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Sample records for affect intracellular trafficking

  1. Intracellular trafficking of nucleic acids.

    PubMed

    Zhou, Rui; Geiger, R Christopher; Dean, David A

    2004-11-01

    Until recently, the attention of most researchers has focused on the first and last steps of gene transfer, namely delivery to the cell and transcription, in order to optimise transfection and gene therapy. However, over the past few years, researchers have realised that the intracellular trafficking of plasmids is more than just a "black box" and is actually one of the major barriers to effective gene delivery. After entering the cytoplasm, following direct delivery or endocytosis, plasmids or other vectors must travel relatively long distances through the mesh of cytoskeletal networks before reaching the nuclear envelope. Once at the nuclear envelope, the DNA must either wait until cell division, or be specifically transported through the nuclear pore complex, in order to reach the nucleoplasm where it can be transcribed. This review focuses on recent developments in the understanding of these intracellular trafficking events as they relate to gene delivery. Hopefully, by continuing to unravel the mechanisms by which plasmids and other gene delivery vectors move throughout the cell, and by understanding the cell biology of gene transfer, superior methods of transfection and gene therapy can be developed.

  2. Cell wall assembly and intracellular trafficking in plant cells are directly affected by changes in the magnitude of gravitational acceleration.

    PubMed

    Chebli, Youssef; Pujol, Lauranne; Shojaeifard, Anahid; Brouwer, Iman; van Loon, Jack J W A; Geitmann, Anja

    2013-01-01

    Plants are able to sense the magnitude and direction of gravity. This capacity is thought to reside in selected cell types within the plant body that are equipped with specialized organelles called statoliths. However, most plant cells do not possess statoliths, yet they respond to changes in gravitational acceleration. To understand the effect of gravity on the metabolism and cellular functioning of non-specialized plant cells, we investigated a rapidly growing plant cell devoid of known statoliths and without gravitropic behavior, the pollen tube. The effects of hyper-gravity and omnidirectional exposure to gravity on intracellular trafficking and on cell wall assembly were assessed in Camellia pollen tubes, a model system with highly reproducible growth behavior in vitro. Using an epi-fluorescence microscope mounted on the Large Diameter Centrifuge at the European Space Agency, we were able to demonstrate that vesicular trafficking is reduced under hyper-gravity conditions. Immuno-cytochemistry confirmed that both in hyper and omnidirectional gravity conditions, the characteristic spatial profiles of cellulose and callose distribution in the pollen tube wall were altered, in accordance with a dose-dependent effect on pollen tube diameter. Our findings suggest that in response to gravity induced stress, the pollen tube responds by modifying cell wall assembly to compensate for the altered mechanical load. The effect was reversible within few minutes demonstrating that the pollen tube is able to quickly adapt to changing stress conditions. PMID:23516452

  3. Cell wall assembly and intracellular trafficking in plant cells are directly affected by changes in the magnitude of gravitational acceleration.

    PubMed

    Chebli, Youssef; Pujol, Lauranne; Shojaeifard, Anahid; Brouwer, Iman; van Loon, Jack J W A; Geitmann, Anja

    2013-01-01

    Plants are able to sense the magnitude and direction of gravity. This capacity is thought to reside in selected cell types within the plant body that are equipped with specialized organelles called statoliths. However, most plant cells do not possess statoliths, yet they respond to changes in gravitational acceleration. To understand the effect of gravity on the metabolism and cellular functioning of non-specialized plant cells, we investigated a rapidly growing plant cell devoid of known statoliths and without gravitropic behavior, the pollen tube. The effects of hyper-gravity and omnidirectional exposure to gravity on intracellular trafficking and on cell wall assembly were assessed in Camellia pollen tubes, a model system with highly reproducible growth behavior in vitro. Using an epi-fluorescence microscope mounted on the Large Diameter Centrifuge at the European Space Agency, we were able to demonstrate that vesicular trafficking is reduced under hyper-gravity conditions. Immuno-cytochemistry confirmed that both in hyper and omnidirectional gravity conditions, the characteristic spatial profiles of cellulose and callose distribution in the pollen tube wall were altered, in accordance with a dose-dependent effect on pollen tube diameter. Our findings suggest that in response to gravity induced stress, the pollen tube responds by modifying cell wall assembly to compensate for the altered mechanical load. The effect was reversible within few minutes demonstrating that the pollen tube is able to quickly adapt to changing stress conditions.

  4. Intracellular parcel service: current issues in intracellular membrane trafficking.

    PubMed

    Herrmann, Johannes M; Spang, Anne

    2015-01-01

    Eukaryotic cells contain a multitude of membrane structures that are connected through a highly dynamic and complex exchange of their constituents. The vibrant instability of these structures challenges the classical view of defined, static compartments that are connected by different types of vesicles. Despite this astonishing complexity, proteins and lipids are accurately transported into the different intracellular membrane systems. Over the past few decades many factors have been identified that either mediate or regulate intracellular membrane trafficking. Like in a modern parcel sorting system of a logistics center, the cargo typically passes through several sequential sorting stations until it finally reaches the location that is specified by its individual address label. While each membrane system employs specific sets of factors, the transport processes typically operate on common principles. With the advent of genome- and proteome-wide screens, the availability of mutant collections, exciting new developments in microscope technology and sophisticated methods to study their dynamics, the future promises a broad and comprehensive picture of the processes by which eukaryotic cells sort their proteins.

  5. Intracellular trafficking mechanism of cationic phospholipids including cationic liposomes in HeLa cells.

    PubMed

    Un, K; Sakai-Kato, K; Goda, Y

    2014-07-01

    The development of gene delivery methods is essential for the achievement of effective gene therapy. Elucidation of the intracellular transfer mechanism for cationic carriers is in progress, but there are few reports regarding the intracellular trafficking processes of the cationic phospholipids taken up into cells. In the present work, the trafficking processes of a cationic phospholipid (1,2-dioleoyl-3-trimethylammonium-propane, DOTAP) were investigated from intracellular uptake to extracellular efflux using cationic liposomes in vitro. Following intracellular transport of liposomes via endocytosis, DOTAP was localized in the endoplasmic reticulum, Golgi apparatus, and mitochondria. Moreover, the proteins involved in DOTAP intracellular trafficking and extracellular efflux were identified. In addition, helper lipids of cationic liposomes were found to partially affect this intracellulartrafficking. These findings might provide valuable information for designing cationic carriers and avoiding unexpected toxic side effects derived from cationic liposomal components.

  6. Intracellular Trafficking Network of Protein Nanocapsules: Endocytosis, Exocytosis and Autophagy

    PubMed Central

    Zhang, Jinxie; Zhang, Xudong; Liu, Gan; Chang, Danfeng; Liang, Xin; Zhu, Xianbing; Tao, Wei; Mei, Lin

    2016-01-01

    The inner membrane vesicle system is a complex transport system that includes endocytosis, exocytosis and autophagy. However, the details of the intracellular trafficking pathway of nanoparticles in cells have been poorly investigated. Here, we investigate in detail the intracellular trafficking pathway of protein nanocapsules using more than 30 Rab proteins as markers of multiple trafficking vesicles in endocytosis, exocytosis and autophagy. We observed that FITC-labeled protein nanoparticles were internalized by the cells mainly through Arf6-dependent endocytosis and Rab34-mediated micropinocytosis. In addition to this classic pathway: early endosome (EEs)/late endosome (LEs) to lysosome, we identified two novel transport pathways: micropinocytosis (Rab34 positive)-LEs (Rab7 positive)-lysosome pathway and EEs-liposome (Rab18 positive)-lysosome pathway. Moreover, the cells use slow endocytosis recycling pathway (Rab11 and Rab35 positive vesicles) and GLUT4 exocytosis vesicles (Rab8 and Rab10 positive) transport the protein nanocapsules out of the cells. In addition, protein nanoparticles are observed in autophagosomes, which receive protein nanocapsules through multiple endocytosis vesicles. Using autophagy inhibitor to block these transport pathways could prevent the degradation of nanoparticles through lysosomes. Using Rab proteins as vesicle markers to investigation the detail intracellular trafficking of the protein nanocapsules, will provide new targets to interfere the cellular behaver of the nanoparticles, and improve the therapeutic effect of nanomedicine.

