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Sample records for acidic vesicular organelles

  1. Mapping organelle motion reveals a vesicular conveyor belt spatially replenishing secretory vesicles in stimulated chromaffin cells.

    PubMed

    Maucort, Guillaume; Kasula, Ravikiran; Papadopulos, Andreas; Nieminen, Timo A; Rubinsztein-Dunlop, Halina; Meunier, Frederic A

    2014-01-01

    How neurosecretory cells spatially adjust their secretory vesicle pools to replenish those that have fused and released their hormonal content is currently unknown. Here we designed a novel set of image analyses to map the probability of tracked organelles undergoing a specific type of movement (free, caged or directed). We then applied our analysis to time-lapse z-stack confocal imaging of secretory vesicles from bovine Chromaffin cells to map the global changes in vesicle motion and directionality occurring upon secretagogue stimulation. We report a defined region abutting the cortical actin network that actively transports secretory vesicles and is dissipated by actin and microtubule depolymerizing drugs. The directionality of this "conveyor belt" towards the cell surface is activated by stimulation. Actin and microtubule networks therefore cooperatively probe the microenvironment to transport secretory vesicles to the periphery, providing a mechanism whereby cells globally adjust their vesicle pools in response to secretagogue stimulation. PMID:24489879

  2. Skin delivery of ferulic acid from different vesicular systems.

    PubMed

    Chen, Ming; Liu, Xiangli; Fahr, Alfred

    2010-10-01

    The aim of the present research is to evaluate the skin delivery capabilities of different vesicular systems, including conventional liposomes (CL), Tween 80-based deformable liposomes (DL), invasomes (INS) and ethosomes bearing ferulic acid (FA) being an antioxidant exhibiting a wide range of therapeutic effects against various diseases. All of the test formulations were characterized for particle size distribution, zeta-potential, vesicular shape and surface morphology, in vitro human skin permeation and skin deposition. Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM) defined that all of liposomal vesicles were almost spherical, displaying unilamellar structures with low polydispersity (PDI < 0.2) and nanometric size range (z-average no more than 150 nm). In addition, all the vesicular systems except conventional liposomes were negatively charged to a certain extent. In vitro skin permeation and skin deposition experiments demonstrated that the permeation profile of ferulic acid through human stratum corneum epidermis membrane (SCE) and the drug deposition in skin were both improved significantly using these vesicular liposomal systems. Permeation and skin deposition enhancing effect was highlighted by the ethosomal system containing 18.0 mg/ml of ferulic acid with an significantly (P < 0.01) enhanced skin flux (267.8 +/- 16.77 microg/cm2/h) and skin drug deposition (51.67 +/- 1.94 microg/cm2), which was 75 times and 7.3 times higher than those of ferulic acid from saturated PBS (pH 7.4) solution, respectively. This study demonstrated that ethosomes are promising vesicular carriers for delivering ferulic acid into or across the skin. PMID:21329050

  3. Fluorogenic Substrates for Visualizing Acidic Organelle Enzyme Activities.

    PubMed

    Harlan, Fiona Karen; Lusk, Jason Scott; Mohr, Breanna Michelle; Guzikowski, Anthony Peter; Batchelor, Robert Hardy; Jiang, Ying; Naleway, John Joseph

    2016-01-01

    Lysosomes are acidic cytoplasmic organelles that are present in all nucleated mammalian cells and are involved in a variety of cellular processes including repair of the plasma membrane, defense against pathogens, cholesterol homeostasis, bone remodeling, metabolism, apoptosis and cell signaling. Defects in lysosomal enzyme activity have been associated with a variety of neurological diseases including Parkinson's Disease, Lysosomal Storage Diseases, Alzheimer's disease and Huntington's disease. Fluorogenic lysosomal staining probes were synthesized for labeling lysosomes and other acidic organelles in a live-cell format and were shown to be capable of monitoring lysosomal metabolic activity. The new targeted substrates were prepared from fluorescent dyes having a low pKa value for optimum fluorescence at the lower physiological pH found in lysosomes. They were modified to contain targeting groups to direct their accumulation in lysosomes as well as enzyme-cleavable functions for monitoring specific enzyme activities using a live-cell staining format. Application to the staining of cells derived from blood and skin samples of patients with Metachromatic Leukodystrophy, Krabbe and Gaucher Diseases as well as healthy human fibroblast and leukocyte control cells exhibited localization to the lysosome when compared with known lysosomal stain LysoTracker® Red DND-99 as well as with anti-LAMP1 Antibody staining. When cell metabolism was inhibited with chloroquine, staining with an esterase substrate was reduced, demonstrating that the substrates can be used to measure cell metabolism. When applied to diseased cells, the intensity of staining was reflective of lysosomal enzyme levels found in diseased cells. Substrates specific to the enzyme deficiencies in Gaucher or Krabbe disease patient cell lines exhibited reduced staining compared to that in non-diseased cells. The new lysosome-targeted fluorogenic substrates should be useful for research, diagnostics and

  4. Fluorogenic Substrates for Visualizing Acidic Organelle Enzyme Activities

    PubMed Central

    Harlan, Fiona Karen; Lusk, Jason Scott; Mohr, Breanna Michelle; Guzikowski, Anthony Peter; Batchelor, Robert Hardy; Jiang, Ying

    2016-01-01

    Lysosomes are acidic cytoplasmic organelles that are present in all nucleated mammalian cells and are involved in a variety of cellular processes including repair of the plasma membrane, defense against pathogens, cholesterol homeostasis, bone remodeling, metabolism, apoptosis and cell signaling. Defects in lysosomal enzyme activity have been associated with a variety of neurological diseases including Parkinson’s Disease, Lysosomal Storage Diseases, Alzheimer's disease and Huntington's disease. Fluorogenic lysosomal staining probes were synthesized for labeling lysosomes and other acidic organelles in a live-cell format and were shown to be capable of monitoring lysosomal metabolic activity. The new targeted substrates were prepared from fluorescent dyes having a low pKa value for optimum fluorescence at the lower physiological pH found in lysosomes. They were modified to contain targeting groups to direct their accumulation in lysosomes as well as enzyme-cleavable functions for monitoring specific enzyme activities using a live-cell staining format. Application to the staining of cells derived from blood and skin samples of patients with Metachromatic Leukodystrophy, Krabbe and Gaucher Diseases as well as healthy human fibroblast and leukocyte control cells exhibited localization to the lysosome when compared with known lysosomal stain LysoTracker® Red DND-99 as well as with anti-LAMP1 Antibody staining. When cell metabolism was inhibited with chloroquine, staining with an esterase substrate was reduced, demonstrating that the substrates can be used to measure cell metabolism. When applied to diseased cells, the intensity of staining was reflective of lysosomal enzyme levels found in diseased cells. Substrates specific to the enzyme deficiencies in Gaucher or Krabbe disease patient cell lines exhibited reduced staining compared to that in non-diseased cells. The new lysosome-targeted fluorogenic substrates should be useful for research, diagnostics and

  5. [High molecular weight protein detected in higher plant cells by antibodies against dynein is associated with vesicular organelles including Golgi apparatus].

    PubMed

    Shanina, N A; Lazareva, E M; Chentsov, Iu S; Smirnova, E A

    2008-01-01

    The cytoplasmic dynein is a multisubunit complex driving organelles along microtubules to their minus-end. We used antibodies against two functional domains (motor and microtubule-binding) of one of principal components of the complex--dynein heavy chain of slime mould Dictyostelium discoideum--to test root meristem cells of wheat Triticum aestivum. The antibodies reacted with a high molecular weight protein (> 500 kDa) in the total cell extract and the band recognized by the antibodies in plant extracts had a lower electrophoretic mobility than the high molecular weight band of mammalian dynein. Antibodies coupled to protein A-Sepharose precipitated the high molecular weight protein from the purified cell extracts. Immunocytochemical analysis demonstrated that the antigen recognized by antibodies against dynein heavy chains is associated with the vesicles whose localization depends on the cell cycle stage. The antigen-positive vesicles were localized to the perinuclear region in interphase and early prophase, to the spindle periphery and to spindle pole region during mitosis, and to the interzonal region in the period of fragmoplast and cell plate formation. Some antigen-positive vesicles also reacted with antibodies against Golgi protein markers. The obtained data indicate that higher plant cells contain a high molecular weight protein interacting with antibodies against the motor and microtubules-binding domains of Dictyostelium dynein heavy chain. The revealed antigen was associated with the vesicular structures in the cytoplasm including the Golgi apparatus. PMID:18409378

  6. Ca2+/H+ exchange by acidic organelles regulates cell migration in vivo.

    PubMed

    Melchionda, Manuela; Pittman, Jon K; Mayor, Roberto; Patel, Sandip

    2016-03-28

    Increasing evidence implicates Ca(2+) in the control of cell migration. However, the underlying mechanisms are incompletely understood. Acidic Ca(2+) stores are fast emerging as signaling centers. But how Ca(2+) is taken up by these organelles in metazoans and the physiological relevance for migration is unclear. Here, we identify a vertebrate Ca(2+)/H(+)exchanger (CAX) as part of a widespread family of homologues in animals. CAX is expressed in neural crest cells and required for their migration in vivo. It localizes to acidic organelles, tempers evoked Ca(2+) signals, and regulates cell-matrix adhesion during migration. Our data provide new molecular insight into how Ca(2+) is handled by acidic organelles and link this to migration, thereby underscoring the role of noncanonical Ca(2+) stores in the control of Ca(2+)-dependent function. PMID:27002171

  7. Inactivation of vesicular stomatitis virus by photosensitization following incubation with a pyrene-fatty acid.

    PubMed

    Rabia, S A; Fibach, E; Kotler, M; Gatt, S

    1990-09-17

    Vesicular stomatitis virus (VSV) was incubated with pyrene dodecanoic acid (P12), a fluorescent derivative of a medium-chain length fatty acid, and subjected to irradiation with a long wavelength ultra-violet light source at 366 nm (UVA). This inactivated the virus, resulting in a drastic decrease of its infectivity. The virus inactivation was proportional to the concentration of the pyrene-fatty acid, the length of exposure of the virus to P12 and the irradiation dose. PMID:2171994

  8. A novel fluorescent vesicular sensor for saccharides based on boronic acid-diol interaction.

    PubMed

    Zhang, Yujian; He, Zhenfeng; Li, Guowen

    2010-04-15

    A novel amphiphile containing two functional groups of both naphthalene and boronic acid, 2-(hexadecyloxy)-naphthalene-6-boronic acid (HNBA), has been synthesized. Scanning electron microscopy (SEM) indicated the formation of bilayer vesicles in the ethanol/water solution (Phi=0.6). Differential scanning calorimetry (DSC) established the presence of crystal-to-liquid crystal transition at 63.36 degrees C. The vesicular fluorescence properties upon binding with carbohydrates have been studied in ethanol/water buffer at pH 7.4. Addition of saccharides to the vesicular solution, the fluorescent intensities of naphthalene in HNBA vesicles centered at 348 nm decreased dramatically with increasing concentration of saccharides. The change tendency of fluorescent intensities of the HNAB vesicles with concentration of saccharides followed in the order of fructose>galactose>maltose>glucose. The pH profiles of the fluorescence intensity were studied in the absence and in the presence of sugars. Also, the urine sample induced spectral changes of the HNBA vesicles were studied. These results suggest that the HNBA vesicles may be developed as a continuous monitoring and implantable fluorescence vesicular sensor, which might be applied in the practical field. PMID:20188967

  9. Membraneless organelles can melt nucleic acid duplexes and act as biomolecular filters

    NASA Astrophysics Data System (ADS)

    Nott, Timothy J.; Craggs, Timothy D.; Baldwin, Andrew J.

    2016-06-01

    Membraneless organelles are cellular compartments made from drops of liquid protein inside a cell. These compartments assemble via the phase separation of disordered regions of proteins in response to changes in the cellular environment and the cell cycle. Here we demonstrate that the solvent environment within the interior of these cellular bodies behaves more like an organic solvent than like water. One of the most-stable biological structures known, the DNA double helix, can be melted once inside the liquid droplet, and simultaneously structures formed from regulatory single-stranded nucleic acids are stabilized. Moreover, proteins are shown to have a wide range of absorption or exclusion from these bodies, and can act as importers for otherwise-excluded nucleic acids, which suggests the existence of a protein-mediated trafficking system. A common strategy in organic chemistry is to utilize different solvents to influence the behaviour of molecules and reactions. These results reveal that cells have also evolved this capability by exploiting the interiors of membraneless organelles.

  10. Membraneless organelles can melt nucleic acid duplexes and act as biomolecular filters.

    PubMed

    Nott, Timothy J; Craggs, Timothy D; Baldwin, Andrew J

    2016-06-01

    Membraneless organelles are cellular compartments made from drops of liquid protein inside a cell. These compartments assemble via the phase separation of disordered regions of proteins in response to changes in the cellular environment and the cell cycle. Here we demonstrate that the solvent environment within the interior of these cellular bodies behaves more like an organic solvent than like water. One of the most-stable biological structures known, the DNA double helix, can be melted once inside the liquid droplet, and simultaneously structures formed from regulatory single-stranded nucleic acids are stabilized. Moreover, proteins are shown to have a wide range of absorption or exclusion from these bodies, and can act as importers for otherwise-excluded nucleic acids, which suggests the existence of a protein-mediated trafficking system. A common strategy in organic chemistry is to utilize different solvents to influence the behaviour of molecules and reactions. These results reveal that cells have also evolved this capability by exploiting the interiors of membraneless organelles. PMID:27219701

  11. Synthesis and Proton NMR Spectroscopy of Intra-Vesicular Gamma-Aminobutyric Acid (GABA)*

    PubMed Central

    Wang, Luke Y.-J.; Tong, Rong; Kohane, Daniel S.

    2014-01-01

    We report the synthesis of vesicles containing gamma-aminobutyric acid (GABA), and their proton nuclear magnetic resonance (1H NMR) spectra. These vesicles were constructed to more closely mimic the intracellular environment wherein GABA exists. For this study, these GABA-containing vesicles were examined under 1H NMR as a potential platform for future studies on the differences between aqueous phantoms, ex vivo brain extracts, and in vivo magnetic resonance spectroscopy results. We found that intra-vesicular GABA faithfully yielded the chemical shifts and J-coupling constants of free aqueous GABA, alongside the chemical shift signals of the vesicle wall. PMID:24109882

  12. Vesicular γ-Aminobutyric Acid Transporter Expression in Amacrine and Horizontal Cells

    PubMed Central

    Cueva, Juan G.; Haverkamp, Silke; Reimer, Richard J.; Edwards, Robert; Wässle, Heinz; Brecha, Nicholas C.

    2010-01-01

    The vesicular γ-aminobutyric acid (GABA) transporter (VGAT), which transports the inhibitory amino acid transmitters GABA and glycine, is localized to synaptic vesicles in axon terminals. The localization of VGAT immunoreactivity to mouse and rat retina was evaluated with light and electron microscopy by using well-characterized VGAT antibodies. Specific VGAT immunoreactivity was localized to numerous varicose processes in all laminae of the inner plexiform layer (IPL) and to the outer plexiform layer (OPL). Amacrine cell somata characterized by weak VGAT immunoreactivity in the cytoplasm were located in the ganglion cell layer and proximal inner nuclear layer (INL) adjacent to the IPL. In rat retina, VGAT-immunoreactive cell bodies also contained GABA, glycine, or parvalbumin (PV) immunoreactivity, suggesting vesicular uptake of GABA or glycine by these cells. A few varicose VGAT-immunoreactive processes entered the OPL from the IPL. VGAT immunoreactivity in the OPL was predominantly localized to horizontal cell processes. VGAT and calcium binding protein-28K immunoreactivities (CaBP; a marker for horizontal cells) were colocalized in processes and terminals distributed to the OPL. Furthermore, VGAT immunoreactivity overlapped or was immediately adjacent to postsynaptic density-95 (PSD-95) immunoreactivity, which is prominent in photoreceptor terminals. Preem-bedding immunoelectron microscopy of mouse and rat retinae showed that VGAT immunoreactivity was localized to horizontal cell processes and their terminals. Immunoreactivity was distributed throughout the cytoplasm of the horizontal cell processes. Taken together, these findings demonstrate VGAT immunoreactivity in both amacrine and horizontal cell processes, suggesting these cells contain vesicles that accumulate GABA and glycine, possibly for vesicular release. PMID:11920703

  13. Lipid Body Organelles within the Parasite Trypanosoma cruzi: A Role for Intracellular Arachidonic Acid Metabolism

    PubMed Central

    Toledo, Daniel A. M.; Roque, Natália R.; Teixeira, Lívia; Milán-Garcés, Erix A.; Carneiro, Alan B.; Almeida, Mariana R.; Andrade, Gustavo F. S.; Martins, Jefferson S.; Pinho, Roberto R.; Freire-de-Lima, Célio G.; Bozza, Patrícia T.; D’Avila, Heloisa

    2016-01-01

    Most eukaryotic cells contain varying amounts of cytosolic lipidic inclusions termed lipid bodies (LBs) or lipid droplets (LDs). In mammalian cells, such as macrophages, these lipid-rich organelles are formed in response to host-pathogen interaction during infectious diseases and are sites for biosynthesis of arachidonic acid (AA)-derived inflammatory mediators (eicosanoids). Less clear are the functions of LBs in pathogenic lower eukaryotes. In this study, we demonstrated that LBs, visualized by light microscopy with different probes and transmission electron microscopy (TEM), are produced in trypomastigote forms of the parasite Trypanosoma cruzi, the causal agent of Chagas’ disease, after both host interaction and exogenous AA stimulation. Quantitative TEM revealed that LBs from amastigotes, the intracellular forms of the parasite, growing in vivo have increased size and electron-density compared to LBs from amastigotes living in vitro. AA-stimulated trypomastigotes released high amounts of prostaglandin E2 (PGE2) and showed PGE2 synthase expression. Raman spectroscopy demonstrated increased unsaturated lipid content and AA incorporation in stimulated parasites. Moreover, both Raman and MALDI mass spectroscopy revealed increased AA content in LBs purified from AA-stimulated parasites compared to LBs from unstimulated group. By using a specific technique for eicosanoid detection, we immunolocalized PGE2 within LBs from AA-stimulated trypomastigotes. Altogether, our findings demonstrate that LBs from the parasite Trypanosoma cruzi are not just lipid storage inclusions but dynamic organelles, able to respond to host interaction and inflammatory events and involved in the AA metabolism. Acting as sources of PGE2, a potent immunomodulatory lipid mediator that inhibits many aspects of innate and adaptive immunity, newly-formed parasite LBs may be implicated with the pathogen survival in its host. PMID:27490663

  14. Lipid Body Organelles within the Parasite Trypanosoma cruzi: A Role for Intracellular Arachidonic Acid Metabolism.

    PubMed

    Toledo, Daniel A M; Roque, Natália R; Teixeira, Lívia; Milán-Garcés, Erix A; Carneiro, Alan B; Almeida, Mariana R; Andrade, Gustavo F S; Martins, Jefferson S; Pinho, Roberto R; Freire-de-Lima, Célio G; Bozza, Patrícia T; D'Avila, Heloisa; Melo, Rossana C N

    2016-01-01

    Most eukaryotic cells contain varying amounts of cytosolic lipidic inclusions termed lipid bodies (LBs) or lipid droplets (LDs). In mammalian cells, such as macrophages, these lipid-rich organelles are formed in response to host-pathogen interaction during infectious diseases and are sites for biosynthesis of arachidonic acid (AA)-derived inflammatory mediators (eicosanoids). Less clear are the functions of LBs in pathogenic lower eukaryotes. In this study, we demonstrated that LBs, visualized by light microscopy with different probes and transmission electron microscopy (TEM), are produced in trypomastigote forms of the parasite Trypanosoma cruzi, the causal agent of Chagas' disease, after both host interaction and exogenous AA stimulation. Quantitative TEM revealed that LBs from amastigotes, the intracellular forms of the parasite, growing in vivo have increased size and electron-density compared to LBs from amastigotes living in vitro. AA-stimulated trypomastigotes released high amounts of prostaglandin E2 (PGE2) and showed PGE2 synthase expression. Raman spectroscopy demonstrated increased unsaturated lipid content and AA incorporation in stimulated parasites. Moreover, both Raman and MALDI mass spectroscopy revealed increased AA content in LBs purified from AA-stimulated parasites compared to LBs from unstimulated group. By using a specific technique for eicosanoid detection, we immunolocalized PGE2 within LBs from AA-stimulated trypomastigotes. Altogether, our findings demonstrate that LBs from the parasite Trypanosoma cruzi are not just lipid storage inclusions but dynamic organelles, able to respond to host interaction and inflammatory events and involved in the AA metabolism. Acting as sources of PGE2, a potent immunomodulatory lipid mediator that inhibits many aspects of innate and adaptive immunity, newly-formed parasite LBs may be implicated with the pathogen survival in its host. PMID:27490663

  15. Plasmalemmal and Vesicular γ-Aminobutyric Acid Transporter Expression in the Developing Mouse Retina

    PubMed Central

    GUO, CHENYING; STELLA, SALVATORE L.; HIRANO, ARLENE A.; BRECHA, NICHOLAS C.

    2009-01-01

    Plasmalemmal and vesicular γ-aminobutyric acid (GABA) transporters influence neurotransmission by regulating high-affinity GABA uptake and GABA release into the synaptic cleft and extracellular space. Postnatal expression of the plasmalemmal GABA transporter-1 (GAT-1), GAT-3, and the vesicular GABA/glycine transporter (VGAT) were evaluated in the developing mouse retina by using immunohistochemistry with affinity-purified antibodies. Weak transporter immunoreactivity was observed in the inner retina at postnatal day 0 (P0). GAT-1 immunostaining at P0 and at older ages was in amacrine and displaced amacrine cells in the inner nuclear layer (INL) and ganglion cell layer (GCL), respectively, and in their processes in the inner plexiform layer (IPL). At P10, weak GAT-1 immunostaining was in Müller cell processes. GAT-3 immunostaining at P0 and older ages was in amacrine cells and their processes, as well as in Müller cells and their processes that extended radially across the retina. At P10, Müller cell somata were observed in the middle of the INL. VGAT immunostaining was present at P0 and older ages in amacrine cells in the INL as well as processes in the IPL. At P5, weak VGAT immunostaining was also observed in horizontal cell somata and processes. By P15, the GAT and VGAT immunostaining patterns appear similar to the adult immunostaining patterns; they reached adult levels by about P20. These findings demonstrate that GABA uptake and release are initially established in the inner retina during the first postnatal week and that these systems subsequently mature in the outer retina during the second postnatal week. PMID:18975268

  16. Polymeric Nucleic Acid Vehicles Exploit Active Inter-Organelle Trafficking Mechanisms

    PubMed Central

    Fichter, Katye M.; Ingle, Nilesh. P.; McLendon, Patrick M.; Reineke, Theresa M.

    2013-01-01

    Materials that self-assemble with nucleic acids into nanocomplexes (polyplexes) are widely used in many fundamental biological and biomedical experiments. However, understanding the intracellular transport mechanisms of these vehicles remains a major hurdle in their effective usage. Here, we investigate two polycation models, Glycofect, (which slowly degrades via hydrolysis) and linear PEI, (which does not rapidly hydrolyze) to determine the impact of polymeric structure on intracellular trafficking. Cells transfected using Glycofect underwent increasing transgene expression over the course of 40 h, and remained benign over the course of 7 days. Transgene expression in cells transfected with PEI peaked at 16 h post-transfection and resulted in less than 10% survival after 7 days. While saccharide-containing Glycofect has a higher buffering capacity than PEI, polyplexes created with Glycofect demonstrate more sustained endosomal release, possibly suggesting an additional or alternative delivery mechanism to the classical “proton sponge mechanism”. PEI appeared to promote release of DNA from acidic organelles more than Glycofect. Immunofluorescence images indicate that both Glycofect and linear PEI traffic oligodeoxynucleotides (ODNs) to the Golgi and endoplasmic reticulum, which may be a route taken for nuclear delivery. However, Glycofect polyplexes demonstrated higher colocalization with the ER than PEI polyplexes and colocalization experiments indicate retrograde transport of polyplexes via COP I vesicles from the Golgi to the ER. We conclude that slow release and unique trafficking behaviors of Glycofect polyplexes may be due to the presence of saccharide units and the degradable nature of the polymer, allowing more efficacious and benign delivery. PMID:23234474

  17. Droplet organelles?

    PubMed

    Courchaine, Edward M; Lu, Alice; Neugebauer, Karla M

    2016-08-01

    Cells contain numerous, molecularly distinct cellular compartments that are not enclosed by lipid bilayers. These compartments are implicated in a wide range of cellular activities, and they have been variously described as bodies, granules, or organelles. Recent evidence suggests that a liquid-liquid phase separation (LLPS) process may drive their formation, possibly justifying the unifying term "droplet organelle". A veritable deluge of recent publications points to the importance of low-complexity proteins and RNA in determining the physical properties of phase-separated structures. Many of the proteins linked to such structures are implicated in human diseases, such as amyotrophic lateral sclerosis (ALS). We provide an overview of the organizational principles that characterize putative "droplet organelles" in healthy and diseased cells, connecting protein biochemistry with cell physiology. PMID:27357569

  18. Vesicular stomatitis.

    PubMed

    Timoney, Peter

    2016-07-30

    More than 800 premises in eight states in the USA have recently reported cases of vesicular stomatitis in their horses. Here, Peter Timoney, of the Gluck Equine Research Center in Kentucky, discusses this zoonotic disease in more detail. PMID:27474058

  19. The endoplasmic reticulum is a target organelle for trivalent dimethylarsinic acid (DMA{sup III})-induced cytotoxicity

    SciTech Connect

    Naranmandura, Hua; Xu, Shi; Koike, Shota; Pan, Li Qiang; Chen, Bin; Wang, Yan Wei; Rehman, Kanwal; Wu, Bin; Chen, Zhe; Suzuki, Noriyuki

    2012-05-01

    The purpose of present study was to characterize the endoplasmic reticulum stress and generation of ROS in rat liver RLC-16 cells by exposing to trivalent dimethylarsinous acid (DMA{sup III}) and compared with that of trivalent arsenite (iAs{sup III}) and monomethylarsonous acid (MMA{sup III}). Protein kinase-like endoplasmic reticulum kinase (PERK) phosphorylation was significantly induced in cells exposed to DMA{sup III}, while there was no change in phosphorylated PERK (P-PERK) detected in cells after exposure to iAs{sup III} or MMA{sup III}. The generation of reactive oxygen species (ROS) after DMA{sup III} exposure was found to take place specifically in the endoplasmic reticulum (ER), while previous reports showed that ROS was generated in mitochondria following exposure to MMA{sup III}. Meanwhile, cycloheximide (CHX) which is an inhibitor of protein biosynthesis strongly inhibited the DMA{sup III}-induced intracellular ROS generation in the ER and the phosphorylation of PERK, suggesting the induction of ER stress probably occurs through the inhibition of the protein folding process. Activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) mRNA were induced by all three arsenic species, however, evidence suggested that they might be induced by different pathways in the case of iAs{sup III} and MMA{sup III}. In addition, ER resident molecular chaperone glucose-regulated protein78 (GRP78) was not affected by trivalent arsenicals, while it was induced in positive control only at high concentration (Thapsigargin;Tg), suggesting the GRP78 is less sensitive to low levels of ER stress. In summary, our findings demonstrate that the endoplasmic reticulum is a target organelle for DMA{sup III}-induced cytotoxicity. Highlights: ►ER is a target organelle for trivalent DMA{sup III}-induced cytotoxicity. ►Generation of ROS in ER can be induced specially by trivalent DMA{sup III}. ►ER-stress and generation of ROS are caused by the increase in

  20. Increased Long Chain acyl-Coa Synthetase Activity and Fatty Acid Import Is Linked to Membrane Synthesis for Development of Picornavirus Replication Organelles

    PubMed Central

    Scott, Alison J.; Ford, Lauren A.; Pei, Zhengtong; Watkins, Paul A.; Ernst, Robert K.; Belov, George A.

    2013-01-01

    All positive strand (+RNA) viruses of eukaryotes replicate their genomes in association with membranes. The mechanisms of membrane remodeling in infected cells represent attractive targets for designing future therapeutics, but our understanding of this process is very limited. Elements of autophagy and/or the secretory pathway were proposed to be hijacked for building of picornavirus replication organelles. However, even closely related viruses differ significantly in their requirements for components of these pathways. We demonstrate here that infection with diverse picornaviruses rapidly activates import of long chain fatty acids. While in non-infected cells the imported fatty acids are channeled to lipid droplets, in infected cells the synthesis of neutral lipids is shut down and the fatty acids are utilized in highly up-regulated phosphatidylcholine synthesis. Thus the replication organelles are likely built from de novo synthesized membrane material, rather than from the remodeled pre-existing membranes. We show that activation of fatty acid import is linked to the up-regulation of cellular long chain acyl-CoA synthetase activity and identify the long chain acyl-CoA syntheatse3 (Acsl3) as a novel host factor required for polio replication. Poliovirus protein 2A is required to trigger the activation of import of fatty acids independent of its protease activity. Shift in fatty acid import preferences by infected cells results in synthesis of phosphatidylcholines different from those in uninfected cells, arguing that the viral replication organelles possess unique properties compared to the pre-existing membranes. Our data show how poliovirus can change the overall cellular membrane homeostasis by targeting one critical process. They explain earlier observations of increased phospholipid synthesis in infected cells and suggest a simple model of the structural development of the membranous scaffold of replication complexes of picorna-like viruses, that may be

  1. Synaptotagmin 1 is required for vesicular Ca²⁺/H⁺-antiport activity.

    PubMed

    Cordeiro, Joao Miguel; Boda, Bernadett; Gonçalves, Paula P; Dunant, Yves

    2013-07-01

    A low-affinity Ca²⁺/H⁺-antiport was described in the membrane of mammalian brain synaptic vesicles. Electrophysiological studies showed that this antiport contributes to the extreme brevity of excitation-release coupling in rapid synapses. Synaptotagmin-1, a vesicular protein interacting with membranes upon low-affinity Ca²⁺-binding, plays a major role in excitation-release coupling, by synchronizing calcium entry with fast neurotransmitter release. Here, we report that synaptotagmin-1 is necessary for expression of the vesicular Ca²⁺/H⁺-antiport. We measured Ca²⁺/H⁺-antiport activity in vesicles and granules of pheochromocytoma PC12 cells by three methods: (i) Ca²⁺-induced dissipation of the vesicular H⁺-gradient; (ii) bafilomycin-sensitive calcium accumulation and (iii) pH-jump-induced calcium accumulation. The results were congruent and highly significant: Ca²⁺/H⁺-antiport activity is detectable only in acidic organelles expressing functional synaptotagmin-1. In contrast, synaptotagmin-1-deficient cells--and cells where transgenically encoded synaptotagmin-1 was acutely photo-inactivated--were devoid of any Ca²⁺/H⁺-antiport activity. Therefore, in addition to its previously described functions, synaptotagmin-1 is involved in a rapid vesicular Ca²⁺ sequestration through a Ca²⁺/H⁺ antiport. PMID:23607712

  2. Two-pool model of cooperative vesicular transport

    NASA Astrophysics Data System (ADS)

    Bressloff, Paul C.

    2012-09-01

    We present a model of bidirectional vesicular transport between two intracellular organelles, which takes into account intermediate stages of transport that occur between vesicular budding from one organelle and subsequent fusion with the other organelle. These are incorporated into the model by associating with each organelle a donor pool of newly budded vesicles and an acceptor pool of transported vesicles ready for fusion. By constructing a system of differential equations that keeps track of the distribution of vesicles and protein concentrations within the various pools and along cytoskeletal tracks, we show how a stable steady state can emerge that consists of organelles that maintain different protein concentrations in spite of the continuous exchange of materials. In particular, exploiting the fact that the surface area of individual vesicles is much smaller than the surface area of organelles, we use an adiabatic approximation to eliminate the vesicular variables. This results in a major simplification of the dynamics and provides a systematic procedure for deriving phenomenological models of cooperative transport.

  3. Membraneless organelles: Phasing in and out

    NASA Astrophysics Data System (ADS)

    Shorter, James

    2016-06-01

    The low-complexity-protein, liquid phases of membraneless organelles have now been established to selectively partition biomolecules. The specialized microenvironment that they provide differs chemically from the surrounding medium and enables specific nucleic-acid remodelling reactions.

  4. First year sugar maple (Acer saccharum, Marsh. ) seedling nutrition, vesicular-arbuscular mycorrhizal colonization, physiology, and growth along an acidic deposition gradient in Michigan

    SciTech Connect

    McLaughlin, J.W.

    1992-01-01

    A field study was conducted to evaluate the use of foliar amino acid and root reducing sugar accumulations to separate acidic deposition from natural (i.e., soil phosphorus, mycorrhizae, and temperature) ecosystem stressors on first-year sugar maple seedling growth in three Michigan forests. Seedling growth was greatest at the sites exposed to highest levels of acidic deposition. However, sites receiving greatest acidic deposition rates also had high available soil phosphorus contents. No significant differences occurred, suggesting increased nitrogen loadings were not reflected in seedling tissue nitrogen. Seedling root or foliar calcium, magnesium, or potassium also were not significantly different, suggesting those elements were not growth limiting. Significant differences, however, occurred for seedling arginine and glutamine concentrations in foliage and reducing sugar concentrations in roots and were negatively correlated with seedling tissue phosphorus concentrations, suggesting phosphorus was limiting seedling growth at the low acidic deposition site. Vesicular-arbuscular mycorrhizal colonization of seedling roots was greater at the low acidic deposition site and positively correlated with seedling amino acid and reducing sugar accumulation but negatively correlated with sucrose concentrations in seedling roots, indicating that the fungal partner may have stimulated sucrose degradation to reducing sugars. Both air and soil temperatures were positively correlated with total sugar and sucrose concentrations in seedling roots. High levels of arginine, glutamine, and reducing sugars were negatively correlated with seedling growth indicating that seedlings at the low acidic deposition site were more stressed than seedlings at the sites receiving higher levels of pollutant loads. The results suggest differences in foliar arginine and glutamine and root reducing sugars in the forests in this study are likely due to natural rather than acidic deposition stress.

  5. Autophagy and proteins involved in vesicular trafficking.

    PubMed

    Amaya, Celina; Fader, Claudio Marcelo; Colombo, María Isabel

    2015-11-14

    Autophagy is an intracellular degradation system that, as a basic mechanism it delivers cytoplasmic components to the lysosomes in order to maintain adequate energy levels and cellular homeostasis. This complex cellular process is activated by low cellular nutrient levels and other stress situations such as low ATP levels, the accumulation of damaged proteins or organelles, or pathogen invasion. Autophagy as a multistep process involves vesicular transport events leading to tethering and fusion of autophagic vesicles with several intracellular compartments. This review summarizes our current understanding of the autophagic pathway with emphasis in the trafficking machinery (i.e. Rabs GTPases and SNAP receptors (SNAREs)) involved in specific steps of the pathway. PMID:26450776

  6. Acid-sensing ion channel (ASIC) 4 predominantly localizes to an early endosome-related organelle upon heterologous expression

    PubMed Central

    Schwartz, Verena; Friedrich, Katharina; Polleichtner, Georg; Gründer, Stefan

    2015-01-01

    Acid-sensing ion channels (ASICs) are voltage-independent proton-gated amiloride sensitive sodium channels, belonging to the DEG/ENaC gene family. Six different ASICs have been identified (ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, ASIC4) that are activated by a drop in extracellular pH, either as homo- or heteromers. An exception is ASIC4, which is not activated by protons as a homomer and which does not contribute to functional heteromeric ASICs. Insensitivity of ASIC4 to protons and its comparatively low sequence identity to other ASICs (45%) raises the question whether ASIC4 may have different functions than other ASICs. In this study, we therefore investigated the subcellular localization of ASIC4 in heterologous cell lines, which revealed a surprising accumulation of the channel in early endosome-related vacuoles. Moreover, we identified an unique amino-terminal motif as important for forward-trafficking from the ER/Golgi to the early endosome-related compartment. Collectively, our results show that heterologously expressed ASIC4 predominantly resides in an intracellular endosomal compartment. PMID:26667795

  7. Acid-sensing ion channel (ASIC) 4 predominantly localizes to an early endosome-related organelle upon heterologous expression.

    PubMed

    Schwartz, Verena; Friedrich, Katharina; Polleichtner, Georg; Gründer, Stefan

    2015-01-01

    Acid-sensing ion channels (ASICs) are voltage-independent proton-gated amiloride sensitive sodium channels, belonging to the DEG/ENaC gene family. Six different ASICs have been identified (ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, ASIC4) that are activated by a drop in extracellular pH, either as homo- or heteromers. An exception is ASIC4, which is not activated by protons as a homomer and which does not contribute to functional heteromeric ASICs. Insensitivity of ASIC4 to protons and its comparatively low sequence identity to other ASICs (45%) raises the question whether ASIC4 may have different functions than other ASICs. In this study, we therefore investigated the subcellular localization of ASIC4 in heterologous cell lines, which revealed a surprising accumulation of the channel in early endosome-related vacuoles. Moreover, we identified an unique amino-terminal motif as important for forward-trafficking from the ER/Golgi to the early endosome-related compartment. Collectively, our results show that heterologously expressed ASIC4 predominantly resides in an intracellular endosomal compartment. PMID:26667795

  8. Organic additive, 5-methylsalicylic acid induces spontaneous structural transformation of aqueous pluronic triblock copolymer solution: a spectroscopic investigation of interaction of curcumin with pluronic micellar and vesicular aggregates.

    PubMed

    Ghosh, Surajit; Kuchlyan, Jagannath; Banik, Debasis; Kundu, Niloy; Roy, Arpita; Banerjee, Chiranjib; Sarkar, Nilmoni

    2014-10-01

    This article presents the interaction of curcumin in the microenvironments provided by aggregation of pluronic triblock copolymer P123 into micellar and vesicular assemblies. The formation of vesicles using triblock copolymer P123 and 5-methylsalicylic acid (5 mS) has been successfully characterized by optical spectroscopy, light scattering measurement, and eventually microscopic techniques. Besides, to make a comparative study between the polymeric micelles, we have also investigated the photophysical changes of curcumin in F127 triblock copolymer micelles having variation in poly(ethylene oxide) (PPO) and poly(propylene oxide) (PEO) unit of polymer chain to that of P123. Time-dependent UV-vis measurement suggests that these polymer micelles are able to stabilize poorly water-soluble curcumin by suppressing the degradation rate in micellar nanocavity. However, experimental observations suggest that P123 micelles are more efficient than F127 to perturb excited state intramolecular proton transfer (ESIPT)-related nonradiative decay of curcumin. We also observed that rigid and confined microenvironment of P123/5 mS vesicles enhances emission intensity and lifetime of curcumin more compared to P123 micelles. All the observations suggest that modulation of photophysics of curcumin is responsible due to its interaction with poly(ethylene oxide) or poly(propylene oxide) unit of triblock copolymer. PMID:25192258

  9. The Amino Acid Substitution Q65H in the 2C Protein of Swine Vesicular Disease Virus Confers Resistance to Golgi Disrupting Drugs

    PubMed Central

    Vázquez-Calvo, Ángela; Caridi, Flavia; González-Magaldi, Mónica; Saiz, Juan-Carlos; Sobrino, Francisco; Martín-Acebes, Miguel A.

    2016-01-01

    Swine vesicular disease virus (SVDV) is a porcine pathogen and a member of the species Enterovirus B within the Picornaviridae family. Brefeldin A (BFA) is an inhibitor of guanine nucleotide exchange factors of Arf proteins that induces Golgi complex disassembly and alters the cellular secretory pathway. Since BFA has been shown to inhibit the RNA replication of different enteroviruses, including SVDV, we have analyzed the effect of BFA and of golgicide A (GCA), another Golgi disrupting drug, on SVDV multiplication. BFA and GCA similarly inhibited SVDV production. To investigate the molecular basis of the antiviral effect of BFA, SVDV mutants with increased resistance to BFA were isolated. A single amino acid substitution, Q65H, in the non-structural protein 2C was found to be responsible for increased resistance to BFA. These results provide new insight into the relationship of enteroviruses with the components of the secretory pathway and on the role of SVDV 2C protein in this process. PMID:27199941

  10. Insights into the mechanisms of sterol transport between organelles.

    PubMed

    Mesmin, Bruno; Antonny, Bruno; Drin, Guillaume

    2013-09-01

    In cells, the levels of sterol vary greatly among organelles. This uneven distribution depends largely on non-vesicular routes of transfer, which are mediated by soluble carriers called lipid-transfer proteins (LTPs). These proteins have a domain with a hydrophobic cavity that accommodates one sterol molecule. However, a demonstration of their role in sterol transport in cells remains difficult. Numerous LTPs also contain membrane-binding elements, but it is not clear how these LTPs couple their ability to target organelles with lipid transport activity. This issue appears critical, since many sterol transporters are thought to act at contact sites between two membrane-bound compartments. Here, we emphasize that biochemical and structural studies provide precious insights into the mode of action of sterol-binding proteins. Recent studies on START, Osh/ORP and NPC proteins suggest models on how these proteins could transport sterol between organelles and, thereby, influence cellular functions. PMID:23283302

  11. Podosomes: Multipurpose organelles?

    PubMed

    Veillat, Veronique; Spuul, Pirjo; Daubon, Thomas; Egaña, Isabel; Kramer, Ijsbrand; Génot, Elisabeth

    2015-08-01

    Thirty years of research have accumulated ample evidence that podosome clusters qualify as genuine cellular organelles that are being found in more and more cell types. A podosome is a dynamic actin-based and membrane-bound microdomain and the organelle consists in an interconnected network of such basic units, forming a cytoskeletal superstructure linked to the plasma membrane. At this strategic location, podosomes are privileged sites of interactions with the pericellular environment that regulates their formation, density, lifetime, distribution, architecture and functioning. Actin polymerization is the driving force behind most podosome characteristics. In contrast to classical organelles, podosomes are not vital at the cell level but rather serve diverse and often intricate functions of which adhesion, matrix degradation and substrate sensing are the most established. These capabilities involve specific molecules, depend on podosome organization and may vary according to the cell type in which they form. Podosome-associated diseases manifest by loss or gain of podosome functions and include genetic diseases affecting podosome components and various cancers where tumor cells ectopically express podosome equivalents (invadopodia). PMID:26028292

  12. New molecular mechanisms of inter-organelle lipid transport.

    PubMed

    Drin, Guillaume; von Filseck, Joachim Moser; Čopič, Alenka

    2016-04-15

    Lipids are precisely distributed in cell membranes, along with associated proteins defining organelle identity. Because the major cellular lipid factory is the endoplasmic reticulum (ER), a key issue is to understand how various lipids are subsequently delivered to other compartments by vesicular and non-vesicular transport pathways. Efforts are currently made to decipher how lipid transfer proteins (LTPs) work either across long distances or confined to membrane contact sites (MCSs) where two organelles are at close proximity. Recent findings reveal that proteins of the oxysterol-binding protein related-proteins (ORP)/oxysterol-binding homology (Osh) family are not all just sterol transporters/sensors: some can bind either phosphatidylinositol 4-phosphate (PtdIns(4)P) and sterol or PtdIns(4)P and phosphatidylserine (PS), exchange these lipids between membranes, and thereby use phosphoinositide metabolism to create cellular lipid gradients. Lipid exchange is likely a widespread mechanism also utilized by other LTPs to efficiently trade lipids between organelle membranes. Finally, the discovery of more proteins bearing a lipid-binding module (SMP or START-like domain) raises new questions on how lipids are conveyed in cells and how the activities of different LTPs are coordinated. PMID:27068959

  13. Chloride in vesicular trafficking and function.

    PubMed

    Stauber, Tobias; Jentsch, Thomas J

    2013-01-01

    Luminal acidification is of pivotal importance for the physiology of the secretory and endocytic pathways and its diverse trafficking events. Acidification by the proton-pumping V-ATPase requires charge compensation by counterion currents that are commonly attributed to chloride. The molecular identification of intracellular chloride transporters and the improvement of methodologies for measuring intraorganellar pH and chloride have facilitated the investigation of the physiology of vesicular chloride transport. New data question the requirement of chloride for pH regulation of various organelles and furthermore ascribe functions to chloride that are beyond merely electrically shunting the proton pump. This review surveys the currently established and proposed intracellular chloride transporters and gives an overview of membrane-trafficking steps that are affected by the perturbation of chloride transport. Finally, potential mechanisms of membrane-trafficking modulation by chloride are discussed and put into the context of organellar ion homeostasis in general. PMID:23092411

  14. Proteomics of Saccharomyces cerevisiae Organelles*

    PubMed Central

    Wiederhold, Elena; Veenhoff, Liesbeth M.; Poolman, Bert; Slotboom, Dirk Jan

    2010-01-01

    Knowledge of the subcellular localization of proteins is indispensable to understand their physiological roles. In the past decade, 18 studies have been performed to analyze the protein content of isolated organelles from Saccharomyces cerevisiae. Here, we integrate the data sets and compare them with other large scale studies on protein localization and abundance. We evaluate the completeness and reliability of the organelle proteomics studies. Reliability depends on the purity of the organelle preparations, which unavoidably contain (small) amounts of contaminants from different locations. Quantitative proteomics methods can be used to distinguish between true organellar constituents and contaminants. Completeness is compromised when loosely or dynamically associated proteins are lost during organelle preparation and also depends on the sensitivity of the analytical methods for protein detection. There is a clear trend in the data from the 18 organelle proteomics studies showing that proteins of low abundance frequently escape detection. Proteins with unknown function or cellular abundance are also infrequently detected, indicating that these proteins may not be expressed under the conditions used. We discuss that the yeast organelle proteomics studies provide powerful lead data for further detailed studies and that methodological advances in organelle preparation and in protein detection may help to improve the completeness and reliability of the data. PMID:19955081

  15. Diverse Bacterial Microcompartment Organelles

    PubMed Central

    Chowdhury, Chiranjit; Sinha, Sharmistha; Chun, Sunny; Yeates, Todd O.

    2014-01-01

    SUMMARY Bacterial microcompartments (MCPs) are sophisticated protein-based organelles used to optimize metabolic pathways. They consist of metabolic enzymes encapsulated within a protein shell, which creates an ideal environment for catalysis and facilitates the channeling of toxic/volatile intermediates to downstream enzymes. The metabolic processes that require MCPs are diverse and widely distributed and play important roles in global carbon fixation and bacterial pathogenesis. The protein shells of MCPs are thought to selectively control the movement of enzyme cofactors, substrates, and products (including toxic or volatile intermediates) between the MCP interior and the cytoplasm of the cell using both passive electrostatic/steric and dynamic gated mechanisms. Evidence suggests that specialized shell proteins conduct electrons between the cytoplasm and the lumen of the MCP and/or help rebuild damaged iron-sulfur centers in the encapsulated enzymes. The MCP shell is elaborated through a family of small proteins whose structural core is known as a bacterial microcompartment (BMC) domain. BMC domain proteins oligomerize into flat, hexagonally shaped tiles, which assemble into extended protein sheets that form the facets of the shell. Shape complementarity along the edges allows different types of BMC domain proteins to form mixed sheets, while sequence variation provides functional diversification. Recent studies have also revealed targeting sequences that mediate protein encapsulation within MCPs, scaffolding proteins that organize lumen enzymes and the use of private cofactor pools (NAD/H and coenzyme A [HS-CoA]) to facilitate cofactor homeostasis. Although much remains to be learned, our growing understanding of MCPs is providing a basis for bioengineering of protein-based containers for the production of chemicals/pharmaceuticals and for use as molecular delivery vehicles. PMID:25184561

  16. Dynein is the motor for retrograde axonal transport of organelles

    SciTech Connect

    Schnapp, B.J.; Reese, T.S.

    1989-03-01

    Vesicular organelles in axons of nerve cells are transported along microtubules either toward their plus ends (fast anterograde transport) or toward their minus ends (retrograde transport). Two microtubule-based motors were previously identified by examining plastic beads induced to move along microtubules by cytosol fractions from the squid giant axon: (i) an anterograde motor, kinesin, and (ii) a retrograde motor, which is characterized here. The retrograde motor, a cytosolic protein previously termed HMW1, was purified from optic lobes and extruded axoplasm by nucleotide-dependent microtubule affinity and release; microtubule gliding was used as the assay of motor activity. The following properties of the retrograde motor suggest that it is cytoplasmic dynein: (i) sedimentation at 20-22 S with a heavy chain of Mr greater than 200,000 that coelectrophoreses with the alpha and beta subunits of axonemal dynein, (ii) cleavage by UV irradiation in the presence of ATP and vanadate, and (iii) a molecular structure resembling two-headed dynein from axonemes. Furthermore, bead movement toward the minus end of microtubules was blocked when axoplasmic supernatants were treated with UV/vanadate. Treatment of axoplasmic supernatant with UV/vanadate also blocks the retrograde movement of purified organelles in vitro without changing the number of anterograde moving organelles, indicating that dynein interacts specifically with a subgroup of organelles programmed to move toward the cell body. However, purified optic lobe dynein, like purified kinesin, does not by itself promote the movement of purified organelles along microtubules, suggesting that additional axoplasmic factors are necessary for retrograde as well as anterograde transport.

  17. Inactivation of Vesicular Stomatitis Virus by Disinfectants

    PubMed Central

    Wright, Herbert S.

    1970-01-01

    Twenty-four chemical disinfectants considered to be viricidal were tested. Ten disinfectants were not viricidal for vesicular stomatitis virus within 10 min at 20 C when an LD50 titer of 108.5 virus units per 0.1 ml were to be inactivated. Quantitative inactivation experiments were done with acid, alkaline, and a substituted phenolic disinfectant to determine the kinetics of the virus inactivation. Substituted phenolic disinfectants, halogens, and cresylic and hydrochloric acids were viricidal. Basic compounds such as lye and sodium metasilicate were not viricidal. PMID:4313317

  18. Individual organelle pH determinations of magnetically-enriched endocytic organelles via laser-induced fluorescence detection

    PubMed Central

    Satori, Chad P.; Kostal, Vratislav; Arriaga, Edgar A.

    2011-01-01

    The analysis of biotransformations that occur in lysosomes and other endocytic organelles is critical to studies on intracellular degradation, nutrient recycling and lysosomal storage disorders. Such analyses require bioactive organelle preparations that are devoid of other contaminating organelles. Commonly used differential centrifugation techniques produce impure fractions and may not compatible with micro-scale separation platforms. Density gradient centrifugation procedures reduce the level of impurities but may compromise bioactivity. Here we report on simple magnetic setup and a procedure that produce highly enriched bioactive organelles based on their magnetic capture as they traveled through open tubes. Following capture, in-line laser-induced fluorecence detection (LIF) determined for the first time that each magnetically retained individual endocytic organelles have an acidic pH. Unlike bulk measurements, this method was suitable to describe the distributions of pH values in endocytic organelles from L6 rat myoblasts treated with dextran-coated iron oxide nanoparticles (for magnetic retention) and fluorescein/TMRM-conjugated dextran (for pH measurements by LIF). Their individual pH values ranged from 4 to 6, which is typical of bioactive endocytic organelles. These analytical procedures are of high relevance to evaluate lysosomal-related degradation pathways in aging, storage disorders and drug development. PMID:21863795

  19. Cell Biology of Prokaryotic Organelles

    PubMed Central

    Murat, Dorothee; Byrne, Meghan; Komeili, Arash

    2010-01-01

    Mounting evidence in recent years has challenged the dogma that prokaryotes are simple and undefined cells devoid of an organized subcellular architecture. In fact, proteins once thought to be the purely eukaryotic inventions, including relatives of actin and tubulin control prokaryotic cell shape, DNA segregation, and cytokinesis. Similarly, compartmentalization, commonly noted as a distinguishing feature of eukaryotic cells, is also prevalent in the prokaryotic world in the form of protein-bounded and lipid-bounded organelles. In this article we highlight some of these prokaryotic organelles and discuss the current knowledge on their ultrastructure and the molecular mechanisms of their biogenesis and maintenance. PMID:20739411

  20. Identification of regions within the Legionella pneumophila VipA effector protein involved in actin binding and polymerization and in interference with eukaryotic organelle trafficking.

    PubMed

    Bugalhão, Joana N; Mota, Luís Jaime; Franco, Irina S

    2016-02-01

    The Legionella pneumophila effector protein VipA is an actin nucleator that co-localizes with actin filaments and early endosomes in infected macrophages and which interferes with organelle trafficking when expressed in yeast. To identify the regions of VipA involved in its subcellular localization and functions, we ectopically expressed specific VipA mutant proteins in eukaryotic cells. This indicated that the characteristic punctate distribution of VipA depends on its NH2 -terminal (amino acid residues 1-133) and central coiled-coil (amino acid residues 133-206) regions, and suggested a role for the COOH-terminal (amino acid residues 206-339) region in association with actin filaments and for the NH2 -terminal in co-localization with early endosomes. Co-immunoprecipitation and in vitro assays showed that the COOH-terminal region of VipA is necessary and sufficient to mediate actin binding, and is essential but insufficient to induce microfilament formation. Assays in yeast revealed that the NH2 and the COOH-terminal regions, and possibly an NPY motif within the NH2 region of VipA, are necessary for interference with organelle trafficking. Overall, this suggests that subversion of eukaryotic vesicular trafficking by VipA involves both its ability to associate with early endosomes via its NH2 -terminal region and its capacity to bind and polymerize actin through its COOH-terminal region. PMID:26626407

  1. Real-time visualization of pH-responsive PLGA hollow particles containing a gas-generating agent targeted for acidic organelles for overcoming multi-drug resistance.

    PubMed

    Ke, Cherng-Jyh; Chiang, Wei-Lun; Liao, Zi-Xian; Chen, Hsin-Lung; Lai, Ping-Shan; Sun, Jui-Sheng; Sung, Hsing-Wen

    2013-01-01

    Chemotherapy research highly prioritizes overcoming the multi-drug resistance (MDR) effect in cancer cells. To overcome the drug efflux mediated by P-glycoprotein (P-gp) transporters, we developed pH-responsive poly(D,L-lactic-co-glycolic acid) hollow particles (PLGA HPs), capable of delivering doxorubicin (DOX) into MDR cells (MCF-7/ADR). The shell wall of PLGA HPs contained DiO (a hydrophobic dye), and their aqueous core carried DOX hydrochloride salt and sodium bicarbonate, a gas-generating agent when present in acidic environments. Both DiO and DOX could serve as fluorescence probes to localize HPs and visualize their intracellular drug release in real-time. Real-time confocal images provided visible evidences of the acid-responsive intracellular release of DOX from PLGA HPs in MDR cells. Via the macropinocytosis pathway, PLGA HPs taken up by cells experienced an increasingly acidic environment as they trafficked through the early endosomes and then matured into more acidic late endosomes/lysosomes. The progressive acidification of the internalized particles in the late endosomes/lysosomes generated CO(2) bubbles, leading to the disruption of HPs, prompt release of DOX, its accumulation in the nuclei, and finally the death of MDR cells. Conversely, taken up via a passive diffusion mechanism, free DOX was found mainly at the perimembrane region and barely reached the cell nuclei; therefore, no apparent cytotoxicity was observed. These results suggest that the developed PLGA HPs were less susceptible to the P-gp-mediated drug efflux in MDR cells and is a highly promising approach in chemotherapy. PMID:23044041

  2. Syntaxin 3 and SNAP-25 pairing, regulated by omega-3 docosahexaenoic acid, controls the delivery of rhodopsin for the biogenesis of cilia-derived sensory organelles, the rod outer segments.

    PubMed

    Mazelova, Jana; Ransom, Nancy; Astuto-Gribble, Lisa; Wilson, Michael C; Deretic, Dusanka

    2009-06-15

    The biogenesis of cilia-derived sensory organelles, the photoreceptor rod outer segments (ROS), is mediated by rhodopsin transport carriers (RTCs). The small GTPase Rab8 regulates ciliary targeting of RTCs, but their specific fusion sites have not been characterized. Here, we report that the Sec6/8 complex, or exocyst, is a candidate effector for Rab8. We also show that the Qa-SNARE syntaxin 3 is present in the rod inner segment (RIS) plasma membrane at the base of the cilium and displays a microtubule-dependent concentration gradient, whereas the Qbc-SNARE SNAP-25 is uniformly distributed in the RIS plasma membrane and the synapse. Treatment with omega-3 docosahexaenoic acid [DHA, 22:6(n-3)] causes increased co-immunoprecipitation and colocalization of SNAP-25 and syntaxin 3 at the base of the cilium, which results in the increased delivery of membrane to the ROS. This is particularly evident in propranolol-treated retinas, in which the DHA-mediated increase in SNARE pairing overcomes the tethering block, including dissociation of Sec8 into the cytosol. Together, our data indicate that the Sec6/8 complex, syntaxin 3 and SNAP-25 regulate rhodopsin delivery, probably by mediating docking and fusion of RTCs. We show further that DHA, an essential polyunsaturated fatty acid of the ROS, increases pairing of syntaxin 3 and SNAP-25 to regulate expansion of the ciliary membrane and ROS biogenesis. PMID:19454479

  3. Co-existence of Functionally Different Vesicular Neurotransmitter Transporters.

    PubMed

    Münster-Wandowski, Agnieszka; Zander, Johannes-Friedrich; Richter, Karin; Ahnert-Hilger, Gudrun

    2016-01-01

    The vesicular transmitter transporters VGLUT, VGAT, VMAT2 and VAChT, define phenotype and physiological properties of neuronal subtypes. VGLUTs concentrate the excitatory amino acid glutamate, VGAT the inhibitory amino acid GABA, VMAT2 monoamines, and VAChT acetylcholine (ACh) into synaptic vesicle (SV). Following membrane depolarization SV release their content into the synaptic cleft. A strict segregation of vesicular transporters is mandatory for the precise functioning of synaptic communication and of neuronal circuits. In the last years, evidence accumulates that subsets of neurons express more than one of these transporters leading to synaptic co-release of different and functionally opposing transmitters and modulation of synaptic plasticity. Synaptic co-existence of transporters may change during pathological scenarios in order to ameliorate misbalances in neuronal activity. In addition, evidence increases that transporters also co-exist on the same vesicle providing another layer of regulation. Generally, vesicular transmitter loading relies on an electrochemical gradient ΔμH(+) driven by the proton ATPase rendering the lumen of the vesicle with respect to the cytosol positive (Δψ) and acidic (ΔpH). While the activity of VGLUT mainly depends on the Δψ component, VMAT, VGAT and VAChT work best at a high ΔpH. Thus, a vesicular synergy of transporters depending on the combination may increase or decrease the filling of SV with the principal transmitter. We provide an overview on synaptic co-existence of vesicular transmitter transporters including changes in the excitatory/inhibitory balance under pathological conditions. Additionally, we discuss functional aspects of vesicular synergy of transmitter transporters. PMID:26909036

  4. Co-existence of Functionally Different Vesicular Neurotransmitter Transporters

    PubMed Central

    Münster-Wandowski, Agnieszka; Zander, Johannes-Friedrich; Richter, Karin; Ahnert-Hilger, Gudrun

    2016-01-01

    The vesicular transmitter transporters VGLUT, VGAT, VMAT2 and VAChT, define phenotype and physiological properties of neuronal subtypes. VGLUTs concentrate the excitatory amino acid glutamate, VGAT the inhibitory amino acid GABA, VMAT2 monoamines, and VAChT acetylcholine (ACh) into synaptic vesicle (SV). Following membrane depolarization SV release their content into the synaptic cleft. A strict segregation of vesicular transporters is mandatory for the precise functioning of synaptic communication and of neuronal circuits. In the last years, evidence accumulates that subsets of neurons express more than one of these transporters leading to synaptic co-release of different and functionally opposing transmitters and modulation of synaptic plasticity. Synaptic co-existence of transporters may change during pathological scenarios in order to ameliorate misbalances in neuronal activity. In addition, evidence increases that transporters also co-exist on the same vesicle providing another layer of regulation. Generally, vesicular transmitter loading relies on an electrochemical gradient ΔμH+ driven by the proton ATPase rendering the lumen of the vesicle with respect to the cytosol positive (Δψ) and acidic (ΔpH). While the activity of VGLUT mainly depends on the Δψ component, VMAT, VGAT and VAChT work best at a high ΔpH. Thus, a vesicular synergy of transporters depending on the combination may increase or decrease the filling of SV with the principal transmitter. We provide an overview on synaptic co-existence of vesicular transmitter transporters including changes in the excitatory/inhibitory balance under pathological conditions. Additionally, we discuss functional aspects of vesicular synergy of transmitter transporters. PMID:26909036

  5. 9 CFR 311.32 - Vesicular diseases.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 2 2014-01-01 2014-01-01 false Vesicular diseases. 311.32 Section 311... CERTIFICATION DISPOSAL OF DISEASED OR OTHERWISE ADULTERATED CARCASSES AND PARTS § 311.32 Vesicular diseases. (a) Any carcass affected with vesicular disease shall be condemned if the condition is acute and if...

  6. 9 CFR 309.15 - Vesicular diseases.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 2 2012-01-01 2012-01-01 false Vesicular diseases. 309.15 Section 309... CERTIFICATION ANTE-MORTEM INSPECTION § 309.15 Vesicular diseases. (a) Immediate notification shall be given by... any livestock is found to be affected with a vesicular disease. (b) No livestock under quarantine...

  7. 9 CFR 311.32 - Vesicular diseases.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 2 2012-01-01 2012-01-01 false Vesicular diseases. 311.32 Section 311... CERTIFICATION DISPOSAL OF DISEASED OR OTHERWISE ADULTERATED CARCASSES AND PARTS § 311.32 Vesicular diseases. (a) Any carcass affected with vesicular disease shall be condemned if the condition is acute and if...

  8. 9 CFR 309.15 - Vesicular diseases.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 2 2011-01-01 2011-01-01 false Vesicular diseases. 309.15 Section 309... CERTIFICATION ANTE-MORTEM INSPECTION § 309.15 Vesicular diseases. (a) Immediate notification shall be given by... any livestock is found to be affected with a vesicular disease. (b) No livestock under quarantine...

  9. 9 CFR 311.32 - Vesicular diseases.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 2 2011-01-01 2011-01-01 false Vesicular diseases. 311.32 Section 311... CERTIFICATION DISPOSAL OF DISEASED OR OTHERWISE ADULTERATED CARCASSES AND PARTS § 311.32 Vesicular diseases. (a) Any carcass affected with vesicular disease shall be condemned if the condition is acute and if...

  10. 9 CFR 309.15 - Vesicular diseases.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 2 2013-01-01 2013-01-01 false Vesicular diseases. 309.15 Section 309... CERTIFICATION ANTE-MORTEM INSPECTION § 309.15 Vesicular diseases. (a) Immediate notification shall be given by... any livestock is found to be affected with a vesicular disease. (b) No livestock under quarantine...

  11. 9 CFR 309.15 - Vesicular diseases.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 2 2010-01-01 2010-01-01 false Vesicular diseases. 309.15 Section 309... CERTIFICATION ANTE-MORTEM INSPECTION § 309.15 Vesicular diseases. (a) Immediate notification shall be given by... any livestock is found to be affected with a vesicular disease. (b) No livestock under quarantine...

  12. 9 CFR 309.15 - Vesicular diseases.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 2 2014-01-01 2014-01-01 false Vesicular diseases. 309.15 Section 309... CERTIFICATION ANTE-MORTEM INSPECTION § 309.15 Vesicular diseases. (a) Immediate notification shall be given by... any livestock is found to be affected with a vesicular disease. (b) No livestock under quarantine...

  13. 9 CFR 311.32 - Vesicular diseases.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 2 2013-01-01 2013-01-01 false Vesicular diseases. 311.32 Section 311... CERTIFICATION DISPOSAL OF DISEASED OR OTHERWISE ADULTERATED CARCASSES AND PARTS § 311.32 Vesicular diseases. (a) Any carcass affected with vesicular disease shall be condemned if the condition is acute and if...

  14. 9 CFR 311.32 - Vesicular diseases.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 2 2010-01-01 2010-01-01 false Vesicular diseases. 311.32 Section 311... CERTIFICATION DISPOSAL OF DISEASED OR OTHERWISE ADULTERATED CARCASSES AND PARTS § 311.32 Vesicular diseases. (a) Any carcass affected with vesicular disease shall be condemned if the condition is acute and if...

  15. The assisted self-association of ATP4- by a poly(amino acid) [poly(Lys)] and its significance for cell organelles that contain high concentrations of nucleotides.

    PubMed

    Sigel, H; Corfù, N A

    1996-09-15

    The occurrence of high concentrations of ATP in certain cell organelles prompted us to study the self-association of ATP via the concentration dependence of the 1H-NMR chemical shifts for H2, H8 and H1' in D2O at pD 8.4 (25 degrees C) in the range 0.0025-0.4 M in the presence and absence of poly(alpha, L-lysine), where [Lys units] was 0.4 M. The experiment in the presence of poly(Lys) was repeated at pD 12.1. At pD 8.4, the poly(amino acid) is protonated, i.e. poly(H.Lys)n+, whereas at pD 12.1 only approximately 10% of the epsilon-amino groups are positively charged. The results in all three systems are consistent with the isodesmic model of indefinite non-cooperative stacking. The stacking tendency follows the series: ATP4- (K = 1.3 M-1; pD 8.4) < ATP4-/poly(H.Lys)n+ (K = 11.5 M-1; pD 8.4) > ATP4-/90% poly(Lys)/10% poly(H.Lys)n+ (K = 3.1 M-1; pD 12.1). It is evident that poly(H.Lys)n+ assists the association of ATP by a factor of approximately 10, and it is suggested that, via its positively charged epsilon-ammonium groups, poly(H.Lys)n+ acts as a matrix by aligning ATP4- ions via ionic interactions with the negatively charged phosphate residues. The intragranular concentrations of various constituents of several storage or secretory cell organelles, as reported in the literature, are tabulated. The chromaffin granules of the adrenal medulla and the dense granules of blood platelets contain particularly high concentrations of nucleotides ([ATP] is approximately 0.14 M in the chromaffin granules and 0.5 M in the dense granules of rabbit blood platelets) and amines, such as epinephrine or 5-hydroxytryptamine. These granules, and probably also the storage vesicles of certain neurons (which seem to have a similar composition), appear, if the total concentrations of the various solutes are considered, to be osmotically unstable, which means that the intragranular solutes must be associated. This aggregation is discussed, especially with regard to the nucleotides. PMID

  16. A single amino acid change resulting in loss of fluorescence of eGFP in a viral fusion protein confers fitness and growth advantage to the recombinant vesicular stomatitis virus

    SciTech Connect

    Dinh, Phat X.; Panda, Debasis; Das, Phani B.; Das, Subash C.; Das, Anshuman; Pattnaik, Asit K.

    2012-10-25

    Using a recombinant vesicular stomatitis virus encoding eGFP fused in-frame with an essential viral replication protein, the phosphoprotein P, we show that during passage in culture, the virus mutates the nucleotide C289 within eGFP of the fusion protein PeGFP to A or T, resulting in R97S/C amino acid substitution and loss of fluorescence. The resultant non-fluorescent virus exhibits increased fitness and growth advantage over its fluorescent counterpart. The growth advantage of the non-fluorescent virus appears to be due to increased transcription and replication activities of the PeGFP protein carrying the R97S/C substitution. Further, our results show that the R97S/C mutation occurs prior to accumulation of mutations that can result in loss of expression of the gene inserted at the G-L gene junction. These results suggest that fitness gain is more important for the recombinant virus than elimination of expression of the heterologous gene.

  17. Mechanisms of Polarized Organelle Distribution in Neurons

    PubMed Central

    Britt, Dylan J.; Farías, Ginny G.; Guardia, Carlos M.; Bonifacino, Juan S.

    2016-01-01

    Neurons are highly polarized cells exhibiting axonal and somatodendritic domains with distinct complements of cytoplasmic organelles. Although some organelles are widely distributed throughout the neuronal cytoplasm, others are segregated to either the axonal or somatodendritic domains. Recent findings show that organelle segregation is largely established at a pre-axonal exclusion zone (PAEZ) within the axon hillock. Polarized sorting of cytoplasmic organelles at the PAEZ is proposed to depend mainly on their selective association with different microtubule motors and, in turn, with distinct microtubule arrays. Somatodendritic organelles that escape sorting at the PAEZ can be subsequently retrieved at the axon initial segment (AIS) by a microtubule- and/or actin-based mechanism. Dynamic sorting along the PAEZ-AIS continuum can thus explain the polarized distribution of cytoplasmic organelles between the axonal and somatodendritic domains. PMID:27065809

  18. Vesicular transport and the Golgi apparatus in yeast.

    PubMed

    Yoda, K; Noda, Y

    2001-01-01

    Eukaryotic cells have developed a complex intracellular membrane system to divide the cell into various compartments where specific biochemical reactions are efficiently conducted locally. They also have developed systems to deliver appropriate materials to each specific compartment. Vesicular transport is a delivery system that also links most of the main organelles in the cell. The Golgi apparatus occupies the central position of the traffic between the endoplasmic reticulum and the endosome/vacuole/plasma membrane by maturating and sorting delivery of materials. Every important feature of vesicular transport has been identified by studying the Golgi apparatus, and the unicellular microorganism Saccharomyces cerevisiae has been an extremely excellent material for this study. Cycles of production and consumption of the transport vesicles by sorting the cargo, budding from the donor, tethering, docking and fusion to the target can now be explained to a large extent at the molecular level. The functional and structural aspects of the Golgi have also been well studied in the last decade. PMID:16232937

  19. Are vesicular neurotransmitter transporters potential treatment targets for temporal lobe epilepsy?

    PubMed Central

    Van Liefferinge, Joeri; Massie, Ann; Portelli, Jeanelle; Di Giovanni, Giuseppe; Smolders, Ilse

    2013-01-01

    The vesicular neurotransmitter transporters (VNTs) are small proteins responsible for packing synaptic vesicles with neurotransmitters thereby determining the amount of neurotransmitter released per vesicle through fusion in both neurons and glial cells. Each transporter subtype was classically seen as a specific neuronal marker of the respective nerve cells containing that particular neurotransmitter or structurally related neurotransmitters. More recently, however, it has become apparent that common neurotransmitters can also act as co-transmitters, adding complexity to neurotransmitter release and suggesting intriguing roles for VNTs therein. We will first describe the current knowledge on vesicular glutamate transporters (VGLUT1/2/3), the vesicular excitatory amino acid transporter (VEAT), the vesicular nucleotide transporter (VNUT), vesicular monoamine transporters (VMAT1/2), the vesicular acetylcholine transporter (VAChT) and the vesicular γ-aminobutyric acid (GABA) transporter (VGAT) in the brain. We will focus on evidence regarding transgenic mice with disruptions in VNTs in different models of seizures and epilepsy. We will also describe the known alterations and reorganizations in the expression levels of these VNTs in rodent models for temporal lobe epilepsy (TLE) and in human tissue resected for epilepsy surgery. Finally, we will discuss perspectives on opportunities and challenges for VNTs as targets for possible future epilepsy therapies. PMID:24009559

  20. The Amyloid Precursor Protein of Alzheimer's Disease Clusters at the Organelle/Microtubule Interface on Organelles that Bind Microtubules in an ATP Dependent Manner.

    PubMed

    Stevenson, James W; Conaty, Eliza A; Walsh, Rylie B; Poidomani, Paul J; Samoriski, Colin M; Scollins, Brianne J; DeGiorgis, Joseph A

    2016-01-01

    The amyloid precursor protein (APP) is a causal agent in the pathogenesis of Alzheimer's disease and is a transmembrane protein that associates with membrane-limited organelles. APP has been shown to co-purify through immunoprecipitation with a kinesin light chain suggesting that APP may act as a trailer hitch linking kinesin to its intercellular cargo, however this hypothesis has been challenged. Previously, we identified an mRNA transcript that encodes a squid homolog of human APP770. The human and squid isoforms share 60% sequence identity and 76% sequence similarity within the cytoplasmic domain and share 15 of the final 19 amino acids at the C-terminus establishing this highly conserved domain as a functionally import segment of the APP molecule. Here, we study the distribution of squid APP in extruded axoplasm as well as in a well-characterized reconstituted organelle/microtubule preparation from the squid giant axon in which organelles bind microtubules and move towards the microtubule plus-ends. We find that APP associates with microtubules by confocal microscopy and co-purifies with KI-washed axoplasmic organelles by sucrose density gradient fractionation. By electron microscopy, APP clusters at a single focal point on the surfaces of organelles and localizes to the organelle/microtubule interface. In addition, the association of APP-organelles with microtubules is an ATP dependent process suggesting that the APP-organelles contain a microtubule-based motor protein. Although a direct kinesin/APP association remains controversial, the distribution of APP at the organelle/microtubule interface strongly suggests that APP-organelles have an orientation and that APP like the Alzheimer's protein tau has a microtubule-based function. PMID:26814888

  1. The Amyloid Precursor Protein of Alzheimer’s Disease Clusters at the Organelle/Microtubule Interface on Organelles that Bind Microtubules in an ATP Dependent Manner

    PubMed Central

    Stevenson, James W.; Conaty, Eliza A.; Walsh, Rylie B.; Poidomani, Paul J.; Samoriski, Colin M.; Scollins, Brianne J.; DeGiorgis, Joseph A.

    2016-01-01

    The amyloid precursor protein (APP) is a causal agent in the pathogenesis of Alzheimer’s disease and is a transmembrane protein that associates with membrane-limited organelles. APP has been shown to co-purify through immunoprecipitation with a kinesin light chain suggesting that APP may act as a trailer hitch linking kinesin to its intercellular cargo, however this hypothesis has been challenged. Previously, we identified an mRNA transcript that encodes a squid homolog of human APP770. The human and squid isoforms share 60% sequence identity and 76% sequence similarity within the cytoplasmic domain and share 15 of the final 19 amino acids at the C-terminus establishing this highly conserved domain as a functionally import segment of the APP molecule. Here, we study the distribution of squid APP in extruded axoplasm as well as in a well-characterized reconstituted organelle/microtubule preparation from the squid giant axon in which organelles bind microtubules and move towards the microtubule plus-ends. We find that APP associates with microtubules by confocal microscopy and co-purifies with KI-washed axoplasmic organelles by sucrose density gradient fractionation. By electron microscopy, APP clusters at a single focal point on the surfaces of organelles and localizes to the organelle/microtubule interface. In addition, the association of APP-organelles with microtubules is an ATP dependent process suggesting that the APP-organelles contain a microtubule-based motor protein. Although a direct kinesin/APP association remains controversial, the distribution of APP at the organelle/microtubule interface strongly suggests that APP-organelles have an orientation and that APP like the Alzheimer’s protein tau has a microtubule-based function. PMID:26814888

  2. Glutamatergic or GABAergic neuron-specific, long-term expression in neocortical neurons from helper virus-free HSV-1 vectors containing the phosphate-activated glutaminase, vesicular glutamate transporter-1, or glutamic acid decarboxylase promoter

    PubMed Central

    Rasmussen, Morten; Kong, Lingxin; Zhang, Guo-rong; Liu, Meng; Wang, Xiaodan; Szabo, Gabor; Curthoys, Norman P.; Geller, Alfred I.

    2009-01-01

    Many potential uses of direct gene transfer into neurons require restricting expression to one of the two major types of forebrain neurons, glutamatergic or GABAergic neurons. Thus, it is desirable to develop virus vectors that contain either a glutamatergic or GABAergic neuron-specific promoter. The brain/kidney phosphate-activated glutaminase (PAG), the product of the GLS1 gene, produces the majority of the glutamate for release as neurotransmitter, and is a marker for glutamatergic neurons. A PAG promoter was partially characterized using a cultured kidney cell line. The three vesicular glutamate transporters (VGLUTs) are expressed in distinct populations of neurons, and VGLUT1 is the predominant VGLUT in the neocortex, hippocampus, and cerebellar cortex. Glutamic acid decarboxylase (GAD) produces GABA; the two molecular forms of the enzyme, GAD65 and GAD67, are expressed in distinct, but largely overlapping, groups of neurons, and GAD67 is the predominant form in the neocortex. In transgenic mice, an ∼9 kb fragment of the GAD67 promoter supports expression in most classes of GABAergic neurons. Here, we constructed plasmid (amplicon) Herpes Simplex Virus (HSV-1) vectors that placed the Lac Z gene under the regulation of putative PAG, VGLUT1, or GAD67 promoters. Helper virus-free vector stocks were delivered into postrhinal cortex, and the rats were sacrificed 4 days or 2 months later. The PAG or VGLUT1 promoters supported ∼90 % glutamatergic neuron-specific expression. The GAD67 promoter supported ∼90 % GABAergic neuron-specific expression. Long-term expression was observed using each promoter. Principles for obtaining long-term expression from HSV-1 vectors, based on these and other results, are discussed. Long-term glutamatergic or GABAergic neuron-specific expression may benefit specific experiments on learning or specific gene therapy approaches. Of note, promoter analyses might identify regulatory elements that determine a glutamatergic or GABAergic

  3. Lead exposure during synaptogenesis alters vesicular proteins and impairs vesicular release: potential role of NMDA receptor-dependent BDNF signaling.

