1 in both axon guidance and subcellular target recognition in the construction of GABAergic circuitry. PMID:27618676

  18. A protocol for the subcellular fractionation of Saccharomyces cerevisiae using nitrogen cavitation and density gradient centrifugation

    PubMed Central

    Wang, Yuchong; Lilley, Kathryn S; Oliver, Stephen G

    2014-01-01

    Most protocols for yeast subcellular fractionation involve the use of mechanical shear forces to lyse the spheroplasts produced by the enzymatic digestion of the Saccharomyces cerevisiae cell wall. These mechanical homogenization procedures often involve the manual use of devices such as the Dounce homogenizer, and so are very operator-dependent and, in consequence, lack reproducibility. Here, we report a highly reproducible method of homogenizing yeast cells based on nitrogen cavitation. This has been optimized to allow efficient release of subcellular compartments that show a high degree of integrity. The protocol remains effective and reproducible across a range of sample volumes and buffer environments. The subsequent separation method, which employs both sucrose and iodixanol density gradients, has been developed to resolve the major membrane-bound compartments of S. cerevisiae. We present an integrated protocol that is fast, facile, robust and efficient and that will enable ‘omics’ studies of the subcellular compartments of S. cerevisiae and other yeasts. PMID:24510422

  19. Human Protein Subcellular Localization with Integrated Source and Multi-label Ensemble Classifier.

    PubMed

    Guo, Xiaotong; Liu, Fulin; Ju, Ying; Wang, Zhen; Wang, Chunyu

    2016-01-01

    Predicting protein subcellular location is necessary for understanding cell function. Several machine learning methods have been developed for computational prediction of primary protein sequences because wet experiments are costly and time consuming. However, two problems still exist in state-of-the-art methods. First, several proteins appear in different subcellular structures simultaneously, whereas current methods only predict one protein sequence in one subcellular structure. Second, most software tools are trained with obsolete data and the latest new databases are missed. We proposed a novel multi-label classification algorithm to solve the first problem and integrated several latest databases to improve prediction performance. Experiments proved the effectiveness of the proposed method. The present study would facilitate research on cellular proteomics. PMID:27323846

  20. Triggering cell detachment from patterned electrode arrays by programmed subcellular release

    PubMed Central

    Wildt, Bridget; Wirtz, Denis; Searson, Peter C

    2010-01-01

    Programmed subcellular release is an in vitro technique for the quantitative study of cell detachment. The dynamics of cell contraction are measured by releasing cells from surfaces to which they are attached with spatial and temporal control. Release of subcellular regions of cells is achieved by plating cells on an electrode array created by standard microfabrication methods. The electrodes are then biochemically functionalized with an arginine-glycine-aspartic acid (RGD)-terminated thiol. Application of a voltage pulse results in electrochemical desorption of the RGD-terminated thiols, triggering cell detachment. This method allows for the study of the full cascade of events from detachment to subsequent subcellular reorganization. Fabrication of the electrode arrays may take 1–2 d. Preparation for experiments, including surface functionalization and cell plating, can be completed in 10 h. A series of cell release experiments on one device may last several hours. PMID:20595956

  1. Human Protein Subcellular Localization with Integrated Source and Multi-label Ensemble Classifier

    NASA Astrophysics Data System (ADS)

    Guo, Xiaotong; Liu, Fulin; Ju, Ying; Wang, Zhen; Wang, Chunyu

    2016-06-01

    Predicting protein subcellular location is necessary for understanding cell function. Several machine learning methods have been developed for computational prediction of primary protein sequences because wet experiments are costly and time consuming. However, two problems still exist in state-of-the-art methods. First, several proteins appear in different subcellular structures simultaneously, whereas current methods only predict one protein sequence in one subcellular structure. Second, most software tools are trained with obsolete data and the latest new databases are missed. We proposed a novel multi-label classification algorithm to solve the first problem and integrated several latest databases to improve prediction performance. Experiments proved the effectiveness of the proposed method. The present study would facilitate research on cellular proteomics.

  2. Triggering cell detachment from patterned electrode arrays by programmed subcellular release.

    PubMed

    Wildt, Bridget; Wirtz, Denis; Searson, Peter C

    2010-07-01

    Programmed subcellular release is an in vitro technique for the quantitative study of cell detachment. The dynamics of cell contraction are measured by releasing cells from surfaces to which they are attached with spatial and temporal control. Release of subcellular regions of cells is achieved by plating cells on an electrode array created by standard microfabrication methods. The electrodes are then biochemically functionalized with an arginine-glycine-aspartic acid (RGD)-terminated thiol. Application of a voltage pulse results in electrochemical desorption of the RGD-terminated thiols, triggering cell detachment. This method allows for the study of the full cascade of events from detachment to subsequent subcellular reorganization. Fabrication of the electrode arrays may take 1-2 d. Preparation for experiments, including surface functionalization and cell plating, can be completed in 10 h. A series of cell release experiments on one device may last several hours. PMID:20595956

  3. Identifying the singleplex and multiplex proteins based on transductive learning for protein subcellular localization prediction.

    PubMed

    Cao, Junzhe; Liu, Wenqi; He, Jianjun; Gu, Hong

    2013-07-01

    A new method is proposed to identify whether a query protein is singleplex or multiplex for improving the quality of protein subcellular localization prediction. Based on the transductive learning technique, this approach utilizes the information from the both query proteins and known proteins to estimate the subcellular location number of every query protein so that the singleplex and multiplex proteins can be recognized and distinguished. Each query protein is then dealt with by a targeted single-label or multi-label predictor to achieve a high-accuracy prediction result. We assess the performance of the proposed approach by applying it to three groups of protein sequences datasets. Simulation experiments show that the proposed approach can effectively identify the singleplex and multiplex proteins. Through a comparison, the reliably of this method for enhancing the power of predicting protein subcellular localization can also be verified.

  4. Echinococcus granulosus fatty acid binding proteins subcellular localization.

    PubMed

    Alvite, Gabriela; Esteves, Adriana

    2016-05-01

    Two fatty acid binding proteins, EgFABP1 and EgFABP2, were isolated from the parasitic platyhelminth Echinococcus granulosus. These proteins bind fatty acids and have particular relevance in flatworms since de novo fatty acids synthesis is absent. Therefore platyhelminthes depend on the capture and intracellular distribution of host's lipids and fatty acid binding proteins could participate in lipid distribution. To elucidate EgFABP's roles, we investigated their intracellular distribution in the larval stage by a proteomic approach. Our results demonstrated the presence of EgFABP1 isoforms in cytosolic, nuclear, mitochondrial and microsomal fractions, suggesting that these molecules could be involved in several cellular processes.

  5. Immunogold labeling reveals subcellular localisation of silica nanoparticles in a human blood-brain barrier model

    NASA Astrophysics Data System (ADS)

    Ye, Dong; Anguissola, Sergio; O'Neill, Tiina; Dawson, Kenneth A.

    2015-05-01

    Subcellular location of nanoparticles has been widely investigated with fluorescence microscopy, via fluorescently labeled antibodies to visualise target antigens in cells. However, fluorescence microscopy, such as confocal or live cell imaging, has generally limited 3D spatial resolution. Conventional electron microscopy can be useful in bridging resolution gap, but still not ideal in resolving subcellular organelle identities. Using the pre-embedding immunogold electron microscopic imaging, we performed accurate examination of the intracellular trafficking and gathered further evidence of transport mechanisms of silica nanoparticles across a human in vitro blood-brain barrier model. Our approach can effectively immunolocalise a variety of intracellular compartments and provide new insights into the uptake and subcellular transport of nanoparticles.Subcellular location of nanoparticles has been widely investigated with fluorescence microscopy, via fluorescently labeled antibodies to visualise target antigens in cells. However, fluorescence microscopy, such as confocal or live cell imaging, has generally limited 3D spatial resolution. Conventional electron microscopy can be useful in bridging resolution gap, but still not ideal in resolving subcellular organelle identities. Using the pre-embedding immunogold electron microscopic imaging, we performed accurate examination of the intracellular trafficking and gathered further evidence of transport mechanisms of silica nanoparticles across a human in vitro blood-brain barrier model. Our approach can effectively immunolocalise a variety of intracellular compartments and provide new insights into the uptake and subcellular transport of nanoparticles. Electronic supplementary information (ESI) available: Nanoparticle characterisation data, preservation of cellular structures, staining controls, optimisation of size amplification via the silver enhancement, and more imaging results from anti-clathrin and anti-caveolin 1

  6. Multilabel learning via random label selection for protein subcellular multilocations prediction.

    PubMed

    Wang, Xiao; Li, Guo-Zheng

    2013-01-01

    Prediction of protein subcellular localization is an important but challenging problem, particularly when proteins may simultaneously exist at, or move between, two or more different subcellular location sites. Most of the existing protein subcellular localization methods are only used to deal with the single-location proteins. In the past few years, only a few methods have been proposed to tackle proteins with multiple locations. However, they only adopt a simple strategy, that is, transforming the multilocation proteins to multiple proteins with single location, which does not take correlations among different subcellular locations into account. In this paper, a novel method named random label selection (RALS) (multilabel learning via RALS), which extends the simple binary relevance (BR) method, is proposed to learn from multilocation proteins in an effective and efficient way. RALS does not explicitly find the correlations among labels, but rather implicitly attempts to learn the label correlations from data by augmenting original feature space with randomly selected labels as its additional input features. Through the fivefold cross-validation test on a benchmark data set, we demonstrate our proposed method with consideration of label correlations obviously outperforms the baseline BR method without consideration of label correlations, indicating correlations among different subcellular locations really exist and contribute to improvement of prediction performance. Experimental results on two benchmark data sets also show that our proposed methods achieve significantly higher performance than some other state-of-the-art methods in predicting subcellular multilocations of proteins. The prediction web server is available at >http://levis.tongji.edu.cn:8080/bioinfo/MLPred-Euk/ for the public usage. PMID:23929867

  7. Subcellular localization and expression of multiple tomato γ-aminobutyrate transaminases that utilize both pyruvate and glyoxylate

    PubMed Central

    Clark, Shawn M.; Di Leo, Rosa; Van Cauwenberghe, Owen R.; Mullen, Robert T.; Shelp, Barry J.

    2009-01-01

    γ-Aminobutyric acid transaminase (GABA-T) catalyses the breakdown of GABA to succinic semialdehyde. In this report, three GABA-T isoforms were identified in the tomato (Solanum lycopersicum L.) plant. The deduced amino acid sequences of the three isoforms are highly similar over most of their coding regions with the exception of their N-terminal regions. Transient expression of the individual full-length GABA-T isoforms fused to the green fluorescent protein in tobacco suspension-cultured cells revealed their distinct subcellular localizations to the mitochondrion, plastid or cytosol, and that the specific targeting of the mitochondrion- and plastid-localized isoforms is mediated by their predicted N-terminal presequences. Removal of the N-terminal targeting presequences from the mitochondrion and plastid GABA-T isoforms yielded good recovery of the soluble recombinant proteins in Escherichia coli when they were co-expressed with the GroES/EL molecular chaperone complex. Activity assays indicated that all three recombinant isoforms possess both pyruvate- and glyoxylate-dependent GABA-T activities, although the mitochondrial enzyme has a specific activity that is significantly higher than that of its plastid and cytosolic counterparts. Finally, differential expression patterns of the three GABA-T isoforms in reproductive tissues, but not vegetative tissues, suggest unique roles for each enzyme in developmental processes. Overall, these findings, together with recent information about rice and pepper GABA-Ts, indicate that the subcellular distribution of GABA-T in the plant kingdom is highly variable. PMID:19470656

  8. Binding of mescaline with subcellular fractions upon incubation of brain cortex slices with [14C] mescaline.

    PubMed

    Datta, R K; Antopol, W; Ghosh, J J

    1977-01-01

    Incubation of brain cortex slices in the presence of glucose resulted in the permeation of about 65% of [14C] mescaline into slices. Of this, about one-third radioactivity was bound with nuclei, mitochondria, microsomes, and ribosomes. Dialysis of subcellular fractions did not markedly reduce the amounts of radioactivity bound to the fractions. The permeation into slices and the binding of mescaline to subcellular fractions were fairly time-dependent, but were inhibited by the presence of potassium cyanide, or by the absence of glucose and by heating to 80 degrees C for 1 min.

  9. Identifying subcellular protein localization with fluorescent protein fusions after transient expression in onion epidermal cells.

    PubMed

    Nebenführ, Andreas

    2014-01-01

    Most biochemical functions of plant cells are carried out by proteins which act at very specific places within these cells, for example, within different organelles. Identifying the subcellular localization of proteins is therefore a useful tool to narrow down the possible functions that a novel or unknown protein may carry out. The discovery of genetically encoded fluorescent markers has made it possible to tag specific proteins and visualize them in vivo under a variety of conditions. This chapter describes a simple method to use transient expression of such fluorescently tagged proteins in onion epidermal cells to determine their subcellular localization relative to known markers.

  10. Calculation of the relative metastabilities of proteins in subcellular compartments of Saccharomyces cerevisiae

    PubMed Central

    Dick, Jeffrey M

    2009-01-01

    Background Protein subcellular localization and differences in oxidation state between subcellular compartments are two well-studied features of the the cellular organization of S. cerevisiae (yeast). Theories about the origin of subcellular organization are assisted by computational models that can integrate data from observations of compositional and chemical properties of the system. Presentation and implications of the hypothesis I adopt the hypothesis that the state of yeast subcellular organization is in a local energy minimum. This hypothesis implies that equilibrium thermodynamic models can yield predictions about the interdependence between populations of proteins and their subcellular chemical environments. Testing the hypothesis Three types of tests are proposed. First, there should be correlations between modeled and observed oxidation states for different compartments. Second, there should be a correspondence between the energy requirements of protein formation and the order the appearance of organelles during cellular development. Third, there should be correlations between the predicted and observed relative abundances of interacting proteins within compartments. Results The relative metastability fields of subcellular homologs of glutaredoxin and thioredoxin indicate a trend from less to more oxidizing as mitochondrion – cytoplasm – nucleus. Representing the overall amino acid compositions of proteins in 23 different compartments each with a single reference model protein suggests that the formation reactions for proteins in the vacuole (in relatively oxidizing conditions), ER and early Golgi (in relatively reducing conditions) are relatively highly favored, while that for the microtubule is the most costly. The relative abundances of model proteins for each compartment inferred from experimental data were found in some cases to correlate with the predicted abundances, and both positive and negative correlations were found for some assemblages

  11. Morphological and chemical studies of pathological human and mice brain at the subcellular level: correlation between light, electron, and nanosims microscopies.

    PubMed

    Quintana, Carmen; Wu, Ting-Di; Delatour, Benoit; Dhenain, Marc; Guerquin-Kern, Jean Luc; Croisy, Alain

    2007-04-01

    Neurodegenerative diseases induce morphological and chemical alterations in well-characterized regions of the brain. Understanding their pathological processes requires the use of methods that assess both morphological and chemical alterations in the tissues. In the past, microprobe approaches such as scanning electron microscopy combined with an X-ray spectrometer, Proton induced X-ray emission, secondary ion mass spectrometry (SIMS), and laser microprobe mass analysis have been used for the study of pathological human brain with limited success. At the present, new SIMS instruments have been developed, such as the NanoSIMS-50 ion microprobe, that allow the simultaneous identification of five elements with high sensitivity, at subcellular spatial resolution (about 50-100 nm with the Cs(+) source and about 150-200 nm with O(-) source). Working in scanning mode, 2D distribution of five elements (elemental maps) can be obtained, thus providing their exact colocalization. The analysis can be performed on semithin or ultrathin embedded sections. The possibility of using transmission electron microscopy and SIMS on the same ultrathin sections allows the correlation between structural and analytical observations at subcellular and ultrastructural level to be established. Our observations on pathological brain areas allow us to establish that the NanoSIMS-50 ion microprobe is a highly useful instrument for the imaging of the morphological and chemical alterations that take place in these brain areas. In the human brain our results put forward the subcellular distribution of iron-ferritin-hemosiderin in the hippocampus of Alzheimer disease patients. In the thalamus of transgenic mice, our results have shown the presence of Ca-Fe mineralized amyloid deposits.

  12. Effects of hypolipidemic agents on lipid synthesis in subcellular fractions from Tetrahymena pyriformis.

    PubMed

    Pan, H Y; Chou, S C; Conklin, K A

    1976-01-01

    Lipid synthesizing systems have been prepared from subcellular fractions of Tetrahymena pyriformis, GL. These fractions, the mitochondrial fraction, the microsomal fraction and the soluble cell fraction, have been characterized as to cofactors and cations required for optimal lipid synthesis. The effects of hypolipidemic agents on lipid synthesis by all fractions are presented.

  13. PSI: A Comprehensive and Integrative Approach for Accurate Plant Subcellular Localization Prediction

    PubMed Central

    Chen, Ming

    2013-01-01

    Predicting the subcellular localization of proteins conquers the major drawbacks of high-throughput localization experiments that are costly and time-consuming. However, current subcellular localization predictors are limited in scope and accuracy. In particular, most predictors perform well on certain locations or with certain data sets while poorly on others. Here, we present PSI, a novel high accuracy web server for plant subcellular localization prediction. PSI derives the wisdom of multiple specialized predictors via a joint-approach of group decision making strategy and machine learning methods to give an integrated best result. The overall accuracy obtained (up to 93.4%) was higher than best individual (CELLO) by ∼10.7%. The precision of each predicable subcellular location (more than 80%) far exceeds that of the individual predictors. It can also deal with multi-localization proteins. PSI is expected to be a powerful tool in protein location engineering as well as in plant sciences, while the strategy employed could be applied to other integrative problems. A user-friendly web server, PSI, has been developed for free access at http://bis.zju.edu.cn/psi/. PMID:24194827

  14. Methods of biomedical optical imaging: from subcellular structures to tissues and organs

    NASA Astrophysics Data System (ADS)

    Turchin, I. V.

    2016-05-01

    Optical bioimaging methods have a wide range of applications in the life sciences, most notably including the molecular resolution study of subcellular structures, small animal molecular imaging, and structural and functional clinical diagnostics of tissue layers and organs. We review fluorescent microscopy, fluorescent macroscopy, optical coherence tomography, optoacoustic tomography, and optical diffuse spectroscopy and tomography from the standpoint of physical fundamentals, applications, and progress.

  15. CellWhere: graphical display of interaction networks organized on subcellular localizations.

    PubMed

    Zhu, Lu; Malatras, Apostolos; Thorley, Matthew; Aghoghogbe, Idonnya; Mer, Arvind; Duguez, Stéphanie; Butler-Browne, Gillian; Voit, Thomas; Duddy, William

    2015-07-01

    Given a query list of genes or proteins, CellWhere produces an interactive graphical display that mimics the structure of a cell, showing the local interaction network organized into subcellular locations. This user-friendly tool helps in the formulation of mechanistic hypotheses by enabling the experimental biologist to explore simultaneously two elements of functional context: (i) protein subcellular localization and (ii) protein-protein interactions or gene functional associations. Subcellular localization terms are obtained from public sources (the Gene Ontology and UniProt-together containing several thousand such terms) then mapped onto a smaller number of CellWhere localizations. These localizations include all major cell compartments, but the user may modify the mapping as desired. Protein-protein interaction listings, and their associated evidence strength scores, are obtained from the Mentha interactome server, or power-users may upload a pre-made network produced using some other interactomics tool. The Cytoscape.js JavaScript library is used in producing the graphical display. Importantly, for a protein that has been observed at multiple subcellular locations, users may prioritize the visual display of locations that are of special relevance to their research domain. CellWhere is at http://cellwhere-myology.rhcloud.com. PMID:25883154

  16. Development of a Charged Particle Microbeam for Targeted and Single Particle Subcellular Irradiation

    SciTech Connect

    Yanch, Jacquelyn C.