  7. Intracellular Trafficking Network of Protein Nanocapsules: Endocytosis, Exocytosis and Autophagy

    PubMed Central

    Zhang, Jinxie; Zhang, Xudong; Liu, Gan; Chang, Danfeng; Liang, Xin; Zhu, Xianbing; Tao, Wei; Mei, Lin

    2016-01-01

    The inner membrane vesicle system is a complex transport system that includes endocytosis, exocytosis and autophagy. However, the details of the intracellular trafficking pathway of nanoparticles in cells have been poorly investigated. Here, we investigate in detail the intracellular trafficking pathway of protein nanocapsules using more than 30 Rab proteins as markers of multiple trafficking vesicles in endocytosis, exocytosis and autophagy. We observed that FITC-labeled protein nanoparticles were internalized by the cells mainly through Arf6-dependent endocytosis and Rab34-mediated micropinocytosis. In addition to this classic pathway: early endosome (EEs)/late endosome (LEs) to lysosome, we identified two novel transport pathways: micropinocytosis (Rab34 positive)-LEs (Rab7 positive)-lysosome pathway and EEs-liposome (Rab18 positive)-lysosome pathway. Moreover, the cells use slow endocytosis recycling pathway (Rab11 and Rab35 positive vesicles) and GLUT4 exocytosis vesicles (Rab8 and Rab10 positive) transport the protein nanocapsules out of the cells. In addition, protein nanoparticles are observed in autophagosomes, which receive protein nanocapsules through multiple endocytosis vesicles. Using autophagy inhibitor to block these transport pathways could prevent the degradation of nanoparticles through lysosomes. Using Rab proteins as vesicle markers to investigation the detail intracellular trafficking of the protein nanocapsules, will provide new targets to interfere the cellular behaver of the nanoparticles, and improve the therapeutic effect of nanomedicine. PMID:27698943

  8. Regulation of dopamine transporter trafficking by intracellular amphetamine.

    PubMed

    Kahlig, Kristopher M; Lute, Brandon J; Wei, Yuqiang; Loland, Claus J; Gether, Ulrik; Javitch, Jonathan A; Galli, Aurelio

    2006-08-01

    The dopamine (DA) transporter (DAT) mediates the removal of released DA. DAT is the major molecular target responsible for the rewarding properties and abuse potential of the psychostimulant amphetamine (AMPH). AMPH has been shown to reduce the number of DATs at the cell surface, and this AMPH-induced cell surface DAT redistribution may result in long-lasting changes in DA homeostasis. The molecular mechanism by which AMPH induces trafficking is not clear. Because AMPH is a substrate, we do not know whether extracellular AMPH stimulates trafficking through its interaction with DAT and subsequent alteration in DAT function, thereby triggering intracellular signaling or whether AMPH must be transported and then act intracellularly. In agreement with our previous studies, extracellular AMPH caused cytosolic redistribution of the wild-type human DAT (WT-hDAT). However, AMPH did not induce cytosolic redistribution in an uptake-impaired hDAT (Y335A-hDAT) that still binds AMPH. The divalent cation zinc (Zn(2+)) inhibits WT-hDAT activity, but it restores Y335A-hDAT uptake. Coadministration of Zn(2+) and AMPH consistently reduced WT-hDAT trafficking but stimulated cytosolic redistribution of Y335A-hDAT. Furthermore, direct intracellular application of AMPH, via a whole-cell patch pipette, stimulated the trafficking of Y335A-hDAT. Taken together, these data suggest that the DAT transport cycle is not required for AMPH-induced down-regulation and that an increase of intracellular AMPH is an essential component of DAT redistribution.

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

    PubMed

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

    2016-08-30

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

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

    PubMed

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

    2016-08-30

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

  11. Protein-coat dynamics and cluster phases in intracellular trafficking

    NASA Astrophysics Data System (ADS)

    Huber, Greg; Wang, Hui; Mukhopadhyay, Ranjan

    2011-09-01

    Clustering of membrane proteins is a hallmark of biological membranes' lateral organization and crucial to their function. However, the physical properties of these protein aggregates remain poorly understood. Ensembles of coat proteins, the example considered here, are necessary for intracellular transport in eukaryotic cells. Assembly and disassembly rates for coat proteins involved in intracellular vesicular trafficking must be carefully controlled: their assembly deforms the membrane patch and drives vesicle formation, yet the protein coat must rapidly disassemble after vesiculation. Motivated by recent experimental findings for protein-coat dynamics, we study a dynamical Ising-type model for coat assembly and disassembly, and demonstrate how simple dynamical rules generate a robust, steady-state distribution of protein clusters (corresponding to intermediate budded shapes) and how cluster sizes are controlled by the kinetics. We interpret the results in terms of both vesiculation and the coupling to cargo proteins.

  12. Signal Transduction and Intracellular Trafficking by the Interleukin 36 Receptor*

    PubMed Central

    Saha, Siddhartha S.; Singh, Divyendu; Raymond, Ernest L.; Ganesan, Rajkumar; Caviness, Gary; Grimaldi, Christine; Woska, Joseph R.; Mennerich, Detlev; Brown, Su-Ellen; Mbow, M. Lamine; Kao, C. Cheng

    2015-01-01

    Improper signaling of the IL-36 receptor (IL-36R), a member of the IL-1 receptor family, has been associated with various inflammation-associated diseases. However, the requirements for IL-36R signal transduction remain poorly characterized. This work seeks to define the requirements for IL-36R signaling and intracellular trafficking. In the absence of cognate agonists, IL-36R was endocytosed and recycled to the plasma membrane. In the presence of IL-36, IL-36R increased accumulation in LAMP1+ lysosomes. Endocytosis predominantly used a clathrin-mediated pathway, and the accumulation of the IL-36R in lysosomes did not result in increased receptor turnover. The ubiquitin-binding Tollip protein contributed to IL-36R signaling and increased the accumulation of both subunits of the IL-36R. PMID:26269592

  13. Intracellular trafficking of silicon particles and logic-embedded vectors

    NASA Astrophysics Data System (ADS)

    Ferrati, Silvia; Mack, Aaron; Chiappini, Ciro; Liu, Xuewu; Bean, Andrew J.; Ferrari, Mauro; Serda, Rita E.

    2010-08-01

    Mesoporous silicon particles show great promise for use in drug delivery and imaging applications as carriers for second-stage nanoparticles and higher order particles or therapeutics. Modulation of particle geometry, surface chemistry, and porosity allows silicon particles to be optimized for specific applications such as vascular targeting and avoidance of biological barriers commonly found between the site of drug injection and the final destination. In this study, the intracellular trafficking of unloaded carrier silicon particles and carrier particles loaded with secondary iron oxide nanoparticles was investigated. Following cellular uptake, membrane-encapsulated silicon particles migrated to the perinuclear region of the cell by a microtubule-driven mechanism. Surface charge, shape (spherical and hemispherical) and size (1.6 and 3.2 μm) of the particle did not alter the rate of migration. Maturation of the phagosome was associated with an increase in acidity and acquisition of markers of late endosomes and lysosomes. Cellular uptake of iron oxide nanoparticle-loaded silicon particles resulted in sorting of the particles and trafficking to unique destinations. The silicon carriers remained localized in phagosomes, while the second stage iron oxide nanoparticles were sorted into multi-vesicular bodies that dissociated from the phagosome into novel membrane-bound compartments. Release of iron from the cells may represent exocytosis of iron oxide nanoparticle-loaded vesicles. These results reinforce the concept of multi-functional nanocarriers, in which different particles are able to perform specific tasks, in order to deliver single- or multi-component payloads to specific sub-cellular compartments.Mesoporous silicon particles show great promise for use in drug delivery and imaging applications as carriers for second-stage nanoparticles and higher order particles or therapeutics. Modulation of particle geometry, surface chemistry, and porosity allows silicon

  14. Uptake and intracellular trafficking of superantigens in dendritic cells.

    PubMed

    Ganem, María B; De Marzi, Mauricio C; Fernández-Lynch, María J; Jancic, Carolina; Vermeulen, Mónica; Geffner, Jorge; Mariuzza, Roy A; Fernández, Marisa M; Malchiodi, Emilio L