    PubMed

    Neal, April P; Stansfield, Kirstie H; Worley, Paul F; Thompson, Richard E; Guilarte, Tomás R

    2010-07-01

    Lead (Pb(2+)) exposure is known to affect presynaptic neurotransmitter release in both in vivo and cell culture models. However, the precise mechanism by which Pb(2+) impairs neurotransmitter release remains unknown. In the current study, we show that Pb(2+) exposure during synaptogenesis in cultured hippocampal neurons produces the loss of synaptophysin (Syn) and synaptobrevin (Syb), two proteins involved in vesicular release. Pb(2+) exposure also increased the number of presynaptic contact sites. However, many of these putative presynaptic contact sites lack Soluble NSF attachment protein receptor complex proteins involved in vesicular exocytosis. Analysis of vesicular release using FM 1-43 dye confirmed that Pb(2+) exposure impaired vesicular release and reduced the number of fast-releasing sites. Because Pb(2+) is a potent N-methyl-D-aspartate receptor (NMDAR) antagonist, we tested the hypothesis that NMDAR inhibition may be producing the presynaptic effects. We show that NMDAR inhibition by aminophosphonovaleric acid mimics the presynaptic effects of Pb(2+) exposure. NMDAR activity has been linked to the signaling of the transsynaptic neurotrophin brain-derived neurotrophic factor (BDNF), and we observed that both the cellular expression of proBDNF and release of BDNF were decreased during the same period of Pb(2+) exposure. Furthermore, exogenous addition of BDNF rescued the presynaptic effects of Pb(2+). We suggest that the presynaptic deficits resulting from Pb(2+) exposure during synaptogenesis are mediated by disruption of NMDAR-dependent BDNF signaling. PMID:20375082

  4. Phospholipids of subcellular organelles isolated from cultured BHK cells.

    PubMed

    Brotherus, J; Renkonen, O

    1977-02-23

    Mitochondrial and nuclei were purified from cultured hamster fibroblasts (BHK21 cells) by centrifugation in sucrose gradients. The phospholipid compositions of the preparations were compared to those of the previously purified plasma membranes, endoplasmic reticulum and lysosomes. The mitochondria had a characteristically high content (approx. 16% of lipid phosphorus) of cardiolipin, which was practically absent from the other purified organelles. The nuclei were enriched in phosphatidylcholine and phosphatidylinositol (approx. 68% and 5% of lipid phosphorus, respectively). Lysobisphosphatidic acid was almost absent from the mitochondria and nuclei, as well as from the plasma membrane and endoplasmic reticulum, which suggests that this phospholipid is confined to the lysosomes of the BHK cell. The nuclei and the mitochondria contained relatively little sphingomyelin, a characteristic lipid of the plasma membrane. The distributions of the total cellular phospholipid and protein between the various organelles were calculated and compared to the corresponding data estimated for the rat liver. The BHK cell contained relatively more phospholipids in the nucleus and the lysosomes than the liver. All the organelles of the BHK cell contained less protein per phospholipid than the equivalent organelles of the liver. PMID:836856

  5. Deficient vesicular storage: A common theme in catecholaminergic neurodegeneration.

    PubMed

    Goldstein, David S; Holmes, Courtney; Sullivan, Patti; Mash, Deborah C; Sidransky, Ellen; Stefani, Alessandro; Kopin, Irwin J; Sharabi, Yehonatan

    2015-09-01

    Several neurodegenerative diseases involve loss of catecholamine neurons--Parkinson's disease (PD) is a prototypical example. Catecholamine neurons are rare in the nervous system, and why they are lost has been mysterious. Accumulating evidence supports the concept of "autotoxicity"--inherent cytotoxicity caused by catecholamine metabolites. Since vesicular sequestration limits the buildup of toxic products of enzymatic and spontaneous oxidation of catecholamines, a vesicular storage defect could play a pathogenic role in the death of catecholaminergic neurons in a variety of neurodegenerative diseases. In putamen, deficient vesicular storage is revealed in vivo by accelerated loss of (18)F-DOPA-derived radioactivity and post-mortem by decreased tissue dopamine (DA):DOPA ratios; in myocardium in vivo by accelerated loss of (18)F-dopamine-derived radioactivity and post-mortem by increased 3,4-dihydroxyphenylglycol:norepinephrine (DHPG:NE) ratios; and in sympathetic noradrenergic nerves overall in vivo by increased plasma F-dihydroxyphenylacetic acid (F-DOPAC):DHPG ratios. We retrospectively analyzed data from 20 conditions with decreased or intact catecholaminergic innervation, involving different etiologies, pathogenetic mechanisms, and lesion locations. All conditions involving parkinsonism had accelerated loss of putamen (18)F-DOPA-derived radioactivity; in those with post-mortem data there were also decreased putamen DA:DOPA ratios. All conditions involving cardiac sympathetic denervation had accelerated loss of myocardial (18)F-dopamine-derived radioactivity; in those with post-mortem data there were increased myocardial DHPG:NE ratios. All conditions involving localized loss of catecholaminergic innervation had evidence of decreased vesicular storage specifically in the denervated regions. Thus, across neurodegenerative diseases, loss of catecholaminergic neurons seems to be associated with decreased vesicular storage in the residual neurons. PMID:26255205

  6. GOBASE: an organelle genome database

    PubMed Central

    O’Brien, Emmet A.; Zhang, Yue; Wang, Eric; Marie, Veronique; Badejoko, Wole; Lang, B. Franz; Burger, Gertraud

    2009-01-01

    The organelle genome database GOBASE, now in its 21st release (June 2008), contains all published mitochondrion-encoded sequences (∼913 000) and chloroplast-encoded sequences (∼250 000) from a wide range of eukaryotic taxa. For all sequences, information on related genes, exons, introns, gene products and taxonomy is available, as well as selected genome maps and RNA secondary structures. Recent major enhancements to database functionality include: (i) addition of an interface for RNA editing data, with substitutions, insertions and deletions displayed using multiple alignments; (ii) addition of medically relevant information, such as haplotypes, SNPs and associated disease states, to human mitochondrial sequence data; (iii) addition of fully reannotated genome sequences for Escherichia coli and Nostoc sp., for reference and comparison; and (iv) a number of interface enhancements, such as the availability of both genomic and gene-coding sequence downloads, and a more sophisticated literature reference search functionality with links to PubMed where available. Future projects include the transfer of GOBASE features to NCBI/GenBank, allowing long-term preservation of accumulated expert information. The GOBASE database can be found at http://gobase.bcm.umontreal.ca/. Queries about custom and large-scale data retrievals should be addressed to gobase@bch.umontreal.ca. PMID:18953030

  7. Laser Surgery: Organelles to Organs

    NASA Astrophysics Data System (ADS)

    Berns, Michael W. D.

    1998-03-01

    Understanding the physical mechanisms of light interaction with biological molecules and structure has resulted in the application of photons to a wide variety of biological and medical problems ranging from subcellular manipulation/surgery to the successful diagnosis and treatment of human disease. Mechanisms such as the generation and transfer of heat, light-driven chemistry (photochemistry), high peak power acoustic-mechanical effects, high photon-energy induced bond breaking, and optical induced forces through momentum transfer, are being utilized in single cells at the microscopic (submicron and micron) level as well as the macroscopic level in tissue and organs. At the subcellular level, focused laser microbeams (laser scissors and tweezers) are being used to cut and move chromosomes to study genetic function as well as to clone and sequence genes. The same laser technology is being used to manipulate a variety of cell organelles such as mitochondria, cell membranes, nucleoli, and mitochondria in order to study their functions in cell physiology. At the tissue level, lasers are being used to diagnose and treat malignancy in combination with light-activated drugs, to ablate cornea and other hard and soft tissue through ultraviolet photoablation, to selectively ablate structures within the skin under controlled heating/cooling conditions, and to differentiate normal from abnormal tissue using a variety of fluorescence detection and light scattering techniques.

  8. Endosymbiotic theory for organelle origins.

    PubMed

    Zimorski, Verena; Ku, Chuan; Martin, William F; Gould, Sven B

    2014-12-01

    Endosymbiotic theory goes back over 100 years. It explains the similarity of chloroplasts and mitochondria to free-living prokaryotes by suggesting that the organelles arose from prokaryotes through (endo)symbiosis. Gene trees provide important evidence in favour of symbiotic theory at a coarse-grained level, but the finer we get into the details of branches in trees containing dozens or hundreds of taxa, the more equivocal evidence for endosymbiotic events sometimes becomes. It seems that either the interpretation of some endosymbiotic events are wrong, or something is wrong with the interpretations of some gene trees having many leaves. There is a need for evidence that is independent of gene trees and that can help outline the course of symbiosis in eukaryote evolution. Protein import is the strongest evidence we have for the single origin of chloroplasts and mitochondria. It is probably also the strongest evidence we have to sort out the number and nature of secondary endosymbiotic events that have occurred in evolution involving the red plastid lineage. If we relax our interpretation of individual gene trees, endosymbiotic theory can tell us a lot. PMID:25306530

  9. Transmission and pathogenesis of vesicular stomatitis viruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Vesicular Stomatitis (VS) is caused by the Vesicular Stomatitis Virus (VSV), a negative single stranded RNA arthropod-borne virus member of the Family Rhabdoviridae. The virion is composed of the host derived plasma membrane, the envelope, and an internal ribonucleoprotein core. The envelope contain...

  10. Cooperative protein transport in cellular organelles

    NASA Astrophysics Data System (ADS)

    Dmitrieff, S.; Sens, P.

    2011-04-01

    Compartmentalization into biochemically distinct organelles constantly exchanging material is one of the hallmarks of eukaryotic cells. In the most naive picture of interorganelle transport driven by concentration gradients, concentration differences between organelles should relax. We determine the conditions under which cooperative transport, i.e., based on molecular recognition, allows for the existence and maintenance of distinct organelle identities. Cooperative transport is also shown to control the flux of material transiting through a compartmentalized system, dramatically increasing the transit time under high incoming flux. By including chemical processing of the transported species, we show that this property provides a strong functional advantage to a system responsible for protein maturation and sorting.

  11. Vesicular monoamine transporter-2 and aromatic L-amino acid decarboxylase gene therapy prevents development of motor complications in parkinsonian rats after chronic intermittent L-3,4-dihydroxyphenylalanine administration.

    PubMed

    Lee, Won Yong; Lee, Eun Ah; Jeon, Mi Young; Kang, Ho Young; Park, Yong Gu

    2006-01-01

    Motor complications after chronic L-3,4-dihydroxyphenylalanine (L-DOPA) therapy occur partly because of the sensitization to dopaminergic agents resulting from pulsatile dopaminergic stimulation. The loss of presynaptic storage contributes to short duration of action by dopamine. Vesicular monoamine transporter-2 (VMAT-2) controls intraneuronal dopamine storage by packaging dopamine into synaptic vesicles, thereby allowing exocytotic release of dopamine. Using primary fibroblast doubly transduced with VMAT-2 and aromatic L-amino acid decarboxylase (AADC) genes, we previously demonstrated the beneficial effects of such double gene transduction in the production, storage, and gradual release of dopamine in vitro and in vivo. In this study, we further evaluate the effect of achieving sustained level of dopamine within the striata by VMAT-2 gene on behavioral response of parkinsonian rats after chronic intermittent L-DOPA administration. Primary fibroblast (PF) cells were genetically modified with AADC and VMAT-2 genes. We grafted primary fibroblast cells, PF with AADC (PFAADC), or doubly transduced PF with AADC and VMAT-2 (PFVMAA) (n = 6 for each group) into parkinsonian rat striata and administered L-DOPA (25 mg/kg/day) intermittently for 4 weeks. For behavioral study, we employed a model of akinesia using forepaw adjusting steps (FAS) that have been well characterized to reflect the effect of the lesion and the antiparkinsonian effect of dopaminergic drugs and transplants. The duration of FAS response to L-DOPA was sustained for a longer duration in rats grafted with PFVMAA cells than in those grafted with either control cells or cells with AADC alone. In PFVMAA-grafted animals, prolonged duration of FAS responses to L-DOPA was sustained even 6 weeks after discontinuation of 4-week intermittent L-DOPA treatment. These findings suggest that the restoration of dopamine storage capacity could enhance the efficacy of L-DOPA therapy and attenuate the motor fluctuations

  12. STEREOLOGICAL ESTIMATES OF THE BASAL FOREBRAIN CELL POPULATION IN THE RAT, INCLUDING NEURONS CONTAINING CHOLINE ACETYLTRANSFERASE (ChAT), GLUTAMIC ACID DECARBOXYLASE (GAD) OR PHOSPHATE-ACTIVATED GLUTAMINASE (PAG) AND COLOCALIZING VESICULAR GLUTAMATE TRANSPORTERS (VGluTs)

    PubMed Central

    GRITTI, I.; HENNY, P.; GALLONI, F.; MAINVILLE, L.; MARIOTTI, M.; JONES, B. E.

    2006-01-01

    The basal forebrain (BF) plays an important role in modulating cortical activity and influencing attention, learning and memory. These activities are fulfilled importantly yet not entirely by cholinergic neurons. Noncholinergic neurons also contribute and are comprised by GABAergic neurons and other possibly glutamatergic neurons. The aim of the present study was to estimate the total number of cells in the BF of the rat and the proportions of that total represented by cholinergic, GABAergic and glutamatergic neurons. For this purpose, cells were counted using unbiased stereological methods within the medial septum, diagonal band, magnocellular preoptic nucleus, substantia innominata and globus pallidus in sections stained for Nissl substance and/or the neurotransmitter enzymes, choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD) or phosphate-activated glutaminase (PAG). In Nissl-stained sections, the total number of neurons in the BF was estimated as ~355,000 and the numbers of ChAT-immuno-positive (+) as ~22,000, GAD+ ~119,000 and PAG+ ~316,000, corresponding to ~5%, ~35% and ~90% of the total. Thus, of the large population of BF neurons, only a small proportion has the capacity to synthesize acetylcholine (ACh), one third to synthesize GABA and the vast majority to synthesize glutamate (Glu). Moreover, through the presence of PAG, a proportion of ACh- and GABA-synthesizing neurons also have the capacity to synthesize Glu. In sections dual fluorescent immunostained for vesicular transporters, VGluT3 and not VGluT2 was present in the cell bodies of most PAG+ and ChAT+ and half the GAD+ cells. Given previous results showing that VGluT2 and not VGluT3 was present in BF axon terminals and not colocalized with VAChT or VGAT, we conclude that the BF cell population influences cortical and subcortical regions through neurons which release ACh, GABA or Glu from their terminals but which in part can also synthesize and release Glu from their soma or

  13. Vesicular transport across the fungal cell wall

    PubMed Central

    Casadevall, Arturo; Nosanchuk, Joshua D.; Williamson, Peter; Rodrigues, Marcio L.

    2014-01-01

    Recent findings indicate that fungi use vesicular transport to deliver substances across their cell walls. Fungal vesicles are similar to mammalian exosomes and could originate from cytoplasmic multivesicular bodies. Vesicular transport enables the export of large molecules across the cell wall, and vesicles contain lipids, proteins and polysaccharides, many of which are associated with virulence. Concentration of fungal products in vesicles could increase their efficiency in food acquisition and/or delivering potentially noxious substances to other cells, such as amoebae or phagocytes. The discovery of vesicular transport in fungi opens many new avenues for investigation in basic cell biology and pathogenesis. PMID:19299133

  14. The different facets of organelle interplay—an overview of organelle interactions

    PubMed Central

    Schrader, Michael; Godinho, Luis F.; Costello, Joseph L.; Islinger, Markus

    2015-01-01

    Membrane-bound organelles such as mitochondria, peroxisomes, or the endoplasmic reticulum (ER) create distinct environments to promote specific cellular tasks such as ATP production, lipid breakdown, or protein export. During recent years, it has become evident that organelles are integrated into cellular networks regulating metabolism, intracellular signaling, cellular maintenance, cell fate decision, and pathogen defence. In order to facilitate such signaling events, specialized membrane regions between apposing organelles bear distinct sets of proteins to enable tethering and exchange of metabolites and signaling molecules. Such membrane associations between the mitochondria and a specialized site of the ER, the mitochondria associated-membrane (MAM), as well as between the ER and the plasma membrane (PAM) have been partially characterized at the molecular level. However, historical and recent observations imply that other organelles like peroxisomes, lysosomes, and lipid droplets might also be involved in the formation of such apposing membrane contact sites. Alternatively, reports on so-called mitochondria derived-vesicles (MDV) suggest alternative mechanisms of organelle interaction. Moreover, maintenance of cellular homeostasis requires the precise removal of aged organelles by autophagy—a process which involves the detection of ubiquitinated organelle proteins by the autophagosome membrane, representing another site of membrane associated-signaling. This review will summarize the available data on the existence and composition of organelle contact sites and the molecular specializations each site uses in order to provide a timely overview on the potential functions of organelle interaction. PMID:26442263

  15. Bupropion increases striatal vesicular monoamine transport.

    PubMed

    Rau, Kristi S; Birdsall, Elisabeth; Hanson, Jarom E; Johnson-Davis, Kamisha L; Carroll, F Ivy; Wilkins, Diana G; Gibb, James W; Hanson, Glen R; Fleckenstein, Annette E

    2005-11-01

    The vesicular monoamine transporter-2 (VMAT-2) is principally involved in regulating cytoplasmic dopamine (DA) concentrations within terminals by sequestering free DA into synaptic vesicles. This laboratory previously identified a correlation between striatal vesicular DA uptake through VMAT-2 and inhibition of the DA transporter (DAT). For example, administration of methylphenidate (MPD), a DAT inhibitor, increases vesicular DA uptake through VMAT-2 in a purified vesicular preparation; an effect associated with a redistribution of VMAT-2 protein within DA terminals. The purpose of this study was to determine if other DAT inhibitors, including bupropion, similarly affect VMAT-2. Results revealed bupropion rapidly, reversibly, and dose-dependently increased vesicular DA uptake; an effect also associated with VMAT-2 protein redistribution. The bupropion-induced increase in vesicular DA uptake was prevented by pretreatment with eticlopride, a DA D2 receptor antagonist, but not by SCH23390, a DA D1 receptor antagonist. We previously reported that MPD post-treatment prevents persistent DA deficits associated with multiple methamphetamine (METH) administrations. Although bupropion attenuated the METH-induced reduction in VMAT-2 activity acutely, it did not prevent the long-term dopaminergic toxicity or the METH-induced redistribution of VMAT-2 protein. The findings from this study demonstrate similarities and differences in the mechanism by which MPD and bupropion affect striatal dopaminergic nerve terminals. PMID:16005476

  16. Regulation of the vesicular monoamine transporter-2: a novel mechanism for cocaine and other psychostimulants.

    PubMed

    Brown, J M; Hanson, G R; Fleckenstein, A E

    2001-03-01

    The plasmalemmal dopamine (DA) transporter (DAT) is a principal site of action for cocaine. This report presents the novel finding that in addition to inhibiting DAT function, cocaine administration rapidly alters vesicular DA transport. Specifically, cocaine treatment abruptly and reversibly increased both the V(max) of DA uptake and the B(max) of vesicular monoamine transporter-2 (VMAT-2) ligand (dihydrotetrabenazine) binding, as assessed ex vivo in purified rat striatal synaptic vesicles. Selective inhibitors of the DAT (amfonelic acid and GBR12935), but not the plasmalemmal serotonin transporter (fluoxetine), also increased vesicular DA uptake. Moreover, DA depletion resulting from administration of the tyrosine hydroxylase inhibitor alpha-methyl-p-tyrosine had cocaine-like effects. Conversely, administration of the DA-releasing agent methamphetamine rapidly decreased vesicular uptake. Taken together, these data demonstrate for the first time ex vivo that cocaine treatment rapidly alters vesicular monoamine transport, and suggest that alterations in cytoplasmic DA concentrations contribute to stimulant-induced changes in vesicular DA uptake. Hence, the VMAT-2 may be an important target for developing strategies to treat not only cocaine addiction but also other disorders involving alterations in neuronal DA disposition, including Parkinson's disease. PMID:11181904

  17. Organelle redox autonomy during environmental stress.

    PubMed

    Bratt, Avishay; Rosenwasser, Shilo; Meyer, Andreas; Fluhr, Robert

    2016-09-01

    Oxidative stress is generated in plants because of inequalities in the rate of reactive oxygen species (ROS) generation and scavenging. The subcellular redox state under various stress conditions was assessed using the redox reporter roGFP2 targeted to chloroplastic, mitochondrial, peroxisomal and cytosolic compartments. In parallel, the vitality of the plant was measured by ion leakage. Our results revealed that during certain physiological stress conditions the changes in roGFP2 oxidation are comparable to application of high concentrations of exogenous H2 O2 . Under each stress, particular organelles were affected. Conditions of extended dark stress, or application of elicitor, impacted chiefly on the status of peroxisomal redox state. In contrast, conditions of drought or high light altered the status of mitochondrial or chloroplast redox state, respectively. Amalgamation of the results from diverse environmental stresses shows cases of organelle autonomy as well as multi-organelle oxidative change. Importantly, organelle-specific oxidation under several stresses proceeded cell death as measured by ion leakage, suggesting early roGFP oxidation as predictive of cell death. The measurement of redox state in multiple compartments enables one to look at redox state connectivity between organelles in relation to oxidative stress as well as assign a redox fingerprint to various types of stress conditions. PMID:27037976

  18. Vesicular transport system in myotubes: ultrastructural study and signposting with vesicle-associated membrane proteins.

    PubMed

    Tajika, Yuki; Takahashi, Maiko; Khairani, Astrid Feinisa; Ueno, Hitoshi; Murakami, Tohru; Yorifuji, Hiroshi

    2014-04-01

    Myofibers have characteristic membrane compartments in their cytoplasm and sarcolemma, such as the sarcoplasmic reticulum, T-tubules, neuromuscular junction, and myotendinous junction. Little is known about the vesicular transport that is believed to mediate the development of these membrane compartments. We determined the locations of organelles in differentiating myotubes. Electron microscopic observation of a whole myotube revealed the arrangement of Golgi apparatus, rough endoplasmic reticulum, autolysosomes, mitochondria, and smooth endoplasmic reticulum from the perinuclear region toward the end of myotubes and the existence of a large number of vesicles near the ends of myotubes. Vesicles in myotubes were further characterized using immunofluorescence microscopy to analyze expression and localization of vesicle-associated membrane proteins (VAMPs). VAMPs are a family of seven proteins that regulate post-Golgi vesicular transport via the fusion of vesicles to the target membranes. Myotubes express five VAMPs in total. Vesicles with VAMP2, VAMP3, or VAMP5 were found near the ends of the myotubes. Some of these vesicles are also positive for caveolin-3, suggesting their participation in the development of T-tubules. Our morphological analyses revealed the characteristic arrangement of organelles in myotubes and the existence of transport vesicles near the ends of the myotubes. PMID:24263617

  19. Coexpression of Tyrosine Hydroxylase, GTP Cyclohydrolase I, Aromatic Amino Acid Decarboxylase, and Vesicular Monoamine Transporter 2 from a Helper Virus-Free Herpes Simplex Virus Type 1 Vector Supports High-Level, Long-Term Biochemical and Behavioral Correction of a Rat Model of Parkinson’s Disease

    PubMed Central

    SUN, MEI; KONG, LINGXIN; WANG, XIAODAN; HOLMES, COURTNEY; GAO, QINGSHENG; ZHANG, GUO-RONG; PFEILSCHIFTER, JOSEF; GOLDSTEIN, DAVID S.; GELLER, ALFRED I.

    2006-01-01

    Parkinson’s disease is due to the selective loss of nigrostriatal dopaminergic neurons. Consequently, many therapeutic strategies have focused on restoring striatal dopamine levels, including direct gene transfer to striatal cells, using viral vectors that express specific dopamine biosynthetic enzymes. The central hypothesis of this study is that coexpression of four dopamine biosynthetic and transporter genes in striatal neurons can support the efficient production and regulated, vesicular release of dopamine: tyrosine hydroxylase (TH) converts tyrosine to l-3,4-dihydroxyphenylalanine (l -DOPA), GTP cyclohydrolase I (GTP CH I) is the rate-limiting enzyme in the biosynthesis of the cofactor for TH, aromatic amino acid decarboxylase (AADC) converts l -DOPA to dopamine, and a vesicular monoamine transporter (VMAT-2) transports dopamine into synaptic vesicles, thereby supporting regulated, vesicular release of dopamine and relieving feedback inhibition of TH by dopamine. Helper virus-free herpes simplex virus type 1 vectors that coexpress the three dopamine biosynthetic enzymes (TH, GTP CH I, and AADC; 3-gene-vector) or these three dopamine biosynthetic enzymes and the vesicular monoamine transporter (TH, GTP CH I, AADC, and VMAT-2; 4-gene-vector) were compared. Both vectors supported production of dopamine in cultured fibroblasts. These vectors were microinjected into the striatum of 6-hydroxydopamine-lesioned rats. These vectors carry a modified neurofilament gene promoter, and γ-aminobutyric acid (GABA)-ergic neuron-specific gene expression was maintained for 14 months after gene transfer. The 4-gene-vector supported higher levels of correction of apomorphine-induced rotational behavior than did the 3-gene-vector, and this correction was maintained for 6 months. Proximal to the injection sites, the 4-gene-vector, but not the 3-gene-vector, supported extracellular levels of dopamine and dihydroxyphenylacetic acid (DOPAC) that were similar to those observed in

  20. Neurogenetics of vesicular transporters in C. elegans.

    PubMed

    Rand, J B; Duerr, J S; Frisby, D L

    2000-12-01

    The nematode Caenorhabditis elegans has a number of advantages for the analysis of synaptic molecules. These include a simple nervous system in which all cells are identified and synaptic connectivity is known and reproducible, a large collection of mutants and powerful methods of genetic analysis, simple methods for the generation and analysis of transgenic animals, and a number of relatively simple quantifiable behaviors. Studies in C. elegans have made major contributions to our understanding of vesicular transmitter transporters. Two of the four classes of vesicular transporters so far identified (VAChT and VGAT) were first described and cloned in C. elegans; in both cases, the genes were first identified and cloned by means of mutations causing a suggestive phenotype (1, 2). The phenotypes of eat-4 mutants and the cell biology of the EAT-4 protein were critical in the identification of this protein as the vesicular glutamate transporter (3, 4). In addition, the unusual gene structure associated with the cholinergic locus was first described in C. elegans (5). The biochemical properties of the nematode transporters are surprisingly similar to their vertebrate counterparts, and they can be assayed under similar conditions using the same types of mammalian cells (6, 7). In addition, mild and severe mutants (including knockouts) are available for each of the four C. elegans vesicular transporters, which has permitted a careful evaluation of the role(s) of vesicular transport in transmitter-specific behaviors. Accordingly, it seems appropriate at this time to present the current status of the field. In this review, we will first discuss the properties of C. elegans vesicular transporters and transporter mutants, and then explore some of the lessons and insights C. elegans research has provided to the field of vesicular transport. PMID:11099459

  1. Organelle morphogenesis by active membrane remodeling

    NASA Astrophysics Data System (ADS)

    Ramakrishnan, N.; Ipsen, John H.; Rao, Madan; Kumar, P. B. Sunil

    Intracellular organelles are subject to a steady flux of lipids and proteins through active, energy consuming transport processes. Active fission and fusion are promoted by GTPases, e.g., Arf-Coatamer and the Rab-Snare complexes, which both sense and generate local membrane curvature. Here we investigate through Dynamical Triangulation Monte Carlo simulations, the role that these active processes play in determining the morphology and compositional segregation in closed membranes. Our results suggest that the ramified morphologies of organelles observed in-vivo are a consequence of driven nonequilibrium processes rather than equilibrium forces.

  2. Designer amphiphilic proteins as building blocks for the intracellular formation of organelle-like compartments

    NASA Astrophysics Data System (ADS)

    Huber, Matthias C.; Schreiber, Andreas; von Olshausen, Philipp; Varga, Balázs R.; Kretz, Oliver; Joch, Barbara; Barnert, Sabine; Schubert, Rolf; Eimer, Stefan; Kele, Péter; Schiller, Stefan M.

    2015-01-01

    Nanoscale biological materials formed by the assembly of defined block-domain proteins control the formation of cellular compartments such as organelles. Here, we introduce an approach to intentionally ‘program’ the de novo synthesis and self-assembly of genetically encoded amphiphilic proteins to form cellular compartments, or organelles, in Escherichia coli. These proteins serve as building blocks for the formation of artificial compartments in vivo in a similar way to lipid-based organelles. We investigated the formation of these organelles using epifluorescence microscopy, total internal reflection fluorescence microscopy and transmission electron microscopy. The in vivo modification of these protein-based de novo organelles, by means of site-specific incorporation of unnatural amino acids, allows the introduction of artificial chemical functionalities. Co-localization of membrane proteins results in the formation of functionalized artificial organelles combining artificial and natural cellular function. Adding these protein structures to the cellular machinery may have consequences in nanobiotechnology, synthetic biology and materials science, including the constitution of artificial cells and bio-based metamaterials.

  3. Designer amphiphilic proteins as building blocks for the intracellular formation of organelle-like compartments.

    PubMed

    Huber, Matthias C; Schreiber, Andreas; von Olshausen, Philipp; Varga, Balázs R; Kretz, Oliver; Joch, Barbara; Barnert, Sabine; Schubert, Rolf; Eimer, Stefan; Kele, Péter; Schiller, Stefan M

    2015-01-01

    Nanoscale biological materials formed by the assembly of defined block-domain proteins control the formation of cellular compartments such as organelles. Here, we introduce an approach to intentionally 'program' the de novo synthesis and self-assembly of genetically encoded amphiphilic proteins to form cellular compartments, or organelles, in Escherichia coli. These proteins serve as building blocks for the formation of artificial compartments in vivo in a similar way to lipid-based organelles. We investigated the formation of these organelles using epifluorescence microscopy, total internal reflection fluorescence microscopy and transmission electron microscopy. The in vivo modification of these protein-based de novo organelles, by means of site-specific incorporation of unnatural amino acids, allows the introduction of artificial chemical functionalities. Co-localization of membrane proteins results in the formation of functionalized artificial organelles combining artificial and natural cellular function. Adding these protein structures to the cellular machinery may have consequences in nanobiotechnology, synthetic biology and materials science, including the constitution of artificial cells and bio-based metamaterials. PMID:25362355

  4. Identification of a mammalian vesicular polyamine transporter

    PubMed Central

    Hiasa, Miki; Miyaji, Takaaki; Haruna, Yuka; Takeuchi, Tomoya; Harada, Yuika; Moriyama, Sawako; Yamamoto, Akitsugu; Omote, Hiroshi; Moriyama, Yoshinori

    2014-01-01

    Spermine and spermidine act as neuromodulators upon binding to the extracellular site(s) of various ionotropic receptors, such as N-methyl-d-aspartate receptors. To gain access to the receptors, polyamines synthesized in neurons and astrocytes are stored in secretory vesicles and released upon depolarization. Although vesicular storage is mediated in an ATP-dependent, reserpine-sensitive fashion, the transporter responsible for this process remains unknown. SLC18B1 is the fourth member of the SLC18 transporter family, which includes vesicular monoamine transporters and vesicular acetylcholine transporter. Proteoliposomes containing purified human SLC18B1 protein actively transport spermine and spermidine by exchange of H+. SLC18B1 protein is predominantly expressed in the hippocampus and is associated with vesicles in astrocytes. SLC18B1 gene knockdown decreased both SLC18B1 protein and spermine/spermidine contents in astrocytes. These results indicated that SLC18B1 encodes a vesicular polyamine transporter (VPAT). PMID:25355561

  5. Different transporter systems regulate extracellular GABA from vesicular and non-vesicular sources

    PubMed Central

    Song, Inseon; Volynski, Kirill; Brenner, Tanja; Ushkaryov, Yuri; Walker, Matthew; Semyanov, Alexey

    2013-01-01

    Tonic GABA type A (GABAA) conductance is a key factor regulating neuronal excitability and computation in neuronal networks. The magnitude of the tonic GABAA conductance depends on the concentration of ambient GABA originating from vesicular and non-vesicular sources and is tightly regulated by GABA uptake. Here we show that the transport system regulating ambient GABA responsible for tonic GABAA conductances in hippocampal CA1 interneurons depends on its source. In mice, GABA from vesicular sources is regulated by mouse GABA transporter 1 (mGAT1), while that from non-vesicular sources by mouse GABA transporters 3/4 (mGAT3/4). This finding suggests that the two transporter systems do not just provide backup for each other, but regulate distinct signaling pathways. This allows individual tuning of the two signaling systems and indicates that drugs designed to act at specific transporters will have distinct therapeutic actions. PMID:23494150

  6. Review on Recent Advances in the Analysis of Isolated Organelles

    PubMed Central

    Satori, Chad P.; Kostal, Vratislav; Arriaga, Edgar A.

    2012-01-01

    The analysis of isolated organelles is one of the pillars of modern bioanalytical chemistry. This review describes recent developments on the isolation and characterization of isolated organelles both from living organisms and cell cultures. Salient reports on methods to release organelles focused on reproducibility and yield, membrane isolation, and integrated devices for organelle release. New developments on organelle fractionation after their isolation were on the topics of centrifugation, immunocapture, free flow electrophoresis, flow field-flow fractionation, fluorescence activated organelle sorting, laser capture microdissection, and dielectrophoresis. New concepts on characterization of isolated organelles included atomic force microscopy, optical tweezers combined with Raman spectroscopy, organelle sensors, flow cytometry, capillary electrophoresis, and microfluidic devices. PMID:23107131

  7. A Eukaryote without a Mitochondrial Organelle.

    PubMed

    Karnkowska, Anna; Vacek, Vojtěch; Zubáčová, Zuzana; Treitli, Sebastian C; Petrželková, Romana; Eme, Laura; Novák, Lukáš; Žárský, Vojtěch; Barlow, Lael D; Herman, Emily K; Soukal, Petr; Hroudová, Miluše; Doležal, Pavel; Stairs, Courtney W; Roger, Andrew J; Eliáš, Marek; Dacks, Joel B; Vlček, Čestmír; Hampl, Vladimír

    2016-05-23

    The presence of mitochondria and related organelles in every studied eukaryote supports the view that mitochondria are essential cellular components. Here, we report the genome sequence of a microbial eukaryote, the oxymonad Monocercomonoides sp., which revealed that this organism lacks all hallmark mitochondrial proteins. Crucially, the mitochondrial iron-sulfur cluster assembly pathway, thought to be conserved in virtually all eukaryotic cells, has been replaced by a cytosolic sulfur mobilization system (SUF) acquired by lateral gene transfer from bacteria. In the context of eukaryotic phylogeny, our data suggest that Monocercomonoides is not primitively amitochondrial but has lost the mitochondrion secondarily. This is the first example of a eukaryote lacking any form of a mitochondrion, demonstrating that this organelle is not absolutely essential for the viability of a eukaryotic cell. PMID:27185558

  8. Mitochondrial fission: rings around the organelle

    PubMed Central

    Pon, Liza A.

    2014-01-01

    Mitochondria form a dynamic network, in which organelles fuse or divide in response to metabolic changes or cellular stress. Inhibition of these processes leads to cell dysfunction and numerous human diseases. New work from several laboratories shows that mitochondria do not divide in isolation from other cellular structures. Rather, they carry out this process in partnership with the endoplasmic reticulum (ER) and actin filaments. PMID:23578876

  9. Ciliary Extracellular Vesicles: Txt Msg Organelles.

    PubMed

    Wang, Juan; Barr, Maureen M

    2016-04-01

    Cilia are sensory organelles that protrude from cell surfaces to monitor the surrounding environment. In addition to its role as sensory receiver, the cilium also releases extracellular vesicles (EVs). The release of sub-micron sized EVs is a conserved form of intercellular communication used by all three kingdoms of life. These extracellular organelles play important roles in both short and long range signaling between donor and target cells and may coordinate systemic responses within an organism in normal and diseased states. EV shedding from ciliated cells and EV-cilia interactions are evolutionarily conserved phenomena, yet remarkably little is known about the relationship between the cilia and EVs and the fundamental biology of EVs. Studies in the model organisms Chlamydomonas and Caenorhabditis elegans have begun to shed light on ciliary EVs. Chlamydomonas EVs are shed from tips of flagella and are bioactive. Caenorhabditis elegans EVs are shed and released by ciliated sensory neurons in an intraflagellar transport-dependent manner. Caenorhabditis elegans EVs play a role in modulating animal-to-animal communication, and this EV bioactivity is dependent on EV cargo content. Some ciliary pathologies, or ciliopathies, are associated with abnormal EV shedding or with abnormal cilia-EV interactions. Until the 21st century, both cilia and EVs were ignored as vestigial or cellular junk. As research interest in these two organelles continues to gain momentum, we envision a new field of cell biology emerging. Here, we propose that the cilium is a dedicated organelle for EV biogenesis and EV reception. We will also discuss possible mechanisms by which EVs exert bioactivity and explain how what is learned in model organisms regarding EV biogenesis and function may provide insight to human ciliopathies. PMID:26983828

  10. Polyadenylation of Vesicular Stomatitis Virus mRNA

    PubMed Central

    Ehrenfeld, Ellie

    1974-01-01

    Vesicular stomatitis virus (VSV) mRNA isolated from infected cell polysomes contains polyadenylic acid [poly(A)] sequences. Detergent-activated purified virions in vitro can transcribe complementary RNA, which has sedimentation properties similar to mRNA, and this RNA also contains poly(A) sequences. Digestion of virion RNA with U2 RNase under conditions where hydrolysis is specific for purine linkages leaves no sequences of polyuridylic acid corresponding in length to the poly(A) on the transcripts. Growth of infectious virus is not inhibited by 3-deoxyadenosine (cordycepin) under conditions in which it inhibits polyadenylation of cellular mRNA. The virus-specific mRNA produced in the presence of cordycepin has poly(A) sequences of the same size distribution as that synthesized in the absence of cordycepin. PMID:4363251

  11. Expression cloning of a reserpine-sensitive vesicular monoamine transporter.

    PubMed Central

    Erickson, J D; Eiden, L E; Hoffman, B J

    1992-01-01

    A cDNA for a rat vesicular monoamine transporter, designated MAT, was isolated by expression cloning in a mammalian cell line (CV-1). The cDNA sequence predicts a protein of 515 amino acids with 12 putative membrane-spanning domains. The characteristics of [3H]serotonin accumulation by CV-1 cells expressing the cDNA clone suggested sequestration by an intracellular compartment. In cells permeabilized with digitonin, uptake was ATP dependent with an apparent Km of 1.3 microM. Uptake was abolished by the proton-translocating ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone and with tri-(n-butyl)tin, an inhibitor of the vacuolar H(+)-ATPase. The rank order of potency to inhibit uptake was reserpine > tetrabenazine > serotonin > dopamine > norepinephrine > epinephrine. Direct comparison of [3H]monoamine uptake indicated that serotonin was the preferred substrate. Photolabeling of membranes prepared from CV-1 cells expressing MAT with 7-azido-8-[125I]iodoketanserin revealed a predominant tetrabenazine-sensitive photolabeled glycoprotein with an apparent molecular mass of approximately 75 kDa. The mRNA that encodes MAT was present specifically in monoamine-containing cells of the locus coeruleus, substantia nigra, and raphe nucleus of rat brain, each of which expresses a unique plasma membrane reuptake transporter. The MAT cDNA clone defines a vesicular monoamine transporter representing a distinct class of neurotransmitter transport molecules. Images PMID:1438304

  12. Organelle communication: signaling crossroads between homeostasis and disease.

    PubMed

    Bravo-Sagua, Roberto; Torrealba, Natalia; Paredes, Felipe; Morales, Pablo E; Pennanen, Christian; López-Crisosto, Camila; Troncoso, Rodrigo; Criollo, Alfredo; Chiong, Mario; Hill, Joseph A; Simmen, Thomas; Quest, Andrew F; Lavandero, Sergio

    2014-05-01

    Cellular organelles do not function as isolated or static units, but rather form dynamic contacts between one another that can be modulated according to cellular needs. The physical interfaces between organelles are important for Ca2+ and lipid homeostasis, and serve as platforms for the control of many essential functions including metabolism, signaling, organelle integrity and execution of the apoptotic program. Emerging evidence also highlights the importance of organelle communication in disorders such as Alzheimer's disease, pulmonary arterial hypertension, cancer, skeletal and cardiac muscle dysfunction. Here, we provide an overview of the current literature on organelle communication and the link to human pathologies. PMID:24534274

  13. Fluorescent Proteins in Cellular Organelles: Serious Pitfalls and Some Solutions

    PubMed Central

    Costantini, Lindsey M.

    2013-01-01

    Fluorescent proteins (FPs) have been powerful tools for cell biologists for over 15 years. The large variety of FPs available rarely comes with an instruction manual or a warning label. The potential pitfalls of the use of FPs in cellular organelles represent a significant concern for investigators. FPs generally did not evolve in the often distinctive physicochemical environments of subcellular organelles. In organelles, FPs can misfold, go dark, and even distort organelle morphology. In this minireview, we describe the issues associated with FPs in organelles and provide solutions to enable investigators to better exploit FP technology in cells. PMID:23971632

  14. Compartmentalization and Organelle Formation in Bacteria

    PubMed Central

    Cornejo, Elias; Abreu, Nicole; Komeili, Arash

    2015-01-01

    A number of bacterial species rely on compartmentalization to gain specific functionalities that will provide them with a selective advantage. Here, we will highlight several of these modes of bacterial compartmentalization with an eye towards describing the mechanisms of their formation and their evolutionary origins. Spore formation in Bacillus subtilis, outer membrane biogenesis in Gram-negative bacteria and protein diffusion barriers of Caulobacter crescentus will be used to demonstrate the physical, chemical and compositional remodeling events that lead to compartmentalization. In addition, magnetosomes and carboxysomes will serve as models to examine the interplay between cytoskeletal systems and the subcellular positioning of organelles. PMID:24440431

  15. Model of reversible vesicular transport with exclusion

    NASA Astrophysics Data System (ADS)

    Bressloff, Paul C.; Karamched, Bhargav R.

    2016-08-01

    A major question in neurobiology concerns the mechanics behind the motor-driven transport and delivery of vesicles to synaptic targets along the axon of a neuron. Experimental evidence suggests that the distribution of vesicles along the axon is relatively uniform and that vesicular delivery to synapses is reversible. A recent modeling study has made explicit the crucial role that reversibility in vesicular delivery to synapses plays in achieving uniformity in vesicle distribution, so called synaptic democracy (Bressloff et al 2015 Phys. Rev. Lett. 114 168101). In this paper we generalize the previous model by accounting for exclusion effects (hard-core repulsion) that may occur between molecular motor-cargo complexes (particles) moving along the same microtubule track. The resulting model takes the form of an exclusion process with four internal states, which distinguish between motile and stationary particles, and whether or not a particle is carrying vesicles. By applying a mean field approximation and an adiabatic approximation we reduce the system of ODEs describing the evolution of occupation numbers of the sites on a 1D lattice to a system of hydrodynamic equations in the continuum limit. We find that reversibility in vesicular delivery allows for synaptic democracy even in the presence of exclusion effects, although exclusion does exacerbate nonuniform distributions of vesicles in an axon when compared with a model without exclusion. We also uncover the relationship between our model and other models of exclusion processes with internal states.

  16. Organelle acidification is important for localisation of vacuolar proteins in Saccharomyces cerevisiae.

    PubMed

    Matsumoto, Risa; Suzuki, Kuninori; Ohya, Yoshikazu

    2013-12-01

    The acidic environments in the vacuole and other acidic organelles are important for many cellular processes in eukaryotic cells. In this study, we comprehensively investigated the roles of organelle acidification in vacuolar protein localisation in Saccharomyces cerevisiae. After repressing the acidification of acidic compartments by treatment with concanamycin A, a specific inhibitor of vacuolar H(+)-ATPase (V-ATPase), we examined the localisation of GFP-fused proteins that were predicted to localise in the vacuolar lumen or on the vacuolar membrane. Of the 73 proteins examined, 19 changed their localisation to the cytoplasmic region. Localisation changes were evaluated quantitatively using the image processing programme CalMorph. The delocalised proteins included vacuolar hydrolases, V-ATPase subunits, transporters and enzymes for membrane biogenesis, as well as proteins required for protein transport. These results suggest that many alterations in the localisation of vacuolar proteins occur after loss of the acidification of acidic compartments. PMID:23708375

  17. The function of genomes in bioenergetic organelles.

    PubMed Central

    Allen, John F

    2003-01-01

    Mitochondria and chloroplasts are energy-transducing organelles of the cytoplasm of eukaryotic cells. They originated as bacterial symbionts whose host cells acquired respiration from the precursor of the mitochondrion, and oxygenic photosynthesis from the precursor of the chloroplast. The host cells also acquired genetic information from their symbionts, eventually incorporating much of it into their own genomes. Genes of the eukaryotic cell nucleus now encode most mitochondrial and chloroplast proteins. Genes are copied and moved between cellular compartments with relative ease, and there is no obvious obstacle to successful import of any protein precursor from the cytosol. So why are any genes at all retained in cytoplasmic organelles? One proposal is that these small but functional genomes provide a location for genes that is close to, and in the same compartment as, their gene products. This co-location facilitates rapid and direct regulatory coupling. Redox control of synthesis de novo is put forward as the common property of those proteins that must be encoded and synthesized within mitochondria and chloroplasts. This testable hypothesis is termed CORR, for co-location for redox regulation. Principles, predictions and consequences of CORR are examined in the context of competing hypotheses and current evidence. PMID:12594916

  18. Coxiella burnetii effector protein subverts clathrin-mediated vesicular trafficking for pathogen vacuole biogenesis

    PubMed Central

    Larson, Charles L.; Beare, Paul A.; Howe, Dale; Heinzen, Robert A.

    2013-01-01

    Successful macrophage colonization by Coxiella burnetii, the cause of human Q fever, requires pathogen-directed biogenesis of a large, growth-permissive parasitophorous vacuole (PV) with phagolysosomal characteristics. The vesicular trafficking pathways co-opted by C. burnetii for PV development are poorly defined; however, it is predicted that effector proteins delivered to the cytosol by a defective in organelle trafficking/intracellular multiplication (Dot/Icm) type 4B secretion system are required for membrane recruitment. Here, we describe involvement of clathrin-mediated vesicular trafficking in PV generation and the engagement of this pathway by the C. burnetii type 4B secretion system substrate Coxiella vacuolar protein A (CvpA). CvpA contains multiple dileucine [DERQ]XXXL[LI] and tyrosine (YXXΦ)-based endocytic sorting motifs like those recognized by the clathrin adaptor protein (AP) complexes AP1, AP2, and AP3. A C. burnetii ΔcvpA mutant exhibited significant defects in replication and PV development, confirming the importance of CvpA in infection. Ectopically expressed mCherry-CvpA localized to tubular and vesicular domains of pericentrosomal recycling endosomes positive for Rab11 and transferrin receptor, and CvpA membrane interactions were lost upon mutation of endocytic sorting motifs. Consistent with CvpA engagement of the endocytic recycling system, ectopic expression reduced uptake of transferrin. In pull-down assays, peptides containing CvpA-sorting motifs and full-length CvpA interacted with AP2 subunits and clathrin heavy chain. Furthermore, depletion of AP2 or clathrin by siRNA treatment significantly inhibited C. burnetii replication. Thus, our results reveal the importance of clathrin-coated vesicle trafficking in C. burnetii infection and define a role for CvpA in subverting these transport mechanisms. PMID:24248335

  19. Genetic Evidence for the Role of the Vacuole in Supplying Secretory Organelles with Ca2+ in Hansenula polymorpha

    PubMed Central

    Fokina, Anastasia V.; Chechenova, Maria B.; Karginov, Azamat V.; Ter-Avanesyan, Michael D.; Agaphonov, Michael O.

    2015-01-01

    Processes taking place in the secretory organelles require Ca2+ and Mn2+, which in yeast are supplied by the Pmr1 ion pump. Here we observed that in the yeast Hansenula polymorpha Ca2+ deficiency in the secretory pathway caused by Pmr1 inactivation is exacerbated by (i) the ret1-27 mutation affecting COPI-mediated vesicular transport, (ii) inactivation of the vacuolar Ca2+ ATPase Pmc1 and (iii) inactivation of Vps35, which is a component of the retromer complex responsible for protein transport between the vacuole and secretory organelles. The ret1-27 mutation also exerted phenotypes indicating alterations in transport between the vacuole and secretory organelles. These data indicate that ret1-27, pmc1 and vps35 affect a previously unknown Pmr1-independent route of the Ca2+ delivery to the secretory pathway. We also observed that the vacuolar protein carboxypeptidase Y receives additional modifications of its glycoside chains if it escapes the Vps10-dependent sorting to the vacuole. PMID:26717478

  20. Optogenetic Control of Molecular Motors and Organelle Distributions in Cells

    PubMed Central

    Duan, Liting; Che, Daphne; Zhang, Kai; Ong, Qunxiang; Guo, Shunling; Cui, Bianxiao

    2015-01-01

    SUMMARY Intracellular transport and distribution of organelles play important roles in diverse cellular functions, including cell polarization, intracellular signaling, cell survival and apoptosis. Here we report an optogenetic strategy to control the transport and distribution of organelles by light. This is achieved by optically recruiting molecular motors onto organelles through the heterodimerization of Arabidopsis thaliana cryptochrome 2 (CRY2) and its interacting partner CIB1. CRY2 and CIB1 dimerize within subseconds upon blue light exposure, which requires no exogenous ligands and low intensity of light. We demonstrate that mitochondria, peroxisomes, and lysosomes can be driven towards the cell periphery upon light-induced recruitment of kinesin, or towards the cell nucleus upon recruitment of dynein. Light-induced motor recruitment and organelle movements are repeatable, reversible and can be achieved at subcellular regions. This light-controlled organelle redistribution provides a new strategy for studying the causal roles of organelle transport and distribution in cellular functions in living cells. PMID:25963241

  1. The Evolution of Per-cell Organelle Number.

    PubMed

    Cole, Logan W

    2016-01-01

    Organelles with their own distinct genomes, such as plastids and mitochondria, are found in most eukaryotic cells. As these organelles and their host cells have evolved, the partitioning of metabolic processes and the encoding of interacting gene products have created an obligate codependence. This relationship has played a role in shaping the number of organelles in cells through evolution. Factors such as stochastic evolutionary forces acting on genes involved in organelle biogenesis, organelle-nuclear gene interactions, and physical limitations may, to varying degrees, dictate the selective constraint that per-cell organelle number is under. In particular, coordination between nuclear and organellar gene expression may be important in maintaining gene product stoichiometry, which may have a significant role in constraining the evolution of this trait. PMID:27588285

  2. Requirements and standards for organelle genome databases

    SciTech Connect

    Boore, Jeffrey L.

    2006-01-09

    Mitochondria and plastids (collectively called organelles)descended from prokaryotes that adopted an intracellular, endosymbioticlifestyle within early eukaryotes. Comparisons of their remnant genomesaddress a wide variety of biological questions, especially when includingthe genomes of their prokaryotic relatives and the many genes transferredto the eukaryotic nucleus during the transitions from endosymbiont toorganelle. The pace of producing complete organellar genome sequences nowmakes it unfeasible to do broad comparisons using the primary literatureand, even if it were feasible, it is now becoming uncommon for journalsto accept detailed descriptions of genome-level features. Unfortunatelyno database is currently useful for this task, since they have littlestandardization and are riddled with error. Here I outline what iscurrently wrong and what must be done to make this data useful to thescientific community.

  3. Synthetic cells and organelles: compartmentalization strategies.

    PubMed

    Roodbeen, Renée; van Hest, Jan C M

    2009-12-01

    The recent development of RNA replicating protocells and capsules that enclose complex biosynthetic cascade reactions are encouraging signs that we are gradually getting better at mastering the complexity of biological systems. The road to truly cellular compartments is still very long, but concrete progress is being made. Compartmentalization is a crucial natural methodology to enable control over biological processes occurring within the living cell. In fact, compartmentalization has been considered by some theories to be instrumental in the creation of life. With the advancement of chemical biology, artificial compartments that can mimic the cell as a whole, or that can be regarded as cell organelles, have recently received much attention. The membrane between the inner and outer environment of the compartment has to meet specific requirements, such as semi-permeability, to allow communication and molecular transport over the border. The membrane can either be built from natural constituents or from synthetic polymers, introducing robustness to the capsule. PMID:19877005

  4. Biogenesis and architecture of arterivirus replication organelles.

    PubMed

    van der Hoeven, Barbara; Oudshoorn, Diede; Koster, Abraham J; Snijder, Eric J; Kikkert, Marjolein; Bárcena, Montserrat

    2016-07-15

    All eukaryotic positive-stranded RNA (+RNA) viruses appropriate host cell membranes and transform them into replication organelles, specialized micro-environments that are thought to support viral RNA synthesis. Arteriviruses (order Nidovirales) belong to the subset of +RNA viruses that induce double-membrane vesicles (DMVs), similar to the structures induced by e.g. coronaviruses, picornaviruses and hepatitis C virus. In the last years, electron tomography has revealed substantial differences between the structures induced by these different virus groups. Arterivirus-induced DMVs appear to be closed compartments that are continuous with endoplasmic reticulum membranes, thus forming an extensive reticulovesicular network (RVN) of intriguing complexity. This RVN is remarkably similar to that described for the distantly related coronaviruses (also order Nidovirales) and sets them apart from other DMV-inducing viruses analysed to date. We review here the current knowledge and open questions on arterivirus replication organelles and discuss them in the light of the latest studies on other DMV-inducing viruses, particularly coronaviruses. Using the equine arteritis virus (EAV) model system and electron tomography, we present new data regarding the biogenesis of arterivirus-induced DMVs and uncover numerous putative intermediates in DMV formation. We generated cell lines that can be induced to express specific EAV replicase proteins and showed that DMVs induced by the transmembrane proteins nsp2 and nsp3 form an RVN and are comparable in topology and architecture to those formed during viral infection. Co-expression of the third EAV transmembrane protein (nsp5), expressed as part of a self-cleaving polypeptide that mimics viral polyprotein processing in infected cells, led to the formation of DMVs whose size was more homogenous and closer to what is observed upon EAV infection, suggesting a regulatory role for nsp5 in modulating membrane curvature and DMV formation. PMID

  5. Organelles on the move: insights from yeast vacuole inheritance.

    PubMed

    Weisman, Lois S

    2006-04-01

    Organelle inheritance is one of several processes that occur during cell division. Recent studies on yeast vacuole inheritance have indicated rules that probably apply to most organelle-inheritance pathways. They have uncovered a molecular mechanism for membrane-cargo transport that is partially conserved from yeast to humans. They have also shown that the transport complex, which is composed of a molecular motor and its receptor, regulates the destination and timing of vacuole movement and might coordinate organelle movement with several other organelle functions. PMID:16607287

  6. Vesicular demyelination induced by raised intracellular calcium.

    PubMed

    Smith, K J; Hall, S M; Schauf, C L

    1985-11-01

    Incubation of nerve with high concentrations of the divalent cation ionophore A23187 produces myelin vesiculation (Schlaepfer 1977). This observation has now been extended using segments of rat ventral or dorsal root incubated with high (19 microM, 10 micrograms/ml) or low (1-1.5 microM) concentrations of A23187, or another divalent ionophore, ionomycin. Low concentrations of A23187 induced no vesiculation within a 2-h period. However, subsequent incubation of these roots in fresh, ionophore-free medium for 20 h, resulted in a prominent vesicular demyelination at the Schmidt-Lanterman incisures and paranodes of many fibres. At this time (22 h) the Schwann cells associated with some demyelinating internodes appeared vital upon ultrastructural examination: the cells also excluded the nuclear dye nigrosin. High concentrations of A23187 induced a similar vesicular demyelination in affected fibres within only 15-20 min. While the Schwann cells continued to exclude nigrosin for a further 4 h, their ultrastructural appearance indicated that they were probably in the early stages of necrosis. Incubation of moribund root with the ionophore produced no myelin vesiculation. At all ionophore concentrations, the myelin vesiculation was dependent upon the presence of extracellular Ca2+, and could be modulated in severity by varying this concentration. Other divalent cations (Ba2+, Co2+, Mg2+, Mn2+, Ni2+, Sr2+) could not substitute for Ca2+. The vesiculation induced by A23187 could be entirely prevented by the addition of Zn2+ (greater than or equal to 1 microM), Ni2+ (greater than or equal to 1-10 microM), Co2+ (greater than or equal to 100 microM) or Mn2+ (greater than or equal to 100 microM) to the bathing medium. A23187 applied to only part of an isolated internode resulted in a localization of the myelin disruption to that region. Ionomycin (greater than or equal to 1 microM), an ionophore with a greater selectivity for Ca2+ than A23187, also induced a prompt Ca2+-dependent

  7. Exocyst-Positive Organelles and Autophagosomes Are Distinct Organelles in Plants1[OPEN

    PubMed Central

    Lin, Youshun; Ding, Yu; Wang, Juan; Shen, Jinbo; Kung, Chun Hong; Zhuang, Xiaohong; Cui, Yong; Yin, Zhao; Xia, Yiji; Lin, Hongxuan; Robinson, David G.; Jiang, Liwen

    2015-01-01

    Autophagosomes are organelles that deliver cytosolic proteins for degradation in the vacuole of the cell. In contrast, exocyst-positive organelles (EXPO) deliver cytosolic proteins to the cell surface and therefore represent a form of unconventional protein secretion. Because both structures have two boundary membranes, it has been suggested that they may have been falsely treated as separate entities. Using suspension culture cells and root tissue cells of transgenic Arabidopsis (Arabidopsis thaliana) plants expressing either the EXPO marker Arabidopsis Exo70E2-GFP or the autophagosome marker yellow fluorescent protein (YFP)-autophagy-related gene 8e/f (ATG8e/f), and using specific antibodies against Exo70E2 and ATG8, we have now established that, in normally growing cells, EXPO and autophagosomes are distinct from one another. However, when cells/roots are subjected to autophagy induction, EXPO as well as autophagosomes fuse with the vacuole. In the presence of concanamycin A, the punctate fluorescent signals from both organelles inside the vacuole remain visible for hours and overlap to a significant degree. Tonoplast staining with FM4-64/YFP-Rab7-like GTPase/YFP-vesicle-associated membrane protein711 confirmed the internalization of tonoplast membrane concomitant with the sequestration of EXPO and autophagosomes. This suggests that EXPO and autophagosomes may be related to one another; however, whereas induction of autophagy led to an increase in the amount of ATG8 recruited to membranes, Exo70E2 did not respond in a similar manner. PMID:26358417

  8. Exocyst-Positive Organelles and Autophagosomes Are Distinct Organelles in Plants.

    PubMed

    Lin, Youshun; Ding, Yu; Wang, Juan; Shen, Jinbo; Kung, Chun Hong; Zhuang, Xiaohong; Cui, Yong; Yin, Zhao; Xia, Yiji; Lin, Hongxuan; Robinson, David G; Jiang, Liwen

    2015-11-01

    Autophagosomes are organelles that deliver cytosolic proteins for degradation in the vacuole of the cell. In contrast, exocyst-positive organelles (EXPO) deliver cytosolic proteins to the cell surface and therefore represent a form of unconventional protein secretion. Because both structures have two boundary membranes, it has been suggested that they may have been falsely treated as separate entities. Using suspension culture cells and root tissue cells of transgenic Arabidopsis (Arabidopsis thaliana) plants expressing either the EXPO marker Arabidopsis Exo70E2-GFP or the autophagosome marker yellow fluorescent protein (YFP)-autophagy-related gene 8e/f (ATG8e/f), and using specific antibodies against Exo70E2 and ATG8, we have now established that, in normally growing cells, EXPO and autophagosomes are distinct from one another. However, when cells/roots are subjected to autophagy induction, EXPO as well as autophagosomes fuse with the vacuole. In the presence of concanamycin A, the punctate fluorescent signals from both organelles inside the vacuole remain visible for hours and overlap to a significant degree. Tonoplast staining with FM4-64/YFP-Rab7-like GTPase/YFP-vesicle-associated membrane protein711 confirmed the internalization of tonoplast membrane concomitant with the sequestration of EXPO and autophagosomes. This suggests that EXPO and autophagosomes may be related to one another; however, whereas induction of autophagy led to an increase in the amount of ATG8 recruited to membranes, Exo70E2 did not respond in a similar manner. PMID:26358417

  9. A vesicular sequestration to oxidative deamination shift in myocardial sympathetic nerves in Parkinson's disease.

    PubMed

    Goldstein, David S; Sullivan, Patricia; Holmes, Courtney; Miller, Gary W; Sharabi, Yehonatan; Kopin, Irwin J

    2014-10-01

    In Parkinson's disease (PD), profound putamen dopamine (DA) depletion reflects denervation and a shift from vesicular sequestration to oxidative deamination of cytoplasmic DA in residual terminals. PD also involves cardiac sympathetic denervation. Whether PD entails myocardial norepinephrine (NE) depletion and a sequestration-deamination shift have been unknown. We measured apical myocardial tissue concentrations of NE, DA, and their neuronal metabolites 3,4-dihydroxyphenylglycol (DHPG), and 3,4-dihydroxyphenylacetic acid (DOPAC) from 23 PD patients and 23 controls and ascertained the extent of myocardial NE depletion in PD. We devised, validated in VMAT2-Lo mice, and applied 5 neurochemical indices of the sequestration-deamination shift-concentration ratios of DOPAC:DA, DA:NE, DHPG:NE, DOPAC:NE, and DHPG:DOPAC-and used a kinetic model to estimate the extent of the vesicular storage defect. The PD group had decreased myocardial NE content (p < 0.0001). The majority of patients (70%) had severe NE depletion (mean 2% of control), and in this subgroup all five indices of a sequestration-deamination shift were increased compared to controls (p < 0.001 for each). Vesicular storage in residual nerves was estimated to be decreased by 84-91% in this subgroup. We conclude that most PD patients have severe myocardial NE depletion, because of both sympathetic denervation and decreased vesicular storage in residual nerves. We found that the majority (70%) of Parkinson's disease (PD) patients have profound (98%) myocardial norepinephrine depletion, because of both cardiac sympathetic denervation and a shift from vesicular sequestration to oxidative deamination of cytoplasmic catecholamines in the residual nerves. This shift may be part of a final common pathogenetic pathway in the loss of catecholaminergic neurons that characterizes PD. PMID:24848581

  10. The Evolution of Per-cell Organelle Number

    PubMed Central

    Cole, Logan W.

    2016-01-01

    Organelles with their own distinct genomes, such as plastids and mitochondria, are found in most eukaryotic cells. As these organelles and their host cells have evolved, the partitioning of metabolic processes and the encoding of interacting gene products have created an obligate codependence. This relationship has played a role in shaping the number of organelles in cells through evolution. Factors such as stochastic evolutionary forces acting on genes involved in organelle biogenesis, organelle–nuclear gene interactions, and physical limitations may, to varying degrees, dictate the selective constraint that per-cell organelle number is under. In particular, coordination between nuclear and organellar gene expression may be important in maintaining gene product stoichiometry, which may have a significant role in constraining the evolution of this trait. PMID:27588285

  11. The number of symbiotic origins of organelles.

    PubMed

    Cavalier-Smith, T

    1992-01-01

    Mitochondria and chloroplasts both originated from bacterial endosymbionts. The available evidence strongly supports a single origin for mitochondria and only somewhat less strongly a single, slightly later, origin for chloroplasts. The arguments and evidence that have sometimes been presented in favor of the alternative theories of the multiple or polyphyletic origins of these two organelles are evaluated and the kinds of data that are needed to test more rigorously the monophyletic theory are discussed. Although chloroplasts probably originated only once, eukaryotic algae are polyphyletic because chloroplasts have been secondarily transferred to new lineages by the permanent incorporation of a photosynthetic eukaryotic algal cell into a phagotrophic protozoan host. How often this has happened is much less clear. It is particularly unclear whether or not the chloroplasts of typical dinoflagellates and euglenoids originated in this way from a eukaryotic symbiont: their direct divergence from the ancestral chloroplast cannot be ruled out and indeed has several arguments in its favor. The evidence for and against the view that the chloroplast of the kingdom Chromista was acquired in a single endosymbiotic event is discussed. The possibility that even the chloroplast of Chlorarachnion might have been acquired during the same symbiosis that created the cryptomonad cell, if the symbiont was a primitive alga that had chlorophyll a, b and c as well as phycobilins, is also considered. An alga with such a combination of pigments might have been ancestral to all eukaryote algae. PMID:1292670

  12. Chemotactic response of plant-growth-promoting bacteria towards roots of vesicular-arbuscular mycorrhizal tomato plants.

    PubMed

    Gupta Sood, Sushma

    2003-08-01

    The chemotactic responses of the plant-growth-promoting rhizobacteria Azotobacter chroococcum and Pseudomonas fluorescens to roots of vesicular-arbuscular mycorrhizal (Glomus fasciculatum) tomato plants were determined. A significantly (P=0.05) greater number of bacterial cells of wild strains were attracted towards vesicular-arbuscular mycorrhizal tomato roots compared to non-vesicular-arbuscular mycorrhizal tomato roots. Substances exuded by roots served as chemoattractants for these bacteria. P. fluorescens was strongly attracted towards citric and malic acids, which were predominant constituents in root exudates of tomato plants. A. chroococcum showed a stronger response towards sugars than amino acids, but the response was weakest towards organic acids. The effects of temperature, pH, and soil water matric potential on bacterial chemotaxis towards roots were also investigated. In general, significantly (P=0.05) greater chemotactic responses of bacteria were observed at higher water matric potentials (0, -1, and -5 kPa), slightly acidic to neutral pH (6, 6.5 and 7), and at 20-30 degrees C (depending on the bacterium) than in other environmental conditions. It is suggested that chemotaxis of P. fluorescens and A. chroococcum towards roots and their exudates is one of the several steps in the interaction process between bacteria and vesicular-arbuscular mycorrhizal roots. PMID:19719591

  13. The Plant Organelles Database 3 (PODB3) update 2014: integrating electron micrographs and new options for plant organelle research.

    PubMed

    Mano, Shoji; Nakamura, Takanori; Kondo, Maki; Miwa, Tomoki; Nishikawa, Shuh-ichi; Mimura, Tetsuro; Nagatani, Akira; Nishimura, Mikio

    2014-01-01

    The Plant Organelles Database 2 (PODB2), which was first launched in 2006 as PODB, provides static image and movie data of plant organelles, protocols for plant organelle research and external links to relevant websites. PODB2 has facilitated plant organellar research and the understanding of plant organelle dynamics. To provide comprehensive information on plant organelles in more detail, PODB2 was updated to PODB3 (http://podb.nibb.ac.jp/Organellome/). PODB3 contains two additional components: the electron micrograph database and the perceptive organelles database. Through the electron micrograph database, users can examine the subcellular and/or suborganellar structures in various organs of wild-type and mutant plants. The perceptive organelles database provides information on organelle dynamics in response to external stimuli. In addition to the extra components, the user interface for access has been enhanced in PODB3. The data in PODB3 are directly submitted by plant researchers and can be freely downloaded for use in further analysis. PODB3 contains all the information included in PODB2, and the volume of data and protocols deposited in PODB3 continue to grow steadily. We welcome contributions of data from all plant researchers to enhance the utility and comprehensiveness of PODB3. PMID:24092884

  14. Role of Intermediate Filaments in Vesicular Traffic

    PubMed Central

    Margiotta, Azzurra; Bucci, Cecilia

    2016-01-01

    Intermediate filaments are an important component of the cellular cytoskeleton. The first established role attributed to intermediate filaments was the mechanical support to cells. However, it is now clear that intermediate filaments have many different roles affecting a variety of other biological functions, such as the organization of microtubules and microfilaments, the regulation of nuclear structure and activity, the control of cell cycle and the regulation of signal transduction pathways. Furthermore, a number of intermediate filament proteins have been involved in the acquisition of tumorigenic properties. Over the last years, a strong involvement of intermediate filament proteins in the regulation of several aspects of intracellular trafficking has strongly emerged. Here, we review the functions of intermediate filaments proteins focusing mainly on the recent knowledge gained from the discovery that intermediate filaments associate with key proteins of the vesicular membrane transport machinery. In particular, we analyze the current understanding of the contribution of intermediate filaments to the endocytic pathway. PMID:27120621

  15. Vesicular exocytosis and microdevices - microelectrode arrays.

    PubMed

    Amatore, Christian; Delacotte, Jérôme; Guille-Collignon, Manon; Lemaître, Frédéric

    2015-06-01

    Among all the analytical techniques capable of monitoring exocytosis in real time at the single cell level, electrochemistry (particularly amperometry at a constant potential) using ultramicroelectrodes has been demonstrated to be an important and convenient tool for more than two decades. Indeed, because the electrochemical sensor is located in the close vicinity of the emitting cell ("artificial synapse" configuration), much data can be gathered from the whole cell activity (secretion frequency) to the individual vesicular release (duration, fluxes or amount of molecules released) with an excellent sensitivity. However, such a single cell analysis and its intrinsic benefits are at the expense of the spatial resolution and/or the number of experiments. The quite recent development of microdevices/microsystems (and mainly the microelectrode arrays (MEAs)) offers in some way a complementary approach either by combining spectroscopy-microscopy or by implementing a multianalysis. Such developments are described and discussed in the present review over the 2005-2014 period. PMID:25803190

  16. Role of Intermediate Filaments in Vesicular Traffic.

    PubMed

    Margiotta, Azzurra; Bucci, Cecilia

    2016-01-01

    Intermediate filaments are an important component of the cellular cytoskeleton. The first established role attributed to intermediate filaments was the mechanical support to cells. However, it is now clear that intermediate filaments have many different roles affecting a variety of other biological functions, such as the organization of microtubules and microfilaments, the regulation of nuclear structure and activity, the control of cell cycle and the regulation of signal transduction pathways. Furthermore, a number of intermediate filament proteins have been involved in the acquisition of tumorigenic properties. Over the last years, a strong involvement of intermediate filament proteins in the regulation of several aspects of intracellular trafficking has strongly emerged. Here, we review the functions of intermediate filaments proteins focusing mainly on the recent knowledge gained from the discovery that intermediate filaments associate with key proteins of the vesicular membrane transport machinery. In particular, we analyze the current understanding of the contribution of intermediate filaments to the endocytic pathway. PMID:27120621

  17. Future of nanotherapeutics: Targeting the cellular sub-organelles.

    PubMed

    Ma, Xiaowei; Gong, Ningqiang; Zhong, Lin; Sun, Jiadong; Liang, Xing-Jie

    2016-08-01

    Many diseases originate from alterations at nanoscale levels. Precise drug delivery should be achieved not only at cell level, but also at organelle level to achieve maximum therapeutic responses as well as avoiding possible toxic side effects of the drugs. However, organelles and subcellular structures are natural barriers that hampering many therapeutics from taking effects. Nanodelivery vehicle is a favorable platform to navigate across physiological barriers and to achieve selective delivery of therapeutic and diagnostic agents to intracellular targets. In this review, we have highlighted recent innovations in organelle-targeted nanomaterials designed to treat a variety of currently challenging diseases. Targeting strategies of four main kinds of organelles: mitochondria, nucleus, lysosomes and endoplasmic reticulum are discussed in detail. This review will help to clarify the intracellular nanomaterial-organelle interactions, and understand the fundamentals of organelle-targeted drug delivery strategies, which is of vital importance for the design and successful biomedical applications of nanomaterials in therapeutic treatments. At the end of this review, challenge and perspectives of organelle-targeted nanotherapy are discussed. PMID:27155363

  18. The bacterial magnetosome: a unique prokaryotic organelle.

    PubMed

    Lower, Brian H; Bazylinski, Dennis A

    2013-01-01

    The bacterial magnetosome is a unique prokaryotic organelle comprising magnetic mineral crystals surrounded by a phospholipid bilayer. These inclusions are biomineralized by the magnetotactic bacteria which are ubiquitous, aquatic, motile microorganisms. Magnetosomes cause cells of magnetotactic bacteria to passively align and swim along the Earth's magnetic field lines, as miniature motile compass needles. These specialized compartments consist of a phospholipid bilayer membrane surrounding magnetic crystals of magnetite (Fe3O4) or greigite (Fe3S4). The morphology of these membrane-bound crystals varies by species with a nominal magnetic domain size between 35 and 120 nm. Almost all magnetotactic bacteria arrange their magnetosomes in a chain within the cell there by maximizing the magnetic dipole moment of the cell. It is presumed that magnetotactic bacteria use magnetotaxis in conjunction with chemotaxis to locate and maintain an optimum position for growth and survival based on chemistry, redox and physiology in aquatic habitats with vertical chemical concentration and redox gradients. The biosynthesis of magnetosomes is a complex process that involves several distinct steps including cytoplasmic membrane modifications, iron uptake and transport, initiation of crystallization, crystal maturation and magnetosome chain formation. While many mechanistic details remain unresolved, magnetotactic bacteria appear to contain the genetic determinants for magnetosome biomineralization within their genomes in clusters of genes that make up what is referred to as the magnetosome gene island in some species. In addition, magnetosomes contain a unique set of proteins, not present in other cellular fractions, which control the biomineralization process. Through the development of genetic systems, proteomic and genomic work, and the use of molecular and biochemical tools, the functions of a number of magnetosome membrane proteins have been demonstrated and the molecular

  19. Programmed death phenomena: from organelle to organism.

    PubMed

    Skulachev, Vladimir P

    2002-04-01

    Programmed death phenomena appear to be inherent not only in living cells (apoptosis), but also in subcellular organelles (e.g., self-elimination of mitochondria, called mitoptosis), organs (organoptosis), and even whole organisms (phenoptosis). In all these cases, the "Samurai law of biology"--it is better to die than to be wrong--seems to be operative. The operation of this law helps complicated living systems avoid the risk of ruin when a system of lower hierarchic position makes a significant mistake. Thus, mitoptosis purifies a cell from damaged and hence unwanted mitochondria; apoptosis purifies a tissue from unwanted cells; and phenoptosis purifies a community from unwanted individuals. Defense against reactive oxygen species (ROS) is probably one of the primary evolutionary functions of programmed death mechanisms. So far, it seems that ROS play a key role in the mito-, apo-, organo-, and phenoptoses, which is consistent with Harman's theory of aging. Here a concept is described that tries to unite Weismann's hypothesis of aging as an adaptive programmed death mechanism and the generally accepted alternative point of view that considers aging as an inevitable result of accumulation in an organism of occasional injuries. It is suggested that injury accumulation is monitored by a system(s) actuating a phenoptotic death program when the number of injuries reaches some critical level. The system(s) in question are organized in such a way that the lethal case appears to be a result of phenoptosis long before the occasional injuries make impossible the functioning of the organism. It is stressed that for humans these cruel regulations look like an atavism that, if overcome, might dramatically prolong the human life span. PMID:11976198

  20. The Biogenesis of Lysosomes and Lysosome-Related Organelles

    PubMed Central

    Luzio, J. Paul; Hackmann, Yvonne; Dieckmann, Nele M.G.; Griffiths, Gillian M.

    2014-01-01

    Lysosomes were once considered the end point of endocytosis, simply used for macromolecule degradation. They are now recognized to be dynamic organelles, able to fuse with a variety of targets and to be re-formed after fusion events. They are also now known to be the site of nutrient sensing and signaling to the cell nucleus. In addition, lysosomes are secretory organelles, with specialized machinery for regulated secretion of proteins in some cell types. The biogenesis of lysosomes and lysosome-related organelles is discussed, taking into account their dynamic nature and multiple roles. PMID:25183830

  1. Organelle size control - increasing vacuole content activates SNAREs to augment organelle volume through homotypic fusion.

    PubMed

    Desfougères, Yann; Neumann, Heinz; Mayer, Andreas

    2016-07-15

    Cells control the size of their compartments relative to cell volume, but there is also size control within each organelle. Yeast vacuoles neither burst nor do they collapse into a ruffled morphology, indicating that the volume of the organellar envelope is adjusted to the amount of content. It is poorly understood how this adjustment is achieved. We show that the accumulating content of yeast vacuoles activates fusion of other vacuoles, thus increasing the volume-to-surface ratio. Synthesis of the dominant compound stored inside vacuoles, polyphosphate, stimulates binding of the chaperone Sec18/NSF to vacuolar SNAREs, which activates them and triggers fusion. SNAREs can only be activated by lumenal, not cytosolic, polyphosphate (polyP). Control of lumenal polyP over SNARE activation in the cytosol requires the cytosolic cyclin-dependent kinase Pho80-Pho85 and the R-SNARE Nyv1. These results suggest that cells can adapt the volume of vacuoles to their content through feedback from the vacuole lumen to the SNAREs on the cytosolic surface of the organelle. PMID:27252384

  2. Nanopreparations for Organelle-Specific Delivery in Cancer

    PubMed Central

    Biswas, Swati; Torchilin, Vladimir P.