    2004-03-12

    The development of a charged particle microbeam for single particle, subcellular irradiations at the Massachusetts Institute of Technology Laboratory for Accelerator Beam Applications (MIT LABA) was initiated under this NEER aeard. The Microbeam apparatus makes use of a pre-existing electrostatic accelerator with a horizontal beam tube.

  17. Substrates with patterned extracellular matrix and subcellular stiffness gradients reveal local biomechanical responses.

    PubMed

    Tseng, Peter; Di Carlo, Dino

    2014-02-26

    A substrate fabrication process is developed to pattern both the extracellular matrix (ECM) and rigidity at sub-cellular spatial resolution. When growing cells on these substrates, it is found that cells respond locally in their cytoskeleton assembly. The presented method allows unique insight into the biological interpretation of mechanical signals, whereas photolithography-based fabrication is amenable to integration with complex microfabricated substructures.

  18. CellWhere: graphical display of interaction networks organized on subcellular localizations

    PubMed Central

    Zhu, Lu; Malatras, Apostolos; Thorley, Matthew; Aghoghogbe, Idonnya; Mer, Arvind; Duguez, Stéphanie; Butler-Browne, Gillian; Voit, Thomas; Duddy, William

    2015-01-01

    Given a query list of genes or proteins, CellWhere produces an interactive graphical display that mimics the structure of a cell, showing the local interaction network organized into subcellular locations. This user-friendly tool helps in the formulation of mechanistic hypotheses by enabling the experimental biologist to explore simultaneously two elements of functional context: (i) protein subcellular localization and (ii) protein–protein interactions or gene functional associations. Subcellular localization terms are obtained from public sources (the Gene Ontology and UniProt—together containing several thousand such terms) then mapped onto a smaller number of CellWhere localizations. These localizations include all major cell compartments, but the user may modify the mapping as desired. Protein–protein interaction listings, and their associated evidence strength scores, are obtained from the Mentha interactome server, or power-users may upload a pre-made network produced using some other interactomics tool. The Cytoscape.js JavaScript library is used in producing the graphical display. Importantly, for a protein that has been observed at multiple subcellular locations, users may prioritize the visual display of locations that are of special relevance to their research domain. CellWhere is at http://cellwhere-myology.rhcloud.com. PMID:25883154

  19. Subcellular Localization of Galloylated Catechins in Tea Plants [Camellia sinensis (L.) O. Kuntze] Assessed via Immunohistochemistry.

    PubMed

    Xu, Huanhuan; Wang, Ya; Chen, Yana; Zhang, Pan; Zhao, Yi; Huang, Yewei; Wang, Xuanjun; Sheng, Jun

    2016-01-01

    Galloylated catechins, as the main secondary metabolites in the tea plant, including (-)-epigallocatechin-3-gallate and (-)-epicatechin-3-gallate, comprise approximately three-quarters of all the tea plant catechins and have stronger effects than non-galloylated catechins, both on the product quality in tea processing and the pharmacological efficacy to human beings. The subcellular localization of galloylated catechins has been the primary focus of studies that assess biosynthesis and physiological functions. Classical histochemical localization staining reagents can not specifically detect galloylated catechins; thus, their subcellular localization remains controversial. In the present study, we generated a monoclonal antibody (mAb) against galloylated catechins, which can be used for the subcellular localization of galloylated catechins in the tea plant by immunohistochemistry. Direct ELISA and ForteBio Octet Red 96 System assay indicated the mAb could recognize the galloylated catechins with high specificities and affinities. In addition, tea bud was ascertained as the optimal tissue for freezing microtomic sections for immunohistochemistry. What's more, the high quality mAbs which exhibited excellent binding capability to galloylated catechins were utilized for the visualization of them via immunohistochemistry. Our findings demonstrated that vacuoles were the primary sites of localization of galloylated catechins at the subcellular level. PMID:27303422

  20. Subcellular Localization of Galloylated Catechins in Tea Plants [Camellia sinensis (L.) O. Kuntze] Assessed via Immunohistochemistry

    PubMed Central

    Xu, Huanhuan; Wang, Ya; Chen, Yana; Zhang, Pan; Zhao, Yi; Huang, Yewei; Wang, Xuanjun; Sheng, Jun

    2016-01-01

    Galloylated catechins, as the main secondary metabolites in the tea plant, including (-)-epigallocatechin-3-gallate and (-)-epicatechin-3-gallate, comprise approximately three-quarters of all the tea plant catechins and have stronger effects than non-galloylated catechins, both on the product quality in tea processing and the pharmacological efficacy to human beings. The subcellular localization of galloylated catechins has been the primary focus of studies that assess biosynthesis and physiological functions. Classical histochemical localization staining reagents can not specifically detect galloylated catechins; thus, their subcellular localization remains controversial. In the present study, we generated a monoclonal antibody (mAb) against galloylated catechins, which can be used for the subcellular localization of galloylated catechins in the tea plant by immunohistochemistry. Direct ELISA and ForteBio Octet Red 96 System assay indicated the mAb could recognize the galloylated catechins with high specificities and affinities. In addition, tea bud was ascertained as the optimal tissue for freezing microtomic sections for immunohistochemistry. What’s more, the high quality mAbs which exhibited excellent binding capability to galloylated catechins were utilized for the visualization of them via immunohistochemistry. Our findings demonstrated that vacuoles were the primary sites of localization of galloylated catechins at the subcellular level. PMID:27303422

  1. Research and teaching with the AFTOL SBD: an informatics resource for fungal subcellular and biochemical data.

    PubMed

    Arun Kumar, T K; Blackwell, Meredith; Letcher, Peter M; Roberson, Robert W; McLaughlin, David J

    2013-12-01

    The Structural and Biochemical Database (SBD), developed as part of the US NSF-funded Assembling the Fungal Tree of Life (AFTOL), is a multi-investigator project. It is a major resource to present and manage morphological and biochemical information on Fungi and serves as a phyloinformatics tool for the scientific community. It also is an important resource for teaching mycology. The database, available at http://aftol.umn.edu, includes new and previously published subcellular data on Fungi, supplemented with images and literature links. Datasets automatically combined in NEXUS format from the site permit independent and combined (with molecular data) phylogenetic analyses. Character lists, a major feature of the site, serve as primary reference documents of subcellular and biochemical characters that distinguish taxa across the major fungal lineages. The character lists illustrated with images and drawings are informative for evolutionary and developmental biologists as well as educators, students and the public. Fungal Subcellular Ontology (FSO), developed as part of this effort is a primary initiative to provide a controlled vocabulary describing subcellular structures unique to Fungi. FSO establishes a full complement of terms that provide an operating ontological framework for the database. Examples are provided for using the database for teaching.

  2. Sub-cellular force microscopy in single normal and cancer cells

    SciTech Connect

    Babahosseini, H.; Carmichael, B.; Strobl, J.S.; Mahmoodi, S.N.; Agah, M.

    2015-08-07

    This work investigates the biomechanical properties of sub-cellular structures of breast cells using atomic force microscopy (AFM). The cells are modeled as a triple-layered structure where the Generalized Maxwell model is applied to experimental data from AFM stress-relaxation tests to extract the elastic modulus, the apparent viscosity, and the relaxation time of sub-cellular structures. The triple-layered modeling results allow for determination and comparison of the biomechanical properties of the three major sub-cellular structures between normal and cancerous cells: the up plasma membrane/actin cortex, the mid cytoplasm/nucleus, and the low nuclear/integrin sub-domains. The results reveal that the sub-domains become stiffer and significantly more viscous with depth, regardless of cell type. In addition, there is a decreasing trend in the average elastic modulus and apparent viscosity of the all corresponding sub-cellular structures from normal to cancerous cells, which becomes most remarkable in the deeper sub-domain. The presented modeling in this work constitutes a unique AFM-based experimental framework to study the biomechanics of sub-cellular structures. - Highlights: • The cells are modeled as a triple-layered structure using Generalized Maxwell model. • The sub-domains include membrane/cortex, cytoplasm/nucleus, and nuclear/integrin. • Biomechanics of corresponding sub-domains are compared among normal and cancer cells. • Viscoelasticity of sub-domains show a decreasing trend from normal to cancer cells. • The decreasing trend becomes most significant in the deeper sub-domain.

  3. Cobalamin derivatives in subcellular fractions of porcine ileal enterocytes.

    PubMed

    Toyoshima, M; Gräsbeck, R

    1987-05-01

    The distribution of cobalamin derivatives in porcine enterocytes was studied using thin-layer chromatography and bioautography. Compared with the original homogenate, adenosyl-cobalamin (Ado-Cbl) was concentrated 5-10 times in the mitochondrial fraction. Cyanocobalamin was found in the lysosomal fraction, but was not detected in the mitochondria. Methyl-cobalamin (Me-Cbl) was found only in the sap fraction and could not be detected in the total homogenate. Hydroxocobalamin was the main form of Cbl in the lysosomal fraction. These results provide indirect evidence that in enterocytes methylmalonyl-CoA mutase, the coenzyme of which is Ado-Cbl, is located in the mitochondrial fraction and that methionine synthetase, the coenzyme of which is Me-Cbl, is located in the sap fraction. These enzymes thus appear to be distributed in the same way as in liver cells. PMID:3589492

  4. Automated Learning of Subcellular Variation among Punctate Protein Patterns and a Generative Model of Their Relation to Microtubules

    PubMed Central

    Johnson, Gregory R.; Li, Jieyue; Shariff, Aabid; Rohde, Gustavo K.; Murphy, Robert F.

    2015-01-01

    Characterizing the spatial distribution of proteins directly from microscopy images is a difficult problem with numerous applications in cell biology (e.g. identifying motor-related proteins) and clinical research (e.g. identification of cancer biomarkers). Here we describe the design of a system that provides automated analysis of punctate protein patterns in microscope images, including quantification of their relationships to microtubules. We constructed the system using confocal immunofluorescence microscopy images from the Human Protein Atlas project for 11 punctate proteins in three cultured cell lines. These proteins have previously been characterized as being primarily located in punctate structures, but their images had all been annotated by visual examination as being simply “vesicular”. We were able to show that these patterns could be distinguished from each other with high accuracy, and we were able to assign to one of these subclasses hundreds of proteins whose subcellular localization had not previously been well defined. In addition to providing these novel annotations, we built a generative approach to modeling of punctate distributions that captures the essential characteristics of the distinct patterns. Such models are expected to be valuable for representing and summarizing each pattern and for constructing systems biology simulations of cell behaviors. PMID:26624011

  5. Altered subcellular localization of ornithine decarboxylase in Alzheimer's disease brain

    SciTech Connect

    Nilsson, Tatjana . E-mail: Tatjana.Nilsson@ki.se; Bogdanovic, Nenad; Volkman, Inga; Winblad, Bengt; Folkesson, Ronnie; Benedikz, Eirikur

    2006-06-02

    The amyloid precursor protein can through ligand-mimicking induce expression of ornithine decarboxylase (ODC), the initial and rate-limiting enzyme in polyamine biosynthesis. We report here the regional distribution and cellular localization of ODC immunoreactivity in Alzheimer's disease (AD) brains. In frontal cortex and hippocampus of control cases, the most pronounced ODC immunoreactivity was found in the nucleus. In possible and definite AD the immunoreactivity had shifted to the cytoplasm. In cerebellum of control cases, ODC staining was found in a small portion of Purkinje cells, mostly in the nucleus. In AD, both possible and definite, the number of stained Purkinje cells increased significantly and immunoreactivity was shifted to the cytoplasm, even though it was still prominent in the nucleus. In conclusion, our study reveals an early shift of the ODC immunoreactivity in AD from the nuclear compartment towards the cytoplasm.

  6. Subcellular transcriptome alterations in a cell culture model of spinal muscular atrophy point to widespread defects in axonal growth and presynaptic differentiation

    PubMed Central

    Saal, Lena; Briese, Michael; Kneitz, Susanne; Glinka, Michael

    2014-01-01

    Neuronal function critically depends on coordinated subcellular distribution of mRNAs. Disturbed mRNA processing and axonal transport has been found in spinal muscular atrophy and could be causative for dysfunction and degeneration of motoneurons. Despite the advances made in characterizing the transport mechanisms of several axonal mRNAs, an unbiased approach to identify the axonal repertoire of mRNAs in healthy and degenerating motoneurons has been lacking. Here we used compartmentalized microfluidic chambers to investigate the somatodendritic and axonal mRNA content of cultured motoneurons by microarray analysis. In axons, transcripts related to protein synthesis and energy production were enriched relative to the somatodendritic compartment. Knockdown of Smn, the protein deficient in spinal muscular atrophy, produced a large number of transcript alterations in both compartments. Transcripts related to immune functions, including MHC class I genes, and with roles in RNA splicing were up-regulated in the somatodendritic compartment. On the axonal side, transcripts associated with axon growth and synaptic activity were down-regulated. These alterations provide evidence that subcellular localization of transcripts with axonal functions as well as regulation of specific transcripts with nonautonomous functions is disturbed in Smn-deficient motoneurons, most likely contributing to the pathophysiology of spinal muscular atrophy. PMID:25246652

  7. Protein synthesis rates in rat brain regions and subcellular fractions during aging

    SciTech Connect

    Avola, R.; Condorelli, D.F.; Ragusa, N.; Renis, M.; Alberghina, M.; Giuffrida Stella, A.M.; Lajtha, A.

    1988-04-01

    In vivo protein synthesis rates in various brain regions (cerebral cortex, cerebellum, hippocampus, hypothalamus, and striatum) of 4-, 12-, and 24-month-old rats were examined after injection of a flooding dose of labeled valine. The incorporation of labeled valine into proteins of mitochondrial, microsomal, and cytosolic fractions from cerebral cortex and cerebellum was also measured. At all ages examined, the incorporation rate was 0.5% per hour in cerebral cortex, cerebellum, hippocampus, and hypothalamus and 0.4% per hour in striatum. Of the subcellular fractions examined, the microsomal proteins were synthesized at the highest rate, followed by cytosolic and mitochondrial proteins. The results obtained indicate that the average synthesis rate of proteins in the various brain regions and subcellular fractions examined is fairly constant and is not significantly altered in the 4 to 24-month period of life of rats.

  8. Direct Imaging of Single Cells and Tissue at Subcellular Spatial Resolution Using Transmission Geometry MALDI MS

    PubMed Central

    Zavalin, Andre; Todd, Erik M.; Rawhouser, Patrick D.; Yang, Junhai; Norris, Jeremy L.; Caprioli, Richard M.

    2012-01-01

    The need of cellular and sub-cellular spatial resolution in LDI / MALDI Imaging Mass Spectrometry (IMS) necessitates micron and sub-micron laser spot sizes at biologically relevant sensitivities, introducing significant challenges for MS technology. To this end we have developed a transmission geometry vacuum ion source that allows the laser beam to irradiate the back side of the sample. This arrangement obviates the mechanical / ion optic complications in the source by completely separating the optical lens and ion optic structures. We have experimentally demonstrated the viability of transmission geometry MALDI MS for imaging biological tissues and cells with sub-cellular spatial resolution. Furthermore, we demonstrate that in conjunction with new sample preparation protocols, the sensitivity of this instrument is sufficient to obtain molecular images at sub-micron spatial resolution. PMID:23147833

  9. Sub-Cellular Resolution Delivery of a Cytokine via Precisely Manipulated Nanowires

    PubMed Central

    Fan, Donglei; Yin, Zhizhong; Cheong, Raymond; Zhu, Frank Q.; Cammarata, Robert C.; Chien, C.L.; Levchenko, Andre

    2010-01-01

    Precise delivery of molecular doses of biologically active chemicals to a pre-specified single cell among many, or a specific sub-cellular location, is still a largely unmet challenge hampering our understanding of cell biology. Overcoming this could allow unprecedented levels of cell manipulation and targeted intervention. Here, we show that gold nanowires conjugated with cytokine, such as tumour necrosis factor-alpha (TNFα), can be transported along any prescribed trajectory or orientation using electrophoretic and dielectrophoretic forces to a specific location with subcellular resolution. The nanowire, 6 μm long and 300 nm in diameter, delivered the cytokine and activated canonical nuclear factor-kappaB signaling in a single cell. Combined computational modeling and experimentation indicated that cell stimulation was highly localized to the nanowire vicinity. This targeted delivery method has profound implications for controlling signaling events on the single cell level. PMID:20543835

  10. Reversible Optogenetic Control of Subcellular Protein Localization in a Live Vertebrate Embryo

    PubMed Central

    Buckley, Clare E.; Moore, Rachel E.; Reade, Anna; Goldberg, Anna R.; Weiner, Orion D.; Clarke, Jonathan D.W.

    2016-01-01

    Summary We demonstrate the utility of the phytochrome system to rapidly and reversibly recruit proteins to specific subcellular regions within specific cells in a living vertebrate embryo. Light-induced heterodimerization using the phytochrome system has previously been used as a powerful tool to dissect signaling pathways for single cells in culture but has not previously been used to reversibly manipulate the precise subcellular location of proteins in multicellular organisms. Here we report the experimental conditions necessary to use this system to manipulate proteins in vivo. As proof of principle, we demonstrate that we can manipulate the localization of the apical polarity protein Pard3 with high temporal and spatial precision in both the neural tube and the embryo’s enveloping layer epithelium. Our optimizations of optogenetic component expression and chromophore purification and delivery should significantly lower the barrier for establishing this powerful optogenetic system in other multicellular organisms. PMID:26766447

  11. High-yield isolation and subcellular proteomic characterization of nuclear and subnuclear structures from trypanosomes.

    PubMed

    DeGrasse, Jeffrey A; Chait, Brian T; Field, Mark C; Rout, Michael P

    2008-01-01

    The vast evolutionary distance between the Opisthokonta (animals and yeast) and the excavata (a major group of protists, including Giardia and Trypanosoma) presents a significant challenge to in silico functional genomics and ortholog identification. Subcellular proteomic identification of the constituents of highly enriched organelles can alleviate this problem by both providing localization evidence and yielding a manageably sized proteome for detailed in silico functional assignment. We describe a method for the high-yield isolation of nuclei from the kinetoplastid Trypanosoma brucei. We also describe the subsequent purification of subnuclear compartments, including the nuclear envelope and nucleolus. Finally, using several proteomic strategies, we survey the proteome of a subcellular structure or organelle, using the nuclear pore complex as an example.

  12. The dynamic subcellular localization of ERK: mechanisms of translocation and role in various organelles.

    PubMed

    Wainstein, Ehud; Seger, Rony

    2016-04-01

    The dynamic subcellular localization of ERK in resting and stimulated cells plays an important role in its regulation. In resting cells, ERK localizes in the cytoplasm, and upon stimulation, it translocates to its target substrates and organelles. ERK signaling initiated from different places in resting cells has distinct outcomes. In this review, we summarize the mechanisms of ERK1/2 translocation to the nucleus and mitochondria, and of ERK1c to the Golgi. We also show that ERK1/2 translocation to the nucleus is a useful anti cancer target. Unraveling the complex subcellular localization of ERK and its dynamic changes upon stimulation provides a better understanding of the regulation of ERK signaling and may result in the development of new strategies to combat ERK-related diseases. PMID:26827288

  13. Apparatus and method for measuring single cell and sub-cellular photosynthetic efficiency

    DOEpatents

    Davis, Ryan Wesley; Singh, Seema; Wu, Huawen

    2013-07-09

    Devices for measuring single cell changes in photosynthetic efficiency in algal aquaculture are disclosed that include a combination of modulated LED trans-illumination of different intensities with synchronized through objective laser illumination and confocal detection. Synchronization and intensity modulation of a dual illumination scheme were provided using a custom microcontroller for a laser beam block and constant current LED driver. Therefore, single whole cell photosynthetic efficiency, and subcellular (diffraction limited) photosynthetic efficiency measurement modes are permitted. Wide field rapid light scanning actinic illumination is provided for both by an intensity modulated 470 nm LED. For the whole cell photosynthetic efficiency measurement, the same LED provides saturating pulses for generating photosynthetic induction curves. For the subcellular photosynthetic efficiency measurement, a switched through objective 488 nm laser provides saturating pulses for generating photosynthetic induction curves. A second near IR LED is employed to generate dark adapted states in the system under study.