    2013-01-01

    Bacterial superantigens (SAgs) are exotoxins produced mainly by Staphylococcus aureus and Streptococcus pyogenes that can cause toxic shock syndrome (TSS). According to current paradigm, SAgs interact directly and simultaneously with T cell receptor (TCR) on the T cell and MHC class II (MHC-II) on the antigen-presenting cell (APC), thereby circumventing intracellular processing to trigger T cell activation. Dendritic cells (DCs) are professional APCs that coat nearly all body surfaces and are the most probable candidate to interact with SAgs. We demonstrate that SAgs are taken up by mouse DCs without triggering DC maturation. SAgs were found in intracellular acidic compartment of DCs as biologically active molecules. Moreover, SAgs co-localized with EEA1, RAB-7 and LAMP-2, at different times, and were then recycled to the cell membrane. DCs loaded with SAgs are capable of triggering in vitro lymphocyte proliferation and, injected into mice, stimulate T cells bearing the proper TCR in draining lymph nodes. Transportation and trafficking of SAgs in DCs might increase the local concentration of these exotoxins where they will produce the highest effect by promoting their encounter with both MHC-II and TCR in lymph nodes, and may explain how just a few SAg molecules can induce the severe pathology associated with TSS. PMID:23799083

  15. Uptake and Intracellular Trafficking of Superantigens in Dendritic Cells

    PubMed Central

    Fernández-Lynch, María J.; Jancic, Carolina; Vermeulen, Mónica; Geffner, Jorge; Mariuzza, Roy A.; Fernández, Marisa M.; Malchiodi, Emilio L.

    2013-01-01

    Bacterial superantigens (SAgs) are exotoxins produced mainly by Staphylococcus aureus and Streptococcus pyogenes that can cause toxic shock syndrome (TSS). According to current paradigm, SAgs interact directly and simultaneously with T cell receptor (TCR) on the T cell and MHC class II (MHC-II) on the antigen-presenting cell (APC), thereby circumventing intracellular processing to trigger T cell activation. Dendritic cells (DCs) are professional APCs that coat nearly all body surfaces and are the most probable candidate to interact with SAgs. We demonstrate that SAgs are taken up by mouse DCs without triggering DC maturation. SAgs were found in intracellular acidic compartment of DCs as biologically active molecules. Moreover, SAgs co-localized with EEA1, RAB-7 and LAMP-2, at different times, and were then recycled to the cell membrane. DCs loaded with SAgs are capable of triggering in vitro lymphocyte proliferation and, injected into mice, stimulate T cells bearing the proper TCR in draining lymph nodes. Transportation and trafficking of SAgs in DCs might increase the local concentration of these exotoxins where they will produce the highest effect by promoting their encounter with both MHC-II and TCR in lymph nodes, and may explain how just a few SAg molecules can induce the severe pathology associated with TSS. PMID:23799083

  16. An adipocentric view of signaling and intracellular trafficking.

    PubMed

    Mora, Silvia; Pessin, Jeffrey E

    2002-01-01

    Adipocytes have traditionally been considered to be the primary site for whole body energy storage mainly in the form of triglycerides and fatty acids. This occurs through the ability of insulin to markedly stimulate both glucose uptake and lipogenesis. Conventional wisdom held that defects in fuel partitioning into adipocytes either because of increased adipose tissue mass and/or increased lipolysis and circulating free fatty acids resulted in dyslipidemia, obesity, insulin resistance and perhaps diabetes. However, it has become increasingly apparent that loss of adipose tissue (lipodystrophies) in both animal models and humans also leads to metabolic disorders that result in severe states of insulin resistance and potential diabetes. These apparently opposite functions can be resolved by the establishment of adipocytes not only as a fuel storage depot but also as a critical endocrine organ that secretes a variety of signaling molecules into the circulation. Although the molecular function of these adipocyte-derived signals are poorly understood, they play a central role in the maintenance of energy homeostasis by regulating insulin secretion, insulin action, glucose and lipid metabolism, energy balance, host defense and reproduction. The diversity of these secretory factors include enzymes (lipoprotein lipase (LPL) and adipsin), growth factors [vascular endothelial growth factor (VEGF)], cytokines (tumor necrosis factor-alpha, interleukin 6) and several other hormones involved in fatty acid and glucose metabolism (leptin, Acrp30, resistin and acylation stimulation protein). Despite the large number of molecules secreted by adipocytes, our understanding of the pathways and mechanisms controlling intracellular trafficking and exocytosis in adipocytes is poorly understood. In this article, we will review the current knowledge of the trafficking and secretion processes that take place in adipocytes, focusing our attention on two of the best characterized adipokine

  17. Ornithine decarboxylase antizyme inhibitor 2 regulates intracellular vesicle trafficking

    SciTech Connect

    Kanerva, Kristiina; Maekitie, Laura T.; Baeck, Nils; Andersson, Leif C.

    2010-07-01

    Antizyme inhibitor 1 (AZIN1) and 2 (AZIN2) are proteins that activate ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis. Both AZINs release ODC from its inactive complex with antizyme (AZ), leading to formation of the catalytically active ODC. The ubiquitously expressed AZIN1 is involved in cell proliferation and transformation whereas the role of the recently found AZIN2 in cellular functions is unknown. Here we report the intracellular localization of AZIN2 and present novel evidence indicating that it acts as a regulator of vesicle trafficking. We used immunostaining to demonstrate that both endogenous and FLAG-tagged AZIN2 localize to post-Golgi vesicles of the secretory pathway. Immuno-electron microscopy revealed that the vesicles associate mainly with the trans-Golgi network (TGN). RNAi-mediated knockdown of AZIN2 or depletion of cellular polyamines caused selective fragmentation of the TGN and retarded the exocytotic release of vesicular stomatitis virus glycoprotein. Exogenous addition of polyamines normalized the morphological changes and reversed the inhibition of protein secretion. Our findings demonstrate that AZIN2 regulates the transport of secretory vesicles by locally activating ODC and polyamine biosynthesis.

  18. Multiscale computational models in physical systems biology of intracellular trafficking

    PubMed Central

    Tourdot, Richard W.; Bradley, Ryan P.; Ramakrishnan, Natesan

    2015-01-01

    In intracellular trafficking, a definitive understanding of the interplay between protein binding and membrane morphology remains incomplete. The authors describe a computational approach by integrating coarse-grained molecular dynamics (CGMD) simulations with continuum Monte Carlo (CM) simulations of the membrane to study protein–membrane interactions and the ensuing membrane curvature. They relate the curvature field strength discerned from the molecular level to its effect at the cellular length-scale. They perform thermodynamic integration on the CM model to describe the free energy landscape of vesiculation in clathrin-mediated endocytosis. The method presented here delineates membrane morphologies and maps out the free energy changes associated with membrane remodeling due to varying coat sizes, coat curvature strengths, membrane bending rigidities, and tensions; furthermore several constraints on mechanisms underlying clathrin-mediated endocytosis have also been identified, Their CGMD simulations have revealed the importance of PIP2 for stable binding of proteins essential for curvature induction in the bilayer and have provided a molecular basis for the positive curvature induction by the epsin N-terminal homology (EIMTH) domain. Calculation of the free energy landscape for vesicle budding has identified the critical size and curvature strength of a clathrin coat required for nucleation and stabilisation of a mature vesicle. PMID:25257021

  19. Heme requirement and intracellular trafficking in Trypanosoma cruzi epimastigotes

    SciTech Connect

    Lara, F.A.; Sant'Anna, C.; Lemos, D.; Laranja, G.A.T.; Coelho, M.G.P.; Reis Salles, I.; Michel, A.; Oliveira, P.L.; Cunha-e-Silva, N.; Salmon, D.; Paes, M.C. . E-mail: mcpaes@uerj.br

    2007-03-30

    Epimastigotes multiplies in the insect midgut by taking up nutrients present in the blood meal including heme bound to hemoglobin of red blood cell. During blood meal digestion by vector proteases in the posterior midgut, hemoglobin is clipped off into amino acids, peptides, and free heme. In this paper, we compared the heme and hemoglobin uptake kinetics and followed their intracellular trafficking. Addition of heme to culture medium increased epimastigote proliferation in a dose-dependent manner, while medium supplemented with hemoglobin enhanced growth after 3-day lag phase. Medium supplemented with globin-derived peptides stimulated cell proliferation in a dose-independent way. Using Palladium mesoporphyrin IX (Pd-mP) as a fluorescent heme-analog, we observed that heme internalization proceeded much faster than that observed by hemoglobin-rhodamine. Binding experiments showed that parasites accumulated the Pd-mP into the posterior region of the cell whereas hemoglobin-rhodamine stained the anterior region. Finally, using different specific inhibitors of ABC transporters we conclude that a P-glycoprotein homologue transporter is probably involved in heme transport through the plasma membrane.