    2014-01-01

    To efficiently deliver therapeutics into cancer cells, a number of strategies have been recently investigated. The toxicity associated with the administration of chemotherapeutic drugs due to their random interactions throughout the body necessitates the development of drug-encapsulating nanopreparations that significantly mask, or reduce, the toxic side effects of the drugs. In addition to reduced side effects associated with drug encapsulation, nanocarriers preferentially accumulate in tumors as a result of its abnormally leaky vasculature via the Enhanced Permeability and Retention (EPR) effect. However, simple passive nanocarrier delivery to the tumor site is unlikely to be enough to elicit a maximum therapeutic response as the drug-loaded carriers must reach the intracellular target sites. Therefore, efficient translocation of the nanocarrier through the cell membrane is necessary for cytosolic delivery of the cargo. However, Crossing the cell membrane barrier and reaching cytosol might still not be enough for achieving maximum therapeutic benefit, which necessitates the delivery of drugs directly to intracellular targets, such as bringing pro-apoptotic drugs to mitochondria, nucleic acid therapeutics to nuclei, and lysosomal enzymes to defective lysosomes. In this review, we discuss the strategies developed for tumor targeting, cytosolic delivery via cell membrane translocation, and finally organelle-specific targeting, which may be applied for developing highly efficacious, truly multifunctional, cancer-targeted nanopreparations. PMID:24270008

  3. Isolation of the envelope of vesicular stomatitis virus.

    PubMed Central

    Taube, S E; Rothfield, L I

    1978-01-01

    Vesicular stomatitis virus was disrupted by a combination of freezing and thawing, osmotic shock, and sonic treatment. Subviral components were separated by isopycnic centrifugation. The low-density, lipid-rich fractions were pooled and shown to contain primarily viral glycoprotein. Further purification of this material resulted in the isolation of a preparation of vesicles which contained only the G protein and the same phospholipids as in the intact virions and exhibited spikelike structures similar to those on intact vesicular stomatitis virions. We conclude that we have isolated fragments of native vesicular stomatitis virus envelopes. Images PMID:209217

  4. Molecular mechanisms regulating secretory organelles and endosomes in neutrophils and their implications for inflammation.

    PubMed

    Ramadass, Mahalakshmi; Catz, Sergio D

    2016-09-01

    Neutrophils constitute the first line of cellular defense against invading microorganisms and modulate the subsequent innate and adaptive immune responses. In order to execute a rapid and precise response to infections, neutrophils rely on preformed effector molecules stored in a variety of intracellular granules. Neutrophil granules contain microbicidal factors, the membrane-bound components of the respiratory burst oxidase, membrane-bound adhesion molecules, and receptors that facilitate the execution of all neutrophil functions including adhesion, transmigration, phagocytosis, degranulation, and neutrophil extracellular trap formation. The rapid mobilization of intracellular organelles is regulated by vesicular trafficking mechanisms controlled by effector molecules that include small GTPases and their interacting proteins. In this review, we focus on recent discoveries of mechanistic processes that are at center stage of the regulation of neutrophil function, highlighting the discrete and selective pathways controlled by trafficking modulators. In particular, we describe novel pathways controlled by the Rab27a effectors JFC1 and Munc13-4 in the regulation of degranulation, reactive oxygen species and neutrophil extracellular trap production, and endolysosomal signaling. Finally, we discuss the importance of understanding these molecular mechanisms in order to design novel approaches to modulate neutrophil-mediated inflammatory processes in a targeted fashion. PMID:27558339

  5. Isolation and characterization of vesicular and non-vesicular microRNAs circulating in sera of partially hepatectomized rats.

    PubMed

    Castoldi, Mirco; Kordes, Claus; Sawitza, Iris; Häussinger, Dieter

    2016-01-01

    Circulating microRNAs are protected from degradation by their association with either vesicles or components of the RNAi machinery. Although increasing evidence indicates that cell-free microRNAs are transported in body fluids by different types of vesicles, current research mainly focuses on the characterization of exosome-associated microRNAs. However, as isolation and characterization of exosomes is challenging, it is yet unclear whether exosomes or other vesicular elements circulating in serum are the most reliable source for discovering disease-associated biomarkers. In this study, circulating microRNAs associated to the vesicular and non-vesicular fraction of sera isolated from partially hepatectomized rats were measured. Here we show that independently from their origin, levels of miR-122, miR-192, miR-194 and Let-7a are up-regulated two days after partial hepatectomy. The inflammation-associated miR-150 and miR-155 are up-regulated in the vesicular-fraction only, while the regeneration-associated miR-21 and miR-33 are up-regulated in the vesicular- and down-regulated in the non-vesicular fraction. Our study shows for the first time the modulation of non-vesicular microRNAs in animals recovering from partial hepatectomy, suggesting that, in the search for novel disease-associated biomarkers, the profiling of either vesicular or non-vesicular microRNAs may be more relevant than the analysis of microRNAs isolated from unfractionated serum. PMID:27535708

  6. Isolation and characterization of vesicular and non-vesicular microRNAs circulating in sera of partially hepatectomized rats

    PubMed Central

    Castoldi, Mirco; Kordes, Claus; Sawitza, Iris; Häussinger, Dieter

    2016-01-01

    Circulating microRNAs are protected from degradation by their association with either vesicles or components of the RNAi machinery. Although increasing evidence indicates that cell-free microRNAs are transported in body fluids by different types of vesicles, current research mainly focuses on the characterization of exosome-associated microRNAs. However, as isolation and characterization of exosomes is challenging, it is yet unclear whether exosomes or other vesicular elements circulating in serum are the most reliable source for discovering disease-associated biomarkers. In this study, circulating microRNAs associated to the vesicular and non-vesicular fraction of sera isolated from partially hepatectomized rats were measured. Here we show that independently from their origin, levels of miR-122, miR-192, miR-194 and Let-7a are up-regulated two days after partial hepatectomy. The inflammation-associated miR-150 and miR-155 are up-regulated in the vesicular-fraction only, while the regeneration-associated miR-21 and miR-33 are up-regulated in the vesicular- and down-regulated in the non-vesicular fraction. Our study shows for the first time the modulation of non-vesicular microRNAs in animals recovering from partial hepatectomy, suggesting that, in the search for novel disease-associated biomarkers, the profiling of either vesicular or non-vesicular microRNAs may be more relevant than the analysis of microRNAs isolated from unfractionated serum. PMID:27535708

  7. Molecular mechanisms of Sar/Arf GTPases in vesicular trafficking in yeast and plants

    PubMed Central

    Yorimitsu, Tomohiro; Sato, Ken; Takeuchi, Masaki

    2014-01-01

    Small GTPase proteins play essential roles in the regulation of vesicular trafficking systems in eukaryotic cells. Two types of small GTPases, secretion-associated Ras-related protein (Sar) and ADP-ribosylation factor (Arf), act in the biogenesis of transport vesicles. Sar/Arf GTPases function as molecular switches by cycling between active, GTP-bound and inactive, GDP-bound forms, catalyzed by guanine nucleotide exchange factors and GTPase-activating proteins, respectively. Activated Sar/Arf GTPases undergo a conformational change, exposing the N-terminal amphipathic α-helix for insertion into membranes. The process triggers the recruitment and assembly of coat proteins to the membranes, followed by coated vesicle formation and scission. In higher plants, Sar/Arf GTPases also play pivotal roles in maintaining the dynamic identity of organelles in the secretory pathway. Sar1 protein strictly controls anterograde transport from the endoplasmic reticulum (ER) through the recruitment of plant COPII coat components onto membranes. COPII vesicle transport is responsible for the organization of highly conserved polygonal ER networks. In contrast, Arf proteins contribute to the regulation of multiple trafficking routes, including transport through the Golgi complex and endocytic transport. These transport systems have diversified in the plant kingdom independently and exhibit several plant-specific features with respect to Golgi organization, endocytic cycling, cell polarity and cytokinesis. The functional diversification of vesicular trafficking systems ensures the multicellular development of higher plants. This review focuses on the current knowledge of Sar/Arf GTPases, highlighting the molecular details of GTPase regulation in vesicle formation in yeast and advances in knowledge of the characteristics of vesicle trafficking in plants. PMID:25191334

  8. Organelle-Specific Activity-Based Protein Profiling in Living Cells

    SciTech Connect

    Wiedner, Susan D.; Anderson, Lindsey N.; Sadler, Natalie C.; Chrisler, William B.; Kodali, Vamsi K.; Smith, Richard D.; Wright, Aaron T.

    2014-02-06

    A multimodal acidic organelle targeting activity-based probe was developed for analysis of subcellular native enzymatic activity of cells by fluorescent microscopy and mass spectrometry. A cathepsin reactive warhead was conjugated to an acidotropic amine, and a clickable alkyne for appendage of AlexaFluor 488 or biotin reporter tags. This probe accumulated in punctate vesicles surrounded by LAMP1, a lysosome marker, as observed by Structured Illumination Microscopy (SIM) in J774 mouse macrophage cells. Biotin conjugation, affinity purification, and analysis of in vivo labeled J774 by mass spectrometry showed that the probe was very selective for Cathepsins B and Z, two lysosomal cysteine proteases. Analysis of starvation induced autophagy, which is an increase in cell component catabolism involving lysosomes, showed a large increase in tagged protein number and an increase in cathepsin activity. Organelle targeting activity-based probes and subsequent analysis of resident proteins by mass spectrometry is enabled by tuning the physicochemical properties of the probe.

  9. Proteomics of secretory and endocytic organelles in Giardia lamblia.

    PubMed

    Wampfler, Petra B; Tosevski, Vinko; Nanni, Paolo; Spycher, Cornelia; Hehl, Adrian B

    2014-01-01

    Giardia lamblia is a flagellated protozoan enteroparasite transmitted as an environmentally resistant cyst. Trophozoites attach to the small intestine of vertebrate hosts and proliferate by binary fission. They access nutrients directly via uptake of bulk fluid phase material into specialized endocytic organelles termed peripheral vesicles (PVs), mainly on the exposed dorsal side. When trophozoites reach the G2/M restriction point in the cell cycle they can begin another round of cell division or encyst if they encounter specific environmental cues. They induce neogenesis of Golgi-like organelles, encystation-specific vesicles (ESVs), for regulated secretion of cyst wall material. PVs and ESVs are highly simplified and thus evolutionary diverged endocytic and exocytic organelle systems with key roles in proliferation and transmission to a new host, respectively. Both organelle systems physically and functionally intersect at the endoplasmic reticulum (ER) which has catabolic as well as anabolic functions. However, the unusually high degree of sequence divergence in Giardia rapidly exhausts phylogenomic strategies to identify and characterize the molecular underpinnings of these streamlined organelles. To define the first proteome of ESVs and PVs we used a novel strategy combining flow cytometry-based organelle sorting with in silico filtration of mass spectrometry data. From the limited size datasets we retrieved many hypothetical but also known organelle-specific factors. In contrast to PVs, ESVs appear to maintain a strong physical and functional link to the ER including recruitment of ribosomes to organelle membranes. Overall the data provide further evidence for the formation of a cyst extracellular matrix with minimal complexity. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000694. PMID:24732305

  10. Proteomics of Secretory and Endocytic Organelles in Giardia lamblia

    PubMed Central

    Wampfler, Petra B.; Tosevski, Vinko; Nanni, Paolo; Spycher, Cornelia; Hehl, Adrian B.

    2014-01-01

    Giardia lamblia is a flagellated protozoan enteroparasite transmitted as an environmentally resistant cyst. Trophozoites attach to the small intestine of vertebrate hosts and proliferate by binary fission. They access nutrients directly via uptake of bulk fluid phase material into specialized endocytic organelles termed peripheral vesicles (PVs), mainly on the exposed dorsal side. When trophozoites reach the G2/M restriction point in the cell cycle they can begin another round of cell division or encyst if they encounter specific environmental cues. They induce neogenesis of Golgi-like organelles, encystation-specific vesicles (ESVs), for regulated secretion of cyst wall material. PVs and ESVs are highly simplified and thus evolutionary diverged endocytic and exocytic organelle systems with key roles in proliferation and transmission to a new host, respectively. Both organelle systems physically and functionally intersect at the endoplasmic reticulum (ER) which has catabolic as well as anabolic functions. However, the unusually high degree of sequence divergence in Giardia rapidly exhausts phylogenomic strategies to identify and characterize the molecular underpinnings of these streamlined organelles. To define the first proteome of ESVs and PVs we used a novel strategy combining flow cytometry-based organelle sorting with in silico filtration of mass spectrometry data. From the limited size datasets we retrieved many hypothetical but also known organelle-specific factors. In contrast to PVs, ESVs appear to maintain a strong physical and functional link to the ER including recruitment of ribosomes to organelle membranes. Overall the data provide further evidence for the formation of a cyst extracellular matrix with minimal complexity. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000694. PMID:24732305

  11. Vesicular nucleotide transport: a brief history and the vesicular nucleotide transporter as a target for drug development.

    PubMed

    Hiasa, Miki; Togawa, Natsuko; Moriyama, Yoshinori

    2014-01-01

    Neurons and neuroendocrine cells store nucleotides in vesicles and release them upon stimulation, leading to intercellular purinergic signaling. The molecular machinery responsible for the vesicular storage of nucleotides was a long standing enigma, however, recently the transporter involving in the process was identified. This article summarizes the history of vesicular storage of nucleotides and the identification of the vesicular nucleotide transporter (VNUT) responsible for the process. The significance of VNUT as a drug target to control purinergic chemical transmission is also discussed. PMID:23886392

  12. Recombination and the maintenance of plant organelle genome stability.

    PubMed

    Maréchal, Alexandre; Brisson, Normand

    2010-04-01

    Like their nuclear counterpart, the plastid and mitochondrial genomes of plants have to be faithfully replicated and repaired to ensure the normal functioning of the plant. Inability to maintain organelle genome stability results in plastid and/or mitochondrial defects, which can lead to potentially detrimental phenotypes. Fortunately, plant organelles have developed multiple strategies to maintain the integrity of their genetic material. Of particular importance among these processes is the extensive use of DNA recombination. In fact, recombination has been implicated in both the replication and the repair of organelle genomes. Revealingly, deregulation of recombination in organelles results in genomic instability, often accompanied by adverse consequences for plant fitness. The recent identification of four families of proteins that prevent aberrant recombination of organelle DNA sheds much needed mechanistic light on this important process. What comes out of these investigations is a partial portrait of the recombination surveillance machinery in which plants have co-opted some proteins of prokaryotic origin but have also evolved whole new factors to keep their organelle genomes intact. These new features presumably optimized the protection of plastid and mitochondrial genomes against the particular genotoxic stresses they face. PMID:20180912

  13. Vesicular carriers for dermal drug delivery.

    PubMed

    Sinico, Chiara; Fadda, Anna Maria

    2009-08-01

    The skin can offer several advantages as a route of drug administration although its barrier nature makes it difficult for most drugs to penetrate into and permeate through it. During the past decades there has been a lot of interest in lipid vesicles as a tool to improve drug topical delivery. Vesicular systems such as liposomes, niosomes, ethosomes and elastic, deformable vesicles provide an alternative for improved skin drug delivery. The function of vesicles as topical delivery systems is controversial with variable effects being reported in relation to the type of vesicles and their composition. In fact, vesicles can act as drug carriers controlling active release; they can provide a localized depot in the skin for dermally active compounds and enhance transdermal drug delivery. A wide variety of lipids and surfactants can be used to prepare vesicles, which are commonly composed of phospholipids (liposomes) or non-ionic surfactants (niosomes). Vesicle composition and preparation method influence their physicochemical properties (size, charge, lamellarity, thermodynamic state, deformability) and therefore their efficacy as drug delivery systems. A review of vesicle value in localizing drugs within the skin at the site of action will be provided with emphasis on their potential mechanism of action. PMID:19569979

  14. Methamphetamine administration reduces hippocampal vesicular monoamine transporter-2 uptake.

    PubMed

    Rau, Kristi S; Birdsall, Elisabeth; Volz, Trent J; Riordan, James A; Baucum, Anthony J; Adair, Brian P; Bitter, Rebecca; Gibb, James W; Hanson, Glen R; Fleckenstein, Annette E

    2006-08-01

    Repeated high-dose injections of methamphetamine (METH) rapidly decrease dopamine uptake by the vesicular monoamine transporter-2 (VMAT-2) associated with dopaminergic nerve terminals, as assessed in nonmembrane-associated vesicles purified from striata of treated rats. The purpose of this study was to determine whether METH similarly affects vesicular uptake in the hippocampus; a region innervated by both serotonergic and noradrenergic neurons and profoundly affected by METH treatment. Results revealed that repeated high-dose METH administrations rapidly (within 1 h) reduced hippocampal vesicular dopamine uptake, as assessed in vesicles purified from treated rats. This reduction was likely associated with serotonergic nerve terminals because METH did not further reduce vesicular monoamine uptake in para-chloroamphetamine-lesioned animals. Pretreatment with the serotonin transporter inhibitor fluoxetine blocked both this acute effect on VMAT-2 and the decrease in serotonin content observed 7 days after METH treatment. In contrast, there was no conclusive evidence that METH affected vesicular dopamine uptake in noradrenergic neurons or caused persistent noradrenergic deficits. These findings suggest a link between METH-induced alterations in serotonergic hippocampal vesicular uptake and the persistent hippocampal serotonergic deficits induced by the stimulant. PMID:16687477

  15. Vesicular uptake blockade generates the toxic dopamine metabolite 3,4-dihydroxyphenylacetaldehyde in PC12 cells: relevance to the pathogenesis of Parkinson's disease.

    PubMed

    Goldstein, David S; Sullivan, Patti; Cooney, Adele; Jinsmaa, Yunden; Sullivan, Rachel; Gross, Daniel J; Holmes, Courtney; Kopin, Irwin J; Sharabi, Yehonatan

    2012-12-01

    Parkinson's disease entails profound loss of nigrostriatal dopaminergic terminals, decreased vesicular uptake of intraneuronal catecholamines, and relatively increased putamen tissue concentrations of the toxic dopamine metabolite, 3,4-dihydroxyphenylacetaldehyde (DOPAL). The objective of this study was to test whether vesicular uptake blockade augments endogenous DOPAL production. We also examined whether intracellular DOPAL contributes to apoptosis and, as α-synuclein oligomers may be pathogenetic in Parkinson's disease, oligomerizes α-synuclein. Catechols were assayed in PC12 cells after reserpine to block vesicular uptake, with or without inhibition of enzymes metabolizing DOPAL-daidzein for aldehyde dehydrogenase and AL1576 for aldehyde reductase. Vesicular uptake was quantified by a method based on 6F- or (13) C-dopamine incubation; DOPAL toxicity by apoptosis responses to exogenous dopamine, with or without daidzein+AL1576; and DOPAL--induced synuclein oligomerization by synuclein dimer production during DOPA incubation, with or without inhibition of L-aromatic-amino-acid decarboxylase or monoamine oxidase. Reserpine inhibited vesicular uptake by 95-97% and rapidly increased cell DOPAL content (p = 0.0008). Daidzein+AL1576 augmented DOPAL responses to reserpine (p = 0.004). Intracellular DOPAL contributed to dopamine-evoked apoptosis and DOPA-evoked synuclein dimerization. The findings fit with the 'catecholaldehyde hypothesis,' according to which decreased vesicular sequestration of cytosolic catecholamines and impaired catecholaldehyde detoxification contribute to the catecholaminergic denervation that characterizes Parkinson's disease. PMID:22906103

  16. Association of a Nonmuscle Myosin II with Axoplasmic Organelles

    PubMed Central

    DeGiorgis, Joseph A.; Reese, Thomas S.; Bearer, Elaine L.

    2002-01-01

    Association of motor proteins with organelles is required for the motors to mediate transport. Because axoplasmic organelles move on actin filaments, they must have associated actin-based motors, most likely members of the myosin superfamily. To gain a better understanding of the roles of myosins in the axon we used the giant axon of the squid, a powerful model for studies of axonal physiology. First, a ∼220 kDa protein was purified from squid optic lobe, using a biochemical protocol designed to isolate myosins. Peptide sequence analysis, followed by cloning and sequencing of the full-length cDNA, identified this ∼220 kDa protein as a nonmuscle myosin II. This myosin is also present in axoplasm, as determined by two independent criteria. First, RT-PCR using sequence-specific primers detected the transcript in the stellate ganglion, which contains the cell bodies that give rise to the giant axon. Second, Western blot analysis using nonmuscle myosin II isotype-specific antibodies detected a single ∼220 kDa band in axoplasm. Axoplasm was fractionated through a four-step sucrose gradient after 0.6 M KI treatment, which separates organelles from cytoskeletal components. Of the total nonmuscle myosin II in axoplasm, 43.2% copurified with organelles in the 15% sucrose fraction, while the remainder (56.8%) was soluble and found in the supernatant. This myosin decorates the cytoplasmic surface of 21% of the axoplasmic organelles, as demonstrated by immunogold electron-microscopy. Thus, nonmuscle myosin II is synthesized in the cell bodies of the giant axon, is present in the axon, and is associated with isolated axoplasmic organelles. Therefore, in addition to myosin V, this myosin is likely to be an axoplasmic organelle motor. PMID:11907281

  17. The Leishmania donovani lipophosphoglycan excludes the vesicular proton-ATPase from phagosomes by impairing the recruitment of synaptotagmin V.

    PubMed

    Vinet, Adrien F; Fukuda, Mitsunori; Turco, Salvatore J; Descoteaux, Albert

    2009-10-01

    We recently showed that the exocytosis regulator Synaptotagmin (Syt) V is recruited to the nascent phagosome and remains associated throughout the maturation process. In this study, we investigated the possibility that Syt V plays a role in regulating interactions between the phagosome and the endocytic organelles. Silencing of Syt V by RNA interference revealed that Syt V contributes to phagolysosome biogenesis by regulating the acquisition of cathepsin D and the vesicular proton-ATPase. In contrast, recruitment of cathepsin B, the early endosomal marker EEA1 and the lysosomal marker LAMP1 to phagosomes was normal in the absence of Syt V. As Leishmania donovani promastigotes inhibit phagosome maturation, we investigated their potential impact on the phagosomal association of Syt V. This inhibition of phagolysosome biogenesis is mediated by the virulence glycolipid lipophosphoglycan, a polymer of the repeating Galbeta1,4Manalpha1-PO(4) units attached to the promastigote surface via an unusual glycosylphosphatidylinositol anchor. Our results showed that insertion of lipophosphoglycan into ganglioside GM1-containing microdomains excluded or caused dissociation of Syt V from phagosome membranes. As a consequence, L. donovani promatigotes established infection in a phagosome from which the vesicular proton-ATPase was excluded and which failed to acidify. Collectively, these results reveal a novel function for Syt V in phagolysosome biogenesis and provide novel insight into the mechanism of vesicular proton-ATPase recruitment to maturing phagosomes. We also provide novel findings into the mechanism of Leishmania pathogenesis, whereby targeting of Syt V is part of the strategy used by L. donovani promastigotes to prevent phagosome acidification. PMID:19834555

  18. A nanobuffer reporter library for fine-scale imaging and perturbation of endocytic organelles

    PubMed Central

    Wang, Chensu; Wang, Yiguang; Li, Yang; Bodemann, Brian; Zhao, Tian; Ma, Xinpeng; Huang, Gang; Hu, Zeping; DeBerardinis, Ralph J.; White, Michael A.; Gao, Jinming

    2015-01-01

    Endosomes, lysosomes and related catabolic organelles are a dynamic continuum of vacuolar structures that impact a number of cell physiological processes such as protein/lipid metabolism, nutrient sensing and cell survival. Here we develop a library of ultra-pH-sensitive fluorescent nanoparticles with chemical properties that allow fine-scale, multiplexed, spatio-temporal perturbation and quantification of catabolic organelle maturation at single organelle resolution to support quantitative investigation of these processes in living cells. Deployment in cells allows quantification of the proton accumulation rate in endosomes; illumination of previously unrecognized regulatory mechanisms coupling pH transitions to endosomal coat protein exchange; discovery of distinct pH thresholds required for mTORC1 activation by free amino acids versus proteins; broad-scale characterization of the consequence of endosomal pH transitions on cellular metabolomic profiles; and functionalization of a context-specific metabolic vulnerability in lung cancer cells. Together, these biological applications indicate the robustness and adaptability of this nanotechnology-enabled ‘detection and perturbation' strategy. PMID:26437053

  19. A pH-independent DNA nanodevice for quantifying chloride transport in organelles of living cells

    NASA Astrophysics Data System (ADS)

    Saha, Sonali; Prakash, Ved; Halder, Saheli; Chakraborty, Kasturi; Krishnan, Yamuna

    2015-07-01

    The concentration of chloride ions in the cytoplasm and subcellular organelles of living cells spans a wide range (5-130 mM), and is tightly regulated by intracellular chloride channels or transporters. Chloride-sensitive protein reporters have been used to study the role of these chloride regulators, but they are limited to a small range of chloride concentrations and are pH-sensitive. Here, we show that a DNA nanodevice can precisely measure the activity and location of subcellular chloride channels and transporters in living cells in a pH-independent manner. The DNA nanodevice, called Clensor, is composed of sensing, normalizing and targeting modules, and is designed to localize within organelles along the endolysosomal pathway. It allows fluorescent, ratiometric sensing of chloride ions across the entire physiological regime. We used Clensor to quantitate the resting chloride concentration in the lumen of acidic organelles in Drosophila melanogaster. We showed that lumenal lysosomal chloride, which is implicated in various lysosomal storage diseases, is regulated by the intracellular chloride transporter DmClC-b.

  20. Systematic Structural Analyses of Attachment Organelle in Mycoplasma pneumoniae

    PubMed Central

    Matsuo, Lisa; Miyata, Makoto

    2015-01-01

    Mycoplasma pneumoniae, a human pathogenic bacterium, glides on host cell surfaces by a unique and unknown mechanism. It forms an attachment organelle at a cell pole as a membrane protrusion composed of surface and internal structures, with a highly organized architecture. In the present study, we succeeded in isolating the internal structure of the organelle by sucrose-gradient centrifugation. The negative-staining electron microscopy clarified the details and dimensions of the internal structure, which is composed of terminal button, paired plates, and bowl complex from the end of cell front. Peptide mass fingerprinting of the structure suggested 25 novel components for the organelle, and 3 of them were suggested for their involvement in the structure through their subcellular localization determined by enhanced yellow fluorescent protein (EYFP) tagging. Thirteen component proteins including the previously reported ones were mapped on the organelle systematically for the first time, in nanometer order by EYFP tagging and immunoelectron microscopy. Two, three, and six specific proteins localized specifically to the terminal button, the paired plates, and the bowl, respectively and interestingly, HMW2 molecules were aligned parallel to form the plate. The integration of these results gave the whole image of the organelle and allowed us to discuss possible gliding mechanisms. PMID:26633540

  1. 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

  2. A role for vesicular glutamate transporter 1 in synaptic vesicle clustering and mobility.

    PubMed

    Siksou, Léa; Silm, Kätlin; Biesemann, Christoph; Nehring, Ralf B; Wojcik, Sonja M; Triller, Antoine; El Mestikawy, Salah; Marty, Serge; Herzog, Etienne

    2013-05-01

    Synaptic vesicles (SVs) from excitatory synapses carry vesicular glutamate transporters (VGLUTs) that fill the vesicles with neurotransmitter. Although the essential function of VGLUTs as glutamate transporters has been well established, the evidence for additional cell-biological functions is more controversial. Both VGLUT1 and VGLUT2 disruptions in mice result in a reduced number of SVs away from release sites, flattening of SVs, and the appearance of tubular structures. Therefore, we analysed the morphology, biochemical composition and trafficking of SVs at synapses of VGLUT1(-/-) mice in order to test for a function of VGLUTs in the formation or clustering of SVs. Analyses with high-pressure freezing immobilisation and electron tomography pointed to a role of VGLUT1 transport function in the tonicity of excitatory SVs, explaining the aldehyde-induced flattening of SVs observed in VGLUT1(-/-) synapses. We confirmed the steep reduction in the number of SVs previously observed in VGLUT1(-/-) presynaptic terminals, but did not observe accumulation of endocytotic intermediates. Furthermore, SV proteins of adult VGLUT1(-/-) mouse brain tissue were expressed at normal levels in all subcellular fractions, suggesting that they were not displaced to another organelle. We thus assessed the mobility of the recently documented superpool of SVs. Synaptobrevin2-enhanced green fluorescent protein time lapse experiments revealed an oversized superpool of SVs in VGLUT1(-/-) neurons. Our results support the idea that, beyond glutamate loading, VGLUT1 enhances the tonicity of excitatory SVs and stabilises SVs at presynaptic terminals. PMID:23581566

  3. Phosphatidylserine flipping enhances membrane curvature and negative charge required for vesicular transport

    PubMed Central

    Xu, Peng; Baldridge, Ryan D.; Chi, Richard J.; Burd, Christopher G.

    2013-01-01

    Vesicle-mediated protein transport between organelles of the secretory and endocytic pathways is strongly influenced by the composition and organization of membrane lipids. In budding yeast, protein transport between the trans-Golgi network (TGN) and early endosome (EE) requires Drs2, a phospholipid translocase in the type IV P-type ATPase family. However, downstream effectors of Drs2 and specific phospholipid substrate requirements for protein transport in this pathway are unknown. Here, we show that the Arf GTPase-activating protein (ArfGAP) Gcs1 is a Drs2 effector that requires a variant of the ArfGAP lipid packing sensor (+ALPS) motif for localization to TGN/EE membranes. Drs2 increases membrane curvature and anionic phospholipid composition of the cytosolic leaflet, both of which are sensed by the +ALPS motif. Using mutant forms of Drs2 and the related protein Dnf1, which alter their ability to recognize phosphatidylserine, we show that translocation of this substrate to the cytosolic leaflet is essential for +ALPS binding and vesicular transport between the EE and the TGN. PMID:24019533

  4. Integrated femtosecond stimulated Raman scattering and two-photon fluorescence imaging of subcellular lipid and vesicular structures

    NASA Astrophysics Data System (ADS)

    Li, Xuesong; Lam, Wen Jiun; Cao, Zhe; Hao, Yan; Sun, Qiqi; He, Sicong; Mak, Ho Yi; Qu, Jianan Y.

    2015-11-01

    The primary goal of this study is to demonstrate that stimulated Raman scattering (SRS) as a new imaging modality can be integrated into a femtosecond (fs) nonlinear optical (NLO) microscope system. The fs sources of high pulse peak power are routinely used in multimodal nonlinear microscopy to enable efficient excitation of multiple NLO signals. However, with fs excitations, the SRS imaging of subcellular lipid and vesicular structures encounters significant interference from proteins due to poor spectral resolution and a lack of chemical specificity, respectively. We developed a unique NLO microscope of fs excitation that enables rapid acquisition of SRS and multiple two-photon excited fluorescence (TPEF) signals. In the in vivo imaging of transgenic C. elegans animals, we discovered that by cross-filtering false positive lipid signals based on the TPEF signals from tryptophan-bearing endogenous proteins and lysosome-related organelles, the imaging system produced highly accurate assignment of SRS signals to lipid. Furthermore, we demonstrated that the multimodal NLO microscope system could sequentially image lipid structure/content and organelles, such as mitochondria, lysosomes, and the endoplasmic reticulum, which are intricately linked to lipid metabolism.

  5. Integrated femtosecond stimulated Raman scattering and two-photon fluorescence imaging of subcellular lipid and vesicular structures.

    PubMed

    Li, Xuesong; Lam, Wen Jiun; Cao, Zhe; Hao, Yan; Sun, Qiqi; He, Sicong; Mak, Ho Yi; Qu, Jianan Y

    2015-11-01

    The primary goal of this study is to demonstrate that stimulated Raman scattering (SRS) as a new imaging modality can be integrated into a femtosecond (fs) nonlinear optical (NLO) microscope system. The fs sources of high pulse peak power are routinely used in multimodal nonlinear microscopy to enable efficient excitation of multiple NLO signals. However, with fs excitations, the SRS imaging of subcellular lipid and vesicular structures encounters significant interference from proteins due to poor spectral resolution and a lack of chemical specificity, respectively. We developed a unique NLO microscope of fs excitation that enables rapid acquisition of SRS and multiple two-photon excited fluorescence (TPEF) signals. In the in vivo imaging of transgenic C. elegans animals, we discovered that by cross-filtering false positive lipid signals based on the TPEF signals from tryptophan-bearing endogenous proteins and lysosome-related organelles, the imaging system produced highly accurate assignment of SRS signals to lipid. Furthermore, we demonstrated that the multimodal NLO microscope system could sequentially image lipid structure/content and organelles, such as mitochondria, lysosomes, and the endoplasmic reticulum, which are intricately linked to lipid metabolism. PMID:26580697

  6. 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

  7. Transient domain formation in membrane-bound organelles undergoing maturation

    NASA Astrophysics Data System (ADS)

    Dmitrieff, Serge; Sens, Pierre

    2013-12-01

    The membrane components of cellular organelles have been shown to segregate into domains as the result of biochemical maturation. We propose that the dynamical competition between maturation and lateral segregation of membrane components regulates domain formation. We study a two-component fluid membrane in which enzymatic reaction irreversibly converts one component into another and phase separation triggers the formation of transient membrane domains. The maximum domain size is shown to depend on the maturation rate as a power law similar to the one observed for domain growth with time in the absence of maturation, despite this time dependence not being verified in the case of irreversible maturation. This control of domain size by enzymatic activity could play a critical role in regulating exchange between organelles or within compartmentalized organelles such as the Golgi apparatus.

  8. 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.

  9. Vesicular trafficking through cortical actin during exocytosis is regulated by the Rab27a effector JFC1/Slp1 and the RhoA-GTPase–activating protein Gem-interacting protein

    PubMed Central

    Johnson, Jennifer L.; Monfregola, Jlenia; Napolitano, Gennaro; Kiosses, William B.; Catz, Sergio D.

    2012-01-01

    Cytoskeleton remodeling is important for the regulation of vesicular transport associated with exocytosis, but a direct association between granular secretory proteins and actin-remodeling molecules has not been shown, and this mechanism remains obscure. Using a proteomic approach, we identified the RhoA-GTPase–activating protein Gem-interacting protein (GMIP) as a factor that associates with the Rab27a effector JFC1 and modulates vesicular transport and exocytosis. GMIP down-regulation induced RhoA activation and actin polymerization. Importantly, GMIP-down-regulated cells showed impaired vesicular transport and exocytosis, while inhibition of the RhoA-signaling pathway induced actin depolymerization and facilitated exocytosis. We show that RhoA activity polarizes around JFC1-containing secretory granules, suggesting that it may control directionality of granule movement. Using quantitative live-cell microscopy, we show that JFC1-containing secretory organelles move in areas near the plasma membrane deprived of polymerized actin and that dynamic vesicles maintain an actin-free environment in their surroundings. Supporting a role for JFC1 in RhoA inactivation and actin remodeling during exocytosis, JFC1 knockout neutrophils showed increased RhoA activity, and azurophilic granules were unable to traverse cortical actin in cells lacking JFC1. We propose that during exocytosis, actin depolymerization commences near the secretory organelle, not the plasma membrane, and that secretory granules use a JFC1- and GMIP-dependent molecular mechanism to traverse cortical actin. PMID:22438581

  10. Brain dopamine-serotonin vesicular transport disease presenting as a severe infantile hypotonic parkinsonian disorder.

    PubMed

    Jacobsen, Jessie C; Wilson, Callum; Cunningham, Vicki; Glamuzina, Emma; Prosser, Debra O; Love, Donald R; Burgess, Trent; Taylor, Juliet; Swan, Brendan; Hill, Rosamund; Robertson, Stephen P; Snell, Russell G; Lehnert, Klaus

    2016-03-01

    Two male siblings from a consanguineous union presented in early infancy with marked truncal hypotonia, a general paucity of movement, extrapyramidal signs and cognitive delay. By mid-childhood they had made little developmental progress and remained severely hypotonic and bradykinetic. They developed epilepsy and had problems with autonomic dysfunction and oculogyric crises. They had a number of orthopaedic problems secondary to their hypotonia. Cerebrospinal fluid (CSF) neurotransmitters were initially normal, apart from mildly elevated 5-hydroxyindolacetic acid, and the children did not respond favourably to a trial of levodopa-carbidopa. The youngest died from respiratory complications at 10 years of age. Repeat CSF neurotransmitters in the older sibling at eight years of age showed slightly low homovanillic acid and 5-hydroxyindoleacetic acid levels. Whole-exome sequencing revealed a novel mutation homozygous in both children in the monoamine transporter gene SLC18A2 (p.Pro237His), resulting in brain dopamine-serotonin vesicular transport disease. This is the second family to be described with a mutation in this gene. Treatment with the dopamine agonist pramipexole in the surviving child resulted in mild improvements in alertness, communication, and eye movements. This case supports the identification of the causal mutation in the original case, expands the clinical phenotype of brain dopamine-serotonin vesicular transport disease and confirms that pramipexole treatment may lead to symptomatic improvement in affected individuals. PMID:26497564

  11. Barley aleurone cells contain two types of vacuoles. Characterization Of lytic organelles by use of fluorescent probes

    PubMed Central

    Swanson, SJ; Bethke, PC; Jones, RL

    1998-01-01

    Light microscopy was used to study the structure and function of vacuoles in living protoplasts of barley (Hordeum vulgare cv Himalaya) aleurone. Light microscopy showed that aleurone protoplasts contain two distinct types of vacuole: the protein storage vacuole and a lysosome-like organelle, which we have called the secondary vacuole. Fluorescence microscopy using pH-sensitive fluorescent probes and a fluorogenic substrate for cysteine proteases showed that both protein storage vacuoles and secondary vacuoles are acidic, lytic organelles. Ratio imaging showed that the pH of secondary vacuoles was lower in aleurone protoplasts incubated in gibberellic acid than in those incubated in abscisic acid. Uptake of fluorescent probes into intact, isolated protein storage vacuoles and secondary vacuoles required ATP and occurred via at least two types of vanadate-sensitive, ATP-dependent tonoplast transporters. One transporter catalyzed the accumulation of glutathione-conjugated probes, and another transported probes not conjugated to glutathione. PMID:9596630

  12. An immunocompetent patient with a vesicular rash and neurological symptomatology.

    PubMed

    Cooper, Chad J; Said, Sarmad; Teleb, Mohamed; Rosa, Paola; Didia, S Claudia

    2013-01-01

    Viral infection is the most common cause of aseptic meningitis with the most frequent virus associated with aseptic meningitis being enteroviruses (coxsackievirus and echovirus). In viral meningitis, cerebrospinal fluid (CSF) shows a mild pleocytosis with a lymphocytic predominance, elevated protein, and normal glucose level. Nucleic acid amplification methods have greatly improved the detection of viral pathogens. In our case, a 47-year-old Caucasian female patient presented with a persistent throbbing headache for six days, localized at the frontal area, associated with photophobia, and exacerbated by bright lights and loud noises. Physical examination revealed nuchal rigidity and a vesicular rash at the right T4-T6 dermatome region. CSF findings were consistent with aseptic meningitis and polymerase chain reaction (PCR) was positive for VZV. Clinical improvement in meningeal signs and symptoms occurred after the initiation of acyclovir to complete a total 10-day course. There are no published data revealing that acyclovir will modify the course of VZV meningitis, but it is important to recognize the potential clinical benefit with the early initiation of antiviral therapy, especially if a zoster rash is discovered on examination. However, this is rarely the case because the majority of VZV meningitis will not present with a rash. Even though the reactivation of VZV is not usually associated with clinical meningitis, it is important to consider VZV in the differential diagnosis of a patient presenting without a rash with CNS disease. PCR has been proven to be a useful and quick diagnostic tool in the early diagnosis of VZV-associated neurological disease. PMID:24367380

  13. Molecular physiology of vesicular glutamate transporters in the digestive system

    PubMed Central

    Li, Tao; Ghishan, Fayez K.; Bai, Liqun

    2005-01-01

    Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system (CNS). Packaging and storage of glutamate into glutamatergic neuronal vesicles require ATP-dependent vesicular glutamate uptake systems, which utilize the electrochemical proton gradient as a driving force. Three vesicular glutamate transporters (VGLUT1-3) have been recently identified from neuronal tissue where they play a key role to maintain the vesicular glutamate level. Recently, it has been demonstrated that glutamate signaling is also functional in peripheral neuronal and non-neuronal tissues, and occurs in sites of pituitary, adrenal, pineal glands, bone, GI tract, pancreas, skin, and testis. The glutamate receptors and VGLUTs in digestive system have been found in both neuronal and endocrinal cells. The glutamate signaling in the digestive system may have significant relevance to diabetes and GI tract motility disorders. This review will focus on the most recent update of molecular physiology of digestive VGLUTs. PMID:15793854

  14. Some Ideas to Improve Pyroclast Density and Vesicularity Data Analysis

    NASA Astrophysics Data System (ADS)

    Bernard, B.; Kueppers, U.; Ortiz, H. D.

    2014-12-01

    Pyroclast density and vesicularity are critical parameters in physical volcanology used to reconstruct eruptive dynamics and feed numerical models. Pyroclastic deposits typically present a wide range of density and vesicularity values, so measurements must be repeated tens of times. These data are generally treated using classical statistical analysis including averages and frequency histograms. One issue in this approach is that density and vesicularity are intensive properties and therefore they cannot be added or averaged directly. We encourage the use of weighted density and vesicularity averages and histograms, which is, until now, done only in few studies. In order to insure an adequate and efficient use of the weighting equations, we introduce an open-source R code to calculate the most common statistical parameters such as range and weighted averages, and produce abundance histograms. An important question when working with statistics is whether or not the sample size is large enough. To address this matter we also included a stability analysis based on a Monte Carlo approach which enables to quantify the reliability of the results. To illustrate this methodology we chose two large datasets from Chachimbiro (Ecuador) and Unzen (Japan) volcanoes. Our first results indicate that the use of weighted analysis instead of frequency analysis can change the density and vesicularity averages up to 4% and the shape of the abundance histogram leading to different interpretations. The stability analysis reveals that the number of measurements required for reliable results depends greatly on the distribution of density and vesicularity values. Therefore the number of measurements must be fixed on an ipso facto basis using a large sample size at the beginning and reducing it to achieve time efficiency.

  15. 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

  16. Vesicular system: Versatile carrier for transdermal delivery of bioactives.

    PubMed

    Singh, Deependra; Pradhan, Madhulika; Nag, Mukesh; Singh, Manju Rawat

    2015-01-01

    The transdermal route of drug delivery has gained immense interest for pharmaceutical researchers. The major hurdle for diffusion of drugs and bioactives through transdermal route is the stratum corneum, the outermost layer of the skin. Currently, various approaches such as physical approach, chemical approach, and delivery carriers have been used to augment the transdermal delivery of bioactives. This review provides a brief overview of mechanism of drug transport across skin, different lipid vesicular systems, with special emphasis on lipid vesicular systems including transfersomes, liposomes, niosomes, ethosomes, virosomes, and pharmacosomes and their application for the delivery of different bioactives. PMID:24564350

  17. Classical Cases of Lymphangioma - As Multiple Vesicular Eruptions.

    PubMed

    Devi, Anju; Narwal, Anjali; Yadav, Achla Bharti; Singh, Virender; Gupta, Ambika

    2016-06-01

    Lymphangiomas are uncommon congenital hamartomas of the lymphatic system, usually diagnosed in infancy and early childhood. They are rarely situated in oral cavity and most commonly identified on the anterior two-thirds of the tongue. Though rarely seen in the oral cavity, lymphangiomas are the entities which should be taken into consideration by the clinician while examining vesicular lesions of the oral cavity. Early recognition is of utmost importance for the initiation of proper treatment and to avoid serious complications. We hereby report two classical cases of lymphangioma of the buccal mucosa with multiple vesicular eruptions, a rare site. PMID:27504428

  18. Classical Cases of Lymphangioma – As Multiple Vesicular Eruptions

    PubMed Central

    Narwal, Anjali; Yadav, Achla Bharti; Singh, Virender; Gupta, Ambika

    2016-01-01

    Lymphangiomas are uncommon congenital hamartomas of the lymphatic system, usually diagnosed in infancy and early childhood. They are rarely situated in oral cavity and most commonly identified on the anterior two-thirds of the tongue. Though rarely seen in the oral cavity, lymphangiomas are the entities which should be taken into consideration by the clinician while examining vesicular lesions of the oral cavity. Early recognition is of utmost importance for the initiation of proper treatment and to avoid serious complications. We hereby report two classical cases of lymphangioma of the buccal mucosa with multiple vesicular eruptions, a rare site. PMID:27504428

  19. Osmotic regulation of Rab-mediated organelle docking

    PubMed Central

    Brett, Christopher L.; Merz, Alexey J.

    2009-01-01

    SUMMARY Osmotic gradients across organelle and plasma membranes modulate the rates of membrane fission and fusion; sufficiently large gradients can cause membrane rupture [1–6]. Hypotonic gradients applied to living yeast cells trigger prompt (within seconds) swelling and fusion of Saccharomyces cerevisiae vacuoles, while hypertonic gradients cause vacuoles to fragment on a slower time scale [7–11]. Here, we analyze the influence of osmotic strength on homotypic fusion of isolated yeast vacuoles. Consistent with previously reported in vivo results, we find that decreases in osmolyte concentration increase the rate and extent of vacuole fusion in vitro, while increases in osmolyte concentration prevent fusion. Unexpectedly, our results reveal that osmolytes regulate fusion by inhibiting early, Rab-dependent docking or predocking events, not late events. Our experiments reveal an organelle-autonomous pathway that may control organelle surface to volume ratio, size and copy number: decreasing the osmolyte concentration in the cytoplasmic compartment accelerates Rab-mediated docking and fusion. Fusion, by altering the organelle surface-to-enclosed volume relationship, in turn reduces the risk of membrane rupture. PMID:18619842

  20. Osmotic regulation of Rab-mediated organelle docking.

    PubMed

    Brett, Christopher L; Merz, Alexey J

    2008-07-22

    Osmotic gradients across organelle and plasma membranes modulate the rates of membrane fission and fusion; sufficiently large gradients can cause membrane rupture [1-6]. Hypotonic gradients applied to living yeast cells trigger prompt (within seconds) swelling and fusion of Saccharomyces cerevisiae vacuoles, whereas hypertonic gradients cause vacuoles to fragment on a slower time scale [7-11]. Here, we analyze the influence of osmotic strength on homotypic fusion of isolated yeast vacuoles. Consistent with previously reported in vivo results, we find that decreases in osmolyte concentration increase the rate and extent of vacuole fusion in vitro, whereas increases in osmolyte concentration prevent fusion. Unexpectedly, our results reveal that osmolytes regulate fusion by inhibiting early Rab-dependent docking or predocking events, not late events. Our experiments reveal an organelle-autonomous pathway that may control organelle surface-to-volume ratio, size, and copy number: Decreasing the osmolyte concentration in the cytoplasmic compartment accelerates Rab-mediated docking and fusion. By altering the relationship between the organelle surface and its enclosed volume, fusion in turn reduces the risk of membrane rupture. PMID:18619842

  1. Renal epithelial cells can release ATP by vesicular fusion

    PubMed Central

    Bjaelde, Randi G.; Arnadottir, Sigrid S.; Overgaard, Morten T.; Leipziger, Jens; Praetorius, Helle A.

    2013-01-01

    Renal epithelial cells have the ability to release nucleotides as paracrine factors. In the intercalated cells of the collecting duct, ATP is released by connexin30 (cx30), which is selectively expressed in this cell type. However, ATP is released by virtually all renal epithelia and the aim of the present study was to identify possible alternative nucleotide release pathways in a renal epithelial cell model. We used MDCK (type1) cells to screen for various potential ATP release pathways. In these cells, inhibition of the vesicular H+-ATPases (bafilomycin) reduced both the spontaneous and hypotonically (80%)-induced nucleotide release. Interference with vesicular fusion using N-ethylamide markedly reduced the spontaneous nucleotide release, as did interference with trafficking from the endoplasmic reticulum to the Golgi apparatus (brefeldin A1) and vesicular transport (nocodazole). These findings were substantiated using a siRNA directed against SNAP-23, which significantly reduced spontaneous ATP release. Inhibition of pannexin and connexins did not affect the spontaneous ATP release in this cell type, which consists of ~90% principal cells. TIRF-microscopy of either fluorescently-labeled ATP (MANT-ATP) or quinacrine-loaded vesicles, revealed that spontaneous release of single vesicles could be promoted by either hypoosmolality (50%) or ionomycin. This vesicular release decreased the overall cellular fluorescence by 5.8 and 7.6% respectively. In summary, this study supports the notion that spontaneous and induced ATP release can occur via exocytosis in renal epithelial cells. PMID:24065923

  2. INFECTIVITY AND PERSISTENCE OF VESICULAR STOMATITIS VIRUS IN CULICOIDES CELLS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The biting midge, Culicoides sonorensis, was recently shown to be a biologically competent vector for the arbovirus, vesicular stomatitis virus (VSV). While arboviruses can be extremely pathogenic to mammalian cells, they typically do not exert deleterious effects on their insect vectors. Infectio...

  3. Vesicularity of basalt erupted at Reykjanes Ridge crest

    USGS Publications Warehouse

    Duffield, W.A.

    1978-01-01

    Average vesicularity of basalt drilled at three sites on the west flank of the Reykjanes Ridge increases with decreasing age. This change apparently records concomitant decrease in water depth at the ridge crest where the basalt was erupted and suggests substantial upward growth of the crest during the past 35 Myr. ?? 1978 Nature Publishing Group.

  4. Three-Dimensional Spot Detection in Ratiometric Fluorescence Imaging For Measurement of Subcellular Organelles

    PubMed Central

    Lau, William W.; Johnson, Calvin A.; Lioi, Sara; Mindell, Joseph A.

    2014-01-01

    Lysosomes are subcellular organelles playing a vital role in the endocytosis process of the cell. Lysosomal acidity is an important factor in assuring proper functioning of the enzymes within the organelle, and can be assessed by labeling the lysosomes with pH-sensitive fluorescence probes. To enhance our understanding of the acidification mechanisms, the goal of this work is to develop a method that can accurately detect and characterize the acidity of each lysosome captured in ratiometric fluorescence images. We present an algorithm that utilizes the h-dome transformation and reconciles spots detected independently from two wavelength channels. We evaluated our algorithm using simulated images for which the exact locations were known. The h-dome algorithm achieved an f-score as high as 0.890. We also computed the fluorescence ratios from lysosomes in live HeLa cell images with known lysosomal pHs. Using leave-one-out cross-validation, we demonstrated that the new algorithm was able to achieve much better pH prediction accuracy than the conventional method. PMID:25621319

  5. Cadmium Stress Disrupts the Endomembrane Organelles and Endocytosis during Picea wilsonii Pollen Germination and Tube Growth

    PubMed Central

    Feng, Yu; Li, Xue; Wei, Qian; Sheng, Xianyong

    2014-01-01

    As one of the most severe pollutants, cadmium has been reported to be harmful to plant cells, but the effects of cadmium on gymnosperm pollen germination and tube growth and the mechanism of this involvement are still unclear. Here, we report that cadmium not only strongly inhibited P. wilsonii pollen germination and tube growth, but also significantly altered tube morphology in a dose-dependent manner. Time-lapse images obtained with a laser scanning confocal microscope revealed that endocytosis was dramatically inhibited by cadmium stress. Further investigation with ER-Tracker dye indicated that cadmium stress reduced the number of the Golgi apparatus, and induced dilation of ER. Additionally, Lyso-Tracker staining showed that cadmium distinctly promoted the formation of acidic organelles in pollen tubes, likely derived from the dilated ER. Taken together, our studies indicated that P. wilsonii pollens were highly susceptible to cadmium stress, and that cadmium stress strongly inhibited pollen germination and tube growth by disrupting the endomembrane organelles, inhibiting endo/exocytosis, and forming acidic vacuoles, resulting in swollen tube tips and irregularly broadened tube diameters. These findings provide a new insight into the effects of cadmium toxicity on the tip growth of pollen tubes. PMID:24722362

  6. Amyloplast sedimentation and organelle saltation in living corn columella cells

    NASA Technical Reports Server (NTRS)

    Sack, F. D.; Suyemoto, M. M.; Leopold, A. C.

    1986-01-01

    Amyloplast sedimentation during gravistimulation and organelle movements was studied in living central rootcap cells of Zea mays L. cv. Merit. Cells from sectioned roots were viewed with a horizontally-mounted videomicroscope. The kinetics of gravity-induced amyloplast sedimentation were comparable to those calculated from experiments using fixed material. Individual amyloplasts fell at an average velocity of 5.5 micrometers min-1; the maximal velocity of fall measured was 18.0 micrometers min-1. Amyloplasts often rotated, sometimes rose in the cytoplasm, and occasionally underwent sudden rapid movements as fast as 58 micrometers min-1. Saltations of other organelles were frequently observed. This appears to be the first report of cytoplasmic streaming in the presumptive statocytes of roots.

  7. Organelle transport along microtubules - the role of KIFs.

    PubMed

    Hirokawa, N

    1996-04-01

    Organelle transporters are very important for cellular morphogenesis and other cellular functions, conveying and targeting important materials to the correct destination, often at considerable velocities. One of the first proteins to be identified as a motor was kinesin, and recently at least 10 new kinesin superfamily proteins (KIFs) have been described. Characterization of some of them reveals that each member can convey a specific organelle or cargo, although there is some redundancy. It has also become clear that there are distinct subclasses of KIFs that form monomeric, heterodimeric and homodimeric motors. Here, Nobutaka Hirokawa reviews what is known about the kinesin superfamily and discusses how a study of the different types of motors is helping to elucidate the mechanism of mechanical force generation. PMID:15157476

  8. Updating Our View of Organelle Genome Nucleotide Landscape

    PubMed Central

    Smith, David Roy

    2012-01-01

    Organelle genomes show remarkable variation in architecture and coding content, yet their nucleotide composition is relatively unvarying across the eukaryotic domain, with most having a high adenine and thymine (AT) content. Recent studies, however, have uncovered guanine and cytosine (GC)-rich mitochondrial and plastid genomes. These sequences come from a small but eclectic list of species, including certain green plants and animals. Here, I review GC-rich organelle DNAs and the insights they have provided into the evolution of nucleotide landscape. I emphasize that GC-biased mitochondrial and plastid DNAs are more widespread than once thought, sometimes occurring together in the same species, and suggest that the forces biasing their nucleotide content can differ both among and within lineages, and may be associated with specific genome architectural features and life history traits. PMID:22973299

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

    DOE PAGESBeta

    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. GOBASE—a database of organelle and bacterial genome information

    PubMed Central

    O'Brien, Emmet A.; Zhang, Yue; Yang, LiuSong; Wang, Eric; Marie, Veronique; Lang, B. Franz; Burger, Gertraud

    2006-01-01

    The organelle genome database GOBASE is now in its twelfth release, and includes 350 000 mitochondrial sequences and 118 000 chloroplast sequences, roughly a 3-fold expansion since previously documented. GOBASE also includes a fully reannotated genome sequence of Rickettsia prowazekii, one of the closest bacterial relatives of mitochondria, and will shortly expand to contain more data from bacteria from which organelles originated. All these sequences are now accessible through a single unified interface. Enhancements to the functionality of GOBASE include addition of pages for RNA structures and a page compiling data about the taxonomic distribution of organelle-encoded genes; incorporation of Gene Ontology terms; addition of features deduced from incomplete annotations to sequences in GenBank; marking of type examples in cases where single genes in single species are oversampled within GenBank; and addition of graphics illustrating gene structure and the position of neighbouring genes on a sequence. The database has been reimplemented in PostgreSQL to facilitate development and maintenance, and structural modifications have been made to speed up queries, particularly those related to taxonomy. The GOBASE database can be queried at and inquiries should be directed to gobase@bch.umontreal.ca. PMID:16381962

  11. Whole-Genome Hitchhiking on an Organelle Mutation.

    PubMed

    Flood, Pádraic J; van Heerwaarden, Joost; Becker, Frank; de Snoo, C Bastiaan; Harbinson, Jeremy; Aarts, Mark G M

    2016-05-23

    Strong selection on a beneficial mutation can cause a selective sweep, which fixes the mutation in the population and reduces the genetic variation in the region flanking the mutation [1-3]. These flanking regions have increased in frequency due to their physical association with the selected loci, a phenomenon called "genetic hitchhiking" [4]. Theoretically, selection could extend the hitchhiking to unlinked parts of the genome, to the point that selection on organelles affects nuclear genome diversity. Such indirect selective sweeps have never been observed in nature. Here we show that strong selection on a chloroplast gene in the wild plant species Arabidopsis thaliana has caused widespread and lasting hitchhiking of the whole nuclear genome. The selected allele spread more than 400 km along the British railway network, reshaping the genetic composition of local populations. This demonstrates that selection on organelle genomes can significantly reduce nuclear genetic diversity in natural populations. We expect that organelle-mediated genetic draft is a more common occurrence than previously realized and needs to be considered when studying genome evolution. PMID:27133865

  12. Photoacoustic “nanobombs” fight against undesirable vesicular compartmentalization of anticancer drugs

    PubMed Central

    Chen, Aiping; Xu, Chun; Li, Min; Zhang, Hailin; Wang, Diancheng; Xia, Mao; Meng, Gang; Kang, Bin; Chen, Hongyuan; Wei, Jiwu

    2015-01-01

    Undesirable intracellular vesicular compartmentalization of anticancer drugs in cancer cells is a common cause of chemoresistance. Strategies aimed at circumventing this problem may improve chemotherapeutic efficacy. We report a novel photophysical strategy for controlled-disruption of vesicular sequestration of the anticancer drug doxorubicin (DOX). Single-walled carbon nanotubes (SWCNTs), modified with folate, were trapped in acidic vesicles after entering lung cancer cells. Upon irradiation by near-infrared pulsed laser, these vesicles were massively broken by the resulting photoacoustic shockwave, and the vesicle-sequestered contents were released, leading to redistribution of DOX from cytoplasm to the target-containing nucleus. Redistribution resulted in 12-fold decrease of the EC50 of DOX in lung cancer cells, and enhanced antitumor efficacy of low-dose DOX in tumor-bearing mice. Side effects were not observed. These findings provide insights of using nanotechnology to improve cancer chemotherapy, i.e. not only for drug delivery, but also for overcoming intracellular drug-transport hurdles. PMID:26483341

  13. Mechanisms of organelle division and inheritance and their implications regarding the origin of eukaryotic cells

    PubMed Central

    KUROIWA, Tsuneyoshi

    2010-01-01

    Mitochondria and plastids have their own DNAs and are regarded as descendants of endosymbiotic prokaryotes. Organellar DNAs are not naked in vivo but are associated with basic proteins to form DNA-protein complexes (called organelle nuclei). The concept of organelle nuclei provides a new approach to explain the origin, division, and inheritance of organelles. Organelles divide using organelle division rings (machineries) after organelle-nuclear division. Organelle division machineries are a chimera of the FtsZ (filamentous temperature sensitive Z) ring of bacterial origin and the eukaryotic mechanochemical dynamin ring. Thus, organelle division machineries contain a key to solve the origin of organelles (eukaryotes). The maternal inheritance of organelles developed during sexual reproduction and it is also probably intimately related to the origin of organelles. The aims of this review are to describe the strategies used to reveal the dynamics of organelle division machineries, and the significance of the division machineries and maternal inheritance in the origin and evolution of eukaryotes. PMID:20467212

  14. Solubilization and reconstitution of vesicular stomatitis virus envelope using octylglucoside.

    PubMed Central

    Paternostre, M; Viard, M; Meyer, O; Ghanam, M; Ollivon, M; Blumenthal, R

    1997-01-01

    Reconstituted vesicular stomatitis virus envelopes or virosomes are formed by detergent removal from solubilized intact virus. We have monitored the solubilization process of the intact vesicular stomatitis virus by the nonionic surfactant octylglucoside at various initial virus concentrations by employing turbidity measurements. This allowed us to determine the phase boundaries between the membrane and the mixed micelles domains. We have also characterized the lipid and protein content of the solubilized material and of the reconstituted envelope. Both G and M proteins and all of the lipids of the envelope were extracted by octylglucoside and recovered in the reconstituted envelope. Fusion activity of the virosomes tested either on Vero cells or on liposomes showed kinetics and pH dependence similar to those of the intact virus. Images FIGURE 4 PMID:9083672

  15. SLC18: Vesicular neurotransmitter transporters for monoamines and acetylcholine ☆

    PubMed Central

    Lawal, Hakeem O.; Krantz, David E.

    2012-01-01

    The exocytotic release of neurotransmitters requires active transport into synaptic vesicles and other types of secretory vesicles. Members of the SLC18 family perform this function for acetylcholine (SLC18A3, the vesicular acetylcholine transporter or VAChT) and monoamines such as dopamine and serotonin (SLC18A1 and 2, the vesicular monoamine transporters VMAT1 and 2, respectively). To date, no specific diseases have been attributed to a mutation in an SLC18 family member; however, polymorphisms in SLC18A1 and SLC18A2 may confer risk for some neuropsychiatric disorders. Additional members of this family include SLC18A4, expressed in insects, and SLC18B1, the function of which is not known. SLC18 is part of the Drug:H+ Antiporter-1 Family (DHA1, TCID 2.A.1.2) within the Major Facilitator Superfamily (MFS, TCID 2.A.1). PMID:23506877

  16. Keeping Wnt Signalosome in Check by Vesicular Traffic

    PubMed Central

    FENG, QIANG; GAO, NAN

    2015-01-01

    Wg/Wnts are paracrine and autocrine ligands that activate distinct signaling pathways while being internalized through surface receptors. Converging and contrasting views are shaping our understanding of whether, where, and how endocytosis may modulate Wnt signaling. We gather considerable amount of evidences to elaborate the point that signal-receiving cells utilize distinct, flexible, and sophisticated vesicular trafficking mechanisms to keep Wnt signaling activity in check. Same molecules in a highly context-dependent fashion serve as regulatory hub for various signaling purposes: amplification, maintenance, inhibition, and termination. Updates are provided for the regulatory mechanisms related to the three critical cell surface complexes, Wnt-Fzd-LRP6, Dkk1-Kremen-LRP6, and R-spondin-LGR5-RNF43, which potently influence Wnt signaling. We pay particular attentions to how cells achieve sustained and delicate control of Wnt signaling strength by employing comprehensive aspects of vesicular trafficking. PMID:25336320

  17. Vesicular thick-walled swollen hyphae in pulmonary zygomycosis.

    PubMed

    Kimura, Masatomo; Ito, Hiroyuki

    2009-03-01

    An autopsy case of pulmonary zygomycosis in a patient with rheumatoid arthritis on immunosuppressive therapy is presented herein. There was a pulmonary cavitated infarct caused by mycotic thrombosis. Thin-walled narrow hyphae and vesicular thick-walled swollen hyphae were found on the pleural surface and in the necrotic tissue at the periphery of the cavity. Findings of such shaped fungal elements may cause erroneous histopathological diagnosis because pauciseptate broad thin-walled hyphae are usually the only detectable fungal elements in zygomycosis tissue. Although immunohistochemistry confirmed these unusual elements to be zygomycetous in the present case, it is important for the differential diagnosis to be aware that zygomycetes can form thin narrow hyphae and vesicular thick-walled swollen hyphae. PMID:19261095

  18. The Journey of the Organelle: Teamwork and Regulation in Intracellular Transport

    PubMed Central

    Barlan, Kari; Rossow, Molly J.; Gelfand, Vladimir I.

    2013-01-01

    Specific subsets of biochemical reactions in eukaryotic cells are restricted to individual membrane compartments, or organelles. Cells, therefore, face the monumental task of moving the products of those reactions between individual organelles. Because of the high density of the cytoplasm and the large size of membrane organelles, simple diffusion is grossly insufficient for this task. Proper trafficking between membrane organelles thus relies on cytoskeletal elements and the activity of motor proteins, that act both in transport of membrane compartments and as tethering agents to ensure their proper distribution and to facilitate organelle interactions. PMID:23510681

  19. Annexin XIIIb: a novel epithelial specific annexin is implicated in vesicular traffic to the apical plasma membrane

    PubMed Central

    1995-01-01

    The sorting of apical and basolateral proteins into vesicular carriers takes place in the trans-Golgi network (TGN) in MDCK cells. We have previously analyzed the protein composition of immunoisolated apical and basolateral transport vesicles and have now identified a component that is highly enriched in apical vesicles. Isolation of the encoding cDNA revealed that this protein, annexin XIIIb, is a new isoform of the epithelial specific annexin XIII sub-family which includes the previously described intestine-specific annexin (annexin XIIIa; Wice, B. M., and J. I. Gordon. 1992. J. Cell Biol. 116:405-422). Annexin XIIIb differs from annexin XIIIa in that it contains a unique insert of 41 amino acids in the NH2 terminus and is exclusively expressed in dog intestine and kidney. Immunofluorescence microscopy demonstrated that annexin XIIIb was localized to the apical plasma membrane and underlying punctate structures. Since annexins have been suggested to play a role in membrane-membrane interactions in exocytosis and endocytosis, we investigated whether annexin XIIIb is involved in delivery to the apical cell surface. To this aim we used permeabilized MDCK cells and a cytosol-dependent in vitro transport assay. Antibodies specific for annexin XIIIb significantly inhibited the transport of influenza virus hemagglutinin from the TGN to the apical plasma membrane while the transport of vesicular stomatitis virus glycoprotein to the basolateral cell surface was unaffected. We propose that annexin XIIIb plays a role in vesicular transport to the apical plasma membrane in MDCK cells. PMID:7896870

  20. A set of SNARE proteins in the contractile vacuole complex of Paramecium regulates cellular calcium tolerance and also contributes to organelle biogenesis.

    PubMed

    Schönemann, Barbara; Bledowski, Alexander; Sehring, Ivonne M; Plattner, Helmut

    2013-03-01

    The contractile vacuole complex (CVC) of freshwater protists serves the extrusion of water and ions, including Ca(2+). No vesicle trafficking based on SNAREs has been detected so far in any CVC. SNAREs (soluble NSF [N-ethylmaleimide sensitive factor] attachment protein receptors) are required for membrane-to-membrane interaction, i.e. docking and fusion also in Paramecium. We have identified three v-/R- and three t/Q-SNAREs selectively in the CVC. Posttranscriptional silencing of Syb2, Syb6 or Syx2 slows down the pumping cycle; silencing of the latter two also causes vacuole swelling. Increase in extracellular Ca(2+) after Syb2, Syb6 or Syx2 silencing causes further swelling of the contractile vacuole and deceleration of its pulsation. Silencing of Syx14 or Syx15 entails lethality in the Ca(2+) stress test. Thus, the effects of silencing strictly depend on the type of the silenced SNARE and on the concentration of Ca(2+) in the medium. This shows the importance of organelle-resident SNARE functions (which may encompass the vesicular delivery of other organelle-resident proteins) for Ca(2+) tolerance. A similar principle may be applicable also to the CVC in widely different unicellular organisms. In addition, in Paramecium, silencing particularly of Syx6 causes aberrant positioning of the CVC during de novo biogenesis before cytokinesis. PMID:23280185

  1. Cytoplasmic dynein is a minus end-directed motor for membranous organelles

    SciTech Connect

    Schroer, T.A.; Steuer, E.R.; Sheetz, M.P.

    1989-03-24

    The role of cytoplasmic dynein in microtubule-based organelle transport was examined using a reconstituted assay developed from chick embryo fibroblasts. Factors present in a high-speed cytosol caused the movement of purified organelles on microtubules predominantly in the minus end direction. Inactivation of cytoplasmic dynein in the high-speed cytosol by vanadate-mediated UV photocleavage inhibited minus end-directed organelle motility by over 90%. Addition of purified cytoplasmic dynein to the inactive cytosol restored minus end-directed organelle motility, although purified cytoplasmic dynein by itself did not support organelle movement. We propose that cytoplasmic dynein is the motor for minus end-directed organelle movement, but that additional cytosolic factors are also required to produce organelle motility.

  2. Scanning ion images; analysis of pharmaceutical drugs at organelle levels

    NASA Astrophysics Data System (ADS)

    Larras-Regard, E.; Mony, M.-C.

    1995-05-01

    With the ion analyser IMS 4F used in microprobe mode, it is possible to obtain images of fields of 10 × 10 [mu]m2, corresponding to an effective magnification of 7000 with lateral resolution of 250 nm, technical characteristics that are appropriate for the size of cell organelles. It is possible to characterize organelles by their relative CN-, P- and S- intensities when the tissues are prepared by freeze fixation and freeze substitution. The recognition of organelles enables correlation of the tissue distribution of ebselen, a pharmaceutical drug containing selenium. The various metabolites characterized in plasma, bile and urine during biotransformation of ebselen all contain selenium, so the presence of the drug and its metabolites can be followed by images of Se. We were also able to detect the endogenous content of Se in tissue, due to the increased sensitivity of ion analysis in microprobe mode. Our results show a natural occurrence of Se in the border corresponding to the basal lamina of cells of proximal but not distal tubules of the kidney. After treatment of rats with ebselen, an additional site of Se is found in the lysosomes. We suggest that in addition to direct elimination of ebselen and its metabolites by glomerular filtration and urinary elimination, a second process of elimination may occur: Se compounds reaching the epithelial cells via the basal lamina accumulate in lysosomes prior to excretion into the tubular fluid. The technical developments of using the IMS 4F instrument in the microprobe mode and the improvement in preparation of samples by freeze fixation and substitution further extend the limit of ion analysis in biology. Direct imaging of trace elements and molecules marked with a tracer make it possible to determine their targets by comparison with images of subcellular structures. This is a promising advance in the study of pathways of compounds within tissues, cells and the whole organism.

  3. KIFC2 is a novel neuron-specific C-terminal type kinesin superfamily motor for dendritic transport of multivesicular body-like organelles.

    PubMed

    Saito, N; Okada, Y; Noda, Y; Kinoshita, Y; Kondo, S; Hirokawa, N

    1997-03-01

    We have cloned two novel C-terminal motor domain-type kinesin superfamily motor proteins (KIFCs) from mouse brain by utilizing a KIFC-specific consensus sequence. The first protein was the murine homologue of CHO2 antigen, a member of the kar3-type mitotic motor subfamily, and we designated this protein KIFC1. The other protein, KIFC2 (792 amino acids), is novel, with no significant similarity to known kinesin superfamily proteins (KIFs). KIFC2 was specifically expressed in adult neurons, and was immunofluorescently localized to punctate structures in cell bodies and dendrites, but was not detected in axons. Electron microscopic analysis of the immunoisolated KIFC2-bound organelles revealed that KIFC2 associates with multivesicular body (mvb)-like organelles, suggesting that KIFC2 functions as the motor for the transport of mvb-like organelles in dendrites. PMID:9115736

  4. Evolution of the bacterial organelle responsible for magnetotaxis.

    PubMed

    Lefèvre, Christopher T; Wu, Long-Fei

    2013-10-01

    There are few examples of protein- and lipid-bounded organelles in bacteria that are encoded by conserved gene clusters and lead to a specific function. The magnetosome chain represents one of these rare examples and is responsible for magnetotaxis in magnetotactic bacteria (MTB), a behavior thought to aid in finding their optimal growth conditions. The origin and evolution of the magnetotaxis is still a matter of debate. Recent breakthroughs in isolation, cultivation, single-cell separation, and whole-genome sequencing have generated abundant data that give new insights into the biodiversity and evolution of MTB. PMID:23948365

  5. Organelle DNA polymorphism in apple cultivars and rootstocks.

    PubMed

    Ishikawa, S; Kato, S; Imakawa, S; Mikami, T; Shimamoto, Y

    1992-05-01

    Restriction fragment length polymorphisms (RFLPs) have been used to detect chloroplast (cp) and mitochondrial (mt) DNA variation among 18 apple cultivars and three rootstocks. The distribution of RFLP patterns allowed the assignment of these genotypes into three groups of cytoplasmic relatedness. Our results also demonstrate maternal inheritance of cp- and mtDNAs in apple. Thus, the organelle DNA assay provides a convenient and reliable method to assess cytoplasmic diversity within the apple germ-plasm collection and to trace the maternal lineages involved in the evolution of apple. PMID:24202920

  6. Ligand-directed profiling of organelles with internalizing phage libraries

    PubMed Central

    Dobroff, Andrey S.; Rangel, Roberto; Guzman-Roja, Liliana; Salmeron, Carolina C.; Gelovani, Juri G.; Sidman, Richard L.; Bologa, Cristian G.; Oprea, Tudor I.; Brinker, C. Jeffrey; Pasqualini, Renata; Arap, Wadih

    2015-01-01

    Phage display is a resourceful tool to, in an unbiased manner, discover and characterize functional protein-protein interactions, to create vaccines, and to engineer peptides, antibodies, and other proteins as targeted diagnostic and/or therapeutic agents. Recently, our group has developed a new class of internalizing phage (iPhage) for ligand-directed targeting of organelles and/or to identify molecular pathways within live cells. This unique technology is suitable for applications ranging from fundamental cell biology to drug development. Here we describe the method for generating and screening the iPhage display system, and explain how to select and validate candidate internalizing homing peptide. PMID:25640897

  7. Lysosome-related organelles: Unusual compartments become mainstream

    PubMed Central

    Marks, Michael S.; Heijnen, Harry F. G.; Raposo, Graça

    2013-01-01

    Lysosome-related organelles (LROs) comprise a group of cell type-specific subcellular compartments with unique composition, morphology and structure that share some features with endosomes and lysosomes and that function in varied processes such as pigmentation, hemostasis, lung plasticity and immunity. In recent years, studies of genetic diseases in which LRO functions are compromised have provided new insights into the mechanisms of LRO biogenesis and the regulated secretion of LRO contents. These insights have revealed previously unappreciated specialized endosomal sorting processes in all cell types, and are expanding our views of the plasticity of the endosomal and secretory systems in adapting to cell type-specific needs. PMID:23726022

  8. The lipid droplet—a well-connected organelle

    PubMed Central

    Gao, Qiang; Goodman, Joel M.

    2015-01-01

    Our knowledge of inter-organellar communication has grown exponentially in recent years. This review focuses on the interactions that cytoplasmic lipid droplets have with other organelles. Twenty-five years ago droplets were considered simply particles of coalesced fat. Ten years ago there were hints from proteomics studies that droplets might interact with other structures to share lipids and proteins. Now it is clear that the droplets interact with many if not most cellular structures to maintain cellular homeostasis and to buffer against insults such as starvation. The evidence for this statement, as well as probes to understand the nature and results of droplet interactions, are presented. PMID:26322308

  9. Disarrangement of actin filaments and Ca²⁺ gradient by CdCl₂ alters cell wall construction in Arabidopsis thaliana root hairs by inhibiting vesicular trafficking.