  14. SherLoc2: a high-accuracy hybrid method for predicting subcellular localization of proteins.

    PubMed

    Briesemeister, Sebastian; Blum, Torsten; Brady, Scott; Lam, Yin; Kohlbacher, Oliver; Shatkay, Hagit

    2009-11-01

    SherLoc2 is a comprehensive high-accuracy subcellular localization prediction system. It is applicable to animal, fungal, and plant proteins and covers all main eukaryotic subcellular locations. SherLoc2 integrates several sequence-based features as well as text-based features. In addition, we incorporate phylogenetic profiles and Gene Ontology (GO) terms derived from the protein sequence to considerably improve the prediction performance. SherLoc2 achieves an overall classification accuracy of up to 93% in 5-fold cross-validation. A novel feature, DiaLoc, allows users to manually provide their current background knowledge by describing a protein in a short abstract which is then used to improve the prediction. SherLoc2 is available both as a free Web service and as a stand-alone version at http://www-bs.informatik.uni-tuebingen.de/Services/SherLoc2.

  15. 'Unite and conquer': enhanced prediction of protein subcellular localization by integrating multiple specialized tools

    PubMed Central

    Shen, Yao Qing; Burger, Gertraud

    2007-01-01

    Background Knowing the subcellular location of proteins provides clues to their function as well as the interconnectivity of biological processes. Dozens of tools are available for predicting protein location in the eukaryotic cell. Each tool performs well on certain data sets, but their predictions often disagree for a given protein. Since the individual tools each have particular strengths, we set out to integrate them in a way that optimally exploits their potential. The method we present here is applicable to various subcellular locations, but tailored for predicting whether or not a protein is localized in mitochondria. Knowledge of the mitochondrial proteome is relevant to understanding the role of this organelle in global cellular processes. Results In order to develop a method for enhanced prediction of subcellular localization, we integrated the outputs of available localization prediction tools by several strategies, and tested the performance of each strategy with known mitochondrial proteins. The accuracy obtained (up to 92%) surpasses by far the individual tools. The method of integration proved crucial to the performance. For the prediction of mitochondrion-located proteins, integration via a two-layer decision tree clearly outperforms simpler methods, as it allows emphasis of biologically relevant features such as the mitochondrial targeting peptide and transmembrane domains. Conclusion We developed an approach that enhances the prediction accuracy of mitochondrial proteins by uniting the strength of specialized tools. The combination of machine-learning based integration with biological expert knowledge leads to improved performance. This approach also alleviates the conundrum of how to choose between conflicting predictions. Our approach is easy to implement, and applicable to predicting subcellular locations other than mitochondria, as well as other biological features. For a trial of our approach, we provide a webservice for mitochondrial protein

  16. Cdc2/cyclin B1 regulates centrosomal Nlp proteolysis and subcellular localization.

    PubMed

    Zhao, Xuelian; Jin, Shunqian; Song, Yongmei; Zhan, Qimin

    2010-11-01

    The formation of proper mitotic spindles is required for appropriate chromosome segregation during cell division. Aberrant spindle formation often causes aneuploidy and results in tumorigenesis. However, the underlying mechanism of regulating spindle formation and chromosome separation remains to be further defined. Centrosomal Nlp (ninein-like protein) is a recently characterized BRCA1-regulated centrosomal protein and plays an important role in centrosome maturation and spindle formation. In this study, we show that Nlp can be phosphorylated by cell cycle protein kinase Cdc2/cyclin B1. The phosphorylation sites of Nlp are mapped at Ser185 and Ser589. Interestingly, the Cdc2/cyclin B1 phosphorylation site Ser185 of Nlp is required for its recognition by PLK1, which enable Nlp depart from centrosomes to allow the establishment of a mitotic scaffold at the onset of mitosis . PLK1 fails to dissociate the Nlp mutant lacking Ser185 from centrosome, suggesting that Cdc2/cyclin B1 might serve as a primary kinase of PLK1 in regulating Nlp subcellular localization. However, the phosphorylation at the site Ser589 by Cdc2/cyclin B1 plays an important role in Nlp protein stability probably due to its effect on protein degradation. Furthermore, we show that deregulated expression or subcellular localization of Nlp lead to multinuclei in cells, indicating that scheduled levels of Nlp and proper subcellular localization of Nlp are critical for successful completion of normal cell mitosis, These findings demonstrate that Cdc2/cyclin B1 is a key regulator in maintaining appropriate degradation and subcellular localization of Nlp, providing novel insights into understanding on the role of Cdc2/cyclin B1 in mitotic progression.

  17. [Subcellular localization of isozymes of NAD-dependent malate dehydrogenase in sugar beet Beta vulgaris L].

    PubMed

    Iudina, R S; Levites, E V

    2008-12-01

    Subcellular localization of isozymes of NAD-dependent malate dehydrogenase (MDH) in sugar beet was studied. Isozymes ss and 11 controlled by loci Mdh2 and Mdh3, respectively, were shown to locate in mitochondria, whereas isozyme pp controlled by locus Mdh1, in microbodies. All examined samples lack hybrid MDH isozymes, which could testify to the interaction between products of nonallelic Mdh genes. This can be explained by the localization of nonallelic isozymes in various compartments of the cell and organelles.

  18. Probing subcellular organic hydroperoxide formation via a genetically encoded ratiometric and reversible fluorescent indicator.

    PubMed

    Zhao, Boxuan Simen; Zhang, Gong; Zeng, Shizhe; He, Chuan; Chen, Peng R

    2013-12-01

    A ratiometric and reversible organic hydroperoxide (OHP) sensor, rOHSer, was developed with high sensitivity and selectivity for subcellular OHP visualization. Through targeting rOHSer to the nucleus, we demonstrated that high levels of D-glucose cause elevated OHP production in this compartment. Further utilization of rOHSer probe may allow more elucidation of unique roles of OHPs in diverse biological processes.

  19. Metabolism of polybrominated diphenyl ethers and tetrabromobisphenol A by fish liver subcellular fractions in vitro.

    PubMed

    Shen, Mengnan; Cheng, Jie; Wu, Ruohan; Zhang, Shenghu; Mao, Liang; Gao, Shixiang

    2012-06-15

    Polybrominated diphenyl ethers (PBDEs) and tetrabromobisphenol A (TBBPA) are two major flame retardants that accumulate in fish tissues and are potentially toxic. Their debrominated and oxidated derivatives were also reported in fish tissues although the sources of theses derivatives were unidentified. Our study was to determine whether PBDEs and TBBPA could be metabolized by fish liver subcellular fractions in vitro and to identify what types of metabolites were formed. Liver microsomes and S9 fractions of crucian carp (Carassius auratus) were exposed to 4,4'-dibromodiphenyl ether (BDE 15), 2,2',4,4'-tetrabromodiphenyl ether (BDE 47) or TBBPA solutions for 4h. Exposure of liver subcellular fractions to BDE 15 resulted in the formation of bromophenol and two monohydroxylated dibromodiphenyl ether metabolites. Neither in microsomes nor in S9 studies has revealed the presence of hydroxylated metabolites with BDE 47 exposure which indicated that the oxidation reactions in vitro were hindered by the increased number of bromine substituents on the PBDEs. TBBPA underwent an oxidative cleavage near the central carbon of the molecule, which led to the production of 2,6-dibromo-4-isopropyl-phenol and three unidentified metabolites. Another metabolite of TBBPA characterized as a hexa-brominated compound with three aromatic rings was also found in the liver subcellular fractions. These results suggest that the biotransformation of BDE 15 and TBBPA in fish liver is mediated by cytochrome P450 (CYP450) enzymes, as revealed by the formation of hydroxylated metabolites and oxidative bond cleavage products. Moreover, further studies on the identification of specific CYP450 isozymes involved in the biotransformation revealed that CYP1A was the major enzyme responsible for the biotransformation of BDE 15 and TBBPA in fish liver subcellular fractions and CYP3A4 also played a major role in metabolism of TBBPA. PMID:22417763

  20. Limited Efficiency of Drug Delivery to Specific Intracellular Organelles Using Subcellularly "Targeted" Drug Delivery Systems.

    PubMed

    Maity, Amit Ranjan; Stepensky, David

    2016-01-01

    Many drugs have been designed to act on intracellular targets and to affect intracellular processes inside target cells. For the desired effects to be exerted, these drugs should permeate target cells and reach specific intracellular organelles. This subcellular drug targeting approach has been proposed for enhancement of accumulation of these drugs in target organelles and improved efficiency. This approach is based on drug encapsulation in drug delivery systems (DDSs) and/or their decoration with specific targeting moieties that are intended to enhance the drug/DDS accumulation in the intracellular organelle of interest. During recent years, there has been a constant increase in interest in DDSs targeted to specific intracellular organelles, and many different approaches have been proposed for attaining efficient drug delivery to specific organelles of interest. However, it appears that in many studies insufficient efforts have been devoted to quantitative analysis of the major formulation parameters of the DDSs disposition (efficiency of DDS endocytosis and endosomal escape, intracellular trafficking, and efficiency of DDS delivery to the target organelle) and of the resulting pharmacological effects. Thus, in many cases, claims regarding efficient delivery of drug/DDS to a specific organelle and efficient subcellular targeting appear to be exaggerated. On the basis of the available experimental data, it appears that drugs/DDS decoration with specific targeting residues can affect their intracellular fate and result in preferential drug accumulation within an organelle of interest. However, it is not clear whether these approaches will be efficient in in vivo settings and be translated into preclinical and clinical applications. Studies that quantitatively assess the mechanisms, barriers, and efficiencies of subcellular drug delivery and of the associated toxic effects are required to determine the therapeutic potential of subcellular DDS targeting.

  1. SLocX: Predicting Subcellular Localization of Arabidopsis Proteins Leveraging Gene Expression Data

    PubMed Central

    Ryngajllo, Malgorzata; Childs, Liam; Lohse, Marc; Giorgi, Federico M.; Lude, Anja; Selbig, Joachim; Usadel, Björn

    2011-01-01

    Despite the growing volume of experimentally validated knowledge about the subcellular localization of plant proteins, a well performing in silico prediction tool is still a necessity. Existing tools, which employ information derived from protein sequence alone, offer limited accuracy and/or rely on full sequence availability. We explored whether gene expression profiling data can be harnessed to enhance prediction performance. To achieve this, we trained several support vector machines to predict the subcellular localization of Arabidopsis thaliana proteins using sequence derived information, expression behavior, or a combination of these data and compared their predictive performance through a cross-validation test. We show that gene expression carries information about the subcellular localization not available in sequence information, yielding dramatic benefits for plastid localization prediction, and some notable improvements for other compartments such as the mitochondrion, the Golgi, and the plasma membrane. Based on these results, we constructed a novel subcellular localization prediction engine, SLocX, combining gene expression profiling data with protein sequence-based information. We then validated the results of this engine using an independent test set of annotated proteins and a transient expression of GFP fusion proteins. Here, we present the prediction framework and a website of predicted localizations for Arabidopsis. The relatively good accuracy of our prediction engine, even in cases where only partial protein sequence is available (e.g., in sequences lacking the N-terminal region), offers a promising opportunity for similar application to non-sequenced or poorly annotated plant species. Although the prediction scope of our method is currently limited by the availability of expression information on the ATH1 array, we believe that the advances in measuring gene expression technology will make our method applicable for all Arabidopsis proteins

  2. Subcellular RNA profiling links splicing and nuclear DICER1 to alternative cleavage and polyadenylation

    PubMed Central

    Neve, Jonathan; Burger, Kaspar; Li, Wencheng; Hoque, Mainul; Patel, Radhika; Tian, Bin; Gullerova, Monika; Furger, Andre

    2016-01-01

    Alternative cleavage and polyadenylation (APA) plays a crucial role in the regulation of gene expression across eukaryotes. Although APA is extensively studied, its regulation within cellular compartments and its physiological impact remains largely enigmatic. Here, we used a rigorous subcellular fractionation approach to compare APA profiles of cytoplasmic and nuclear RNA fractions from human cell lines. This approach allowed us to extract APA isoforms that are subjected to differential regulation and provided us with a platform to interrogate the molecular regulatory pathways that shape APA profiles in different subcellular locations. Here, we show that APA isoforms with shorter 3′ UTRs tend to be overrepresented in the cytoplasm and appear to be cell-type–specific events. Nuclear retention of longer APA isoforms occurs and is partly a result of incomplete splicing contributing to the observed cytoplasmic bias of transcripts with shorter 3′ UTRs. We demonstrate that the endoribonuclease III, DICER1, contributes to the establishment of subcellular APA profiles not only by expected cytoplasmic miRNA-mediated destabilization of APA mRNA isoforms, but also by affecting polyadenylation site choice. PMID:26546131

  3. Birth and rapid subcellular adaptation of a hominoid-specific CDC14 protein.

    PubMed

    Rosso, Lia; Marques, Ana Claudia; Weier, Manuela; Lambert, Nelle; Lambot, Marie-Alexandra; Vanderhaeghen, Pierre; Kaessmann, Henrik

    2008-06-10

    Gene duplication was prevalent during hominoid evolution, yet little is known about the functional fate of new ape gene copies. We characterized the CDC14B cell cycle gene and the functional evolution of its hominoid-specific daughter gene, CDC14Bretro. We found that CDC14B encodes four different splice isoforms that show different subcellular localizations (nucleus or microtubule-associated) and functional properties. A microtubular CDC14B variant spawned CDC14Bretro through retroposition in the hominoid ancestor 18-25 million years ago (Mya). CDC14Bretro evolved brain-/testis-specific expression after the duplication event and experienced a short period of intense positive selection in the African ape ancestor 7-12 Mya. Using resurrected ancestral protein variants, we demonstrate that by virtue of amino acid substitutions in distinct protein regions during this time, the subcellular localization of CDC14Bretro progressively shifted from the association with microtubules (stabilizing them) to an association with the endoplasmic reticulum. CDC14Bretro evolution represents a paradigm example of rapid, selectively driven subcellular relocalization, thus revealing a novel mode for the emergence of new gene function.

  4. Subcellular localisations of the CPTI collection of YFP-tagged proteins in Drosophila embryos

    PubMed Central

    Lye, Claire M.; Naylor, Huw W.; Sanson, Bénédicte

    2014-01-01

    A key challenge in the post-genomic area is to identify the function of the genes discovered, with many still uncharacterised in all metazoans. A first step is transcription pattern characterisation, for which we now have near whole-genome coverage in Drosophila. However, we have much more limited information about the expression and subcellular localisation of the corresponding proteins. The Cambridge Protein Trap Consortium generated, via piggyBac transposition, over 600 novel YFP-trap proteins tagging just under 400 Drosophila loci. Here, we characterise the subcellular localisations and expression patterns of these insertions, called the CPTI lines, in Drosophila embryos. We have systematically analysed subcellular localisations at cellularisation (stage 5) and recorded expression patterns at stage 5, at mid-embryogenesis (stage 11) and at late embryogenesis (stages 15-17). At stage 5, 31% of the nuclear lines (41) and 26% of the cytoplasmic lines (67) show discrete localisations that provide clues on the function of the protein and markers for organelles or regions, including nucleoli, the nuclear envelope, nuclear speckles, centrosomes, mitochondria, the endoplasmic reticulum, Golgi, lysosomes and peroxisomes. We characterised the membranous/cortical lines (102) throughout stage 5 to 10 during epithelial morphogenesis, documenting their apico-basal position and identifying those secreted in the extracellular space. We identified the tricellular vertices as a specialized membrane domain marked by the integral membrane protein Sidekick. Finally, we categorised the localisation of the membranous/cortical proteins during cytokinesis. PMID:25294944

  5. Using Fluorescent Protein Fusions to Study Protein Subcellular Localization and Dynamics in Plant Cells.

    PubMed

    Cui, Yong; Gao, Caiji; Zhao, Qiong; Jiang, Liwen

    2016-01-01

    Studies of protein subcellular localization and dynamics are helpful in understanding the cellular functions of proteins in an organism. In the past decade, the use of green fluorescent protein (GFP) as a fusion tag has dramatically extended our knowledge in this field. Transient expression and stable transformation of GFP-tagged proteins have been wildly used to study protein localization in vivo in different systems. Although GFP-based tags provide a fast and convenient way to characterize protein properties in living cells, several reports have demonstrated that GFP fusions might not accurately reflect the localization of the native protein as GFP tags may alter the protein properties. To facilitate proper usage of GFP tags in plant cell biology study, we describe detailed protocols to identify possible inhibitory effects of fluorescent tags on protein subcellular localization and to determine if a fluorescently tagged protein is localized to the correct subcellular compartment. Using Arabidopsis Endomembrane protein 12 (EMP12) as an example, we first show the possible inhibitory effect of GFP tags on proper protein localization and then describe the immunofluorescence labeling method to verify the correct localization of GFP fusion proteins. Next, a method is presented using the ImageJ program with the Pearson-Spearman correlation (PSC) colocalization plug-in for statistical quantification of colocalization ratios of two fluorophores. Finally we provide a detailed method for protein dynamics studies using spinning disk confocal microscopy in Arabidopsis cells. PMID:27515077

  6. A Comprehensive Subcellular Proteomic Survey of Salmonella Grown under Phagosome-Mimicking versus Standard Laboratory Conditions

    SciTech Connect

    Brown, Roslyn N.; Sanford, James A.; Park, Jea H.; Deatherage, Brooke L.; Champion, Boyd L.; Smith, Richard D.; Heffron, Fred; Adkins, Joshua N.

    2012-06-01

    Towards developing a systems-level pathobiological understanding of Salmonella enterica, we performed a subcellular proteomic analysis of this pathogen grown under standard laboratory and infection-mimicking conditions in vitro. Analysis of proteins from cytoplasmic, inner membrane, periplasmic, and outer membrane fractions yielded coverage of over 30% of the theoretical proteome. Confident subcellular location could be assigned to over 1000 proteins, with good agreement between experimentally observed location and predicted/known protein properties. Comparison of protein location under the different environmental conditions provided insight into dynamic protein localization and possible moonlighting (multiple function) activities. Notable examples of dynamic localization were the response regulators of two-component regulatory systems (e.g., ArcB, PhoQ). The DNA-binding protein Dps that is generally regarded as cytoplasmic was significantly enriched in the outer membrane for all growth conditions examined, suggestive of moonlighting activities. These observations imply the existence of unknown transport mechanisms and novel functions for a subset of Salmonella proteins. Overall, this work provides a catalog of experimentally verified subcellular protein location for Salmonella and a framework for further investigations using computational modeling.

  7. Subcellular location for the formation of the retinol/retinol-binding protein complex in rat liver

    SciTech Connect

    Crumbaugh, L.M.; Green, E.L.; Smith, J.E.