  20. A new ATP-binding cassette protein is involved in intracellular haem trafficking in Leishmania.

    PubMed

    Campos-Salinas, Jenny; Cabello-Donayre, María; García-Hernández, Raquel; Pérez-Victoria, Ignacio; Castanys, Santiago; Gamarro, Francisco; Pérez-Victoria, José M

    2011-03-01

    The characterization of LABCG5, a new intracellular ATP-binding cassette protein in Leishmania donovani, is described. Unlike other ABCG half-transporters, LABCG5 is not involved in either drug resistance or phospholipid efflux. However, we provide evidence suggesting that this protein is involved in intracellular haem trafficking. Thus, downregulation of LABCG5 function produced upon overexpression of an inactive version of the protein caused a dramatic growth arrest unless a haemin supplement was added or the mutated gene was eliminated. Supplementation with haemoglobin, an upstream metabolite normally sufficient to meet parasite haem requirements, was unable to rescue the growth defect phenotype. Haemoglobin endocytosis was not hampered in dominant-negative parasites and neither was haem uptake, a process that we show here to be dependent on a specific transporter. In contrast, LABCG5 function was required for the correct intracellular trafficking of haemoglobin-bound porphyrins to the mitochondria, not affecting the routing of free haem. Finally, LABCG5 binds haem through hydrophobic and electrostatic interactions. Altogether, these data suggest that LABCG5 is involved in the salvage of the haem released after the breakdown of internalized haemoglobin. As Leishmania is auxotrophic for haem, the pharmacological targeting of this route could represent a novel approach to control fatal visceral leishmaniasis. PMID:21255121

  1. Methods to analyze subcellular localization and intracellular trafficking of Claudin-16.

    PubMed

    Kausalya, P Jaya; Hunziker, Walter

    2011-01-01

    The integral tight junction protein Claudin-16 (Cldn16) is predominantly expressed in renal epithelial cells of the thick ascending limb of Henle's loop where, together with claudin-19, it forms a cation-selective pore that allows influx of Na+ from the interstitial fluid into the lumen of the kidney tubule. This leads to an electrochemical gradient that drives the reabsorbtion of Mg2+ and Ca2+ ions from the renal filtrate. Mutations in the Cldn16 gene have been identified in patients suffering from familial hypomagnesemia with hypercalciuria and nephrocalcinosis, with excessive renal wastage of Mg2+ and Ca2+ being a hallmark of this condition. Studies into the mechanism by which mutations impair Cldn16 function have shown that although several mutations affect paracellular ion transport, many interfere with intracellular trafficking of Cldn16, ultimately compromising its localization to TJs. Here, we describe the experimental approaches that can be used to monitor intracellular localization and trafficking of Cldn16. These methods can easily be adapted to study other claudins, provided suitable antibodies are available.

  2. Endocytosis and Intracellular Trafficking of Human Natural Killer Cell Receptors

    PubMed Central

    Masilamani, Madhan; Peruzzi, Giovanna; Borrego, Francisco; Coligan, John E.

    2009-01-01

    Natural killer (NK) cells play a vital role in the defense against viral infections and tumor development. NK cell function is primarily regulated by the sum of signals from a broad array of activation and inhibitory receptors. Key to generating the input level of either activating or inhibitory signals is the maintenance of receptor expression levels on the cell surface. Although the mechanisms of endocytosis and trafficking for some cell surface receptors, such as transferrin receptor, and certain immune receptors, are very well known, that is not the situation for receptors expressed by NK cells. Recent studies have uncovered that endocytosis and trafficking routes characteristic for specific activation and inhibitory receptors can regulate the functional responses of NK cells. In this review, we summarize the current knowledge of receptor endocytosis and trafficking, and integrate this with our current understanding of NK cell receptor trafficking. PMID:19719476

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

    PubMed

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

    2014-09-01

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

  4. Cellular Uptake and Intracellular Trafficking of Oligonucleotides: Implications for Oligonucleotide Pharmacology

    PubMed Central

    Ming, Xin; Carver, Kyle; Laing, Brian

    2014-01-01

    One of the major constraints on the therapeutic use of oligonucleotides is inefficient delivery to their sites of action in the cytosol or nucleus. Recently it has become evident that the pathways of cellular uptake and intracellular trafficking of oligonucleotides can strongly influence their pharmacological actions. Here we provide background information on the basic processes of endocytosis and trafficking and then review recent literature on targeted delivery and subcellular trafficking of oligonucleotides in that context. A variety of approaches including molecular scale ligand-oligonucleotide conjugates, ligand-targeted nanocarriers, and the use of small molecules to enhance oligonucleotide effects are discussed. PMID:24383421

  5. Microtubule and Actin Interplay Drive Intracellular c-Src Trafficking.

    PubMed

    Arnette, Christopher; Frye, Keyada; Kaverina, Irina

    2016-01-01

    The proto-oncogene c-Src is involved in a variety of signaling processes. Therefore, c-Src spatiotemporal localization is critical for interaction with downstream targets. However, the mechanisms regulating this localization have remained elusive. Previous studies have shown that c-Src trafficking is a microtubule-dependent process that facilitates c-Src turnover in neuronal growth cones. As such, microtubule depolymerization lead to the inhibition of c-Src recycling. Alternatively, c-Src trafficking was also shown to be regulated by RhoB-dependent actin polymerization. Our results show that c-Src vesicles primarily exhibit microtubule-dependent trafficking; however, microtubule depolymerization does not inhibit vesicle movement. Instead, vesicular movement becomes both faster and less directional. This movement was associated with actin polymerization directly at c-Src vesicle membranes. Interestingly, it has been shown previously that c-Src delivery is an actin polymerization-dependent process that relies on small GTPase RhoB at c-Src vesicles. In agreement with this finding, microtubule depolymerization induced significant activation of RhoB, together with actin comet tail formation. These effects occurred downstream of GTP-exchange factor, GEF-H1, which was released from depolymerizing MTs. Accordingly, GEF-H1 activity was necessary for actin comet tail formation at the Src vesicles. Our results indicate that regulation of c-Src trafficking requires both microtubules and actin polymerization, and that GEF-H1 coordinates c-Src trafficking, acting as a molecular switch between these two mechanisms. PMID:26866809

  6. Microtubule and Actin Interplay Drive Intracellular c-Src Trafficking

    PubMed Central

    Arnette, Christopher; Frye, Keyada; Kaverina, Irina

    2016-01-01

    The proto-oncogene c-Src is involved in a variety of signaling processes. Therefore, c-Src spatiotemporal localization is critical for interaction with downstream targets. However, the mechanisms regulating this localization have remained elusive. Previous studies have shown that c-Src trafficking is a microtubule-dependent process that facilitates c-Src turnover in neuronal growth cones. As such, microtubule depolymerization lead to the inhibition of c-Src recycling. Alternatively, c-Src trafficking was also shown to be regulated by RhoB-dependent actin polymerization. Our results show that c-Src vesicles primarily exhibit microtubule-dependent trafficking; however, microtubule depolymerization does not inhibit vesicle movement. Instead, vesicular movement becomes both faster and less directional. This movement was associated with actin polymerization directly at c-Src vesicle membranes. Interestingly, it has been shown previously that c-Src delivery is an actin polymerization-dependent process that relies on small GTPase RhoB at c-Src vesicles. In agreement with this finding, microtubule depolymerization induced significant activation of RhoB, together with actin comet tail formation. These effects occurred downstream of GTP-exchange factor, GEF-H1, which was released from depolymerizing MTs. Accordingly, GEF-H1 activity was necessary for actin comet tail formation at the Src vesicles. Our results indicate that regulation of c-Src trafficking requires both microtubules and actin polymerization, and that GEF-H1 coordinates c-Src trafficking, acting as a molecular switch between these two mechanisms. PMID:26866809

  7. Systematic analysis of intracellular trafficking motifs located within the cytoplasmic domain of simian immunodeficiency virus glycoprotein gp41.