    PubMed

    Fan, Jun-Ling; Wei, Xue-Zhi; Wan, Li-Chuan; Zhang, Ling-Yun; Zhao, Xue-Qin; Liu, Wei-Zhong; Hao, Huai-Qin; Zhang, Hai-Yan

    2011-07-15

    Cadmium (Cd), one of the most toxic heavy metals, inhibits many cellular and physiological processes in plants. Here, the involvement of cytoplasmic Ca²⁺ gradient and actin filaments (AFs) in vesicular trafficking, cell wall deposition and tip growth was investigated during root (hair) development of Arabidopsis thaliana in response to CdCl₂ treatment. Seed germination and root elongation were prevented in a dose- and time-dependent manner by CdCl₂ treatment. Fluorescence labelling and non-invasive detection showed that CdCl₂ inhibited extracellular Ca²⁺ influx, promoted intracellular Ca²⁺ efflux, and disturbed the cytoplasmic tip-focused Ca²⁺ gradient. In vivo labelling revealed that CdCl₂ modified actin organization, which subsequently contributed to vesicle trafficking. Transmission electron microscopy revealed that CdCl₂ induced cytoplasmic vacuolization and was detrimental to organelles such as mitochondria and endoplasmic reticulum (ER). Finally, immunofluorescent labelling and Fourier transform infrared (FTIR) analysis indicated that configuration/distribution of cell wall components such as pectins and cellulose was significantly altered in response to CdCl₂. Our results indicate that CdCl₂ induces disruption of Ca²⁺ gradient and AFs affects the distribution of cell wall components in root hairs by disturbing vesicular trafficking in A. thaliana. PMID:21497412

  10. The organelle of differentiation in embryos: the cell state splitter.

    PubMed

    Gordon, Natalie K; Gordon, Richard

    2016-01-01

    The cell state splitter is a membraneless organelle at the apical end of each epithelial cell in a developing embryo. It consists of a microfilament ring and an intermediate filament ring subtending a microtubule mat. The microtubules and microfilament ring are in mechanical opposition as in a tensegrity structure. The cell state splitter is bistable, perturbations causing it to contract or expand radially. The intermediate filament ring provides metastability against small perturbations. Once this snap-through organelle is triggered, it initiates signal transduction to the nucleus, which changes gene expression in one of two readied manners, causing its cell to undergo a step of determination and subsequent differentiation. The cell state splitter also triggers the cell state splitters of adjacent cells to respond, resulting in a differentiation wave. Embryogenesis may be represented then as a bifurcating differentiation tree, each edge representing one cell type. In combination with the differentiation waves they propagate, cell state splitters explain the spatiotemporal course of differentiation in the developing embryo. This review is excerpted from and elaborates on "Embryogenesis Explained" (World Scientific Publishing, Singapore, 2016). PMID:26965444

  11. Targeting mammalian organelles with internalizing phage (iPhage) libraries

    PubMed Central

    Rangel, Roberto; Dobroff, Andrey S.; Guzman-Rojas, Liliana; Salmeron, Carolina C.; Gelovani, Juri G.; Sidman, Richard L.; Pasqualini, Renata; Arap, Wadih

    2015-01-01

    Techniques largely used for protein interaction studies and discovery of intracellular receptors, such as affinity capture complex purification and yeast two-hybrid, may produce inaccurate datasets due to protein insolubility, transient or weak protein interactions, or irrelevant intracellular context. A versatile tool to overcome these limitations as well as to potentially create vaccines and engineer peptides and antibodies as targeted diagnostic and therapeutic agents, is the phage display technique. We have recently developed a new technology for screening internalizing phage (iPhage) vectors and libraries utilizing a ligand/receptor-independent mechanism to penetrate eukaryotic cells. iPhage particles provide a unique discovery platform for combinatorial intracellular targeting of organelle ligands along with their corresponding receptors and to fingerprint functional protein domains in living cells. Here we explain the design, cloning, construction, and production of iPhage-based vectors and libraries, along with basic ligand-receptor identification and validation methodologies for organelle receptors. An iPhage library screening can be performed in ~8 weeks. PMID:24030441

  12. Is Spontaneous Translocation of Polar Lipids Between Cellular Organelles Negligible?

    PubMed Central

    Somerharju, Pentti

    2015-01-01

    In most reviews addressing intracellular lipid trafficking, spontaneous diffusion of lipid monomers between the cellular organelles is considered biologically irrelevant because it is thought to be far too slow to significantly contribute to organelle biogenesis. This view is based on intervesicle transfer experiments carried out in vitro with few lipids as well as on the view that lipids are highly hydrophobic and thus cannot undergo spontaneous intermembrane diffusion at a significant rate. However, besides that single-chain lipids can translocate between vesicles in seconds, it has been demonstrated that the rate of spontaneous transfer of two-chain polar lipids can vary even 1000-fold, depending on the number of carbons and double bonds in the acyl chains. In addition, the rate of spontaneous lipid transfer can strongly depend on the experimental conditions such as vesicle composition and concentration. This review examines the studies suggesting that spontaneous lipid transfer is probably more relevant to intracellular trafficking of amphipathic lipids than commonly thought. PMID:27147824

  13. Detecting Bacterial Surface Organelles on Single Cells Using Optical Tweezers.

    PubMed

    Zakrisson, Johan; Singh, Bhupender; Svenmarker, Pontus; Wiklund, Krister; Zhang, Hanqing; Hakobyan, Shoghik; Ramstedt, Madeleine; Andersson, Magnus

    2016-05-10

    Bacterial cells display a diverse array of surface organelles that are important for a range of processes such as intercellular communication, motility and adhesion leading to biofilm formation, infections, and bacterial spread. More specifically, attachment to host cells by Gram-negative bacteria are mediated by adhesion pili, which are nanometers wide and micrometers long fibrous organelles. Since these pili are significantly thinner than the wavelength of visible light, they cannot be detected using standard light microscopy techniques. At present, there is no fast and simple method available to investigate if a single cell expresses pili while keeping the cell alive for further studies. In this study, we present a method to determine the presence of pili on a single bacterium. The protocol involves imaging the bacterium to measure its size, followed by predicting the fluid drag based on its size using an analytical model, and thereafter oscillating the sample while a single bacterium is trapped by an optical tweezer to measure its effective fluid drag. Comparison between the predicted and the measured fluid drag thereby indicate the presence of pili. Herein, we verify the method using polymer coated silica microspheres and Escherichia coli bacteria expressing adhesion pili. Our protocol can in real time and within seconds assist single cell studies by distinguishing between piliated and nonpiliated bacteria. PMID:27088225

  14. 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

  15. Correlative video-light-electron microscopy of mobile organelles.

    PubMed

    Beznoussenko, Galina V; Mironov, Alexander A

    2015-01-01

    Correlative microscopy is a method when for the analysis of the very same cell or tissue area, several different methods of light microscopy (LM) and then electron microscopy (EM) are used consecutively. The combination of LM and EM allows researchers to study phenomena at a global scale and then to look for unique or rare events for their subsequent EM examination. Unfortunately, the observation of living cells under EM is still impossible. LM provides the possibility to examine quickly many live cells, whereas EM provides the high level of resolution. On the other side, the final goal of any morphological analysis of a biological sample, whether it is an organism, organ, tissue, cell, organelle, or molecule, is to get an averaged three-dimensional model of the structure examined and to determine the chemical composition of it. This chapter describes the methodology of imaging with the help of CVLEM. The guidelines presented herein enable researchers to analyze structure of organelles and to obtain the three-dimensional model of the structure examined, and in particular rare events captured by low-resolution imaging of a population or transient events captured by live imaging can now also be studied at high resolution by EM. PMID:25702127

  16. Comparative Bioinformatics Analyses and Profiling of Lysosome-Related Organelle Proteomes

    PubMed Central

    Hu, Zhang-Zhi; Valencia, Julio C.; Huang, Hongzhan; Chi, An; Shabanowitz, Jeffrey; Hearing, Vincent J.; Appella, Ettore; Wu, Cathy

    2007-01-01

    Complete and accurate profiling of cellular organelle proteomes, while challenging, is important for the understanding of detailed cellular processes at the organelle level. Mass spectrometry technologies coupled with bioinformatics analysis provide an effective approach for protein identification and functional interpretation of organelle proteomes. In this study, we have compiled human organelle reference datasets from large-scale proteomic studies and protein databases for 7 lysosome-related organelles (LROs), as well as the endoplasmic reticulum and mitochondria, for comparative organelle proteome analysis. Heterogeneous sources of human organelle proteins and rodent homologs are mapped to human UniProtKB protein entries based on ID and/or peptide mappings, followed by functional annotation and categorization using the iProXpress proteomic expression analysis system. Cataloging organelle proteomes allows close examination of both shared and unique proteins among various LROs and reveals their functional relevance. The proteomic comparisons show that LROs are a closely related family of organelles. The shared proteins indicate the dynamic and hybrid nature of LROs, while the unique transmembrane proteins may represent additional candidate marker proteins for LROs. This comparative analysis, therefore, provides a basis for hypothesis formulation and experimental validation of organelle proteins and their functional roles. PMID:17375895

  17. Comparative bioinformatics analyses and profiling of lysosome-related organelle proteomes

    NASA Astrophysics Data System (ADS)

    Hu, Zhang-Zhi; Valencia, Julio C.; Huang, Hongzhan; Chi, An; Shabanowitz, Jeffrey; Hearing, Vincent J.; Appella, Ettore; Wu, Cathy

    2007-01-01

    Complete and accurate profiling of cellular organelle proteomes, while challenging, is important for the understanding of detailed cellular processes at the organelle level. Mass spectrometry technologies coupled with bioinformatics analysis provide an effective approach for protein identification and functional interpretation of organelle proteomes. In this study, we have compiled human organelle reference datasets from large-scale proteomic studies and protein databases for seven lysosome-related organelles (LROs), as well as the endoplasmic reticulum and mitochondria, for comparative organelle proteome analysis. Heterogeneous sources of human organelle proteins and rodent homologs are mapped to human UniProtKB protein entries based on ID and/or peptide mappings, followed by functional annotation and categorization using the iProXpress proteomic expression analysis system. Cataloging organelle proteomes allows close examination of both shared and unique proteins among various LROs and reveals their functional relevance. The proteomic comparisons show that LROs are a closely related family of organelles. The shared proteins indicate the dynamic and hybrid nature of LROs, while the unique transmembrane proteins may represent additional candidate marker proteins for LROs. This comparative analysis, therefore, provides a basis for hypothesis formulation and experimental validation of organelle proteins and their functional roles.

  18. PRIMARY PEPTIDE SEQUENCES FROM SQUID MUSCLE AND OPTIC LOBE MYOSIN IIs: A STRATEGY TO IDENTIFY AN ORGANELLE MYOSIN

    PubMed Central

    MEDEIROS, NELSON A.; REESE, THOMAS S.; JAFFE, HOWARD; DEGIORGIS, JOSEPH A.; BEARER, ELAINE L.

    2013-01-01

    The squid giant axon provides an excellent model system for the study of actin-based organelle transport likely to be mediated by myosins, but the identification of these motors has proven to be difficult. Here the authors purified and obtained primary peptide sequence of squid muscle myosin as a first step in a strategy designed to identify myosins in the squid nervous system. Limited digestion yielded fourteen peptides derived from the muscle myosin which possess high amino acid sequence identities to myosin II from scallop (60–95%) and chick pectoralis muscle (31–83%). Antibodies generated to this purified muscle myosin were used to isolate a potential myosin from squid optic lobe which yielded 11 peptide fragments. Sequences from six of these fragments identified this protein as a myosin II. The other five sequences matched myosin II (50–60%, identities), and some also matched unconventional myosins (33–50%). A single band that has a molecular weight similar to the myosin purified from optic lobe copurifies with axoplasmic organelles, and, like the optic lobe myosin, this band is also recognized by the antibodies raised against squid muscle myosin II. Hence, this strategy provides an approach to the identification of a myosin associated with motile axoplasmic organelles. PMID:9878103

  19. Single-prolonged stress induce different change in the cell organelle of the hippocampal cells: A study of ultrastructure.

    PubMed

    Wan, JunLai; Liu, Dongjuan; Zhang, Jie; Shi, Yuxiu; Han, Fang

    2016-01-01

    MRI studies have revealed structural and functional changes in the hippocampus of post-traumatic stress disorder (PTSD) patients. Previous studies conducted by us in a PTSD animal model found that single prolonged stress (SPS) induced abnormal morphological changes in hippocampal cells. The effects of SPS on cellular organelles of the hippocampal neurons remain unknown; however, these changes have been involved in SPS-induced abnormal hippocampal function. The aim of the present study is to examine ultrastructural changes in cellular organelles, including the lysosomes, mitochondria (Mit), Golgi apparatus, and endoplasmic reticulum (ER), following SPS exposure using transmission electron microscopy, enzyme histochemistry, and enzyme cytochemistry. First, morphological changes of the hippocampal cells and ultrastructural changes in cellular organelles, including lysosomes, ER, and Mit-induced by SPS were observed. Results from histo- and cytochemistry demonstrated that the Mit marker enzyme, cytochrome c oxidase (COX), and the lysosomal enzyme acid phosphatase, (ACP), increased following exposure to SPS. SPS induced COX release from Mit and led to a wider distribution of ACP in round lysosomes, NLY, and the Golgi. In addition, we found that SPS increased the presence of autophagosomes and induced changes in the autophagy-related protein, Beclin. These results indicated the differential effects of SPS on cellular organelles, that is, a positive effect on lysosomes as well as a negative effect on the Mit and ER. Increased lysosomal function may serve as protection against SPS-induced cell damage. Structural changes in the Mit and ER may be involved in SPS-induced disorders of energy metabolism and protein synthesis and export. PMID:26589383

  20. Gas transport and vesicularity in low-viscosity liquids

    NASA Astrophysics Data System (ADS)

    Pioli, Laura; Bonadonna, Costanza; Abdulkareem, Lokman; Azzopardi, Barry; Phillips, Jeremy

    2010-05-01

    Vesicle textures of basaltic scoria preserve information on magma bubble content at fragmentation and are commonly used to constrain degassing, vesiculation and magma permeability. These studies are based on the assumption that microscale textures are representative of the conduit-scale structures and processes. However, the conditions for which this assumption is valid have not been investigated in detail. We have investigated conduit-scale structures by performing a series of experiments of separate two-phase flows in a 6.5-m high cylindrical bubble column using a combination of air with pure glucose syrup, water-syrup mixtures and pure water to reproduce open-system degassing and strombolian activity conditions in the upper volcanic conduit (i.e. at very low or zero liquid fluxes). We have varied gas fluxes, initial liquid height, gas inlet configuration and liquid viscosity and analyzed flow regimes and properties. Temperature and pressure were measured at several heights along the pipe and vesicularity was calculated using pressure data, liquid level measurements and an Electrical Capacitance tomography (ECT) system, which measures instantaneous vesicularity and phase distribution from capacitance measurements between pairs of electrodes placed uniformly around the pipe circumference. The aim of the experiments was to identify the effect of gas-flow rates on the flow regimes (i.e. bubbly, slug, churn and annular), the main degassing structures and the total gas content of the column. The effect of increasing and decreasing gas flow rates was also studied to check hysteresis effects. Results indicate that the vesicularity of the liquid column depends primarily on gas flux, whereas flow regimes exert a minor control. In fact, vesicularity increases with gas flux following a power-law trend whose exponent depends on the viscosity of the liquid. In addition, distributions of instantaneous gas fraction in the column cross section during syrup experiments have shown

  1. Retromer-Mediated Protein Sorting and Vesicular Trafficking.

    PubMed

    Liu, Jia-Jia

    2016-04-20

    Retromer is an evolutionarily conserved multimeric protein complex that mediates intracellular transport of various vesicular cargoes and functions in a wide variety of cellular processes including polarized trafficking, developmental signaling and lysosome biogenesis. Through its interaction with the Rab GTPases and their effectors, membrane lipids, molecular motors, the endocytic machinery and actin nucleation promoting factors, retromer regulates sorting and trafficking of transmembrane proteins from endosomes to the trans-Golgi network (TGN) and the plasma membrane. In this review, I highlight recent progress in the understanding of retromer-mediated protein sorting and vesicle trafficking and discuss how retromer contributes to a diverse set of developmental, physiological and pathological processes. PMID:27157806

  2. Restricted Replication of Vesicular Stomatitis Virus in Human Lymphoblastoid Cells

    PubMed Central

    Nowakowski, Maja; Bloom, Barry R.; Ehrenfeld, Ellie; Summers, Donald F.

    1973-01-01

    Replication of vesicular stomatitis virus (VSV) is restricted in one human lymphoblastoid cell line (Raji), but not in another similar cell line (Wil-2), compared with growth in HeLa cells. This restriction is characterized by a low proportion of cells yielding infectious virus and is associated with limited production of 42S virion RNA. Primary transcription of 13S and 26S VSV-specific RNA is not restricted in Raji cells, and the 13S RNA produced contains adenylate-rich sequences. This suggests that the block in Raji cells involves some step required for the replication of virion RNA. PMID:4357508

  3. Artificial Organelles: Reactions inside Protein-Polymer Supramolecular Assemblies.

    PubMed

    Garni, Martina; Einfalt, TomaŽ; Lomora, Mihai; Car, Anja; Meier, Wolfgang; Palivan, Cornelia G

    2016-01-01

    Reactions inside confined compartments at the nanoscale represent an essential step in the development of complex multifunctional systems to serve as molecular factories. In this respect, the biomimetic approach of combining biomolecules (proteins, enzymes, mimics) with synthetic membranes is an elegant way to create functional nanoreactors, or even simple artificial organelles, that function inside cells after uptake. Functionality is provided by the specificity of the biomolecule(s), whilst the synthetic compartment provides mechanical stability and robustness. The availability of a large variety of biomolecules and synthetic membranes allows the properties and functionality of these reaction spaces to be tailored and adjusted for building complex self-organized systems as the basis for molecular factories. PMID:27363371

  4. Photoacoustic Tomography: In Vivo Imaging from Organelles to Organs

    NASA Astrophysics Data System (ADS)

    Wang, Lihong V.; Hu, Song

    2012-03-01

    Photoacoustic tomography (PAT) can create multiscale multicontrast images of living biological structures ranging from organelles to organs. This emerging technology overcomes the high degree of scattering of optical photons in biological tissue by making use of the photoacoustic effect. Light absorption by molecules creates a thermally induced pressure jump that launches ultrasonic waves, which are received by acoustic detectors to form images. Different implementations of PAT allow the spatial resolution to be scaled with the desired imaging depth in tissue while a high depth-to-resolution ratio is maintained. As a rule of thumb, the achievable spatial resolution is on the order of 1/200 of the desired imaging depth, which can reach up to 7 centimeters. PAT provides anatomical, functional, metabolic, molecular, and genetic contrasts of vasculature, hemodynamics, oxygen metabolism, biomarkers, and gene expression. We review the state of the art of PAT for both biological and clinical studies and discuss future prospects.

  5. Intracellular partitioning of cell organelles and extraneous nanoparticles during mitosis.

    PubMed

    Symens, Nathalie; Soenen, Stefaan J; Rejman, Joanna; Braeckmans, Kevin; De Smedt, Stefaan C; Remaut, Katrien

    2012-01-01

    The nucleocytoplasmic partitioning of nanoparticles as a result of cell division is highly relevant to the field of nonviral gene delivery. We reviewed the literature on the intracellular distribution of cell organelles (the endosomal vesicles, Golgi apparatus, endoplasmic reticulum and nucleus), foreign macromolecules (dextrans and plasmid DNA) and inorganic nanoparticles (gold, quantum dot and iron oxide) during mitosis. For nonviral gene delivery particles (lipid- or polymer-based), indirect proof of nuclear entry during mitosis is provided. We also describe how retroviruses and latent DNA viruses take advantage of mitosis to transfer their viral genome and segregate their episomes into the host daughter nuclei. Based on this knowledge, we propose strategies to improve nonviral gene delivery in dividing cells with the ultimate goal of designing nonviral gene delivery systems that are as efficient as their viral counterparts but non-immunogenic, non-oncogenic and easy and inexpensive to prepare. PMID:22210278

  6. Phase transitions and size scaling of membrane-less organelles

    PubMed Central

    2013-01-01

    The coordinated growth of cells and their organelles is a fundamental and poorly understood problem, with implications for processes ranging from embryonic development to oncogenesis. Recent experiments have shed light on the cell size–dependent assembly of membrane-less cytoplasmic and nucleoplasmic structures, including ribonucleoprotein (RNP) granules and other intracellular bodies. Many of these structures behave as condensed liquid-like phases of the cytoplasm/nucleoplasm. The phase transitions that appear to govern their assembly exhibit an intrinsic dependence on cell size, and may explain the size scaling reported for a number of structures. This size scaling could, in turn, play a role in cell growth and size control. PMID:24368804

  7. Mitochondria as signaling organelles in the vascular endothelium

    PubMed Central

    Quintero, Marisol; Colombo, Sergio L.; Godfrey, Andrew; Moncada, Salvador

    2006-01-01

    Vascular endothelial cells are highly glycolytic and consume relatively low amounts of oxygen (O2) compared with other cells. We have confirmed that oxidative phosphorylation is not the main source of ATP generation in these cells. We also show that at a low O2 concentration (<1%) endogenous NO plays a key role in preventing the accumulation of the α-subunit of hypoxia-inducible factor 1. At higher O2 concentrations (1–3%) NO facilitates the production of mitochondrial reactive oxygen species. This production activates the AMP-activated protein kinase by a mechanism independent of nucleotide concentrations. Thus, the primary role of mitochondria in vascular endothelial cells may not be to generate ATP but, under the control of NO, to act as signaling organelles using either O2 or O2-derived species as signaling molecules. Diversion of O2 away from endothelial cell mitochondria by NO might also facilitate oxygenation of vascular smooth muscle cells. PMID:16565215

  8. The microsporidian polar tube: A highly specialised invasion organelle

    PubMed Central

    Xu, Yanji; Weiss, Louis M.

    2011-01-01

    All of the members of the Microsporidia possess a unique, highly specialised structure, the polar tube. This article reviews the available data on the organisation, structure and function of this invasion organelle. It was over 100 years ago that Thelohan accurately described the microsporidian polar tube and the triggering of its discharge. In the spore, the polar tube is connected at the anterior end, and then coils around the sporoplasm. Upon appropriate environmental stimulation the polar tube rapidly discharges out of the spore pierces a cell membrane and serves as a conduit for sporoplasm passage into the new host cell. The mechanism of germination of spores, however, remains to be definitively determined. In addition, further studies on the characterisation of the early events in the rupture of the anterior attachment complex, eversion of the polar tube as well as the mechanism of host cell attachment and penetration are needed in order to clarify the function and assembly of this structure. The application of immunological and molecular techniques has resulted in the identification of three polar tube proteins referred to as PTP1, PTP2 and PTP3. The interactions of these identified proteins in the formation and function of the polar tube remain to be determined. Data suggest that PTP1 is an O-mannosylated glycoprotein, a post-translational modification that may be important for its function. With the availability of the Encephalitozoon cuniculi genome it is now possible to apply proteomic techniques to the characterisation of the components of the microsporidian spore and invasion organelle. PMID:16005007

  9. An Intracellular Nanotrap Redirects Proteins and Organelles in Live Bacteria

    PubMed Central

    Borg, Sarah; Popp, Felix; Hofmann, Julia; Leonhardt, Heinrich; Rothbauer, Ulrich

    2015-01-01

    ABSTRACT  Owing to their small size and enhanced stability, nanobodies derived from camelids have previously been used for the construction of intracellular “nanotraps,” which enable redirection and manipulation of green fluorescent protein (GFP)-tagged targets within living plant and animal cells. By taking advantage of intracellular compartmentalization in the magnetic bacterium Magnetospirillum gryphiswaldense, we demonstrate that proteins and even entire organelles can be retargeted also within prokaryotic cells by versatile nanotrap technology. Expression of multivalent GFP-binding nanobodies on magnetosomes ectopically recruited the chemotaxis protein CheW1-GFP from polar chemoreceptor clusters to the midcell, resulting in a gradual knockdown of aerotaxis. Conversely, entire magnetosome chains could be redirected from the midcell and tethered to one of the cell poles. Similar approaches could potentially be used for building synthetic cellular structures and targeted protein knockdowns in other bacteria. Importance   Intrabodies are commonly used in eukaryotic systems for intracellular analysis and manipulation of proteins within distinct subcellular compartments. In particular, so-called nanobodies have great potential for synthetic biology approaches because they can be expressed easily in heterologous hosts and actively interact with intracellular targets, for instance, by the construction of intracellular “nanotraps” in living animal and plant cells. Although prokaryotic cells also exhibit a considerable degree of intracellular organization, there are few tools available equivalent to the well-established methods used in eukaryotes. Here, we demonstrate the ectopic retargeting and depletion of polar membrane proteins and entire organelles to distinct compartments in a magnetotactic bacterium, resulting in a gradual knockdown of magneto-aerotaxis. This intracellular nanotrap approach has the potential to be applied in other bacteria for

  10. Chemical Shift Images of Organelles in Leydig cells of Mice Testes

    NASA Astrophysics Data System (ADS)

    Ejima, T.; Neichi, Y.; Yanagihara, M.; Kado, M.; Ishino, M.; Yasuda, K.; Tamotsu, S.

    2013-10-01

    Soft X-ray transmission images of Leydig cells of mice testes changing incident wavelength were observed with the use of a contact microscope. After normalization of transmission images, absorbance images were obtained and compared with a visible differential interference image. Some organelles were identified by the image comparison, and absorption spectra of the organelles were obtained from the absorbance images. The absorption spectra show that peak structures are different depending on the observed organelles. The structures and the positions of organelles were clearly identified at C-K absorption.

  11. Characterization of the soluble glycoprotein released from vesicular stomatitis virus-infected cells.

    PubMed Central

    Chatis, P A; Morrison, T G

    1983-01-01

    Vesicular stomatitis virus-infected Chinese hamster ovary cells release into the extracellular medium a soluble form of the vesicular stomatitis virus glycoprotein (G protein) termed Gs (Kang and Prevec, Virology 46:678-680, 1971). The properties of this molecule and the cellular site at which it is generated were characterized. By comparing the sizes and the peptide maps of the unglycosylated forms of G and Gs, we found that between 5,000 and 6,000 daltons of the carboxy-terminal end of the G protein is cleaved to generate the Gs molecule. This truncated molecule contains no fatty acid. Gs released from cells grown at 39 degrees C migrated on polyacrylamide gels slightly slower than Gs released at 30 degrees C. The unglycosylated form of Gs also showed this size difference. Furthermore, unglycosylated Gs was resolved into two species upon isoelectric focusing: the relative amounts of the two species depended upon the temperature at which infected cells were incubated. Full-sized unglycosylated virus-associated G also was resolved into two species, but the more basic form predominated at both 30 and 39 degrees C. The appearance of Gs in the extracellular medium depended upon the presence of stable, full-sized G at the cell surface. The amount of Gs released was quantitated in seven different situations in which the migration of G to the cell surface was inhibited. In all cases, the amount of Gs released was also decreased. In addition, incubation of cells surface labeled with 125I resulted in the release of 125I-labeled Gs protein, as well as full-sized G protein. These results suggest that Gs is generated primarily by proteolytic cleavage of plasma membrane-associated G at a site in the molecule just amino terminal to the membrane-spanning region of the molecule. Images PMID:6296461

  12. Vesicularity variation to pyroclasts from silicic eruptions at Laguna del Maule volcanic complex, Chile

    NASA Astrophysics Data System (ADS)

    Wright, H. M. N.; Fierstein, J.; Amigo, A.; Miranda, J.

    2014-12-01

    Crystal-poor rhyodacitic to rhyolitic volcanic eruptions at Laguna del Maule volcanic complex, Chile have produced an astonishing range of textural variation to pyroclasts. Here, we focus on eruptive deposits from two Quaternary eruptions from vents on the northwestern side of the Laguna del Maule basin: the rhyolite of Loma de Los Espejos and the rhyodacite of Laguna Sin Puerto. Clasts in the pyroclastic fall and pyroclastic flow deposits from the rhyolite of Loma de Los Espejos range from dense, non-vesicular (obsidian) to highly vesicular, frothy (coarsely vesicular reticulite); where vesicularity varies from <1% to >90%. Bulk compositions range from 75.6-76.7 wt.% SiO2. The highest vesicularity clasts are found in early fall deposits and widely dispersed pyroclastic flow deposits; the frothy carapace to lava flows is similarly highly vesicular. Pyroclastic deposits also contain tube pumice, and macroscopically folded, finely vesicular, breadcrusted, and heterogeneously vesiculated textures. We speculate that preservation of the highest vesicularities requires relatively low decompression rates or open system degassing such that relaxation times were sufficient to allow extensive vesiculation. Such an inference is in apparent contradiction to documentation of Plinian dispersal to the eruption. Clasts in the pyroclastic fall deposit of the rhyodacite (68-72 wt.% SiO2) of Laguna Sin Puerto are finely vesicular, with vesicularity modes at ~50% and ~68% corresponding to gray and white pumice colors, respectively. Some clasts are banded in color (and vesicularity). All clasts were fragmented into highly angular particles, with subplanar to slightly concave exterior surfaces (average Wadell Roundness of clast margins between 0.32 and 0.39), indicating brittle fragmentation. In contrast to Loma de Los Espejos, high bubble number densities to Laguna Sin Puerto rhyodacite imply high decompression rates.

  13. Organelle-mimicking liposome dissociates G-quadruplexes and facilitates transcription

    PubMed Central

    Pramanik, Smritimoy; Tateishi-Karimata, Hisae; Sugimoto, Naoki

    2014-01-01

    Important biological reactions involving nucleic acids occur near the surface of membranes such as the nuclear membrane (NM) and rough endoplasmic reticulum (ER); however, the interactions between biomembranes and nucleic acids are poorly understood. We report here that transcription was facilitated in solution with liposomes, which mimic a biomembrane surface, relative to the reaction in a homogeneous aqueous solution when the template was able to form a G-quadruplex. The G-quadruplex is known to be an inhibitor of transcription, but the stability of the G-quadruplex was decreased at the liposome surface because of unfavourable enthalpy. The destabilization of the G-quadruplex was greater at the surface of NM- and ER-mimicking liposomes than at the surfaces of liposomes designed to mimic other organelles. Thermodynamic analyses revealed that the G-rich oligonucleotides adopted an extended structure at the liposome surface, whereas in solution the compact G-quadruplex was formed. Our data suggest that changes in structure and stability of nucleic acids regulate biological reactions at membrane surfaces. PMID:25336617

  14. Lycopene from tomatoes: vesicular nanocarrier formulations for dermal delivery.

    PubMed

    Ascenso, Andreia; Pinho, Sónia; Eleutério, Carla; Praça, Fabíola Garcia; Bentley, Maria Vitória Lopes Badra; Oliveira, Helena; Santos, Conceição; Silva, Olga; Simões, Sandra

    2013-07-31

    This experimental work aimed to develop a simple, fast, economic, and environmentally friendly process for the extraction of lycopene from tomato and incorporate this lycopene-rich extract into ultradeformable vesicular nanocarriers suitable for topical application. Lycopene extraction was conducted without a cosolvent for 30 min. The extracts were analyzed and incorporated in transfersomes and ethosomes. These formulations were characterized, and the cellular uptake was observed by confocal microscopy. Dermal delivery of lycopene formulations was tested under in vitro and in vivo conditions. Lycopene extraction proved to be quite safe and selective. The vesicular formulation was taken up by the cells, being more concentrated around the nucleus. Epicutaneous application of lycopene formulations decreased the level of anthralin-induced ear swelling by 97 and 87%, in a manner nonstatistically different from the positive control. These results support the idea that the lycopene-rich extract may be a good alternative to the expensive commercial lycopene for incorporation into advanced topical delivery systems. PMID:23826819

  15. Chromosomal localization of the human vesicular amine transporter genes

    SciTech Connect

    Peter, D.; Finn, P.; Liu, Y.; Roghani, A.; Edwards, R.H.; Klisak, I.; Kojis, T.; Heinzmann, C.; Sparkes, R.S. )

    1993-12-01

    The physiologic and behavioral effects of pharmacologic agents that interfere with the transport of monoamine neurotransmitters into vesicles suggest that vesicular amine transport may contribute to human neuropsychiatric disease. To determine whether an alteration in the genes that encode vesicular amine transport contributes to the inherited component of these disorders, the authors have isolated a human cDNA for the brain transporter and localized the human vesciular amine transporter genes. The human brain synaptic vesicle amine transporter (SVAT) shows unexpected conservation with rat SVAT in the regions that diverge extensively between rat SVAT and the rat adrenal chromaffin granule amine transporter (CGAT). Using the cloned sequences with a panel of mouse-human hybrids and in situ hybridization for regional localization, the adrenal CGAT gene (or VAT1) maps to human chromosome 8p21.3 and the brain SVAT gene (or VAT2) maps to chromosome 10q25. Both of these sites occur very close to if not within previously described deletions that produce severe but viable phenotypes. 26 refs., 3 figs., 1 tab.

  16. Distribution of vesicular glutamate transporters in the human brain

    PubMed Central

    Vigneault, Érika; Poirel, Odile; Riad, Mustapha; Prud'homme, Josée; Dumas, Sylvie; Turecki, Gustavo; Fasano, Caroline; Mechawar, Naguib; El Mestikawy, Salah

    2015-01-01

    Glutamate is the major excitatory transmitter in the brain. Vesicular glutamate transporters (VGLUT1-3) are responsible for uploading glutamate into synaptic vesicles. VGLUT1 and VGLUT2 are considered as specific markers of canonical glutamatergic neurons, while VGLUT3 is found in neurons previously shown to use other neurotransmitters than glutamate. Although there exists a rich literature on the localization of these glutamatergic markers in the rodent brain, little is currently known about the distribution of VGLUT1-3 in the human brain. In the present study, using subtype specific probes and antisera, we examined the localization of the three vesicular glutamate transporters in the human brain by in situ hybridization, immunoautoradiography and immunohistochemistry. We found that the VGLUT1 transcript was highly expressed in the cerebral cortex, hippocampus and cerebellum, whereas VGLUT2 mRNA was mainly found in the thalamus and brainstem. VGLUT3 mRNA was localized in scarce neurons within the cerebral cortex, hippocampus, striatum and raphe nuclei. Following immunoautoradiographic labeling, intense VGLUT1- and VGLUT2-immunoreactivities were observed in all regions investigated (cerebral cortex, hippocampus, caudate-putamen, cerebellum, thalamus, amygdala, substantia nigra, raphe) while VGLUT3 was absent from the thalamus and cerebellum. This extensive mapping of VGLUT1-3 in human brain reveals distributions that correspond for the most part to those previously described in rodent brains. PMID:25798091

  17. SDS-resistant aggregation of membrane proteins: application to the purification of the vesicular monoamine transporter.

    PubMed Central

    Sagné, C; Isambert, M F; Henry, J P; Gasnier, B

    1996-01-01

    The vesicular monoamine transporter, which catalyses a H+/ monoamine antiport in monoaminergic vesicle membrane, is a very hydrophobic intrinsic membrane protein. After solubilization, this protein was found to have a high tendency to aggregate, as shown by SDS/PAGE, especially when samples were boiled in the classical Laemmli buffer before electrophoresis. This behavior was analysed in some detail. The aggregation was promoted by high temperatures, organic solvents and acidic pH, suggesting that it resulted from the unfolding of structure remaining in SDS. The aggregates were very stable and could be dissociated only by suspension in anhydrous trifluoroacetic acid. This SDS-resistant aggregation behaviour was shared by very few intrinsic proteins of the chromaffin granule membrane. Consequently, a purification procedure was based on this property. A detergent extract of chromaffin granule membranes enriched in monoamine transporter was heated and the aggregates were isolated by size-exclusion HPLC in SDS. The aggregates, containing the transporter, were dissociated in the presence of trifluoroacetic acid and analysed on the same HPLC column. This strategy might be of general interest for the purification of membrane proteins that exhibit SDS-resistant aggregation. PMID:8670158

  18. Short-range inversions: rethinking organelle genome stability: template switching events during DNA replication destabilize organelle genomes.

    PubMed

    Tremblay-Belzile, Samuel; Lepage, Étienne; Zampini, Éric; Brisson, Normand

    2015-10-01

    In the organelles of plants and mammals, recent evidence suggests that genomic instability stems in large part from template switching events taking place during DNA replication. Although more than one mechanism may be responsible for this, some similarities exist between the different proposed models. These can be separated into two main categories, depending on whether they involve a single-strand-switching or a reciprocal-strand-switching event. Single-strand-switching events lead to intermediates containing Y junctions, whereas reciprocal-strand-switching creates Holliday junctions. Common features in all the described models include replication stress, fork stalling and the presence of inverted repeats, but no single element appears to be required in all cases. We review the field, and examine the ideas that several mechanisms may take place in any given genome, and that the presence of palindromes or inverted repeats in certain regions may favor specific rearrangements. PMID:26222836

  19. Viral Surveillance during the 2006 Vesicular Stomatitis Outbreak in Natrona County, Wyoming

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In 2006, we collected 12203 biting flies from a vesicular stomatitis outbreak in Natrona County, Wyoming. Flies were identified to the species level and viruses were isolated and identified by RT-PCR. We detected vesicular stomatitis virus-New Jersey serotype in two pools of Simulium bivittatum, W...

  20. Experimental infection of Didelphis marsupialis with Vesicular Stomatitis New Jersey Virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Although vesicular stomatitis has been present for many years in the Americas, many aspects of its natural history remain undefined. In this study we challenged five adult Virginia opossums (Didelphis marsupialis) with vesicular stomatitis New Jersey serotype virus (VSNJV). Opossums had no detecta...

  1. Spatial and phylogenetic analysis of vesicular stomatitus virus overwintering in the United States

    Technology Transfer Automated Retrieval System (TEKTRAN)

    From 2004 through 2006, 751 vesicular stomatitis (VS) outbreaks caused by vesicular stomatitis virus serotype New Jersey (VSNJV) were reported in nine states of the southwestern United States. The normal model of the space scan statistic and phylogenetic techniques were used to test the hypothesis t...

  2. Vesicular Disease in 9-Week-Old Pigs Experimentally Infected with Senecavirus A

    PubMed Central

    Montiel, Nestor; Buckley, Alexandra; Guo, Baoqing; Kulshreshtha, Vikas; VanGeelen, Albert; Hoang, Hai; Rademacher, Christopher; Yoon, Kyoung-Jin

    2016-01-01

    Senecavirus A has been infrequently associated with vesicular disease in swine since 1988. However, clinical disease has not been reproduced after experimental infection with this virus. We report vesicular disease in 9-week-old pigs after Sencavirus A infection by the intranasal route under experimental conditions. PMID:27315363

  3. 9 CFR 94.14 - Swine from regions where swine vesicular disease exists; importations prohibited.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Swine from regions where swine... SWINE FEVER, CLASSICAL SWINE FEVER, SWINE VESICULAR DISEASE, AND BOVINE SPONGIFORM ENCEPHALOPATHY: PROHIBITED AND RESTRICTED IMPORTATIONS § 94.14 Swine from regions where swine vesicular disease...

  4. 9 CFR 94.12 - Pork and pork products from regions where swine vesicular disease exists.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... citations affecting § 94.12, see the List of CFR Sections Affected, which appears in the Finding Aids... where swine vesicular disease exists. 94.12 Section 94.12 Animals and Animal Products ANIMAL AND PLANT... AVIAN INFLUENZA, AFRICAN SWINE FEVER, CLASSICAL SWINE FEVER, SWINE VESICULAR DISEASE, AND...

  5. 9 CFR 94.14 - Swine from regions where swine vesicular disease exists; importations prohibited.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Swine from regions where swine... SWINE FEVER, CLASSICAL SWINE FEVER, SWINE VESICULAR DISEASE, AND BOVINE SPONGIFORM ENCEPHALOPATHY: PROHIBITED AND RESTRICTED IMPORTATIONS § 94.14 Swine from regions where swine vesicular disease...

  6. 9 CFR 94.14 - Swine from regions where swine vesicular disease exists; importations prohibited.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Swine from regions where swine... SWINE FEVER, CLASSICAL SWINE FEVER, SWINE VESICULAR DISEASE, AND BOVINE SPONGIFORM ENCEPHALOPATHY: PROHIBITED AND RESTRICTED IMPORTATIONS § 94.14 Swine from regions where swine vesicular disease...

  7. 9 CFR 94.14 - Swine from regions where swine vesicular disease exists; importations prohibited.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Swine from regions where swine... SWINE FEVER, CLASSICAL SWINE FEVER, SWINE VESICULAR DISEASE, AND BOVINE SPONGIFORM ENCEPHALOPATHY: PROHIBITED AND RESTRICTED IMPORTATIONS § 94.14 Swine from regions where swine vesicular disease...

  8. Diagnostic evaluation of a reverse transcribed real time PCR assay for vesicular stomatitis in horses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Vesicular stomatitis (VS) is a viral infection that affects a variety of domesticated species including cattle, sheep, pigs, and horses. The symptoms vary among species, but VS usually manifests as vesicles on the epithelium of the nose, tongue, mouth, udder, and coronary bands. Vesicular stomatit...

  9. Vesicular Disease in 9-Week-Old Pigs Experimentally Infected with Senecavirus A

    DOE PAGESBeta

    Montiel, Nestor; Buckley, Alexandra; Guo, Baoqing; Kulshreshtha, Vikas; VanGeelen, Albert; Hoang, Hai; Rademacher, Christopher; Yoon, Kyoung-Jin; Lager, Kelly

    2016-07-01

    Senecavirus A has been infrequently associated with vesicular disease in swine since 1988. However, clinical disease has not been reproduced after experimental infection with this virus. Here we report vesicular disease in 9-week-old pigs after Sencavirus A infection by the intranasal route under experimental conditions.

  10. Vesicular Disease in 9-Week-Old Pigs Experimentally Infected with Senecavirus A.

    PubMed

    Montiel, Nestor; Buckley, Alexandra; Guo, Baoqing; Kulshreshtha, Vikas; VanGeelen, Albert; Hoang, Hai; Rademacher, Christopher; Yoon, Kyoung-Jin; Lager, Kelly

    2016-07-01

    Senecavirus A has been infrequently associated with vesicular disease in swine since 1988. However, clinical disease has not been reproduced after experimental infection with this virus. We report vesicular disease in 9-week-old pigs after Sencavirus A infection by the intranasal route under experimental conditions. PMID:27315363

  11. Organelle interactions and possible degradation pathways visualized in high-pressure frozen algal cells.

    PubMed

    Aichinger, N; Lütz-Meindl, U

    2005-08-01

    Summary Organelle interactions, although essential for both anabolic and catabolic pathways in plant cells have not been examined in detail so far. In the present study the structure of different organelle-organelle, organelle-vesicle and organelle-membrane interactions were investigated in growing and nongrowing cells of the green alga Micrasterias denticulata by use of high pressure freeze fixation and energy filtering transmission electron microscopy. It became clear that contacts between mitochondria always occur by formation of a cone-shaped protuberance of one of the mitochondria which penetrates into its fusion partner. In the same way, structural interactions between mitochondria and mucilage vesicles and between microbodies and mucilage vesicles are achieved. Lytic compartments contact mitochondria or mucilage vesicles again by forming protuberances and by extending their contents into the respective compartment. Detached portions of mitochondria are found inside lytic compartments as a consequence of such interactions. Mitochondria found in contact with the plasma membrane reveal structural disintegration. Our study shows that interactions of organelles and vesicles are frequent events in Micrasterias cells of different ages. The interactive contacts between lytic compartments and organelles or vesicles suggest a degradation pathway different from autophagy processes described in the literature. Both the interactions between vesicles and organelles and the degradation pathways occur independently from cytoskeleton function as demonstrated by use of cytochalasin D and the microtubule inhibitor amiprophos-methyl. PMID:16159344

  12. Proteomic Analysis of the Acidocalcisome, an Organelle Conserved from Bacteria to Human Cells

    PubMed Central

    Huang, Guozhong; Ulrich, Paul N.; Storey, Melissa; Johnson, Darryl; Tischer, Julie; Tovar, Javier A.; Moreno, Silvia N. J.; Orlando, Ron; Docampo, Roberto

    2014-01-01

    Acidocalcisomes are acidic organelles present in a diverse range of organisms from bacteria to human cells. In this study acidocalcisomes were purified from the model organism Trypanosoma brucei, and their protein composition was determined by mass spectrometry. The results, along with those that we previously reported, show that acidocalcisomes are rich in pumps and transporters, involved in phosphate and cation homeostasis, and calcium signaling. We validated the acidocalcisome localization of seven new, putative, acidocalcisome proteins (phosphate transporter, vacuolar H+-ATPase subunits a and d, vacuolar iron transporter, zinc transporter, polyamine transporter, and acid phosphatase), confirmed the presence of six previously characterized acidocalcisome proteins, and validated the localization of five novel proteins to different subcellular compartments by expressing them fused to epitope tags in their endogenous loci or by immunofluorescence microscopy with specific antibodies. Knockdown of several newly identified acidocalcisome proteins by RNA interference (RNAi) revealed that they are essential for the survival of the parasites. These results provide a comprehensive insight into the unique composition of acidocalcisomes of T. brucei, an important eukaryotic pathogen, and direct evidence that acidocalcisomes are especially adapted for the accumulation of polyphosphate. PMID:25503798

  13. Modeling organelle transport in branching dendrites with a variable cross-sectional area

    PubMed Central

    2010-01-01

    The purpose of this paper is to develop a method for calculating organelle transport in dendrites with a non-uniform cross-sectional area that depends on the distance from the neuron soma. The model is based on modified Smith–Simmons equations governing molecular motor-assisted organelle transport. The developed method is then applied to simulating organelle transport in branching dendrites with two particular microtubule (MT) orientations reported from experiments. It is found that the rate of organelle transport toward a dendrite’s growth cone heavily depends on the MT orientation, and since there is experimental evidence that the MT orientation in a particular region of a dendrite may depend on the dendrite’s developmental stage, the obtained results suggest that a rearrangement of the MT structure may depend on the amount of organelles needed at the growth cone. PMID:21886345

  14. Mouse oocytes differentiate through organelle enrichment from sister cyst germ cells.

    PubMed

    Lei, Lei; Spradling, Allan C

    2016-04-01

    Oocytes differentiate in diverse species by receiving organelles and cytoplasm from sister germ cells while joined in germline cysts or syncytia. Mouse primordial germ cells form germline cysts, but the role of cysts in oogenesis is unknown. We find that mouse germ cells receive organelles from neighboring cyst cells and build a Balbiani body to become oocytes, whereas nurselike germ cells die. Organelle movement, Balbiani body formation, and oocyte fate determination are selectively blocked by low levels of microtubule-dependent transport inhibitors. Membrane breakdown within the cyst and an apoptosis-like process are associated with organelle transfer into the oocyte, events reminiscent of nurse cell dumping in Drosophila We propose that cytoplasmic and organelle transport plays an evolutionarily conserved and functionally important role in mammalian oocyte differentiation. PMID:26917595

  15. Stability of a Vesicular Stomatitis Virus-Vectored Ebola Vaccine.

    PubMed

    Arnemo, Marianne; Viksmoen Watle, Sara Sofie; Schoultz, Kristin Merete; Vainio, Kirsti; Norheim, Gunnstein; Moorthy, Vasee; Fast, Patricia; Røttingen, John-Arne; Gjøen, Tor

    2016-03-15

    The live attenuated vesicular stomatitis virus-vectored Ebola vaccine rVSV-ZEBOV is currently undergoing clinical trials in West Africa. The vaccine is to be stored at -70°C or less. Since maintaining the cold chain is challenging in rural areas, the rVSV-ZEBOV vaccine's short-term and long-term stability at different temperatures was examined. Different dilutions were tested since the optimal vaccine dosage had not yet been determined at the start of this experiment. The results demonstrate that the original vaccine formulation was stable for 1 week at 4°C and for 24 hours at 25°C. The stability of the vaccine was compromised by both high temperatures and dilution. PMID:26563239

  16. Investigation of vesicular rashes for HSV and VZV by PCR.

    PubMed

    Beards, G; Graham, C; Pillay, D

    1998-03-01

    Vesicular fluid from rashes of 132 patients was tested by a multiplex PCR shown to be specific for herpes simplex virus (HSV) type 1 and 2, and varicella zoster virus (VZV) genomic DNA. The results were compared with those obtained by examination by electron microscopy and virus isolation by cell culture. The PCR did not differentiate between HSV 1 and 2. By PCR, 64 HSV infections and 53 VZV infections were identified, with presumed 100% sensitivity and specificity. Fifteen specimens tested negative by PCR, electron microscopy, and virus isolation for herpes viruses. The sensitivities of virus isolation and electron microscopy for detection of herpes simplex virus were 56% and 80%. For varicella zoster virus, the sensitivities of virus isolation and electron microscopy were 47% and 60%. These data illustrate the advantage of rapid PCR diagnosis of herpes simplex virus and varicella zoster virus in vesicle fluids. PMID:9515761

  17. Asymmetric packaging of polymerases within vesicular stomatitis virus

    PubMed Central

    Hodges, Jeffery; Tang, Xiaolin; Landesman, Michael B.; Ruedas, John B.; Ghimire, Anil; Gudheti, Manasa V.; Perrault, Jacques; Jorgensen, Erik M.; Gerton, Jordan M.; Saffarian, Saveez

    2015-01-01

    Vesicular stomatitis virus (VSV) is a prototypic negative sense single-stranded RNA virus. The bullet-shape appearance of the virion results from tightly wound helical turns of the nucleoprotein encapsidated RNA template (N-RNA) around a central cavity. Transcription and replication require polymerase complexes, which include a catalytic subunit L and a template-binding subunit P. L and P are inferred to be in the cavity, however lacking direct observation, their exact position has remained unclear. Using super-resolution fluorescence imaging and atomic force microscopy (AFM) on single VSV virions, we show that L and P are packaged asymmetrically towards the blunt end of the virus. The number of L and P proteins varies between individual virions and they occupy 57 ± 12 nm of the 150 nm central cavity of the virus. Our finding positions the polymerases at the opposite end of the genome with respect to the only transcriptional promoter. PMID:24055706

  18. Antiviral activity of doxycycline against vesicular stomatitis virus in vitro.

    PubMed

    Wu, Zhuan-Chang; Wang, Xin; Wei, Jian-Chao; Li, Bei-Bei; Shao, Dong-Hua; Li, Yu-Ming; Liu, Ke; Shi, Yuan-Yuan; Zhou, Bin; Qiu, Ya-Feng; Ma, Zhi-Yong

    2015-11-01

    Doxycycline (Dox) is a tetracycline derivative with broad-spectrum antimicrobial activities that is used as an effector substance in inducible gene-expression systems. We investigated the antiviral activity of Dox against vesicular stomatitis virus (VSV) infection in cultured H1299 cells. Dox at concentrations of 1.0-2.0 μg ml(-1) significantly inhibited VSV replication and the VSV-induced cytopathic effect in dose-dependent manners, suggesting that Dox may have broader activity in inhibiting viral replication, in addition to its well-defined bacteriostatic activity. Dox exerted its antiviral effect at the early-mid stage of VSV infection, suggesting that it did not interfere with VSV infectivity, adsorption, or entry into target cells. These results indicate that Dox can inhibit VSV infection and may therefore have potential applications for the treatment of viral infections. PMID:26459887

  19. Vesicular Nucleotide Transporter-Mediated ATP Release Regulates Insulin Secretion

    PubMed Central

    Geisler, Jessica C.; Corbin, Kathryn L.; Li, Qin; Feranchak, Andrew P.; Nunemaker, Craig S.

    2013-01-01

    Extracellular ATP plays a critical role in regulating insulin secretion in pancreatic β cells. The ATP released from insulin secretory vesicles has been proposed to be a major source of extracellular ATP. Currently, the mechanism by which ATP accumulates into insulin secretory granules remains elusive. In this study, the authors identified the expression of a vesicular nucleotide transporter (VNUT) in mouse pancreas, isolated mouse islets, and MIN6 cells, a mouse β cell line. Immunohistochemistry and immunofluorescence revealed that VNUT colocalized extensively with insulin secretory granules. Functional studies showed that suppressing endogenous VNUT expression in β cells by small hairpin RNA knockdown greatly reduced basal- and glucose-induced ATP release. Importantly, knocking down VNUT expression by VNUT small hairpin RNA in MIN6 cells and isolated mouse islets dramatically suppressed basal insulin release and glucose-stimulated insulin secretion (GSIS). Moreover, acute pharmacologic blockade of VNUT with Evans blue, a VNUT antagonist, greatly attenuated GSIS in a dose-dependent manner. Exogenous ATP treatment effectively reversed the insulin secretion defect induced by both VNUT knockdown and functional inhibition, indicating that VNUT-mediated ATP release is essential for maintaining normal insulin secretion. In contrast to VNUT knockdown, overexpression of VNUT in β cells resulted in excessive ATP release and enhanced basal insulin secretion and GSIS. Elevated insulin secretion induced by VNUT overexpression was reversed by pharmacologic inhibition of P2X but not P2Y purinergic receptors. This study reveals VNUT is expressed in pancreatic β cells and plays an essential and novel role in regulating insulin secretion through vesicular ATP release and extracellular purinergic signaling. PMID:23254199

  20. The Gas Vacuole - an Early Organelle of Prokaryote Motility

    NASA Astrophysics Data System (ADS)

    Staley, James T.

    1980-06-01

    Several lines of evidence suggest that the gas vesicle may have been an early organelle of prokaryote motility. First, it is found in bacteria that are thought to be representatives of primitive groups. Second, it is a simple structure, and the structure alone imparts the function of motility. Thirdly, it is widely distributed amongst prokaryotes, having been found in the purple and green sulfur photosynthetic bacteria, cyanobacteria, methanogenic bacteria, obligate and facultative anaerobic heterotrophic bacteria, as well as aerobic heterotrophic bacteria that divide by budding and binary transverse fission. Recent evidence suggests that in some bacteria the genes for gas vesicle synthesis occur on plasmids. Thus, the wide distribution of this characteristic could be due to recent evolution and rapid dispersal, though early evolution is not precluded. Though the gas vesicle structure itself appears to be highly conserved among the various groups of bacteria, it seems doubtful that the regulatory mechanism to control its synthesis could be the same for the diverse gas vacuolate bacterial groups.

  1. Modularity of a carbon-fixing protein organelle

    PubMed Central

    Bonacci, Walter; Teng, Poh K.; Afonso, Bruno; Niederholtmeyer, Henrike; Grob, Patricia; Silver, Pamela A.; Savage, David F.

    2012-01-01

    Bacterial microcompartments are proteinaceous complexes that catalyze metabolic pathways in a manner reminiscent of organelles. Although microcompartment structure is well understood, much less is known about their assembly and function in vivo. We show here that carboxysomes, CO2-fixing microcompartments encoded by 10 genes, can be heterologously produced in Escherichia coli. Expression of carboxysomes in E. coli resulted in the production of icosahedral complexes similar to those from the native host. In vivo, the complexes were capable of both assembling with carboxysomal proteins and fixing CO2. Characterization of purified synthetic carboxysomes indicated that they were well formed in structure, contained the expected molecular components, and were capable of fixing CO2 in vitro. In addition, we verify association of the postulated pore-forming protein CsoS1D with the carboxysome and show how it may modulate function. We have developed a genetic system capable of producing modular carbon-fixing microcompartments in a heterologous host. In doing so, we lay the groundwork for understanding these elaborate protein complexes and for the synthetic biological engineering of self-assembling molecular structures. PMID:22184212

  2. Structure, Function, and Assembly of Adhesive Organelles by Uropathogenic Bacteria

    PubMed Central

    Chahales, Peter; Thanassi, David G.

    2015-01-01

    Bacteria assemble a wide range of adhesive proteins, termed adhesins, to mediate binding to receptors and colonization of surfaces. For pathogenic bacteria, adhesins are critical for early stages of infection, allowing the bacteria to initiate contact with host cells, colonize different tissues, and establish a foothold within the host. The adhesins expressed by a pathogen are also critical for bacterial-bacterial interactions and the formation of bacterial communities such as biofilms. The ability to adhere to host tissues is particularly important for bacteria that colonize sites such as the urinary tract, where the flow of urine functions to maintain sterility by washing away non-adherent pathogens. Adhesins vary from monomeric proteins that are directly anchored to the bacterial surface to polymeric, hairlike fibers that extend out from the cell surface. These latter fibers are termed pili or fimbriae, and were among the first identified virulence factors of uropathogenic Escherichia coli. Studies since then have identified a range of both pilus and non-pilus adhesins that contribute to bacterial colonization of the urinary tract, and have revealed molecular details of the structures, assembly pathways, and functions of these adhesive organelles. In this review, we describe the different types of adhesins expressed by both Gram-negative and Gram-positive uropathogens, what is known about their structures, how they are assembled on the bacterial surface, and the functions of specific adhesins in the pathogenesis of urinary tract infections. PMID:26542038

  3. The murine cardiac 26S proteasome: an organelle awaiting exploration.

    PubMed

    Gomes, Aldrin V; Zong, Chenggong; Edmondson, Ricky D; Berhane, Beniam T; Wang, Guang-Wu; Le, Steven; Young, Glen; Zhang, Jun; Vondriska, Thomas M; Whitelegge, Julian P; Jones, Richard C; Joshua, Irving G; Thyparambil, Sheeno; Pantaleon, Dawn; Qiao, Joe; Loo, Joseph; Ping, Peipei

    2005-06-01

    Multiprotein complexes have been increasingly recognized as essential functional units for a variety of cellular processes, including the protein degradation system. Selective degradation of proteins in eukaryotes is primarily conducted by the ubiquitin proteasome system. The current knowledge base, pertaining to the proteasome complexes in mammalian cells, relies largely upon information gained in the yeast system, where the 26S proteasome is hypothesized to contain a 20S multiprotein core complex and one or two 19S regulatory complexes. To date, the molecular structure of the proteasome system, the proteomic composition of the entire 26S multiprotein complexes, and the specific designated function of individual components within this essential protein degradation system in the heart remain virtually unknown. A functional proteomic approach, employing multidimensional chromatography purification combined with liquid chromatography tandem mass spectrometry and protein chemistry, was utilized to explore the murine cardiac 26S proteasome system. This article presents an overview on the subject of protein degradation in mammalian cells. In addition, this review shares the limited information that has been garnered thus far pertaining to the molecular composition, function, and regulation of this important organelle in the cardiac cells. PMID:16093497

  4. Modularity of a carbon-fixing protein organelle.

    PubMed

    Bonacci, Walter; Teng, Poh K; Afonso, Bruno; Niederholtmeyer, Henrike; Grob, Patricia; Silver, Pamela A; Savage, David F

    2012-01-10

    Bacterial microcompartments are proteinaceous complexes that catalyze metabolic pathways in a manner reminiscent of organelles. Although microcompartment structure is well understood, much less is known about their assembly and function in vivo. We show here that carboxysomes, CO(2)-fixing microcompartments encoded by 10 genes, can be heterologously produced in Escherichia coli. Expression of carboxysomes in E. coli resulted in the production of icosahedral complexes similar to those from the native host. In vivo, the complexes were capable of both assembling with carboxysomal proteins and fixing CO(2). Characterization of purified synthetic carboxysomes indicated that they were well formed in structure, contained the expected molecular components, and were capable of fixing CO(2) in vitro. In addition, we verify association of the postulated pore-forming protein CsoS1D with the carboxysome and show how it may modulate function. We have developed a genetic system capable of producing modular carbon-fixing microcompartments in a heterologous host. In doing so, we lay the groundwork for understanding these elaborate protein complexes and for the synthetic biological engineering of self-assembling molecular structures. PMID:22184212

  5. MLT1 links cytoskeletal asymmetry to organelle placement in Chlamydomonas

    PubMed Central

    Mittelmeier, Telsa M.; Thompson, Mark D.; Lamb, Mary Rose; Lin, Huawen; Dieckmann, Carol L.

    2015-01-01

    Asymmetric placement of the photosensory eyespot organelle in Chlamydomonas is patterned by mother-daughter differences between the two basal bodies, which template the anterior flagella. Each basal body is associated with two bundled microtubule rootlets, one with two microtubules and one with four, forming a cruciate pattern. In wild type cells, the single eyespot is positioned at the equator in close proximity to the plus end of the daughter rootlet comprising four microtubules, the D4. Here we identify mutations in two linked loci, MLT1 and MLT2, which cause multiple eyespots. Antiserum raised against MLT1 localized the protein along the D4 rootlet microtubules, from the basal bodies to the eyespot. MLT1 associates immediately with the new D4 as it extends during cell division, before microtubule acetylation. MLT1 is a low-complexity protein of over 300,000 daltons. The expression or stability of MLT1 is dependent on MLT2, predicted to encode a second large, low-complexity protein. MLT1 was not restricted to the D4 rootlet in cells with the vfl2-220 mutation in the gene encoding the basal body-associated protein centrin. The cumulative data highlight the role of mother-daughter basal body differences in establishing asymmetry in associated rootlets, and suggest that eyespot components are directed to the correct location by MLT1 on the D4 microtubules. PMID:25809438

  6. Lung Surfactant and Organelles after an Exposure to Dibenzoxazepine (CR)

    PubMed Central

    Pattle, R. E.; Schock, C.; Dirnhuber, P.; Creasey, J. M.

    1974-01-01

    Rats were exposed to a heavy dosage of the sensory irritant dibenz (b.f.)-1,4 oxazepine (CR). No change in the lung surfactant could be detected by the methods used. Electron micrography showed that the ordinary lamellated osmiophilic bodies (LOPBs) and their precursors were unaffected. Bodies containing both mitochondrial cristae and dense osmiophilic whorls (“mitochondrial lamellated bodies”, or MLBs) were found in the type II cells of some animals up to 15 days after the exposure. These whorls originate from the bounding membranes and cristae; serial sections show that they usually abut on the boundary of the organelle. A large proportion of the mitochondria in any cell may be affected by this process. Unequivocal evidence that the MLBs finally evolve into LOPBs without cristae was not obtained in this series; the ultimate fate of the MLBs and the cells containing them is uncertain. The MLBs may perhaps act as an emergency source of surfactant. ImagesFigs. 9-10Figs. 6-8Figs. 1-2Figs. 3-5 PMID:4479334

  7. Tetrapyrrole signal as a cell-cycle coordinator from organelle to nuclear DNA replication in plant cells

    PubMed Central

    Kobayashi, Yuki; Kanesaki, Yu; Tanaka, Ayumi; Kuroiwa, Haruko; Kuroiwa, Tsuneyoshi; Tanaka, Kan

    2009-01-01

    Eukaryotic cells arose from an ancient endosymbiotic association of prokaryotes, with plant cells harboring 3 genomes as the remnants of such evolution. In plant cells, plastid and mitochondrial DNA replication [organelle DNA replication (ODR)] occurs in advance of the subsequent cell cycles composed of nuclear DNA replication (NDR) and cell division. However, the mechanism by which replication of these genomes with different origins is coordinated is largely unknown. Here, we show that NDR is regulated by a tetrapyrrole signal in plant cells, which has been suggested as an organelle-to-nucleus retrograde signal. In synchronized cultures of the primitive red alga Cyanidioschyzon merolae, specific inhibition of A-type cyclin-dependent kinase (CDKA) prevented NDR but not ODR after onset of the cell cycle. In contrast, inhibition of ODR by nalidixic acid also resulted in inhibition of NDR, indicating a strict dependence of NDR on ODR. The requirement of ODR for NDR was bypassed by addition of the tetrapyrrole intermediates protoporphyrin IX (ProtoIX) or Mg-ProtoIX, both of which activated CDKA without inducing ODR. This scheme was also observed in cultured tobacco cells (BY-2), where inhibition of ODR by nalidixic acid prevented CDKA activation and NDR, and these inhibitions were circumvented by Mg-ProtoIX without inducing ODR. We thus show that tetrapyrrole-mediated organelle–nucleus replicational coupling is an evolutionary conserved process among plant cells. PMID:19141634

  8. A Set of Organelle-Localizable Reactive Molecules for Mitochondrial Chemical Proteomics in Living Cells and Brain Tissues.

    PubMed

    Yasueda, Yuki; Tamura, Tomonori; Fujisawa, Alma; Kuwata, Keiko; Tsukiji, Shinya; Kiyonaka, Shigeki; Hamachi, Itaru

    2016-06-22

    Protein functions are tightly regulated by their subcellular localization in live cells, and quantitative evaluation of dynamically altered proteomes in each organelle should provide valuable information. Here, we describe a novel method for organelle-focused chemical proteomics using spatially limited reactions. In this work, mitochondria-localizable reactive molecules (MRMs) were designed that penetrate biomembranes and spontaneously concentrate in mitochondria, where protein labeling is facilitated by the condensation effect. The combination of this selective labeling and liquid chromatography-mass spectrometry (LC-MS) based proteomics technology facilitated identification of mitochondrial proteomes and the profile of the intrinsic reactivity of amino acids tethered to proteins expressed in live cultured cells, primary neurons and brain slices. Furthermore, quantitative profiling of mitochondrial proteins whose expression levels change significantly during an oxidant-induced apoptotic process was performed by combination of this MRMs-based method with a standard quantitative MS technique (SILAC: stable isotope labeling by amino acids in cell culture). The use of a set of MRMs represents a powerful tool for chemical proteomics to elucidate mitochondria-associated biological events and diseases. PMID:27228550

  9. Nucleotide sequence analysis of the L gene of Newcastle disease virus: homologies with Sendai and vesicular stomatitis viruses.

    PubMed Central

    Yusoff, K; Millar, N S; Chambers, P; Emmerson, P T

    1987-01-01

    The nucleotide sequence of the L gene of the Beaudette C strain of Newcastle disease virus (NDV) has been determined. The L gene is 6704 nucleotides long and encodes a protein of 2204 amino acids with a calculated molecular weight of 248822. Mung bean nuclease mapping of the 5' terminus of the L gene mRNA indicates that the transcription of the L gene is initiated 11 nucleotides upstream of the translational start site. Comparison with the amino acid sequences of the L genes of Sendai virus and vesicular stomatitis virus (VSV) suggests that there are several regions of homology between the sequences. These data provide further evidence for an evolutionary relationship between the Paramyxoviridae and the Rhabdoviridae. A non-coding sequence of 46 nucleotides downstream of the presumed polyadenylation site of the L gene may be part of a negative strand leader RNA. Images PMID:3035486

  10. Ion Channels in Plant Bioenergetic Organelles, Chloroplasts and Mitochondria: From Molecular Identification to Function.

    PubMed

    Carraretto, Luca; Teardo, Enrico; Checchetto, Vanessa; Finazzi, Giovanni; Uozumi, Nobuyuki; Szabo, Ildiko

    2016-03-01

    Recent technical advances in electrophysiological measurements, organelle-targeted fluorescence imaging, and organelle proteomics have pushed the research of ion transport a step forward in the case of the plant bioenergetic organelles, chloroplasts and mitochondria, leading to the molecular identification and functional characterization of several ion transport systems in recent years. Here we focus on channels that mediate relatively high-rate ion and water flux and summarize the current knowledge in this field, focusing on targeting mechanisms, proteomics, electrophysiology, and physiological function. In addition, since chloroplasts evolved from a cyanobacterial ancestor, we give an overview of the information available about cyanobacterial ion channels and discuss the evolutionary origin of chloroplast channels. The recent molecular identification of some of these ion channels allowed their physiological functions to be studied using genetically modified Arabidopsis plants and cyanobacteria. The view is emerging that alteration of chloroplast and mitochondrial ion homeostasis leads to organelle dysfunction, which in turn significantly affects the energy metabolism of the whole organism. Clear-cut identification of genes encoding for channels in these organelles, however, remains a major challenge in this rapidly developing field. Multiple strategies including bioinformatics, cell biology, electrophysiology, use of organelle-targeted ion-sensitive probes, genetics, and identification of signals eliciting specific ion fluxes across organelle membranes should provide a better understanding of the physiological role of organellar channels and their contribution to signaling pathways in plants in the future. PMID:26751960

  11. Trans-Membrane Area Asymmetry Controls the Shape of Cellular Organelles

    PubMed Central

    Beznoussenko, Galina V.; Pilyugin, Sergei S.; Geerts, Willie J. C.; Kozlov, Michael M.; Burger, Koert N. J.; Luini, Alberto; Derganc, Jure; Mironov, Alexander A.

    2015-01-01

    Membrane organelles often have complicated shapes and differ in their volume, surface area and membrane curvature. The ratio between the surface area of the cytosolic and luminal leaflets (trans-membrane area asymmetry (TAA)) determines the membrane curvature within different sites of the organelle. Thus, the shape of the organelle could be critically dependent on TAA. Here, using mathematical modeling and stereological measurements of TAA during fast transformation of organelle shapes, we present evidence that suggests that when organelle volume and surface area are constant, TAA can regulate transformation of the shape of the Golgi apparatus, endosomal multivesicular bodies, and microvilli of brush borders of kidney epithelial cells. Extraction of membrane curvature by small spheres, such as COPI-dependent vesicles within the Golgi (extraction of positive curvature), or by intraluminal vesicles within endosomes (extraction of negative curvature) controls the shape of these organelles. For instance, Golgi tubulation is critically dependent on the fusion of COPI vesicles with Golgi cisternae, and vice versa, for the extraction of membrane curvature into 50–60 nm vesicles, to induce transformation of Golgi tubules into cisternae. Also, formation of intraluminal ultra-small vesicles after fusion of endosomes allows equilibration of their TAA, volume and surface area. Finally, when microvilli of the brush border are broken into vesicles and microvilli fragments, TAA of these membranes remains the same as TAA of the microvilli. Thus, TAA has a significant role in transformation of organelle shape when other factors remain constant. PMID:25761238

  12. High Speed Size Sorting of Subcellular Organelles by Flow Field-Flow Fractionation.

    PubMed

    Yang, Joon Seon; Lee, Ju Yong; Moon, Myeong Hee

    2015-06-16

    Separation/isolation of subcellular species, such as mitochondria, lysosomes, peroxisomes, Golgi apparatus, and others, from cells is important for gaining an understanding of the cellular functions performed by specific organelles. This study introduces a high speed, semipreparative scale, biocompatible size sorting method for the isolation of subcellular organelle species from homogenate mixtures of HEK 293T cells using flow field-flow fractionation (FlFFF). Separation of organelles was achieved using asymmetrical FlFFF (AF4) channel system at the steric/hyperlayer mode in which nuclei, lysosomes, mitochondria, and peroxisomes were separated in a decreasing order of hydrodynamic diameter without complicated preprocessing steps. Fractions in which organelles were not clearly separated were reinjected to AF4 for a finer separation using the normal mode, in which smaller sized species can be well fractionated by an increasing order of diameter. The subcellular species contained in collected AF4 fractions were examined with scanning electron microscopy to evaluate their size and morphology, Western blot analysis using organelle specific markers was used for organelle confirmation, and proteomic analysis was performed with nanoflow liquid chromatography-tandem mass spectrometry (nLC-ESI-MS/MS). Since FlFFF operates with biocompatible buffer solutions, it offers great flexibility in handling subcellular components without relying on a high concentration sucrose solution for centrifugation or affinity- or fluorescence tag-based sorting methods. Consequently, the current study provides an alternative, competitive method for the isolation/purification of subcellular organelle species in their intact states. PMID:26005782

  13. Magnetic Fractionation and Proteomic Dissection of Cellular Organelles Occupied by the Late Replication Complexes of Semliki Forest Virus

    PubMed Central

    Varjak, Margus; Saul, Sirle; Arike, Liisa; Lulla, Aleksei; Peil, Lauri

    2013-01-01

    Alphavirus replicase complexes are initially formed at the plasma membrane and are subsequently internalized by endocytosis. During the late stages of infection, viral replication organelles are represented by large cytopathic vacuoles, where replicase complexes bind to membranes of endolysosomal origin. In addition to viral components, these organelles harbor an unknown number of host proteins. In this study, a fraction of modified lysosomes carrying functionally intact replicase complexes was obtained by feeding Semliki Forest virus (SFV)-infected HeLa cells with dextran-covered magnetic nanoparticles and later magnetically isolating the nanoparticle-containing lysosomes. Stable isotope labeling with amino acids in cell culture combined with quantitative proteomics was used to reveal 78 distinct cellular proteins that were at least 2.5-fold more abundant in replicase complex-carrying vesicles than in vesicles obtained from noninfected cells. These host components included the RNA-binding proteins PCBP1, hnRNP M, hnRNP C, and hnRNP K, which were shown to colocalize with the viral replicase. Silencing of hnRNP M and hnRNP C expression enhanced the replication of SFV, Chikungunya virus (CHIKV), and Sindbis virus (SINV). PCBP1 silencing decreased SFV-mediated protein synthesis, whereas hnRNP K silencing increased this synthesis. Notably, the effect of hnRNP K silencing on CHIKV- and SINV-mediated protein synthesis was opposite to that observed for SFV. This study provides a new approach for analyzing the proteome of the virus replication organelle of positive-strand RNA viruses and helps to elucidate how host RNA-binding proteins exert important but diverse functions during positive-strand RNA viral infection. PMID:23864636

  14. Hypoxia signaling pathways: modulators of oxygen-related organelles

    PubMed Central

    Schönenberger, Miriam J.; Kovacs, Werner J.

    2015-01-01

    Oxygen (O2) is an essential substrate in cellular metabolism, bioenergetics, and signaling and as such linked to the survival and normal function of all metazoans. Low O2 tension (hypoxia) is a fundamental feature of physiological processes as well as pathophysiological conditions such as cancer and ischemic diseases. Central to the molecular mechanisms underlying O2 homeostasis are the hypoxia-inducible factors-1 and -2 alpha (HIF-1α and EPAS1/HIF-2α) that function as master regulators of the adaptive response to hypoxia. HIF-induced genes promote characteristic tumor behaviors, including angiogenesis and metabolic reprogramming. The aim of this review is to critically explore current knowledge of how HIF-α signaling regulates the abundance and function of major O2-consuming organelles. Abundant evidence suggests key roles for HIF-1α in the regulation of mitochondrial homeostasis. An essential adaptation to sustained hypoxia is repression of mitochondrial respiration and induction of glycolysis. HIF-1α activates several genes that trigger mitophagy and represses regulators of mitochondrial biogenesis. Several lines of evidence point to a strong relationship between hypoxia, the accumulation of misfolded proteins in the endoplasmic reticulum, and activation of the unfolded protein response. Surprisingly, although peroxisomes depend highly on molecular O2 for their function, there has been no evidence linking HIF signaling to peroxisomes. We discuss our recent findings that establish HIF-2α as a negative regulator of peroxisome abundance and suggest a mechanism by which cells attune peroxisomal function with O2 availability. HIF-2α activation augments peroxisome turnover by pexophagy and thereby changes lipid composition reminiscent of peroxisomal disorders. We discuss potential mechanisms by which HIF-2α might trigger pexophagy and place special emphasis on the potential pathological implications of HIF-2α-mediated pexophagy for human health. PMID:26258123

  15. Bubble development in explosive silicic eruptions: insights from pyroclast vesicularity textures from Raoul volcano (Kermadec arc)

    NASA Astrophysics Data System (ADS)

    Rotella, Melissa D.; Wilson, Colin J. N.; Barker, Simon J.; Cashman, Katharine V.; Houghton, Bruce F.; Wright, Ian C.

    2014-08-01

    Critical to understanding explosive eruptions is establishing how accurately representative pyroclasts are of processes during magma vesiculation and fragmentation. Here, we present data on densities, and vesicle size and number characteristics, for representative pyroclasts from six silicic eruptions of contrasting size and style from Raoul volcano (Kermadec arc). We use these data to evaluate histories of bubble nucleation, coalescence, and growth in explosive eruptions and to provide comparisons with pumiceous dome carapace material. Density/vesicularity distributions show a scarcity of pyroclasts with ˜65-75 % vesicularity; however, pyroclasts closest to this vesicularity range have the highest bubble number density (BND) values regardless of eruptive intensity or style. Clasts with vesicularities greater than this 65-75 % "pivotal" vesicularity range have decreasing BNDs with increasing vesicularities, interpreted to reflect continuing bubble growth and coalescence. Clasts with vesicularities less than the pivotal range have BNDs that decrease with decreasing vesicularity and preserve textures indicative of processes such as stalling and open system degassing prior to vesiculation in a microlite-rich magma, or vesiculation during slow ascent of degassing magma. Bubble size distributions (BSDs) and BNDs show variations consistent with 65-75 % representing the vesicularity at which vesiculating magma is most likely to undergo fragmentation, consistent with the closest packing of spheres. We consider that the observed vesicularity range may reflect the development of permeability in the magma through shearing as it flows through the conduit. These processes can act in concert with multiple nucleation events, generating a situation of heterogeneous bubble populations that permit some regions of the magma to expand and bubbles to coalesce with other regions in which permeable networks are formed. Fragmentation preserves the range in vesicularity seen as well as

  16. Effects of altered cytoplasmic domains on transport of the vesicular stomatitis virus glycoprotein are transferable to other proteins.

    PubMed Central

    Guan, J L; Ruusala, A; Cao, H; Rose, J K

    1988-01-01

    Alterations of the cytoplasmic domain of the vesicular stomatitis virus glycoprotein (G protein) were shown previously to affect transport of the protein from the endoplasmic reticulum, and recent studies have shown that this occurs without detectable effects on G protein folding and trimerization (R. W. Doms et al., J. Cell Biol., in press). Deletions within this domain slowed exit of the mutant proteins from the endoplasmic reticulum, and replacement of this domain with a foreign 12-amino-acid sequence blocked all transport out of the endoplasmic reticulum. To extend these studies, we determined whether such effects of cytoplasmic domain changes were transferable to other proteins. Three different assays showed that the effects of the mutations on transport of two membrane-anchored secretory proteins were the same as those observed with vesicular stomatitis virus G protein. In addition, possible effects on oligomerization were examined for both transported and nontransported forms of membrane-anchored human chorionic gonadotropin-alpha. These membrane-anchored forms, like the nonanchored human chorionic gonadotropin-alpha, had sedimentation coefficients consistent with a monomeric structure. Taken together, our results provide strong evidence that these cytoplasmic mutations affect transport by affecting interactions at or near the cytoplasmic side of the membrane. Images PMID:2841589

  17. Prolonged starvation drives reversible sequestration of lipid biosynthetic enzymes and organelle reorganization in Saccharomyces cerevisiae

    PubMed Central

    Suresh, Harsha Garadi; da Silveira dos Santos, Aline Xavier; Kukulski, Wanda; Tyedmers, Jens; Riezman, Howard; Bukau, Bernd; Mogk, Axel

    2015-01-01

    Cells adapt to changing nutrient availability by modulating a variety of processes, including the spatial sequestration of enzymes, the physiological significance of which remains controversial. These enzyme deposits are claimed to represent aggregates of misfolded proteins, protein storage, or complexes with superior enzymatic activity. We monitored spatial distribution of lipid biosynthetic enzymes upon glucose depletion in Saccharomyces cerevisiae. Several different cytosolic-, endoplasmic reticulum–, and mitochondria-localized lipid biosynthetic enzymes sequester into distinct foci. Using the key enzyme fatty acid synthetase (FAS) as a model, we show that FAS foci represent active enzyme assemblies. Upon starvation, phospholipid synthesis remains active, although with some alterations, implying that other foci-forming lipid biosynthetic enzymes might retain activity as well. Thus sequestration may restrict enzymes' access to one another and their substrates, modulating metabolic flux. Enzyme sequestrations coincide with reversible drastic mitochondrial reorganization and concomitant loss of endoplasmic reticulum–mitochondria encounter structures and vacuole and mitochondria patch organelle contact sites that are reflected in qualitative and quantitative changes in phospholipid profiles. This highlights a novel mechanism that regulates lipid homeostasis without profoundly affecting the activity status of involved enzymes such that, upon entry into favorable growth conditions, cells can quickly alter lipid flux by relocalizing their enzymes. PMID:25761633

  18. Structure and Mechanisms of a Protein-Based Organelle in Escherichia coli

    SciTech Connect

    Tanaka, Shiho; Sawaya, Michael R.; Yeates, Todd O.