    1986-03-01

    Retinol complexes with retinol-binding protein (RBP) within the hepatocyte, however the subcellular location where complex formation occurs has not previously been identified. A model similar to that of lipoproteins formation has been hypothesized. The authors have identified the initial site of retinol/RBP complex formation. Furthermore, the authors have elucidated the progression of the complex through the subcellular organelles. Intravenous injections of /sup 3/H-retinol suspended in Tween 40 were administered to vitamin A depleted rats. After intervals of 2, 3, 4, 5, 10, 15, 20, and 30 minutes the rat livers were removed and fractions enriched in rough and smooth microsomes and Golgi apparatus were prepared. Extracts of these subcellular fractions were chromatographed on Sephadex G-100. Simultaneous elution of /sup 3/H-retinol and immunoreactive RBP indicated the presence of the complex. The retinol/RBP complex was observed in rough microsomes 2 minute after the injection of /sup 3/H-retinal. The complex appeared subsequently in smooth microsomes and Golgi apparatus. The complex was first detected serum around 10 minutes after injection. Based on the data, they believe that the retinol/RBP complex formation occurs in rough microsomes.

  8. Origin and evolution of metabolic sub-cellular compartmentalization in eukaryotes

    PubMed Central

    Gabaldón, Toni; Pittis, Alexandros A.

    2015-01-01

    A high level of subcellular compartmentalization is a hallmark of eukaryotic cells. This intricate internal organization was present already in the common ancestor of all extant eukaryotes, and the determination of the origins and early evolution of the different organelles remains largely elusive. Organellar proteomes are determined through regulated pathways that target proteins produced in the cytosol to their final subcellular destinations. This internal sorting of proteins can vary across different physiological conditions, cell types and lineages. Evolutionary retargeting – the alteration of a subcellular localization of a protein in the course of evolution – has been rampant in eukaryotes and involves any possible combination of organelles. This fact adds another layer of difficulty to the reconstruction of the origins and evolution of organelles. In this review we discuss current themes in relation to the origin and evolution of organellar proteomes. Throughout the text, a special focus is set on the evolution of mitochondrial and peroxisomal proteomes, which are two organelles for which extensive proteomic and evolutionary studies have been performed. PMID:25869000

  9. Zymogen Activation and Subcellular Activity of Subtilisin Kexin Isozyme 1/Site 1 Protease*

    PubMed Central

    da Palma, Joel Ramos; Burri, Dominique Julien; Oppliger, Joël; Salamina, Marco; Cendron, Laura; de Laureto, Patrizia Polverino; Seidah, Nabil Georges; Kunz, Stefan; Pasquato, Antonella

    2014-01-01

    The proprotein convertase subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P) plays crucial roles in cellular homeostatic functions and is hijacked by pathogenic viruses for the processing of their envelope glycoproteins. Zymogen activation of SKI-1/S1P involves sequential autocatalytic processing of its N-terminal prodomain at sites B′/B followed by the herein newly identified C′/C sites. We found that SKI-1/S1P autoprocessing results in intermediates whose catalytic domain remains associated with prodomain fragments of different lengths. In contrast to other zymogen proprotein convertases, all incompletely matured intermediates of SKI-1/S1P showed full catalytic activity toward cellular substrates, whereas optimal cleavage of viral glycoproteins depended on B′/B processing. Incompletely matured forms of SKI-1/S1P further process cellular and viral substrates in distinct subcellular compartments. Using a cell-based sensor for SKI-1/S1P activity, we found that 9 amino acid residues at the cleavage site (P1–P8) and P1′ are necessary and sufficient to define the subcellular location of processing and to determine to what extent processing of a substrate depends on SKI-1/S1P maturation. In sum, our study reveals novel and unexpected features of SKI-1/S1P zymogen activation and subcellular specificity of activity toward cellular and pathogen-derived substrates. PMID:25378398

  10. Subcellular RNA profiling links splicing and nuclear DICER1 to alternative cleavage and polyadenylation.

    PubMed

    Neve, Jonathan; Burger, Kaspar; Li, Wencheng; Hoque, Mainul; Patel, Radhika; Tian, Bin; Gullerova, Monika; Furger, Andre

    2016-01-01

    Alternative cleavage and polyadenylation (APA) plays a crucial role in the regulation of gene expression across eukaryotes. Although APA is extensively studied, its regulation within cellular compartments and its physiological impact remains largely enigmatic. Here, we used a rigorous subcellular fractionation approach to compare APA profiles of cytoplasmic and nuclear RNA fractions from human cell lines. This approach allowed us to extract APA isoforms that are subjected to differential regulation and provided us with a platform to interrogate the molecular regulatory pathways that shape APA profiles in different subcellular locations. Here, we show that APA isoforms with shorter 3' UTRs tend to be overrepresented in the cytoplasm and appear to be cell-type-specific events. Nuclear retention of longer APA isoforms occurs and is partly a result of incomplete splicing contributing to the observed cytoplasmic bias of transcripts with shorter 3' UTRs. We demonstrate that the endoribonuclease III, DICER1, contributes to the establishment of subcellular APA profiles not only by expected cytoplasmic miRNA-mediated destabilization of APA mRNA isoforms, but also by affecting polyadenylation site choice. PMID:26546131

  11. Plant species and organ influence the structure and subcellular localization of recombinant glycoproteins.

    PubMed

    Arcalis, Elsa; Stadlmann, Johannes; Rademacher, Thomas; Marcel, Sylvain; Sack, Markus; Altmann, Friedrich; Stoger, Eva

    2013-09-01

    Many plant-based systems have been developed as bioreactors to produce recombinant proteins. The choice of system for large-scale production depends on its intrinsic expression efficiency and its propensity for scale-up, post-harvest storage and downstream processing. Factors that must be considered include the anticipated production scale, the value and intended use of the product, the geographical production area, the proximity of processing facilities, intellectual property, safety and economics. It is also necessary to consider whether different species and organs affect the subcellular trafficking, structure and qualitative properties of recombinant proteins. In this article we discuss the subcellular localization and N-glycosylation of two commercially-relevant recombinant glycoproteins (Aspergillus niger phytase and anti-HIV antibody 2G12) produced in different plant species and organs. We augment existing data with novel results based on the expression of the same recombinant proteins in Arabidopsis and tobacco seeds, focusing on similarities and subtle differences in N-glycosylation that often reflect the subcellular trafficking route and final destination, as well as differences generated by unique enzyme activities in different species and tissues. We discuss the potential consequences of such modifications on the stability and activity of the recombinant glycoproteins.

  12. Subcellular localization and complements of GABA(A) and GABA(C) receptors on bullfrog retinal bipolar cells.

    PubMed

    Du, J L; Yang, X L

    2000-08-01

    gamma-Aminobutyric acid (GABA) receptors on retinal bipolar cells (BCs) are highly relevant to spatial and temporal integration of visual signals in the outer and inner retina. In the present work, subcellular localization and complements of GABA(A) and GABA(C) receptors on BCs were investigated by whole cell recordings and local drug application via multi-barreled puff pipettes in the bullfrog retinal slice preparation. Four types of the BCs (types 1-4) were identified morphologically by injection of Lucifer yellow. According to the ramification levels of the axon terminals and the responses of these cells to glutamate (or kainate) applied at their dendrites, types 1 and 2 of BCs were supposed to be OFF type, whereas types 3 and 4 of BCs might be ON type. Bicuculline (BIC), a GABA(A) receptor antagonist, and imidazole-4-acetic acid (I4AA), a GABA(C) receptor antagonist, were used to distinguish GABA receptor-mediated responses. In all BCs tested, not only the axon terminals but also the dendrites showed high GABA sensitivity mediated by both GABA(A) and GABA(C) receptors. Subcellular localization and complements of GABA(A) and GABA(C) receptors at the dendrites and axon terminals were highly related to the dichotomy of OFF and ON BCs. In the case of OFF BCs, GABA(A) receptors were rather evenly distributed at the dendrites and axon terminals, but GABA(C) receptors were predominantly expressed at the axon terminals. Moreover, the relative contribution of GABA(C) receptors to the axon terminals was prevalent over that of GABA(A) receptors, while the situation was reversed at the dendrites. In the case of ON BCs, GABA(A) and GABA(C) receptors both preferred to be expressed at the axon terminals; relative contributions of these two GABA receptor subtypes to both the sites were comparable, while GABA(C) receptors were much less expressed than GABA(A) receptors. GABA(A), but not GABA(C) receptors, were expressed clusteringly at axons of a population of BCs. In a

  13. Subcellular Raman Microspectroscopy Imaging of Nucleic Acids and Tryptophan for Distinction of Normal Human Skin Cells and Tumorigenic Keratinocytes.

    PubMed

    Piredda, Paola; Berning, Manuel; Boukamp, Petra; Volkmer, Andreas

    2015-07-01

    At present, tumor diagnostic imaging is commonly based on hematoxylin and eosin or immunohistochemical staining of biopsies, which requires tissue excision, fixation, and staining with exogenous marker molecules. Here, we report on label-free tumor imaging using confocal spontaneous Raman scattering microspectroscopy, which exploits the intrinsic vibrational contrast of endogenous biomolecular species. We present a chemically specific and quantitative approach to monitoring normal human skin cells (keratinocytes and fibroblasts) as well as the human HaCaT in vitro skin carcinogenesis model and the tumor-derived MET in vivo skin cancer progression model. Mapping the amplitudes of two spectrally well isolated Raman bands at 752 and 785 cm(-1) allowed for direct visualization of the distributions representative of tryptophan-rich proteins and nucleic acids, respectively, with subcellular spatial resolution. Using these Raman markers, it was feasible to discriminate between normal human epidermal keratinocytes (NHEK) and dermal fibroblasts (NHDF) and to confine all tumorigenic cells from both the NHEK and NHDF. First evidence for the successful application of the proposed intracellular nucleic acid and tryptophan Raman signatures for skin cancer diagnosis was further demonstrated in an organotypic cutaneous squamous cell carcinomas model, allowing for the identification of tumor cells and their surrounding stroma in the tissue context.

  14. Outside or inside: role of the subcellular localization of DP4-like enzymes for substrate conversion and inhibitor effects.

    PubMed

    Bank, Ute; Heimburg, Anke; Wohlfarth, Astrid; Koch, Gudrun; Nordhoff, Karsten; Julius, Heiko; Helmuth, Martin; Breyer, Doreen; Reinhold, Dirk; Täger, Michael; Ansorge, Siegfried

    2011-03-01

    The discovery of the DP4-related enzymes DP8 and DP9 raised controversial discussion regarding the physiological and pathophysiological function of distinct members of the DP4 family. Particularly with regard to their potential relevance in regulating immune functions, it is of interest to know which role the subcellular distribution of the enzymes play. Synthetic substrates as well as low molecular weight inhibitors are widely used as tools, but little is yet known regarding their features in cell experiments, such as their plasma membrane penetration capacity. The fluorogenic substrates Gly-Pro-AMC or (Ala-Pro)₂-R110 predominantly detect plasma membrane-bound activities of viable cells (less than 0.1% of fluorochromes R110 or AMC inside viable cells after 1 h incubation). Additionally, the selective and non-selective DP8/9 inhibitors allo-Ile-isoindoline and Lys[Z(NO₂)]-pyrrolidide were found to be incapable of passing the plasma membrane easily. This suggests that previously reported cellular effects are not due to inhibition of the cytosolic enzymes DP8 or DP9. Moreover, our enzymatic studies with viable cells provided evidence that DP8 and/or DP9 are also present on the surface of immune cells under certain circumstances and could gain relevance particularly in the absence of DP4 expression. In summary, in cells which do express DP4 on the surface, this archetypical member of the DP4 family is the most relevant peptidase in the regulation of cellular functions.

  15. Phosphatidate Kinase, a Novel Enzyme in Phospholipid Metabolism (Purification, Subcellular Localization, and Occurrence in the Plant Kingdom).

    PubMed Central

    Wissing, J. B.; Behrbohm, H.

    1993-01-01

    Microsomal membranes from suspension-cultured Catharanthus roseus cells possess an enzymic activity that catalyzes the ATP-dependent phosphorylation of phosphatidic acid (PA) to form diacylglycerol pyrophosphate (H. Behrbohm, J.B. Wissing [1993] FEBS Lett 315: 95-99). This enzyme activity, PA kinase, was purified and characterized. Plasma membranes, obtained from C. roseus microsomes by aqueous two-phase partitioning, were extracted, and PA kinase was purified 3200-fold by applying different chromatographic steps that resulted in a specific activity of about 10 [mu]mol min-1 mg-1. Sodium dodecyl sulfate-gel electrophoresis of the fractions obtained from the final chromatographic step revealed a 39-kD protein that correlated with the enzyme activity; PA kinase activity could be eluted from this protein band. Subcellular localization, investigated with C. roseus cells, showed that the activity was confined to membrane fractions, and at least 80% was associated with plasma membranes. The data revealed the same distribution within the cellular membranes of PA kinase as reported for diacylglycerol kinase, which is a typical plasma membrane-located enzyme. Furthermore, PA kinase activity was detected in the calli of 16 different plant species and in the different organs of C. roseus plants and obviously occurs ubiquitously in the plant kingdom. PMID:12231900

  16. Subcellular localization of the b-cytochrome component of the human neutrophil microbicidal oxidase: translocation during activation

    PubMed Central

    1983-01-01

    We describe a new method for subcellular fractionation of human neutrophils. Neutrophils were disrupted by nitrogen cavitation and the nuclei removed by centrifugation. The postnuclear supernatant was applied on top of a discontinuous Percoll density gradient. Centrifugation for 15 min at 48,000 g resulted in complete separation of plasma membranes, azurophil granules, and specific granules. As determined by ultrastructure and the distribution of biochemical markers of these organelles, approximately 90% of the b-cytochrome in unstimulated cells was recovered from the band containing the specific granules and was shown to be in or tightly associated with the membrane. During stimulation of intact neutrophils with phorbol myristate acetate or the ionophore A23187, we observed translocation of 40-75% of the b-cytochrome to the plasma membrane. The extent of this translocation closely paralleled release of the specific granule marker, vitamin B12-binding protein. These data indicate that the b- cytochrome is in the membrane of the specific granules of unstimulated neutrophils and that stimulus-induced fusion of these granules with the plasma membrane results in a translocation of the cytochrome. Our observations provide a basis for the assembly of the microbicidal oxidase of the human neutrophil. PMID:6408102

  17. Cloning, characterization and sub-cellular localization of gamma subunit of T-complex protein-1 (chaperonin) from Leishmania donovani.

    PubMed

    Bhaskar; Kumari, Neeti; Goyal, Neena

    2012-12-01

    T-complex protein-1 (TCP1) complex, a chaperonin class of protein, ubiquitous in all genera of life, is involved in intracellular assembly and folding of various proteins. The gamma subunit of TCP1 complex (TCP1γ), plays a pivotal role in the folding and assembly of cytoskeleton protein(s) as an individual or complexed with other subunits. Here, we report for the first time cloning, characterization and expression of the TCP1γ of Leishmania donovani (LdTCP1γ), the causative agent of Indian Kala-azar. Primary sequence analysis of LdTCP1γ revealed the presence of all the characteristic features of TCP1γ. However, leishmanial TCP1γ represents a distinct kinetoplastid group, clustered in a separate branch of the phylogenic tree. LdTCP1γ exhibited differential expression in different stages of promastigotes. The non-dividing stationary phase promastigotes exhibited 2.5-fold less expression of LdTCP1γ as compared to rapidly dividing log phase parasites. The sub-cellular distribution of LdTCP1γ was studied in log phase promastigotes by employing indirect immunofluorescence microscopy. The protein was present not only in cytoplasm but it was also localized in nucleus, peri-nuclear region, flagella, flagellar pocket and apical region. Co-localization of LdTCP1γ with actin suggests that, this gene may have a role in maintaining the structural dynamics of cytoskeleton of parasite. PMID:23137535

  18. Subcellular Fractionation and Localization Studies Reveal a Direct Interaction of the Fragile X Mental Retardation Protein (FMRP) with Nucleolin

    PubMed Central

    Taha, Mohamed S.; Nouri, Kazem; Milroy, Lech G.; Moll, Jens M.; Herrmann, Christian; Brunsveld, Luc; Piekorz, Roland P.; Ahmadian, Mohammad R.

    2014-01-01

    Fragile X mental Retardation Protein (FMRP) is a well-known regulator of local translation of its mRNA targets in neurons. However, despite its ubiquitous expression, the role of FMRP remains ill-defined in other cell types. In this study we investigated the subcellular distribution of FMRP and its protein complexes in HeLa cells using confocal imaging as well as detergent-free fractionation and size exclusion protocols. We found FMRP localized exclusively to solid compartments, including cytosolic heavy and light membranes, mitochondria, nuclear membrane and nucleoli. Interestingly, FMRP was associated with nucleolin in both a high molecular weight ribosomal and translation-associated complex (≥6 MDa) in the cytosol, and a low molecular weight complex (∼200 kDa) in the nucleoli. Consistently, we identified two functional nucleolar localization signals (NoLSs) in FMRP that are responsible for a strong nucleolar colocalization of the C-terminus of FMRP with nucleolin, and a direct interaction of the N-terminus of FMRP with the arginine-glycine-glycine (RGG) domain of nucleolin. Taken together, we propose a novel mechanism by which a transient nucleolar localization of FMRP underlies a strong nucleocytoplasmic translocation, most likely in a complex with nucleolin and possibly ribosomes, in order to regulate translation of its target mRNAs. PMID:24658146

  19. Two rhodamine lactam modulated lysosome-targetable fluorescence probes for sensitively and selectively monitoring subcellular organelle pH change.

    PubMed

    Li, Hongmei; Wang, Cuiling; She, Mengyao; Zhu, Yuelu; Zhang, Jidong; Yang, Zheng; Liu, Ping; Wang, Yaoyu; Li, Jianli

    2015-11-01

    Be a powerful technique for convenient detection of pH change in living cells, especially at subcellular level, fluorescent probes has attracted more and more attention. In this work, we designed and synthesized three rhodamine lactam modulated fluorescent probes RS1, RS2 and RS3, which all respond sensitively toward weak acidity (pH range 4-6) via the photophysical property in buffer solution without interference from the other metal ions, and they also show ideal pKa values and excellent reversibility. Particularly, by changing the lone pair electrons distribution of lactam-N atom with different conjugations, RS2 and RS3 exhibit high quantum yield, negligible cytotoxicity and excellent permeability. They are suitable to stain selectively lysosomes of tumor cells and monitor its pH changes sensitively via optical molecular imaging. The above findings suggest that the probes we designed could act as ideal and easy method for investigating the pivotal role of H(+) in lysosomes and are potential pH detectors in disease diagnosis through direct intracellular imaging.

  20. Effect of cadmium on the physiological parameters and the subcellular cadmium localization in the potato (Solanum tuberosum L.).