    PubMed

    Postler, Thomas S; Bixby, Jacqueline G; Desrosiers, Ronald C; Yuste, Eloísa

    2014-01-01

    Previous studies have shown that truncation of the cytoplasmic-domain sequences of the simian immunodeficiency virus (SIV) envelope glycoprotein (Env) just prior to a potential intracellular-trafficking signal of the sequence YIHF can strongly increase Env protein expression on the cell surface, Env incorporation into virions and, at least in some contexts, virion infectivity. Here, all 12 potential intracellular-trafficking motifs (YXXΦ or LL/LI/IL) in the gp41 cytoplasmic domain (gp41CD) of SIVmac239 were analyzed by systematic mutagenesis. One single and 7 sequential combination mutants in this cytoplasmic domain were characterized. Cell-surface levels of Env were not significantly affected by any of the mutations. Most combination mutations resulted in moderate 3- to 8-fold increases in Env incorporation into virions. However, mutation of all 12 potential sites actually decreased Env incorporation into virions. Variant forms with 11 or 12 mutated sites exhibited 3-fold lower levels of inherent infectivity, while none of the other single or combination mutations that were studied significantly affected the inherent infectivity of SIVmac239. These minor effects of mutations in trafficking motifs form a stark contrast to the strong increases in cell-surface expression and Env incorporation which have previously been reported for large truncations of gp41CD. Surprisingly, mutation of potential trafficking motifs in gp41CD of SIVmac316, which differs by only one residue from gp41CD of SIVmac239, effectively recapitulated the increases in Env incorporation into virions observed with gp41CD truncations. Our results indicate that increases in Env surface expression and virion incorporation associated with truncation of SIVmac239 gp41CD are not fully explained by loss of consensus trafficking motifs. PMID:25479017

  8. Systematic Analysis of Intracellular Trafficking Motifs Located within the Cytoplasmic Domain of Simian Immunodeficiency Virus Glycoprotein gp41

    PubMed Central

    Postler, Thomas S.; Bixby, Jacqueline G.; Desrosiers, Ronald C.; Yuste, Eloísa

    2014-01-01

    Previous studies have shown that truncation of the cytoplasmic-domain sequences of the simian immunodeficiency virus (SIV) envelope glycoprotein (Env) just prior to a potential intracellular-trafficking signal of the sequence YIHF can strongly increase Env protein expression on the cell surface, Env incorporation into virions and, at least in some contexts, virion infectivity. Here, all 12 potential intracellular-trafficking motifs (YXXΦ or LL/LI/IL) in the gp41 cytoplasmic domain (gp41CD) of SIVmac239 were analyzed by systematic mutagenesis. One single and 7 sequential combination mutants in this cytoplasmic domain were characterized. Cell-surface levels of Env were not significantly affected by any of the mutations. Most combination mutations resulted in moderate 3- to 8-fold increases in Env incorporation into virions. However, mutation of all 12 potential sites actually decreased Env incorporation into virions. Variant forms with 11 or 12 mutated sites exhibited 3-fold lower levels of inherent infectivity, while none of the other single or combination mutations that were studied significantly affected the inherent infectivity of SIVmac239. These minor effects of mutations in trafficking motifs form a stark contrast to the strong increases in cell-surface expression and Env incorporation which have previously been reported for large truncations of gp41CD. Surprisingly, mutation of potential trafficking motifs in gp41CD of SIVmac316, which differs by only one residue from gp41CD of SIVmac239, effectively recapitulated the increases in Env incorporation into virions observed with gp41CD truncations. Our results indicate that increases in Env surface expression and virion incorporation associated with truncation of SIVmac239 gp41CD are not fully explained by loss of consensus trafficking motifs. PMID:25479017

  9. Intracellular trafficking pathway of BK virus in human renal proximal tubular epithelial cells

    PubMed Central

    Moriyama, Takahito; Sorokin, Andrey

    2009-01-01

    Intracellular trafficking of BK Virus (BKV) in human renal proximal tubular epithelial cells (HRPTEC) is critical for BKV nephritis. However, the major trafficking components utilized by BKV remain unknown. Co-incubation of HRPTEC with BKV and microtubule disrupting agents prevented BKV infection as detected by immunofluorescence and western blot analysis with antibodies which recognize BKV large T antigen. However, inhibition of a dynein, cellular motor protein, did not interfere with BKV infection in HRPTEC. A colocalization study of BKV with the markers of the endoplasmic reticulum (ER) and the Golgi apparatus (GA), indicated that BKV reached the ER from 6 to 10 hours, while bypassing the GA or passing through the GA too transiently to be detected. This study contributes to the understanding of mechanisms of intracellular trafficking used by BKV in the infection of HRPTEC. PMID:17976677

  10. Alpha-arrestins Aly1 and Aly2 regulate intracellular trafficking in response to nutrient signaling.

    PubMed

    O'Donnell, Allyson F; Apffel, Alex; Gardner, Richard G; Cyert, Martha S

    2010-10-15

    Extracellular signals regulate trafficking events to reorganize proteins at the plasma membrane (PM); however, few effectors of this regulation have been identified. β-Arrestins relay signaling cues to the trafficking machinery by controlling agonist-stimulated endocytosis of G-protein-coupled receptors. In contrast, we show that yeast α-arrestins, Aly1 and Aly2, control intracellular sorting of Gap1, the general amino acid permease, in response to nutrients. These studies are the first to demonstrate association of α-arrestins with clathrin and clathrin adaptor proteins (AP) and show that Aly1 and Aly2 interact directly with the γ-subunit of AP-1, Apl4. Aly2-dependent trafficking of Gap1 requires AP-1, which mediates endosome-to-Golgi transport, and the nutrient-regulated kinase, Npr1, which phosphorylates Aly2. During nitrogen starvation, Npr1 phosphorylation of Aly2 may stimulate Gap1 incorporation into AP-1/clathrin-coated vesicles to promote Gap1 trafficking from endosomes to the trans-Golgi network. Ultimately, increased Aly1-/Aly2-mediated recycling of Gap1 from endosomes results in higher Gap1 levels within cells and at the PM by diverting Gap away from trafficking pathways that lead to vacuolar degradation. This work defines a new role for arrestins in membrane trafficking and offers insight into how α-arrestins coordinate signaling events with protein trafficking.

  11. Live cell imaging of duplex siRNA intracellular trafficking

    PubMed Central

    Hirsch, Markus; Helm, Mark

    2015-01-01

    Intracellular distribution of siRNA after in vitro transfection typically depends on lipopolyplexes, which must release the siRNA into the cytosol. Here, the fate of siRNAs was monitored by FRET-based live cell imaging. Subsequent to in situ observation of uptake and release processes, this approach allowed the observation of a number of hitherto uncharacterized intracellular distribution and degradation processes, commencing with a burst of endosomal releases, followed, in some cases, by fast siRNA influx into the nucleus. The continued observation of intact siRNA against a background of free fluorophores resulting from advanced degradation was possible by a specifically developed imaging algorithm, which identified populations of intact siRNA in pixels based on FRET. This proved to be essential in the end point definition of siRNA distribution, which typically featured partially degraded siRNA pools in perinuclear structures. Our results depict the initial 4 h as a critical time window, characterized by fast initial burst release into the cytosol, which lay the foundations for subsequent intracellular distribution of siRNA. Combination with a subsequent slower, but sustained release from endosomal reservoirs may contribute to the efficiency and duration of RNAi, and explain the success of lipopolyplexes in RNAi experiments in cell culture. PMID:25870407