    2010-08-18

    Many bacterial cells contain proteinaceous microcompartments that act as simple organelles by sequestering specific metabolic processes involving volatile or toxic metabolites. Here we report the three-dimensional (3D) crystal structures, with resolutions between 1.65 and 2.5 angstroms, of the four homologous proteins (EutS, EutL, EutK, and EutM) that are thought to be the major shell constituents of a functionally complex ethanolamine utilization (Eut) microcompartment. The Eut microcompartment is used to sequester the metabolism of ethanolamine in bacteria such as Escherichia coli and Salmonella enterica. The four Eut shell proteins share an overall similar 3D fold, but they have distinguishing structural features that help explain the specific roles they play in the microcompartment. For example, EutL undergoes a conformational change that is probably involved in gating molecular transport through shell protein pores, whereas structural evidence suggests that EutK might bind a nucleic acid component. Together these structures give mechanistic insight into bacterial microcompartments.

  19. Exosomes as Intercellular Signaling Organelles Involved in Health and Disease: Basic Science and Clinical Applications

    PubMed Central

    Corrado, Chiara; Raimondo, Stefania; Chiesi, Antonio; Ciccia, Francesco; De Leo, Giacomo; Alessandro, Riccardo

    2013-01-01

    Cell to cell communication is essential for the coordination and proper organization of different cell types in multicellular systems. Cells exchange information through a multitude of mechanisms such as secreted growth factors and chemokines, small molecules (peptides, ions, bioactive lipids and nucleotides), cell-cell contact and the secretion of extracellular matrix components. Over the last few years, however, a considerable amount of experimental evidence has demonstrated the occurrence of a sophisticated method of cell communication based on the release of specialized membranous nano-sized vesicles termed exosomes. Exosome biogenesis involves the endosomal compartment, the multivesicular bodies (MVB), which contain internal vesicles packed with an extraordinary set of molecules including enzymes, cytokines, nucleic acids and different bioactive compounds. In response to stimuli, MVB fuse with the plasma membrane and vesicles are released in the extracellular space where they can interact with neighboring cells and directly induce a signaling pathway or affect the cellular phenotype through the transfer of new receptors or even genetic material. This review will focus on exosomes as intercellular signaling organelles involved in a number of physiological as well as pathological processes and their potential use in clinical diagnostics and therapeutics. PMID:23466882

  20. Nondisruptive micropatterning of fluid membranes through selective vesicular adsorption and rupture by nanotopography.

    PubMed

    Lee, Sang-Wook; Na, Yu-Jin; Lee, Sin-Doo

    2009-05-19

    We report on a nondisruptive method of patterning fluid membranes into micrometer-scale arrays through a selective vesicular rupture pathway by nanotopography. The site- and pathway-selective formation of supported lipid bilayers (SLBs) was achieved by different vesicular adsorption and rupture processes between nanocorrugated and nanosmooth topographies. The SLBs were first developed in the nanocorrugated region due to fast vesicular adsorption and then grew into the nanosmooth region through bilayer edge-induced vesicular rupture. Our topographic approach provides a viable scheme, yet unattainable in conventional ways, of actively controlling the position and the coverage of the SLBs on a variety of substrates without disrupting two-dimensional fluidity for highly integrated membrane devices. PMID:19368337

  1. A plausible mechanism of biosorption in dual symbioses by vesicular-arbuscular mycorrhizal in plants.

    PubMed

    Azmat, Rafia; Hamid, Neelofer

    2015-03-01

    Dual symbioses of vesicular-arbuscular mycorrhizal (VAM) fungi with growth of Momordica charantia were elucidated in terms of plausible mechanism of biosorption in this article. The experiment was conducted in green house and mixed inoculum of the VAM fungi was used in the three replicates. Results demonstrated that the starch contents were the main source of C for the VAM to builds their hyphae. The increased plant height and leaves surface area were explained in relation with an increase in the photosynthetic rates to produce rapid sugar contents for the survival of plants. A decreased in protein, and amino acid contents and increased proline and protease activity in VAM plants suggested that these contents were the main bio-indicators of the plants under biotic stress. The decline in protein may be due to the degradation of these contents, which later on converted into dextrose where it can easily be absorbed by for the period of symbioses. A mechanism of C chemisorption in relation with physiology and morphology of plant was discussed. PMID:25730809

  2. Interactions of normal and mutant vesicular stomatitis virus matrix proteins with the plasma membrane and nucleocapsids.

    PubMed Central

    Chong, L D; Rose, J K

    1994-01-01

    We demonstrated recently that a fraction of the matrix (M) protein of vesicular stomatitis virus (VSV) binds tightly to cellular membranes in vivo when expressed in the absence of other VSV proteins. This membrane-associated M protein was functional in binding purified VSV nucleocapsids in vitro. Here we show that the membrane-associated M protein is largely associated with a membrane fraction having the density of plasma membranes, indicating membrane specificity in the binding. In addition, we analyzed truncated forms of M protein to identify regions responsible for membrane association and nucleocapsid binding. Truncated M protein lacking the amino-terminal basic domain still associated with cellular membranes, although not as tightly as wild-type M protein, and could not bind nucleocapsids. In contrast, deletion of the carboxy-terminal 14 amino acids did not disrupt stable membrane association or nucleocapsid interaction. These results suggest that the amino terminus of M protein either interacts directly with membranes and nucleocapsids or stabilizes a conformation that is required for M protein to mediate both of these interactions. Images PMID:8254754

  3. Glycoprotein-Dependent Acidification of Vesicular Stomatitis Virus Enhances Release of Matrix Protein▿

    PubMed Central

    Mire, Chad E.; Dube, Derek; Delos, Sue E.; White, Judith M.; Whitt, Michael A.

    2009-01-01

    To study vesicular stomatitis virus (VSV) entry and uncoating, we generated a recombinant VSV encoding a matrix (M) protein containing a C-terminal tetracysteine Lumio tag (rVSV-ML) that could be fluorescently labeled using biarsenical compounds. Quantitative confocal microscopy showed that there is a transient loss of fluorescence at early times after the initiation of endocytosis of rVSV-ML-Green (rVSV-MLG) virions, which did not occur when cells were treated with bafilomycin A1. The reduction in fluorescence occurred 5 to 10 min postentry, followed by a steady increase in fluorescence intensity from 15 to 60 min postentry. A similar loss of fluorescence was observed in vitro when virions were exposed to acidic pH. The reduction in fluorescence required G protein since “bald” ΔG-MLG particles did not show a similar loss of fluorescence at low pH. Based on the pH-dependent fluorescence properties of Lumio Green, we hypothesize that the loss of fluorescence of rVSV-MLG virions during virus entry is due to a G ectodomain-dependent acidification of the virion interior. Biochemical analysis indicated that low pH also resulted in an enhancement of M protein dissociation from partially permeabilized, but otherwise intact, wild-type virions. From these data we propose that low-pH conformational changes in G protein promote acidification of the virus interior, which facilitates the release of M from ribonucleoprotein particles during uncoating. PMID:19776119

  4. Vesicular uptake of N-acetylaspartylglutamate is catalysed by sialin (SLC17A5).

    PubMed

    Lodder-Gadaczek, Julia; Gieselmann, Volkmar; Eckhardt, Matthias

    2013-08-15

    NAAG (N-acetylaspartylglutamate) is an abundant neuropeptide in the vertebrate nervous system. It is released from synaptic terminals in a calcium-dependent manner and has been shown to act as an agonist at the type II metabotropic glutamate receptor mGluR3. It has been proposed that NAAG may also be released from axons. So far, however, it has remained unclear how NAAG is transported into synaptic or other vesicles before it is secreted. In the present study, we demonstrate that uptake of NAAG and the related peptide NAAG2 (N-acetylaspartylglutamylglutamate) into vesicles depends on the sialic acid transporter sialin (SLC17A5). This was demonstrated using cell lines expressing a cell surface variant of sialin and by functional reconstitution of sialin in liposomes. NAAG uptake into sialin-containing proteoliposomes was detectable in the presence of an active H+-ATPase or valinomycin, indicating that transport is driven by membrane potential rather than H+ gradient. We also show that sialin is most probably the major and possibly only vesicular transporter for NAAG and NAAG2, because ATP-dependent transport of both peptides was not detectable in vesicles isolated from sialin-deficient mice. PMID:23889254

  5. Structure and Function of the N-Terminal Domain of the Vesicular Stomatitis Virus RNA Polymerase

    PubMed Central

    Qiu, Shihong; Ogino, Minako; Luo, Ming

    2015-01-01

    ABSTRACT Viruses have various mechanisms to duplicate their genomes and produce virus-specific mRNAs. Negative-strand RNA viruses encode their own polymerases to perform each of these processes. For the nonsegmented negative-strand RNA viruses, the polymerase is comprised of the large polymerase subunit (L) and the phosphoprotein (P). L proteins from members of the Rhabdoviridae, Paramyxoviridae, and Filoviridae share sequence and predicted secondary structure homology. Here, we present the structure of the N-terminal domain (conserved region I) of the L protein from a rhabdovirus, vesicular stomatitis virus, at 1.8-Å resolution. The strictly and strongly conserved residues in this domain cluster in a single area of the protein. Serial mutation of these residues shows that many of the amino acids are essential for viral transcription but not for mRNA capping. Three-dimensional alignments show that this domain shares structural homology with polymerases from other viral families, including segmented negative-strand RNA and double-stranded RNA (dsRNA) viruses. IMPORTANCE Negative-strand RNA viruses include a diverse set of viral families that infect animals and plants, causing serious illness and economic impact. The members of this group of viruses share a set of functionally conserved proteins that are essential to their replication cycle. Among this set of proteins is the viral polymerase, which performs a unique set of reactions to produce genome- and subgenome-length RNA transcripts. In this article, we study the polymerase of vesicular stomatitis virus, a member of the rhabdoviruses, which has served in the past as a model to study negative-strand RNA virus replication. We have identified a site in the N-terminal domain of the polymerase that is essential to viral transcription and that shares sequence homology with members of the paramyxoviruses and the filoviruses. Newly identified sites such as that described here could prove to be useful targets in the

  6. Multiple organelle-targeting signals in the N-terminal portion of peroxisomal membrane protein PMP70.

    PubMed

    Iwashita, Shohei; Tsuchida, Masashi; Tsukuda, Miwa; Yamashita, Yukari; Emi, Yoshikazu; Kida, Yuichiro; Komori, Masayuki; Kashiwayama, Yoshinori; Imanaka, Tsuneo; Sakaguchi, Masao

    2010-04-01

    Most membrane proteins are recognized by a signal recognition particle and are cotranslationally targeted to the endoplasmic reticulum (ER) membrane, whereas almost all peroxisomal membrane proteins are posttranslationally targeted to the destination. Here we examined organelle-targeting properties of the N-terminal portions of the peroxisomal isoform of the ABC transporter PMP70 (ABCD3) using enhanced green fluorescent protein (EGFP) fusion. When the N-terminal 80 amino acid residue (N80)-segment preceding transmembrane segment (TM) 1 was deleted and the TM1-TM2 region was fused to EGFP, the TM1 segment induced ER-targeting and integration in COS cells. When the N80-segment was fused to EGFP, the fusion protein was targeted to the outer mitochondrial membrane. When both the N80-segment and the following TM1-TM2 region were present, the fusion located exclusively to the peroxisome. The full-length PMP70 molecule was clearly located in the ER in the absence of the N80-segment, even when multiple peroxisome-targeting signals were retained. We concluded that the TM1 segment possesses a sufficient ER-targeting function and that the N80-segment is critical for suppressing the ER-targeting function to allow the TM1-TM2 region to localize to the peroxisome. Cooperation of the organelle-targeting signals enables PMP70 to correctly target to peroxisomal membranes. PMID:20007743

  7. A divergent ADP/ATP carrier in the hydrogenosomes of Trichomonas gallinae argues for an independent origin of these organelles.

    PubMed

    Tjaden, Joachim; Haferkamp, Ilka; Boxma, Brigitte; Tielens, Aloysius G M; Huynen, Martijn; Hackstein, Johannes H P

    2004-03-01

    The evolution of mitochondrial ADP and ATP exchanging proteins (AACs) highlights a key event in the evolution of the eukaryotic cell, as ATP exporting carriers were indispensable in establishing the role of mitochondria as ATP-generating cellular organelles. Hydrogenosomes, i.e. ATP- and hydrogen-generating organelles of certain anaerobic unicellular eukaryotes, are believed to have evolved from the same ancestral endosymbiont that gave rise to present day mitochondria. Notably, the hydrogenosomes of the parasitic anaerobic flagellate Trichomonas seemed to be deficient in mitochondrial-type AACs. Instead, HMP 31, a different member of the mitochondrial carrier family (MCF) with a hitherto unknown function, is abundant in the hydrogenosomal membranes of Trichomonas vaginalis. Here we show that the homologous HMP 31 of closely related Trichomonas gallinae specifically transports ADP and ATP with high efficiency, as do genuine mitochondrial AACs. However, phylogenetic analysis and its resistance against bongkrekic acid (BKA, an efficient inhibitor of mitochondrial-type AACs) identify HMP 31 as a member of the mitochondrial carrier family that is distinct from all mitochondrial and hydrogenosomal AACs studied so far. Thus, our data support the hypothesis that the various hydrogenosomes evolved repeatedly and independently. PMID:14982636

  8. Transferosomes - A vesicular transdermal delivery system for enhanced drug permeation

    PubMed Central

    Rajan, Reshmy; Jose, Shoma; Mukund, V. P. Biju; Vasudevan, Deepa T.

    2011-01-01

    Transdermal administration of drugs is generally limited by the barrier function of the skin. Vesicular systems are one of the most controversial methods for transdermal delivery of active substances. The interest in designing transdermal delivery systems was relaunched after the discovery of elastic vesicles like transferosomes, ethosomes, cubosomes, phytosomes, etc. This paper presents the composition, mechanisms of penetration, manufacturing and characterization methods of transferosomes as transdermal delivery systems of active substances. For a drug to be absorbed and distributed into organs and tissues and eliminated from the body, it must pass through one or more biological membranes/barriers at various locations. Such a movement of drug across the membrane is called as drug transport. For the drugs to be delivered to the body, they should cross the membranous barrier. The concept of these delivery systems was designed in an attempt to concentrate the drug in the tissues of interest, while reducing the amount of drug in the remaining tissues. Hence, surrounding tissues are not affected by the drug. In addition, loss of drug does not happen due to localization of drug, leading to get maximum efficacy of the medication. Therefore, the phospholipid based carrier systems are of considerable interest in this era. PMID:22171309

  9. Asymmetric packaging of polymerases within vesicular stomatitis virus

    SciTech Connect

    Hodges, Jeffery; Tang, Xiaolin; Landesman, Michael B.; Ruedas, John B.; Ghimire, Anil; Gudheti, Manasa V.; Perrault, Jacques; Jorgensen, Erik M.; Gerton, Jordan M.; Saffarian, Saveez

    2013-10-18

    Highlights: •The VSV polymerases (L proteins) are localized to the blunt end of the virus. •The VSV phosphoproteins (P proteins) are localized to the blunt end of the virus. •Each VSV virion packages a variable number of P and L proteins. -- Abstract: Vesicular stomatitis virus (VSV) is a prototypic negative sense single-stranded RNA virus. The bullet-shape appearance of the virion results from tightly wound helical turns of the nucleoprotein encapsidated RNA template (N-RNA) around a central cavity. Transcription and replication require polymerase complexes, which include a catalytic subunit L and a template-binding subunit P. L and P are inferred to be in the cavity, however lacking direct observation, their exact position has remained unclear. Using super-resolution fluorescence imaging and atomic force microscopy (AFM) on single VSV virions, we show that L and P are packaged asymmetrically towards the blunt end of the virus. The number of L and P proteins varies between individual virions and they occupy 57 ± 12 nm of the 150 nm central cavity of the virus. Our finding positions the polymerases at the opposite end of the genome with respect to the only transcriptional promoter.

  10. Differential vesicular sorting of AMPA and GABAA receptors

    PubMed Central

    Gu, Yi; Chiu, Shu-Ling; Liu, Bian; Wu, Pei-Hsun; Delannoy, Michael; Lin, Da-Ting; Wirtz, Denis; Huganir, Richard L.

    2016-01-01

    In mature neurons AMPA receptors cluster at excitatory synapses primarily on dendritic spines, whereas GABAA receptors cluster at inhibitory synapses mainly on the soma and dendritic shafts. The molecular mechanisms underlying the precise sorting of these receptors remain unclear. By directly studying the constitutive exocytic vesicles of AMPA and GABAA receptors in vitro and in vivo, we demonstrate that they are initially sorted into different vesicles in the Golgi apparatus and inserted into distinct domains of the plasma membrane. These insertions are dependent on distinct Rab GTPases and SNARE complexes. The insertion of AMPA receptors requires SNAP25–syntaxin1A/B–VAMP2 complexes, whereas insertion of GABAA receptors relies on SNAP23–syntaxin1A/B–VAMP2 complexes. These SNARE complexes affect surface targeting of AMPA or GABAA receptors and synaptic transmission. Our studies reveal vesicular sorting mechanisms controlling the constitutive exocytosis of AMPA and GABAA receptors, which are critical for the regulation of excitatory and inhibitory responses in neurons. PMID:26839408

  11. Transferosomes - A vesicular transdermal delivery system for enhanced drug permeation.

    PubMed

    Rajan, Reshmy; Jose, Shoma; Mukund, V P Biju; Vasudevan, Deepa T

    2011-07-01

    Transdermal administration of drugs is generally limited by the barrier function of the skin. Vesicular systems are one of the most controversial methods for transdermal delivery of active substances. The interest in designing transdermal delivery systems was relaunched after the discovery of elastic vesicles like transferosomes, ethosomes, cubosomes, phytosomes, etc. This paper presents the composition, mechanisms of penetration, manufacturing and characterization methods of transferosomes as transdermal delivery systems of active substances. For a drug to be absorbed and distributed into organs and tissues and eliminated from the body, it must pass through one or more biological membranes/barriers at various locations. Such a movement of drug across the membrane is called as drug transport. For the drugs to be delivered to the body, they should cross the membranous barrier. The concept of these delivery systems was designed in an attempt to concentrate the drug in the tissues of interest, while reducing the amount of drug in the remaining tissues. Hence, surrounding tissues are not affected by the drug. In addition, loss of drug does not happen due to localization of drug, leading to get maximum efficacy of the medication. Therefore, the phospholipid based carrier systems are of considerable interest in this era. PMID:22171309

  12. Role of matrix protein in cytopathogenesis of vesicular stomatitis virus.

    PubMed Central

    Blondel, D; Harmison, G G; Schubert, M

    1990-01-01

    The matrix (M) protein of vesicular stomatitis virus (VSV) plays an important structural role in viral assembly, and it also has a regulatory role in viral transcription. We demonstrate here that the M protein has an additional function. It causes visible cytopathic effects (CPE), as evidenced by the typical rounding of polygonal cells after VSV infection. We have analyzed a temperature-sensitive mutant of the M protein of VSV (tsG33) which is defective in viral assembly and which fails to cause morphological changes of the cells after infection at the nonpermissive temperature (40 degrees C). Interestingly, this defect in viral assembly as well as the CPE were reversible. Microinjection of antisense oligonucleotides which specifically inhibit M protein translation also inhibited the occurrence of CPE. Most importantly, when cells were transfected with a cDNA encoding the temperature-sensitive M protein of tsG33, no CPE was observed at the nonpermissive temperature. However, when these cells were shifted to the permissive temperature (32 degrees C), they rounded up and detached from the dish. These results demonstrate that M protein in the absence of the other viral proteins causes rounding of the cells, probably through a disorganization of the cytoskeleton. The absence of CPE at the nonpermissive temperature is correlated with an abnormal dotted staining pattern of M in these cells, suggesting that the mutant M protein may self-aggregate or associate with membranes rather than interact with cytoskeletal elements. Images PMID:2157054

  13. Understanding and altering cell tropism of vesicular stomatitis virus

    PubMed Central

    Hastie, Eric; Cataldi, Marcela; Marriott, Ian; Grdzelishvili, Valery Z.

    2013-01-01

    Vesicular stomatitis virus (VSV) is a prototypic nonsegmented negative-strand RNA virus. VSV’s broad cell tropism makes it a popular model virus for many basic research applications. In addition, a lack of preexisting human immunity against VSV, inherent oncotropism and other features make VSV a widely used platform for vaccine and oncolytic vectors. However, VSV’s neurotropism that can result in viral encephalitis in experimental animals needs to be addressed for the use of the virus as a safe vector. Therefore, it is very important to understand the determinants of VSV tropism and develop strategies to alter it. VSV glycoprotein (G) and matrix (M) protein play major roles in its cell tropism. VSV G protein is responsible for VSV broad cell tropism and is often used for pseudotyping other viruses. VSV M affects cell tropism via evasion of antiviral responses, and M mutants can be used to limit cell tropism to cell types defective in interferon signaling. In addition, other VSV proteins and host proteins may function as determinants of VSV cell tropism. Various approaches have been successfully used to alter VSV tropism to benefit basic research and clinically relevant applications. PMID:23796410

  14. Impact of probe compound in MRP2 vesicular transport assays.

    PubMed

    Kidron, Heidi; Wissel, Gloria; Manevski, Nenad; Häkli, Marika; Ketola, Raimo A; Finel, Moshe; Yliperttula, Marjo; Xhaard, Henri; Urtti, Arto

    2012-05-12

    MRP2 is an efflux transporter that is expressed mainly in the canalicular membrane of hepatocytes, where it expels polar and ionic compounds into the bile. MRP2 is also present in the apical membrane of enterocytes and epithelial cells of proximal tubules of the kidney. Inhibition of MRP2 transport can lead to the accumulation of metabolites and other MRP2 substrates up to toxic levels in these cells. The transport properties of MRP2 are frequently studied with the vesicular transport assay. The assay identifies compounds that interact with MRP2 by measuring the effect of a compound on the transport of a radioactively labeled or fluorescent probe. We have compared the effect of eight selected test compounds (quercetin, disopyramide, paracetamol, indomethacin, diclofenac, estrone-3-sulfate, budesonide, and thioridazine) on the MRP2-mediated transport of three commonly used probes: 5(6)-carboxy-2,7-dichlorofluorescein, leukotriene C4 and estradiol-17-β-d-glucuronide (E217βG). Five of the test compounds had different probe-dependent effects on the MRP2-mediated transport, suggesting differences in the transport mechanism of the probes. Our results underline the complexity of substrate recognition by these efflux transporters and the difficulties in directly comparing results obtained with different assays, especially when different probes are used. PMID:22406294

  15. The formation of vesicular cylinders in pahoehoe lava flows

    NASA Astrophysics Data System (ADS)

    Fowler, A. C.; Rust, Alison C.; Vynnycky, M.

    2015-01-01

    Vertical cylinders of bubble-enriched, chemically evolved volcanic rock are found in many inflated pahoehoe lava flows. We provide a putative theoretical explanation for their formation, based on a description of a crystallising three-phase (liquid, solid, gas) crystal pile in which the water-saturated silicate melt exsolves steam and becomes more silica-rich as it crystallises anhydrous minerals. These cylinders resemble pipes that form in solidifying binary alloys as a result of sufficiently vigorous porous medium convection within the mush. A convection model with the addition of gas bubbles that provide the buoyancy source indicates that the effective Rayleigh number is too low for convection to occur in the mush of a basalt lava flow. However, the formation of gas bubbles during crystallisation means that the base state includes fluid migration up through the crystal mush even without convection. Stability considerations suggest that it is plausible to form a positive feedback where increased local porosity causes increased upwards fluid flow, which brings more silicic melt up and lowers the liquidus temperature, promoting locally higher porosity. Numerical solutions show that there are steady solutions in which cylinders form, and we conclude that this model provides a viable explanation for vesicular cylinder formation in inflated basalt lava flows.

  16. Entry of Vesicular Stomatitis Virus into L Cells

    PubMed Central

    Heine, Jochen W.; Schnaitman, Carl A.

    1971-01-01

    Early stages of the entry of vesicular stomatitis (VS) virus into L cells were followed by electron microscopy with the aid of ferritin antibody labeling. Cells which were infected at 0 C and incubated for 10 min at 37 C were reacted first with antiviral-antiferritin hybrid antibody and then with ferritin or fluorescein-labeled apoferritin. Extensive ferritin labeling of the cell surface was detected by both electron and fluorescence microscopy. The labeled regions of the cell surface were continuous with and indistinguishable from the rest of the host cell membrane, suggesting incorporation of viral antigens into the cell surface during viral penetration. Fusion of parental viral membrane with host cell membrane was further demonstrated by examining the localization of 3H-labeled viral structural proteins in cells infected at 0 C and incubated for short periods at 37 C. Viral nucleoprotein was found in a soluble fraction of the cells which was derived primarily from the cytoplasm, whereas a particulate fraction from the cells was enriched in viral envelope proteins. Cytoplasmic membrane was isolated from these cells, and this membrane contained viral envelope proteins. These results suggest that penetration by VS virus occurs by fusion of the viral and cellular membranes followed by release of nucleo-protein into the cytoplasm. Images PMID:4332145

  17. Expression of Vesicular Nucleotide Transporter in Rat Odontoblasts

    PubMed Central

    Ikeda, Erina; Goto, Tetsuya; Gunjigake, Kaori; Kuroishi, Kayoko; Ueda, Masae; Kataoka, Shinji; Toyono, Takashi; Nakatomi, Mitsushiro; Seta, Yuji; Kitamura, Chiaki; Nishihara, Tatsuji; Kawamoto, Tatsuo

    2016-01-01

    Several theories have been proposed regarding pain transmission mechanisms in tooth. However, the exact signaling mechanism from odontoblasts to pulp nerves remains to be clarified. Recently, ATP-associated pain transmission has been reported, but it is unclear whether ATP is involved in tooth pain transmission. In the present study, we focused on the vesicular nucleotide transporter (VNUT), a transporter of ATP into vesicles, and examined whether VNUT was involved in ATP release from odontoblasts. We examined the expression of VNUT in rat pulp by RT-PCR and immunostaining. ATP release from cultured odontoblast-like cells with heat stimulation was evaluated using ATP luciferase methods. VNUT was expressed in pulp tissue, and the distribution of VNUT-immunopositive vesicles was confirmed in odontoblasts. In odontoblasts, some VNUT-immunopositive vesicles were colocalized with membrane fusion proteins. Additionally P2X3, an ATP receptor, immunopositive axons were distributed between odontoblasts. The ATP release by thermal stimulation from odontoblast-like cells was inhibited by the addition of siRNA for VNUT. These findings suggest that cytosolic ATP is transported by VNUT and that the ATP in the vesicles is then released from odontoblasts to ATP receptors on axons. ATP vesicle transport in odontoblasts seems to be a key mechanism for signal transduction from odontoblasts to axons in the pulp. PMID:27006518

  18. Expression of Vesicular Nucleotide Transporter in Rat Odontoblasts.

    PubMed

    Ikeda, Erina; Goto, Tetsuya; Gunjigake, Kaori; Kuroishi, Kayoko; Ueda, Masae; Kataoka, Shinji; Toyono, Takashi; Nakatomi, Mitsushiro; Seta, Yuji; Kitamura, Chiaki; Nishihara, Tatsuji; Kawamoto, Tatsuo

    2016-02-27

    Several theories have been proposed regarding pain transmission mechanisms in tooth. However, the exact signaling mechanism from odontoblasts to pulp nerves remains to be clarified. Recently, ATP-associated pain transmission has been reported, but it is unclear whether ATP is involved in tooth pain transmission. In the present study, we focused on the vesicular nucleotide transporter (VNUT), a transporter of ATP into vesicles, and examined whether VNUT was involved in ATP release from odontoblasts. We examined the expression of VNUT in rat pulp by RT-PCR and immunostaining. ATP release from cultured odontoblast-like cells with heat stimulation was evaluated using ATP luciferase methods. VNUT was expressed in pulp tissue, and the distribution of VNUT-immunopositive vesicles was confirmed in odontoblasts. In odontoblasts, some VNUT-immunopositive vesicles were colocalized with membrane fusion proteins. Additionally P2X3, an ATP receptor, immunopositive axons were distributed between odontoblasts. The ATP release by thermal stimulation from odontoblast-like cells was inhibited by the addition of siRNA for VNUT. These findings suggest that cytosolic ATP is transported by VNUT and that the ATP in the vesicles is then released from odontoblasts to ATP receptors on axons. ATP vesicle transport in odontoblasts seems to be a key mechanism for signal transduction from odontoblasts to axons in the pulp. PMID:27006518

  19. Evolution of Fitness in Experimental Populations of Vesicular Stomatitis Virus

    PubMed Central

    Elena, S. F.; Gonzalez-Candelas, F.; Novella, I. S.; Duarte, E. A.; Clarke, D. K.; Domingo, E.; Holland, J. J.; Moya, A.

    1996-01-01

    The evolution of fitness in experimental clonal populations of vesicular stomatitis virus (VSV) has been compared under different genetic (fitness of initial clone) and demographic (population dynamics) regimes. In spite of the high genetic heterogeneity among replicates within experiments, there is a clear effect of population dynamics on the evolution of fitness. Those populations that went through strong periodic bottlenecks showed a decreased fitness in competition experiments with wild type. Conversely, mutant populations that were transferred under the dynamics of continuous population expansions increased their fitness when compared with the same wild type. The magnitude of the observed effect depended on the fitness of the original viral clone. Thus, high fitness clones showed a larger reduction in fitness than low fitness clones under dynamics with included periodic bottleneck. In contrast, the gain in fitness was larger the lower the initial fitness of the viral clone. The quantitative genetic analysis of the trait ``fitness'' in the resulting populations shows that genetic variation for the trait is positively correlated with the magnitude of the change in the same trait. The results are interpreted in terms of the operation of MULLER's ratchet and genetic drift as opposed to the appearance of beneficial mutations. PMID:8849878

  20. Vesicular stomatitis virus P function depends on cellular growth cycle.

    PubMed

    Stanners, C P; Kennedy, S; Poliquin, L

    1987-09-01

    The P function of vesicular stomatitis virus (VSV) is defined as the viral function which results in a reduced rate of total protein synthesis (viral plus cellular) arising from a nonspecific reduction in the efficiency of the translational machinery in infected cells. The existence of P function has been challenged by Lodish and Porter who were unable to detect it in L-strain mouse cells infected with wild-type VSV (HR) or, as expected, with the P- mutant, T1026-R1. Although other groups have subsequently confirmed the existence of P function and the difference between HR and T1026-R1, we have sought an explanation for the difference between Lodish and Porter's results and those of other laboratories. We show that the VSV P function depends on the phase of the growth cycle of infected L-cell cultures. In very early exponential phase, as used by Lodish and Porter, HR has very little demonstrable P function; as the growth cycle proceeds toward stationary phase, P function becomes more and more manifest. Under the same conditions, T1026-R1 shows no P function throughout the growth cycle. Furthermore we show that the VSV M protein mutant tsG31 has a P++ phenotype reducing total protein synthesis below that seen with wild-type HR. P function can be observed in cells infected with tsG31, even early in the exponential phase of the cellular growth cycle. PMID:2820132

  1. Dendrisomes: vesicular structures derived from a cationic lipidic dendron.

    PubMed

    Al-Jamal, Khuloud T; Sakthivel, Thiagarajan; Florence, Alexander T

    2005-01-01

    The behavior of a novel synthetic lipidic cationic lysine-based dendron (partial dendrimer) in aqueous media and its ability, with and without cholesterol, to self-assemble into higher order structures was studied to gain an understanding of these structures as potential drug carriers. The dendron was prepared by solid-phase peptide synthesis. A reverse-phase evaporation (REV) technique was used to prepare cationic vesicular aggregates of the dendron with different molar ratios of cholesterol. The size and zeta potential of these supramolecular aggregates or "dendrisomes" was determined by photon correlation spectroscopy (PCS). Dendrisome morphology and thermotropic properties were studied by transmission electron microscopy (TEM) and differential scanning calorimetry (DSC). Radiolabeled penicillin G was used as a model of a negatively charged water-soluble compound to investigate the encapsulation efficiency of the dendrisomes. In vitro release of the drug was determined using as a comparator a REV liposome formulation. Dendrisomes of all compositions have higher encapsulation efficiencies and slower release rates compared to the comparator. Cholesterol was found both to increase the size of the aggregates from around 310 to 560 nm and to increase shape irregularities, but did not change the positive zeta potential, in the order of +50 mV, of the dendrisomes. Cholesterol decreases penicillin G entrapment efficiency but increases solute leakage at 25 degrees C. PMID:15761934

  2. Inter-organelle ER-endolysosomal contact sites in metabolism and disease across evolution.

    PubMed

    Hariri, Hanaa; Ugrankar, Rupali; Liu, Yang; Henne, W Mike

    2016-01-01

    Since their initial observation, contact sites formed between different organelles have transitioned from ignored curiosities to recognized centers for the exchange of metabolites and lipids. Contact formed between the ER and endomembrane system (eg. the plasma membrane, endosomes, and lysosomes) is of particular biomedical interest, as it governs aspects of lipid metabolism, organelle identity, and cell signaling. Here, we review the field of ER-endolysosomal communication from the perspective of three model systems: budding yeast, the fruit fly D. melanogaster, and mammals. From this broad perspective, inter-organelle communication displays a consistent role in metabolic regulation that was differentially tuned during the development of complex metazoan life. We also examine the current state of understanding of lipid exchange between organelles, and discuss molecular mechanisms by which this occurs. PMID:27489577

  3. Inter-organelle ER-endolysosomal contact sites in metabolism and disease across evolution

    PubMed Central

    Hariri, Hanaa; Ugrankar, Rupali; Liu, Yang; Henne, W. Mike

    2016-01-01

    ABSTRACT Since their initial observation, contact sites formed between different organelles have transitioned from ignored curiosities to recognized centers for the exchange of metabolites and lipids. Contact formed between the ER and endomembrane system (eg. the plasma membrane, endosomes, and lysosomes) is of particular biomedical interest, as it governs aspects of lipid metabolism, organelle identity, and cell signaling. Here, we review the field of ER-endolysosomal communication from the perspective of three model systems: budding yeast, the fruit fly D. melanogaster, and mammals. From this broad perspective, inter-organelle communication displays a consistent role in metabolic regulation that was differentially tuned during the development of complex metazoan life. We also examine the current state of understanding of lipid exchange between organelles, and discuss molecular mechanisms by which this occurs. PMID:27489577

  4. Increased vesicular monoamine transporter enhances dopamine release and opposes Parkinson disease-related neurodegeneration in vivo

    PubMed Central

    Lohr, Kelly M.; Bernstein, Alison I.; Stout, Kristen A.; Dunn, Amy R.; Lazo, Carlos R.; Alter, Shawn P.; Wang, Minzheng; Li, Yingjie; Fan, Xueliang; Hess, Ellen J.; Yi, Hong; Vecchio, Laura M.; Goldstein, David S.; Guillot, Thomas S.; Salahpour, Ali; Miller, Gary W.

    2014-01-01

    Disruption of neurotransmitter vesicle dynamics (transport, capacity, release) has been implicated in a variety of neurodegenerative and neuropsychiatric conditions. Here, we report a novel mouse model of enhanced vesicular function via bacterial artificial chromosome (BAC)-mediated overexpression of the vesicular monoamine transporter 2 (VMAT2; Slc18a2). A twofold increase in vesicular transport enhances the vesicular capacity for dopamine (56%), dopamine vesicle volume (33%), and basal tissue dopamine levels (21%) in the mouse striatum. The elevated vesicular capacity leads to an increase in stimulated dopamine release (84%) and extracellular dopamine levels (44%). VMAT2-overexpressing mice show improved outcomes on anxiety and depressive-like behaviors and increased basal locomotor activity (41%). Finally, these mice exhibit significant protection from neurotoxic insult by the dopaminergic toxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), as measured by reduced dopamine terminal damage and substantia nigra pars compacta cell loss. The increased release of dopamine and neuroprotection from MPTP toxicity in the VMAT2-overexpressing mice suggest that interventions aimed at enhancing vesicular capacity may be of therapeutic benefit in Parkinson disease. PMID:24979780

  5. Increased vesicular monoamine transporter enhances dopamine release and opposes Parkinson disease-related neurodegeneration in vivo.

    PubMed

    Lohr, Kelly M; Bernstein, Alison I; Stout, Kristen A; Dunn, Amy R; Lazo, Carlos R; Alter, Shawn P; Wang, Minzheng; Li, Yingjie; Fan, Xueliang; Hess, Ellen J; Yi, Hong; Vecchio, Laura M; Goldstein, David S; Guillot, Thomas S; Salahpour, Ali; Miller, Gary W

    2014-07-01

    Disruption of neurotransmitter vesicle dynamics (transport, capacity, release) has been implicated in a variety of neurodegenerative and neuropsychiatric conditions. Here, we report a novel mouse model of enhanced vesicular function via bacterial artificial chromosome (BAC)-mediated overexpression of the vesicular monoamine transporter 2 (VMAT2; Slc18a2). A twofold increase in vesicular transport enhances the vesicular capacity for dopamine (56%), dopamine vesicle volume (33%), and basal tissue dopamine levels (21%) in the mouse striatum. The elevated vesicular capacity leads to an increase in stimulated dopamine release (84%) and extracellular dopamine levels (44%). VMAT2-overexpressing mice show improved outcomes on anxiety and depressive-like behaviors and increased basal locomotor activity (41%). Finally, these mice exhibit significant protection from neurotoxic insult by the dopaminergic toxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), as measured by reduced dopamine terminal damage and substantia nigra pars compacta cell loss. The increased release of dopamine and neuroprotection from MPTP toxicity in the VMAT2-overexpressing mice suggest that interventions aimed at enhancing vesicular capacity may be of therapeutic benefit in Parkinson disease. PMID:24979780

  6. Nanomanipulation-Coupled Matrix-Assisted Laser Desorption/ Ionization-Direct Organelle Mass Spectrometry: A Technique for the Detailed Analysis of Single Organelles

    NASA Astrophysics Data System (ADS)

    Phelps, Mandy S.; Sturtevant, Drew; Chapman, Kent D.; Verbeck, Guido F.

    2016-02-01

    We describe a novel technique combining precise organelle microextraction with deposition and matrix-assisted laser desorption/ionization (MALDI) for a rapid, minimally invasive mass spectrometry (MS) analysis of single organelles from living cells. A dual-positioner nanomanipulator workstation was utilized for both extraction of organelle content and precise co-deposition of analyte and matrix solution for MALDI-direct organelle mass spectrometry (DOMS) analysis. Here, the triacylglycerol (TAG) profiles of single lipid droplets from 3T3-L1 adipocytes were acquired and results validated with nanoelectrospray ionization (NSI) MS. The results demonstrate the utility of the MALDI-DOMS technique as it enabled longer mass analysis time, higher ionization efficiency, MS imaging of the co-deposited spot, and subsequent MS/MS capabilities of localized lipid content in comparison to NSI-DOMS. This method provides selective organellar resolution, which complements current biochemical analyses and prompts for subsequent subcellular studies to be performed where limited samples and analyte volume are of concern.

  7. Target Biological Structures: The Cell, Organelles, DNA and RNA

    NASA Astrophysics Data System (ADS)

    van Holst, Marcelis; Grant, Maxine P.; Aldrich-Wright, Janice

    Living organisms are self replicating molecular factories of staggering complexity [1]. As a result, we are often overwhelmed when trying to identify potential targets for therapeutics. Water, inorganic ions and a large array of relatively small organic molecules (e.g., sugars, vitamins and fatty acids) account for approximately 80% of living matter, with water being the most abundant. Macromolecules such as proteins, polysaccharides, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) constitute the rest. The majority of potential therapeutic targets are found within the cell. Small molecules which are vital for cellular function are imported into the cell by a variety of mechanisms but unlike smaller molecules, macromolecules are assembled within the cell itself. Drugs are usually designed to target cellular macromolecules, as they perform very specific roles in the metabolic processes.

  8. In Vitro Protein-Synthesizing Activity of Vesicular Stomatitis Virus-Infected Cell Extracts

    PubMed Central

    Grubman, Marvin J.; Summers, Donald F.

    1973-01-01

    Crude cytoplasmic extracts from vesicular stomatitis virus (VSV)-infected HeLa cells incorporate radioactive amino acids into hot trichloroacetic acid-precipitable material linearly for 10 to 20 min. The material synthesized in vitro corresponds in molecular weight to four of the five VSV structural proteins. However, synthesis of the viral glycoprotein (G) is significantly reduced, whereas the relative amounts of viral structural proteins L and NS synthesized are increased compared with the ratio of the proteins found in the virion. Fractionation of a VSV-infected crude cytoplasmic extract into a cytoplasmic pellet (20,000 × g for 30 min) and a cytoplasmic supernatant results in a significant reduction in protein synthesizing activity of both fractions, although both contain polysomes. The products synthesized by a cytoplasmic supernatant-directed system included all the VSV structural proteins except the glycoprotein, whereas in an in vitro system directed by the cytoplasmic pellet there is a marked reduction in synthesis of the nucleoprotein (N) and also a small relative increase in synthesis of the glycoprotein. Addition of uninfected, preincubated HeLa or L-cell S10 or a HeLa ribosomal fraction to the VSV-infected cytoplasmic pellet results in a 30- to 60-fold stimulation of 35S-methionine incorporation. However, these uninfected extracts do not stimulate 35S-methionine incorporation by the infected crude cytoplasmic extract or the cytoplasmic supernatant. The products synthesized by the stimulated cytoplasmic pellet now include sizeable amounts of the glycoprotein in addition to the other VSV structural proteins. PMID:4355931

  9. Extreme heterogeneity in populations of vesicular stomatitis virus.

    PubMed Central

    Steinhauer, D A; de la Torre, J C; Meier, E; Holland, J J

    1989-01-01

    Vesicular stomatitis virus (VSV) sequence evolution and population heterogeneity were examined by T1 oligonucleotide mapping. Individual clones isolated from clonal pools of wild-type Indiana serotype VSV displayed identical T1 maps. This was observed even after one passage at high concentrations of the potent viral mutagen 5-fluorouracil. Under low-multiplicity passage conditions, the consensus T1 fingerprint of this virus remained unchanged after 523 passages. Interestingly, however, individual clones from this population (passage 523) differed significantly from each other and from consensus sequence. When virus population equilibria were disrupted by high-multiplicity passage (in which defective interfering particle interference is maximized) or passage in the presence of mutagenic levels of 5-fluorouracil, rapid consensus sequence evolution occurred and extreme population heterogeneity was observed (with some members of these population differing from others at hundreds of genome positions). A limited sampling of clones at one stage during high-multiplicity passages suggested the presence of at least several distinct master sequences, the related subpopulations of which exhibit at least transient competitive fitness within the total virus population (M. Eigen and C.K. Biebricher, p. 211-245, in E. Domingo, J.J. Holland, P. Ahlquist, ed., RNA Genetics, vol. 3, 1988). These studies further demonstrate the important role of selective pressure in determining the genetic composition of RNA virus populations. This is true under equilibrium conditions in which little consensus sequence evolution is observed owing to stabilizing selection as well as under conditions in which selective pressure is driving rapid RNA virus genome evolution. Images PMID:2539503

  10. Preferential targeting of vesicular stomatitis virus to breast cancer cells

    SciTech Connect

    Bergman, Ira . E-mail: ira.bergman@chp.edu; Whitaker-Dowling, Patricia; Gao Yanhua; Griffin, Judith A.

    2004-12-05

    Vesicular stomatitis virus (VSV) is a candidate for development for cancer therapy. We created a recombinant replicating VSV (rrVSV) with an altered surface protein that targeted preferentially to breast cancer cells. The rrVSV genome contained a single glycoprotein (gp) gene derived from Sindbis virus. This gene expressed a chimeric Sindbis E2 binding gp and the native Sindbis E1 fusion gp. The chimeric E2 binding gp, called Sindbis-SCA-erbb2, was modified to reduce its native binding function and to contain a single chain antibody (SCA) with specificity for the human epidermal growth factor receptor Her2/neu protein, erbb2. These viruses selectively infected, replicated in and killed cells expressing erbb2. The titer of rrVSV on SKBR3 cells, a human breast cancer cell line which highly expresses erbb2 was 3.1 x 10{sup 7}/ml compared with a titer of 7.3 x 10{sup 5}/ml on 143 cells, a human osteosarcoma cell line which does not express erbb2. The titer of rrVSV on D2F2/E2 cells, a mouse mammary cancer cell line stably transfected to express human erbb2 was 2.46 x 10{sup 6}/ml compared with a titer of 5 x 10{sup 4}/ml on the parent D2F2 cells which do not express erbb2. When titered on erbb2-negative cells, non-replicating pseudotype VSV coated with Sindbis-SCA-erbb2 had <3% the titer of pseudotype VSV coated with wild type Sindbis gp indicating that the chimeric Sindbis gp had severely impaired binding to the natural receptor. Analysis of the protein composition of the rrVSV found low expression of the modified Sindbis gp on the virus.

  11. Guinea Pig Horizontal Cells Express GABA, the GABA-Synthesizing Enzyme GAD65, and the GABA Vesicular Transporter

    PubMed Central

    Guo, Chenying; Hirano, Arlene A.; Stella, Salvatore L.; Bitzer, Michaela; Brecha, Nicholas C.

    2013-01-01

    γ-Aminobutyric acid (GABA) is likely expressed in horizontal cells of all species, although conflicting physiological findings have led to considerable controversy regarding its role as a transmitter in the outer retina. This study has evaluated key components of the GABA system in the outer retina of guinea pig, an emerging retinal model system. The presence of GABA, its rate-limiting synthetic enzyme glutamic acid decarboxylase (GAD65 and GAD67 isoforms), the plasma membrane GABA transporters (GAT-1 and GAT-3), and the vesicular GABA transporter (VGAT) was evaluated by using immunohistochemistry with well-characterized antibodies. The presence of GAD65 mRNA was also evaluated by using laser capture microdissection and reverse transcriptase-polymerase chain reaction. Specific GABA, GAD65, and VGAT immunostaining was localized to horizontal cell bodies, as well as to their processes and tips in the outer plexiform layer. Furthermore, immunostaining of retinal whole mounts and acutely dissociated retinas showed GAD65 and VGAT immunoreactivity in both A-type and B-type horizontal cells. However, these cells did not contain GAD67, GAT-1, or GAT-3 immunoreactivity. GAD65 mRNA was detected in horizontal cells, and sequencing of the amplified GAD65 fragment showed approximately 85% identity with other mammalian GAD65 mRNAs. These studies demonstrate the presence of GABA, GAD65, and VGAT in horizontal cells of the guinea pig retina, and support the idea that GABA is synthesized from GAD65, taken up into synaptic vesicles by VGAT, and likely released by a vesicular mechanism from horizontal cells. PMID:20235161

  12. Vesicular stomatitis virus glycoprotein mutations that affect membrane fusion activity and abolish virus infectivity.

    PubMed Central

    Fredericksen, B L; Whitt, M A

    1995-01-01

    We have introduced amino acid substitutions into two regions of the extracellular domain of the vesicular stomatitis virus (VSV) glycoprotein (G protein) and examined the effect of these mutations on protein transport, low-pH-induced stability of G protein oligomers, and membrane fusion activity. We suggested previously that the region between amino acids 118 and 139 may be important for the membrane fusion activity of G protein, on the basis of the characterization of a fusion-defective G protein mutant (M. A. Whitt, P. Zagouras, B. Crise, and J. K. Rose, J. Virol. 64:4907-4913, 1990). It has also been postulated by others that this region as well as the region between amino acids 181 and 212 may constitute putative internal fusion domains of VSV G protein. In this report, we show that three different amino acids substitutions between residues 118 and 139 (G-124-->E, P-127-->D, and A-133-->K) either altered or abolished low-pH-dependent membrane fusion activity. In contrast, substitutions between residues 192 and 212 resulted either in G proteins that had wild-type fusion activity or in mutant proteins in which the mutation prevented transport of G protein to the cell surface. Two of the substitutions between residues 118 and 139 (G-124-->E and P-127-->D) resulted in G proteins that were fusion defective at pH 5.7, although syncytia were observed after cells were treated with fusion buffer at pH 5.5, albeit at levels significantly less than that induced by wild-type G protein. Interestingly, when either G-124-->E or P-127-->D was incorporated into tsO45 virions, the resulting particles were not infectious, presumably because the viral envelope was not able to fuse with the proper intracellular membrane. These results support the hypothesis that the region between amino acids 118 and 139 is important for the membrane fusion activity of VSV G protein and may constitute an internal fusion domain. PMID:7853475

  13. Ursodeoxycholic and deoxycholic acids: Differential effects on intestinal Ca(2+) uptake, apoptosis and autophagy of rat intestine.

    PubMed

    Rodríguez, Valeria A; Rivoira, María A; Pérez, Adriana del V; Marchionatti, Ana M; Tolosa de Talamoni, Nori G

    2016-02-01

    The aim of this work was to study the effect of sodium deoxycholate (NaDOC) and ursodeoxycholic acid (UDCA) on Ca(2+) uptake by enterocytes and the underlying mechanisms. Rats were divided into four groups: a) controls, b) treated with NaDOC, c) treated with UDCA d) treated with NaDOC and UDCA. Ca(2+) uptake was studied in enterocytes with different degrees of maturation. Apoptosis, autophagy and NO content and iNOS protein expression were evaluated. NaDOC decreased and UDCA increased Ca(2+) uptake only in mature enterocytes. The enhancement of protein expression of Fas, FasL, caspase-8 and caspase-3 activity by NaDOC indicates triggering of the apoptotic extrinsic pathway, which was blocked by UDCA. NO content and iNOS protein expression were enhanced by NaDOC, and avoided by UDCA. The increment of acidic vesicular organelles and LC3 II produced by NaDOC was also prevented by UDCA. In conclusion, the inhibitory effects of NaDOC on intestinal Ca(2+) absorption occur by decreasing the Ca(2+) uptake by mature enterocytes. NaDOC triggers apoptosis and autophagy, in part as a result of nitrosative stress. In contrast, UDCA increases the Ca(2+) uptake by mature enterocytes, and in combination with NaDOC acts as an antiapoptotic and antiautophagic agent normalizing the transcellular Ca(2+) pathway. PMID:26707246

  14. Foot & Mouth Disease & Ulcerative/Vesicular Rule-outs: Challenges Encountered in Recent Outbreaks

    SciTech Connect

    Hullinger, P

    2008-01-28

    . It has been shown that the African Cape buffalo are the major maintenance host of SAT serotypes. FMDV transmission can occur by either direct or indirect contact. Indirect transmission can occur via contaminated animate vectors (humans, etc.), inanimate vectors (vehicles, implements) or airborne transmission. Indirect disease transmission via animate or inanimate vectors can play a major role in disease transmission. Good biosecurity can significantly reduce this type of transmission. Airborne transmission is often debated and is known to be serotype and species specific as well as require specific environmental conditions to occur. Airborne transmission is favored in temperate zones and has been postulated to occur over distances of up to 60 km overland and 300 km by sea. Foot and mouth disease virus is an unenveloped virus which is preserved by refrigeration and freezing and progressively inactivated by temperatures above 50 C. FMDV is highly sensitive to pH change and is inactivated by pH < 6.0 or > 9.0. There are many disinfectants which are effective against FMDV including sodium hydroxide (2%), sodium carbonate (4%), and citric acid (0.2%). FMDV is resistant to iodophores, quaternary ammonium compounds, hypochlorite and phenol, especially in the presence of organic matter. The virus can survive in lymph nodes and bone marrow at neutral pH, but is destroyed in muscle when is pH < 6.0 i.e. after rigor mortis. FMDV can persist in contaminated feed/commodities and the environment for over to 1 month, depending on the temperature and pH conditions. The incubation period for FMD is 2-14 days. Animals transition through latent (infected but not infectious), subclinically infected (infectious but lacking clinical signs) clinically infected and recovered disease states. In cattle clinical signs include pyrexia, reluctance to eat, bruxism, drooling, lameness, treading or stamping of the feet and decreased milk production. Most clinical signs are related to the

  15. Disulfide-bonded discontinuous epitopes on the glycoprotein of vesicular stomatitis virus (New Jersey serotype).

    PubMed

    Grigera, P R; Keil, W; Wagner, R R

    1992-06-01

    Intrachain disulfide bonds between paired cysteines in the glycoprotein (G) of vesicular stomatitis virus (VSV) are required for the recognition of discontinuous epitopes by specific monoclonal antibodies (MAbs) (W. Keil and R. R. Wagner, Virology 170:392-407, 1989). Cleavage by Staphylococcus aureus V8 protease of the 517-amino-acid VSV-New Jersey G protein, limited to the glutamic acid at residue 110, resulted in a protein (designated GV8) with greatly retarded migration by polyacrylamide gel electrophoresis (PAGE) under nonreducing conditions. By Western blot (immunoblot) analysis, protein GV8 was found to lose discontinuous epitope IV, which maps within the first 193 NH2-terminal amino acids. These data, coupled with those obtained by PAGE migration of a vector-expressed, truncated protein (G1-193) under reducing and nonreducing conditions, lead us to postulate the existence of a major loop structure within the first 193 NH2-terminal amino acids of the G protein, possibly anchored by a disulfide bond between cysteine 108 and cysteine 169, encompassing epitope IV. Site-directed mutants in which 10 of the 12 cysteines were individually converted to serines in vaccinia virus-based vectors expressing these single-site mutant G proteins were also constructed, each of which was then tested by immunoprecipitation for its capacity to recognize epitope-specific MAbs. These results showed that mutations in NH2-terminal cysteines 130, 174, and, to a lesser extent, 193 all resulted in the loss of neutralization epitope VIII. A mutation at NH2-terminal cysteine 130 also resulted in the loss of neutralization epitope VII, as did a mutation at cysteine 108 to a lesser extent. Both epitopes VII and VIII disappeared when mutations were made in COOH-distal cysteine 235, 240, or 273, the general map locations of epitopes VII and VIII. These studies also reveal that distal, as well as proximal, cysteine residues markedly influence the disulfide-bond secondary structure, which

  16. Mitochondria and hydrogenosomes are two forms of the same fundamental organelle.

    PubMed Central

    Embley, T Martin; van der Giezen, Mark; Horner, David S; Dyal, Patricia L; Foster, Peter

    2003-01-01

    Published data suggest that hydrogenosomes, organelles found in diverse anaerobic eukaryotes that make energy and hydrogen, were once mitochondria. As hydrogenosomes generally lack a genome, the conversion is probably one way. The sources of the key hydrogenosomal enzymes, pyruvate : ferredoxin oxidoreductase (PFO) and hydrogenase, are not resolved by current phylogenetic analyses, but it is likely that both were present at an early stage of eukaryotic evolution. Once thought to be restricted to a few unusual anaerobic eukaryotes, the proteins are intimately integrated into the fabric of diverse eukaryotic cells, where they are targeted to different cell compartments, and not just hydrogenosomes. There is no evidence supporting the view that PFO and hydrogenase originated from the mitochondrial endosymbiont, as posited by the hydrogen hypothesis for eukaryogenesis. Other organelles derived from mitochondria have now been described in anaerobic and parasitic microbial eukaryotes, including species that were once thought to have diverged before the mitochondrial symbiosis. It thus seems possible that all eukaryotes may eventually be shown to contain an organelle of mitochondrial ancestry, to which different types of biochemistry can be targeted. It remains to be seen if, despite their obvious differences, this family of organelles shares a common function of importance for the eukaryotic cell, other than energy production, that might provide the underlying selection pressure for organelle retention. PMID:12594927

  17. Synthesis of cellular organelles containing nano-magnets stunts growth of magnetotactic bacteria.

    PubMed

    Naresh, Mohit; Hasija, Vivek; Sharma, Megha; Mittal, Aditya

    2010-07-01

    Magnetotactic bacteria are unique prokaryotes possessing the feature of cellular organelles called magnetosomes (membrane bound 40-50 nm vesicles entrapping a magnetic nano-crystal of magnetite or greigite). The obvious energetic impact of sophisticated eukaryotic-like membrane-bound organelle assembly on a presumably simpler prokaryotic system is not addressed in literature. In this work, while presenting evidence of direct coupling of carbon source consumption to synthesis of magnetosomes, we provide the first experimentally derived estimate of energy for organelle synthesis by Magnetospirillum gryphiswaldense as approximately 5 nJoules per magnetosome. Considering our estimate of approximately 0.2 microJoules per bacterial cell as the energy required for growth, we show that the energetic load of organelle synthesis results in stunting of cell growth. We also show that removal of soluble iron or sequestration by exogenous compounds in the bacterial cell cultures reverses the impact of the excess metabolic load exerted during magnetosomal synthesis. Thus, by taking advantage of the magnetotactic bacterial system we present the first experimental evidence for the presumed energy consumption during assembly of naturally occurring sub-100 nm intra-cellular organelles. PMID:21128392

  18. Lipid droplet organelle distribution in populations of dividing cells studied by simulation

    NASA Astrophysics Data System (ADS)

    Dalhaimer, Paul

    2013-06-01

    One of the key questions in cell biology is how organelles are passed from parent to daughter cells. To help address this question, I used Brownian dynamics to simulate lipid droplets as model organelles in populations of dividing cells. Lipid droplets are dynamic bodies that can form both de novo and by fission, they can also be depleted. The quantitative interplay among these three events is unknown but would seem crucial for controlling droplet distribution in populations of dividing cells. Surprisingly, of the three main events studied: biogenesis, fission, and depletion, the third played the key role in maintaining droplet organelle number—and to a lesser extent volume—in populations of dividing cells where formation events would have seemed paramount. In the case of lipid droplets, this provides computational evidence that they must be sustained, most likely through contacts with the endoplasmic reticulum. The findings also agree with video microscopy experiments over much shorter timescales where droplet depletion in fission yeast cells was not observed. In general, this work shows that organelle maintenance is invaluable and lack thereof cannot necessarily be compensated for by organelle formation. This study provides a time-accurate, physical-based template for long-term cell division studies.

  19. Robust organelle size extractions from elastic scattering measurements of single cells (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Cannaday, Ashley E.; Draham, Robert; Berger, Andrew J.

    2016-04-01

    The goal of this project is to estimate non-nuclear organelle size distributions in single cells by measuring angular scattering patterns and fitting them with Mie theory. Simulations have indicated that the large relative size distribution of organelles (mean:width≈2) leads to unstable Mie fits unless scattering is collected at polar angles less than 20 degrees. Our optical system has therefore been modified to collect angles down to 10 degrees. Initial validations will be performed on polystyrene bead populations whose size distributions resemble those of cell organelles. Unlike with the narrow bead distributions that are often used for calibration, we expect to see an order-of-magnitude improvement in the stability of the size estimates as the minimum angle decreases from 20 to 10 degrees. Scattering patterns will then be acquired and analyzed from single cells (EMT6 mouse cancer cells), both fixed and live, at multiple time points. Fixed cells, with no changes in organelle sizes over time, will be measured to determine the fluctuation level in estimated size distribution due to measurement imperfections alone. Subsequent measurements on live cells will determine whether there is a higher level of fluctuation that could be attributed to dynamic changes in organelle size. Studies on unperturbed cells are precursors to ones in which the effects of exogenous agents are monitored over time.

  20. New Organelles by Gene Duplication in a Biophysical Model of Eukaryote Endomembrane Evolution

    PubMed Central

    Ramadas, Rohini; Thattai, Mukund

    2013-01-01

    Extant eukaryotic cells have a dynamic traffic network that consists of diverse membrane-bound organelles exchanging matter via vesicles. This endomembrane system arose and diversified during a period characterized by massive expansions of gene families involved in trafficking after the acquisition of a mitochondrial endosymbiont by a prokaryotic host cell >1.8 billion years ago. Here we investigate the mechanistic link between gene duplication and the emergence of new nonendosymbiotic organelles, using a minimal biophysical model of traffic. Our model incorporates membrane-bound compartments, coat proteins and adaptors that drive vesicles to bud and segregate cargo from source compartments, and SNARE proteins and associated factors that cause vesicles to fuse into specific destination compartments. In simulations, arbitrary numbers of compartments with heterogeneous initial compositions segregate into a few compositionally distinct subsets that we term organelles. The global structure of the traffic system (i.e., the number, composition, and connectivity of organelles) is determined completely by local molecular interactions. On evolutionary timescales, duplication of the budding and fusion machinery followed by loss of cross-interactions leads to the emergence of new organelles, with increased molecular specificity being necessary to maintain larger organellar repertoires. These results clarify potential modes of early eukaryotic evolution as well as more recent eukaryotic diversification. PMID:23746528

  1. Systematic study of subcellular localization of Arabidopsis PPR proteins confirms a massive targeting to organelles

    PubMed Central

    Colcombet, Jean; Lopez-Obando, Mauricio; Heurtevin, Laure; Bernard, Clément; Martin, Karine; Berthomé, Richard; Lurin, Claire

    2013-01-01

    Four hundred and fifty-eight genes coding for PentatricoPeptide Repeat (PPR) proteins are annotated in the Arabidopsis thaliana genome. Over the past 10 years, numerous reports have shown that many of these proteins function in organelles to target specific transcripts and are involved in post-transcriptional regulation. Therefore, they are thought to be important players in the coordination between nuclear and organelle genome expression. Only four of these proteins have been described to be addressed outside organelles, indicating that some PPRs could function in post-transcriptional regulations of nuclear genes. In this work, we updated and improved our current knowledge on the localization of PPR proteins of Arabidopsis within the plant cell. We particularly investigated the subcellular localization of 166 PPR proteins whose targeting predictions were ambiguous, using a combination of high-throughput cloning and microscopy. Through systematic localization experiments and data integration, we confirmed that PPR proteins are largely targeted to organelles and showed that dual targeting to both the mitochondria and plastid occurs more frequently than expected. These results allow us to speculate that dual-targeted PPR proteins could be important for the fine coordination of gene expressions in both organelles. PMID:24037373

  2. Impairment of vesicular ATP release affects glucose metabolism and increases insulin sensitivity

    PubMed Central

    Sakamoto, Shohei; Miyaji, Takaaki; Hiasa, Miki; Ichikawa, Reiko; Uematsu, Akira; Iwatsuki, Ken; Shibata, Atsushi; Uneyama, Hisayuki; Takayanagi, Ryoichi; Yamamoto, Akitsugu; Omote, Hiroshi; Nomura, Masatoshi; Moriyama, Yoshinori

    2014-01-01

    Neuroendocrine cells store ATP in secretory granules and release it along with hormones that may trigger a variety of cellular responses in a process called purinergic chemical transmission. Although the vesicular nucleotide transporter (VNUT) has been shown to be involved in vesicular storage and release of ATP, its physiological relevance in vivo is far less well understood. In Vnut knockout (Vnut−/−) mice, we found that the loss of functional VNUT in adrenal chromaffin granules and insulin granules in the islets of Langerhans led to several significant effects. Vesicular ATP accumulation and depolarization-dependent ATP release were absent in the chromaffin granules of Vnut−/− mice. Glucose-responsive ATP release was also absent in pancreatic β-cells in Vnut−/− mice, while glucose-responsive insulin secretion was enhanced to a greater extent than that in wild-type tissue. Vnut−/− mice exhibited improved glucose tolerance and low blood glucose upon fasting due to increased insulin sensitivity. These results demonstrated an essential role of VNUT in vesicular storage and release of ATP in neuroendocrine cells in vivo and suggest that vesicular ATP and/or its degradation products act as feedback regulators in catecholamine and insulin secretion, thereby regulating blood glucose homeostasis. PMID:25331291

  3. Mapping the distribution of vesicular textures on silicic lavas using the Thermal Infrared Multispectral Scanner

    NASA Technical Reports Server (NTRS)

    Ondrusek, Jaime; Christensen, Philip R.; Fink, Jonathan H.

    1993-01-01

    To investigate the effect of vesicularity on TIMS (Thermal Infrared Multispectral Scanner) imagery independent of chemical variations, we studied a large rhyolitic flow of uniform composition but textural heterogeneity. The imagery was recalibrated so that the digital number values for a lake in the scene matched a calculated ideal spectrum for water. TIMS spectra for the lava show useful differences in coarsely and finely vesicular pumice data, particularly in TIMS bands 3 and 4. Images generated by ratioing these bands accurately map out those areas known from field studies to be coarsely vesicular pumice. These texture-related emissivity variations are probably due to the larger vesicles being relatively deeper and separated by smaller septa leaving less smooth glass available to give the characteristic emission of the lava. In studies of inaccessible lava flows (as on Mars) areas of coarsely vesicular pumice must be identified and avoided before chemical variations can be interpreted. Remotely determined distributions of vesicular and glassy textures can also be related to the volatile contents and potential hazards associated with the emplacement of silicic lava flows on Earth.

  4. Impairment of vesicular ATP release affects glucose metabolism and increases insulin sensitivity.

    PubMed

    Sakamoto, Shohei; Miyaji, Takaaki; Hiasa, Miki; Ichikawa, Reiko; Uematsu, Akira; Iwatsuki, Ken; Shibata, Atsushi; Uneyama, Hisayuki; Takayanagi, Ryoichi; Yamamoto, Akitsugu; Omote, Hiroshi; Nomura, Masatoshi; Moriyama, Yoshinori

    2014-01-01

    Neuroendocrine cells store ATP in secretory granules and release it along with hormones that may trigger a variety of cellular responses in a process called purinergic chemical transmission. Although the vesicular nucleotide transporter (VNUT) has been shown to be involved in vesicular storage and release of ATP, its physiological relevance in vivo is far less well understood. In Vnut knockout (Vnut(-/-)) mice, we found that the loss of functional VNUT in adrenal chromaffin granules and insulin granules in the islets of Langerhans led to several significant effects. Vesicular ATP accumulation and depolarization-dependent ATP release were absent in the chromaffin granules of Vnut(-/-) mice. Glucose-responsive ATP release was also absent in pancreatic β-cells in Vnut(-/-) mice, while glucose-responsive insulin secretion was enhanced to a greater extent than that in wild-type tissue. Vnut(-/-) mice exhibited improved glucose tolerance and low blood glucose upon fasting due to increased insulin sensitivity. These results demonstrated an essential role of VNUT in vesicular storage and release of ATP in neuroendocrine cells in vivo and suggest that vesicular ATP and/or its degradation products act as feedback regulators in catecholamine and insulin secretion, thereby regulating blood glucose homeostasis. PMID:25331291

  5. Self-assembled cylindrical and vesicular molecular templates for polyaniline nanofibers and nanotapes.

    PubMed

    Anilkumar, P; Jayakannan, M

    2009-08-27

    We report a soft template approach based on a custom-designed novel surfactant-cum-dopant for size and shape tuning of polyaniline nanomaterials such as nanofibers and nanotapes via emulsion and dispersion polymerization routes. A new amphiphilic 4-(3-dodecyl-8-enylphenyloxy) butane sulfonic acid was synthesized by ring-opening of butanesultone with renewable resource cardanol. The new amphiphilic dopant forms spherical micelles in water and its critical micelle concentration was determined by dye encapsulation and surface tension methods. In the emulsion route, the amphiphilic dopant complexed with aniline to produce cylindrical micellar aggregates that template exclusively for polyaniline nanofibers. The dispersion of aniline+dopant in water/toluene solvent mixture produces vesicles that selectively template for polyaniline nanotapes. The mechanism of the polyaniline nanomaterials formation was investigated by dynamic light scattering (DLS) and high-resolution transmission electron microscopy (HR-TEM). DLS of the polymerization templates in water proved the presence of micrometer range aggregates, and TEM images confirmed the shape of the cylindrical and vesicular templates. The polyaniline nanomaterials were found soluble in water and polar organic solvents for structural characterization and composition analysis by 1H NMR spectroscopy. Absorbance spectra of the nanomaterials showed free carrier tail above 900 nm in the near IR region for the delocalization of electrons in the polaron band corresponding to expanded conformation of polyaniline chains. Wide angle X-ray diffraction showed two new peaks at low angle region with d-spacing of 26.5 and 13.6 A corresponding to lamellar ordering of polyaniline chains followed by interdigitations of the amphiphilic dopant in the nanomaterials. PMID:19642663

  6. Membrane contact sites between pathogen-containing compartments and host organelles.

    PubMed

    Dumoux, Maud; Hayward, Richard D

    2016-08-01

    Intracellular pathogens survive and replicate within specialised membrane-bound compartments that can be considered as pseudo-organelles. Using the obligate intracellular bacterium Chlamydia as an illustrative example, we consider the modes of lipid transport between pathogen-containing compartments and host organelles, including the formation of static membrane contact sites. We discuss how lipid scavenging can be mediated via the reprogramming of cellular transporters at these interfaces and describe recent data suggesting that pathogen effectors modulate the formation of specific membrane contacts. Further study of these emerging mechanisms is likely to yield new insights into the cell biology of lipid transport and organelle communication, which highlights potential new targets and strategies for future therapeutics. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon. PMID:26825687

  7. Artificially-induced organelles are optimal targets for optical trapping experiments in living cells.

    PubMed

    López-Quesada, C; Fontaine, A-S; Farré, A; Joseph, M; Selva, J; Egea, G; Ludevid, M D; Martín-Badosa, E; Montes-Usategui, M

    2014-07-01

    Optical trapping supplies information on the structural, kinetic or rheological properties of inner constituents of the cell. However, the application of significant forces to intracellular objects is notoriously difficult due to a combination of factors, such as the small difference between the refractive indices of the target structures and the cytoplasm. Here we discuss the possibility of artificially inducing the formation of spherical organelles in the endoplasmic reticulum, which would contain densely packed engineered proteins, to be used as optimized targets for optical trapping experiments. The high index of refraction and large size of our organelles provide a firm grip for optical trapping and thereby allow us to exert large forces easily within safe irradiation limits. This has clear advantages over alternative probes, such as subcellular organelles or internalized synthetic beads. PMID:25071944

  8. Artificially-induced organelles are optimal targets for optical trapping experiments in living cells

    PubMed Central

    López-Quesada, C.; Fontaine, A.-S.; Farré, A.; Joseph, M.; Selva, J.; Egea, G.; Ludevid, M. D.; Martín-Badosa, E.; Montes-Usategui, M.

    2014-01-01

    Optical trapping supplies information on the structural, kinetic or rheological properties of inner constituents of the cell. However, the application of significant forces to intracellular objects is notoriously difficult due to a combination of factors, such as the small difference between the refractive indices of the target structures and the cytoplasm. Here we discuss the possibility of artificially inducing the formation of spherical organelles in the endoplasmic reticulum, which would contain densely packed engineered proteins, to be used as optimized targets for optical trapping experiments. The high index of refraction and large size of our organelles provide a firm grip for optical trapping and thereby allow us to exert large forces easily within safe irradiation limits. This has clear advantages over alternative probes, such as subcellular organelles or internalized synthetic beads. PMID:25071944

  9. Phosphorylation-mediated RNA/peptide complex coacervation as a model for intracellular liquid organelles

    NASA Astrophysics Data System (ADS)

    Aumiller, William M.; Keating, Christine D.

    2016-02-01

    Biological cells are highly organized, with numerous subcellular compartments. Phosphorylation has been hypothesized as a means to control the assembly/disassembly of liquid-like RNA- and protein-rich intracellular bodies, or liquid organelles, that lack delimiting membranes. Here, we demonstrate that charge-mediated phase separation, or complex coacervation, of RNAs with cationic peptides can generate simple model liquid organelles capable of reversibly compartmentalizing biomolecules. Formation and dissolution of these liquid bodies was controlled by changes in peptide phosphorylation state using a kinase/phosphatase enzyme pair. The droplet-generating phase transition responded to modification of even a single serine residue. Electrostatic interactions between the short cationic peptides and the much longer polyanionic RNAs drove phase separation. Coacervates were also formed on silica beads, a primitive model for localization at specific intracellular sites. This work supports phosphoregulation of complex coacervation as a viable mechanism for dynamic intracellular compartmentalization in membraneless organelles.

  10. Lipidomics Analyses Reveal Temporal and Spatial Lipid Organization and Uncover Daily Oscillations in Intracellular Organelles.

    PubMed

    Aviram, Rona; Manella, Gal; Kopelman, Naama; Neufeld-Cohen, Adi; Zwighaft, Ziv; Elimelech, Meytar; Adamovich, Yaarit; Golik, Marina; Wang, Chunyan; Han, Xianlin; Asher, Gad

    2016-05-19

    Cells have evolved mechanisms to handle incompatible processes through temporal organization by circadian clocks and by spatial compartmentalization within organelles defined by lipid bilayers. Recent advances in lipidomics have led to identification of plentiful lipid species, yet our knowledge regarding their spatiotemporal organization is lagging behind. In this study, we quantitatively characterized the nuclear and mitochondrial lipidome in mouse liver throughout the day, upon different feeding regimens, and in clock-disrupted mice. Our analyses revealed potential connections between lipid species within and between lipid classes. Remarkably, we uncovered diurnal oscillations in lipid accumulation in the nucleus and mitochondria. These oscillations exhibited opposite phases and readily responded to feeding time. Furthermore, we found that the circadian clock coordinates the phase relation between the organelles. In summary, our study provides temporal and spatial depiction of lipid organization and reveals the presence and coordination of diurnal rhythmicity in intracellular organelles. PMID:27161994

  11. Fat(al) attraction: Picornaviruses Usurp Lipid Transfer at Membrane Contact Sites to Create Replication Organelles.

    PubMed

    van der Schaar, Hilde M; Dorobantu, Cristina M; Albulescu, Lucian; Strating, Jeroen R P M; van Kuppeveld, Frank J M

    2016-07-01

    All viruses that carry a positive-sense RNA genome (+RNA), such as picornaviruses, hepatitis C virus, dengue virus, and SARS- and MERS-coronavirus, confiscate intracellular membranes of the host cell to generate new compartments (i.e., replication organelles) for amplification of their genome. Replication organelles (ROs) are membranous structures that not only harbor viral proteins but also contain a specific array of hijacked host factors that create a unique lipid microenvironment optimal for genome replication. While some lipids may be locally synthesized de novo, other lipids are shuttled towards ROs. In picornavirus-infected cells, lipids are exchanged at membrane contact sites between ROs and other organelles. In this paper, we review recent advances in our understanding of how picornaviruses exploit host membrane contact site machinery to generate ROs, a mechanism that is used by some other +RNA viruses as well. PMID:27020598

  12. Cryptic organelle homology in Apicomplexan parasites: Insights from evolutionary cell biology

    PubMed Central

    Klinger, Christen M.; Nisbet, R. Ellen; Ouologuem, Dinkorma T.; Roos, David S.; Dacks, Joel B.

    2013-01-01

    The economic and clinical significance of apicomplexan parasites drives interest in their many evolutionary novelties. Distinctive intracellular organelles play key roles in parasite motility, invasion, metabolism, and replication, and understanding their relationship with the organelles of better-studied eukaryotic systems suggests potential targets for therapeutic intervention. Recent work has demonstrated divergent aspects of canonical eukaryotic components in the apicomplexa, including Golgi bodies and mitochondria. The apicoplast is a relict plastid of secondary endosymbiotic origin, harboring metabolic pathways distinct from those of host species. The inner membrane complex is derived from the cortical alveoli defining the superphylum Alveolata, but in apicomplexans functions in parasite motility and replication. Micronemes and rhoptries are associated with establishment of the intracellular niche, and define the apical complex for which the phylum is named. Morphological, cell biological and molecular evidence strongly suggest that these organelles are derived from the endocytic pathway. PMID:23932202

  13. Protein kinase Darkener of apricot and its substrate EF1γ regulate organelle transport along microtubules.

    PubMed

    Serpinskaya, Anna S; Tuphile, Karine; Rabinow, Leonard; Gelfand, Vladimir I

    2014-01-01

    Regulation of organelle transport along microtubules is important for proper distribution of membrane organelles and protein complexes in the cytoplasm. RNAi-mediated knockdown in cultured Drosophila S2 cells demonstrates that two microtubule-binding proteins, a unique isoform of Darkener of apricot (DOA) protein kinase, and its substrate, translational elongation factor EF1γ, negatively regulate transport of several classes of membrane organelles along microtubules. Inhibition of transport by EF1γ requires its phosphorylation by DOA on serine 294. Together, our results indicate a new role for two proteins that have not previously been implicated in regulation of the cytoskeleton. These results further suggest that the biological role of some of the proteins binding to the microtubule track is to regulate cargo transport along these tracks. PMID:24163433

  14. A nanobuffer reporter library for fine-scale imaging and perturbation of endocytic organelles | Office of Cancer Genomics

    Cancer.gov

    Endosomes, lysosomes and related catabolic organelles are a dynamic continuum of vacuolar structures that impact a number of cell physiological processes such as protein/lipid metabolism, nutrient sensing and cell survival. Here we develop a library of ultra-pH-sensitive fluorescent nanoparticles with chemical properties that allow fine-scale, multiplexed, spatio-temporal perturbation and quantification of catabolic organelle maturation at single organelle resolution to support quantitative investigation of these processes in living cells.