    PubMed

    Xu, Dongyu; Chen, Zhifan; Sun, Ke; Yan, Dong; Kang, Mingjie; Zhao, Ye

    2013-11-01

    The pollution of agricultural soils with cadmium (Cd) has become a serious problem worldwide. The potato (Solanum tuberosum L.) was used to investigate how different concentrations of Cd (1, 5, and 25mgkg(-1)) affected the physiological parameters and the subcellular distribution of Cd in the potato. The analyses were conducted using scanning electron microscopy coupled with energy dispersive X-ray (SEM-EDX). The results suggest that the leaf is the organ with the highest accumulation of Cd. The malondialdehyde (MDA) content increased and the chlorophyll content decreased in response to high level of Cd. The SEM-EDX microanalysis revealed that Cd was primarily deposited in the spongy and palisade tissues of the leaf. Furthermore, Cd was also detected in the cortex and the adjacent phloem and was observed inside the intercellular space, the interior surface of the plasma membrane, and on the surface of the elliptical starch granules in the tubers of the potato. Although low concentrations of Cd migrated from the root to the tuber, the accumulation of Cd in the tuber exceeded the standard for food security. Therefore, the planting of potato plants in farmland containing Cd should be seriously evaluated because Cd-containing potatoes might present high health risk to humans.

  1. Subcellular post-transcriptional targeting: delivery of an intracellular protein to the extracellular leaflet of the plasma membrane using a glycosyl-phosphatidylinositol (GPI) membrane anchor in neurons and polarised epithelial cells.

    PubMed

    Brown, O; Cowen, R L; Preston, C M; Castro, M G; Lowenstein, P R

    2000-11-01

    The effectiveness of viral vector-mediated gene transfer depends on the expression of therapeutic transgenes in the correct target cell types. So far, however, little attention has been given to targeted subcellular distribution of expressed transgenes. Targeting individual transgenes to particular subcellular compartments will provide various advantages in increasing the safety, efficacy, and specificity of viral vector-mediated gene delivery. Viruses normally hijack the cellular protein synthesis machinery for their own advantages. It is thus unknown whether cells infected with viral vectors will be able to target proteins to the correct subcellular organelles, or whether the subcellular targeting machinery would be selectively disrupted by viral infection. In this article we explored whether a herpes simplex virus type 1-derived vector could be used to deliver a transgene engineered to be targeted to the extracellular membrane of target cells. To do so we constructed a temperature-sensitive mutant HSV-1 vector, tsK-TT21 expressing a recombinant marker protein, tissue inhibitor of metalloproteinases (TIMP), linked to sequence encoding a signal for the addition of a glycosyl-phosphatidylinositol (GPI)-anchor within the endoplasmic reticulum. Our results demonstrate that HSV1-derived viral vectors can be used to target transgenes as GPI anchored proteins to the outside leaflet of plasma membranes, without disrupting the targeting machinery of host epithelial cells or neurons. This approach could then be used to target specific proteins to the cell membrane to modify cell-cell interactions, the function of specific plasma membrane proteins, or their interactions with other membrane proteins, and also to target a prodrug converting enzyme to the plasma membrane of target cells, therefore enhancing its cell killing effects.

  2. Aldehyde dehydrogenase (ALDH) 2 associates with oxidation of methoxyacetaldehyde; in vitro analysis with liver subcellular fraction derived from human and Aldh2 gene targeting mouse.

    PubMed

    Kitagawa, K; Kawamoto, T; Kunugita, N; Tsukiyama, T; Okamoto, K; Yoshida, A; Nakayama, K; Nakayama, K

    2000-07-01

    A principal pathway of 2-methoxyethanol (ME) metabolism is to the toxic oxidative product, methoxyacetaldehyde (MALD). To assess the role of aldehyde dehydrogenase (ALDH) in MALD metabolism, in vitro MALD oxidation was examined with liver subcellular fractions from Japanese subjects who carried three different ALDH2 genotypes and Aldh2 knockout mice, which were generated in this study. The activity was distributed in mitochondrial fractions of ALDH2*1/*1 and wild type (Aldh2+/+) mice but not ALDH2*1/*2, *2/*2 subjects or Aldh2 homozygous mutant (Aldh2-/-) mice. These data suggest that ALDH2 is a key enzyme for MALD oxidation and ME susceptibility may be influenced by the ALDH2 genotype. PMID:10913633

  3. Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

    PubMed Central

    Masedunskas, Andrius; Porat-Shliom, Natalie; Tora, Muhibullah; Milberg, Oleg; Weigert, Roberto

    2013-01-01

    Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level. In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton. PMID:24022089

  4. Brain glycogen—new perspectives on its metabolic function and regulation at the subcellular level

    PubMed Central

    Obel, Linea F.; Müller, Margit S.; Walls, Anne B.; Sickmann, Helle M.; Bak, Lasse K.; Waagepetersen, Helle S.; Schousboe, Arne

    2012-01-01

    Glycogen is a complex glucose polymer found in a variety of tissues, including brain, where it is localized primarily in astrocytes. The small quantity found in brain compared to e.g., liver has led to the understanding that brain glycogen is merely used during hypoglycemia or ischemia. In this review evidence is brought forward highlighting what has been an emerging understanding in brain energy metabolism: that glycogen is more than just a convenient way to store energy for use in emergencies—it is a highly dynamic molecule with versatile implications in brain function, i.e., synaptic activity and memory formation. In line with the great spatiotemporal complexity of the brain and thereof derived focus on the basis for ensuring the availability of the right amount of energy at the right time and place, we here encourage a closer look into the molecular and subcellular mechanisms underlying glycogen metabolism. Based on (1) the compartmentation of the interconnected second messenger pathways controlling glycogen metabolism (calcium and cAMP), (2) alterations in the subcellular location of glycogen-associated enzymes and proteins induced by the metabolic status and (3) a sequential component in the intermolecular mechanisms of glycogen metabolism, we suggest that glycogen metabolism in astrocytes is compartmentalized at the subcellular level. As a consequence, the meaning and importance of conventional terms used to describe glycogen metabolism (e.g., turnover) is challenged. Overall, this review represents an overview of contemporary knowledge about brain glycogen and its metabolism and function. However, it also has a sharp focus on what we do not know, which is perhaps even more important for the future quest of uncovering the roles of glycogen in brain physiology and pathology. PMID:22403540

  5. Linking sub-cellular biomarkers to embryo aberrations in the benthic amphipod Monoporeia affinis.

    PubMed

    Reutgard, Martin; Furuhagen, Sara

    2016-04-01

    To adequately assess and monitor environmental status in the aquatic environment a broad approach is needed that integrates physical variables, chemical analyses and biological effects at different levels of the biological organization. Embryo aberrations in the Baltic Sea key species Monoporeia affinis can be induced by both metals and organic substances as well as by hypoxia, increasing temperatures and malnutrition. This amphipod has therefore been used for more than three decades as a biological effect indicator in monitoring and assessment of chemical pollution and environmental stress. However, little is known about the sub-cellular mechanisms underlying embryo aberrations. An improved mechanistic understanding may open up the possibility of including sub-cellular alterations as sensitive warning signals of stress-induced embryo aberrations. In the present study, M. affinis was exposed in microcosms to 4 different sediments from the Baltic Sea. After 88-95 days of exposure, survival and fecundity were determined as well as the frequency and type of embryo aberrations. Moreover, oxygen radical absorption capacity (ORAC) was assayed as a proxy for antioxidant defense, thiobarbituric acid reactive substances (TBARS) level as a measure of lipid peroxidation and acetylcholinesterase (AChE) activity as an indicator of neurotoxicity. The results show that AChE and ORAC can be linked to the frequency of malformed embryos and arrested embryo development. The occurrence of dead broods was significantly associated with elevated TBARS levels. It can be concluded that these sub-cellular biomarkers are indicative of effects that could affect Darwinian fitness and that oxidative stress is a likely mechanism in the development of aberrant embryos in M. affinis. PMID:26836507

  6. Subcellular real-time in vivo imaging of intralymphatic and intravascular cancer-cell trafficking

    NASA Astrophysics Data System (ADS)

    McElroy, M.; Hayashi, K.; Kaushal, S.; Bouvet, M.; Hoffman, Robert M.

    2008-02-01

    With the use of fluorescent cells labeled with green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm and a highly sensitive small animal imaging system with both macro-optics and micro-optics, we have developed subcellular real-time imaging of cancer cell trafficking in live mice. Dual-color cancer cells were injected by a vascular route in an abdominal skin flap in nude mice. The mice were imaged with an Olympus OV100 small animal imaging system with a sensitive CCD camera and four objective lenses, parcentered and parfocal, enabling imaging from macrocellular to subcellular. We observed the nuclear and cytoplasmic behavior of cancer cells in real time in blood vessels as they moved by various means or adhered to the vessel surface in the abdominal skin flap. During extravasation, real-time dual-color imaging showed that cytoplasmic processes of the cancer cells exited the vessels first, with nuclei following along the cytoplasmic projections. Both cytoplasm and nuclei underwent deformation during extravasation. Different cancer cell lines seemed to strongly vary in their ability to extravasate. We have also developed real-time imaging of cancer cell trafficking in lymphatic vessels. Cancer cells labeled with GFP and/or RFP were injected into the inguinal lymph node of nude mice. The labeled cancer cells trafficked through lymphatic vessels where they were imaged via a skin flap in real-time at the cellular level until they entered the axillary lymph node. The bright dual-color fluorescence of the cancer cells and the real-time microscopic imaging capability of the Olympus OV100 enabled imaging the trafficking cancer cells in both blood vessels and lymphatics. With the dual-color cancer cells and the highly sensitive imaging system described here, the subcellular dynamics of cancer metastasis can now be observed in live mice in real time.

  7. Metabolic Interplay between Peroxisomes and Other Subcellular Organelles Including Mitochondria and the Endoplasmic Reticulum

    PubMed Central

    Wanders, Ronald J. A.; Waterham, Hans R.; Ferdinandusse, Sacha

    2016-01-01

    Peroxisomes are unique subcellular organelles which play an indispensable role in several key metabolic pathways which include: (1.) etherphospholipid biosynthesis; (2.) fatty acid beta-oxidation; (3.) bile acid synthesis; (4.) docosahexaenoic acid (DHA) synthesis; (5.) fatty acid alpha-oxidation; (6.) glyoxylate metabolism; (7.) amino acid degradation, and (8.) ROS/RNS metabolism. The importance of peroxisomes for human health and development is exemplified by the existence of a large number of inborn errors of peroxisome metabolism in which there is an impairment in one or more of the metabolic functions of peroxisomes. Although the clinical signs and symptoms of affected patients differ depending upon the enzyme which is deficient and the extent of the deficiency, the disorders involved are usually (very) severe diseases with neurological dysfunction and early death in many of them. With respect to the role of peroxisomes in metabolism it is clear that peroxisomes are dependent on the functional interplay with other subcellular organelles to sustain their role in metabolism. Indeed, whereas mitochondria can oxidize fatty acids all the way to CO2 and H2O, peroxisomes are only able to chain-shorten fatty acids and the end products of peroxisomal beta-oxidation need to be shuttled to mitochondria for full oxidation to CO2 and H2O. Furthermore, NADH is generated during beta-oxidation in peroxisomes and beta-oxidation can only continue if peroxisomes are equipped with a mechanism to reoxidize NADH back to NAD+, which is now known to be mediated by specific NAD(H)-redox shuttles. In this paper we describe the current state of knowledge about the functional interplay between peroxisomes and other subcellular compartments notably the mitochondria and endoplasmic reticulum for each of the metabolic pathways in which peroxisomes are involved. PMID:26858947

  8. Linking sub-cellular biomarkers to embryo aberrations in the benthic amphipod Monoporeia affinis.

    PubMed

    Reutgard, Martin; Furuhagen, Sara

    2016-04-01

    To adequately assess and monitor environmental status in the aquatic environment a broad approach is needed that integrates physical variables, chemical analyses and biological effects at different levels of the biological organization. Embryo aberrations in the Baltic Sea key species Monoporeia affinis can be induced by both metals and organic substances as well as by hypoxia, increasing temperatures and malnutrition. This amphipod has therefore been used for more than three decades as a biological effect indicator in monitoring and assessment of chemical pollution and environmental stress. However, little is known about the sub-cellular mechanisms underlying embryo aberrations. An improved mechanistic understanding may open up the possibility of including sub-cellular alterations as sensitive warning signals of stress-induced embryo aberrations. In the present study, M. affinis was exposed in microcosms to 4 different sediments from the Baltic Sea. After 88-95 days of exposure, survival and fecundity were determined as well as the frequency and type of embryo aberrations. Moreover, oxygen radical absorption capacity (ORAC) was assayed as a proxy for antioxidant defense, thiobarbituric acid reactive substances (TBARS) level as a measure of lipid peroxidation and acetylcholinesterase (AChE) activity as an indicator of neurotoxicity. The results show that AChE and ORAC can be linked to the frequency of malformed embryos and arrested embryo development. The occurrence of dead broods was significantly associated with elevated TBARS levels. It can be concluded that these sub-cellular biomarkers are indicative of effects that could affect Darwinian fitness and that oxidative stress is a likely mechanism in the development of aberrant embryos in M. affinis.

  9. Integrating Subcellular Location for Improving Machine Learning Models of Remote Homology Detection in Eukaryotic Organisms

    SciTech Connect

    Shah, Anuj R.; Oehmen, Chris S.; Harper, Jill K.; Webb-Robertson, Bobbie-Jo M.

    2007-02-23

    Motivation: At the center of bioinformatics, genomics, and pro-teomics is the need for highly accurate genome annotations. Producing high-quality reliable annotations depends on identifying sequences which are related evolutionarily (homologs) on which to infer function. Homology detection is one of the oldest tasks in bioinformatics, however most approaches still fail when presented with sequences that have low residue similarity despite a distant evolutionary relationship (remote homology). Recently, discriminative approaches, such as support vector machines (SVMs) have demonstrated a vast improvement in sensitivity for remote homology detection. These methods however have only focused on one aspect of the sequence at a time, e.g., sequence similarity or motif based scores. However, supplementary information, such as the sub-cellular location of a protein within the cell would give further clues as to possible homologous pairs, additionally eliminating false relationships due to simple functional roles that cannot exist due to location. We have developed a method, SVM-SimLoc that integrates sub-cellular location with sequence similarity information into a pro-tein family classifier and compared it to one of the most accurate sequence based SVM approaches, SVM-Pairwise. Results: The SCOP 1.53 benchmark data set was utilized to assess the performance of SVM-SimLoc. As cellular location prediction is dependent upon the type of sequence, eukaryotic or prokaryotic, the analysis is restricted to the 2630 eukaryotic sequences in the benchmark dataset, evaluating a total of 27 protein families. We demonstrate that the integration of sequence similarity and sub-cellular location yields notably more accurate results than using sequence similarity independently at a significance level of 0.006.

  10. Raman micro spectroscopy for in vitro drug screening: subcellular localisation and interactions of doxorubicin.

    PubMed

    Farhane, Z; Bonnier, F; Casey, A; Byrne, H J

    2015-06-21

    Vibrational spectroscopy, including Raman micro spectroscopy, has been widely used over the last few years to explore potential biomedical applications. Indeed, Raman micro spectroscopy has been demonstrated to be a powerful non-invasive tool in cancer diagnosis and monitoring. In confocal microscopic mode, the technique is also a molecularly specific analytical tool with optical resolution which has potential applications in subcellular analysis of biochemical processes, and therefore as an in vitro screening tool of the efficacy and mode of action of, for example, chemotherapeutic agents. In order to demonstrate and explore the potential in this field, established, model chemotherapeutic agents can be valuable. In this study paper, Raman micro spectroscopy coupled with confocal microscopy were used for the localization and tracking of the commercially available drug, doxorubicin (DOX), in the intracellular environment of the lung cancer cell line, A549. Cytotoxicity assays were employed to establish clinically relevant drug doses for 24 h exposure, and Confocal Laser Scanning Fluorescence Microscopy was conducted in parallel with Raman micro spectroscopy profiling to confirm the drug internalisation and localisation. Multivariate statistical analysis, consisting of PCA (principal components analysis) was used to highlight doxorubicin interaction with cancer cells and spectral variations due to its effects before and after DOX spectral features subtraction from nuclear and nucleolar spectra, were compared to non-exposed control spectra. Results show that Raman micro spectroscopy is not only able to detect doxorubicin inside cells and profile its specific subcellular localisation, but, it is also capable of elucidating the local biomolecular changes elicited by the drug, differentiating the responses in different sub cellular regions. Further analysis clearly demonstrates the early apoptotic effect in the nuclear regions and the initial responses of cells to this

  11. Subcellular Localization of Arabidopsis 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase1

    PubMed Central

    Leivar, Pablo; González, Víctor M.; Castel, Susanna; Trelease, Richard N.; López-Iglesias, Carmen; Arró, Montserrat; Boronat, Albert; Campos, Narciso; Ferrer, Albert; Fernàndez-Busquets, Xavier

    2005-01-01

    Plants produce diverse isoprenoids, which are synthesized in plastids, mitochondria, endoplasmic reticulum (ER), and the nonorganellar cytoplasm. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) catalyzes the synthesis of mevalonate, a rate-limiting step in the cytoplasmic pathway. Several branches of the pathway lead to the synthesis of structurally and functionally varied, yet essential, isoprenoids. Several HMGR isoforms have been identified in all plants examined. Studies based on gene expression and on fractionation of enzyme activity suggested that subcellular compartmentalization of HMGR is an important intracellular channeling mechanism for the production of the specific classes of isoprenoids. Plant HMGR has been shown previously to insert in vitro into the membrane of microsomal vesicles, but the final in vivo subcellular localization(s) remains controversial. To address the latter in Arabidopsis (Arabidopsis thaliana) cells, we conducted a multipronged microscopy and cell fractionation approach that included imaging of chimeric HMGR green fluorescent protein localizations in transiently transformed cell leaves, immunofluorescence confocal microscopy in wild-type and stably transformed seedlings, immunogold electron microscopy examinations of endogenous HMGR in seedling cotyledons, and sucrose density gradient analyses of HMGR-containing organelles. Taken together, the results reveal that endogenous Arabidopsis HMGR is localized at steady state within ER as expected, but surprisingly also predominantly within spherical, vesicular structures that range from 0.2- to 0.6-μm diameter, located in the cytoplasm and within the central vacuole in differentiated cotyledon cells. The N-terminal region, including the transmembrane domain of HMGR, was found to be necessary and sufficient for directing HMGR to ER and the spherical structures. It is believed, although not directly demonstrated, that these vesicle-like structures are derived from segments of HMGR

  12. Brain glycogen-new perspectives on its metabolic function and regulation at the subcellular level.

    PubMed

    Obel, Linea F; Müller, Margit S; Walls, Anne B; Sickmann, Helle M; Bak, Lasse K; Waagepetersen, Helle S; Schousboe, Arne

    2012-01-01

    Glycogen is a complex glucose polymer found in a variety of tissues, including brain, where it is localized primarily in astrocytes. The small quantity found in brain compared to e.g., liver has led to the understanding that brain glycogen is merely used during hypoglycemia or ischemia. In this review evidence is brought forward highlighting what has been an emerging understanding in brain energy metabolism: that glycogen is more than just a convenient way to store energy for use in emergencies-it is a highly dynamic molecule with versatile implications in brain function, i.e., synaptic activity and memory formation. In line with the great spatiotemporal complexity of the brain and thereof derived focus on the basis for ensuring the availability of the right amount of energy at the right time and place, we here encourage a closer look into the molecular and subcellular mechanisms underlying glycogen metabolism. Based on (1) the compartmentation of the interconnected second messenger pathways controlling glycogen metabolism (calcium and cAMP), (2) alterations in the subcellular location of glycogen-associated enzymes and proteins induced by the metabolic status and (3) a sequential component in the intermolecular mechanisms of glycogen metabolism, we suggest that glycogen metabolism in astrocytes is compartmentalized at the subcellular level. As a consequence, the meaning and importance of conventional terms used to describe glycogen metabolism (e.g., turnover) is challenged. Overall, this review represents an overview of contemporary knowledge about brain glycogen and its metabolism and function. However, it also has a sharp focus on what we do not know, which is perhaps even more important for the future quest of uncovering the roles of glycogen in brain physiology and pathology.