  12. The emerging role of phosphoinositide clustering in intracellular trafficking and signal transduction

    PubMed Central

    Picas, Laura; Gaits-Iacovoni, Frederique; Goud, Bruno

    2016-01-01

    Phosphoinositides are master regulators of multiple cellular processes: from vesicular trafficking to signaling, cytoskeleton dynamics, and cell growth. They are synthesized by the spatiotemporal regulated activity of phosphoinositide-metabolizing enzymes. The recent observation that some protein modules are able to cluster phosphoinositides suggests that alternative or complementary mechanisms might operate to stabilize the different phosphoinositide pools within cellular compartments. Herein, we discuss the different known and potential molecular players that are prone to engage phosphoinositide clustering and elaborate on how such a mechanism might take part in the regulation of intracellular trafficking and signal transduction. PMID:27092250

  13. Intracellular delivery and trafficking dynamics of a lymphoma-targeting antibody-polymer conjugate.

    PubMed

    Berguig, Geoffrey Y; Convertine, Anthony J; Shi, Julie; Palanca-Wessels, Maria Corinna; Duvall, Craig L; Pun, Suzie H; Press, Oliver W; Stayton, Patrick S

    2012-12-01

    Ratiometric fluorescence and cellular fractionation studies were employed to characterize the intracellular trafficking dynamics of antibody-poly(propylacrylic acid) (PPAA) conjugates in CD22+ RAMOS-AW cells. The HD39 monoclonal antibody (mAb) directs CD22-dependent, receptor-mediated uptake in human B-cell lymphoma cells, where it is rapidly trafficked to the lysosomal compartment. To characterize the intracellular-release dynamics of the polymer-mAb conjugates, HD39-streptavidin (HD39/SA) was dual-labeled with pH-insensitive Alexa Fluor 488 and pH-sensitive pHrodo fluorophores. The subcellular pH distribution of the HD39/SA-polymer conjugates was quantified as a function of time by live-cell fluorescence microscopy, and the average intracellular pH value experienced by the conjugates was also characterized as a function of time by flow cytometry. PPAA was shown to alter the intracellular trafficking kinetics strongly relative to HD39/SA alone or HD39/SA conjugates with a control polymer, poly(methacryclic acid) (PMAA). Subcellular trafficking studies revealed that after 6 h, only 11% of the HD39/SA-PPAA conjugates had been trafficked to acidic lysosomal compartments with values at or below pH 5.6. In contrast, the average intracellular pH of HD39/SA alone dropped from 6.7 ± 0.2 at 1 h to 5.6 ± 0.5 after 3 h and 4.7 ± 0.6 after 6 h. Conjugation of the control polymer PMAA to HD39/SA showed an average pH drop similar to that of HD39/SA. Subcellular fractionation studies with tritium-labeled HD39/SA demonstrated that after 6 h, 89% of HD39/SA was associated with endosomes (Rab5+) and lysosomes (Lamp2+), while 45% of HD39/SA-PPAA was translocated to the cytosol (lactate dehydrogenase+). These results demonstrate the endosomal-releasing properties of PPAA with antibody-polymer conjugates and detail their intracellular trafficking dynamics and subcellular compartmental distributions over time.

  14. Abrin Immunotoxin: Targeted Cytotoxicity and Intracellular Trafficking Pathway

    PubMed Central

    Gadadhar, Sudarshan; Karande, Anjali A.

    2013-01-01

    Background Immunotherapy is fast emerging as one of the leading modes of treatment of cancer, in combination with chemotherapy and radiation. Use of immunotoxins, proteins bearing a cell-surface receptor-specific antibody conjugated to a toxin, enhances the efficacy of cancer treatment. The toxin Abrin, isolated from the Abrus precatorius plant, is a type II ribosome inactivating protein, has a catalytic efficiency higher than any other toxin belonging to this class of proteins but has not been exploited much for use in targeted therapy. Methods Protein synthesis assay using 3[H] L-leucine incorporation; construction and purification of immunotoxin; study of cell death using flow cytometry; confocal scanning microscopy and sub-cellular fractionation with immunoblot analysis of localization of proteins. Results We used the recombinant A chain of abrin to conjugate to antibodies raised against the human gonadotropin releasing hormone receptor. The conjugate inhibited protein synthesis and also induced cell death specifically in cells expressing the receptor. The conjugate exhibited differences in the kinetics of inhibition of protein synthesis, in comparison to abrin, and this was attributed to differences in internalization and trafficking of the conjugate within the cells. Moreover, observations of sequestration of the A chain into the nucleus of cells treated with abrin but not in cells treated with the conjugate reveal a novel pathway for the movement of the conjugate in the cells. Conclusions This is one of the first reports on nuclear localization of abrin, a type II RIP. The immunotoxin mAb F1G4-rABRa-A, generated in our laboratory, inhibits protein synthesis specifically on cells expressing the gonadotropin releasing hormone receptor and the pathway of internalization of the protein is distinct from that seen for abrin. PMID:23472175

  15. DNA uptake, intracellular trafficking and gene transfection after ultrasound exposure.

    PubMed

    Liu, Ying; Yan, Jing; Santangelo, Philip J; Prausnitz, Mark R

    2016-07-28

    Ultrasound has been studied as a promising tool for intracellular gene delivery. In this work, we studied gene transfection of a human prostate cancer cell line exposed to megahertz pulsed ultrasound in the presence of contrast agent and assessed the efficiency of fluorescently labelled DNA delivery into cell nuclei, which is necessary for gene transfection. At the sonication conditions studied, ~30% of cells showed DNA uptake 30min after sonication, but that fraction decreased over time to ~10% of cells after 24h. Most cells containing DNA had DNA in their nuclei, but the amount varied significantly. Transfection efficiency peaked at ~10% at 8h post sonication. Among those cells containing DNA, ~30% of DNA was localized in the cell nuclei, ~30% was in autophagosomes/autophagolysosomes and the remainder was "free" in the cytoplasm 30min after sonication. At later times up to 24h, ~30% of DNA continued to be found in the nuclei and most or all of the rest of the DNA was in autophagosomes/autophagolysosomes. These results demonstrate that ultrasound can deliver DNA into cell nuclei shortly after sonication and that the rest of the DNA can be cleared by autophagosomes/autophagolysosomes. PMID:27165808

  16. Neisseria meningitidis subverts the polarized organization and intracellular trafficking of host cells to cross the epithelial barrier.

    PubMed

    Barrile, Riccardo; Kasendra, Magdalena; Rossi-Paccani, Silvia; Merola, Marcello; Pizza, Mariagrazia; Baldari, Cosima; Soriani, Marco; Aricò, Beatrice

    2015-09-01

    Translocation of the nasopharyngeal barrier by Neisseria meningitidis occurs via an intracellular microtubule-dependent pathway and represents a crucial step in its pathogenesis. Despite this fact, the interaction of invasive meningococci with host subcellular compartments and the resulting impact on their organization and function have not been investigated. The influence of serogroup B strain MC58 on host cell polarity and intracellular trafficking system was assessed by confocal microscopy visualization of different plasma membrane-associated components (such as E-cadherin, ZO-1 and transferrin receptor) and evaluation of the transferrin uptake and recycling in infected Calu-3 monolayers. Additionally, the association of N. meningitidis with different endosomal compartments was evaluated through the concomitant staining of bacteria and markers specific for Rab11, Rab22a, Rab25 and Rab3 followed by confocal microscopy imaging. Subversion of the host cell architecture and intracellular trafficking system, denoted by mis-targeting of cell plasma membrane components and perturbations of transferrin transport, was shown to occur in response to N. meningitidis infection. Notably, the appearance of all of these events seems to positively correlate with the efficiency of N. meningitidis to cross the epithelial barrier. Our data reveal for the first time that N. meningitidis is able to modulate the host cell architecture and function, which might serve as a strategy of this pathogen for overcoming the nasopharyngeal barrier without affecting the monolayer integrity.