  15. Cryogenic transmission electron microscopy study: preparation of vesicular dispersions by quenching microemulsions.

    PubMed

    Lee, H S; Morrison, E D; Zhang, Q; McCormick, A V

    2016-09-01

    We previously showed that long-lived nanoemulsions, seeming initially vesicular, might be prepared simply by diluting and cooling (quenching) warm microemulsions with n-hexadecane with precooled water. In this paper, we confirm that these systems are vesicular dispersions when fresh, and they can be made with similar structures and compositional dependence using alkanes with chain lengths ranging from octane to hexadecane. The nanostructures of fresh nanoemulsions are imaged with cryogenic transmission electron microscopy (cryo-TEM). We confirm that water-continuous microemulsions give simple dispersions of vesicles (sometimes unilamellar), typically less than 100 nm in diameter; these systems can avoid separation for over 2 months. Selected samples were also prepared using halogenated alkanes to create additional contrast in the cryo-TEM, allowing us to confirm that the oil is located in the observed vesicular structures. PMID:26937849

  16. Transcriptional changes of mouse splenocyte organelle components following acute infection with Toxoplasma gondii.

    PubMed

    He, Jun-Jun; Ma, Jun; Li, Fa-Cai; Song, Hui-Qun; Xu, Min-Jun; Zhu, Xing-Quan

    2016-08-01

    Toxoplasmosis is a globally spread zoonosis. The pathogen Toxoplasma gondii can hijack cellular organelles of host for replication. Although a number of important cellular life events are controlled by cell organelles, very little is known of the transcriptional changes of host cellular organelles after infection with T. gondii. Herein, we performed RNA-sequencing (RNA-seq) and bioinformatics analyses to study the global organelle component changes. It was found that many transcripts of the mouse spleen cellular organelle components were altered by acute T. gondii infection with the RH strain (Type I). Most differentially expressed transcripts of mitochondrial components were downregulated, especially those involved in biosynthetic and metabolic processes. Moreover, mitochondria based apoptosis process was downregulated. In terms of cytoskeleton, most differentially expressed transcript of cytoskeleton components were also downregulated, including septin cytoskeleton, cytoskeleton organization, centrosome and myosin. For endolysosomal system, ion transporters were downregulated at mRNA level, whereas the cytolytic components were increased, such as granzymes, Rab27a and perforin1 (Prf1). The main transcripts of Golgi apparatus components involved in sialylation or vesicle-mediated transportation were downregulated, while immune related components were upregulated. For endoplasmic reticulum (ER), posttranslational modification, drug metabolism and material transportation related transcripts were downregulated. In addition, T. gondii antigen cross-presentation by MHC-I complex could be downregulated by the downregulation of CD76 and ubiquitination related transcripts. The present study, for the first time, described the transcriptional changes of the mouse spleen cellular organelles following acute T. gondii infection, which provides a foundation to study the interaction between T. gondii and host cells at the sub-cellular level. PMID:27132051

  17. Real-Time Reverse Transcription PCR Assay for Detection of Senecavirus A in Swine Vesicular Diagnostic Specimens

    PubMed Central

    Fabian, Andrew W.; Barrette, Roger W.; Sayed, Abu

    2016-01-01

    Senecavirus A (SV-A), formerly, Seneca Valley virus (SVV), has been detected in swine with vesicular lesions and is thought to be associated with swine idiopathic vesicular disease (SIVD), a vesicular disease syndrome that lacks a defined causative agent. The clinical presentation of SIVD resembles that of other more contagious and economically devastating vesicular diseases, such as foot-and-mouth disease (FMD), swine vesicular disease (SVD), and vesicular stomatitis (VS), that typically require immediate rule out diagnostics to lift restrictions on animal quarantine, movement, and trade. This study presents the development of a sensitive, SYBR Green RT-qPCR assay suitable for detection of SV-A in diagnostic swine specimens. After testing 50 pigs with clinical signs consistent with vesicular disease, 44 (88%) were found to be positive for SV-A by RT-qPCR as compared to none from a negative cohort of 35 animals without vesicular disease, indicating that the assay is able to successfully detect the virus in an endemic population. SV-A RNA was also detectable at a low level in sera from a subset of pigs that presented with (18%) or without (6%) vesicular signs. In 2015, there has been an increase in the occurrence of SV-A in the US, and over 200 specimens submitted to our laboratory for vesicular investigation have tested positive for the virus using this method. SV-A RNA was detectable in all common types of vesicular specimens including swabs and tissue from hoof lesions, oral and snout epithelium, oral swabs, scabs, and internal organ tissues such as liver and lymph node. Genome sequencing analysis from recent virus isolates was performed to confirm target amplicon specificity and was aligned to previous isolates. PMID:26757142

  18. Real-Time Reverse Transcription PCR Assay for Detection of Senecavirus A in Swine Vesicular Diagnostic Specimens.

    PubMed

    Bracht, Alexa J; O'Hearn, Emily S; Fabian, Andrew W; Barrette, Roger W; Sayed, Abu

    2016-01-01

    Senecavirus A (SV-A), formerly, Seneca Valley virus (SVV), has been detected in swine with vesicular lesions and is thought to be associated with swine idiopathic vesicular disease (SIVD), a vesicular disease syndrome that lacks a defined causative agent. The clinical presentation of SIVD resembles that of other more contagious and economically devastating vesicular diseases, such as foot-and-mouth disease (FMD), swine vesicular disease (SVD), and vesicular stomatitis (VS), that typically require immediate rule out diagnostics to lift restrictions on animal quarantine, movement, and trade. This study presents the development of a sensitive, SYBR Green RT-qPCR assay suitable for detection of SV-A in diagnostic swine specimens. After testing 50 pigs with clinical signs consistent with vesicular disease, 44 (88%) were found to be positive for SV-A by RT-qPCR as compared to none from a negative cohort of 35 animals without vesicular disease, indicating that the assay is able to successfully detect the virus in an endemic population. SV-A RNA was also detectable at a low level in sera from a subset of pigs that presented with (18%) or without (6%) vesicular signs. In 2015, there has been an increase in the occurrence of SV-A in the US, and over 200 specimens submitted to our laboratory for vesicular investigation have tested positive for the virus using this method. SV-A RNA was detectable in all common types of vesicular specimens including swabs and tissue from hoof lesions, oral and snout epithelium, oral swabs, scabs, and internal organ tissues such as liver and lymph node. Genome sequencing analysis from recent virus isolates was performed to confirm target amplicon specificity and was aligned to previous isolates. PMID:26757142

  19. Influence of Vesicular-Arbuscular Mycorrhizal Fungi on the Response of Potato to Phosphorus Deficiency.

    PubMed Central

    McArthur, DAJ.; Knowles, N. R.

    1993-01-01

    Morphological and biochemical interactions between a vesicular-arbuscular mycorrhizal (VAM) fungus (Glomus fasciculatum [Thaxt. sensu Gerdemann] Gerdemann and Trappe) and potato (Solanum tuberosum L.) plants during the development of P deficiency were characterized. Nonmycorrhizal (NM) plants grown for 63 d with low abiotic P supply (0.5 mM) produced 34, 52, and 73% less root, shoot, and tuber dry matter, respectively, than plants grown with high P (2.5 mM). The total leaf area and the leaf area:plant dry weight ratio of low-P plants were substantially lower than those of high-P plants. Moreover, a lower shoot:root dry weight ratio and tuber:plant dry weight ratio in low-P plants than in high-P plants characterized a major effect of P deficiency stress on dry matter partitioning. In addition to a slower rate of growth, low-P plants accumulated nonreducing sugars and nitrate. Furthermore, root respiration and leaf nitrate reductase activity were lower in low-P plants than in high-P plants. Low abiotic P supply also induced physiological changes that contributed to the greater efficiency of P acquisition by low-P plants than by high-P plants. For example, allocation of dry matter and P to root growth was less restricted by P deficiency stress than to shoot and tuber growth. Also, the specific activities of root acid phosphatases and vanadate-sensitive microsomal ATPases were enhanced in P-deficient plants. The establishment of a VAM symbiosis by low-P plants was essential for efficient P acquisition, and a greater root infection level for P-stressed plants indicated increased compatibility to the VAM fungus. By 63 d after planting, low-P VAM plants had recovered 42% more of the available soil P than low-P NM plants. However, the VAM fungus only partially alleviated P deficiency stress and did not completely compensate for inadequate abiotic P supply. Although the specific activities of acid phosphatases and microsomal ATPases were only marginally influenced by VAM

  20. Characterization of the major proteins in gamma particles, cytoplasmic organelles in Blastocladiella emersonii zoospores.

    PubMed

    Hohn, T M; Lovett, J S; Bracker, C E

    1984-04-01

    The gamma particles of Blastocladiella emersonii are 0.5-micron (diameter), electron dense, membrane-enclosed organelles in the cytoplasm of zoospores that have been reported (E.C. Cantino and G.L. Mills, in P. Lemke, ed., Viruses and Plasmids in Fungi, 1979, and R.B. Myers and E.C. Cantino, in A. Wolsky (ed.), Monographs in Developmental Biology, 1974) to store the enzyme chitin synthetase. These particles were isolated from zoospores, and the two major proteins were purified for an analysis of their composition and function. The lower-molecular-weight protein (apparent molecular weight, 41,000) was insoluble in aqueous buffers, had an unusual, very basic amino acid composition, and comprised the characteristic electron-dense inclusions seen in micrographs of sections of fixed and stained gamma particles. After dispersal of the gamma particle membranes with detergent, the higher-molecular-weight protein (apparent molecular weight, 43,000) and a third minor protein (apparent molecular weight, 45,000) sedimented through sucrose cushions with the 41 kilodalton inclusion body protein but were dissociated from it by sonication in buffer containing 7 M urea. Together, the two major proteins represent 60 to 70% of the total protein in the gamma particle and 2.9% of the total protein in zoospores. Tests with specific antisera showed that the two major proteins were not antigenically related, a result consistent with the differences in amino acid composition. When zoospore lysates were centrifuged in sucrose density gradients, the major gamma particle proteins and chitin synthetase activity migrated to regions of different density. Proteins from sporulating thalli and germinating zoospores were separated by gel electrophoresis, and the two major gamma particle proteins were detected by reaction with specific antisera after electrophoretic transfer to nitrocellulose filters. Neither protein could be found in growth phase cells; the appearance and disappearances of both

  1. Brief communication: A 61-year-old woman with vesicular eruption after varicella zoster vaccination

    PubMed Central

    Spriet, Sarah; Banks, Taylor A.

    2016-01-01

    Background: Vesicular rashes are associated with a variety of infectious and noninfectious causes. Objective: To discuss the differential diagnoses of vesicular rashes. Methods: We present the clinical case of an adult woman who was immunocompetent and who developed several clear fluid-filled vesicles on her upper extremity within days of receiving the varicella zoster vaccine. Over the next several days, the skin eruption generalized, and she developed new lesions in various stages of healing. Results: After a detailed history and further studies were obtained, a final diagnosis was made. Conclusion: In patients who have recently been vaccinated, a high index of suspicion for an adverse vaccine reaction should be maintained. PMID:27349562

  2. Pramipexole increases vesicular dopamine uptake: implications for treatment of Parkinson's neurodegeneration.

    PubMed

    Truong, Jannine G; Rau, Kristi S; Hanson, Glen R; Fleckenstein, Annette E

    2003-08-01

    Pramipexole is a dopamine D2/D3 receptor agonist used to treat Parkinson's disease. Both human and animal studies suggest that pramipexole may exhibit neuroprotective properties involving dopamine neurons. However, mechanisms underlying its neuroprotective effects remain uncertain. The present results reveal a novel cellular action of this agent. Specifically, pramipexole rapidly increases vesicular dopamine uptake in synaptic vesicles prepared from striata of treated rats. This effect is: (1) associated with a redistribution of vesicular monoamine transporter-2 (VMAT-2) immunoreactivity within nerve terminals; and, (2) prevented by pretreatment with the dopamine D2 receptor antagonist, eticlopride. The implications of this finding relevant to the treatment of neurodegenerative disorders are discussed. PMID:12921866

  3. 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.

  4. Cytopathogenesis of Vesicular Stomatitis virus is regulated by the PSAP motif of M protein in a species-dependent manner

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Vesicular stomatitis virus (VSV) is an important vector-borne pathogen of bovine and equine species, causing a reportable vesicular disease. The matrix (M) protein of VSV is multifunctional and plays a key role in cytopathogenesis, apoptosis, host protein shut-off, and virion assembly/budding. Our ...

  5. Organelle acidification negatively regulates vacuole membrane fusion in vivo

    PubMed Central

    Desfougères, Yann; Vavassori, Stefano; Rompf, Maria; Gerasimaite, Ruta; Mayer, Andreas

    2016-01-01

    The V-ATPase is a proton pump consisting of a membrane-integral V0 sector and a peripheral V1 sector, which carries the ATPase activity. In vitro studies of yeast vacuole fusion and evidence from worms, flies, zebrafish and mice suggested that V0 interacts with the SNARE machinery for membrane fusion, that it promotes the induction of hemifusion and that this activity requires physical presence of V0 rather than its proton pump activity. A recent in vivo study in yeast has challenged these interpretations, concluding that fusion required solely lumenal acidification but not the V0 sector itself. Here, we identify the reasons for this discrepancy and reconcile it. We find that acute pharmacological or physiological inhibition of V-ATPase pump activity de-acidifies the vacuole lumen in living yeast cells within minutes. Time-lapse microscopy revealed that de-acidification induces vacuole fusion rather than inhibiting it. Cells expressing mutated V0 subunits that maintain vacuolar acidity were blocked in this fusion. Thus, proton pump activity of the V-ATPase negatively regulates vacuole fusion in vivo. Vacuole fusion in vivo does, however, require physical presence of a fusion-competent V0 sector. PMID:27363625

  6. Castor Bean Organelle Genome Sequencing and Worldwide Genetic Diversity Analysis

    PubMed Central

    Chan, Agnes P.; Williams, Amber L.; Rice, Danny W.; Liu, Xinyue; Melake-Berhan, Admasu; Huot Creasy, Heather; Puiu, Daniela; Rosovitz, M. J.; Khouri, Hoda M.; Beckstrom-Sternberg, Stephen M.; Allan, Gerard J.; Keim, Paul; Ravel, Jacques; Rabinowicz, Pablo D.

    2011-01-01

    Castor bean is an important oil-producing plant in the Euphorbiaceae family. Its high-quality oil contains up to 90% of the unusual fatty acid ricinoleate, which has many industrial and medical applications. Castor bean seeds also contain ricin, a highly toxic Type 2 ribosome-inactivating protein, which has gained relevance in recent years due to biosafety concerns. In order to gain knowledge on global genetic diversity in castor bean and to ultimately help the development of breeding and forensic tools, we carried out an extensive chloroplast sequence diversity analysis. Taking advantage of the recently published genome sequence of castor bean, we assembled the chloroplast and mitochondrion genomes extracting selected reads from the available whole genome shotgun reads. Using the chloroplast reference genome we used the methylation filtration technique to readily obtain draft genome sequences of 7 geographically and genetically diverse castor bean accessions. These sequence data were used to identify single nucleotide polymorphism markers and phylogenetic analysis resulted in the identification of two major clades that were not apparent in previous population genetic studies using genetic markers derived from nuclear DNA. Two distinct sub-clades could be defined within each major clade and large-scale genotyping of castor bean populations worldwide confirmed previously observed low levels of genetic diversity and showed a broad geographic distribution of each sub-clade. PMID:21750729

  7. Organelle acidification negatively regulates vacuole membrane fusion in vivo.

    PubMed

    Desfougères, Yann; Vavassori, Stefano; Rompf, Maria; Gerasimaite, Ruta; Mayer, Andreas

    2016-01-01

    The V-ATPase is a proton pump consisting of a membrane-integral V0 sector and a peripheral V1 sector, which carries the ATPase activity. In vitro studies of yeast vacuole fusion and evidence from worms, flies, zebrafish and mice suggested that V0 interacts with the SNARE machinery for membrane fusion, that it promotes the induction of hemifusion and that this activity requires physical presence of V0 rather than its proton pump activity. A recent in vivo study in yeast has challenged these interpretations, concluding that fusion required solely lumenal acidification but not the V0 sector itself. Here, we identify the reasons for this discrepancy and reconcile it. We find that acute pharmacological or physiological inhibition of V-ATPase pump activity de-acidifies the vacuole lumen in living yeast cells within minutes. Time-lapse microscopy revealed that de-acidification induces vacuole fusion rather than inhibiting it. Cells expressing mutated V0 subunits that maintain vacuolar acidity were blocked in this fusion. Thus, proton pump activity of the V-ATPase negatively regulates vacuole fusion in vivo. Vacuole fusion in vivo does, however, require physical presence of a fusion-competent V0 sector. PMID:27363625

  8. High-throughput imaging of heterogeneous cell organelles with an X-ray laser

    SciTech Connect

    Hantke, Max, F.

    2014-11-17

    Preprocessed detector images that were used for the paper "High-throughput imaging of heterogeneous cell organelles with an X-ray laser". The CXI file contains the entire recorded data - including both hits and blanks. It also includes down-sampled images and LCLS machine parameters. Additionally, the Cheetah configuration file is attached that was used to create the pre-processed data.

  9. An organelle-specific protein landscape identifies novel diseases and molecular mechanisms

    PubMed Central

    Boldt, Karsten; van Reeuwijk, Jeroen; Lu, Qianhao; Koutroumpas, Konstantinos; Nguyen, Thanh-Minh T.; Texier, Yves; van Beersum, Sylvia E. C.; Horn, Nicola; Willer, Jason R.; Mans, Dorus A.; Dougherty, Gerard; Lamers, Ideke J. C.; Coene, Karlien L. M.; Arts, Heleen H.; Betts, Matthew J.; Beyer, Tina; Bolat, Emine; Gloeckner, Christian Johannes; Haidari, Khatera; Hetterschijt, Lisette; Iaconis, Daniela; Jenkins, Dagan; Klose, Franziska; Knapp, Barbara; Latour, Brooke; Letteboer, Stef J. F.; Marcelis, Carlo L.; Mitic, Dragana; Morleo, Manuela; Oud, Machteld M.; Riemersma, Moniek; Rix, Susan; Terhal, Paulien A.; Toedt, Grischa; van Dam, Teunis J. P.; de Vrieze, Erik; Wissinger, Yasmin; Wu, Ka Man; Apic, Gordana; Beales, Philip L.; Blacque, Oliver E.; Gibson, Toby J.; Huynen, Martijn A.; Katsanis, Nicholas; Kremer, Hannie; Omran, Heymut; van Wijk, Erwin; Wolfrum, Uwe; Kepes, François; Davis, Erica E.; Franco, Brunella; Giles, Rachel H.; Ueffing, Marius; Russell, Robert B.; Roepman, Ronald; Al-Turki, Saeed; Anderson, Carl; Antony, Dinu; Barroso, Inês; Bentham, Jamie; Bhattacharya, Shoumo; Carss, Keren; Chatterjee, Krishna; Cirak, Sebahattin; Cosgrove, Catherine; Danecek, Petr; Durbin, Richard; Fitzpatrick, David; Floyd, Jamie; Reghan Foley, A.; Franklin, Chris; Futema, Marta; Humphries, Steve E.; Hurles, Matt; Joyce, Chris; McCarthy, Shane; Mitchison, Hannah M.; Muddyman, Dawn; Muntoni, Francesco; O'Rahilly, Stephen; Onoufriadis, Alexandros; Payne, Felicity; Plagnol, Vincent; Raymond, Lucy; Savage, David B.; Scambler, Peter; Schmidts, Miriam; Schoenmakers, Nadia; Semple, Robert; Serra, Eva; Stalker, Jim; van Kogelenberg, Margriet; Vijayarangakannan, Parthiban; Walter, Klaudia; Whittall, Ros; Williamson, Kathy

    2016-01-01

    Cellular organelles provide opportunities to relate biological mechanisms to disease. Here we use affinity proteomics, genetics and cell biology to interrogate cilia: poorly understood organelles, where defects cause genetic diseases. Two hundred and seventeen tagged human ciliary proteins create a final landscape of 1,319 proteins, 4,905 interactions and 52 complexes. Reverse tagging, repetition of purifications and statistical analyses, produce a high-resolution network that reveals organelle-specific interactions and complexes not apparent in larger studies, and links vesicle transport, the cytoskeleton, signalling and ubiquitination to ciliary signalling and proteostasis. We observe sub-complexes in exocyst and intraflagellar transport complexes, which we validate biochemically, and by probing structurally predicted, disruptive, genetic variants from ciliary disease patients. The landscape suggests other genetic diseases could be ciliary including 3M syndrome. We show that 3M genes are involved in ciliogenesis, and that patient fibroblasts lack cilia. Overall, this organelle-specific targeting strategy shows considerable promise for Systems Medicine. PMID:27173435

  10. A one-step organelle capture: gynogenetic kiwifruits with paternal chloroplasts.

    PubMed

    Chat, Joëlle; Decroocq, Stéphane; Petit, Rémy J

    2003-04-22

    Androgenesis, the development of a haploid embryo from a male nucleus, has been shown to result in the instantaneous uncoupling of the transmission of the organelle and nuclear genomes (with the nuclear genome originating from the male parent only and the organelle genomes from the female parent). We report, for the first time, uncoupling resulting from gynogenesis, in Actinidia deliciosa (kiwifruit), a plant species known for its paternal mode of chloroplast inheritance. After pollen irradiation, transmission of nuclear genes from the pollen parent to the progeny was inhibited, but transmission of the chloroplast genome was not. This demonstrates that plastids can be discharged from the pollen tube into the egg with little or no concomitant transmission of paternal nuclear genes. Such events of opposite inheritance of the organelle and nuclear genomes must be very rare in nature and are unlikely to endanger the long-term stability of the association between the different genomes of the cell. However, they could lead to incongruences between organelle gene trees and species trees and may constitute an alternative to the hybridization/introgression scenario commonly invoked to account for such incongruences. PMID:12737655

  11. A one-step organelle capture: gynogenetic kiwifruits with paternal chloroplasts.

    PubMed Central

    Chat, Joëlle; Decroocq, Stéphane; Petit, Rémy J

    2003-01-01

    Androgenesis, the development of a haploid embryo from a male nucleus, has been shown to result in the instantaneous uncoupling of the transmission of the organelle and nuclear genomes (with the nuclear genome originating from the male parent only and the organelle genomes from the female parent). We report, for the first time, uncoupling resulting from gynogenesis, in Actinidia deliciosa (kiwifruit), a plant species known for its paternal mode of chloroplast inheritance. After pollen irradiation, transmission of nuclear genes from the pollen parent to the progeny was inhibited, but transmission of the chloroplast genome was not. This demonstrates that plastids can be discharged from the pollen tube into the egg with little or no concomitant transmission of paternal nuclear genes. Such events of opposite inheritance of the organelle and nuclear genomes must be very rare in nature and are unlikely to endanger the long-term stability of the association between the different genomes of the cell. However, they could lead to incongruences between organelle gene trees and species trees and may constitute an alternative to the hybridization/introgression scenario commonly invoked to account for such incongruences. PMID:12737655

  12. An organelle-specific protein landscape identifies novel diseases and molecular mechanisms.

    PubMed

    Boldt, Karsten; van Reeuwijk, Jeroen; Lu, Qianhao; Koutroumpas, Konstantinos; Nguyen, Thanh-Minh T; Texier, Yves; van Beersum, Sylvia E C; Horn, Nicola; Willer, Jason R; Mans, Dorus A; Dougherty, Gerard; Lamers, Ideke J C; Coene, Karlien L M; Arts, Heleen H; Betts, Matthew J; Beyer, Tina; Bolat, Emine; Gloeckner, Christian Johannes; Haidari, Khatera; Hetterschijt, Lisette; Iaconis, Daniela; Jenkins, Dagan; Klose, Franziska; Knapp, Barbara; Latour, Brooke; Letteboer, Stef J F; Marcelis, Carlo L; Mitic, Dragana; Morleo, Manuela; Oud, Machteld M; Riemersma, Moniek; Rix, Susan; Terhal, Paulien A; Toedt, Grischa; van Dam, Teunis J P; de Vrieze, Erik; Wissinger, Yasmin; Wu, Ka Man; Apic, Gordana; Beales, Philip L; Blacque, Oliver E; Gibson, Toby J; Huynen, Martijn A; Katsanis, Nicholas; Kremer, Hannie; Omran, Heymut; van Wijk, Erwin; Wolfrum, Uwe; Kepes, François; Davis, Erica E; Franco, Brunella; Giles, Rachel H; Ueffing, Marius; Russell, Robert B; Roepman, Ronald

    2016-01-01

    Cellular organelles provide opportunities to relate biological mechanisms to disease. Here we use affinity proteomics, genetics and cell biology to interrogate cilia: poorly understood organelles, where defects cause genetic diseases. Two hundred and seventeen tagged human ciliary proteins create a final landscape of 1,319 proteins, 4,905 interactions and 52 complexes. Reverse tagging, repetition of purifications and statistical analyses, produce a high-resolution network that reveals organelle-specific interactions and complexes not apparent in larger studies, and links vesicle transport, the cytoskeleton, signalling and ubiquitination to ciliary signalling and proteostasis. We observe sub-complexes in exocyst and intraflagellar transport complexes, which we validate biochemically, and by probing structurally predicted, disruptive, genetic variants from ciliary disease patients. The landscape suggests other genetic diseases could be ciliary including 3M syndrome. We show that 3M genes are involved in ciliogenesis, and that patient fibroblasts lack cilia. Overall, this organelle-specific targeting strategy shows considerable promise for Systems Medicine. PMID:27173435

  13. In situ observation of cellular organelles with a contact x-ray microscope

    NASA Astrophysics Data System (ADS)

    Kado, M.; Kishimoto, M.; Tamotsu, S.; Yasuda, K.; Shinohara, K.

    2013-10-01

    A contact x-ray microscope coupled with a highly intense laser plasma soft x-ray source has been developed and in situ observations of cellular organelles have been conducted. The soft x-rays were generated by irradiating a high power laser pulse onto a thin-foiled gold target and about 1.3×1015 photons/sr were obtained, which allowed the inner structures of live wet biological cells to be imaged. Single shot flash imaging is a key technique to image cellular organelles of live biological cells avoiding degradation of the spatial resolution of the images resulting from motion blur and radiation damage. The use of a fluorescence microscope to identify cellular organelles in conjunction with the soft x-ray microscope has allowed several cellular organelles to be identified precisely in the soft x-ray images. Combining the fluorescence microscope with the soft x-ray microscope will be very useful and will establish the technique as a powerful tool to analyze living function of biological cells.

  14. Phase Transition of a Disordered Nuage Protein Generates Environmentally Responsive Membraneless Organelles

    PubMed Central

    Nott, Timothy J.; Petsalaki, Evangelia; Farber, Patrick; Jervis, Dylan; Fussner, Eden; Plochowietz, Anne; Craggs, Timothy D.; Bazett-Jones, David P.; Pawson, Tony; Forman-Kay, Julie D.; Baldwin, Andrew J.

    2015-01-01

    Summary Cells chemically isolate molecules in compartments to both facilitate and regulate their interactions. In addition to membrane-encapsulated compartments, cells can form proteinaceous and membraneless organelles, including nucleoli, Cajal and PML bodies, and stress granules. The principles that determine when and why these structures form have remained elusive. Here, we demonstrate that the disordered tails of Ddx4, a primary constituent of nuage or germ granules, form phase-separated organelles both in live cells and in vitro. These bodies are stabilized by patterned electrostatic interactions that are highly sensitive to temperature, ionic strength, arginine methylation, and splicing. Sequence determinants are used to identify proteins found in both membraneless organelles and cell adhesion. Moreover, the bodies provide an alternative solvent environment that can concentrate single-stranded DNA but largely exclude double-stranded DNA. We propose that phase separation of disordered proteins containing weakly interacting blocks is a general mechanism for forming regulated, membraneless organelles. PMID:25747659

  15. Biogenesis of the crystalloid organelle in Plasmodium involves microtubule-dependent vesicle transport and assembly

    PubMed Central

    Saeed, Sadia; Tremp, Annie Z.; Dessens, Johannes T.

    2015-01-01

    Malaria parasites possess unique subcellular structures and organelles. One of these is the crystalloid, a multivesicular organelle that forms during the parasite’s development in vector mosquitoes. The formation and function of these organelles remain poorly understood. A family of six conserved and modular proteins named LCCL-lectin adhesive-like proteins (LAPs), which have essential roles in sporozoite transmission, localise to the crystalloids. In this study we analyse crystalloid formation using transgenic Plasmodium berghei parasites expressing GFP-tagged LAP3. We show that deletion of the LCCL domain from LAP3 causes retarded crystalloid development, while knockout of LAP3 prevents formation of the organelle. Our data reveal that the process of crystalloid formation involves active relocation of endoplasmic reticulum-derived vesicles to common assembly points via microtubule-dependent transport. Inhibition of microtubule-dependent cargo transport disrupts this process and replicates the LCCL domain deletion mutant phenotype in wildtype parasites. These findings provide the first clear insight into crystalloid biogenesis, demonstrating a fundamental role for the LAP family in this process, and identifying the crystalloid and its formation as potential targets for malaria transmission control. PMID:25900212

  16. Lens fibre cell differentiation and organelle loss: many paths lead to clarity

    PubMed Central

    Wride, Michael A.

    2011-01-01

    The programmed removal of organelles from differentiating lens fibre cells contributes towards lens transparency through formation of an organelle-free zone (OFZ). Disruptions in OFZ formation are accompanied by the persistence of organelles in lens fibre cells and can contribute towards cataract. A great deal of work has gone into elucidating the nature of the mechanisms and signalling pathways involved. It is apparent that multiple, parallel and redundant pathways are involved in this process and that these pathways form interacting networks. Furthermore, it is possible that the pathways can functionally compensate for each other, for example in mouse knockout studies. This makes sense given the importance of lens clarity in an evolutionary context. Apoptosis signalling and proteolytic pathways have been implicated in both lens fibre cell differentiation and organelle loss, including the Bcl-2 and inhibitor of apoptosis families, tumour necrosis factors, p53 and its regulators (such as Mdm2) and proteolytic enzymes, including caspases, cathepsins, calpains and the ubiquitin–proteasome pathway. Ongoing approaches being used to dissect the molecular pathways involved, such as transgenics, lens-specific gene deletion and zebrafish mutants, are discussed here. Finally, some of the remaining unresolved issues and potential areas for future studies are highlighted. PMID:21402582

  17. Membrane trafficking and organelle biogenesis in Giardia lamblia: use it or lose it.

    PubMed

    Faso, Carmen; Hehl, Adrian B

    2011-04-01

    The secretory transport capacity of Giardia trophozoites is perfectly adapted to the changing environment in the small intestine of the host and is able to deploy essential protective surface coats as well as molecules which act on epithelia. These lumen-dwelling parasites take up nutrients by bulk endocytosis through peripheral vesicles or by receptor-mediated transport. The environmentally-resistant cyst form is quiescent but poised for activation following stomach passage. Its versatility and fidelity notwithstanding, the giardial trafficking systems appear to be the product of a general secondary reduction process geared towards minimization of all components and machineries identified to date. Since membrane transport is directly linked to organelle biogenesis and maintenance, less complexity also means loss of organelle structures and functions. A case in point is the Golgi apparatus which is missing as a steady-state organelle system. Only a few basic Golgi functions have been experimentally demonstrated in trophozoites undergoing encystation. Similarly, mitochondrial remnants have reached a terminally minimized state and appear to be functionally restricted to essential iron-sulfur protein maturation processes. Giardia's minimized organization combined with its genetic tractability provides unique opportunities to study basic principles of secretory transport in an uncluttered cellular environment. Not surprisingly, Giardia is gaining increasing attention as a model for the investigation of gene regulation, organelle biogenesis, and export of simple but highly protective cell wall biopolymers, a hallmark of all perorally transmitted protozoan and metazoan parasites. PMID:21296082

  18. Differential expression of vesicular glutamate transporters by vagal afferent terminals in rat nucleus of the solitary tract: projections from the heart preferentially express vesicular glutamate transporter 1.

    PubMed

    Corbett, E K A; Sinfield, J K; McWilliam, P N; Deuchars, J; Batten, T F C

    2005-01-01

    The central projections and neurochemistry of vagal afferent neurones supplying the heart in the rat were investigated by injecting cholera toxin B-subunit into the pericardium. Transganglionically transported cholera toxin B-subunit was visualized in the medulla oblongata in axons and varicosities that were predominantly aggregated in the dorsomedial, dorsolateral, ventrolateral and commissural subnuclei of the caudal nucleus of the solitary tract. Unilateral vagal section in control rats prevented cholera toxin B-subunit labeling on the ipsilateral side of the nucleus of the solitary tract. Fluorescent and electron microscopic dual labeling showed colocalization of immunoreactivity for vesicular glutamate transporter 1, but only rarely vesicular glutamate transporters 2 or 3 with cholera toxin B-subunit in terminals in nucleus of the solitary tract, suggesting that cardiac vagal axons release glutamate as a neurotransmitter. In contrast, populations of vagal afferent fibers labeled by injection of cholera toxin B-subunit, tetra-methylrhodamine dextran or biotin dextran amine into the aortic nerve, stomach or nodose ganglion colocalized vesicular glutamate transporter 2 more frequently than vesicular glutamate transporter 1. The presence of other neurochemical markers of primary afferent neurones was examined in nucleus of the solitary tract axons and nodose ganglion cells labeled by pericardial cholera toxin B-subunit injections. Immunoreactivity for a 200-kDa neurofilament protein in many large, cholera toxin B-subunit-labeled nodose ganglion cells indicated that the cardiac afferent fibers labeled are mostly myelinated, whereas binding of Griffonia simplicifolia isolectin B4 to fewer small cholera toxin B-subunit-labeled ganglion cells suggested that tracer was also taken up by some non-myelinated axons. A few labeled nucleus of the solitary tract axons and ganglion cells were positive for substance P and calcitonin gene-related peptide, which are considered as

  19. Association of six YFP-myosin XI-tail fusions with mobile plant cell organelles

    PubMed Central

    Reisen, Daniel; Hanson, Maureen R

    2007-01-01

    Background Myosins are molecular motors that carry cargo on actin filaments in eukaryotic cells. Seventeen myosin genes have been identified in the nuclear genome of Arabidopsis. The myosin genes can be divided into two plant-specific subfamilies, class VIII with four members and class XI with 13 members. Class XI myosins are related to animal and fungal myosin class V that are responsible for movement of particular vesicles and organelles. Organelle localization of only one of the 13 Arabidopsis myosin XI (myosin XI-6; At MYA2), which is found on peroxisomes, has so far been reported. Little information is available concerning the remaining 12 class XI myosins. Results We investigated 6 of the 13 class XI Arabidopsis myosins. cDNAs corresponding to the tail region of 6 myosin genes were generated and incorporated into a vector to encode YFP-myosin tail fusion proteins lacking the motor domain. Chimeric genes incorporating tail regions of myosin XI-5 (At MYA1), myosin XI-6 (At MYA2), myosin XI-8 (At XI-B), myosin XI-15 (At XI-I), myosin XI-16 (At XI-J) and myosin XI-17 (At XI-K) were expressed transiently. All YFP-myosin-tail fusion proteins were targeted to small organelles ranging in size from 0.5 to 3.0 μm. Despite the absence of a motor domain, the fluorescently-labeled organelles were motile in most cells. Tail cropping experiments demonstrated that the coiled-coil region was required for specific localization and shorter tail regions were inadequate for targeting. Myosin XI-6 (At MYA2), previously reported to localize to peroxisomes by immunofluorescence, labeled both peroxisomes and vesicles when expressed as a YFP-tail fusion. None of the 6 YFP-myosin tail fusions interacted with chloroplasts, and only one YFP-tail fusion appeared to sometimes co-localize with fluorescent proteins targeted to Golgi and mitochondria. Conclusion 6 myosin XI tails, extending from the coiled-coil region to the C-terminus, label specific vesicles and/or organelles when

  20. Quantitative analysis of organelle distribution and dynamics in Physcomitrella patens protonemal cells

    PubMed Central

    2012-01-01

    Background In the last decade, the moss Physcomitrella patens has emerged as a powerful plant model system, amenable for genetic manipulations not possible in any other plant. This moss is particularly well suited for plant polarized cell growth studies, as in its protonemal phase, expansion is restricted to the tip of its cells. Based on pollen tube and root hair studies, it is well known that tip growth requires active secretion and high polarization of the cellular components. However, such information is still missing in Physcomitrella patens. To gain insight into the mechanisms underlying the participation of organelle organization in tip growth, it is essential to determine the distribution and the dynamics of the organelles in moss cells. Results We used fluorescent protein fusions to visualize and track Golgi dictyosomes, mitochondria, and peroxisomes in live protonemal cells. We also visualized and tracked chloroplasts based on chlorophyll auto-fluorescence. We showed that in protonemata all four organelles are distributed in a gradient from the tip of the apical cell to the base of the sub-apical cell. For example, the density of Golgi dictyosomes is 4.7 and 3.4 times higher at the tip than at the base in caulonemata and chloronemata respectively. While Golgi stacks are concentrated at the extreme tip of the caulonemata, chloroplasts and peroxisomes are totally excluded. Interestingly, caulonemata, which grow faster than chloronemata, also contain significantly more Golgi dictyosomes and fewer chloroplasts than chloronemata. Moreover, the motility analysis revealed that organelles in protonemata move with low persistency and average instantaneous speeds ranging from 29 to 75 nm/s, which are at least three orders of magnitude slower than those of pollen tube or root hair organelles. Conclusions To our knowledge, this study reports the first quantitative analysis of organelles in Physcomitrella patens and will make possible comparisons of the distribution

  1. Genome Sequences of Nine Vesicular Stomatitis Virus Isolates from South America

    PubMed Central

    King, David J.; Howson, Emma L. A.; Madi, Mikidache; Pauszek, Steven J.; Rodriguez, Luis L.; Knowles, Nick J.; Mioulet, Valérie; King, Donald P.

    2016-01-01

    We report nine full-genome sequences of vesicular stomatitis virus obtained by Illumina next-generation sequencing of RNA, isolated from either cattle epithelial suspensions or cell culture supernatants. Seven of these viral genomes belonged to the New Jersey serotype/species (clade III), while two isolates belonged to the Indiana serotype/species. PMID:27081129

  2. Potential for Transovarial Transmission of Vesicular Stomatitis Virus in the biting midge, Culicoides sonorensis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Vesicular stomatitis virus (VSV) is an insect transmitted rhabdovirus which causes economically devastating disease in cattle and horses in the western U.S. Important insect vectors identified thus far include Lutzomyia shannoni sand flies, Simulium vittatum black flies, and Culicoides sonorensis bi...

  3. Characterization of the Full-Length Genomic Sequence of Vesicular Stomatitis Cocal and Alagoas Viruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Vesicular stomatitis (VS) is an important viral disease of livestock throughout the Americas caused by members of the vesiculovirus genus of the family Rhabdoviridae. VS outbreaks between northern South America and North America are caused principally by the New Jersey serotype (VSNJV) and to a les...

  4. Vesicular Trafficking and Signaling for Cytokine and Chemokine Secretion in Mast Cells

    PubMed Central

    Blank, Ulrich; Madera-Salcedo, Iris Karina; Danelli, Luca; Claver, Julien; Tiwari, Neeraj; Sánchez-Miranda, Elizabeth; Vázquez-Victorio, Genaro; Ramírez-Valadez, Karla Alina; Macias-Silva, Marina; González-Espinosa, Claudia

    2014-01-01

    Upon activation mast cells (MCs) secrete numerous inflammatory compounds stored in their cytoplasmic secretory granules by a process called anaphylactic degranulation, which is responsible for type I hypersensitivity responses. Prestored mediators include histamine and MC proteases but also some cytokines and growth factors making them available within minutes for a maximal biological effect. Degranulation is followed by the de novo synthesis of lipid mediators such as prostaglandins and leukotrienes as well as a vast array of cytokines, chemokines, and growth factors, which are responsible for late phase inflammatory responses. While lipid mediators diffuse freely out of the cell through lipid bilayers, both anaphylactic degranulation and secretion of cytokines, chemokines, and growth factors depends on highly regulated vesicular trafficking steps that occur along the secretory pathway starting with the translocation of proteins to the endoplasmic reticulum. Vesicular trafficking in MCs also intersects with endocytic routes, notably to form specialized cytoplasmic granules called secretory lysosomes. Some of the mediators like histamine reach granules via specific vesicular monoamine transporters directly from the cytoplasm. In this review, we try to summarize the available data on granule biogenesis and signaling events that coordinate the complex steps that lead to the release of the inflammatory mediators from the various vesicular carriers in MCs. PMID:25295038

  5. Infection of Melanoplus Sanguinipes Grasshoppers Following Ingestion of Rangeland Plant Species Harboring Vesicular Stomatitis Virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Knowledge of the many mechanisms of vesicular stomatitis virus (VSV) transmission is critical to understanding the epidemiology of sporadic disease outbreaks in the western U.S. Migratory grasshoppers (Melanoplus sanguinipes, Fabricius) have been implicated as reservoirs and mechanical vectors of VS...

  6. Blood Feeding Behavior of Vesicular Stomatitis Virus Infected Culicoides Sonorensis (Diptera: Ceratopogonidae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Culicoides sonorensis (Diptera: Ceratopogonidae) is the primary vector of Bluetongue virus in North America and a competent vector of Vesicular Stomatitis virus (VSV). Little is known about how viral infection of this midge affects blood feeding behavior and how this might affect virus transmission....

  7. 9 CFR 94.12 - Pork and pork products from regions where swine vesicular disease exists.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... citations affecting § 94.12, see the List of CFR Sections Affected, which appears in the Finding Aids... where swine vesicular disease exists. 94.12 Section 94.12 Animals and Animal Products ANIMAL AND PLANT... POULTRY) AND ANIMAL PRODUCTS RINDERPEST, FOOT-AND-MOUTH DISEASE, EXOTIC NEWCASTLE DISEASE, AFRICAN...

  8. 9 CFR 94.12 - Pork and pork products from regions where swine vesicular disease exists.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... List of CFR Sections Affected, which appears in the Finding Aids section of the printed volume and on... where swine vesicular disease exists. 94.12 Section 94.12 Animals and Animal Products ANIMAL AND PLANT... POULTRY) AND ANIMAL PRODUCTS RINDERPEST, FOOT-AND-MOUTH DISEASE, EXOTIC NEWCASTLE DISEASE, AFRICAN...

  9. 9 CFR 94.12 - Pork and pork products from regions where swine vesicular disease exists.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... citations affecting § 94.12, see the List of CFR Sections Affected, which appears in the Finding Aids... where swine vesicular disease exists. 94.12 Section 94.12 Animals and Animal Products ANIMAL AND PLANT... POULTRY) AND ANIMAL PRODUCTS RINDERPEST, FOOT-AND-MOUTH DISEASE, EXOTIC NEWCASTLE DISEASE, AFRICAN...

  10. 9 CFR 94.12 - Pork and pork products from regions where swine vesicular disease exists.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... citations affecting § 94.12, see the List of CFR Sections Affected, which appears in the Finding Aids... where swine vesicular disease exists. 94.12 Section 94.12 Animals and Animal Products ANIMAL AND PLANT... POULTRY) AND ANIMAL PRODUCTS RINDERPEST, FOOT-AND-MOUTH DISEASE, EXOTIC NEWCASTLE DISEASE, AFRICAN...

  11. 9 CFR 94.14 - Swine from regions where swine vesicular disease exists; importations prohibited.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Swine from regions where swine... PATHOGENIC AVIAN INFLUENZA, AFRICAN SWINE FEVER, CLASSICAL SWINE FEVER, SWINE VESICULAR DISEASE, AND BOVINE SPONGIFORM ENCEPHALOPATHY: PROHIBITED AND RESTRICTED IMPORTATIONS § 94.14 Swine from regions where...

  12. Genome Sequences of Nine Vesicular Stomatitis Virus Isolates from South America.

    PubMed

    Fowler, Veronica L; King, David J; Howson, Emma L A; Madi, Mikidache; Pauszek, Steven J; Rodriguez, Luis L; Knowles, Nick J; Mioulet, Valérie; King, Donald P

    2016-01-01

    We report nine full-genome sequences of vesicular stomatitis virus obtained by Illumina next-generation sequencing of RNA, isolated from either cattle epithelial suspensions or cell culture supernatants. Seven of these viral genomes belonged to the New Jersey serotype/species (clade III), while two isolates belonged to the Indiana serotype/species. PMID:27081129

  13. Infection of Guinea Pigs with Vesicular Stomatitis New Jersey Virus Transmitted by Culicoides sonorensis (Diptera: Ceratopogonidae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Interpretive Biting midges,Culicoides sonorensis were shown to be capable of transmitting vesicular stomatitis New Jersey virus (VSNJV) to guinea pigs. Despite seroconversion for VSNJV, none of the guinea pigs developed clinical signs when infected in the abdomen by either infected insects or by nee...

  14. Domestic cattle as a non-conventional amplifying host of vesicular stomatitis New Jersey virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The role of vertebrates as amplifying and maintenance hosts for vesicular stomatitis New Jersey virus (VSNJV) remains unclear. Livestock have been considered dead-end hosts because detectable viremia is absent in VSNJV-infected animals. We demonstrated two situations where cattle can serve as a so...

  15. Infection of grasshoppers following ingestion of grassland plant species harboring vesicular stomatitis virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Vesicular stomatitis virus (VSV) causes sporadic, re-emerging disease outbreaks in horses and cattle in the western United States. Lesions in the oral cavity result in excessive salivation and significant virus shedding. This results in efficient direct contact transmission within the herd, and vira...

  16. First Complete Coding Sequence of a Spanish Isolate of Swine Vesicular Disease Virus

    PubMed Central

    Vázquez-Calvo, Ángela; Saiz, Juan-Carlos; Martín-Acebes, Miguel A.

    2016-01-01

    Swine vesicular disease virus (SVDV) is a porcine pathogen and a member of the Enterovirus genus within the Picornaviridae family. The SVDV genome is composed of a single-stranded RNA molecule of positive polarity. Here, we report the first complete sequence of the coding region of a Spanish SVDV isolate (SPA/1/'93). PMID:26941157

  17. Bovine neuronal vesicular glutamate transporter activity is inhibited by ergovaline and other ergopeptines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    L-Glutamate (Glu) is the major excitatory neurotransmitter responsible for neurotransmission in the vertebrate central nervous system, including the gastrointestinal tract (GIT) of cattle. Vesicular Glu transporters VGLUT1 and VGLUT2 concentrate (50 mM) Glu (Km = 1 to 4 mM) into synaptic vesicles (S...

  18. Genome sequences of nine Vesicular Stomatitis Virus isolates from South America

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We report nine full-genome sequences of vesicular stomatitis virus obtrained by Illumina next-generation sequencing of RNA, isolated from either cattle epithelial suspensions or cell culture supernatants. Seven of these viral genomes belonged to the New Jersey serotype/species, clade III, while two...

  19. Mitochondria-derived organelles in the diplomonad fish parasite Spironucleus vortens.

    PubMed

    Millet, Coralie O M; Williams, Catrin F; Hayes, Anthony J; Hann, Anthony C; Cable, Joanne; Lloyd, David

    2013-10-01

    In some eukaryotes, mitochondria have become modified during evolution to yield derived organelles (MDOs) of a similar size (hydrogenosomes), or extremely reduced to produce tiny cellular vesicles (mitosomes). The current study provides evidence for the presence of MDOs in the highly infectious fish pathogen Spironucleus vortens, an organism that produces H₂ and is shown here to have no detectable cytochromes. Transmission electron microscopy (TEM) reveals that S. vortens trophozoites contain electron-dense, membranous structures sometimes with an electron-dense core (200 nm-1 μm), resembling the hydrogenosomes previously described in other protists from habitats deficient in O₂. Confocal microscopy establishes that these organelles exhibit autofluorescence emission spectra similar to flavoprotein constituents previously described for mitochondria and also present in hydrogenosomes. These organelles possess a membrane potential and are labelled by a fluorescently labeled antibody against Fe-hydrogenase from Blastocystis hominis. Heterologous antibodies raised to mitochondrial proteins frataxin and Isu1, also exhibit a discrete punctate pattern of localization in S. vortens; however these labelled structures are distinctly smaller (90-150 nm) than hydrogenosomes as observed previously in other organisms. TEM confirms the presence of double-membrane bounded organelles of this smaller size. In addition, strong background immunostaining occurs in the cytosol for frataxin and Isu1, and labelling by anti-ferredoxin antibody is generally distributed and not specifically localized except for at the anterior polar region. This suggests that some of the functions traditionally attributed to such MDOs may also occur elsewhere. The specialized parasitic life-style of S. vortens may necessitate more complex intracellular compartmentation of redox reactions than previously recognized. Control of infection requires biochemical characterization of redox-related organelles

  20. Organelle Size Scaling of the Budding Yeast Vacuole by Relative Growth and Inheritance.

    PubMed

    Chan, Yee-Hung M; Reyes, Lorena; Sohail, Saba M; Tran, Nancy K; Marshall, Wallace F

    2016-05-01

    It has long been noted that larger animals have larger organs compared to smaller animals of the same species, a phenomenon termed scaling [1]. Julian Huxley proposed an appealingly simple model of "relative growth"-in which an organ and the whole body grow with their own intrinsic rates [2]-that was invoked to explain scaling in organs from fiddler crab claws to human brains. Because organ size is regulated by complex, unpredictable pathways [3], it remains unclear whether scaling requires feedback mechanisms to regulate organ growth in response to organ or body size. The molecular pathways governing organelle biogenesis are simpler than organogenesis, and therefore organelle size scaling in the cell provides a more tractable case for testing Huxley's model. We ask the question: is it possible for organelle size scaling to arise if organelle growth is independent of organelle or cell size? Using the yeast vacuole as a model, we tested whether mutants defective in vacuole inheritance, vac8Δ and vac17Δ, tune vacuole biogenesis in response to perturbations in vacuole size. In vac8Δ/vac17Δ, vacuole scaling increases with the replicative age of the cell. Furthermore, vac8Δ/vac17Δ cells continued generating vacuole at roughly constant rates even when they had significantly larger vacuoles compared to wild-type. With support from computational modeling, these results suggest there is no feedback between vacuole biogenesis rates and vacuole or cell size. Rather, size scaling is determined by the relative growth rates of the vacuole and the cell, thus representing a cellular version of Huxley's model. PMID:27151661

  1. The antinociceptive effects of intracerebroventricular administration of Chicago sky blue 6B, a vesicular glutamate transporter inhibitor.

    PubMed

    Yu, Gang; Yi, Shoupu; Wang, Meiliang; Yan, Hui; Yan, Lingdi; Su, Ruibin; Gong, Zehui

    2013-12-01

    Accumulating evidence suggests that vesicular glutamate transporters (VGLUTs), which control the storage and release of glutamate, may play a role in pain processing. Chicago sky blue 6B (CSB6B), which is structurally related to glutamate, is a competitive VGLUT inhibitor without affecting plasma membrane transporters. The present study was designed to investigate the antinociceptive effects of CSB6B in a number of pain models. The hot-plate test was used as an acute thermal pain test. Inflammatory pain was evaluated using acetic acid writhing, formalin, and complete Freund's adjuvant tests. Intracerebroventricular administration of CSB6B did not affect acute thermal pain responses in 50 or 55°C hot plate tests. However, CSB6B attenuated acetic acid-induced writhing in a dose-dependent and time-dependent manner. In addition, CSB6B reduced licking/biting behavior during the second phase, but not during the first phase, following an intraplantar injection of formalin. In the complete Freund's adjuvant test, a significant attenuation of thermal hyperalgesia was also observed in CSB6B-treated mice. At antinociceptive doses, CSB6B did not affect mice spontaneous locomotor activity. The present study shows that pharmacological inhibition of VGLUT activity was sufficient to attenuate experimental inflammatory pain and suggests that regulation of VGLUTs might be a novel therapeutic strategy for the treatment of pain. PMID:24128751

  2. Overexpressing OsPIN2 enhances aluminium internalization by elevating vesicular trafficking in rice root apex

    PubMed Central

    Wu, Daoming; Shen, Hong; Yokawa, Ken; Baluška, František

    2015-01-01

    Aluminium (Al) sequestration is required for internal detoxification of Al in plant cells. In this study, it was found that the rice OsPIN2 overexpression line (OX1) had significantly reduced Al content in its cell wall and increased Al concentration in cell sap only in rice root tips relative to the wild-type (WT). In comparison with WT, OX1 reduced morin staining of cytosolic Al, enhanced FM 4–64 staining of membrane vesicular trafficking in root tip sections (0–1mm), and showed morin-FM 4–64 fluorescence overlap. Recovery treatment showed that cell-wall-bound Al was internalized into vacuoles via endocytic vesicular trafficking after removal of external Al. In this process, OX1 showed a higher rate of Al internalization than WT. Brefeldin A (BFA) interfered with vesicular trafficking and resulted in inhibition of Al internalization. This inhibitory effect could be alleviated when BFA was washed out, and the process of alleviation was slower in the cells of WT than in those of OX1. Microscopic observations revealed that, upon Al exposure, numerous multilamellar endosomes were detected between the cell wall and plasma membrane in the cells of OX1. Moreover, more vesicles enriched with Al complexes accumulated in the cells of OX1 than in those of WT, and these vesicles transformed into larger structures in the cells of OX1. Taken together, the data indicate that endocytic vesicular trafficking might contribute to Al internalization, and that overexpressing OsPIN2 enhances rice Al tolerance via elevated endocytic vesicular trafficking and Al internalization. PMID:26254327

  3. Overexpressing OsPIN2 enhances aluminium internalization by elevating vesicular trafficking in rice root apex.

    PubMed

    Wu, Daoming; Shen, Hong; Yokawa, Ken; Baluška, František

    2015-11-01

    Aluminium (Al) sequestration is required for internal detoxification of Al in plant cells. In this study, it was found that the rice OsPIN2 overexpression line (OX1) had significantly reduced Al content in its cell wall and increased Al concentration in cell sap only in rice root tips relative to the wild-type (WT). In comparison with WT, OX1 reduced morin staining of cytosolic Al, enhanced FM 4-64 staining of membrane vesicular trafficking in root tip sections (0-1mm), and showed morin-FM 4-64 fluorescence overlap. Recovery treatment showed that cell-wall-bound Al was internalized into vacuoles via endocytic vesicular trafficking after removal of external Al. In this process, OX1 showed a higher rate of Al internalization than WT. Brefeldin A (BFA) interfered with vesicular trafficking and resulted in inhibition of Al internalization. This inhibitory effect could be alleviated when BFA was washed out, and the process of alleviation was slower in the cells of WT than in those of OX1. Microscopic observations revealed that, upon Al exposure, numerous multilamellar endosomes were detected between the cell wall and plasma membrane in the cells of OX1. Moreover, more vesicles enriched with Al complexes accumulated in the cells of OX1 than in those of WT, and these vesicles transformed into larger structures in the cells of OX1. Taken together, the data indicate that endocytic vesicular trafficking might contribute to Al internalization, and that overexpressing OsPIN2 enhances rice Al tolerance via elevated endocytic vesicular trafficking and Al internalization. PMID:26254327

  4. Astrocytes as secretory cells of the central nervous system: idiosyncrasies of vesicular secretion.

    PubMed

    Verkhratsky, Alexei; Matteoli, Michela; Parpura, Vladimir; Mothet, Jean-Pierre; Zorec, Robert

    2016-02-01

    Astrocytes are housekeepers of the central nervous system (CNS) and are important for CNS development, homeostasis and defence. They communicate with neurones and other glial cells through the release of signalling molecules. Astrocytes secrete a wide array of classic neurotransmitters, neuromodulators and hormones, as well as metabolic, trophic and plastic factors, all of which contribute to the gliocrine system. The release of neuroactive substances from astrocytes occurs through several distinct pathways that include diffusion through plasmalemmal channels, translocation by multiple transporters and regulated exocytosis. As in other eukaryotic cells, exocytotic secretion from astrocytes involves divergent secretory organelles (synaptic-like microvesicles, dense-core vesicles, lysosomes, exosomes and ectosomes), which differ in size, origin, cargo, membrane composition, dynamics and functions. In this review, we summarize the features and functions of secretory organelles in astrocytes. We focus on the biogenesis and trafficking of secretory organelles and on the regulation of the exocytotic secretory system in the context of healthy and diseased astrocytes. PMID:26758544

  5. The Dunaliella salina organelle genomes: large sequences, inflated with intronic and intergenic DNA

    SciTech Connect

    Smith, David R.; Lee, Robert W.; Cushman, John C.; Magnuson, Jon K.; Tran, Duc; Polle, Juergen E.

    2010-05-07

    Abstract Background: Dunaliella salina Teodoresco, a unicellular, halophilic green alga belonging to the Chlorophyceae, is among the most industrially important microalgae. This is because D. salina can produce massive amounts of β-carotene, which can be collected for commercial purposes, and because of its potential as a feedstock for biofuels production. Although the biochemistry and physiology of D. salina have been studied in great detail, virtually nothing is known about the genomes it carries, especially those within its mitochondrion and plastid. This study presents the complete mitochondrial and plastid genome sequences of D. salina and compares them with those of the model green algae Chlamydomonas reinhardtii and Volvox carteri. Results: The D. salina organelle genomes are large, circular-mapping molecules with ~60% noncoding DNA, placing them among the most inflated organelle DNAs sampled from the Chlorophyta. In fact, the D. salina plastid genome, at 269 kb, is the largest complete plastid DNA (ptDNA) sequence currently deposited in GenBank, and both the mitochondrial and plastid genomes have unprecedentedly high intron densities for organelle DNA: ~1.5 and ~0.4 introns per gene, respectively. Moreover, what appear to be the relics of genes, introns, and intronic open reading frames are found scattered throughout the intergenic ptDNA regions -- a trait without parallel in other characterized organelle genomes and one that gives insight into the mechanisms and modes of expansion of the D. salina ptDNA. Conclusions: These findings confirm the notion that chlamydomonadalean algae have some of the most extreme organelle genomes of all eukaryotes. They also suggest that the events giving rise to the expanded ptDNA architecture of D. salina and other Chlamydomonadales may have occurred early in the evolution of this lineage. Although interesting from a genome evolution standpoint, the D. salina organelle DNA sequences will aid in the development of a viable

  6. Synaptic connections of amacrine cells containing vesicular glutamate transporter 3 in baboon retinas

    PubMed Central

    MARSHAK, DAVID W.; CHUANG, ALICE Z.; DOLINO, DREW M.; JACOBY, ROY A.; LIU, WEILEY S.; LONG, YE; SHERMAN, MICHAEL B.; SUH, JAE M.; VILA, ALEJANDRO; MILLS, STEPHEN L.

    2016-01-01

    The goals of these experiments were to describe the morphology and synaptic connections of amacrine cells in the baboon retina that contain immunoreactive vesicular glutamate transporter 3 (vGluT3). These amacrine cells had the morphology characteristic of knotty bistratified type 1 cells, and their dendrites formed two plexuses on either side of the center of the inner plexiform layer. The primary dendrites received large synapses from amacrine cells, and the higher-order dendrites were both pre- and postsynaptic to other amacrine cells. Based on light microscopic immunolabeling results, these include AII cells and starburst cells, but not the polyaxonal amacrine cells tracer-coupled to ON parasol ganglion cells. The vGluT3 cells received input from ON bipolar cells at ribbon synapses and made synapses onto OFF bipolar cells, including the diffuse DB3a type. Many synapses from vGluT3 cells onto retinal ganglion cells were observed in both plexuses. At synapses where vGluT3 cells were presynaptic, two types of postsynaptic densities were observed; there were relatively thin ones characteristic of inhibitory synapses and relatively thick ones characteristic of excitatory synapses. In the light microscopic experiments with Neurobiotin-injected ganglion cells, vGluT3 cells made contacts with midget and parasol ganglion cells, including both ON and OFF types. Puncta containing immunoreactive gephyrin, an inhibitory synapse marker, were found at appositions between vGluT3 cells and each of the four types of labeled ganglion cells. The vGluT3 cells did not have detectable levels of immunoreactive γ-aminobutyric acid (GABA) or immunoreactive glycine transporter 1. Thus, the vGluT3 cells would be expected to have ON responses to light and make synapses onto neurons in both the ON and the OFF pathways. Taken with previous results, these findings suggest that vGluT3 cells release glycine at some of their output synapses and glutamate at others. PMID:26241195

  7. p115 is a general vesicular transport factor related to the yeast endoplasmic reticulum to Golgi transport factor Uso1p.

    PubMed Central

    Sapperstein, S K; Walter, D M; Grosvenor, A R; Heuser, J E; Waters, M G

    1995-01-01

    A recently discovered vesicular transport factor, termed p115, is required along with N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment proteins for in vitro Golgi transport. p115 is a peripheral membrane protein found predominantly on the Golgi. Biochemical and electron microscopic analyses indicate that p115 is an elongated homodimer with two globular "heads" and an extended "tail" reminiscent of myosin II. We have cloned and sequenced cDNAs for bovine and rat p115. The predicted translation products are 90% identical, and each can be divided into three domains. The predicted 108-kDa bovine protein consists of an N-terminal 73-kDa globular domain followed by a 29-kDa coiled-coil dimerization domain, a linker segment of 4 kDa, and a highly acidic domain of 3 kDa. p115 is related to Uso1p, a protein required for endoplasmic reticulum to Golgi vesicular transport in Saccharomyces cerevisiae, which has a similar "head-coil-acid" domain structure. The p115 and Uso1p heads are similar in size, have approximately 25% sequence identity, and possess two highly homologous regions (62% and 60% identity over 34 and 53 residues, respectively). There is a third region of homology (50% identity over 28 residues) between the coiled-coil and acidic domains. Although the acidic nature of the p115 and Uso1p C termini is conserved, the primary sequence is not. We discuss these results in light of the proposed function of p115 in membrane targeting and/or fusion. Images Fig. 1 Fig. 3 PMID:7831323

  8. Multiple vacuoles in impaired tonoplast trafficking3 mutants are independent organelles.

    PubMed

    Zheng, Jiameng; Won Han, Sang; Munnik, Teun; Rojas-Pierce, Marcela

    2014-08-13

    Plant vacuoles are essential and dynamic organelles, and mechanisms of vacuole biogenesis and fusion are not well characterized. We recently demonstrated that Wortmannin, an inhibitor of Phosphatidylinositol-3-Kinase (PI3K), induces the fusion of plant vacuoles both in roots of itt3/vti11 mutant alleles and in guard cells of wild type Arabidopsis and Fava bean. Here we used Fluorescence Recovery After Photobleaching (FRAP) to demonstrate that the vacuoles in itt3/vti11 are independent organelles. Furthermore, we used fluorescent protein reporters that bind specifically to Phosphatidylinositol-3-Phosphate (PtdIns(3)P) or PtdIns(4)P to show that Wortmannin treatments that induce the fusion of vti11 vacuoles result in the loss of PtdIns(3)P from cellular membranes. These results provided supporting evidence for a critical role of PtdIns(3)P in vacuole fusion in roots and guard cells. PMID:25119109

  9. Multiple vacuoles in impaired tonoplast trafficking3 mutants are independent organelles

    PubMed Central

    Zheng, Jiameng; Han, Sang Won; Munnik, Teun; Rojas-Pierce, Marcela

    2014-01-01

    Plant vacuoles are essential and dynamic organelles, and mechanisms of vacuole biogenesis and fusion are not well characterized. We recently demonstrated that Wortmannin, an inhibitor of Phosphatidylinositol 3-Kinase (PI3K), induces the fusion of plant vacuoles both in roots of itt3/vti11 mutant alleles and in guard cells of wild type Arabidopsis and Fava bean. Here we used Fluorescence Recovery After Photobleaching (FRAP) to demonstrate that the vacuoles in itt3/vti11 are independent organelles. Furthermore, we used fluorescent protein reporters that bind specifically to Phosphatidylinositol 3-Phosphate (PtdIns(3)P) or PtdIns(4)P to show that Wortmannin treatments that induce the fusion of vti11 vacuoles result in the loss of PtdIns(3)P from cellular membranes. These results provided supporting evidence for a critical role of PtdIns(3)P in vacuole fusion in roots and guard cells. PMID:25482812

  10. Multiple vacuoles in impaired tonoplast trafficking3 mutants are independent organelles.

    PubMed

    Zheng, Jiameng; Han, Sang Won; Munnik, Teun; Rojas-Pierce, Marcela

    2014-01-01

    Plant vacuoles are essential and dynamic organelles, and mechanisms of vacuole biogenesis and fusion are not well characterized. We recently demonstrated that Wortmannin, an inhibitor of Phosphatidylinositol 3-Kinase (PI3K), induces the fusion of plant vacuoles both in roots of itt3/vti11 mutant alleles and in guard cells of wild type Arabidopsis and Fava bean. Here we used Fluorescence Recovery After Photobleaching (FRAP) to demonstrate that the vacuoles in itt3/vti11 are independent organelles. Furthermore, we used fluorescent protein reporters that bind specifically to Phosphatidylinositol 3-Phosphate (PtdIns(3)P) or PtdIns(4)P to show that Wortmannin treatments that induce the fusion of vti11 vacuoles result in the loss of PtdIns(3)P from cellular membranes. These results provided supporting evidence for a critical role of PtdIns(3)P in vacuole fusion in roots and guard cells. PMID:25482812

  11. Biogenesis and subcellular organization of the magnetosome organelles of magnetotactic bacteria

    PubMed Central

    Greene, Shannon E.; Komeili, Arash

    2013-01-01

    Bacterial cells, like their eukaryotic counterparts, are capable of constructing lipid-based organelles that carry out essential biochemical functions. The magnetosomes of magnetotactic bacteria are one such compartment that is quickly becoming a model for exploring the process of organelle biogenesis in bacteria. Magnetosomes consist of a lipid-bilayer compartment that houses a magnetic crystal. By arranging magnetosomes into chains within the cell, magnetotactic bacteria create an internal compass that is used for navigation along magnetic fields. Over the past decade, a number of studies have elucidated the possible factors involved in the formation of the magnetosome membrane and biomineralization of magnetic minerals. Here, we highlight some of these recent advances with a particular focus on the cell biology of magnetosome formation. PMID:22726584

  12. Resonance Raman Probes for Organelle-Specific Labeling in Live Cells.

    PubMed

    Kuzmin, Andrey N; Pliss, Artem; Lim, Chang-Keun; Heo, Jeongyun; Kim, Sehoon; Rzhevskii, Alexander; Gu, Bobo; Yong, Ken-Tye; Wen, Shangchun; Prasad, Paras N

    2016-01-01

    Raman microspectroscopy provides for high-resolution non-invasive molecular analysis of biological samples and has a breakthrough potential for dissection of cellular molecular composition at a single organelle level. However, the potential of Raman microspectroscopy can be fully realized only when novel types of molecular probes distinguishable in the Raman spectroscopy modality are developed for labeling of specific cellular domains to guide spectrochemical spatial imaging. Here we report on the design of a next generation Raman probe, based on BlackBerry Quencher 650 compound, which provides unprecedentedly high signal intensity through the Resonance Raman (RR) enhancement mechanism. Remarkably, RR enhancement occurs with low-toxic red light, which is close to maximum transparency in the biological optical window. The utility of proposed RR probes was validated for targeting lysosomes in live cultured cells, which enabled identification and subsequent monitoring of dynamic changes in this organelle by Raman imaging. PMID:27339882

  13. Active diffusion and microtubule-based transport oppose myosin forces to position organelles in cells

    PubMed Central

    Lin, Congping; Schuster, Martin; Guimaraes, Sofia Cunha; Ashwin, Peter; Schrader, Michael; Metz, Jeremy; Hacker, Christian; Gurr, Sarah Jane; Steinberg, Gero

    2016-01-01

    Even distribution of peroxisomes (POs) and lipid droplets (LDs) is critical to their role in lipid and reactive oxygen species homeostasis. How even distribution is achieved remains elusive, but diffusive motion and directed motility may play a role. Here we show that in the fungus Ustilago maydis ∼95% of POs and LDs undergo diffusive motions. These movements require ATP and involve bidirectional early endosome motility, indicating that microtubule-associated membrane trafficking enhances diffusion of organelles. When early endosome transport is abolished, POs and LDs drift slowly towards the growing cell end. This pole-ward drift is facilitated by anterograde delivery of secretory cargo to the cell tip by myosin-5. Modelling reveals that microtubule-based directed transport and active diffusion support distribution, mobility and mixing of POs. In mammalian COS-7 cells, microtubules and F-actin also counteract each other to distribute POs. This highlights the importance of opposing cytoskeletal forces in organelle positioning in eukaryotes. PMID:27251117

  14. Toxoplasma Rhoptries: Unique Secretory Organelles and Source of Promising Vaccine Proteins for Immunoprevention of Toxoplasmosis

    PubMed Central

    Dlugonska, Henryka

    2008-01-01

    Toxoplasma gondii is an obligate intracellular protozoan parasite classified in the phylum Apicomplexa, which includes numerous notable human and animal pathogens (Plasmodium species, Cryptosporidium species, Neospora caninum, etc.). The invasive stages of apicomplexans are characterized by the presence of an apical complex composed of specialized cytoskeletal and secretory organelles, including rhoptries. Rhoptries, unique apical secretory organelles shared exclusively by all apicomplexan parasites, are known to be involved in an active parasite's penetration into the host cell associated with the biogenesis of specific intracellular compartment, parasitophorous vacuole in which the parasite multiplies intensively, avoiding intracellular killing. Due to the key biological role of rhoptries, rhoptry proteins have recently become vaccine candidates for the prevention of several parasitoses, toxoplasmosis among them. The article presents current data on T. gondii rhoptries biology and new approaches to the development of effective vaccines against toxoplasmosis using rhoptry antigens. PMID:18670609

  15. Organelle biogenesis and interorganellar connections: Better in contact than in isolation.

    PubMed

    Daniele, Tiziana; Schiaffino, Maria Vittoria

    2014-01-01

    Membrane contact sites (MCSs) allow the exchange of molecules and information between organelles, even when their membranes cannot fuse directly. In recent years, a number of functions have been attributed to these contacts, highlighting their critical role in cell homeostasis. Although inter-organellar connections typically involve the endoplasmic reticulum (ER), we recently reported the presence of a novel MCSs between melanosomes and mitochondria. Melanosome-mitochondrion contacts appear mediated by fibrillar bridges resembling the protein tethers linking mitochondria and the ER, both for their ultrastructural features and the involvement of Mitofusin 2. The frequency of these connections correlates spatially and timely with melanosome biogenesis, suggesting a functional link between the 2 processes and in general that organelle biogenesis in the secretory pathway requires interorganellar crosstalks at multiple steps. Here, we summarize the different functions attributed to MCSs, and discuss their possible relevance for the newly identified melanosome-mitochondrion liaison. PMID:25346798

  16. Active diffusion and microtubule-based transport oppose myosin forces to position organelles in cells

    NASA Astrophysics Data System (ADS)

    Lin, Congping; Schuster, Martin; Guimaraes, Sofia Cunha; Ashwin, Peter; Schrader, Michael; Metz, Jeremy; Hacker, Christian; Gurr, Sarah Jane; Steinberg, Gero

    2016-06-01

    Even distribution of peroxisomes (POs) and lipid droplets (LDs) is critical to their role in lipid and reactive oxygen species homeostasis. How even distribution is achieved remains elusive, but diffusive motion and directed motility may play a role. Here we show that in the fungus Ustilago maydis ~95% of POs and LDs undergo diffusive motions. These movements require ATP and involve bidirectional early endosome motility, indicating that microtubule-associated membrane trafficking enhances diffusion of organelles. When early endosome transport is abolished, POs and LDs drift slowly towards the growing cell end. This pole-ward drift is facilitated by anterograde delivery of secretory cargo to the cell tip by myosin-5. Modelling reveals that microtubule-based directed transport and active diffusion support distribution, mobility and mixing of POs. In mammalian COS-7 cells, microtubules and F-actin also counteract each other to distribute POs. This highlights the importance of opposing cytoskeletal forces in organelle positioning in eukaryotes.

  17. Evaluation of predictions of the stochastic model of organelle production based on exact distributions

    PubMed Central

    Craven, C Jeremy

    2016-01-01

    We present a reanalysis of the stochastic model of organelle production and show that the equilibrium distributions for the organelle numbers predicted by this model can be readily calculated in three different scenarios. These three distributions can be identified as standard distributions, and the corresponding exact formulae for their mean and variance can therefore be used in further analysis. This removes the need to rely on stochastic simulations or approximate formulae (derived using the fluctuation dissipation theorem). These calculations allow for further analysis of the predictions of the model. On the basis of this we question the extent to which the model can be used to conclude that peroxisome biogenesis is dominated by de novo production when Saccharomyces cerevisiae cells are grown on glucose medium. DOI: http://dx.doi.org/10.7554/eLife.10167.001 PMID:26783763

  18. Active diffusion and microtubule-based transport oppose myosin forces to position organelles in cells.

    PubMed

    Lin, Congping; Schuster, Martin; Guimaraes, Sofia Cunha; Ashwin, Peter; Schrader, Michael; Metz, Jeremy; Hacker, Christian; Gurr, Sarah Jane; Steinberg, Gero

    2016-01-01

    Even distribution of peroxisomes (POs) and lipid droplets (LDs) is critical to their role in lipid and reactive oxygen species homeostasis. How even distribution is achieved remains elusive, but diffusive motion and directed motility may play a role. Here we show that in the fungus Ustilago maydis ∼95% of POs and LDs undergo diffusive motions. These movements require ATP and involve bidirectional early endosome motility, indicating that microtubule-associated membrane trafficking enhances diffusion of organelles. When early endosome transport is abolished, POs and LDs drift slowly towards the growing cell end. This pole-ward drift is facilitated by anterograde delivery of secretory cargo to the cell tip by myosin-5. Modelling reveals that microtubule-based directed transport and active diffusion support distribution, mobility and mixing of POs. In mammalian COS-7 cells, microtubules and F-actin also counteract each other to distribute POs. This highlights the importance of opposing cytoskeletal forces in organelle positioning in eukaryotes. PMID:27251117

  19. Quantitatively Mapping Cellular Viscosity with Detailed Organelle Information via a Designed PET Fluorescent Probe

    NASA Astrophysics Data System (ADS)

    Liu, Tianyu; Liu, Xiaogang; Spring, David R.; Qian, Xuhong; Cui, Jingnan; Xu, Zhaochao

    2014-06-01

    Viscosity is a fundamental physical parameter that influences diffusion in biological processes. The distribution of intracellular viscosity is highly heterogeneous, and it is challenging to obtain a full map of cellular viscosity with detailed organelle information. In this work, we report 1 as the first fluorescent viscosity probe which is able to quantitatively map cellular viscosity with detailed organelle information based on the PET mechanism. This probe exhibited a significant ratiometric fluorescence intensity enhancement as solvent viscosity increases. The emission intensity increase was attributed to combined effects of the inhibition of PET due to restricted conformational access (favorable for FRET, but not for PET), and the decreased PET efficiency caused by viscosity-dependent twisted intramolecular charge transfer (TICT). A full map of subcellular viscosity was successfully constructed via fluorescent ratiometric detection and fluorescence lifetime imaging; it was found that lysosomal regions in a cell possess the highest viscosity, followed by mitochondrial regions.

  20. Resonance Raman Probes for Organelle-Specific Labeling in Live Cells

    NASA Astrophysics Data System (ADS)

    Kuzmin, Andrey N.; Pliss, Artem; Lim, Chang-Keun; Heo, Jeongyun; Kim, Sehoon; Rzhevskii, Alexander; Gu, Bobo; Yong, Ken-Tye; Wen, Shangchun; Prasad, Paras N.

    2016-06-01

    Raman microspectroscopy provides for high-resolution non-invasive molecular analysis of biological samples and has a breakthrough potential for dissection of cellular molecular composition at a single organelle level. However, the potential of Raman microspectroscopy can be fully realized only when novel types of molecular probes distinguishable in the Raman spectroscopy modality are developed for labeling of specific cellular domains to guide spectrochemical spatial imaging. Here we report on the design of a next generation Raman probe, based on BlackBerry Quencher 650 compound, which provides unprecedentedly high signal intensity through the Resonance Raman (RR) enhancement mechanism. Remarkably, RR enhancement occurs with low-toxic red light, which is close to maximum transparency in the biological optical window. The utility of proposed RR probes was validated for targeting lysosomes in live cultured cells, which enabled identification and subsequent monitoring of dynamic changes in this organelle by Raman imaging.

  1. Organelle DNA haplotypes reflect crop-use characteristics and geographic origins of Cannabis sativa.

    PubMed

    Gilmore, Simon; Peakall, Rod; Robertson, James

    2007-10-25

    Comparative sequencing of cannabis individuals across 12 chloroplast and mitochondrial DNA loci revealed 7 polymorphic sites, including 5 length variable regions and 2 single nucleotide polymorphisms. Simple PCR assays were developed to assay these polymorphisms, and organelle DNA haplotypes were obtained for 188 cannabis individuals from 76 separate populations, including drug-type, fibre-type and wild populations. The haplotype data were analysed using parsimony, UPGMA and neighbour joining methods. Three haplotype groups were recovered by each analysis method, and these groups are suggestive of the crop-use characteristics and geographical origin of the populations, although not strictly diagnostic. We discuss the relationship between our haplotype data and taxonomic opinions of cannabis, and the implications of organelle DNA haplotyping to forensic investigations of cannabis. PMID:17293071

  2. Organelle RNA recognition motif-containing (ORRM) proteins are plastid and mitochondrial editing factors in Arabidopsis

    PubMed Central

    Shi, Xiaowen; Bentolila, Stephane; Hanson, Maureen R.