  13. Calcification and measurements of net proton and oxygen flux reveal subcellular domains in Acetabularia acetabulum.

    PubMed

    Serikawa, K A; Porterfield, D M; Smith, P J; Mandoli, D F

    2000-09-01

    Vegetative adults of Acetabularia acetabulum (L.) Silva were studied as a model system for subcellular patterning in plants, and a description of several phenotypic and physiological characteristics that reveal patterns of subcellular differentiation in this unicellular macroalga was undertaken. Initially, calcification patterns were studied. Under favorable conditions, the rhizoid and most of the stalk calcified. Only the apical 10-20% of the stalk and a small region adjacent to the rhizoid remained uncalcified. Calcification in algae has been reported to result from a biologically mediated local increase in alkalinity. To test this model extracellular pH and extracellular hydrogen ion gradients were examined with ion-selective, self-referencing, electrodes. In the light, A. acetabulum displayed a general pattern of extracellular alkalinity around the entire alga, although in some individuals the region near the rhizoid and the rhizoid itself displayed extracellular acidity. Acetabularia acetabulum also displayed net hydrogen ion influx at the rhizoid and the apical half of the stalk, variable flux in the lower part of the stalk, and net hydrogen ion efflux at the base of the stalk next to the rhizoid. The lack of complete correlation between external pH patterns and calcification suggests that other factors contribute to the control of calcification in this alga. To examine whether net hydrogen ion flux patterns correlated with photosynthetic or respiration patterns, oxygen flux was measured along the stalk using self-referencing O2 electrodes. Photosynthetic oxygen evolution occurred at comparable levels throughout the stalk, with less evolution in the rhizoid. Respiration mainly occurred near and in the rhizoid, with less O2 consumption occurring more apically along the stalk. Our studies of calcification patterns, net hydrogen ion flux and O2 flux revealed several overlapping patterns of subcellular differentiation in A. acetabulum.

  14. Transformation of tobacco plants by Yali PPO-GFP fusion gene and observation of subcellular localization.

    PubMed

    Qi, Jing; Li, Gui-Qin; Dong, Zhen; Zhou, Wei

    2016-01-01

    To explore the subcellular localization of Polyphenol oxidase (PPO) from Pyrus bretschneideri, the 1779 bp cDNA of PPO gene excluding the termination codon TAA was cloned and fused with GFP to construct a binary vector pBI121-PPO-GFP. Then, the binary vector was transformed into Nicotiana tabacum by the tumefanciens-mediated method. Using confocal laser scanning microscopy, green fluorescent signals were localized in chloroplasts of the transformed Nicotiana tabacum cell, suggesting that the Polyphenol oxidase from Pyrus bretschneideri was a chloroplast protein. PMID:27158362

  15. Subcellular compartmentalization of Cd and Zn in two bivalves. II. Significance of trophically available metal (TAM)

    USGS Publications Warehouse

    Wallace, W.G.; Luoma, S.N.

    2003-01-01

    This paper examines how the subcellular partitioning of Cd and Zn in the bivalves Macoma balthica and Potamocorbula amurensis may affect the trophic transfer of metal to predators. Results show that the partitioning of metals to organelles, 'enzymes' and metallothioneins (MT) comprise a subcellular compartment containing trophically available metal (TAM; i.e. metal trophically available to predators), and that because this partitioning varies with species, animal size and metal, TAM is similarly influenced. Clams from San Francisco Bay, California, were exposed for 14 d to 3.5 ??g 1-1 Cd and 20.5 ??g 1-1 Zn, including 109Cd and 65Zn as radiotracers, and were used in feeding experiments with grass shrimp Palaemon macrodatylus, or used to investigate the subcellular partitioning of metal. Grass shrimp fed Cd-contaminated P. amurensis absorbed ???60% of ingested Cd, which was in accordance with the partitioning of Cd to the bivalve's TAM compartment (i.e. Cd associated with organelles, 'enzymes' and MT); a similar relationship was found in previous studies with grass shrimp fed Cd-contaminated oligochaetes. Thus, TAM may be used as a tool to predict the trophic transfer of at least Cd. Subcellular fractionation revealed that ???34% of both the Cd and Zn accumulated by M. balthica was associated with TAM, while partitioning to TAM in P. amurensis was metal-dependent (???60% for TAM-Cd%, ???73% for TAM-Zn%). The greater TAM-Cd% of P. amurensis than M. balthica is due to preferential binding of Cd to MT and 'enzymes', while enhanced TAM-Zn% of P. amurensis results from a greater binding of Zn to organelles. TAM for most species-metal combinations was size-dependent, decreasing with increased clam size. Based on field data, it is estimated that of the 2 bivalves, P. amurensis poses the greater threat of Cd exposure to predators because of higher tissue concentrations and greater partitioning as TAM; exposure of Zn to predators would be similar between these species.

  16. Subcellular chemical and morphological analysis by stimulated Raman scattering microscopy and image analysis techniques.

    PubMed

    D'Arco, Annalisa; Brancati, Nadia; Ferrara, Maria Antonietta; Indolfi, Maurizio; Frucci, Maria; Sirleto, Luigi

    2016-05-01

    The visualization of heterogeneous morphology, segmentation and quantification of image features is a crucial point for nonlinear optics microscopy applications, spanning from imaging of living cells or tissues to biomedical diagnostic. In this paper, a methodology combining stimulated Raman scattering microscopy and image analysis technique is presented. The basic idea is to join the potential of vibrational contrast of stimulated Raman scattering and the strength of imaging analysis technique in order to delineate subcellular morphology with chemical specificity. Validation tests on label free imaging of polystyrene-beads and of adipocyte cells are reported and discussed. PMID:27231626

  17. The role of Cysteine 227 in subcellular localization, water permeability, and multimerization of aquaporin-11.

    PubMed

    Takahashi, Saki; Muta, Kanako; Sonoda, Hiroko; Kato, Ayaka; Abdeen, Ahmed; Ikeda, Masahiro

    2014-01-01

    Aquaporin-11 (AQP11) is the latest member of the mammalian water channel protein family to be described. Recent in vivo studies have shown that mutation at Cys(227) causes renal failure. However the importance of Cys(227) for the molecular function of AQP11 is largely unknown. In this study, we examined the subcellular localization, water permeability, and multimerization of AQP11 with a mutation at Cys(227). Interestingly, cells expressing the mutants had significantly higher osmotic water permeability. In contrast, the mutation lowered the cell surface expression and multimerization levels. Our observations suggest that Cys(227) is crucial for the proper molecular function of AQP11. PMID:24918044

  18. Subcellular chemical and morphological analysis by stimulated Raman scattering microscopy and image analysis techniques

    PubMed Central

    D’Arco, Annalisa; Brancati, Nadia; Ferrara, Maria Antonietta; Indolfi, Maurizio; Frucci, Maria; Sirleto, Luigi

    2016-01-01

    The visualization of heterogeneous morphology, segmentation and quantification of image features is a crucial point for nonlinear optics microscopy applications, spanning from imaging of living cells or tissues to biomedical diagnostic. In this paper, a methodology combining stimulated Raman scattering microscopy and image analysis technique is presented. The basic idea is to join the potential of vibrational contrast of stimulated Raman scattering and the strength of imaging analysis technique in order to delineate subcellular morphology with chemical specificity. Validation tests on label free imaging of polystyrene-beads and of adipocyte cells are reported and discussed. PMID:27231626

  19. The Subcellular Compartmentalization of Arginine Metabolizing Enzymes and Their Role in Endothelial Dysfunction

    PubMed Central

    Chen, Feng; Lucas, Rudolf; Fulton, David

    2013-01-01

    The endothelial production of nitric oxide (NO) mediates endothelium-dependent vasorelaxation and restrains vascular inflammation, smooth muscle cell proliferation, and platelet aggregation. Impaired production of NO is a hallmark of endothelial dysfunction and promotes the development of cardiovascular disease. In endothelial cells, NO is generated by endothelial nitric oxide synthase (eNOS) through the conversion of its substrate, l-arginine to l-citrulline. Reduced access to l-arginine has been proposed as a major mechanism underlying reduced eNOS activity and NO production in cardiovascular disease. The arginases (Arg1 and Arg2) metabolize l-arginine to generate l-ornithine and urea and increased expression of arginase has been proposed as a mechanism of reduced eNOS activity secondary to the depletion of l-arginine. Indeed, supplemental l-arginine and suppression of arginase activity has been shown to improve endothelium-dependent relaxation and ameliorate cardiovascular disease. However, this simple relationship is complicated by observations that l-arginine concentrations in endothelial cells remain sufficiently high to support NO synthesis. Accordingly, the subcellular compartmentalization of intracellular l-arginine into poorly interchangeable pools has been proposed to allow for the local depletion of pools or pockets of l-arginine. In agreement with this, there is considerable evidence supporting the importance of the subcellular localization of l-arginine metabolizing enzymes. In endothelial cells in vitro and in vivo, eNOS is found in discrete intracellular locations and the capacity to generate NO is heavily influenced by its localization inside the cell. Arg1 and Arg2 also reside in different subcellular environments and are thought to differentially influence endothelial function. The plasma membrane solute transporter, CAT-1 and the arginine recycling enzyme, arginosuccinate lyase, co-localize with eNOS and facilitate NO release. Herein, we

  20. Non-Linear Dynamics of Cardiac Alternans: Subcellular to Tissue-Level Mechanisms of Arrhythmia

    PubMed Central

    Gaeta, Stephen A.; Christini, David J.

    2012-01-01

    Cardiac repolarization alternans is a rhythm disturbance of the heart in which rapid stimulation elicits a beat-to-beat alternation in the duration of action potentials and magnitude of intracellular calcium transients in individual cardiac myocytes. Although this phenomenon has been identified as a potential precursor to dangerous reentrant arrhythmias and sudden cardiac death, significant uncertainty remains regarding its mechanism and no clinically practical means of halting its occurrence or progression currently exists. Cardiac alternans has well-characterized tissue, cellular, and subcellular manifestations, the mechanisms and interplay of which are an active area of research. PMID:22783195

  1. Subcellular chemical and morphological analysis by stimulated Raman scattering microscopy and image analysis techniques.

    PubMed

    D'Arco, Annalisa; Brancati, Nadia; Ferrara, Maria Antonietta; Indolfi, Maurizio; Frucci, Maria; Sirleto, Luigi

    2016-05-01

    The visualization of heterogeneous morphology, segmentation and quantification of image features is a crucial point for nonlinear optics microscopy applications, spanning from imaging of living cells or tissues to biomedical diagnostic. In this paper, a methodology combining stimulated Raman scattering microscopy and image analysis technique is presented. The basic idea is to join the potential of vibrational contrast of stimulated Raman scattering and the strength of imaging analysis technique in order to delineate subcellular morphology with chemical specificity. Validation tests on label free imaging of polystyrene-beads and of adipocyte cells are reported and discussed.

  2. SUBA3: a database for integrating experimentation and prediction to define the SUBcellular location of proteins in Arabidopsis

    PubMed Central

    Tanz, Sandra K.; Castleden, Ian; Hooper, Cornelia M.; Vacher, Michael; Small, Ian; Millar, Harvey A.

    2013-01-01

    The subcellular location database for Arabidopsis proteins (SUBA3, http://suba.plantenergy.uwa.edu.au) combines manual literature curation of large-scale subcellular proteomics, fluorescent protein visualization and protein–protein interaction (PPI) datasets with subcellular targeting calls from 22 prediction programs. More than 14 500 new experimental locations have been added since its first release in 2007. Overall, nearly 650 000 new calls of subcellular location for 35 388 non-redundant Arabidopsis proteins are included (almost six times the information in the previous SUBA version). A re-designed interface makes the SUBA3 site more intuitive and easier to use than earlier versions and provides powerful options to search for PPIs within the context of cell compartmentation. SUBA3 also includes detailed localization information for reference organelle datasets and incorporates green fluorescent protein (GFP) images for many proteins. To determine as objectively as possible where a particular protein is located, we have developed SUBAcon, a Bayesian approach that incorporates experimental localization and targeting prediction data to best estimate a protein’s location in the cell. The probabilities of subcellular location for each protein are provided and displayed as a pictographic heat map of a plant cell in SUBA3. PMID:23180787

  3. Effects of macronutrient additions on nickel uptake and distribution in the dinoflagellate Prorocentrum donghaiense Lu.

    PubMed

    Hong, Hua-Sheng; Wang, Ming-Hua; Huang, Xu-Guang; Wang, Da-Zhi

    2009-06-01

    The influences of macronutrient additions on nickel (Ni) uptake and distribution in the subcellular structures and macromolecular components of the dinoflagellate Prorocentrum donghaiense Lu were examined using a radioisotope tracer method. The results showed that nitrate addition enhanced the uptake of Ni by P. donghaiense, whereas phosphate addition inhibited Ni uptake at high-Ni concentration. Nitrate or phosphate addition significantly affected Ni distribution in the subcellular structures and components. The majority of Ni was found in the soluble substances (>70%) and in the proteins (55.0-79.6%) of the algal cells. Urea reduced the Ni content in the amino acid-carbohydrate but elevated its content in proteins, and shown significantly correlated with the protein content of the algal cells. Thus, nutrient enrichment could influence both metal uptake and its distribution in the subcellular structures and components of the phytoplankton, as well as its subsequent transfer in marine food chains. PMID:19217194

  4. Competition of Spontaneous Protein Folding and Mitochondrial Import Causes Dual Subcellular Location of Major Adenylate Kinase

    PubMed Central

    Strobel, Gertrud; Zollner, Alfred; Angermayr, Michaela; Bandlow, Wolfhard

    2002-01-01

    Sorting of cytoplasmically synthesized proteins to their target compartments usually is highly efficient so that cytoplasmic precursor pools are negligible and a particular gene product occurs at one subcellular location only. Yeast major adenylate kinase (Adk1p/Aky2p) is one prominent exception to this rule. In contrast to most mitochondrial proteins, only a minor fraction (6–8%) is taken up into the mitochondrial intermembrane space, whereas the bulk of the protein remains in the cytosol in sequence-identical form. We demonstrate that Adk1p/Aky2p uses a novel mechanism for subcellular partitioning between cytoplasm and mitochondria, which is based on competition between spontaneous protein folding and mitochondrial targeting and import. Folding is spontaneous and rapid and can dispense with molecular chaperons. After denaturation, enzymatic activity of Adk1p/Aky2p returns within a few minutes and, once folded, the protein is thermally and proteolytically very stable. In an uncoupled cell-free organellar import system, uptake of Adk1p/Aky2p is negligible, but can be improved by previous chaotropic denaturation. Import ensues independently of Hsp70 or membrane potential. Thus, nascent Adk1p/Aky2p has two options: either it is synthesized to completion and folds into an enzymatically active import-incompetent conformation that remains in the cytosol; or, during synthesis and before commencement of significant tertiary structure formation, it reaches a mitochondrial surface receptor and is internalized. PMID:12006643

  5. Differential Spatial Expression and Subcellular Localization of CtBP Family Members in Rodent Brain

    PubMed Central

    Richter, Karin; Lazarevic, Vesna; Altrock, Wilko D.; Fischer, Klaus-Dieter; Gundelfinger, Eckart D.; Fejtova, Anna

    2012-01-01

    C-terminal binding proteins (CtBPs) are well-characterized nuclear transcriptional co-regulators. In addition, cytoplasmic functions were discovered for these ubiquitously expressed proteins. These include the involvement of the isoform CtBP1-S/BARS50 in cellular membrane-trafficking processes and a role of the isoform RIBEYE as molecular scaffolds in ribbons, the presynaptic specializations of sensory synapses. CtBPs were suggested to regulate neuronal differentiation and they were implied in the control of gene expression during epileptogenesis. However, the expression patterns of CtBP family members in specific brain areas and their subcellular localizations in neurons in situ are largely unknown. Here, we performed comprehensive assessment of the expression of CtBP1 and CtBP2 in mouse brain at the microscopic and the ultra-structural levels using specific antibodies. We quantified and compared expression levels of both CtBPs in biochemically isolated brain fractions containing cellular nuclei or synaptic compartment. Our study demonstrates differential regional and subcellular expression patterns for the two CtBP family members in brain and reveals a previously unknown synaptic localization for CtBP2 in particular brain regions. Finally, we propose a mechanism of differential synapto-nuclear targeting of its splice variants CtBP2-S and CtBP2-L in neurons. PMID:22745816

  6. Differential spatial expression and subcellular localization of CtBP family members in rodent brain.

    PubMed

    Hübler, Diana; Rankovic, Marija; Richter, Karin; Lazarevic, Vesna; Altrock, Wilko D; Fischer, Klaus-Dieter; Gundelfinger, Eckart D; Fejtova, Anna

    2012-01-01

    C-terminal binding proteins (CtBPs) are well-characterized nuclear transcriptional co-regulators. In addition, cytoplasmic functions were discovered for these ubiquitously expressed proteins. These include the involvement of the isoform CtBP1-S/BARS50 in cellular membrane-trafficking processes and a role of the isoform RIBEYE as molecular scaffolds in ribbons, the presynaptic specializations of sensory synapses. CtBPs were suggested to regulate neuronal differentiation and they were implied in the control of gene expression during epileptogenesis. However, the expression patterns of CtBP family members in specific brain areas and their subcellular localizations in neurons in situ are largely unknown. Here, we performed comprehensive assessment of the expression of CtBP1 and CtBP2 in mouse brain at the microscopic and the ultra-structural levels using specific antibodies. We quantified and compared expression levels of both CtBPs in biochemically isolated brain fractions containing cellular nuclei or synaptic compartment. Our study demonstrates differential regional and subcellular expression patterns for the two CtBP family members in brain and reveals a previously unknown synaptic localization for CtBP2 in particular brain regions. Finally, we propose a mechanism of differential synapto-nuclear targeting of its splice variants CtBP2-S and CtBP2-L in neurons.

  7. Slit2 expression and its correlation with subcellular localization of β-catenin in gastric cancer.

    PubMed

    Shi, Rongliang; Liu, Weiyan; Liu, Bingya; Xu, Ziping; Chen, Liping; Zhang, Ziping

    2013-10-01

    Gastric cancer is the fourth most common cancer worldwide. Several signaling pathways are involved in gastric cancer development and progression. Slit2 was recently found to be involved in cancer; however, its expression pattern in gastric cancer has not been discovered yet. In the present study, we investigated the expression of Slit2 in human gastric cancer and its correlation with the expression and subcellular localization of β-catenin. Immunohistochemistry (IHC) staining revealed that Slit2 was highly expressed in human gastric cancer tissues, while it was low or weakly expressed in normal gastric tissues. The differences in clinicopathological features between different groups were determined using Pearson's χ2 test. Slit2 levels were significantly associated with differentiation, Lauren's classification, lymph node metastasis and TNM staging. Slit2 levels were positively correlated with β-catenin level in gastric cancer tissues and cell lines. High levels of Slit2 were correlated with the membrane localization of β-catenin, and low levels of Slit2 were correlated with nuclear translocation of β-catenin in both gastric cancer tissues and cell lines assayed by IHC and immunofluorescence staining, respectively. Our data suggest that Slit2 was highly expressed in gastric cancer patients with less advanced clinicopathological features. Slit2 levels were correlated with β-catenin level and subcellular localization.