  17. Galectin-3 regulates intracellular trafficking of epidermal growth factor receptor through Alix and promotes keratinocyte migration

    PubMed Central

    Liu, Wei; Hsu, Daniel K.; Chen, Huan-Yuan; Yang, Ri-Yao; Carraway, Kermit L.; Isseroff, Roslyn R.; Liu, Fu-Tong

    2012-01-01

    The epidermal growth factor receptor (EGFR)-mediated signaling pathways are important in a variety of cellular processes, including cell migration and wound re-epithelialization. Intracellular trafficking of EGFR is critical for maintaining EGFR surface expression. Galectin-3, a member of an animal lectin family, has been implicated in a number of physiological and pathological processes. Through studies of galectin-3-deficient mice and cells isolated from these mice, we demonstrated that absence of galectin-3 impairs keratinocyte migration and skin wound re-epithelialization. We have linked this pro-migratory function to a crucial role of cytosolic galectin-3 in controlling intracellular trafficking and cell surface expression of EGFR after EGF stimulation. Without galectin-3, the surface levels of EGFR are dramatically reduced and the receptor accumulates diffusely in the cytoplasm. This is associated with reduced rates of both endocytosis and recycling of the receptor. We have provided evidence that this novel function of galectin-3 may be mediated through interaction with its binding partner Alix, which is a protein component of the endosomal sorting complex required for transport (ESCRT) machinery. Our results suggest that galectin-3 is potentially a critical regulator of a number of important cellular responses through its intracellular control of trafficking of cell surface receptors. PMID:22785133

  18. Multifunctional envelope-type nano device for controlled intracellular trafficking and selective targeting in vivo.

    PubMed

    Kajimoto, Kazuaki; Sato, Yusuke; Nakamura, Takashi; Yamada, Yuma; Harashima, Hideyoshi

    2014-09-28

    Nanomedicine is expected to be a basic technology for using nucleic acids as a drug, in which treating the cause of diseases represent the ultimate therapy. However, a sophisticated delivery system is required for efficient delivery of RNA/DNA, since these compounds need precise control of intracellular trafficking as well as biodistribution. Here we report on the use of a multifunctional envelope-type nano device (MEND) which is capable of intracellular trafficking such as endosomal escape, delivery to mitochondria, as well as active targeting to selective tissues/cells in vivo. In this review, we focused on the controlled intracellular trafficking of antigens for advanced immunotherapy, and then introduced a mitochondrial delivery system as an organelle targeting system for unmet medical needs. We also provide a successful in vivo delivery of siRNA to the liver based on a newly designed pH-responsive cationic lipid. Finally we will discuss an important role of an active targeting system using a peptide ligand to adipose vasculature. These progresses in drug delivery system will break through the barriers exist in our body, tissues and cells and open a window for future Nanomedicine.

  19. High-throughput quantitation of intracellular trafficking and organelle disruption by flow cytometry.

    PubMed

    Chia, Pei Zhi Cheryl; Ramdzan, Yasmin M; Houghton, Fiona J; Hatters, Danny M; Gleeson, Paul A

    2014-05-01

    Current methods for the quantitation of membrane protein trafficking rely heavily on microscopy, which has limited quantitative capacity for analyses of cell populations and is cumbersome to perform. Here we describe a simple flow cytometry-based method that circumvents these limitations. The method utilizes fluorescent pulse-width measurements as a highly sensitive indicator to monitor the changes in intracellular distributions of a fluorescently labelled molecule in a cell. Pulse-width analysis enabled us to discriminate cells with target proteins in different intracellular locations including Golgi, lyso-endosomal network and the plasma membrane, as well as detecting morphological changes in organelles such as Golgi perturbation. The movement of endogenous and exogenous retrograde cargo was tracked from the plasma membrane-to-endosomes-to-Golgi, by decreasing pulse-width values. A block in transport upon RNAi-mediated ablation of transport machinery was readily quantified, demonstrating the versatility of this technique to identify pathway inhibitors. We also showed that pulse-width can be exploited to sort and recover cells based on different intracellular staining patterns, e.g. early endosomes and Golgi, opening up novel downstream applications. Overall, the method provides new capabilities for viewing membrane transport in thousands of cells per minute, unbiased analysis of the trafficking of cargo, and the potential for rapid screening of inhibitors of trafficking pathways.

  20. FYVE1 is essential for vacuole biogenesis and intracellular trafficking in Arabidopsis.

    PubMed

    Kolb, Cornelia; Nagel, Marie-Kristin; Kalinowska, Kamila; Hagmann, Jörg; Ichikawa, Mie; Anzenberger, Franziska; Alkofer, Angela; Sato, Masa H; Braun, Pascal; Isono, Erika

    2015-04-01

    The plant vacuole is a central organelle that is involved in various biological processes throughout the plant life cycle. Elucidating the mechanism of vacuole biogenesis and maintenance is thus the basis for our understanding of these processes. Proper formation of the vacuole has been shown to depend on the intracellular membrane trafficking pathway. Although several mutants with altered vacuole morphology have been characterized in the past, the molecular basis for plant vacuole biogenesis has yet to be fully elucidated. With the aim to identify key factors that are essential for vacuole biogenesis, we performed a forward genetics screen in Arabidopsis (Arabidopsis thaliana) and isolated mutants with altered vacuole morphology. The vacuolar fusion defective1 (vfd1) mutant shows seedling lethality and defects in central vacuole formation. VFD1 encodes a Fab1, YOTB, Vac1, and EEA1 (FYVE) domain-containing protein, FYVE1, that has been implicated in intracellular trafficking. FYVE1 localizes on late endosomes and interacts with Src homology-3 domain-containing proteins. Mutants of FYVE1 are defective in ubiquitin-mediated protein degradation, vacuolar transport, and autophagy. Altogether, our results show that FYVE1 is essential for plant growth and development and place FYVE1 as a key regulator of intracellular trafficking and vacuole biogenesis.

  1. Ultrasound-targeted Bubble Liposome Destruction Enhances AG73-mediated Gene Transfer by Improvement of Intracellular Trafficking

    NASA Astrophysics Data System (ADS)

    Omata, Daiki; Negishi, Yoichi; Endo-Takahashi, Yoko; Suzuki, Ryo; Maruyama, Kazuo; Nomizu, Motoyoshi; Aramaki, Yukihiko

    2011-09-01

    For selective cancer gene therapy, we have developed AG73-labeled polyethyleneglycol-modified liposomes (AG73-PEG liposomes) capable of encapsulating a gene condensed by poly-l-lysine. The present study examined whether echo-contrast gas-entrapping liposomes, also known as Bubble liposomes (BLs), and ultrasound (US) exposure affect not only the cell membrane but also intracellular vesicles, and enhance the release of pDNA from endosomes into the cytoplasm to achieve efficient gene transfection. AG73-mediated liposomal gene transfection efficiency was enhanced when BLs and US exposure were used. Furthermore, confocal microscopic analysis revealed that the BLs and US exposure promoted intracellular trafficking of the AG73-PEG liposomes during gene transfection. Thus, the use of AG73-PEG liposomes together with BLs and US exposure may be a promising way to achieve selective and efficient gene delivery.

  2. Tyrosine-phosphorylation of AAV2 vectors and its consequences on viral intracellular trafficking and transgene expression

    SciTech Connect

    Zhong Li; Li Baozheng; Jayandharan, Giridhararao; Mah, Cathryn S.; Govindasamy, Lakshmanan; Agbandje-McKenna, Mavis; Herzog, Roland W.

    2008-11-25

    We have documented that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects intracellular trafficking and transduction efficiency of recombinant adeno-associated virus 2 (AAV2) vectors. Specifically, inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsid proteins, which in turn, facilitates viral nuclear transport by limiting proteasome-mediated degradation of AAV2 vectors. In the present studies, we observed that AAV capsids can indeed be phosphorylated at tyrosine residues by EGFR-PTK in in vitro phosphorylation assays and that phosphorylated AAV capsids retain their structural integrity. However, although phosphorylated AAV vectors enter cells as efficiently as their unphosphorylated counterparts, their transduction efficiency is significantly reduced. This reduction is not due to impaired viral second-strand DNA synthesis since transduction efficiency of both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors is decreased by {approx} 68% and {approx} 74%, respectively. We also observed that intracellular trafficking of tyrosine-phosphorylated AAV vectors from cytoplasm to nucleus is significantly decreased, which results from ubiquitination of AAV capsids followed by proteasome-mediated degradation, although downstream consequences of capsid ubiquitination may also be affected by tyrosine-phosphorylation. These studies provide new insights into the role of tyrosine-phosphorylation of AAV capsids in various steps in the virus life cycle, which has implications in the optimal use of recombinant AAV vectors in human gene therapy.