    2016-01-01

    ABSTRACT Post-transcriptional C-to-U RNA editing occurs at specific sites in plastid and plant mitochondrial transcripts. Members of the Arabidopsis pentatricopeptide repeat (PPR) motif-containing protein family and RNA-editing factor Interacting Protein (RIP, also known as MORF) family have been characterized as essential components of the RNA editing apparatus. Recent studies reveal that several organelle-targeted RNA recognition motif (RRM)-containing proteins are involved in either plastid or mitochondrial RNA editing. ORRM1 (Organelle RRM protein 1) is essential for plastid editing, whereas ORRM2, ORRM3 and ORRM4 are involved in mitochondrial RNA editing. The RRM domain of ORRM1, ORRM3 and ORRM4 is required for editing activity, whereas the auxiliary RIP and Glycine-Rich (GR) domains mediate the ORRM proteins' interactions with other editing factors. The identification of the ORRM proteins as RNA editing factors further expands our knowledge of the composition of the editosome. PMID:27082488

  3. Quantitatively Mapping Cellular Viscosity with Detailed Organelle Information via a Designed PET Fluorescent Probe

    PubMed Central

    Liu, Tianyu; Liu, Xiaogang; Spring, David R.; Qian, Xuhong; Cui, Jingnan; Xu, Zhaochao

    2014-01-01

    Viscosity is a fundamental physical parameter that influences diffusion in biological processes. The distribution of intracellular viscosity is highly heterogeneous, and it is challenging to obtain a full map of cellular viscosity with detailed organelle information. In this work, we report 1 as the first fluorescent viscosity probe which is able to quantitatively map cellular viscosity with detailed organelle information based on the PET mechanism. This probe exhibited a significant ratiometric fluorescence intensity enhancement as solvent viscosity increases. The emission intensity increase was attributed to combined effects of the inhibition of PET due to restricted conformational access (favorable for FRET, but not for PET), and the decreased PET efficiency caused by viscosity-dependent twisted intramolecular charge transfer (TICT). A full map of subcellular viscosity was successfully constructed via fluorescent ratiometric detection and fluorescence lifetime imaging; it was found that lysosomal regions in a cell possess the highest viscosity, followed by mitochondrial regions. PMID:24957323

  4. Resonance Raman Probes for Organelle-Specific Labeling in Live Cells

    PubMed Central

    Kuzmin, Andrey N.; Pliss, Artem; Lim, Chang-Keun; Heo, Jeongyun; Kim, Sehoon; Rzhevskii, Alexander; Gu, Bobo; Yong, Ken-Tye; Wen, Shangchun; Prasad, Paras N.

    2016-01-01

    Raman microspectroscopy provides for high-resolution non-invasive molecular analysis of biological samples and has a breakthrough potential for dissection of cellular molecular composition at a single organelle level. However, the potential of Raman microspectroscopy can be fully realized only when novel types of molecular probes distinguishable in the Raman spectroscopy modality are developed for labeling of specific cellular domains to guide spectrochemical spatial imaging. Here we report on the design of a next generation Raman probe, based on BlackBerry Quencher 650 compound, which provides unprecedentedly high signal intensity through the Resonance Raman (RR) enhancement mechanism. Remarkably, RR enhancement occurs with low-toxic red light, which is close to maximum transparency in the biological optical window. The utility of proposed RR probes was validated for targeting lysosomes in live cultured cells, which enabled identification and subsequent monitoring of dynamic changes in this organelle by Raman imaging. PMID:27339882

  5. Organelle RNA recognition motif-containing (ORRM) proteins are plastid and mitochondrial editing factors in Arabidopsis.

    PubMed

    Shi, Xiaowen; Bentolila, Stephane; Hanson, Maureen R

    2016-05-01

    Post-transcriptional C-to-U RNA editing occurs at specific sites in plastid and plant mitochondrial transcripts. Members of the Arabidopsis pentatricopeptide repeat (PPR) motif-containing protein family and RNA-editing factor Interacting Protein (RIP, also known as MORF) family have been characterized as essential components of the RNA editing apparatus. Recent studies reveal that several organelle-targeted RNA recognition motif (RRM)-containing proteins are involved in either plastid or mitochondrial RNA editing. ORRM1 (Organelle RRM protein 1) is essential for plastid editing, whereas ORRM2, ORRM3 and ORRM4 are involved in mitochondrial RNA editing. The RRM domain of ORRM1, ORRM3 and ORRM4 is required for editing activity, whereas the auxiliary RIP and Glycine-Rich (GR) domains mediate the ORRM proteins' interactions with other editing factors. The identification of the ORRM proteins as RNA editing factors further expands our knowledge of the composition of the editosome. PMID:27082488

  6. A stereological study on organelle distribution in human oocytes at prophase I.

    PubMed

    Pires-Luís, Ana Sílvia; Rocha, Eduardo; Bartosch, Carla; Oliveira, Elsa; Silva, Joaquina; Barros, Alberto; Sá, Rosália; Sousa, Mário

    2016-06-01

    The ultrastructural analysis of human oocytes at different maturation stages has only been descriptive. The aim of this study was to use a stereological approach to quantify the distribution of organelles in oocytes at prophase I (GV). Seven immature GV oocytes were processed for transmission electron microscopy and a classical manual stereological technique based on point-counting with an adequate stereological grid was used. The Kruskal-Wallis test and Mann-Whitney U-test with Bonferroni correction were used to compare the means of the relative volumes occupied by organelles in oocyte regions: cortex (C), subcortex (SC) and inner cytoplasm (IC). Here we first describe in GV oocytes very large vesicles of the smooth endoplasmic reticulum (SER), vesicles containing zona pellucida-like materials and coated vesicles. The most abundant organelles were the very large vesicles of the SER (6.9%), mitochondria (6.3%) and other SER vesicles (6.1%). Significant differences in organelle distribution were observed between ooplasm regions: cortical vesicles (C: 1.3% versus SC: 0.1%, IC: 0.1%, P = 0.001) and medium-sized vesicles containing zona pellucida-like materials (C: 0.2% versus SC: 0.02%, IC: 0%, P = 0.004) were mostly observed at the oocyte cortex, whereas mitochondria (C: 3.6% versus SC: 6.0%, IC: 7.2%, P = 0.005) were preferentially located in the subcortex and inner cytoplasm, and SER very large vesicles (IC: 10.1% versus C: 0.9%, SC: 1.67%, P = 0.001) in the oocyte inner cytoplasm. Further quantitative studies are needed in immature metaphase-I and mature metaphase-II oocytes, as well as analysis of correlations between ultrastructural and molecular data, to better understand human oocyte in vitro maturation. PMID:26170179

  7. Nanohole Array-Directed Trapping of Mammalian Mitochondria Enabling Single Organelle Analysis.

    PubMed

    Kumar, Shailabh; Wolken, Gregory G; Wittenberg, Nathan J; Arriaga, Edgar A; Oh, Sang-Hyun

    2015-12-15

    We present periodic nanohole arrays fabricated in free-standing metal-coated nitride films as a platform for trapping and analyzing single organelles. When a microliter-scale droplet containing mitochondria is dispensed above the nanohole array, the combination of evaporation and capillary flow directs individual mitochondria to the nanoholes. Mammalian mitochondria arrays were rapidly formed on chip using this technique without any surface modification steps, microfluidic interconnects, or external power sources. The trapped mitochondria were depolarized on chip using an ionophore with results showing that the organelle viability and behavior were preserved during the on-chip assembly process. Fluorescence signal related to mitochondrial membrane potential was obtained from single mitochondria trapped in individual nanoholes revealing statistical differences between the behavior of polarized vs depolarized mammalian mitochondria. This technique provides a fast and stable route for droplet-based directed localization of organelles-on-a-chip with minimal limitations and complexity, as well as promotes integration with other optical or electrochemical detection techniques. PMID:26593329

  8. Axonal transport of organelles visualized by light microscopy: cinemicrographic and computer analysis.

    PubMed

    Forman, D S; Padjen, A L; Siggins, G R

    1977-11-11

    Rapid movements of intra-axonal organelles in acutely isolated single myelinated fibers from bullfrog sciatic nerve were visualized by dark-field microscopy. The movements were recorded by cinemicrography, and analyzed by computer-based methods. The movements are saltatory and bidirectional, but each particle moves mainly in a single direction. For more than 90% of the particles, the predominant movement direction is retrograde, i.e. toward the cell body. Quantitative measurements on a variety of parameters of the organelle movements are presented. Different particles in the same axon show a broad range of mean speeds. The average mean speed of movement in the retrograde direction at 28 degrees C was 1.08 micrometer/sec (S.D. - 0.41), equivalent to an axonal transport rate of 93 mm/day. Disperse distributions were also found for other parameters such as the instantaneous velocities of individual particles. Quantal velocities, periodic movement patterns, and specific 'channels' were not detected. When the data from a population of particles is treated statistically, the average mean speed, the distribution of velocities, and other statistical parameters are found to be similar in different axons studied at the same temperature. Direct microscopical observation of axonal organelle movement is a technique which provides information about axonal transport which is different from and complementary to that obtained from enzyme accumulation of radioactive tracer methods. PMID:72584

  9. Viral Reorganization of the Secretory Pathway Generates Distinct Organelles for RNA Replication

    PubMed Central

    Hsu, Nai-Yun; Ilnytska, Olha; Belov, Georgiy; Santiana, Marianita; Chen, Ying-Han; Takvorian, Peter M.; Pau, Cyrilla; van der Schaar, Hilde; Kaushik-Basu, Neerja; Balla, Tamas; Cameron, Craig E.; Ehrenfeld, Ellie; van Kuppeveld, Frank J.M.; Altan-Bonnet, Nihal

    2010-01-01

    SUMMARY Many RNA viruses remodel intracellular membranes to generate specialized sites for RNA replication. How membranes are remodeled and what properties make them conducive for replication are unknown. Here we show how RNA viruses can manipulate multiple components of the cellular secretory pathway to generate organelles specialized for replication that are distinct in protein and lipid composition from the host cell. Specific viral proteins modulate effector recruitment by Arf1 GTPase and its guanine nucleotide exchange factor GBF1, promoting preferential recruitment of phosphatidylinositol-4-kinase IIIβ (PI4KIIIβ) to membranes over coat proteins, yielding uncoated phosphatidylinositol-4-phosphate (PI4P) lipid-enriched organelles. The PI4P-rich lipid micro-environment is essential for both enteroviral and flaviviral RNA replication; PI4KIIIβ inhibition interferes with this process; and enteroviral RNA polymerases specifically bind PI4P. These findings reveal how RNA viruses can selectively exploit specific elements of the host to form specialized organelles where cellular phosphoinositide lipids are key to regulating viral RNA replication. PMID:20510927

  10. New Insights Into Roles of Ubiquitin Modification in Regulating Plastids and Other Endosymbiotic Organelles.

    PubMed

    Broad, W; Ling, Q; Jarvis, P

    2016-01-01

    Recent findings have revealed important and diverse roles for the ubiquitin modification of proteins in the regulation of endosymbiotic organelles, which include the primary plastids of plants as well as complex plastids: the secondary endosymbiotic organelles of cryptophytes, alveolates, stramenopiles, and haptophytes. Ubiquitin modifications have a variety of potential consequences, both to the modified protein itself and to cellular regulation. The ubiquitin-proteasome system (UPS) can target individual proteins for selective degradation by the cytosolic 26S proteasome. Ubiquitin modifications can also signal the removal of whole endosymbiotic organelles, for example, via autophagy as has been well characterized in mitochondria. As plastids must import over 90% of their proteins from the cytosol, the observation that the UPS selectively targets the plastid protein import machinery is particularly significant. In this way, the UPS may influence the development and interconversions of different plastid types, as well as plastid responses to stress, by reconfiguring the organellar proteome. In complex plastids, the Symbiont-derived ERAD-Like Machinery (SELMA) has coopted the protein transport capabilities of the ER-Associated Degradation (ERAD) system, whereby misfolded proteins are retrotranslocated from ER for proteasomal degradation, uncoupling them from proteolysis: SELMA components have been retargeted to the second outermost plastid membrane to mediate protein import. In spite of this wealth of new information, there still remain a large number of unanswered questions and a need to define the roles of ubiquitin modification further in the regulation of plastids. PMID:27241217

  11. Towards understanding the evolution and functional diversification of DNA-containing plant organelles.

    PubMed

    Leister, Dario

    2016-01-01

    Plastids and mitochondria derive from prokaryotic symbionts that lost most of their genes after the establishment of endosymbiosis. In consequence, relatively few of the thousands of different proteins in these organelles are actually encoded there. Most are now specified by nuclear genes. The most direct way to reconstruct the evolutionary history of plastids and mitochondria is to sequence and analyze their relatively small genomes. However, understanding the functional diversification of these organelles requires the identification of their complete protein repertoires - which is the ultimate goal of organellar proteomics. In the meantime, judicious combination of proteomics-based data with analyses of nuclear genes that include interspecies comparisons and/or predictions of subcellular location is the method of choice. Such genome-wide approaches can now make use of the entire sequences of plant nuclear genomes that have emerged since 2000. Here I review the results of these attempts to reconstruct the evolution and functions of plant DNA-containing organelles, focusing in particular on data from nuclear genomes. In addition, I discuss proteomic approaches to the direct identification of organellar proteins and briefly refer to ongoing research on non-coding nuclear DNAs of organellar origin (specifically, nuclear mitochondrial DNA and nuclear plastid DNA). PMID:26998248

  12. Helical repeats modular proteins are major players for organelle gene expression.

    PubMed

    Hammani, Kamel; Bonnard, Géraldine; Bouchoucha, Ayoub; Gobert, Anthony; Pinker, Franziska; Salinas, Thalia; Giegé, Philippe

    2014-05-01

    Mitochondria and chloroplasts are often described as semi-autonomous organelles because they have retained a genome. They thus require fully functional gene expression machineries. Many of the required processes going all the way from transcription to translation have specificities in organelles and arose during eukaryote history. Most factors involved in these RNA maturation steps have remained elusive for a long time. The recent identification of a number of novel protein families including pentatricopeptide repeat proteins, half-a-tetratricopeptide proteins, octotricopeptide repeat proteins and mitochondrial transcription termination factors has helped to settle long-standing questions regarding organelle gene expression. In particular, their functions have been related to replication, transcription, RNA processing, RNA editing, splicing, the control of RNA turnover and translation throughout eukaryotes. These families of proteins, although evolutionary independent, seem to share a common overall architecture. For all of them, proteins contain tandem arrays of repeated motifs. Each module is composed of two to three α-helices and their succession forms a super-helix. Here, we review the features characterising these protein families, in particular, their distribution, the identified functions and mode of action and propose that they might share similar substrate recognition mechanisms. PMID:24021622

  13. Involvement of Rab6a in organelle rearrangement and cytoskeletal organization during mouse oocyte maturation

    PubMed Central

    Ma, Rujun; Zhang, Jiaqi; Liu, Xiaohui; Li, Ling; Liu, Honglin; Rui, Rong; Gu, Ling; Wang, Qiang

    2016-01-01

    Rab GTPases have been reported to define the identity and transport routes of vesicles. Rab6 is one of the most extensively studied Rab proteins involved in regulating organelle trafficking and integrity maintenance. However, to date, the function of Rab6 in mammalian oocytes has not been addressed. Here we report severe disorganization of endoplasmic reticulum upon specific knockdown of Rab6a in mouse oocytes. In line with this finding, intracellular Ca2+ stores are accordingly reduced in Rab6a-depleted oocytes. Furthermore, in these oocytes, we observe the absence of cortical granule free domain, which is a kind of special organelle in matured oocytes and its exocytosis is calcium dependent. On the other hand, following Rab6a knockdown, the prominent defects of cytoskeletal structures are detected during oocyte meiosis. In particular, the majority of Rab6a-depleted oocytes fail to form the actin cap, and the frequency of spindle defects and chromosome misalignment is significantly elevated. In summary, our data reveal that Rab6a not only participates in modulating the organization of oocyte organelles, but also is a novel regulator of meiotic apparatus in mammalian oocytes. PMID:27030207

  14. Crystal Structures of DNA-Whirly Complexes and Their Role in Arabidopsis Organelle Genome Repair

    SciTech Connect

    Cappadocia, Laurent; Maréchal, Alexandre; Parent, Jean-Sébastien; Lepage, Étienne; Sygusch, Jurgen; Brisson, Normand

    2010-09-07

    DNA double-strand breaks are highly detrimental to all organisms and need to be quickly and accurately repaired. Although several proteins are known to maintain plastid and mitochondrial genome stability in plants, little is known about the mechanisms of DNA repair in these organelles and the roles of specific proteins. Here, using ciprofloxacin as a DNA damaging agent specific to the organelles, we show that plastids and mitochondria can repair DNA double-strand breaks through an error-prone pathway similar to the microhomology-mediated break-induced replication observed in humans, yeast, and bacteria. This pathway is negatively regulated by the single-stranded DNA (ssDNA) binding proteins from the Whirly family, thus indicating that these proteins could contribute to the accurate repair of plant organelle genomes. To understand the role of Whirly proteins in this process, we solved the crystal structures of several Whirly-DNA complexes. These reveal a nonsequence-specific ssDNA binding mechanism in which DNA is stabilized between domains of adjacent subunits and rendered unavailable for duplex formation and/or protein interactions. Our results suggest a model in which the binding of Whirly proteins to ssDNA would favor accurate repair of DNA double-strand breaks over an error-prone microhomology-mediated break-induced replication repair pathway.

  15. Surface Organelles Assembled by Secretion Systems of Gram-Negative Bacteria: Diversity in Structure and Function

    PubMed Central

    Thanassi, David G.; Bliska, James B.; Christie, Peter J.

    2012-01-01

    Gram-negative bacteria express a wide variety of organelles on their cell surface. These surface structures may be the end products of secretion systems, such as the hair-like fibers assembled by the chaperone/usher and type IV pilus pathways, which generally function in adhesion to surfaces and bacterial-bacterial and bacterial-host interactions. Alternatively, the surface organelles may be integral components of the secretion machinery itself, such as the needle complex and pilus extensions formed by the type III and type IV secretion systems, which function in the delivery of bacterial effectors inside host cells. Bacterial surface structures perform functions critical for pathogenesis and have evolved to withstand forces exerted by the external environment and cope with defenses mounted by the host immune system. Given their essential roles in pathogenesis and exposed nature, bacterial surface structures also make attractive targets for therapeutic intervention. This review will describe the structure and function of surface organelles assembled by four different Gram-negative bacterial secretion systems: the chaperone/usher pathway, the type IV pilus pathway, and the type III and type IV secretion systems. PMID:22545799

  16. Organelle sedimentation in gravitropic roots of Limnobium is restricted to the elongation zone

    NASA Technical Reports Server (NTRS)

    Sack, F. D.; Kim, D.; Stein, B.

    1994-01-01

    Roots of the aquatic angiosperm Limnobium spongia (Bosc) Steud. were evaluated by light and electron microscopy to determine the distribution of organelle sedimentation towards gravity. Roots of Limnobium are strongly gravitropic. The rootcap consists of only two layers of cells. Although small amyloplasts are present in the central cap cells, no sedimentation of any organelle, including amyloplasts, was found. In contrast, both amyloplasts and nuclei sediment consistently and completely in cells of the elongation zone. Sedimentation occurs in one cell layer of the cortex just outside the endodermis. Sedimentation of both amyloplasts and nuclei begins in cells that are in their initial stages of elongation and persists at least to the level of the root where root hairs emerge. This is the first modern report of the presence of sedimentation away from, but not in, the rootcap. It shows that sedimentation in the rootcap is not necessary for gravitropic sensing in at least one angiosperm. If amyloplast sedimentation is responsible for gravitropic sensing, then the site of sensing in Limnobium roots is the elongation zone and not the rootcap. These data do not necessarily conflict with the hypothesis that sensing occurs in the cap in other roots, since Limnobium roots are exceptional in rootcap origin and structure, as well as in the distribution of organelle sedimentation. Similarly, if nuclear sedimentation is involved in gravitropic sensing, then nuclear mass would function in addition to, not instead of, that of amyloplasts.

  17. Towards understanding the evolution and functional diversification of DNA-containing plant organelles

    PubMed Central

    Leister, Dario

    2016-01-01

    Plastids and mitochondria derive from prokaryotic symbionts that lost most of their genes after the establishment of endosymbiosis. In consequence, relatively few of the thousands of different proteins in these organelles are actually encoded there. Most are now specified by nuclear genes. The most direct way to reconstruct the evolutionary history of plastids and mitochondria is to sequence and analyze their relatively small genomes. However, understanding the functional diversification of these organelles requires the identification of their complete protein repertoires – which is the ultimate goal of organellar proteomics. In the meantime, judicious combination of proteomics-based data with analyses of nuclear genes that include interspecies comparisons and/or predictions of subcellular location is the method of choice. Such genome-wide approaches can now make use of the entire sequences of plant nuclear genomes that have emerged since 2000. Here I review the results of these attempts to reconstruct the evolution and functions of plant DNA-containing organelles, focusing in particular on data from nuclear genomes. In addition, I discuss proteomic approaches to the direct identification of organellar proteins and briefly refer to ongoing research on non-coding nuclear DNAs of organellar origin (specifically, nuclear mitochondrial DNA and nuclear plastid DNA). PMID:26998248

  18. Polyvinyl Pyrrolidone-Assisted Solvothermal Synthesis of Fe3O4 Vesicular Nanospheres.

    PubMed

    Song, Hongfei; Liu, Meiying; Li, Sainan; Chen, Linlin; Lin, Chunming; Zhang, Liqing

    2015-05-01

    Monodispersed Fe3O4 vesicular nanospheres with a diameter of 160 nm have been fabricated solvothermally in the mixed solution of ethylene glycol (EG) and ethylenediamine (en) with the surfactant polyvinyl pyrrolidone (PVP). The microstructure and magnetic properties of the products were characterized by XRD, Raman, SEM, TEM, HRTEM, N2 adsorption-desorption and SQUID techniques. The HRTEM result shows that spherical Fe3O4 nanoparticles are structurally uniform with a distinct lattice spacing of 2.6 Å, which can be assigned to the (311) crystal facet of cubic Fe3O4. Besides, the as-obtained Fe3O4 vesicular nanospheres are ferromagnetic with a saturation magnetization of 86.9 emu/g as high as its bulk counterpart, demonstrating its promising applications in advanced magnetic materials and biomedicine. PMID:26505038

  19. Exclusion of close linkage between the synaptic vesicular monoamine transporter locus and schizophrenia spectrum disorders

    SciTech Connect

    Persico, A.M.; Uhl, G.R.; Wang, Zhe Wu

    1995-12-18

    The principal brain synaptic vesicular monoamine transporter (VMAT2) is responsible for the reuptake of serotonin, dopamine, norepinephrine, epinephrine, and histamine from the cytoplasm into synaptic vesicles, thus contributing to determination of the size of releasable neurotransmitter vesicular pools. Potential involvement of VMAT2 gene variants in the etiology of schizophrenia and related disorders was tested using polymorphic VMAT2 gene markers in 156 subjects from 16 multiplex pedigrees with schizophrenia, schizophreniform, schizoaffective, and schizotypal disorders and mood incongruent psychotic depression. Assuming genetic homogeneity, complete ({theta} = 0.0) linkage to the schizophrenia spectrum was excluded under both dominant and recessive models. Allelic variants at the VMAT2 locus do not appear to provide major genetic contributions to the etiology of schizophrenia spectrum disorders in these pedigrees. 16 refs.

  20. Rheumatoid arthritis and pseudo-vesicular skin plaques: rheumatoid neutrophilic dermatosis*

    PubMed Central

    Manriquez, Juan; Giesen, Laura; del Puerto, Constanza; Gonzalez, Sergio

    2016-01-01

    A 54 year-old woman with a 3-year history of rheumatoid arthritis (RA) consulted us because of weight loss, fever and skin eruption. On physical examination, erythematous plaques with a pseudo-vesicular appearance were seen on the back of both shoulders. Histological examination was consistent with rheumatoid neutrophilic dermatosis (RND). After 3 days of prednisone treatment, the skin eruption resolved. RND is a rare cutaneous manifestation of seropositive RA, characterized by asymptomatic, symmetrical erythematous plaques with a pseudo-vesicular appearance. Histology characteristically reveals a dense, neutrophilic infiltrate with leucocitoclasis but without other signs of vasculitis. Lesions may resolve spontaneously or with RA treatment. This case illustrates an uncommon skin manifestation of active rheumatoid arthritis. PMID:27579747

  1. High-Density Reconstitution of Functional Water Channels into Vesicular and Planar Block Copolymer Membranes

    PubMed Central

    2012-01-01

    The exquisite selectivity and unique transport properties of membrane proteins can be harnessed for a variety of engineering and biomedical applications if suitable membranes can be produced. Amphiphilic block copolymers (BCPs), developed as stable lipid analogs, form membranes that functionally incorporate membrane proteins and are ideal for such applications. While high protein density and planar membrane morphology are most desirable, BCP–membrane protein aggregates have so far been limited to low protein densities in either vesicular or bilayer morphologies. Here, we used dialysis to reproducibly form planar and vesicular BCP membranes with a high density of reconstituted aquaporin-0 (AQP0) water channels. We show that AQP0 retains its biological activity when incorporated at high density in BCP membranes, and that the morphology of the BCP–protein aggregates can be controlled by adjusting the amount of incorporated AQP0. We also show that BCPs can be used to form two-dimensional crystals of AQP0. PMID:23082933

  2. Vesicular Glutamate Transport Promotes Dopamine Storage and Glutamate Corelease In Vivo

    PubMed Central

    Hnasko, Thomas S.; Chuhma, Nao; Zhang, Hui; Goh, Germaine Y.; Sulzer, David; Palmiter, Richard D.; Rayport, Stephen; Edwards, Robert H.

    2010-01-01

    SUMMARY Dopamine neurons in the ventral tegmental area (VTA) play an important role in the motivational systems underlying drug addiction, and recent work has suggested that they also release the excitatory neurotransmitter glutamate. To assess a physiological role for glutamate corelease, we disrupted the expression of vesicular glutamate transporter 2 selectively in dopamine neurons. The conditional knockout abolishes glutamate release from midbrain dopamine neurons in culture and severely reduces their excitatory synaptic output in mesoaccumbens slices. Baseline motor behavior is not affected, but stimulation of locomotor activity by cocaine is impaired, apparently through a selective reduction of dopamine stores in the projection of VTA neurons to ventral striatum. Glutamate co-entry promotes monoamine storage by increasing the pH gradient that drives vesicular monoamine transport. Remarkably, low concentrations of glutamate acidify synaptic vesicles more slowly but to a greater extent than equimolar Cl−, indicating a distinct, presynaptic mechanism to regulate quantal size. PMID:20223200

  3. Cholinergic neurons and terminal fields revealed by immunohistochemistry for the vesicular acetylcholine transporter. II. The peripheral nervous system.

    PubMed

    Schäfer, M K; Eiden, L E; Weihe, E

    1998-05-01

    The peripheral sympathetic and parasympathetic cholinergic innervation was investigated with antibodies directed against the C-terminus of the rat vesicular acetylcholine transporter. Immunohistochemistry for the vesicular acetylcholine transporter resulted in considerably more detailed visualization of cholinergic terminal fields in the peripheral nervous system than reported previously and was well suited to also identify cholinergic perikarya. Vesicular acetylcholine transporter immunoreactivity completely delineated the preganglionic sympathetic terminals in pre- and paravertebral sympathetic ganglia, and in the adrenal medulla as well as postganglionic cholinergic neurons in the paravertebral chain. Cholinergic terminals of sudomotor and vasomotor nerves of skeletal muscle were optimally visualized. Mixed peripheral ganglia, including periprostatic and uterovaginal ganglia, exhibited extensive preganglionic cholinergic innervation of both noradrenergic and cholinergic postganglionic principal neurons which were intermingled in these ganglia. Varicose vesicular acetylcholine transporter-positive fibres and terminals, representing the cranial parasympathetic innervation of the cerebral vasculature, of salivary and lacrimal glands, of the eye, of the respiratory tract and of the upper digestive tract innervated various target structures including seromucous gland epithelium and myoepithelium, respiratory epithelium, and smooth muscle of the tracheobronchial tree. The only macrovascular elements receiving vesicular acetylcholine transporter-positive innervation were the cerebral arteries. The microvasculature throughout the viscera, with the exception of lymphoid tissues, the liver and kidney, received vesicular acetylcholine transporter-positive innervation while the microvasculature of limb and trunk skeletal muscle appeared to be the only relevant somatic target of vesicular acetylcholine transporter innervation. Vesicular acetylcholine transporter

  4. Continuous Microfluidic Self-Assembly of Hybrid Janus-Like Vesicular Motors: Autonomous Propulsion and Controlled Release.

    PubMed

    Wang, Lei; Liu, Yijing; He, Jie; Hourwitz, Matthew J; Yang, Yunlong; Fourkas, John T; Han, Xiaojun; Nie, Zhihong

    2015-08-01

    A microfluidic strategy is developed for the continuous fabrication of hybrid Janus vesicular motors that uniquely combine the capability of autonomous propulsion and externally controlled delivery of encapsulated payload. PMID:25925707

  5. The pollen organelle membrane proteome reveals highly spatial-temporal dynamics during germination and tube growth of lily pollen.

    PubMed

    Pertl, Heidi; Schulze, Waltraud X; Obermeyer, Gerhard

    2009-11-01

    As a first step in understanding the membrane-related dynamics during pollen grain germination and subsequent tube growth, the changes in protein abundance of membrane and membrane-associated proteins of 5 different membrane/organelle fractions were studied at physiologically important stages (0, 10, 30, 60, and 240 min) of Lilium longiflorum pollen in vitro culture. Proteins of each fraction and time point were identified by 'shot-gun' proteomics (LC-MS/MS). Analysis of more than 270 identified proteins revealed an increase in the abundance of proteins involved in cytoskeleton, carbohydrate and energy metabolism, as well as ion transport before pollen grain germination (10-30 min), whereas proteins involved in membrane/protein trafficking, signal transduction, stress response and protein biosynthesis decreased in abundance during this time. Proteins of amino acids and lipids/steroids metabolism, proteolysis, transcription, cell wall biosynthesis as well as nutrient transport showed a time-independent abundance profile. These spatiotemporal patterns were confirmed by immunodetection of specific proteins of the cellular processes membrane/protein trafficking and ion transport. Our results reveal major protein rearrangements at endomembranes and the plasma membrane before and as the pollen grains start tube growth. The spatiotemporal protein abundance changes correlate with the underlying developmental and physiological processes of the germinating pollen grain. PMID:19799449

  6. Axonal Segregation and Role of the Vesicular Glutamate Transporter VGLUT3 in Serotonin Neurons

    PubMed Central

    Voisin, Aurore N.; Mnie-Filali, Ouissame; Giguère, Nicolas; Fortin, Guillaume M.; Vigneault, Erika; El Mestikawy, Salah; Descarries, Laurent; Trudeau, Louis-Éric

    2016-01-01

    A subset of monoamine neurons releases glutamate as a cotransmitter due to presence of the vesicular glutamate transporters VGLUT2 or VGLUT3. In addition to mediating vesicular loading of glutamate, it has been proposed that VGLUT3 enhances serotonin (5-HT) vesicular loading by the vesicular monoamine transporter (VMAT2) in 5-HT neurons. In dopamine (DA) neurons, glutamate appears to be released from specialized subsets of terminals and it may play a developmental role, promoting neuronal growth and survival. The hypothesis of a similar developmental role and axonal localization of glutamate co-release in 5-HT neurons has not been directly examined. Using postnatal mouse raphe neurons in culture, we first observed that in contrast to 5-HT itself, other phenotypic markers of 5-HT axon terminals such as the 5-HT reuptake transporter (SERT) show a more restricted localization in the axonal arborization. Interestingly, only a subset of SERT- and 5-HT-positive axonal varicosities expressed VGLUT3, with SERT and VGLUT3 being mostly segregated. Using VGLUT3 knockout mice, we found that deletion of this transporter leads to reduced survival of 5-HT neurons in vitro and also decreased the density of 5-HT-immunoreactivity in terminals in the dorsal striatum and dorsal part of the hippocampus in the intact brain. Our results demonstrate that raphe 5-HT neurons express SERT and VGLUT3 mainly in segregated axon terminals and that VGLUT3 regulates the vulnerability of these neurons and the neurochemical identity of their axonal domain, offering new perspectives on the functional connectivity of a cell population involved in anxiety disorders and depression. PMID:27147980

  7. Efficacy study of vesicular gel containing methotrexate and menthol combination on parakeratotic rat skin model.

    PubMed

    Nagle, Amrita; Goyal, Amit K; Kesarla, Rajesh; Murthy, Rayasa R

    2011-06-01

    Methotrexate (MTX) is indicated in the symptomatic control of severe, recalcitrant, and disabling psoriasis. The oral or parenteral route of administration causes systemic toxicity. The topical route of delivery, though, reduces systemic toxicity and has limited applicability due to restricted permeability. Liposomal and niosomal MTX topical formulations have also been investigated with limited success to achieve drug localization in the skin. Menthol has been suggested in conditions of psoriasis, in addition to its skin-penetration-enhancing effect on drugs. The present work aimed at investigating the potential benefits of combining menthol with MTX in a vesicular gel base for not only improving the penetration and dermal availability of MTX, but also to render such a formulation more effective with greater patient acceptability. MTX liposomes were prepared by thin-film hydration, and the vesicles were characterized for drug-entrapment efficiency, size, and morphology. These liposomal vesicles were incorporated in a gel base, and this vesicular gel was evaluated for transdermal drug permeation and extent of drug accumulation in the skin, using a rat skin ex vivo model. Skin histology studies were carried out to investigate any structural changes caused by the permeation enhancers. Antipsoriatic efficacy of the formulations was tested in vivo, using the rat tail model. The results indicated that the vesicular gel containing menthol could cause maximum drug retention in the skin. The skin treated with menthol had a disrupted epidermis and microcavities. The in vivo studies also ascertained the effectiveness of the formulation in inducing a normal pattern of differentiation in the rat tail skin that initially showed parakeratosis, which is also characteristic of psoriatic epidermis. These results show the potential of vesicular gel containing MTX and menthol to improve penetration into the skin and cause drug retention in skin appendages. PMID:20557280

  8. Purification of Regucalcin from the Seminal Vesicular Fluid: A Calcium Binding Multi-Functional Protein.

    PubMed

    Harikrishna, P; Shende, A M; Reena, K K; Thomas, Jobin; Bhure, S K

    2016-08-01

    Regucalcin is a multi-functional protein having roles in calcium homeostasis as well as in anti-apoptotic, anti-prolific and anti-oxidative functions. Recently, it has been reported from the male reproductive tract, but its role in male reproduction needs further investigation; for which the native regucalcin of reproductive origin will be more appropriate. The gel exclusion chromatography followed by diethyl aminoethane cellulose chromatography and two-dimentional cellulose acetate membrane electrophoresis used for its purification are time consuming and less specific. Here, the regucalcin gene from buffalo testis has been cloned, expressed and purified in recombinant form, and subsequently used for raising hyper-immune serum. The Western blot of seminal vesicular fluid probed with anti-regucalcin polyclonal and monoclonal antibodies showed the presence of 28 and 34 kDa bands specific to regucalcin. Further, an affinity matrix has been prepared using anti-regucalcin polyclonal antibodies. An immuno-affinity chromatography method has been standardized to isolate regucalcin from seminal vesicular fluid. The initial complexity of the protein mixture in the seminal vesicular fluid has been reduced by a heat coagulation step. The purified protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band at 68 kDa that has been further confirmed as regucalcin by Liquid chromatography-mass spectrometry/mass spectrometry. The RGN purified from seminal vesicular fluid will be more appropriate for studying its possible role in male reproduction, especially sperm cell capacitation, hyperactivation, acrosome reaction and cryopreservation. The study can be applied in purifying regucalcin from different tissues or species with minor modifications in the methodology. PMID:27460579

  9. Inhibition of skin inflammation in mice by diclofenac in vesicular carriers: liposomes, ethosomes and PEVs.

    PubMed

    Caddeo, Carla; Sales, Octavio Diez; Valenti, Donatella; Saurí, Amparo Ruiz; Fadda, Anna Maria; Manconi, Maria

    2013-02-25

    Diclofenac-loaded phospholipid vesicles, namely conventional liposomes, ethosomes and PEVs (penetration enhancer-containing vesicles) were developed and their efficacy in TPA (phorbol ester) induced skin inflammation was examined. Vesicles were made from a cheap and unpurified mixture of phospholipids and diclofenac sodium; Transcutol P and propylene glycol were added to obtain PEVs, and ethanol to produce ethosomes. The structure and lamellar organization of the vesicle bilayer were investigated by transmission electron microscopy and small and wide angle X-ray scattering, as well as the main physico-chemical features. The formulations, along with a diclofenac solution and commercial Voltaren Emulgel, were tested in a comparative trial for anti-inflammatory efficacy on TPA-treated mice dorsal skin. Vesicles were around 100 nm, negatively charged, able to encapsulate diclofenac in good yields, and disclosed different lamellarity, as a function of the formulation composition. Vesicular formulations promoted drug accumulation and reduced the permeation. Administration of vesicular diclofenac on TPA-inflamed skin resulted in marked attenuation of oedema and leucocyte infiltration, especially using PEVs. Histology confirmed the effectiveness of vesicles, since they provided an amelioration of the tissual damage induced by TPA. The proposed approach based on vesicular nanocarriers may hold promising therapeutic value for treating a variety of inflammatory skin disorders. PMID:23299087

  10. Diagnostic value of Tzanck smear in various erosive, vesicular, and bullous skin lesions

    PubMed Central

    Yaeen, Atiya; Ahmad, Qazi Masood; Farhana, Anjum; Shah, Parveen; Hassan, Iffat

    2015-01-01

    Background: Cutaneous cytology has long been shown to be useful in the diagnosis of several erosive, vesicular, and bullous skin lesions. The Tzanck smear although an old tool, still remains a simple, rapid, easily applied, and inexpensive test for these skin lesions. Aims and Objectives: The aim of this study was to evaluate the diagnostic value of Tzanck smear by determining its sensitivity and specificity in various erosive, vesicular, and bullous skin lesions. Materials and Methods: One hundred and forty-two patients with erosive, vesicular, and/or bullous skin lesions were included in the study. Four groups of disorders were identified: infections, immunologic disorders, genodermatosis, and spongiotic dermatitis. All the study cases were evaluated by Tzanck smear. Definitive diagnosis was established by standard diagnostic techniques (including when appropriate, viral serology, bacterial culture, histopathology, direct immunoflourescence). Results: The sensitivity and specificity of cytologic findings was respectively 86.36% and 91.30% for viral infections; for bacterial infections, it was 85.7% and 66.6%. The sensitivity and specificity of Tzanck smear was respectively 85.0% and 83.33% for pemphigus; for bullous pemhigoid it was 11.11% and 100.0%. Tzanck smear sensitivity in genodermatoses was 100%. The sensitivity and specificity of the test in spongiotic dermatitis could not be calculated due to an insufficient number of patients. Conclusion: The Tzanck smear is a quick and reliable tool for the evaluation of various erosive and vesiculobullous skin lesions. PMID:26751561

  11. Experimental infection of Didelphis marsupialis with vesicular stomatitis New Jersey virus.

    PubMed

    Trujillo, Carlos M; Rodriguez, Luis; Rodas, Juan D; Arboleda, John Jairo

    2010-01-01

    Although vesicular stomatitis has been present for many years in the Americas, many aspects of its natural history remain undefined. In this study, we challenged five adult Virginia opossums (Didelphis marsupialis) with vesicular stomatitis New Jersey serotype virus (VSNJV). Opossums had no detectable antibodies against VSNJV prior to being inoculated with 10(6.5) median tissue culture infective doses (TCID(50)) of VSNJV by two routes; intraepithelial/subepithelial (IE/SE) inoculation and scarification in the muzzle (SM). Clinical response was monitored daily and animals were tested for viral shedding. All infected animals developed vesicles and ulcers on the tongue and inflammation of the nasal alar folds. Virus was isolated from esophagus-pharynx, nasal, and from ocular swabs and lesions samples. The failure to detect viremia in these animals indicates that a source other than blood may be required for transmission to insect vectors. Our results suggest that D. marsupialis could play a role in the maintenance of VSNJV outside of domestic animal populations and could provide a model to study vesicular stomatitis virus pathogenesis. PMID:20090034

  12. Vesicular erythema multiforme-like reaction to para-phenylenediamine in a henna tattoo.

    PubMed

    Sidwell, Rachel U; Francis, Nick D; Basarab, Tamara; Morar, Nilesh

    2008-01-01

    Allergic contact dermatitis reaction to topical "black henna" tattoo is usually described secondary to the organic dye para-phenylenediamine, a derivative of analine. Allergic contact dermatitis reactions to para-phenylenediamine are well recognized and most commonly involve an eczematous reaction that may become generalized and an acute angio-edema. Only four previous instances have been reported of an erythema multiforme-like reaction to para-phenylenediamine and its derivatives, including only one mild reaction to a tattoo. A vesicular erythema multiforme-like reaction has not been reported. An erythema multiforme-like reaction to contact allergens is usually caused by potent allergens including plant quinolones in Compositae and sesquiterpene lactones in exotic woods, and it is also reported to topical drugs, epoxy resin, metals (particularly nickel), and various chemicals. A generalized vesicular erythema multiforme-like reaction is unusual, and rarely reported. We describe a 6-year-old boy who developed a localized, eczematous and severe generalized vesicular erythema multiforme-like contact allergy to para-phenylenediamine secondary to a henna tattoo. As henna tattoos are becoming increasingly popular, one should be aware of the possibility of such a reaction. This presentation also highlights the call to ban the use of para-phenylenediamine and its derivatives in dyes. PMID:18429780

  13. In vitro percutaneous permeation and skin accumulation of finasteride using vesicular ethosomal carriers.

    PubMed

    Rao, Yuefeng; Zheng, Feiyue; Zhang, Xingguo; Gao, Jianqing; Liang, Wenquan

    2008-01-01

    In order to develop a novel transdermal drug delivery system that facilitates the skin permeation of finasteride encapsulated in novel lipid-based vesicular carriers (ethosomes)finasteride ethosomes were constructed and the morphological characteristics were studied by transmission electron microscopy. The particle size, zeta potential and the entrapment capacity of ethosome were also determined. In contrast to liposomes ethosomes were of more condensed vesicular structure and they were found to be oppositely charged. Ethosomes were found to be more efficient delivery carriers with high encapsulation capacities. In vitro percutaneous permeation experiments demonstrated that the permeation of finasteride through human cadaver skin was significantly increased when ethosomes were used. The finasteride transdermal fluxes from ethosomes containing formulation (1.34 +/- 0.11 microg/cm(2)/h) were 7.4, 3.2 and 2.6 times higher than that of finasteride from aqueous solution, conventional liposomes and hydroethanolic solution respectively (P < 0.01).Furthermore, ethosomes produced a significant (P < 0.01) finasteride accumulation in the skin, especially in deeper layers, for instance in dermis it reached to 18.2 +/- 1.8 microg/cm(2). In contrast, the accumulation of finasteride in the dermis was only 2.8 +/- 1.3 microg/cm(2) with liposome formulation. The study demonstrated that ethosomes are promising vesicular carriers for enhancing percutaneous absorption of finasteride. PMID:18649143

  14. Astrocytic vesicles and gliotransmitters: Slowness of vesicular release and synaptobrevin2-laden vesicle nanoarchitecture.

    PubMed

    Zorec, R; Verkhratsky, A; Rodríguez, J J; Parpura, V

    2016-05-26

    Neurotransmitters released at synapses activate neighboring astrocytes, which in turn, modulate neuronal activity by the release of diverse neuroactive substances that include classical neurotransmitters such as glutamate, GABA or ATP. Neuroactive substances are released from astrocytes through several distinct molecular mechanisms, for example, by diffusion through membrane channels, by translocation via plasmalemmal transporters or by vesicular exocytosis. Vesicular release regulated by a stimulus-mediated increase in cytosolic calcium involves soluble N-ethyl maleimide-sensitive fusion protein attachment protein receptor (SNARE)-dependent merger of the vesicle membrane with the plasmalemma. Up to 25 molecules of synaptobrevin 2 (Sb2), a SNARE complex protein, reside at a single astroglial vesicle; an individual neuronal, i.e. synaptic, vesicle contains ∼70 Sb2 molecules. It is proposed that this paucity of Sb2 molecules in astrocytic vesicles may determine the slow secretion. In the present essay we shall overview multiple aspects of vesicular architecture and types of vesicles based on their cargo and dynamics in astroglial cells. PMID:25727638

  15. Detection of multiple viral infections in cattle and buffalo with suspected vesicular disease in Brazil.

    PubMed

    Laguardia-Nascimento, Mateus; Sales, Érica Bravo; Gasparini, Marcela Ribeiro; de Souza, Natália Mendes; da Silva, Josiane Aparecida Gonçalina; Souza, Giovana Gonçalves; Carani, Fernanda Rezek; Dos Santos, Alyane Figueiredo; Rivetti Júnior, Anselmo Vasconcelos; Camargos, Marcelo Fernandes; Fonseca Júnior, Antônio Augusto

    2016-07-01

    Vesicular diseases are of high importance for livestock, primarily because of foot-and-mouth disease (FMD), which is a high-morbidity disease that generates direct losses caused by low milk production, weight loss, and indirect losses because of the need for sanitary barriers. Other vesicular diseases are also of importance for livestock because of direct impacts or because their clinical signs may be confused with those of FMD. We report herein the detection of multiple infections in cattle with suspected vesicular disease in the Brazilian states of Amazonas (AM), Mato Grosso (MT), and Roraima. Thirty-seven epithelial samples from cattle and 1 sample from a buffalo were sent to the laboratory for testing for FMDV and similar disease agents. All samples from MT were positive for parapoxvirus (Pseudocowpox virus and Bovine papular stomatitis virus). In addition, 3 samples were positive for Bluetongue virus, and 5 samples were positive for Bovine herpesvirus 1 Among these samples, 1 was positive for all of these 3 agents. Only 2 samples from AM were negative for parapoxvirus. The molecular tests conducted in this study detected multiple infections, with a high prevalence of parapoxvirus. PMID:27154321

  16. Effects of vesicular acetylcholine uptake blockers on frequency augmentation-potentiation in frog neuromuscular transmission.

    PubMed

    Maeno, T; Enomoto, K

    1994-03-01

    Vesamicol inhibits the vesicular loading of acetylcholine molecules. The effects of vesamicol and similarly acting compounds on neuromuscular transmission in frogs were investigated to determine whether these inhibitors-inhibit the frequency augmentation-potentiation of transmitter release. Various vesicular acetylcholine transport blockers suppressed the stimulation frequency-related release parameter, k, in a dose-dependent manner. Artane, cetiedil, chloroquine, ethodin, quinacrine, vesamicol and its benzyl-analogue, 2-(4-benzylpiperidino)cyclohexanol, had strong effects, while those of aminacrine, chlorpromazine, fluphenazine, imipramine, pyrilamine and thioridazine were weak. A significant correlation was observed between the biochemically reported values of IC50 and the electrophysiological inhibitory potencies on k at 20 microM. Contrary to expectations from the biochemical data, however, vesamicol and its benzyl-analogue showed equipotent inhibitory actions on the electrophysiological frequency augmentation-potentiation relation. Low sensitivity and low selectivity of the frequency augmentation-potentiation for vesamicol and its benzyl-analogue lead us to conclude that the vesicular acetylcholine transporter is not the site of the electrophysiological action of vesamicol and similarly acting chemicals. PMID:8008203

  17. Vesicular Monoamine and Glutamate Transporters Select Distinct Synaptic Vesicle Recycling Pathways

    PubMed Central

    Onoa, Bibiana; Li, Haiyan; Gagnon-Bartsch, Johann A.; Elias, Laura A. B.; Edwards, Robert H.

    2011-01-01

    Previous work has characterized the properties of neurotransmitter release at excitatory and inhibitory synapses, but we know remarkably little about the properties of monoamine release because these neuromodulators do not generally produce a fast ionotropic response. Since dopamine and serotonin neurons can also release glutamate in vitro and in vivo, we have used the vesicular monoamine transporter VMAT2 and the vesicular glutamate transporter VGLUT1 to compare the localization and recycling of synaptic vesicles that store, respectively, monoamines and glutamate. First, VMAT2 segregates partially from VGLUT1 in the boutons of midbrain dopamine neurons, indicating the potential for distinct release sites. Second, endocytosis after stimulation is slower for VMAT2 than VGLUT1. During the stimulus, however, the endocytosis of VMAT2 (but not VGLUT1) accelerates dramatically in midbrain dopamine but not hippocampal neurons, indicating a novel, cell-specific mechanism to sustain high rates of release. On the other hand, we find that in both midbrain dopamine and hippocampal neurons, a substantially smaller proportion of VMAT2 than VGLUT1 is available for evoked release, and VMAT2 shows considerably more dispersion along the axon after exocytosis than VGLUT1. Even when expressed in the same neuron, the two vesicular transporters thus target to distinct populations of synaptic vesicles, presumably due to their selection of distinct recycling pathways. PMID:20534840

  18. Spiroindolines Identify the Vesicular Acetylcholine Transporter as a Novel Target for Insecticide Action

    PubMed Central

    Sluder, Ann; Shah, Sheetal; Cassayre, Jérôme; Clover, Ralph; Maienfisch, Peter; Molleyres, Louis-Pierre; Hirst, Elizabeth A.; Flemming, Anthony J.; Shi, Min; Cutler, Penny; Stanger, Carole; Roberts, Richard S.; Hughes, David J.; Flury, Thomas; Robinson, Michael P.; Hillesheim, Elke; Pitterna, Thomas; Cederbaum, Fredrik; Worthington, Paul A.; Crossthwaite, Andrew J.; Windass, John D.; Currie, Richard A.; Earley, Fergus G. P.

    2012-01-01

    The efficacy of all major insecticide classes continues to be eroded by the development of resistance mediated, in part, by selection of alleles encoding insecticide insensitive target proteins. The discovery of new insecticide classes acting at novel protein binding sites is therefore important for the continued protection of the food supply from insect predators, and of human and animal health from insect borne disease. Here we describe a novel class of insecticides (Spiroindolines) encompassing molecules that combine excellent activity against major agricultural pest species with low mammalian toxicity. We confidently assign the vesicular acetylcholine transporter as the molecular target of Spiroindolines through the combination of molecular genetics in model organisms with a pharmacological approach in insect tissues. The vesicular acetylcholine transporter can now be added to the list of validated insecticide targets in the acetylcholine signalling pathway and we anticipate that this will lead to the discovery of novel molecules useful in sustaining agriculture. In addition to their potential as insecticides and nematocides, Spiroindolines represent the only other class of chemical ligands for the vesicular acetylcholine transporter since those based on the discovery of vesamicol over 40 years ago, and as such, have potential to provide more selective tools for PET imaging in the diagnosis of neurodegenerative disease. They also provide novel biochemical tools for studies of the function of this protein family. PMID:22563457

  19. A Ribbon-like Structure in the Ejective Organelle of the Green Microalga Pyramimonas parkeae (Prasinophyceae) Consists of Core Histones and Polymers Containing N-acetyl-glucosamine.

    PubMed

    Yamagishi, Takahiro; Kurihara, Akira; Kawai, Hiroshi

    2015-11-01

    The green microalga, Pyramimonas parkeae (Prasinophyceae) has an ejective organelle containing a coiled ribbon structure resembling the ejectisome in Cryptophyta. This structure is discharged from the cell by a stimulus and extends to form a tube-like structure, but the molecular components of the structure have not been identified. Tricine-SDS-PAGE analysis indicated that the ribbon-like structure of P. parkeae contains some proteins and low molecular acidic polymers. Edman degradation, LC/MS/MS analyses and immunological studies demonstrated that their proteins are core histones (H3, H2A, H2B and H4). In addition, monosaccharide composition analysis of the ribbon-like structures and degradation by lysozyme strongly indicated that the ribbon-like structure consist of β (1-4) linked polymers containing N-acetyl-glucosamine. Purified polymers and recombinant histones formed glob-like or filamentous structures. Therefore we conclude that the ribbon-like structure of P. parkeae mainly consists of a complex of core histones (H3, H2A, H2B and H4) and polymers containing N-acetyl-glucosamine, and suggest to name the ejective organelle in P. parkeae the "histrosome" to distinguish it from the ejectisome in Cryptophyta. PMID:26398336

  20. The molecular mechanism of organelle transport along microtubules: the identification and characterization of KIFs (kinesin superfamily proteins).

    PubMed

    Hirokawa, N

    1996-10-01

    In the cells various kinds of organelles are transported and distributed to their proper destinations in the cell. Organelle transports are very important for cellular morphogenesis and functions, with the conveying and targeting of essential materials to their correct destination being conducted, often at considerable velocities. Recently we have identified at least 10 new microtubule-associated motor proteins named as KIFs (kinesin superfamily proteins). Their characterization reveals that each member can convey a specific organelle or cargo, although there is some redundancy. It has also become clear that there are distinct subclasses of KIFs that form monomeric, heterodimeric and homodimeric motors. Molecular cell biological approaches combining multidisciplinary methods such as new electron microscopy, biochemistry, immunocytochemistry, biophysics, molecular biology and molecular genetics have revealed precise mechanisms of organelle transports in the cells by KIFs. PMID:9118241

  1. Characterization of a novel organelle in Toxoplasma gondii with similar composition and function to the plant vacuole

    PubMed Central

    Miranda, Kildare; Pace, Douglas A.; Cintron, Roxana; Rodrigues, Juliany C.F.; Fang, Jianmin; Smith, Alyssa; Rohloff, Peter; Coelho, Elvis; de Haas, Felix; de Souza, Wanderley; Coppens, Isabelle; Sibley, L. David; Moreno, Silvia N. J.

    2010-01-01

    Toxoplasma gondii belongs to the phylum Apicomplexa and is an important cause of congenital disease and infection in immunocompromised patients. Like most apicomplexans, Toxoplasma gondii possesses several plant-like features, such as the chloroplast-like organelle, the apicoplast. We describe and characterize a novel organelle in T. gondii tachyzoites, which is visible by light microscopy, and possesses a broad similarity to the plant vacuole. Electron tomography shows the interaction of this vacuole with other organelles. The presence of a plant-like vacuolar proton pyrophosphatase (TgVP1), a vacuolar proton ATPase, a cathepsin L-like protease (TgCPL), an aquaporin (TgAQP1), as well as Ca2+/H+ and Na+/H+ exchange activities, supports similarity to the plant vacuole. Biochemical characterization of TgVP1 in enriched fractions shows a functional similarity to the respective plant enzyme. The organelle is a Ca2+ store and appears to have protective effects against salt stress potentially linked to its sodium transport activity. In intracellular parasites, the organelle fragments, with some markers co-localizing with the late endosomal marker, Rab7, suggesting its involvement with the endocytic pathway. Studies on the characterization of this novel organelle will be relevant to the identification of novel targets for chemotherapy against T. gondii and other apicomplexan parasites as well. PMID:20398214

  2. Off to the Organelles - Killing Cancer Cells with Targeted Gold Nanoparticles

    PubMed Central

    Kodiha, Mohamed; Wang, Yi Meng; Hutter, Eliza; Maysinger, Dusica; Stochaj, Ursula

    2015-01-01

    Gold nanoparticles (AuNPs) are excellent tools for cancer cell imaging and basic research. However, they have yet to reach their full potential in the clinic. At present, we are only beginning to understand the molecular mechanisms that underlie the biological effects of AuNPs, including the structural and functional changes of cancer cells. This knowledge is critical for two aspects of nanomedicine. First, it will define the AuNP-induced events at the subcellular and molecular level, thereby possibly identifying new targets for cancer treatment. Second, it could provide new strategies to improve AuNP-dependent cancer diagnosis and treatment. Our review summarizes the impact of AuNPs on selected subcellular organelles that are relevant to cancer therapy. We focus on the nucleus, its subcompartments, and mitochondria, because they are intimately linked to cancer cell survival, growth, proliferation and death. While non-targeted AuNPs can damage tumor cells, concentrating AuNPs in particular subcellular locations will likely improve tumor cell killing. Thus, it will increase cancer cell damage by photothermal ablation, mechanical injury or localized drug delivery. This concept is promising, but AuNPs have to overcome multiple hurdles to perform these tasks. AuNP size, morphology and surface modification are critical parameters for their delivery to organelles. Recent strategies explored all of these variables, and surface functionalization has become crucial to concentrate AuNPs in subcellular compartments. Here, we highlight the use of AuNPs to damage cancer cells and their organelles. We discuss current limitations of AuNP-based cancer research and conclude with future directions for AuNP-dependent cancer treatment. PMID:25699096

  3. Optical tweezers for single molecule force spectroscopy on bacterial adhesion organelles

    NASA Astrophysics Data System (ADS)

    Andersson, Magnus; Axner, Ove; Uhlin, Bernt Eric; Fällman, Erik

    2006-08-01

    Instrumentation and methodologies for single molecule force spectroscopy on bacterial adhesion organelles by the use of force measuring optical tweezers have been developed. A thorough study of the biomechanical properties of fimbrial adhesion organelles expressed by uropathogenic E. coli, so-called pili, is presented. Steady-state as well as dynamic force measurements on P pili, expressed by E. coli causing pyelonephritis, have revealed, among other things, various unfolding and refolding properties of the helical structure of P pili, the PapA rod. Based on these properties an energy landscape model has been constructed by which specific biophysical properties of the PapA rod have been extracted, e.g. the number of subunits, the length of a single pilus, bond lengths and activation energies for bond opening and closure. Moreover, long time repetitive measurements have shown that the rod can be unfolded and refolded repetitive times without losing its intrinsic properties. These properties are believed to be of importance for the bacteria's ability to maintain close contact with host cells during initial infections. The results presented are considered to be of importance for the field of biopolymers in general and the development of new pharmaceuticals towards urinary tract infections in particular. The results show furthermore that the methodology can be used to gain knowledge of the intrinsic biomechanical function of adhesion organelles. The instrumentation is currently used for characterization of type 1 pili, expressed by E. coli causing cystitis, i.e. infections in the bladder. The first force spectrometry investigations of these pili will be presented.

  4. Threshold-free method for three-dimensional segmentation of organelles

    NASA Astrophysics Data System (ADS)

    Chan, Yee-Hung M.; Marshall, Wallace F.

    2012-03-01

    An ongoing challenge in the field of cell biology is to how to quantify the size and shape of organelles within cells. Automated image analysis methods often utilize thresholding for segmentation, but the calculated surface of objects depends sensitively on the exact threshold value chosen, and this problem is generally worse at the upper and lower zboundaries because of the anisotropy of the point spread function. We present here a threshold-independent method for extracting the three-dimensional surface of vacuoles in budding yeast whose limiting membranes are labeled with a fluorescent fusion protein. These organelles typically exist as a clustered set of 1-10 sphere-like compartments. Vacuole compartments and center points are identified manually within z-stacks taken using a spinning disk confocal microscope. A set of rays is defined originating from each center point and radiating outwards in random directions. Intensity profiles are calculated at coordinates along these rays, and intensity maxima are taken as the points the rays cross the limiting membrane of the vacuole. These points are then fit with a weighted sum of basis functions to define the surface of the vacuole, and then parameters such as volume and surface area are calculated. This method is able to determine the volume and surface area of spherical beads (0.96 to 2 micron diameter) with less than 10% error, and validation using model convolution methods produce similar results. Thus, this method provides an accurate, automated method for measuring the size and morphology of organelles and can be generalized to measure cells and other objects on biologically relevant length-scales.

  5. Redistribution of Golgi Stacks and Other Organelles during Mitosis and Cytokinesis in Plant Cells1[w

    PubMed Central

    Nebenführ, Andreas; Frohlick, Jennifer A.; Staehelin, L. Andrew

    2000-01-01

    We have followed the redistribution of Golgi stacks during mitosis and cytokinesis in living tobacco BY-2 suspension culture cells by means of a green fluorescent protein-tagged soybean α-1,2 mannosidase, and correlated the findings to cytoskeletal rearrangements and to the redistribution of endoplasmic reticulum, mitochondria, and plastids. In preparation for cell division, when the general streaming of Golgi stacks stops, about one-third of the peripheral Golgi stacks redistributes to the perinuclear cytoplasm, the phragmosome, thereby reversing the ratio of interior to cortical Golgi from 2:3 to 3:2. During metaphase, approximately 20% of all Golgi stacks aggregate in the immediate vicinity of the mitotic spindle and a similar number becomes concentrated in an equatorial region under the plasma membrane. This latter localization, the “Golgi belt,” accurately predicts the future site of cell division, and thus forms a novel marker for this region after the disassembly of the preprophase band. During telophase and cytokinesis, many Golgi stacks redistribute around the phragmoplast where the cell plate is formed. At the end of cytokinesis, the daughter cells have very similar Golgi stack densities. The sites of preferential Golgi stack localization are specific for this organelle and largely exclude mitochondria and plastids, although some mitochondria can approach the phragmoplast. This segregation of organelles is first observed in metaphase and persists until completion of cytokinesis. Maintenance of the distinct localizations does not depend on intact actin filaments or microtubules, although the mitotic spindle appears to play a major role in organizing the organelle distribution patterns. The redistribution of Golgi stacks during mitosis and cytokinesis is consistent with the hypothesis that Golgi stacks are repositioned to ensure equal partitioning between daughter cells as well as rapid cell plate assembly. PMID:10982429

  6. Monte Carlo analysis of obstructed diffusion in three dimensions: application to molecular diffusion in organelles.

    PubMed

    Olveczky, B P; Verkman, A S

    1998-05-01

    Molecular transport in the aqueous lumen of organelles involves diffusion in a confined compartment with complex geometry. Monte Carlo simulations of particle diffusion in three dimensions were carried out to evaluate the influence of organelle structure on diffusive transport and to relate experimental photobleaching data to intrinsic diffusion coefficients. Two organelle structures were modeled: a mitochondria-like long closed cylinder containing fixed luminal obstructions of variable number and size, and an endoplasmic reticulum-like network of interconnected cylinders of variable diameter and density. Trajectories were computed in each simulation for >10(5) particles, generally for >10(5) time steps. Computed time-dependent concentration profiles agreed quantitatively with analytical solutions of the diffusion equation for simple geometries. For mitochondria-like cylinders, significant slowing of diffusion required large or wide single obstacles, or multiple obstacles. In simulated spot photobleaching experiments, a approximately 25% decrease in apparent diffusive transport rate (defined by the time to 75% fluorescence recovery) was found for a single thin transverse obstacle occluding 93% of lumen area, a single 53%-occluding obstacle of width 16 lattice points (8% of cylinder length), 10 equally spaced 53% obstacles alternately occluding opposite halves of the cylinder lumen, or particle binding to walls (with mean residence time = 10 time steps). Recovery curve shape with obstacles showed long tails indicating anomalous diffusion. Simulations also demonstrated the utility of measurement of fluorescence depletion at a spot distant from the bleach zone. For a reticulum-like network, particle diffusive transport was mildly reduced from that in unobstructed three-dimensional space. In simulated photobleaching experiments, apparent diffusive transport was decreased by 39-60% in reticular structures in which 90-97% of space was occluded. These computations provide

  7. Myosin-Va and dynamic actin oppose microtubules to drive long-range organelle transport.

    PubMed

    Evans, Richard D; Robinson, Christopher; Briggs, Deborah A; Tooth, David J; Ramalho, Jose S; Cantero, Marta; Montoliu, Lluis; Patel, Shyamal; Sviderskaya, Elena V; Hume, Alistair N

    2014-08-01

    In animal cells, microtubule and actin tracks and their associated motors (dynein, kinesin, and myosin) are thought to regulate long- and short-range transport, respectively. Consistent with this, microtubules extend from the perinuclear centrosome to the plasma membrane and allow bidirectional cargo transport over long distances (>1 μm). In contrast, actin often comprises a complex network of short randomly oriented filaments, suggesting that myosin motors move cargo short distances. These observations underpin the "highways and local roads" model for transport along microtubule and actin tracks. The "cooperative capture" model exemplifies this view and suggests that melanosome distribution in melanocyte dendrites is maintained by long-range transport on microtubules followed by actin/myosin-Va-dependent tethering. In this study, we used cell normalization technology to quantitatively examine the contribution of microtubules and actin/myosin-Va to organelle distribution in melanocytes. Surprisingly, our results indicate that microtubules are essential for centripetal, but not centrifugal, transport. Instead, we find that microtubules retard a centrifugal transport process that is dependent on myosin-Va and a population of dynamic F-actin. Functional analysis of mutant proteins indicates that myosin-Va works as a transporter dispersing melanosomes along actin tracks whose +/barbed ends are oriented toward the plasma membrane. Overall, our data highlight the role of myosin-Va and actin in transport, and not tethering, and suggest a new model in which organelle distribution is determined by the balance between microtubule-dependent centripetal and myosin-Va/actin-dependent centrifugal transport. These observations appear to be consistent with evidence coming from other systems showing that actin/myosin networks can drive long-distance organelle transport and positioning. PMID:25065759

  8. Disorders of Lysosome-related Organelle Biogenesis: Clinical and Molecular Genetics

    PubMed Central

    Huizing, Marjan; Helip-Wooley, Amanda; Westbroek, Wendy; Gunay-Aygun, Meral; Gahl, William A.

    2009-01-01

    Lysosome-related organelles (LROs) are a heterogeneous group of vesicles that share various features with lysosomes, but are distinct in function, morphology, and composition. The biogenesis of LROs employs a common machinery, and genetic defects in this machinery can affect all LROs or only an individual LRO, resulting in a variety of clinical features. In this review, we discuss the main components in LRO biogenesis. We also address the function, composition and resident cell type of the major LROs. Finally, we describe the clinical characteristics of the major human LRO disorders. PMID:18544035

  9. Endocytic control of growth factor signalling: multivesicular bodies as signalling organelles

    PubMed Central

    Dobrowolski, Radek; De Robertis, Edward M.

    2012-01-01

    Signal transduction and endocytosis are intertwined processes. The internalization of ligand-activated receptors by endocytosis has classically been thought to attenuate signals by targeting receptors for degradation in lysosomes, but it can also maintain signals in early signalling endosomes. In both cases, localization to multivesicular endosomes en route to lysosomes is thought to terminate signalling. However, during WNT signal transduction, sequestration of the enzyme glycogen synthase kinase 3 (GSK3) inside multivesicular endosomes results in the stabilization of many cytosolic proteins. Thus, the role of endocytosis during signal transduction may be more diverse than anticipated, and multivesicular endosomes may constitute a crucial signalling organelle. PMID:22108513

  10. Organelle-Specific Nitric Oxide Detection in Living Cells via HaloTag Protein Labeling

    PubMed Central

    Zhu, Qian; Du, Zengmin; Hu, Aiguo; Yang, Yi

    2015-01-01

    Nitric oxide (NO) is a membrane-permeable signaling molecule that is constantly produced, transferred, and consumed in vivo. NO participates and plays important roles in multiple biological processes. However, spatiotemporal imaging of NO in living cells is challenging. To fill the gap in currently used techniques, we exploited the versatility of HaloTag technology and synthesized a novel organelle-targetable fluorescent probe called HTDAF-2DA. We demonstrate the utility of the probe by monitoring subcellular NO dynamics. The developed strategy enables precise determination of local NO function. PMID:25923693

  11. Organelle-Specific Nitric Oxide Detection in Living Cells via HaloTag Protein Labeling.

    PubMed

    Wang, Jianhua; Zhao, Yuzheng; Wang, Chao; Zhu, Qian; Du, Zengmin; Hu, Aiguo; Yang, Yi

    2015-01-01

    Nitric oxide (NO) is a membrane-permeable signaling molecule that is constantly produced, transferred, and consumed in vivo. NO participates and plays important roles in multiple biological processes. However, spatiotemporal imaging of NO in living cells is challenging. To fill the gap in currently used techniques, we exploited the versatility of HaloTag technology and synthesized a novel organelle-targetable fluorescent probe called HTDAF-2DA. We demonstrate the utility of the probe by monitoring subcellular NO dynamics. The developed strategy enables precise determination of local NO function. PMID:25923693

  12. Observation of organelle by a laser plasma x-ray microscope

    NASA Astrophysics Data System (ADS)

    Kado, Masataka; Kishimoto, Maki; Ishino, Masahiko; Tamotsu, Satoshi; Yasuda, Keiko; Shinohara, Kunio

    2012-07-01

    Contact x-ray microscopy has a potential to image wet biological specimens in natural condition. It is very important to identify obtained features in the x-ray images, since x-ray microscopes have potential to image features that have not been visualized yet. We have proposed to compare the x-ray images of the biological specimens with the fluorescence images and to identify the features found in the x-ray images. We have succeeded to observe fine structures of the cellular organelles such as mitochondria by the soft x-ray microscope.

  13. Kinesin and dynein superfamily proteins in organelle transport and cell division.

    PubMed

    Hirokawa, N; Noda, Y; Okada, Y

    1998-02-01

    Microtubule-associated motor proteins of the kinesin and dynein superfamilies play important roles in cellular mechanisms such as organelle transport and mitosis. Identification and characterization of new family members (in particular KIFC2, 16 new KIFs, XKlp2 and XKCM1 of the kinesin superfamily, and DHC2 and DHC3 of the dynein superfamily) and further characterization of known family members have improved our understanding of these cellular mechanisms. Sophisticated biophysical and structural analyses of monomeric and dimeric motor proteins have contributed to elucidating the mechanisms behind motor protein motility and polarity. PMID:9484596

  14. Multiclassifier combinatorial proteomics of organelle shadows at the example of mitochondria in chromatin data

    PubMed Central

    Kustatscher, Georg; Grabowski, Piotr

    2016-01-01

    Subcellular localization is an important aspect of protein function, but the protein composition of many intracellular compartments is poorly characterized. For example, many nuclear bodies are challenging to isolate biochemically and thus remain inaccessible to proteomics. Here, we explore covariation in proteomics data as an alternative route to subcellular proteomes. Rather than targeting a structure of interest biochemically, we target it by machine learning. This becomes possible by taking data obtained for one organelle and searching it for traces of another organelle. As an extreme example and proof‐of‐concept we predict mitochondrial proteins based on their covariation in published interphase chromatin data. We detect about ⅓ of the known mitochondrial proteins in our chromatin data, presumably most as contaminants. However, these proteins are not present at random. We show covariation of mitochondrial proteins in chromatin proteomics data. We then exploit this covariation by multiclassifier combinatorial proteomics to define a list of mitochondrial proteins. This list agrees well with different databases on mitochondrial composition. This benchmark test raises the possibility that, in principle, covariation proteomics may also be applicable to structures for which no biochemical isolation procedures are available. PMID:26510496

  15. A Non-sulfated Chondroitin Stabilizes Membrane Tubulation in Cnidarian Organelles*

    PubMed Central

    Adamczyk, Patrizia; Zenkert, Claudia; Balasubramanian, Prakash G.; Yamada, Shuhei; Murakoshi, Saori; Sugahara, Kazuyuki; Hwang, Jung Shan; Gojobori, Takashi; Holstein, Thomas W.; Özbek, Suat

    2010-01-01

    Membrane tubulation is generally associated with rearrangements of the cytoskeleton and other cytoplasmic factors. Little is known about the contribution of extracellular matrix components to this process. Here, we demonstrate an essential role of proteoglycans in the tubulation of the cnidarian nematocyst vesicle. The morphogenesis of this extrusive organelle takes place inside a giant post-Golgi vesicle, which topologically represents extracellular space. This process includes the formation of a complex collagenous capsule structure that elongates into a long tubule, which invaginates after its completion. We show that a non-sulfated chondroitin appears as a scaffold in early morphogenesis of all nematocyst types in Hydra and Nematostella. It accompanies the tubulation of the vesicle membrane forming a provisional tubule structure, which after invagination matures by collagen incorporation. Inhibition of chondroitin synthesis by β-xylosides arrests nematocyst morphogenesis at different stages of tubule outgrowth resulting in retention of tubule material and a depletion of mature capsules in the tentacles of hydra. Our data suggest a conserved role of proteoglycans in the stabilization of a membrane protrusion as an essential step in organelle morphogenesis. PMID:20538610

  16. Extension of the concept of an anomalous water component to images of T-cell organelles.

    PubMed

    Tychinsky, Vladimir P

    2014-12-01

    Microscopic images of a living cell are the main source of information on its functional state. Modern interference microscopy techniques allow the numerical parameters of cell images to be obtained with an accuracy not available with other methods. Quantitative analysis of phase images of T lymphocytes (TCs) in different functional states demonstrated that variations of the properties of intracellular water should be taken into account. This conclusion agrees with the current view that the physical parameters of water, including the refractive index (RI) of a water layer, depend on the hydrophilicity and other characteristics of the adjacent surface. Application of this concept to phase images of TCs showed that the contribution of the fourth phase of water (4-water) or the structured water component, which has an increased RI, should be considered. The proportion of 4-water depends on the functional state of the cell determined by the culture medium composition. Normally, the proportion of 4-water in organelles is as high as 30%; it is considerably lower in organelles of cells with inhibited metabolism. PMID:25500678

  17. Cytosolic organelles shape calcium signals and exo-endocytotic responses of chromaffin cells.

    PubMed

    García, Antonio G; Padín, Fernando; Fernández-Morales, José C; Maroto, Marcos; García-Sancho, Javier

    2012-01-01

    The concept of stimulus-secretion coupling was born from experiments performed in chromaffin cells 50 years ago. Stimulation of these cells with acetylcholine enhances calcium (Ca(2+)) entry and this generates a transient elevation of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) that triggers the exocytotic release of catecholamines. The control of the [Ca(2+)](c) signal is complex and depends on various classes of plasmalemmal calcium channels, cytosolic calcium buffers, the uptake and release of Ca(2+) from cytoplasmic organelles, such as the endoplasmic reticulum, mitochondria, chromaffin vesicles and the nucleus, and Ca(2+) extrusion mechanisms, such as the plasma membrane Ca(2+)-stimulated ATPase, and the Na(+)/Ca(2+) exchanger. Computation of the rates of Ca(2+) fluxes between the different cell compartments support the proposal that the chromaffin cell has developed functional calcium tetrads formed by calcium channels, cytosolic calcium buffers, the endoplasmic reticulum, and mitochondria nearby the exocytotic plasmalemmal sites. These tetrads shape the Ca(2+) transients occurring during cell activation to regulate early and late steps of exocytosis, and the ensuing endocytotic responses. The different patterns of catecholamine secretion in response to stress may thus depend on such local [Ca(2+)](c) transients occurring at different cell compartments, and generated by redistribution and release of Ca(2+) by cytoplasmic organelles. In this manner, the calcium tetrads serve to couple the variable energy demands due to exo-endocytotic activities with energy production and protein synthesis. PMID:22209033

  18. Detection, imaging, and kinetics of sub-micron organelles of chondrocytes by multiple beam interference microscopy

    NASA Astrophysics Data System (ADS)

    Joshi, Narahari V.; Medina, Honorio; Barboza, J. M.; Colantuoni, Gladys; Quintero, Maritza

    2004-07-01

    Chondrocytes, obtained from testosterone treated human articular cartilage, were examined by a recently developed Multiple Beam Interference Microscopy (MBIM) attached to a confocal set up, Video-enhanced differential interference microphotography and also by cinematography. In the MBIM, the intensity of the transmitted pattern is given by the Airy function which increases the contrast dramatically as the coefficient of the reflectance of the parallel plates increases. Moreover, in this configuration, the beam passes several times through a specific organelle and increases its optical path difference both because of the increase in the trajectory and refractive index (high density) of the organelle. The improved contrast enhances the resolving power of the system and makes visible several structural details of sub micron dimensions like nucleolus, retraction fibers, podia, etc. which are not possible to reveal with such a clarity by conventional techniques such as bright field, phase contrast or DIC. This technique permits to detect the oscillatory and rotational motions of unstained cilia for the first time. The frequency of oscillations was found to be 0.8 Hz.