  8. An Improved Procedure for Subcellular Spatial Alignment during Live-Cell CLEM

    PubMed Central

    Padman, Benjamin S.; Bach, Markus; Ramm, Georg

    2014-01-01

    Live-cell correlative light and electron microscopy (CLEM) offers unique insights into the ultrastructure of dynamic cellular processes. A critical and technically challenging part of CLEM is the 3-dimensional relocation of the intracellular region of interest during sample processing. We have developed a simple CLEM procedure that uses toner particles from a laser printer as orientation marks. This facilitates easy tracking of a region of interest even by eye throughout the whole procedure. Combined with subcellular fluorescence markers for the plasma membrane and nucleus, the toner particles allow for precise subcellular spatial alignment of the optical and electron microscopy data sets. The toner-based reference grid is printed and transferred onto a polymer film using a standard office printer and laminator. We have also designed a polymer film holder that is compatible with most inverted microscopes, and have validated our strategy by following the ultrastructure of mitochondria that were selectively photo-irradiated during live-cell microscopy. In summary, our inexpensive and robust CLEM procedure simplifies optical imaging, without limiting the choice of optical microscope. PMID:24755651

  9. A Silicone-Based Stretchable Micropost Array Membrane for Monitoring Live-Cell Subcellular Cytoskeletal Response

    PubMed Central

    Mann, Jennifer M.; Lam, Raymond H. W.; Weng, Shinuo; Sun, Yubing; Fu, Jianping

    2014-01-01

    External forces are increasingly recognized as major regulators of cellular structure and function, yet the underlying mechanism by which cells sense forces and transduce them into intracellular biochemical signals and behavioral responses (‘mechanotransduction’) is largely undetermined. To aid in the mechanistic study of mechanotransduction, herein we devised a cell stretch device that allowed for quantitative control and real-time measurements of mechanical stimuli and cellular biomechanical responses. Our strategy involved a microfabricated array of silicone elastomeric microposts integrated onto a stretchable elastomeric membrane. By using a computer-controlled vacuum, this micropost array membrane (mPAM) was activated to apply equibiaxial cell stretching forces to adherent cells attached on the microposts. Using the mPAM, we studied live-cell subcellular dynamic responses of contractile forces of vascular smooth muscle cells (VSMCs) to sustained static equibiaxial cell stretches. Our data showed that in response to sustained cell stretches, VSMCs regulated their cytoskeletal (CSK) contractility in a biphasic manner: they first acutely enhanced their contraction to resist rapid cell deformation (‘stiffening’) before they allowed slow adaptive inelastic CSK reorganization to release their contractility (‘softening’). The contractile response across entire single VSMCs was spatially inhomogeneous and force-dependent. Our mPAM device and live-cell subcellular contractile measurements will help elucidate the mechanotransductive system in VSMCs and thus contribute to our understanding of pressure-induced vascular disease processes. PMID:22193351

  10. Subcellular localization and mechanisms of nucleocytoplasmic trafficking of steroid receptor coactivator-1.

    PubMed

    Amazit, Larbi; Alj, Youssef; Tyagi, Rakesh Kumar; Chauchereau, Anne; Loosfelt, Hugues; Pichon, Christophe; Pantel, Jacques; Foulon-Guinchard, Emmanuelle; Leclerc, Philippe; Milgrom, Edwin; Guiochon-Mantel, Anne

    2003-08-22

    Steroid hormone receptors are ligand-stimulated transcription factors that modulate gene transcription by recruiting coregulators to gene promoters. Subcellular localization and dynamic movements of transcription factors have been shown to be one of the major means of regulating their transcriptional activity. In the present report we describe the subcellular localization and the dynamics of intracellular trafficking of steroid receptor coactivator 1 (SRC-1). After its synthesis in the cytoplasm, SRC-1 is imported into the nucleus, where it activates transcription and is subsequently exported back to the cytoplasm. In both the nucleus and cytoplasm, SRC-1 is localized in speckles. The characterization of SRC-1 nuclear localization sequence reveals that it is a classic bipartite signal localized in the N-terminal region of the protein, between amino acids 18 and 36. This sequence is highly conserved within the other members of the p160 family. Additionally, SRC-1 nuclear export is inhibited by leptomycin B. The region involved in its nuclear export is localized between amino acids 990 and 1038. It is an unusually large domain differing from the classic leucine-rich NES sequences. Thus SRC-1 nuclear export involves either an alternate type of NES or is dependent on the interaction of SRC-1 with a protein, which is exported through the crm1/exportin pathway. Overall, the intracellular trafficking of SRC-1 might be a mechanism to regulate the termination of hormone action, the interaction with other signaling pathways in the cytoplasm and its degradation. PMID:12791702

  11. Active migration into the subcellular space precedes Campylobacter jejuni invasion of epithelial cells.

    PubMed

    van Alphen, Lieke B; Bleumink-Pluym, Nancy M C; Rochat, Klazina D; van Balkom, Bas W M; Wösten, Marc M S M; van Putten, Jos P M

    2008-01-01

    The bacterial pathogen Campylobacter jejuni invades mucosal cells via largely undefined and rather inefficient (0.01-2 bacteria per cell) mechanisms. Here we report a novel, highly efficient C. jejuni infection pathway resulting in 10-15 intracellular bacteria per cell within 3 h of infection. Electron microscopy, pulse-chase infection assays and time-lapse multiphoton laser confocal microscopy demonstrated that the mechanism involved active and rapid migration of the pathogen into the subcellular space (termed 'subvasion'), followed by bacterial entry ('invasion') at the cell basis. Efficient subvasion was maximal after repeated rounds of selection for the subvasive phenotype. Targeted mutagenesis indicated that the CadF, JlpA or PEB1 adhesins were not required. Dissection of the selected and parental phenotypes by SDS-PAGE yielded comparable capsule polysaccharide and lipooligosaccharide profiles. Proteomics revealed reduced amounts of the chemotaxis protein CheW for the subvasive phenotype. Swarming assays confirmed that the selected phenotype exhibited altered migration behaviour. Introduction of a plasmid carrying chemotaxis genes into the subvasive strain yielded wild-type subvasion levels and migration behaviour. These results indicate that alterations in the bacterial migration machinery enable C. jejuni to actively penetrate the subcellular space and gain access to the cell interior with unprecedented efficiency. PMID:18052944

  12. Improved prediction of subcellular location for apoptosis proteins by the dual-layer support vector machine.

    PubMed

    Zhou, X-B; Chen, C; Li, Z-C; Zou, X-Y

    2008-08-01

    Apoptosis proteins play an important role in the development and homeostasis of an organism. The accurate prediction of subcellular location for apoptosis proteins is very helpful for understanding the mechanism of apoptosis and their biological functions. However, most of the existing predictive methods are designed by utilizing a single classifier, which would limit the further improvement of their performances. In this paper, a novel predictive method, which is essentially a multi-classifier system, has been proposed by combing a dual-layer support vector machine (SVM) with multiple compositions including amino acid composition (AAC), dipeptide composition (DPC) and amphiphilic pseudo amino acid composition (Am-Pse-AAC). As a demonstration, the predictive performance of our method was evaluated on two datasets of apoptosis proteins, involving the standard dataset ZD98 generated by Zhou and Doctor, and a larger dataset ZW225 generated by Zhang et al. With the jackknife test, the overall accuracies of our method on the two datasets reach 94.90% and 88.44%, respectively. The promising results indicate that our method can be a complementary tool for the prediction of subcellular location.

  13. Long-Term Growth of Moss in Microfluidic Devices Enables Subcellular Studies in Development.

    PubMed

    Bascom, Carlisle S; Wu, Shu-Zon; Nelson, Katherine; Oakey, John; Bezanilla, Magdalena

    2016-09-01

    Key developmental processes that occur on the subcellular and cellular level or occur in occluded tissues are difficult to access, let alone image and analyze. Recently, culturing living samples within polydimethylsiloxane (PDMS) microfluidic devices has facilitated the study of hard-to-reach developmental events. Here, we show that an early diverging land plant, Physcomitrella patens, can be continuously cultured within PDMS microfluidic chambers. Because the PDMS chambers are bonded to a coverslip, it is possible to image P. patens development at high resolution over long time periods. Using PDMS chambers, we report that wild-type protonemal tissue grows at the same rate as previously reported for growth on solid medium. Using long-term imaging, we highlight key developmental events, demonstrate compatibility with high-resolution confocal microscopy, and obtain growth rates for a slow-growing mutant. By coupling the powerful genetic tools available to P. patens with long-term growth and imaging provided by PDMS microfluidic chambers, we demonstrate the capability to study cellular and subcellular developmental events in plants directly and in real time.

  14. MAP kinase subcellular localization controls both pattern and proliferation in the developing Drosophila wing

    PubMed Central

    Marenda, Daniel R.; Vrailas, Alysia D.; Rodrigues, Aloma B.; Cook, Summer; Powers, Maureen A.; Lorenzen, James A.; Perkins, Lizabeth A.; Moses, Kevin

    2007-01-01

    Mitogen-activated protein kinases (MAPKs) phosphorylate target proteins in both the cytoplasm and nucleus, and a strong correlation exists between the subcellular localization of MAPK and resulting cellular responses. It was thought that MAPK phosphorylation was always followed by rapid nuclear translocation. However, we and others have found that MAPK phosphorylation is not always sufficient for nuclear translocation in vivo. In the developing Drosophila wing, MAPK-mediated signaling is required both for patterning and for cell proliferation, although the mechanism of this differential control is not fully understood. Here, we show that phosphorylated MAPK (pMAPK) is held in the cytoplasm in differentiating larval and pupal wing vein cells, and we show that this cytoplasmic hold is required for vein cell fate. At the same time, we show that MAPK does move into the nucleus of other wing cells where it promotes cell proliferation. We propose a novel Ras pathway bifurcation in Drosophila and our results suggest a mechanism by which MAPK phosphorylation can signal two different cellular outcomes (differentiation versus proliferation) based on the subcellular localization of MAPK. PMID:16308331

  15. Subcellular localization and rearrangement of endoplasmic reticulum by Brome mosaic virus capsid protein.

    PubMed

    Bamunusinghe, Devinka; Seo, Jang-Kyun; Rao, A L N

    2011-03-01

    Genome packaging in the plant-infecting Brome mosaic virus (BMV), a member of the alphavirus-like superfamily, as well as in other positive-strand RNA viruses pathogenic to humans (e.g., poliovirus) and animals (e.g., Flock House virus), is functionally coupled to replication. Although the subcellular localization site of BMV replication has been identified, that of the capsid protein (CP) has remained elusive. In this study, the application of immunofluorescence confocal microscopy to Nicotiana benthamiana leaves expressing replication-derived BMV CP as a green fluorescent protein (GFP) fusion, in conjunction with antibodies to the CP and double-stranded RNA, a presumed marker of RNA replication, revealed that the subcellular localization sites of replication and CP overlap. Our temporal analysis by transmission electron microscopy of ultrastructural modifications induced in BMV-infected N. benthamiana leaves revealed a reticulovesicular network of modified endoplasmic reticulum (ER) incorporating large assemblies of vesicles derived from ER accumulated in the cytoplasm during BMV infection. Additionally, for the first time, we have found by ectopic expression experiments that BMV CP itself has the intrinsic property of modifying ER to induce vesicles similar to those present in BMV infections. The significance of CP-induced vesicles in relation to CP-organized viral functions that are linked to replication-coupled packaging is discussed.

  16. The Salmonella effector SteA binds phosphatidylinositol 4-phosphate for subcellular targeting within host cells.

    PubMed

    Domingues, Lia; Ismail, Ahmad; Charro, Nuno; Rodríguez-Escudero, Isabel; Holden, David W; Molina, María; Cid, Víctor J; Mota, Luís Jaime

    2016-07-01

    Many bacterial pathogens use specialized secretion systems to deliver virulence effector proteins into eukaryotic host cells. The function of these effectors depends on their localization within infected cells, but the mechanisms determining subcellular targeting of each effector are mostly elusive. Here, we show that the Salmonella type III secretion effector SteA binds specifically to phosphatidylinositol 4-phosphate [PI(4)P]. Ectopically expressed SteA localized at the plasma membrane (PM) of eukaryotic cells. However, SteA was displaced from the PM of Saccharomyces cerevisiae in mutants unable to synthesize the local pool of PI(4)P and from the PM of HeLa cells after localized depletion of PI(4)P. Moreover, in infected cells, bacterially translocated or ectopically expressed SteA localized at the membrane of the Salmonella-containing vacuole (SCV) and to Salmonella-induced tubules; using the PI(4)P-binding domain of the Legionella type IV secretion effector SidC as probe, we found PI(4)P at the SCV membrane and associated tubules throughout Salmonella infection of HeLa cells. Both binding of SteA to PI(4)P and the subcellular localization of ectopically expressed or bacterially translocated SteA were dependent on a lysine residue near the N-terminus of the protein. Overall, this indicates that binding of SteA to PI(4)P is necessary for its localization within host cells.

  17. Long-Term Growth of Moss in Microfluidic Devices Enables Subcellular Studies in Development1[OPEN

    PubMed Central

    2016-01-01

    Key developmental processes that occur on the subcellular and cellular level or occur in occluded tissues are difficult to access, let alone image and analyze. Recently, culturing living samples within polydimethylsiloxane (PDMS) microfluidic devices has facilitated the study of hard-to-reach developmental events. Here, we show that an early diverging land plant, Physcomitrella patens, can be continuously cultured within PDMS microfluidic chambers. Because the PDMS chambers are bonded to a coverslip, it is possible to image P. patens development at high resolution over long time periods. Using PDMS chambers, we report that wild-type protonemal tissue grows at the same rate as previously reported for growth on solid medium. Using long-term imaging, we highlight key developmental events, demonstrate compatibility with high-resolution confocal microscopy, and obtain growth rates for a slow-growing mutant. By coupling the powerful genetic tools available to P. patens with long-term growth and imaging provided by PDMS microfluidic chambers, we demonstrate the capability to study cellular and subcellular developmental events in plants directly and in real time. PMID:27406170

  18. Evaluation of methods for detection of fluorescence labeled subcellular objects in microscope images

    PubMed Central

    2010-01-01

    Background Several algorithms have been proposed for detecting fluorescently labeled subcellular objects in microscope images. Many of these algorithms have been designed for specific tasks and validated with limited image data. But despite the potential of using extensive comparisons between algorithms to provide useful information to guide method selection and thus more accurate results, relatively few studies have been performed. Results To better understand algorithm performance under different conditions, we have carried out a comparative study including eleven spot detection or segmentation algorithms from various application fields. We used microscope images from well plate experiments with a human osteosarcoma cell line and frames from image stacks of yeast cells in different focal planes. These experimentally derived images permit a comparison of method performance in realistic situations where the number of objects varies within image set. We also used simulated microscope images in order to compare the methods and validate them against a ground truth reference result. Our study finds major differences in the performance of different algorithms, in terms of both object counts and segmentation accuracies. Conclusions These results suggest that the selection of detection algorithms for image based screens should be done carefully and take into account different conditions, such as the possibility of acquiring empty images or images with very few spots. Our inclusion of methods that have not been used before in this context broadens the set of available detection methods and compares them against the current state-of-the-art methods for subcellular particle detection. PMID:20465797

  19. Subcellular Investigation of Photosynthesis-Driven Carbon Assimilation in the Symbiotic Reef Coral Pocillopora damicornis

    PubMed Central

    Domart-Coulon, Isabelle; Escrig, Stephane; Humbel, Bruno M.; Hignette, Michel

    2015-01-01

    ABSTRACT  Reef-building corals form essential, mutualistic endosymbiotic associations with photosynthetic Symbiodinium dinoflagellates, providing their animal host partner with photosynthetically derived nutrients that allow the coral to thrive in oligotrophic waters. However, little is known about the dynamics of these nutritional interactions at the (sub)cellular level. Here, we visualize with submicrometer spatial resolution the carbon and nitrogen fluxes in the intact coral-dinoflagellate association from the reef coral Pocillopora damicornis by combining nanoscale secondary ion mass spectrometry (NanoSIMS) and transmission electron microscopy with pulse-chase isotopic labeling using [13C]bicarbonate and [15N]nitrate. This allows us to observe that (i) through light-driven photosynthesis, dinoflagellates rapidly assimilate inorganic bicarbonate and nitrate, temporarily storing carbon within lipid droplets and starch granules for remobilization in nighttime, along with carbon and nitrogen incorporation into other subcellular compartments for dinoflagellate growth and maintenance, (ii) carbon-containing photosynthates are translocated to all four coral tissue layers, where they accumulate after only 15 min in coral lipid droplets from the oral gastroderm and within 6 h in glycogen granules from the oral epiderm, and (iii) the translocation of nitrogen-containing photosynthates is delayed by 3 h. PMID:25670779

  20. An improved procedure for subcellular spatial alignment during live-cell CLEM.

    PubMed

    Padman, Benjamin S; Bach, Markus; Ramm, Georg

    2014-01-01

    Live-cell correlative light and electron microscopy (CLEM) offers unique insights into the ultrastructure of dynamic cellular processes. A critical and technically challenging part of CLEM is the 3-dimensional relocation of the intracellular region of interest during sample processing. We have developed a simple CLEM procedure that uses toner particles from a laser printer as orientation marks. This facilitates easy tracking of a region of interest even by eye throughout the whole procedure. Combined with subcellular fluorescence markers for the plasma membrane and nucleus, the toner particles allow for precise subcellular spatial alignment of the optical and electron microscopy data sets. The toner-based reference grid is printed and transferred onto a polymer film using a standard office printer and laminator. We have also designed a polymer film holder that is compatible with most inverted microscopes, and have validated our strategy by following the ultrastructure of mitochondria that were selectively photo-irradiated during live-cell microscopy. In summary, our inexpensive and robust CLEM procedure simplifies optical imaging, without limiting the choice of optical microscope. PMID:24755651

  1. Subcellular optogenetic activation of Cdc42 controls local and distal signaling to drive immune cell migration

    PubMed Central

    O’Neill, Patrick R.; Kalyanaraman, Vani; Gautam, N.

    2016-01-01

    Migratory immune cells use intracellular signaling networks to generate and orient spatially polarized responses to extracellular cues. The monomeric G protein Cdc42 is believed to play an important role in controlling the polarized responses, but it has been difficult to determine directly the consequences of localized Cdc42 activation within an immune cell. Here we used subcellular optogenetics to determine how Cdc42 activation at one side of a cell affects both cell behavior and dynamic molecular responses throughout the cell. We found that localized Cdc42 activation is sufficient to generate polarized signaling and directional cell migration. The optically activated region becomes the leading edge of the cell, with Cdc42 activating Rac and generating membrane protrusions driven by the actin cytoskeleton. Cdc42 also exerts long-range effects that cause myosin accumulation at the opposite side of the cell and actomyosin-mediated retraction of the cell rear. This process requires the RhoA-activated kinase ROCK, suggesting that Cdc42 activation at one side of a cell triggers increased RhoA signaling at the opposite side. Our results demonstrate how dynamic, subcellular perturbation of an individual signaling protein can help to determine its role in controlling polarized cellular responses. PMID:26941336

  2. Modelling platelet–blood flow interaction using the subcellular element Langevin method

    PubMed Central

    Sweet, Christopher R.; Chatterjee, Santanu; Xu, Zhiliang; Bisordi, Katharine; Rosen, Elliot D.; Alber, Mark

    2011-01-01

    In this paper, a new three-dimensional modelling approach is described for studying fluid–viscoelastic cell interaction, the subcellular element Langevin (SCEL) method, with cells modelled by subcellular elements (SCEs) and SCE cells coupled with fluid flow and substrate models by using the Langevin equation. It is demonstrated that: (i) the new method is computationally efficient, scaling as 𝒪(N) for N SCEs; (ii) cell geometry, stiffness and adhesivity can be modelled by directly relating parameters to experimentally measured values; (iii) modelling the fluid–platelet interface as a surface leads to a very good correlation with experimentally observed platelet flow interactions. Using this method, the three-dimensional motion of a viscoelastic platelet in a shear blood flow was simulated and compared with experiments on tracking platelets in a blood chamber. It is shown that the complex platelet-flipping dynamics under linear shear flows can be accurately recovered with the SCEL model when compared with the experiments. All experimental details and electronic supplementary material are archived at http://biomath.math.nd.edu/scelsupplementaryinformation/. PMID:21593027

  3. Top Down Proteomics Reveals Mature Proteoforms Expressed in Subcellular Fractions of the Echinococcus granulosus Preadult Stage.