  19. A histidine protein kinase is involved in polar organelle development in Caulobacter crescentus.

    PubMed Central

    Wang, S P; Sharma, P L; Schoenlein, P V; Ely, B

    1993-01-01

    Mutations having pleiotropic effects on polar organelle development (pod) in Caulobacter crescentus have been identified and shown to occur in at least 13 genes scattered throughout the genome. Mutations at each locus affect a unique combination of polar traits, suggesting that complex interactions occur among these genes. The DNA sequence of one of these genes, pleC, indicates that it is homologous to members of the family of histidine protein kinase genes. Membes of this family include the senor components of the bacterial two-component regulatory systems. Furthermore, in vitro experiments demonstrated that the PleC protein was capable of autophosphorylation. These results suggest that the PleC protein (and perhaps the proteins encoded by the other pod genes as well) regulates the expression of genes involved in polar organelle development through the phosphorylation of key regulatory proteins. The use of a phosphorelay system cued to internal changes in the cell would provide a mechanism for coordinating major changes in gene expression with the completion of specific cell cycle events. Images PMID:8421698

  20. Combinatorial targeting and discovery of ligand-receptors in organelles of mammalian cells

    PubMed Central

    Rangel, Roberto; Guzman-Rojas, Liliana; le Roux, Lucia G.; Staquicini, Fernanda I.; Hosoya, Hitomi; Barbu, E. Magda; Ozawa, Michael G.; Nie, Jing; Jr, Kenneth Dunner; Langley, Robert R.; Sage, E. Helene; Koivunen, Erkki; Gelovani, Juri G.; Lobb, Roy R.; Sidman, Richard L.; Pasqualini, Renata; Arap, Wadih

    2012-01-01

    Phage display screening allows the study of functional protein–protein interactions at the cell surface, but investigating intracellular organelles remains a challenge. Here we introduce internalizing-phage libraries to identify clones that enter mammalian cells through a receptor-independent mechanism and target-specific organelles as a tool to select ligand peptides and identify their intracellular receptors. We demonstrate that penetratin, an antennapedia-derived peptide, can be displayed on the phage envelope and mediate receptor-independent uptake of internalizing phage into cells. We also show that an internalizing-phage construct displaying an established mitochondria-specific localization signal targets mitochondria, and that an internalizing-phage random peptide library selects for peptide motifs that localize to different intracellular compartments. As a proof-of-concept, we demonstrate that one such peptide, if chemically fused to penetratin, is internalized receptor-independently, localizes to mitochondria, and promotes cell death. This combinatorial platform technology has potential applications in cell biology and drug development. PMID:22510693

  1. Oxidative Stress in the Healthy and Wounded Hepatocyte: A Cellular Organelles Perspective

    PubMed Central

    Mello, Tommaso; Zanieri, Francesca; Ceni, Elisabetta; Galli, Andrea

    2016-01-01

    Accurate control of the cell redox state is mandatory for maintaining the structural integrity and physiological functions. This control is achieved both by a fine-tuned balance between prooxidant and anti-oxidant molecules and by spatial and temporal confinement of the oxidative species. The diverse cellular compartments each, although structurally and functionally related, actively maintain their own redox balance, which is necessary to fulfill specialized tasks. Many fundamental cellular processes such as insulin signaling, cell proliferation and differentiation and cell migration and adhesion, rely on localized changes in the redox state of signal transducers, which is mainly mediated by hydrogen peroxide (H2O2). Therefore, oxidative stress can also occur long before direct structural damage to cellular components, by disruption of the redox circuits that regulate the cellular organelles homeostasis. The hepatocyte is a systemic hub integrating the whole body metabolic demand, iron homeostasis and detoxification processes, all of which are redox-regulated processes. Imbalance of the hepatocyte's organelles redox homeostasis underlies virtually any liver disease and is a field of intense research activity. This review recapitulates the evolving concept of oxidative stress in the diverse cellular compartments, highlighting the principle mechanisms of oxidative stress occurring in the healthy and wounded hepatocyte. PMID:26788252

  2. Evolution and significance of the Lon gene family in Arabidopsis organelle biogenesis and energy metabolism

    PubMed Central

    Rigas, Stamatis; Daras, Gerasimos; Tsitsekian, Dikran; Alatzas, Anastasios; Hatzopoulos, Polydefkis

    2014-01-01

    Lon is the first identified ATP-dependent protease highly conserved across all kingdoms. Model plant species Arabidopsis thaliana has a small Lon gene family of four members. Although these genes share common structural features, they have distinct properties in terms of gene expression profile, subcellular targeting and substrate recognition motifs. This supports the notion that their functions under different environmental conditions are not necessarily redundant. This article intends to unravel the biological role of Lon proteases in energy metabolism and plant growth through an evolutionary perspective. Given that plants are sessile organisms exposed to diverse environmental conditions and plant organelles are semi-autonomous, it is tempting to suggest that Lon genes in Arabidopsis are paralogs. Adaptive evolution through repetitive gene duplication events of a single archaic gene led to Lon genes with complementing sets of subfunctions providing to the organism rapid adaptability for canonical development under different environmental conditions. Lon1 function is adequately characterized being involved in mitochondrial biogenesis, modulating carbon metabolism, oxidative phosphorylation and energy supply, all prerequisites for seed germination and seedling establishment. Lon is not a stand-alone proteolytic machine in plant organelles. Lon in association with other nuclear-encoded ATP-dependent proteases builds up an elegant nevertheless, tight interconnected circuit. This circuitry channels properly and accurately, proteostasis and protein quality control among the distinct subcellular compartments namely mitochondria, chloroplasts, and peroxisomes. PMID:24782883

  3. Release from myosin V via regulated recruitment of an E3 Ub ligase controls organelle localization

    PubMed Central

    Yau, Richard G.; Peng, Yutian; Valiathan, Rajeshwari R.; Birkeland, Shanda R.; Wilson, Thomas E.; Weisman, Lois S.

    2014-01-01

    Summary Molecular motors transport organelles to specific subcellular locations. Upon arrival at their correct locations, motors release organelles via unknown mechanisms. The yeast myosin-V, Myo2, binds the vacuole specific adaptor, Vac17, to transport the vacuole from the mother cell to the bud. Here, we show that vacuole detachment from Myo2 occurs in multiple regulated steps along the entire pathway of vacuole transport. Detachment initiates in the mother cell with the phosphorylation of Vac17 which recruits the E3 ligase, Dma1, to the vacuole. However, Dma1 recruitment also requires the assembly of the vacuole transport complex and is first observed after the vacuole enters the bud. Dma1 remains on the vacuole until the bud and mother vacuoles separate. Subsequently, Dma1 targets Vac17 for proteasomal degradation. Notably, we find that the termination of peroxisome transport also requires Dma1. We predict that this is a general mechanism which detaches myosin-V from select cargoes. PMID:24636257

  4. Oxidative Stress in the Healthy and Wounded Hepatocyte: A Cellular Organelles Perspective.

    PubMed

    Mello, Tommaso; Zanieri, Francesca; Ceni, Elisabetta; Galli, Andrea

    2016-01-01

    Accurate control of the cell redox state is mandatory for maintaining the structural integrity and physiological functions. This control is achieved both by a fine-tuned balance between prooxidant and anti-oxidant molecules and by spatial and temporal confinement of the oxidative species. The diverse cellular compartments each, although structurally and functionally related, actively maintain their own redox balance, which is necessary to fulfill specialized tasks. Many fundamental cellular processes such as insulin signaling, cell proliferation and differentiation and cell migration and adhesion, rely on localized changes in the redox state of signal transducers, which is mainly mediated by hydrogen peroxide (H2O2). Therefore, oxidative stress can also occur long before direct structural damage to cellular components, by disruption of the redox circuits that regulate the cellular organelles homeostasis. The hepatocyte is a systemic hub integrating the whole body metabolic demand, iron homeostasis and detoxification processes, all of which are redox-regulated processes. Imbalance of the hepatocyte's organelles redox homeostasis underlies virtually any liver disease and is a field of intense research activity. This review recapitulates the evolving concept of oxidative stress in the diverse cellular compartments, highlighting the principle mechanisms of oxidative stress occurring in the healthy and wounded hepatocyte. PMID:26788252

  5. Interrelations between the Parasitophorous Vacuole of Toxoplasma gondii and Host Cell Organelles

    NASA Astrophysics Data System (ADS)

    Cardoso Magno, Rodrigo; Cobra Straker, Lorian; de Souza, Wanderley; Attias, Marcia

    2005-04-01

    Toxoplasma gondii, the causative agent of toxoplasmosis, is capable of actively penetrating and multiplying in any nucleated cell of warm-blooded animals. Its survival strategies include escape from fusion of the parasitophorous vacuole with host cell lysosomes and rearrangement of host cell organelles in relation to the parasitophorous vacuole. In this article we report the rearrangement of host cell organelles and elements of the cytoskeleton of LLCMK2 cells, a lineage derived from green monkey kidney epithelial cells, in response to infection by T. gondii tachyzoites. Transmission electron microscopy made on flat embedded monolayers cut horizontally to the apical side of the cells or field emission scanning electron microscopy of monolayers scraped with scotch tape before sputtering showed that association of mitochondria to the vacuole is much less frequent than previously described. On the other hand, all parasitophorous vacuoles were surrounded by elements of the endoplasmic reticulum. These data were complemented by observations by laser scanning microscopy using fluorescent probes from mitochondria and endoplasmic reticulum and reinforced by three-dimensional reconstruction from serial sections observed by transmission electron microscopy and labeling of mitochondria and endoplasmic reticulum by fluorescent probes.

  6. Cell organelles at uncoated cryofractured surfaces as viewed with the scanning electron microscope.

    PubMed

    Woods, P S; Ledbetter, M C

    1976-06-01

    A method of direct visualization of cell organelles by scanning electron microscopy (SEM) is described. Plant and animal tissues fixed in glutaraldehyde and osmium tetroxide are treated with the ligand thiocarbohydrazide and a second osmium tetroxide solution, to increase their osmium content. Tissues are then dehydrated, infiltrated with an epoxy monomer, and together solidified with dry ice and fractured. The pieces are transferred to pure acetone, critical-point dried, attached to stubs with silver paint and viewed by SEM. The ligating procedure increases the osmium concentration at its original bonding site sufficiently to render the tissue electrically conductive, thus obviating the need for metallic coating. he organelles at the fractured surface are revaled in relation to their osmium incorporation rather than by surface irregularities as with coating methods. The image derived from the uncoated surface approaches in resolution that of transmission electron micrographs of thin sections. A protion of the image arising from a small distance below the surface, while at progressively lower resolution, provides some 3-dimensional information about cell fine structure. PMID:777015

  7. Extension of the concept of an anomalous water component to images of T-cell organelles

    NASA Astrophysics Data System (ADS)

    Tychinsky, Vladimir P.

    2014-12-01

    Microscopic images of a living cell are the main source of information on its functional state. Modern interference microscopy techniques allow the numerical parameters of cell images to be obtained with an accuracy not available with other methods. Quantitative analysis of phase images of T lymphocytes (TCs) in different functional states demonstrated that variations of the properties of intracellular water should be taken into account. This conclusion agrees with the current view that the physical parameters of water, including the refractive index (RI) of a water layer, depend on the hydrophilicity and other characteristics of the adjacent surface. Application of this concept to phase images of TCs showed that the contribution of the fourth phase of water (4-water) or the structured water component, which has an increased RI, should be considered. The proportion of 4-water depends on the functional state of the cell determined by the culture medium composition. Normally, the proportion of 4-water in organelles is as high as 30% it is considerably lower in organelles of cells with inhibited metabolism.

  8. From Endosymbiont to Host-Controlled Organelle: The Hijacking of Mitochondrial Protein Synthesis and Metabolism

    PubMed Central

    Gabaldón, Toni; Huynen, Martijn A

    2007-01-01

    Mitochondria are eukaryotic organelles that originated from the endosymbiosis of an alpha-proteobacterium. To gain insight into the evolution of the mitochondrial proteome as it proceeded through the transition from a free-living cell to a specialized organelle, we compared a reconstructed ancestral proteome of the mitochondrion with the proteomes of alpha-proteobacteria as well as with the mitochondrial proteomes in yeast and man. Overall, there has been a large turnover of the mitochondrial proteome during the evolution of mitochondria. Early in the evolution of the mitochondrion, proteins involved in cell envelope synthesis have virtually disappeared, whereas proteins involved in replication, transcription, cell division, transport, regulation, and signal transduction have been replaced by eukaryotic proteins. More than half of what remains from the mitochondrial ancestor in modern mitochondria corresponds to translation, including post-translational modifications, and to metabolic pathways that are directly, or indirectly, involved in energy conversion. Altogether, the results indicate that the eukaryotic host has hijacked the proto-mitochondrion, taking control of its protein synthesis and metabolism. PMID:17983265

  9. Apical Organelle Secretion by Toxoplasma Controls Innate and Adaptive Immunity and Mediates Long-Term Protection.

    PubMed

    Sloves, Pierre-Julien; Mouveaux, Thomas; Ait-Yahia, Saliha; Vorng, Han; Everaere, Laetitia; Sangare, Lamba Omar; Tsicopoulos, Anne; Tomavo, Stanislas

    2015-11-01

    Apicomplexan parasites have unique apical rhoptry and microneme secretory organelles that are crucial for host infection, although their role in protection against Toxoplasma gondii infection is not thoroughly understood. Here, we report a novel function of the endolysosomal T. gondii sortilin-like receptor (TgSORTLR), which mediates trafficking to functional apical organelles and their subsequent secretion of virulence factors that are critical to the induction of sterile immunity against parasite reinfection. We further demonstrate that the T. gondii armadillo repeats-only protein (TgARO) mutant, which is deficient only in apical secretion of rhoptries, is also critical in mounting protective immunity. The lack of TgSORTLR and TgARO proteins completely inhibited T-helper 1-dependent adaptive immunity and compromised the function of natural killer T-cell-mediated innate immunity. Our findings reveal an essential role for apical secretion in promoting sterile protection against T. gondii and provide strong evidence for rhoptry-regulated discharge of antigens as a key effector for inducing protective immunity. PMID:25910629

  10. Visualization of volatile substances in different organelles with an atmospheric-pressure mass microscope.

    PubMed

    Harada, Takahiro; Yuba-Kubo, Akiko; Sugiura, Yuki; Zaima, Nobuhiro; Hayasaka, Takahiro; Goto-Inoue, Naoko; Wakui, Masatoshi; Suematsu, Makoto; Takeshita, Kengo; Ogawa, Kiyoshi; Yoshida, Yoshikazu; Setou, Mitsutoshi

    2009-11-01

    We have developed a mass microscope (mass spectrometry imager with spatial resolution higher than the naked eye) equipped with an atmospheric pressure ion-source chamber for laser desorption/ionization (AP-LDI) and a quadrupole ion trap time-of-flight (QIT-TOF) analyzer. The optical microscope combined with the mass spectrometer permitted us to precisely determine the relevant tissue region prior to performing imaging mass spectrometry (IMS). An ultraviolet laser tightly focused with a triplet lens was used to achieve high spatial resolution. An atmospheric pressure ion-source chamber enables us to analyze fresh samples with minimal loss of intrinsic water or volatile compounds. Mass-microscopic AP-LDI imaging of freshly cut ginger rhizome sections revealed that 6-gingerol ([M + K](+)at m/z 333.15, positive mode; [M - H](-) at m/z 293.17, negative mode) and the monoterpene ([M + K](+) at m/z 191.09), which are the compounds related to pungency and flavor, respectively, were localized in oil drop-containing organelles. AP-LDI-tandem MS/MS analyses were applied to compare authentic signals from freshly cut ginger directly with the standard reagent. Thus, our atmosphere-imaging mass spectrometer enabled us to monitor a quality of plants at the organelle level. PMID:19788281

  11. The Neurospora organelle motor: a distant relative of conventional kinesin with unconventional properties.

    PubMed Central

    Steinberg, G; Schliwa, M

    1995-01-01

    The "conventional" kinesins comprise a conserved family of molecular motors for organelle transport that have been identified in various animal species. Organelle motors from other phyla have not yet been analyzed at the molecular level. Here we report the identification, biochemical and immunological characterization, and molecular cloning of a cytoplasmic motor in a "lower" eukaryote, the Ascomycete fungus Neurospora crassa. This motor, termed Nkin (for Neurospora kinesin), exhibits several unique structural and functional features, including a high rate of microtubule transport, a lack of copurifying light chains, a second P-loop motif, and an overall sequence organization reminiscent of a kinesin-like protein. However, a greater than average sequence homology in the motor domain and the presence of a highly conserved region in the C-terminus identify Nkin as a distant relative of the family of conventional kinesins. A molecular phylogenetic analysis suggests Nkin to have diverged early in the evolution of this family of motors. The discovery of Nkin may help identify domains important for specific biological functions in conventional kinesins. Images PMID:8589459

  12. Function of metabolic and organelle networks in crowded and organized media

    PubMed Central

    Aon, Miguel A.; Cortassa, Sonia

    2015-01-01

    (Macro)molecular crowding and the ability of the ubiquitous cytoskeleton to dynamically polymerize–depolymerize are prevalent cytoplasmic conditions in prokaryotic and eukaryotic cells. Protein interactions, enzymatic or signaling reactions - single, sequential or in complexes - whole metabolic pathways and organelles can be affected by crowding, the type and polymeric status of cytoskeletal proteins (e.g., tubulin, actin), and their imparted organization. The self-organizing capability of the cytoskeleton can orchestrate metabolic fluxes through entire pathways while its fractal organization can frame the scaling of activities in several levels of organization. The intracellular environment dynamics (e.g., biochemical reactions) is dominated by the orderly cytoskeleton and the intrinsic randomness of molecular crowding. Existing evidence underscores the inherent capacity of intracellular organization to generate emergent global behavior. Yet unknown is the relative impact on cell function provided by organelle or functional compartmentation based on transient proteins association driven by weak interactions (quinary structures) under specific environmental challenges or functional conditions (e.g., hypoxia, division, differentiation). We propose a qualitative, integrated structural–functional model of cytoplasmic organization based on a modified version of the Sierspinsky–Menger–Mandelbrot sponge, a 3D representation of a percolation cluster, and examine its capacity to accommodate established experimental facts. PMID:25653618

  13. Increased Vesicular Monoamine Transporter 2 (VMAT2; Slc18a2) Protects against Methamphetamine Toxicity

    PubMed Central

    Lohr, Kelly M.; Stout, Kristen A.; Dunn, Amy R.; Wang, Minzheng; Salahpour, Ali; Guillot, Thomas S.; Miller, Gary W.

    2015-01-01

    The psychostimulant methamphetamine (METH) is highly addictive and neurotoxic to dopamine terminals. METH toxicity has been suggested to be due to the release and accumulation of dopamine in the cytosol of these terminals. The vesicular monoamine transporter 2 (VMAT2; SLC18A2) is a critical mediator of dopamine handling. Mice overexpressing VMAT2 (VMAT2-HI) have an increased vesicular capacity to store dopamine, thus augmenting striatal dopamine levels and dopamine release in the striatum. Based on the altered compartmentalization of intracellular dopamine in the VMAT2-HI mice, we assessed whether enhanced vesicular function was capable of reducing METH-induced damage to the striatal dopamine system. While wildtype mice show significant losses in striatal levels of the dopamine transporter (65% loss) and tyrosine hydroxylase (46% loss) following a 4 × 10 mg/kg METH dosing regimen, VMAT2-HI mice were protected from this damage. VMAT2-HI mice were also spared from the inflammatory response that follows METH treatment, showing an increase in astroglial markers that was approximately one-third of that of wildtype animals (117% vs 36% increase in GFAP, wildtype vs VMAT2-HI). Further analysis also showed that elevated VMAT2 level does not alter the ability of METH to increase core body temperature, a mechanism integral to the toxicity of the drug. Finally, the VMAT2-HI mice showed no difference from wildtype littermates on both METH-induced conditioned place preference and in METH-induced locomotor activity (1 mg/kg METH). These results demonstrate that elevated VMAT2 protects against METH toxicity without enhancing the rewarding effects of the drug. Since the VMAT2-HI mice are protected from METH despite higher basal dopamine levels, this study suggests that METH toxicity depends more on the proper compartmentalization of synaptic dopamine than on the absolute amount of dopamine in the brain. PMID:25746685

  14. Evolution of desert pavements and the vesicular layer in soils of the Transantarctic Mountains

    NASA Astrophysics Data System (ADS)

    Bockheim, James G.

    2010-06-01

    Compared to mid-latitude deserts, the properties, formation and evolution of desert pavements and the underlying vesicular layer in Antarctica are poorly understood. This study examines the desert pavements and the vesicular layer from seven soil chronosequences in the Transantarctic Mountains that have developed on two contrasting parent materials: sandstone-dolerite and granite-gneiss. The pavement density commonly ranges from 63 to 92% with a median value of 80% and does not vary significantly with time of exposure or parent material composition. The dominant size range of clasts decreases with time of exposure, ranging from 16-64 mm on Holocene and late Quaternary surfaces to 8-16 mm on surfaces of middle Quaternary and older age. The proportion of clasts with ventifaction increases progressively through time from 20% on drifts of Holocene and late Quaternary age to 35% on Miocene-aged drifts. Desert varnish forms rapidly, especially on dolerite clasts, with nearly 100% cover on surfaces of early Quaternary and older age. Macropitting occurs only on clasts that have been exposed since the Miocene. A pavement development index, based on predominant clast-size class, pavement density, and the proportion of clasts with ventifaction, varnish, and pits, readily differentiated pavements according to relative age. From these findings we judge that desert pavements initially form from a surficial concentration of boulders during till deposition followed by a short period of deflation and a longer period of progressive chemical and physical weathering of surface clasts. The vesicular layer that underlies the desert pavement averages 4 cm in thickness and is enriched in silt, which is contributed primarily by weathering rather than eolian deposition. A comparison is made between desert pavement properties in mid-latitude deserts and Antarctic deserts.

  15. Reduced vesicular monoamine transport disrupts serotonin signaling but does not cause serotonergic degeneration

    PubMed Central

    Alter, Shawn P.; Stout, Kristen A.; Lohr, Kelly M.; Taylor, Tonya N.; Shepherd, Kennie R.; Wang, Minzheng; Guillot, Thomas S.; Miller, Gary W.

    2016-01-01

    We previously demonstrated that mice with reduced expression of the vesicular monoamine transporter 2 (VMAT2 LO) undergo age-related degeneration of the catecholamine-producing neurons of the substantia nigra pars compacta and locus ceruleus and exhibit motor disturbances and depressive-like behavior. In this work, we investigated the effects of reduced vesicular transport on the function and viability of serotonin neurons in these mice. Adult (4–6 months of age), VMAT2 LO mice exhibit dramatically reduced (90%) serotonin release capacity, as measured by fast scan cyclic voltammetry. We observed changes in serotonin receptor responsivity in in vivo pharmacological assays. Aged (months) VMAT2 LO mice exhibited abolished 5-HT1A autoreceptor sensitivity, as determined by 8-OH-DPAT (0.1 mg/kg) induction of hypothermia. When challenged with the 5HT2 agonist, 2,5-dimethoxy-4-iodoamphetamine (1 mg/kg), VMAT2 LO mice exhibited a marked increase (50%) in head twitch responses. We observed sparing of serotonergic terminals in aged mice (18–24 months) throughout the forebrain by SERT immunohistochemistry and [3H]-paroxetine binding in striatal homogenates of aged VMAT2 LO mice. In contrast to their loss of catecholamine neurons of the substantia nigra and locus ceruleus, aged VMAT2 LO mice do not exhibit a change in the number of serotonergic (TPH2 +) neurons within the dorsal raphe, as measured by unbiased stereology at 26–30 months. Collectively, these data indicate that reduced vesicular monoamine transport significantly disrupts serotonergic signaling, but does not drive degeneration of serotonin neurons. PMID:26428905

  16. A papulopustular, vesicular, crusted rash in a 4-week-old neonate.

    PubMed

    Subramaniam, Sathyaseelan; Rutman, Maia S; Wenger, Jodi K

    2013-11-01

    Scabies is commonly seen worldwide, in its usual classic form when afflicting older children and adults. However, neonatal scabies is described as its own entity in the literature. We present a case of a 4-week-old infant with a generalized papulopustular, vesicular, and crusted rash who was diagnosed with scabies. We contrast the differing clinical features of neonatal and classic scabies, describe possible mimickers of this diagnostic dilemma, and review current treatment options available for scabies in this very young age group. PMID:24196092

  17. Vesicular stomatitis virus M protein may be inside the ribonucleocapsid coil.

    PubMed Central

    Barge, A; Gaudin, Y; Coulon, P; Ruigrok, R W

    1993-01-01

    Vesicular stomatitis virus is an enveloped virus with an external glycoprotein G and a nucleocapsid that form, together with the M protein, a tight helically coiled structure: the skeleton. Negative staining and immunoelectron microscopy studies on skeleton preparations were performed to determine the localization of the M protein. These studies have resulted in a new model for the structure of rhabdoviruses in which the nucleocapsid is wound around a core containing the M protein. This model predicts contact between M and lipid only at the extreme ends of the skeleton, which is confirmed by skeleton-liposome binding studies. Images PMID:8230447

  18. Isolation of vesicular stomatitis virus defective interfering genomes with different amounts of 5'-terminal complementarity.

    PubMed Central

    Kolakofsky, D

    1982-01-01

    I isolated at least 30 different vesicular stomatitis virus defective interfering (DI) genomes, distinguished by chain length, by five independent undiluted passages of a repeatedly cloned virus plaque. Labeling of the 3' hydroxyl ends of these DI genomes and RNase digestion studies demonstrated that the ends of these DI genomes were terminally complementary to different extents (approximately 46 to 200 nucleotides). Mapping studies showed that the complementary ends of all of the DI genomes were derived from the 5' ends of the nondefective minus-strand genome. Regardless of the extent of terminal complementarity, all of the DI genomes synthesized the same 46-nucleotide minus-strand leader RNA. Images PMID:6281468

  19. Synthesis, structure, and excimer formation of vesicular assemblies carrying 1- or 2-naphthyl chromophores

    SciTech Connect

    Sisido, Masahiki; Sato, Yasuhiko; Sasaki, Hiroki; Imanishi, Yukio )

    1990-01-01

    New chromophoric amphiphiles consisting of optically active 1- or 2-naphthylalanines, each carrying two long alkyl chains and an ammonium ion, were synthesized. These amphiphiles were found to form vesicular structures in aqueous dispersion, and those having two octadecyl chains showed a gel-liquid crystalline transition around room temperature. UV and CD spectra showed exciton-type interactions for the 2-naphthyl amphiphiles in a high-energy excited state but no dimers or higher aggregates of the naphthyl groups in the ground state. Fluorescence spectra showed monomer and excimer emissions. The circularly polarized fluorescence spectra showed a positive signal at the excimer emission, indicating a chiral excimer configuration.

  20. The management of VA (vesicular-arbuscular) mycorrhizae in semi-arid environments

    SciTech Connect

    Miller, R.M.

    1987-01-01

    The need for management of vesicular-arbuscular mycorrhizae in semi-arid ecosystems represent an important challenge to belowground researchers especially as we increase our utilization of these stressed habitats. Within the laser couple of years several reviews have been prepared on the effects of disturbance to shrub and grasslands and their mycorrhizae. The purpose of this presentation is to discuss some research findings and management needs using examples from a high elevation cold desert, and from research in mid-grass and tallgrass prairies.

  1. Multi Vesicular Osseous Hydatid Disease of the Mandible- A Case Report

    PubMed Central

    Shahoon, H; Esmaeili, M; Mobedi, I; Nematollahi, M

    2010-01-01

    Hydatid disease is a common and major public health issue caused by parasite Echinococcus granulosus. The highest prevalence of the parasite can be found in different parts of world like Africa, Australia, and South America. This infection can occurs in almost any part of the body. Here we present clinical, radiological, histological features and treatment of a multi vesicular osseous hydatid disease of the mandible in an Afghan 5 year old boy with a firm swelling in the right side of mandible. PMID:22347236

  2. Concomitant TLR/RLH signaling of radioresistant and radiosensitive cells is essential for protection against vesicular stomatitis virus infection.

    PubMed

    Spanier, Julia; Lienenklaus, Stefan; Paijo, Jennifer; Kessler, Annett; Borst, Katharina; Heindorf, Sabrina; Baker, Darren P; Kröger, Andrea; Weiss, Siegfried; Detje, Claudia N; Staeheli, Peter; Kalinke, Ulrich

    2014-09-15

    Several studies indicated that TLR as well as retinoic acid-inducible gene I-like helicase (RLH) signaling contribute to vesicular stomatitis virus (VSV)-mediated triggering of type I IFN (IFN-I) responses. Nevertheless, TLR-deficient MyD88(-/-)Trif(-/-) mice and RLH-deficient caspase activation and recruitment domain adaptor inducing IFN-β (Cardif)(-/-) mice showed only marginally enhanced susceptibility to lethal VSV i.v. infection. Therefore, we addressed whether concomitant TLR and RLH signaling, or some other additional mechanism, played a role. To this end, we generated MyD88(-/-)Trif(-/-)Cardif(-/-) (MyTrCa(-/-)) mice that succumbed to low-dose i.v. VSV infection with similar kinetics as IFN-I receptor-deficient mice. Three independent approaches (i.e., analysis of IFN-α/β serum levels, experiments with IFN-β reporter mice, and investigation of local IFN-stimulated gene induction) revealed that MyTrCa(-/-) mice did not mount IFN-I responses following VSV infection. Of note, treatment with rIFN-α protected the animals, qualifying MyTrCa(-/-) mice as a model to study the contribution of different immune cell subsets to the production of antiviral IFN-I. Upon adoptive transfer of wild-type plasmacytoid dendritic cells and subsequent VSV infection, MyTrCa(-/-) mice displayed significantly reduced viral loads in peripheral organs and showed prolonged survival. On the contrary, adoptive transfer of wild-type myeloid dendritic cells did not have such effects. Analysis of bone marrow chimeric mice revealed that TLR and RLH signaling of radioresistant and radiosensitive cells was required for efficient protection. Thus, upon VSV infection, plasmacytoid dendritic cell-derived IFN-I primarily protects peripheral organs, whereas concomitant TLR and RLH signaling of radioresistant stroma cells as well as of radiosensitive immune cells is needed to effectively protect against lethal disease. PMID:25127863

  3. In Vitro Synthesis of Proteins by Membrane-Bound Polyribosomes from Vesicular Stomatitis Virus-Infected HeLa Cells

    PubMed Central

    Grubman, Marvin J.; Ehrenfeld, Ellie; Summers, Donald F.

    1974-01-01

    Membrane-bound polysomes from vesicular stomatitis virus (VSV)-infected HeLa cells synthesize predominantly three proteins in an in vitro protein synthesizing system. These three proteins have different molecular weights than the viral structural proteins, i.e., 115,000, 88,000, and 72,000. Addition of preincubated L or HeLa cell S10 or HeLa cell crude initiation factors stimulates amino acid incorporation and, furthermore, alters the pattern of proteins synthesized. Stimulated membrane-bound polysomes synthesize predominantly viral protein G and lesser amounts of N, NS, and M. In vitro synthesized proteins G and N are very similar to virion proteins G and N based on analysis of tryptic methionine-labeled peptides. Most methionine-labeled tryptic peptides of virion G protein contain no carbohydrate moieties, since about 90% of sugar-labeled peptides co-chromatograph with only about 10% of methionine-labeled peptides. Sucrose gradient analysis of the labeled RNA present in VSV-infected membrane-bound polysomes reveals a relative enrichment in a class of viral RNA sedimenting slightly faster than the total population of the 13 to 15S mRNA, as compared to a VSV-infected crude cytoplasmic extract. A number of proteins, other than the viral structural proteins, are synthesized in the cytoplasm of five lines of VSV-infected cells. One of these proteins has the same molecular weight as the major in vitro synthesized protein, P88. In vitro synthesized protein P88 does not appear to be a precursor of viral structural proteins G, N, or M based on pulse-chase experiments and tryptic peptide mapping. Nonstimulated membrane-bound polysomes from uninfected HeLa cells synthesize the same size distribution of proteins as nonstimulated VSV-infected membrane-bound polysomes. Images PMID:4368799

  4. Vesicular zinc promotes presynaptic and inhibits postsynaptic long term potentiation of mossy fiber-CA3 synapse

    PubMed Central

    Pan, Enhui; Zhang, Xiao-an; Huang, Zhen; Krezel, Artur; Zhao, Min; Tin-berg, Christine E.; Lippard, Stephen J.; McNamara, James O.

    2011-01-01

    The presence of zinc in glutamatergic synaptic vesicles of excitatory neurons of mammalian cerebral cortex suggests that zinc might regulate plasticity of synapses formed by these neurons. Long term potentiation (LTP) is a form of synaptic plasticity that may underlie learning and memory. We tested the hypothesis that zinc within vesicles of mossy fibers (mf) contributes to mf-LTP, a classical form of presynaptic LTP. We synthesized an extracellular zinc chelator with selectivity and kinetic properties suitable for study of the large transient of zinc in the synaptic cleft induced by mf stimulation. We found that vesicular zinc is required for presynaptic mf-LTP. Unexpectedly, vesicular zinc also inhibits a novel form of postsynaptic mf-LTP. Because the mf-CA3 synapse provides a major source of excitatory input to the hippocampus, regulating its efficacy by these dual actions of vesicular zinc is critical to proper function of hippocampal circuitry in health and disease. PMID:21943607

  5. Chloroplast DNA Copy Number Changes during Plant Development in Organelle DNA Polymerase Mutants

    PubMed Central

    Morley, Stewart A.; Nielsen, Brent L.

    2016-01-01

    Chloroplast genome copy number is very high in leaf tissue, with upwards of 10,000 or more copies of the chloroplast DNA (ctDNA) per leaf cell. This is often promoted as a major advantage for engineering the plastid genome, as it provides high gene copy number and thus is expected to result in high expression of foreign proteins from integrated genes. However, it is also known that ctDNA copy number and ctDNA integrity decrease as cells age. Quantitative PCR (qPCR) allows measurement of organelle DNA levels relative to a nuclear gene target. We have used this approach to determine changes in copy number of ctDNA relative to the nuclear genome at different ages of Arabidopsis plant growth and in organellar DNA polymerase mutants. The mutant plant lines have T-DNA insertions in genes encoding the two organelle localized DNA polymerases (PolIA and PolIB). Each of these mutant lines exhibits some delay in plant growth and development as compared to wild-type plants, with the PolIB plants having a more pronounced delay. Both mutant lines develop to maturity and produce viable seeds. Mutants for both proteins were observed to have a reduction in ctDNA and mtDNA copy number relative to wild type plants at all time points as measured by qPCR. Both DNA polymerase mutants had a fairly similar decrease in ctDNA copy number, while the PolIB mutant had a greater effect of reduction in mtDNA levels. However, despite similar decreases in genome copy number, RT-PCR analysis of PolIA mutants show that PolIB expression remains unchanged, suggesting that PolIA may not be essential to plant survival. Furthermore, genotypic analysis of plants from heterozygous parents display a strong pressure to maintain two functioning copies of PolIB. These results indicate that the two DNA polymerases are both important in ctDNA replication, and they are not fully redundant to each other, suggesting each has a specific function in plant organelles. PMID:26870072

  6. Nannochloropsis plastid and mitochondrial phylogenomes reveal organelle diversification mechanism and intragenus phylotyping strategy in microalgae

    PubMed Central

    2013-01-01

    Background Microalgae are promising feedstock for production of lipids, sugars, bioactive compounds and in particular biofuels, yet development of sensitive and reliable phylotyping strategies for microalgae has been hindered by the paucity of phylogenetically closely-related finished genomes. Results Using the oleaginous eustigmatophyte Nannochloropsis as a model, we assessed current intragenus phylotyping strategies by producing the complete plastid (pt) and mitochondrial (mt) genomes of seven strains from six Nannochloropsis species. Genes on the pt and mt genomes have been highly conserved in content, size and order, strongly negatively selected and evolving at a rate 33% and 66% of nuclear genomes respectively. Pt genome diversification was driven by asymmetric evolution of two inverted repeats (IRa and IRb): psbV and clpC in IRb are highly conserved whereas their counterparts in IRa exhibit three lineage-associated types of structural polymorphism via duplication or disruption of whole or partial genes. In the mt genomes, however, a single evolution hotspot varies in copy-number of a 3.5 Kb-long, cox1-harboring repeat. The organelle markers (e.g., cox1, cox2, psbA, rbcL and rrn16_mt) and nuclear markers (e.g., ITS2 and 18S) that are widely used for phylogenetic analysis obtained a divergent phylogeny for the seven strains, largely due to low SNP density. A new strategy for intragenus phylotyping of microalgae was thus proposed that includes (i) twelve sequence markers that are of higher sensitivity than ITS2 for interspecies phylogenetic analysis, (ii) multi-locus sequence typing based on rps11_mt-nad4, rps3_mt and cox2-rrn16_mt for intraspecies phylogenetic reconstruction and (iii) several SSR loci for identification of strains within a given species. Conclusion This first comprehensive dataset of organelle genomes for a microalgal genus enabled exhaustive assessment and searches of all candidate phylogenetic markers on the organelle genomes. A new strategy

  7. Vesicular systems for delivering conventional small organic molecules and larger macromolecules to and through human skin.

    PubMed

    El Maghraby, G M; Williams, A C

    2009-02-01

    The history of using vesicular systems for drug delivery to and through skin started nearly three decades ago with a study utilising phospholipid liposomes to improve skin deposition and reduce systemic effects of triamcinolone acetonide. Subsequently, many researchers evaluated liposomes with respect to skin delivery, with the majority of them recording localised effects and relatively few studies showing transdermal delivery effects. Shortly after this, transfersomes were developed with claims about their ability to deliver their payload into and through the skin with efficiencies similar to subcutaneous administration. Since these vesicles are ultradeformable, they were thought to penetrate intact skin deep enough to reach the systemic circulation. Their mechanisms of action remain controversial, with diverse processes being reported. Parallel to this development, other classes of vesicles were produced, with ethanol being included into the vesicles to provide flexibility (as in ethosomes); vesicles were constructed from surfactants and cholesterol (as in niosomes). The ultradeformable vesicles showed variable efficiency in delivering low-molecular-weight and macromolecular drugs. This article will critically evaluate vesicular systems for dermal and transdermal delivery of drugs, considering both their efficacy and their potential mechanisms of action. PMID:19239387

  8. Duration of effect of interferon aerosol prophylaxis of vesicular stomatitis virus infection in mice.

    PubMed Central

    Wyde, P R; Sun, C S; Wilson, S Z; Knight, V

    1985-01-01

    Mice were exposed for 8 h to continuous small-particle aerosols containing natural mouse alpha interferon (estimated dosage 100 U per mouse) or one of two concentrations of hybrid recombinant alpha interferon A/D bgl (estimated dosages of 100 and 10,000 U per mouse, respectively). On days 1, 3, 5, 7, and 9 after exposure to these interferons, three mice from each group were inoculated intranasally with 100 PFU of vesicular stomatitis virus. Control mice were exposed to aerosols of saline or inoculated intraperitoneally with either natural mouse alpha interferon (350 U) or one of two doses of hybrid recombinant alpha interferon A/D bgl (350 or 35,000 U) and challenged similarly. Of mice injected intraperitoneally, only those given 35,000 U of hybrid recombinant alpha interferon A/D bgl 24 h before virus challenge were protected from pulmonary infection, compared with the saline-treated control mice. Of mice given 100 U of either interferon by small-particle aerosol, only those exposed 24 h before inoculation of vesicular stomatitis virus had reduced pulmonary titers of the virus. However, of mice given ca. 10,000 U of hybrid recombinant alpha interferon A/D bgl by small-particle aerosol, all groups except those exposed 9 days before virus inoculation had significantly reduced lung virus titers. PMID:2984982

  9. Transient phenomena in vesicular lava flows based on laboratory experiments with analogue materials

    NASA Astrophysics Data System (ADS)

    Bagdassarov, N.; Pinkerton, H.

    2004-04-01

    Realistic lava flow models require a comprehensive understanding of the rheological properties of lava under a range of stress conditions. Previous measurements have shown that at typical eruption temperatures lavas are non-Newtonian. This is commonly attributed to the formation and destruction of crystal networks. In the present study, the effects of bubbles on the time-dependent, non-Newtonian properties of vesicular melts are investigated experimentally using analogue materials. The shear-thinning behaviour of bubbly liquids is shown to be dependent on the previous shearing history. This thixotropic behaviour, which was investigated using a rotational vane-viscometer, is caused by delayed bubble deformation and recovery when subjected to changes in shear stress. The viscoelastic transition and the transient flow behaviour of analogue fluids were studied using both a rotational vane-viscometer and oscillatory shear apparatus. These experiments have shown that vesicular suspensions are viscoelastic fluids with a yield strength, power law rheology, and a non-zero shear modulus. These properties are also found in polymer fluids commonly used as analogue materials for lava such as gum rosin. We show that, when materials with this rheology are accelerated in channels, they may be fragmented, and when they flow through a narrowing conduit, pulsating flow can develop as a consequence of a transition from slip to non-slip conditions at the conduit wall. This has important implications both for effusive and explosive volcanic eruptions.

  10. DAYSLEEPER: a nuclear and vesicular-localized protein that is expressed in proliferating tissues

    PubMed Central

    2013-01-01

    Background DAYSLEEPER is a domesticated transposase that is essential for development in Arabidopsis thaliana [Nature, 436:282–284, 2005]. It is derived from a hAT-superfamily transposon and contains many of the features found in the coding sequence of these elements [Nature, 436:282–284, 2005, Genetics, 158:949–957, 2001]. This work sheds light on the expression of this gene and localization of its product in protoplasts and in planta. Using deletion constructs, important domains in the protein were identified. Results DAYSLEEPER is predominantly expressed in meristems, developing flowers and siliques. The protein is mainly localized in the nucleus, but can also be seen in discrete foci in the cytoplasm. Using several vesicular markers, we found that these foci belong to vesicular structures of the trans-golgi network, multivesicular bodies (MVB’s) and late endosomes. The central region as well as both the N- and the C-terminus are essential to DAYSLEEPER function, since versions of DAYSLEEPER deleted for these regions are not able to complement the daysleeper phenotype. Like hAT-transposases, we show that DAYSLEEPER has a functionally conserved dimerization domain [J Biol Chem, 282:7563–7575, 2007]. Conclusions DAYSLEEPER has retained the global structure of hAT transposases and it seems that most of these conserved features are essential to DAYSLEEPER’s cellular function. Although structurally similar, DAYSLEEPER seems to have broadened its range of action beyond the nucleus in comparison to transposases. PMID:24330683

  11. Interaction of Rhizosphere Bacteria, Fertilizer, and Vesicular-Arbuscular Mycorrhizal Fungi with Sea Oats †

    PubMed Central

    Will, M. E.; Sylvia, D. M.

    1990-01-01

    Plants must be established quickly on replenished beaches in order to stabilize the sand and begin the dune-building process. The objective of this research was to determine whether inoculation of sea oats (Uniola paniculata L.) with bacteria (indigenous rhizosphere bacteria and N2 fixers) alone or in combination with vesicular-arbuscular mycorrhizal fungi would enhance plant growth in beach sand. At two fertilizer-N levels, Klebsiella pneumoniae and two Azospirillum spp. did not provide the plants with fixed atmospheric N; however, K. pneumoniae increased root and shoot growth. When a sparingly soluble P source (CaHPO4) was added to two sands, K. pneumoniae increased plant growth in sand with a high P content. The phosphorus content of shoots was not affected by bacterial inoculation, indicating that a mechanism other than bacterially enhanced P availability to plants was responsible for the growth increases. When sea oats were inoculated with either K. pneumoniae or Acaligenes denitrificans and a mixed Glomus inoculum, there was no consistent evidence of a synergistic effect on plant growth. Nonetheless, bacterial inoculation increased root colonization by vesicular-arbuscular mycorrhizal fungi when the fungal inoculum consisted of colonized roots but had no effect on colonization when the inoculum consisted of spores alone. K. pneumoniae was found to increase spore germination and hyphal growth of Glomus deserticola compared with the control. The use of bacterial inoculants to enhance establishment of pioneer dune plants warrants further study. PMID:16348236

  12. Role of the atypical vesicular glutamate transporter VGLUT3 in l-DOPA-induced dyskinesia.

    PubMed

    Gangarossa, Giuseppe; Guzman, Monica; Prado, Vania F; Prado, Marco A M; Daumas, Stephanie; El Mestikawy, Salah; Valjent, Emmanuel

    2016-03-01

    Parkinson's disease (PD) is characterized by the degeneration of dopaminergic neurons. The gold standard therapy relies on dopamine (DA) replacement by the administration of levodopa (l-DOPA). However, with time l-DOPA treatment induces severe motor side effects characterized by abnormal and involuntary movements, or dyskinesia. Although earlier studies point to a role of striatal cholinergic interneurons, also known as striatal tonically active neurons (TANs), in l-DOPA-induced dyskinesia (LID), the underlying mechanisms remain to be fully characterized. Here, we find that DA depletion is accompanied by increased expression of choline acetyltransferase (ChAT), the vesicular acetylcholine transporter (VAChT) as well as the atypical vesicular glutamate transporter type 3 (VGLUT3). TANs number and soma size are not changed. In dyskinetic mice, the VAChT levels remain high whereas the expression of VGLUT3 decreases. LID is attenuated in VGLUT3-deficient mice but not in mice bearing selective inactivation of VAChT in TANs. Finally, the absence of VGLUT3 is accompanied by a reduction of l-DOPA-induced phosphorylation of ERK1/2, ribosomal subunit (rpS6) and GluA1. Our results reveal that VGLUT3 plays an important role in the development of LID and should be considered as a potential and promising therapeutic target for prevention of LID. PMID:26711621

  13. Heterogeneous transport of digitalis-like compounds by P-glycoprotein in vesicular and cellular assays.

    PubMed

    Gozalpour, Elnaz; Wilmer, Martijn J; Bilos, Albert; Masereeuw, Rosalinde; Russel, Frans G M; Koenderink, Jan B

    2016-04-01

    Digitalis-like compounds (DLCs), the ancient medication of heart failure and Na,K-ATPase inhibitors, are characterized by their toxicity. Drug-drug interactions (DDIs) at absorption and excretion levels play a key role in their toxicity, hence, knowledge about the transporters involved might prevent these unwanted interactions. In the present study, the transport of fourteen DLCs with human P-glycoprotein (P-gp; ABCB1) was studied using a liquid chromatography-mass spectrometry (LC-MS) quantification method. DLC transport by P-gp overexpressing Madin-Darby canine kidney (MDCK) and immortalized human renal cells (ciPTEC) was compared to vesicular DLC transport. Previously, we identified convallatoxin as a substrate using membrane vesicles overexpressing P-gp; however, we could not measure transport of other DLCs in this assay (Gozalpour et al., 2014a). Here, we showed that lipophilic digitoxin, digoxigenin, strophanthidin and proscillaridin A are P-gp substrates in cellular accumulation assays, whereas the less lipophilic convallatoxin was not. P-gp function in the cellular accumulation assays depends on the entrance of lipophilic compounds by passive diffusion, whereas the vesicular transport assay is more appropriate for hydrophilic substrates. In conclusion, we identified digitoxin, digoxigenin, strophanthidin and proscillaridin A as P-gp substrates using cellular accumulation assays and recognized lipophilicity as an important factor in selecting a suitable transport assay. PMID:26708294

  14. Vibrio tapetis isolated from vesicular skin lesions in Dover sole Solea solea.

    PubMed

    Declercq, A M; Chiers, K; Soetaert, M; Lasa, A; Romalde, J L; Polet, H; Haesebrouck, F; Decostere, A

    2015-06-29

    Vibrio tapetis is primarily known as the causative agent for brown ring disease in bivalves, although it has been isolated from cultivated fish during mortalities on farms. Here we describe the first isolation of V. tapetis from wild-caught and subsequently captive-held Dover sole Solea solea. Pathological features consisted of multifocal circular greyish-white skin discolourations evolving into vesicular lesions and subsequent ulcerations on the pigmented side. On the non-pigmented side, multiple circular lesions-white at the center and red at the edges-were evident. Histological examination of the vesicular lesions revealed dermal fluid-filled spaces, collagen tissue necrosis and a mixed inflammatory infiltrate, with large numbers of small rod-shaped bacteria. In the deep skin lesions, loss of scales and dermal connective tissue, with degeneration and fragmentation of the myofibres bordering the ulceration, were noted. Serotyping, DNA-DNA hybridization and REP- and ERIC-PCR techniques showed that the retrieved isolates displayed a profile similar to the representative strain of genotype/serotype O2 which originally was isolated from carpet-shell clam Venerupis decussata and to which isolates obtained from wedge sole Dicologoglossa cuneata were also closely related. PMID:26119302

  15. Presynaptic Mechanisms of Lead Neurotoxicity: Effects on Vesicular Release, Vesicle Clustering and Mitochondria Number

    PubMed Central

    McGlothan, Jennifer L.; Stansfield, Kirstie H.; Stanton, Patric K.; Guilarte, Tomás R.

    2015-01-01

    Childhood lead (Pb2+) intoxication is a global public health problem and accounts for 0.6% of the global burden of disease associated with intellectual disabilities. Despite the recognition that childhood Pb2+ intoxication contributes significantly to intellectual disabilities, there is a fundamental lack of knowledge on presynaptic mechanisms by which Pb2+ disrupts synaptic function. In this study, using a well-characterized rodent model of developmental Pb2+ neurotoxicity, we show that Pb2+ exposure markedly inhibits presynaptic vesicular release in hippocampal Schaffer collateral-CA1 synapses in young adult rats. This effect was associated with ultrastructural changes which revealed a reduction in vesicle number in the readily releasable/docked vesicle pool, disperse vesicle clusters in the resting pool, and a reduced number of presynaptic terminals with multiple mitochondria with no change in presynaptic calcium influx. These studies provide fundamental knowledge on mechanisms by which Pb2+ produces profound inhibition of presynaptic vesicular release that contribute to deficits in synaptic plasticity and intellectual development. PMID:26011056

  16. Presynaptic mechanisms of lead neurotoxicity: effects on vesicular release, vesicle clustering and mitochondria number.

    PubMed

    Zhang, Xiao-Lei; Guariglia, Sara R; McGlothan, Jennifer L; Stansfield, Kirstie H; Stanton, Patric K; Guilarte, Tomás R

    2015-01-01

    Childhood lead (Pb2+) intoxication is a global public health problem and accounts for 0.6% of the global burden of disease associated with intellectual disabilities. Despite the recognition that childhood Pb2+ intoxication contributes significantly to intellectual disabilities, there is a fundamental lack of knowledge on presynaptic mechanisms by which Pb2+ disrupts synaptic function. In this study, using a well-characterized rodent model of developmental Pb2+ neurotoxicity, we show that Pb2+ exposure markedly inhibits presynaptic vesicular release in hippocampal Schaffer collateral-CA1 synapses in young adult rats. This effect was associated with ultrastructural changes which revealed a reduction in vesicle number in the readily releasable/docked vesicle pool, disperse vesicle clusters in the resting pool, and a reduced number of presynaptic terminals with multiple mitochondria with no change in presynaptic calcium influx. These studies provide fundamental knowledge on mechanisms by which Pb2+ produces profound inhibition of presynaptic vesicular release that contribute to deficits in synaptic plasticity and intellectual development. PMID:26011056

  17. GLTP mediated non-vesicular GM1 transport between native membranes.

    PubMed

    Lauria, Ines; van Üüm, Jan; Mjumjunov-Crncevic, Esmina; Walrafen, David; Spitta, Luis; Thiele, Christoph; Lang, Thorsten

    2013-01-01

    Lipid transfer proteins (LTPs) are emerging as key players in lipid homeostasis by mediating non-vesicular transport steps between two membrane surfaces. Little is known about the driving force that governs the direction of transport in cells. Using the soluble LTP glycolipid transfer protein (GLTP), we examined GM1 (monosialotetrahexosyl-ganglioside) transfer to native membrane surfaces. With artificial GM1 donor liposomes, GLTP can be used to increase glycolipid levels over natural levels in either side of the membrane leaflet, i.e., external or cytosolic. In a system with native donor- and acceptor-membranes, we find that GLTP balances highly variable GM1 concentrations in a population of membranes from one cell type, and in addition, transfers lipids between membranes from different cell types. Glycolipid transport is highly efficient, independent of cofactors, solely driven by the chemical potential of GM1 and not discriminating between the extra- and intracellular membrane leaflet. We conclude that GLTP mediated non-vesicular lipid trafficking between native membranes is driven by simple thermodynamic principles and that for intracellular transport less than 1 µM GLTP would be required in the cytosol. Furthermore, the data demonstrates the suitability of GLTP as a tool for artificially increasing glycolipid levels in cellular membranes. PMID:23555818

  18. Herpes Simplex Virus Capsid-Organelle Association in the Absence of the Large Tegument Protein UL36p

    PubMed Central

    Kharkwal, Himanshu; Furgiuele, Sara Shanda; Smith, Caitlin G.

    2015-01-01

    ABSTRACT UL36p (VP1/2) is the largest protein encoded by herpes simplex virus 1 (HSV-1) and resides in the innermost layer of the viral tegument, lying between the capsid and the envelope. UL36p performs multiple functions in the HSV life cycle, including an essential role in cytoplasmic envelopment. We earlier described the isolation of a virion-associated cytoplasmic membrane fraction from HSV-infected cells. Biochemical and ultrastructural analyses showed that the organelles in this buoyant fraction contain enveloped infectious HSV particles in their lumens and naked capsids docked to their cytoplasmic surfaces. These organelles can also recruit molecular motors and transport their cargo virions along microtubules in vitro. Here we examine the properties of these HSV-associated organelles in the absence of UL36p. We find that while capsid envelopment is clearly defective, a subpopulation of capsids nevertheless still associate with the cytoplasmic faces of these organelles. The existence of these capsid-membrane structures was confirmed by subcellular fractionation, immunocytochemistry, lipophilic dye fluorescence microscopy, thin-section electron microscopy, and correlative light and electron microscopy. We conclude that capsid-membrane binding can occur in the absence of UL36p and propose that this association may precede the events of UL36p-driven envelopment. IMPORTANCE Membrane association and envelopment of the HSV capsid are essential for the assembly of an infectious virion. Envelopment involves the complex interplay of a large number of viral and cellular proteins; however, the function of most of them is unknown. One example of this is the viral protein UL36p, which is clearly essential for envelopment but plays a poorly understood role. Here we demonstrate that organelles utilized for HSV capsid envelopment still accumulate surface-bound capsids in the absence of UL36p. We propose that UL36p-independent binding of capsids to organelles occurs prior to

  19. Development and laboratory evaluation of two lateral flow devices for the detection of vesicular stomatitis virus in clinical samples.

    PubMed

    Ferris, Nigel P; Clavijo, Alfonso; Yang, Ming; Velazquez-Salinas, Lauro; Nordengrahn, Ann; Hutchings, Geoffrey H; Kristersson, Therese; Merza, Malik

    2012-03-01

    Two lateral flow devices (LFD) for the detection of vesicular stomatitis (VS) virus (VSV), types Indiana (VSV-IND) and New Jersey (VSV-NJ) were developed using monoclonal antibodies C1 and F25VSVNJ-45 to the respective VSV serotypes. The performance of the LFDs was evaluated in the laboratory on suspensions of vesicular epithelia and cell culture passage derived supernatants of VSV. The collection of test samples included 105 positive for VSV-IND (92 vesicular epithelial suspensions and 13 cell culture antigens; encompassing 93 samples of subtype 1 [VSV-IND-1], 9 of subtype 2 [VSV-IND-2] and 3 of subtype 3 [VSV-IND-3]) and 189 positive for VSV-NJ (162 vesicular epithelial suspensions and 27 cell culture antigens) from suspected cases of vesicular disease in cattle and horses collected from 11 countries between 1937 and 2008 or else were derived from experimental infection and 777 samples that were either shown to be positive or negative for foot-and-mouth disease (FMD) virus (FMDV) and swine vesicular disease virus (SVDV) or else collected from healthy cattle or pigs and collected from 68 countries between 1965 and 2011. The diagnostic sensitivity of the VSV-IND (for reaction with VSV-IND-1) and VSV-NJ LFDs was either similar or identical at 94.6% (VSV-IND) and 97.4% (VSV-NJ) compared to 92.5% and 97.4% obtained by the reference method of antigen ELISA. The VSV-IND LFD failed to react with viruses of VSV-IND-2 and 3, while the VSV-NJ device recognized all VSV-NJ virus strains. The diagnostic specificities of the VSV-IND and VSV-NJ LFDs were 99.1% and 100, respectively, compared to 99.6% and 99.8% for the ELISA. Reactions with FMDV which can produce indistinguishable syndromes clinically in cattle, pigs and sheep and SVDV (vesicular disease in pigs) did not occur. These data illustrate the potential for the LFDs to be used next to the animal for providing rapid and objective support to veterinarians in their clinical judgment of vesicular disease and for the subtype

  20. The Role of Organelle Stresses in Diabetes Mellitus and Obesity: Implication for Treatment

    PubMed Central

    Chang, Yi-Cheng; Hee, Siow-Wey; Hsieh, Meng-Lun; Jeng, Yung-Ming; Chuang, Lee-Ming

    2015-01-01

    The type 2 diabetes pandemic in recent decades is a huge global health threat. This pandemic is primarily attributed to the surplus of nutrients and the increased prevalence of obesity worldwide. In contrast, calorie restriction and weight reduction can drastically prevent type 2 diabetes, indicating a central role of nutrient excess in the development of diabetes. Recently, the molecular links between excessive nutrients, organelle stress, and development of metabolic disease have been extensively studied. Specifically, excessive nutrients trigger endoplasmic reticulum stress and increase the production of mitochondrial reactive oxygen species, leading to activation of stress signaling pathway, inflammatory response, lipogenesis, and pancreatic beta-cell death. Autophagy is required for clearance of hepatic lipid clearance, alleviation of pancreatic beta-cell stress, and white adipocyte differentiation. ROS scavengers, chemical chaperones, and autophagy activators have demonstrated promising effects for the treatment of insulin resistance and diabetes in preclinical models. Further results from clinical trials are eagerly awaited. PMID:26613076

  1. Taking organelles apart, putting them back together and creating new ones: lessons from the endoplasmic reticulum.

    PubMed

    Lavoie, Christine; Roy, Line; Lanoix, Joël; Taheri, Mariam; Young, Robin; Thibault, Geneviève; Farah, Carol Abi; Leclerc, Nicole; Paiement, Jacques

    2011-06-01

    The endoplasmic reticulum (ER) is a highly dynamic organelle. It is composed of four subcompartments including nuclear envelope (NE), rough ER (rER), smooth ER (sER) and transitional ER (tER). The subcompartments are interconnected, can fragment and dissociate and are able to reassemble again. They coordinate with cell function by way of protein regulators in the surrounding cytosol. The activity of the many associated molecular machines of the ER as well as the fluid nature of the limiting membrane of the ER contribute extensively to the dynamics of the ER. This review examines the properties of the ER that permit its isolation and purification and the physiological conditions that permit reconstitution both in vitro and in vivo in normal and in disease conditions. PMID:21536318

  2. [Methods of substances and organelles introduction in living cell for cell engineering technologies].

    PubMed

    Nikitin, V A

    2007-01-01

    We have presented the classification of more than 40 methods of genetic material, substances and organelles introduction into a living cell. Each of them has its characteristic advantages, disadvantages and limitations with respect to cell viability, transfer efficiency, general applicability, and technical requirements. It this article we have enlarged on the description of our developments of several new and improved approaches, methods and devices of the direct microinjection into a single cell and cell microsurgery with the help of glass micropipettes. The problem of low efficiency of mammalian cloning is discussed with emphasis on the necessity of expertizing of each step of single cell reconstruction to begin with microsurgical manipulations and necessity of the development of such methods of single cell resonstruction that could minimize the possible damage of the cell. PMID:17926558

  3. Muscle intermediate filaments and their links to membranes and membranous organelles

    SciTech Connect

    Capetanaki, Yassemi . E-mail: ycapetanaki@bioacademy.gr; Bloch, Robert J.; Kouloumenta, Asimina; Mavroidis, Manolis; Psarras, Stelios

    2007-06-10

    Intermediate filaments (IFs) play a key role in the integration of structure and function of striated muscle, primarily by mediating mechanochemical links between the contractile apparatus and mitochondria, myonuclei, the sarcolemma and potentially the vesicle trafficking apparatus. Linkage of all these membranous structures to the contractile apparatus, mainly through the Z-disks, supports the integration and coordination of growth and energy demands of the working myocyte, not only with force transmission, but also with de novo gene expression, energy production and efficient protein and lipid trafficking and targeting. Desmin, the most abundant and intensively studied muscle intermediate filament protein, is linked to proper costamere organization, myoblast and stem cell fusion and differentiation, nuclear shape and positioning, as well as mitochondrial shape, structure, positioning and function. Similar links have been established for lysosomes and lysosome-related organelles, consistent with the presence of widespread links between IFs and membranous structures and the regulation of their fusion, morphology and stabilization necessary for cell survival.

  4. Biochemistry of the Phagosome: The Challenge to Study a Transient Organelle

    PubMed Central

    Nüsse, Oliver

    2011-01-01

    Phagocytes are specialized cells of the immune system, designed to engulf and destroy harmful microorganisms inside the newly formed phagosome. The latter is an intracellular organelle that is transformed into a toxic environment within minutes and disappears once the pathogen is destroyed. Reactive oxygen species and reactive nitrogen species are produced inside the phagosome. Intracellular granules or lysosomes of the phagocyte fuse with the phagosome and liberate their destructive enzymes. This process of phagocytosis efficiently protects against most infections; however, some microorganisms avoid their destruction and cause severe damage. To understand such failure of phagosomal killing, we need to learn more about the actual destruction process in the phagosome. This paper summarizes methods to investigate the biochemistry of the phagosome and discusses some of their limitations. In accordance with the nature of the phagosome, the issue of localization and temporal dynamics is emphasized, and recent developments are highlighted. PMID:22194668

  5. B Chromosomes of Aegilops speltoides Are Enriched in Organelle Genome-Derived Sequences

    PubMed Central

    Ruban, Alevtina; Fuchs, Jörg; Marques, André; Schubert, Veit; Soloviev, Alexander; Raskina, Olga; Badaeva, Ekaterina; Houben, Andreas

    2014-01-01

    B chromosomes (Bs) are dispensable components of the genome exhibiting non-Mendelian inheritance. Chromosome counts and flow cytometric analysis of the grass species Aegilops speltoides revealed a tissue-type specific distribution of the roughly 570 Mbp large B chromosomes. To address the question whether organelle-to-nucleus DNA transfer is a mechanism that drives the evolution of Bs, in situ hybridization was performed with labelled organellar DNA. The observed B-specific accumulation of chloroplast- and mitochondria-derived sequences suggests a reduced selection against the insertion of organellar DNA in supernumerary chromosomes. The distribution of B-localised organellar-derived sequences and other sequences differs between genotypes of different geographical origins. PMID:24587288

  6. Organelle-specific injury to melanin-containing cells in human skin by pulsed laser irradiation

    SciTech Connect

    Murphy, G.F.; Shepard, R.S.; Paul, B.S.; Menkes, A.; Anderson, R.R.; Parrish, J.A.

    1983-12-01

    Physical models predict that ultraviolet laser radiation of appropriately brief pulses can selectively alter melanin-containing cellular targets in human skin. Skin of normal human volunteers was exposed to brief (20 nanosecond) 351-nm wave length pulses from a XeF excimer laser, predicting that those cells containing the greatest quantities of melanized melanosomes (lower half of the epidermis) would be selectively damaged. Transmission electron microscopy revealed the earliest cellular alteration to be immediate disruption of melanosomes, both within melanocytes and basal keratinocytes. This disruption was dose dependent and culminated in striking degenerative changes in these cells. Superficial keratinocytes and Langerhans cells were not affected. It was concluded that the XeF excimer laser is capable of organelle-specific injury to melanosomes. These findings may have important clinical implications in the treatment of both benign and malignant pigmented lesions by laser radiations of defined wave lengths and pulse durations.

  7. Influence of organelle geometry on the apparent binding kinetics of peripheral membrane proteins

    NASA Astrophysics Data System (ADS)

    Hoffmann, Julia; Fickentscher, Rolf; Weiss, Matthias

    2015-02-01

    Information processing in living cells frequently involves an exchange of peripheral membrane proteins between the cytosol and organelle membranes. The typical time scale τ of these association-dissociation cycles is commonly quantified in vivo via fluorescence recovery after photobleaching (FRAP). Contrary to common assumptions, we show here that τ values determined by FRAP depend on the size and number of target structures. Hence, FRAP times alone are insufficient to draw conclusions about the proteins' binding kinetics. In contrast, extracting primary molecular association and dissociation rates from FRAP approaches provides a size-independent and therefore robust measure for the proteins' binding kinetics. We support our theoretical considerations with experiments on the small GTPase Arf-1 that transiently associates with Golgi membranes: While Arf-1 recovery times in untreated cells and in cells with disrupted microtubules are significantly different, the molecular kinetic rates are shown to be the same in both cases.

  8. 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

  9. Symbiotic theory of the origin of eukaryotic organelles; criteria for proof.

    PubMed

    Margulis, L

    1975-01-01

    The purpose of a scientific theory is to unite apparently disparate observations into a coherent set of generalizations with predictive power. Historical theories, which necessarily treat complex irreversible events, can never be directly tested. However they certainly can lead to predictions. The 'extreme' version of the serial endosymbiotic theory argues that three classes of eukaryotic organelles had free-living ancestors: mitochondria, basal bodies/flagella/cilia [(9 + 2) homologues] and photosynthetic plastids. Many lines of evidence support this theory and can be interpreted in relation to one another on the basis of this theory. Even if this theory should eventually be proved wrong it has the real advantage of generating a large number of unique experimentally verifiable hypotheses. PMID:822529

  10. MicroRNAs in the intracellular space, regulation of organelle specific pathways in health and disease.

    PubMed

    Nguyen, Thao T; Brenu, Ekua W; Staines, Don R; Marshall-Gradisnik, Sonya M

    2014-01-01

    MicroRNAs (miRNA) are small (~22 nucleotide] non-coding RNA molecules originally characterised as nonsense or junk DNA. Emerging research suggests that these molecules have diverse regulatory roles in an array of molecular, cellular and physiological processes. MiRNAs are versatile and highly stable molecules, therefore, they are able to exist as intracellular or extracellular miRNAs. The purpose of this paper is to review the function and role of miRNAs in the intracellular space with specific focus on the interactions between miRNAs and organelles such as the mitochondria and the rough endoplasmic reticulum. Understanding the role of miRNAs in the intracellular space may be vital in understanding the mechanism of certain diseases. PMID:25541912

  11. Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes

    PubMed Central

    Shim, Sang-Hee; Xia, Chenglong; Zhong, Guisheng; Babcock, Hazen P.; Vaughan, Joshua C.; Huang, Bo; Wang, Xun; Xu, Cheng; Bi, Guo-Qiang; Zhuang, Xiaowei

    2012-01-01

    Imaging membranes in live cells with nanometer-scale resolution promises to reveal ultrastructural dynamics of organelles that are essential for cellular functions. In this work, we identified photoswitchable membrane probes and obtained super-resolution fluorescence images of cellular membranes. We demonstrated the photoswitching capabilities of eight commonly used membrane probes, each specific to the plasma membrane, mitochondria, the endoplasmic recticulum (ER) or lysosomes. These small-molecule probes readily label live cells with high probe densities. Using these probes, we achieved dynamic imaging of specific membrane structures in living cells with 30–60 nm spatial resolution at temporal resolutions down to 1–2 s. Moreover, by using spectrally distinguishable probes, we obtained two-color super-resolution images of mitochondria and the ER. We observed previously obscured details of morphological dynamics of mitochondrial fusion/fission and ER remodeling, as well as heterogeneous membrane diffusivity on neuronal processes. PMID:22891300

  12. Maternally supplied S-acyl-transferase is required for crystalloid organelle formation and transmission of the malaria parasite.

    PubMed

    Santos, Jorge M; Duarte, Neuza; Kehrer, Jessica; Ramesar, Jai; Avramut, M Cristina; Koster, Abraham J; Dessens, Johannes T; Frischknecht, Friedrich; Chevalley-Maurel, Séverine; Janse, Chris J; Franke-Fayard, Blandine; Mair, Gunnar R

    2016-06-28

    Transmission of the malaria parasite from the mammalian host to the mosquito vector requires the formation of adequately adapted parasite forms and stage-specific organelles. Here we show that formation of the crystalloid-a unique and short-lived organelle of the Plasmodium ookinete and oocyst stage required for sporogony-is dependent on the precisely timed expression of the S-acyl-transferase DHHC10. DHHC10, translationally repressed in female Plasmodium berghei gametocytes, is activated translationally during ookinete formation, where the protein is essential for the formation of the crystalloid, the correct targeting of crystalloid-resident protein LAP2, and malaria parasite transmission. PMID:27303037

  13. Developmental changes and organelle biogenesis in the reproductive organs of thermogenic skunk cabbage (Symplocarpus renifolius)

    PubMed Central

    Ito-Inaba, Yasuko; Sato, Mayuko; Masuko, Hiromi; Hida, Yamato; Toyooka, Kiminori; Watanabe, Masao; Inaba, Takehito

    2009-01-01

    Sex-dependent thermogenesis during reproductive organ development in the inflorescence is a characteristic feature of some of the protogynous arum species. One such plant, skunk cabbage (Symplocarpus renifolius), can produce massive heat during the female stage but not during the subsequent male stage in which the stamen completes development, the anthers dehisce, and pollen is released. Unlike other thermogenic species, skunk cabbage belongs to the bisexual flower group. Although recent studies have identified the spadix as the thermogenic organ, it remains unclear how individual tissues or intracellular structures are involved in thermogenesis. In this study, reproductive organ development and organelle biogenesis were examined during the transition from the female to the male stage. During the female stage, the stamens exhibit extensive structural changes including changes in organelle structure and density. They accumulate high levels of mitochondrial proteins, including possible thermogenic factors, alternative oxidase, and uncoupling protein. By contrast, the petals and pistils do not undergo extensive changes during the female stage. However, they contain a larger number of mitochondria than during the male stage in which they develop large cytoplasmic vacuoles. Comparison between female and male spadices suggests that mitochondrial number rather than their level of activity correlates with thermogenesis. Their spadices, even in the male, contain a larger amount of mitochondria that had greater oxygen consumption, compared with non-thermogenic plants. Taken together, our data suggest that the extensive maturation process in stamens produces massive heat through increased metabolic activities. The possible mechanisms by which petal and pistil metabolism may affect thermogenesis are also discussed. PMID:19640927

  14. A repeat protein links Rubisco to form the eukaryotic carbon-concentrating organelle

    PubMed Central

    Mackinder, Luke C. M.; Meyer, Moritz T.; Mettler-Altmann, Tabea; Chen, Vivian K.; Mitchell, Madeline C.; Caspari, Oliver; Freeman Rosenzweig, Elizabeth S.; Pallesen, Leif; Reeves, Gregory; Itakura, Alan; Roth, Robyn; Sommer, Frederik; Geimer, Stefan; Mühlhaus, Timo; Schroda, Michael; Goodenough, Ursula; Stitt, Mark; Griffiths, Howard; Jonikas, Martin C.

    2016-01-01

    Biological carbon fixation is a key step in the global carbon cycle that regulates the atmosphere's composition while producing the food we eat and the fuels we burn. Approximately one-third of global carbon fixation occurs in an overlooked algal organelle called the pyrenoid. The pyrenoid contains the CO2-fixing enzyme Rubisco and enhances carbon fixation by supplying Rubisco with a high concentration of CO2. Since the discovery of the pyrenoid more that 130 y ago, the molecular structure and biogenesis of this ecologically fundamental organelle have remained enigmatic. Here we use the model green alga Chlamydomonas reinhardtii to discover that a low-complexity repeat protein, Essential Pyrenoid Component 1 (EPYC1), links Rubisco to form the pyrenoid. We find that EPYC1 is of comparable abundance to Rubisco and colocalizes with Rubisco throughout the pyrenoid. We show that EPYC1 is essential for normal pyrenoid size, number, morphology, Rubisco content, and efficient carbon fixation at low CO2. We explain the central role of EPYC1 in pyrenoid biogenesis by the finding that EPYC1 binds Rubisco to form the pyrenoid matrix. We propose two models in which EPYC1’s four repeats could produce the observed lattice arrangement of Rubisco in the Chlamydomonas pyrenoid. Our results suggest a surprisingly simple molecular mechanism for how Rubisco can be packaged to form the pyrenoid matrix, potentially explaining how Rubisco packaging into a pyrenoid could have evolved across a broad range of photosynthetic eukaryotes through convergent evolution. In addition, our findings represent a key step toward engineering a pyrenoid into crops to enhance their carbon fixation efficiency. PMID:27166422

  15. A repeat protein links Rubisco to form the eukaryotic carbon-concentrating organelle.

    PubMed

    Mackinder, Luke C M; Meyer, Moritz T; Mettler-Altmann, Tabea; Chen, Vivian K; Mitchell, Madeline C; Caspari, Oliver; Freeman Rosenzweig, Elizabeth S; Pallesen, Leif; Reeves, Gregory; Itakura, Alan; Roth, Robyn; Sommer, Frederik; Geimer, Stefan; Mühlhaus, Timo; Schroda, Michael; Goodenough, Ursula; Stitt, Mark; Griffiths, Howard; Jonikas, Martin C

    2016-05-24

    Biological carbon fixation is a key step in the global carbon cycle that regulates the atmosphere's composition while producing the food we eat and the fuels we burn. Approximately one-third of global carbon fixation occurs in an overlooked algal organelle called the pyrenoid. The pyrenoid contains the CO2-fixing enzyme Rubisco and enhances carbon fixation by supplying Rubisco with a high concentration of CO2 Since the discovery of the pyrenoid more that 130 y ago, the molecular structure and biogenesis of this ecologically fundamental organelle have remained enigmatic. Here we use the model green alga Chlamydomonas reinhardtii to discover that a low-complexity repeat protein, Essential Pyrenoid Component 1 (EPYC1), links Rubisco to form the pyrenoid. We find that EPYC1 is of comparable abundance to Rubisco and colocalizes with Rubisco throughout the pyrenoid. We show that EPYC1 is essential for normal pyrenoid size, number, morphology, Rubisco content, and efficient carbon fixation at low CO2 We explain the central role of EPYC1 in pyrenoid biogenesis by the finding that EPYC1 binds Rubisco to form the pyrenoid matrix. We propose two models in which EPYC1's four repeats could produce the observed lattice arrangement of Rubisco in the Chlamydomonas pyrenoid. Our results suggest a surprisingly simple molecular mechanism for how Rubisco can be packaged to form the pyrenoid matrix, potentially explaining how Rubisco packaging into a pyrenoid could have evolved across a broad range of photosynthetic eukaryotes through convergent evolution. In addition, our findings represent a key step toward engineering a pyrenoid into crops to enhance their carbon fixation efficiency. PMID:27166422

  16. Experimental Transmission of Vesicular Stomatitis New Jersey Virus From Black Flies (Simulium vittatum) To Cattle: Clinical Outcome Is Determined By Site of Insect Feeding

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Vesicular stomatitis New Jersey virus (VSNJV) is an insect-transmitted Rhabdovirus causing vesicular disease in domestic livestock including cattle, horses and pigs. The natural transmission of VSV during epidemics remains poorly understood. Transmission of VSNJV from experimentally infected black f...

  17. Molecular Epidemiology of Vesicular Stomatitis New Jersey Virus from the 2004-2005 United States Outbreak Indicates a Common Origin with Mexico Strains

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Outbreaks of vesicular stomatitis occur at 8-10 year intervals in the southwestern United States (US) with the most recent outbreak starting in 2004 and continuing in 2005. Phylogenetic relationships among 116 vesicular stomatitis-New Jersey virus (VSNJV) isolates obtained from this outbreak and end...

  18. Lesion development and replication kinetics during early infection in cattle inoculated with vesicular stomatitis New Jersey virus via scarification and black fly (Simulium vittatum) bite

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Vesicular stomatitis viruses are the causative agents of vesicular stomatitis, an economically important contagious disease of livestock that occurs in North, Central, and South America. Little is known regarding the early stages of infection in natural hosts. Twelve adult Holstein steers were inocu...

  19. Development of a reverse transcription loop-mediated isothermal amplification assay for the detection of vesicular stomatitis New Jersey virus: use of rapid molecular assay to differentiate between vesicular disease viruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Vesicular stomatitis (VS) is endemic in Central America and northern regions of South America. Sporadic outbreaks of VS can occur in cattle and pigs where the clinical presentation can be similar to foot-and-mouth disease (FMD). There is therefore a pressing need for rapid, sensitive and specific d...

  20. Vesicular sorting controls the polarity of expanding membranes in the C. elegans intestine

    PubMed Central

    Zhang, Hongjie; Kim, Ahlee; Abraham, Nessy; Khan, Liakot A.; Göbel, Verena

    2013-01-01

    Biological tubes consist of polarized epithelial cells with apical membranes building the central lumen and basolateral membranes contacting adjacent cells or the extracellular matrix. Cellular polarity requires distinct inputs from outside the cell, e.g., the matrix, inside the cell, e.g., vesicular trafficking and the plasma membrane and its junctions.1 Many highly conserved polarity cues have been identified, but their integration during the complex process of polarized tissue and organ morphogenesis is not well understood. It is assumed that plasma-membrane-associated polarity determinants, such as the partitioning-defective (PAR) complex, define plasma membrane domain identities, whereas vesicular trafficking delivers membrane components to these domains, but lacks the ability to define them. In vitro studies on lumenal membrane biogenesis in mammalian cell lines now indicate that trafficking could contribute to defining membrane domains by targeting the polarity determinants, e.g., the PARs, themselves.2 This possibility suggests a mechanism for PARs’ asymmetric distribution on membranes and places vesicle-associated polarity cues upstream of membrane-associated polarity determinants. In such an upstream position, trafficking might even direct multiple membrane components, not only polarity determinants, an original concept of polarized plasma membrane biogenesis3,4that was largely abandoned due to the failure to identify a molecularly defined intrinsic vesicular sorting mechanism. Our two recent studies on C. elegans intestinal tubulogenesis reveal that glycosphingolipids (GSLs) and the well-recognized vesicle components clathrin and its AP-1 adaptor are required for targeting multiple apical molecules, including polarity regulators, to the expanding apical/lumenal membrane.5,6 These findings support GSLs’ long-proposed role in in vivo polarized epithelial membrane biogenesis and development and identify a novel function in apical polarity for classical