    PubMed

    Lorenzatto, Karina R; Kim, Kyunggon; Ntai, Ioanna; Paludo, Gabriela P; Camargo de Lima, Jeferson; Thomas, Paul M; Kelleher, Neil L; Ferreira, Henrique B

    2015-11-01

    Echinococcus granulosus is the causative agent of cystic hydatid disease, a neglected zoonosis responsible for high morbidity and mortality. Several molecular mechanisms underlying parasite biology remain poorly understood. Here, E. granulosus subcellular fractions were analyzed by top down and bottom up proteomics for protein identification and characterization of co-translational and post-translational modifications (CTMs and PTMs, respectively). Nuclear and cytosolic extracts of E. granulosus protoscoleces were fractionated by 10% GELFrEE and proteins under 30 kDa were analyzed by LC-MS/MS. By top down analysis, 186 proteins and 207 proteoforms were identified, of which 122 and 52 proteoforms were exclusively detected in nuclear and cytosolic fractions, respectively. CTMs were evident as 71% of the proteoforms had methionine excised and 47% were N-terminal acetylated. In addition, in silico internal acetylation prediction coupled with top down MS allowed the characterization of 9 proteins differentially acetylated, including histones. Bottom up analysis increased the overall number of identified proteins in nuclear and cytosolic fractions to 154 and 112, respectively. Overall, our results provided the first description of the low mass proteome of E. granulosus subcellular fractions and highlighted proteoforms with CTMs and PTMS whose characterization may lead to another level of understanding about molecular mechanisms controlling parasitic flatworm biology.

  4. Characterization of Subcellular Responses Induced by Exposure of Microbubbles to Astrocytes.

    PubMed

    Sun, Shan; Kanagaraj, Johnwesly; Cho, Lindsey; Kang, Dongkoo; Xiao, Shu; Cho, Michael

    2015-10-01

    Blast traumatic brain injury (bTBI) has now been identified to associate with adverse health consequences among combat veterans. Post-traumatic stress disorder linked with explosive blasts, for example, may result from such brain injury. The fundamental questions about the nature, diagnosis, and long-term consequences of bTBI and causative relationship to post-traumatic stress disorder remain elusive, however. A better understanding of brain tissue injury requires elucidation of potential mechanisms. One such mechanism may be generation of microcavitation bubbles in the brain after an explosive blast and their subsequent interaction with brain cells. Using a controlled electrical discharge system, we have successfully generated shock waves (∼10 MPa) and microbubbles (20-30 μm) in the cell culture of mouse astrocytes. Detachment of astrocytes from the substrate after exposure to microbubbles was observed, and it depended on repetitive exposures. Of the cells that survived the initial assault, several subcellular changes were monitored and determined using fluorescent microscopy, including cell viability, cytoskeletal reorganization, changes in focal adhesion, membrane permeability, and potential onset of apoptosis. While the astrocytes impacted by the shock wave only demonstrated essentially unaltered cellular behavior, the astrocytes exposed to microbubbles exhibited significantly different responses, including production of reactive oxygen species by collapse of microbubbles. In the present study, we characterized and report for the first time the altered biophysical and subcellular properties in astrocytes in response to exposure to the combination of shock waves and microbubbles. PMID:25843567

  5. Mono- and Dinuclear Phosphorescent Rhenium(I) Complexes: Impact of Subcellular Localization on Anticancer Mechanisms.

    PubMed

    Ye, Rui-Rong; Tan, Cai-Ping; Chen, Mu-He; Hao, Liang; Ji, Liang-Nian; Mao, Zong-Wan

    2016-06-01

    Elucidation of relationship among chemical structure, cellular uptake, localization, and biological activity of anticancer metal complexes is important for the understanding of their mechanisms of action. Organometallic rhenium(I) tricarbonyl compounds have emerged as potential multifunctional anticancer drug candidates that can integrate therapeutic and imaging capabilities in a single molecule. Herein, two mononuclear phosphorescent rhenium(I) complexes (Re1 and Re2), along with their corresponding dinuclear complexes (Re3 and Re4), were designed and synthesized as potent anticancer agents. The subcellular accumulation of Re1-Re4 was conveniently analyzed by confocal microscopy in situ in live cells by utilizing their intrinsic phosphorescence. We found that increased lipophilicity of the bidentate ligands could enhance their cellular uptake, leading to improved anticancer efficacy. The dinuclear complexes were more potent than the mononuclear counterparts. The molecular anticancer mechanisms of action evoked by Re3 and Re4 were explored in detail. Re3 with a lower lipophilicity localizes to lysosomes and induces caspase-independent apoptosis, whereas Re4 with higher lipophilicity specially accumulates in mitochondria and induces caspase-independent paraptosis in cancer cells. Our study demonstrates that subcellular localization is crucial for the anticancer mechanisms of these phosphorescent rhenium(I) complexes. PMID:27106876

  6. Mono- and Dinuclear Phosphorescent Rhenium(I) Complexes: Impact of Subcellular Localization on Anticancer Mechanisms.

    PubMed

    Ye, Rui-Rong; Tan, Cai-Ping; Chen, Mu-He; Hao, Liang; Ji, Liang-Nian; Mao, Zong-Wan

    2016-06-01

    Elucidation of relationship among chemical structure, cellular uptake, localization, and biological activity of anticancer metal complexes is important for the understanding of their mechanisms of action. Organometallic rhenium(I) tricarbonyl compounds have emerged as potential multifunctional anticancer drug candidates that can integrate therapeutic and imaging capabilities in a single molecule. Herein, two mononuclear phosphorescent rhenium(I) complexes (Re1 and Re2), along with their corresponding dinuclear complexes (Re3 and Re4), were designed and synthesized as potent anticancer agents. The subcellular accumulation of Re1-Re4 was conveniently analyzed by confocal microscopy in situ in live cells by utilizing their intrinsic phosphorescence. We found that increased lipophilicity of the bidentate ligands could enhance their cellular uptake, leading to improved anticancer efficacy. The dinuclear complexes were more potent than the mononuclear counterparts. The molecular anticancer mechanisms of action evoked by Re3 and Re4 were explored in detail. Re3 with a lower lipophilicity localizes to lysosomes and induces caspase-independent apoptosis, whereas Re4 with higher lipophilicity specially accumulates in mitochondria and induces caspase-independent paraptosis in cancer cells. Our study demonstrates that subcellular localization is crucial for the anticancer mechanisms of these phosphorescent rhenium(I) complexes.

  7. Long-Term Growth of Moss in Microfluidic Devices Enables Subcellular Studies in Development.

    PubMed

    Bascom, Carlisle S; Wu, Shu-Zon; Nelson, Katherine; Oakey, John; Bezanilla, Magdalena

    2016-09-01

    Key developmental processes that occur on the subcellular and cellular level or occur in occluded tissues are difficult to access, let alone image and analyze. Recently, culturing living samples within polydimethylsiloxane (PDMS) microfluidic devices has facilitated the study of hard-to-reach developmental events. Here, we show that an early diverging land plant, Physcomitrella patens, can be continuously cultured within PDMS microfluidic chambers. Because the PDMS chambers are bonded to a coverslip, it is possible to image P. patens development at high resolution over long time periods. Using PDMS chambers, we report that wild-type protonemal tissue grows at the same rate as previously reported for growth on solid medium. Using long-term imaging, we highlight key developmental events, demonstrate compatibility with high-resolution confocal microscopy, and obtain growth rates for a slow-growing mutant. By coupling the powerful genetic tools available to P. patens with long-term growth and imaging provided by PDMS microfluidic chambers, we demonstrate the capability to study cellular and subcellular developmental events in plants directly and in real time. PMID:27406170

  8. Characterization and sub-cellular localization of GalNAc-binding proteins isolated from human hepatic stellate cells.

    PubMed

    Zhong, Yaogang; Zhang, Jing; Yu, Hanjie; Zhang, Jiaxu; Sun, Xiu-Xuan; Chen, Wentian; Bian, Huijie; Li, Zheng

    2015-12-25

    Although the expression levels of total GalNAc-binding proteins (GNBPs) were up-regulated significantly in human hepatic stellate cells (HSCs) activated with transforming growth factor-β1(TGF-β1), yet little is known about the precise types, distribution and sub-cellular localization of the GNBPs in HSCs. Here, 264 GNBPs from the activated HSCs and 257 GNBPs from the quiescent HSCs were identified and annotated. A total of 46 GNBPs were estimated to be significantly up-regulated and 40 GNBPs were estimated to be significantly down-regulated in the activated HSCs. For example, the GNBPs (i.e. BTF3, COX17, and ATP5A1) responsible for the regulation of protein binding were up-regulated, and those (i.e. FAM114A1, ENO3, and TKT) responsible for the regulation of protein binding were down-regulated in the activated HSCs. The motifs of the isolated GNBPs showed that Proline residue had the maximum preference in consensus sequences. The western blotting showed the expression levels of COX17, and PRMT1 were significantly up-regulated, while, the expression level of CLIC1(B5) was down-regulated in the activated HSCs and liver cirrhosis tissues. Moreover, the GNBPs were sub-localized in the Golgi apparatus of HSCs. In conclusion, the precision alteration of the GNBPs referred to pathological changes in liver fibrosis/cirrhosis may provide useful information to find new molecular mechanism of HSC activation and discover the biomarkers for diagnosis of liver fibrosis/cirrhosis as well as development of new anti-fibrotic strategies.

  9. The subcellular localization of an aquaporin-2 tetramer depends on the stoichiometry of phosphorylated and nonphosphorylated monomers.

    PubMed

    Kamsteeg, E J; Heijnen, I; van Os, C H; Deen, P M

    2000-11-13

    In renal principal cells, vasopressin regulates the shuttling of the aquaporin (AQP)2 water channel between intracellular vesicles and the apical plasma membrane. Vasopressin-induced phosphorylation of AQP2 at serine 256 (S256) by protein kinase A (PKA) is essential for its localization in the membrane. However, phosphorylated AQP2 (p-AQP2) has also been detected in intracellular vesicles of noninduced principal cells. As AQP2 is expressed as homotetramers, we hypothesized that the number of p-AQP2 monomers in a tetramer might be critical for the its steady state distribution. Expressed in oocytes, AQP2-S256D and AQP2-S256A mimicked p-AQP2 and non-p-AQP2, respectively, as routing and function of AQP2-S256D and wild-type AQP2 (wt-AQP2) were identical, whereas AQP2-S256A was retained intracellularly. In coinjection experiments, AQP2-S256A and AQP2-S256D formed heterotetramers. Coinjection of different ratios of AQP2-S256A and AQP2-S256D cRNAs revealed that minimally three AQP2-S256D monomers in an AQP2 tetramer were essential for its plasma membrane localization. Therefore, our results suggest that in principal cells, minimally three monomers per AQP2 tetramer have to be phosphorylated for its steady state localization in the apical membrane. As other multisubunit channels are also regulated by phosphorylation, it is anticipated that the stoichiometry of their phosphorylated and nonphosphorylated subunits may fine-tune the activity or subcellular localization of these complexes. PMID:11076974

  10. A Novel Function for the PAR Complex in Subcellular Morphogenesis of Tracheal Terminal Cells in Drosophila melanogaster

    PubMed Central

    Jones, Tiffani A.; Metzstein, Mark M.

    2011-01-01

    The processes that generate cellular morphology are not well understood. To investigate this problem, we use Drosophila melanogaster tracheal terminal cells, which undergo two distinct morphogenetic processes: subcellular branching morphogenesis and subcellular apical lumen formation. Here we show these processes are regulated by components of the PAR-polarity complex. This complex, composed of the proteins Par-6, Bazooka (Par-3), aPKC, and Cdc42, is best known for roles in asymmetric cell division and apical/basal polarity. We find Par-6, Bazooka, and aPKC, as well as known interactions between them, are required for subcellular branch initiation, but not for branch outgrowth. By analysis of single and double mutants, and isolation of two novel alleles of Par-6, one of which specifically truncates the Par-6 PDZ domain, we conclude that dynamic interactions between apical PAR-complex members control the branching pattern of terminal cells. These data suggest that canonical apical PAR-complex activity is required for subcellular branching morphogenesis. In addition, we find the PAR proteins are downstream of the FGF pathway that controls terminal cell branching. In contrast, we find that while Par-6 and aPKC are both required for subcellular lumen formation, neither Bazooka nor a direct interaction between Par-6 and aPKC is needed for this process. Thus a novel, noncanonical role for the polarity proteins Par-6 and aPKC is used in formation of this subcellular apical compartment. Our results demonstrate that proteins from the PAR complex can be deployed independently within a single cell to control two different morphogenetic processes. PMID:21750259

  11. Comparison of helper component-protease RNA silencing suppression activity, subcellular localization, and aggregation of three Korean isolates of Turnip mosaic virus.

    PubMed

    Han, Jae-Yeong; Chung, Jinsoo; Kim, Jungkyu; Seo, Eun-Young; Kilcrease, James P; Bauchan, Gary R; Lim, Seungmo; Hammond, John; Lim, Hyoun-Sub

    2016-08-01

    In 2014, we performed a nationwide survey in Korean radish fields to investigate the distribution and variability of Turnip mosaic virus (TuMV). Brassica rapa ssp. pekinensis sap-inoculated with three isolates of TuMV from infected radish tissue showed different symptom severities, whereas symptoms in Raphanus sativus were similar for each isolate. The helper component-protease (HC-Pro) genes of each isolate were sequenced, and phylogenetic analysis showed that the three Korean isolates were clustered into the basal-BR group. The HC-Pro proteins of these isolates were tested for their RNA silencing suppressor (VSR) activity and subcellular localization in Nicotiana benthamiana. A VSR assay by co-agroinfiltration of HC-Pro with soluble-modified GFP (smGFP) showed that HC-Pro of isolate R007 and R041 showed stronger VSR activity than R065. The HC-Pros showed 98.25 % amino acid identity, and weak VSR isolate (R065) has a single variant residue in the C-terminal domain associated with protease activity and self-interaction compared to isolates with strong VSR activity. Formation of large subcellular aggregates of GFP:HC-Pro fusion proteins in N. benthamiana was only observed for HC-Pro from isolates with strong VSR activity, suggesting that R065 'weak' HC-Pro may have diminished self-association; substitution of the variant C-terminal residue largely reversed the HC-Pro aggregation and silencing suppressor characteristics. The lack of correlation between VSR efficiency and induction of systemic necrosis (SN) suggests that differences in viral accumulation due to HC-Pro are not responsible for SN.

  12. Mobility and subcellular localization of endogenous, gene-edited Tau differs from that of over-expressed human wild-type and P301L mutant Tau

    PubMed Central

    Di Xia; Gutmann, Julia M.; Götz, Jürgen

    2016-01-01

    Alzheimer’s disease (AD) and a subset of frontotemporal dementia termed FTLD-Tau are characterized by a massive, yet incompletely characterized and understood redistribution of Tau. To establish a framework for understanding this pathology, we used the genome-editing tool TALEN and generated Tau-mEOS2 knock-in mice to determine the mobility and subcellular localization of endogenous Tau in hippocampal cultures. We analysed Tau in axons, dendrites and spines at three stages of maturation using live-cell imaging, photo-conversion and FRAP assays. Tau-mEOS2 cultures were compared with those over-expressing EGFP-tagged forms of human wild-type (hWT-Tau) and P301L mutant Tau (hP301L-Tau), modelling Tau accumulation in AD and FTLD-Tau, respectively. In developing neurons, Tau-mEOS2 followed a proximo-distal gradient in axons and a subcellular distribution similar to that of endogenous Tau in neurons obtained from wild-type mice, which were abolished, when either hWT-Tau or hP301L-Tau was over-expressed. For the three conditions, FRAP analysis revealed a similar mobility in dendrites compared with axons; however, Tau-mEOS2 was less mobile than hWT-Tau and hP301L-Tau and the mobile fraction was smaller, possibly reflecting less efficient microtubule binding of Tau when over-expressed. Together, our study presents Tau-mEOS2 mice as a novel tool for the study of Tau in a physiological and a pathological context. PMID:27378256

  13. Comparison of helper component-protease RNA silencing suppression activity, subcellular localization, and aggregation of three Korean isolates of Turnip mosaic virus.

    PubMed

    Han, Jae-Yeong; Chung, Jinsoo; Kim, Jungkyu; Seo, Eun-Young; Kilcrease, James P; Bauchan, Gary R; Lim, Seungmo; Hammond, John; Lim, Hyoun-Sub

    2016-08-01

    In 2014, we performed a nationwide survey in Korean radish fields to investigate the distribution and variability of Turnip mosaic virus (TuMV). Brassica rapa ssp. pekinensis sap-inoculated with three isolates of TuMV from infected radish tissue showed different symptom severities, whereas symptoms in Raphanus sativus were similar for each isolate. The helper component-protease (HC-Pro) genes of each isolate were sequenced, and phylogenetic analysis showed that the three Korean isolates were clustered into the basal-BR group. The HC-Pro proteins of these isolates were tested for their RNA silencing suppressor (VSR) activity and subcellular localization in Nicotiana benthamiana. A VSR assay by co-agroinfiltration of HC-Pro with soluble-modified GFP (smGFP) showed that HC-Pro of isolate R007 and R041 showed stronger VSR activity than R065. The HC-Pros showed 98.25 % amino acid identity, and weak VSR isolate (R065) has a single variant residue in the C-terminal domain associated with protease activity and self-interaction compared to isolates with strong VSR activity. Formation of large subcellular aggregates of GFP:HC-Pro fusion proteins in N. benthamiana was only observed for HC-Pro from isolates with strong VSR activity, suggesting that R065 'weak' HC-Pro may have diminished self-association; substitution of the variant C-terminal residue largely reversed the HC-Pro aggregation and silencing suppressor characteristics. The lack of correlation between VSR efficiency and induction of systemic necrosis (SN) suggests that differences in viral accumulation due to HC-Pro are not responsible for SN. PMID:27059238

  14. How a (sub)Cellular Coincidence Detection Mechanism Featuring Layer-5 Pyramidal Cells May Help Produce Various Visual Phenomena.

    PubMed

    Bachmann, Talis

    2015-01-01

    Perceptual phenomena such as spatio-temporal illusions and masking are typically explained by psychological (cognitive) processing theories or large-scale neural theories involving inter-areal connectivity and neural circuits comprising of hundreds or more interconnected single cells. Subcellular mechanisms are hardly used for such purpose. Here, a mechanistic theoretical view is presented on how a subcellular brain mechanism of integration of presynaptic signals that arrive at different compartments of layer-5 pyramidal neurons could explain a couple of spatiotemporal visual-phenomenal effects unfolding along very brief time intervals within the range of the sub-second temporal scale. PMID:26733926