  3. Intracellular trafficking of factor VIII to von Willebrand factor storage granules.

    PubMed Central

    Rosenberg, J B; Foster, P A; Kaufman, R J; Vokac, E A; Moussalli, M; Kroner, P A; Montgomery, R R

    1998-01-01

    In plasma, von Willebrand factor (vWf) associates with Factor VIII (FVIII); however, the site at which these proteins first interact has not been defined. Administration of 1-desamino-8-D-arginine vasopressin (DDAVP) causes a rapid, concomitant elevation in plasma levels of both vWf and FVIII, suggesting the existence of a DDAVP-releasable storage pool for both proteins. To determine whether vWf and FVIII can associate intracellularly and colocalize to storage vesicles, we transfected AtT-20 cells with vWf and FVIII expression plasmids. FVIII alone was not detectable within storage granules; however, transfection of vWf cDNA into the same cell caused FVIII to alter its intracellular trafficking and to undergo granular storage, colocalizing to the vWf-containing granules. In contrast, colocalization of FVIII was not observed when these cells were transfected with plasmids encoding defective FVIII-binding vWf mutants. Transfection of bovine endothelial cells with FVIII further demonstrated vesicular storage of FVIII with vWf in Weibel-Palade bodies. Since gene therapy of hemophilia A may ultimately target endothelium or hematopoietic stem cells, the interaction between vWf and FVIII within a secretory cell is important. Thus, vWf can alter the intracellular trafficking of FVIII from a constitutive to a regulated secretory pathway, thereby producing an intracellular storage pool of both proteins. PMID:9449695

  4. Intracellular Trafficking of Plasmids for Gene Therapy: Mechanisms of Cytoplasmic Movement and Nuclear Import

    PubMed Central

    Dean, David A.

    2015-01-01

    Under physiologically relevant conditions, the levels of non-viral gene transfer are low at best. The reason for this is that many barriers exist for the efficient transfer of genes to cells, even before any gene expression can occur. While many transfection strategies focus on DNA condensation and overcoming the plasma membrane, events associated with the intracellular trafficking of the DNA complexes have not been as extensively studied. Once internalized, plasmids must travel potentially long distances through the cytoplasm to reach their next barrier, the nuclear envelope. This review summarizes the current progress on the cytoplasmic trafficking and nuclear transport of plasmids used for gene therapy applications. Both of these processes utilize specific and defined mechanisms to facilitate movement of DNA complexes through the cell. The continued elucidation and exploitation of these mechanisms will lead to improved strategies for transfection and successful gene therapy. PMID:17168698

  5. Neurocutaneous syndrome with mental delay, autism, blockage in intracellular vescicular trafficking and melanosome defects.

    PubMed

    Buoni, S; Zannolli, R; de Santi, M; Macucci, F; Hayek, J; Orsi, A; Scarinci, R; Buscalferri, A; Cuccia, A; Zappella, M; Miracco, C

    2006-08-01

    We evaluated a 11-year-old male patient with mental delay, autism and brownish and whitish skin spots. The former resembled those of neurofibromatosis, the latter those of tuberous sclerosis. The patient received a complete clinical work-up to exclude neurofibromatosis, tuberous sclerosis, or any other known neurocutaneous disease, with biochemistry, chromosome analysis and analysis of skin specimens. Being all the other tests not significant, two main ultrastructural defects were observed. The first was a blockage in intracellular vescicular trafficking with sparing of the mitochondria; the second an aberrant presence of melanosomes in vacuoles of several cell lines and abnormal transfer of these organelles to keratinocytes. This patient presented with a unique clinical picture distinct from neurofibromatosis or tuberous sclerosis or any other known neurocutaneous disease. The ultrastructural abnormalities suggested a defect in cell trafficking involving several cell lines and compartments.

  6. Modulation of hydrogel nanoparticle intracellular trafficking by multivalent surface engineering with tumor targeting peptide.

    PubMed

    Karamchand, Leshern; Kim, Gwangseong; Wang, Shouyan; Hah, Hoe Jin; Ray, Aniruddha; Jiddou, Ruba; Koo Lee, Yong-Eun; Philbert, Martin A; Kopelman, Raoul

    2013-11-01

    Surface engineering of a hydrogel nanoparticle (NP) with the tumor-targeting ligand, F3 peptide, enhances both the NP's binding affinity for, and internalization by, nucleolin overexpressing tumor cells. Remarkably, the F3-functionalized NPs consistently exhibited significantly lower trafficking to the degradative lysosomes than the non-functionalized NPs, in the tumor cells, after internalization. This is attributed to the non-functionalized NPs, but not the F3-functionalized NPs, being co-internalized with Lysosome-associated Membrane Protein-1 (LAMP1) from the surface of the tumor cells. Furthermore, it is shown that the intracellular trafficking of the F3-functionalized NPs differs significantly from that of the molecular F3 peptides (untethered to NPs). This has important implications for designing effective, chemically-responsive, controlled-release and multifunctional nanodrugs for multi-drug-resistant cancers.

  7. Tight junction targeting and intracellular trafficking of occludin in polarized epithelial cells.

    PubMed

    Subramanian, Veedamali S; Marchant, Jonathan S; Ye, Dongmei; Ma, Thomas Y; Said, Hamid M

    2007-11-01

    Occludin, a transmembrane (TM)-spanning protein, is an integral component of the tight junctional (TJ) complexes that regulate epithelial integrity and paracellular barrier function. However, the molecular determinants that dictate occludin targeting and delivery to the TJs remain unclear. Here, using live cell imaging of yellow fluorescent protein-labeled occludin fragments, we resolved the intracellular trafficking of occludin-fusion proteins in polarized Madin-Darby canine kidney and Caco-2 cells to delineate the regions within the occludin polypeptide that are important for occludin targeting to the TJs. Live cell confocal imaging showed that complete or partial truncation of the COOH-terminal tail of the occludin polypeptide did not prevent occludin targeting to the TJs in epithelial cell lines. Progressive truncations into the COOH-terminal tail decreased the efficiency of occludin expression; after the removal of the regions proximal to the fourth transmembrane domain (TM4), the efficiency of expression increased. However, further deletions into the TM4 abolished TJ targeting, which resulted in constructs that were retained intracellularly within the endoplasmic reticulum. The full-length occludin polypeptide trafficked to the cell surface within a heterogenous population of intracellular vesicles that delivered occludin to the plasma membrane in a microtubule- and temperature-dependent manner. In contrast, the steady-state localization of occludin at the cell surface was dependent on intact microfilaments but not microtubules.

  8. Intracellular trafficking of superparamagnetic iron oxide nanoparticles conjugated with TAT peptide: 3-dimensional electron tomography analysis

    SciTech Connect

    Nair, Baiju G.; Fukuda, Takahiro; Mizuki, Toru; Hanajiri, Tatsuro; Maekawa, Toru

    2012-05-18

    Highlights: Black-Right-Pointing-Pointer We study the intracellular localisation of TAT-SPIONs using 3-D electron tomography. Black-Right-Pointing-Pointer 3-D images of TAT-SPIONs in a cell are clearly shown. Black-Right-Pointing-Pointer Release of TAT-SPIONs from endocytic vesicles into the cytoplasm is clearly shown. -- Abstract: Internalisation of nanoparticles conjugated with cell penetrating peptides is a promising approach to various drug delivery applications. Cell penetrating peptides such as transactivating transcriptional activator (TAT) peptides derived from HIV-1 proteins are effective intracellular delivery vectors for a wide range of nanoparticles and pharmaceutical agents thanks to their amicable ability to enter cells and minimum cytotoxicity. Although different mechanisms of intracellular uptake and localisation have been proposed for TAT conjugated nanoparticles, it is necessary to visualise the particles on a 3-D plane in order to investigate the actual intracellular uptake and localisation. Here, we study the intracellular localisation and trafficking of TAT peptide conjugated superparamagnetic ion oxide nanoparticles (TAT-SPIONs) using 3-D electron tomography. 3-D tomograms clearly show the location of TAT-SPIONs in a cell and their slow release from the endocytic vesicles into the cytoplasm. The present methodology may well be utilised for further investigations of the behaviours of nanoparticles in cells and eventually for the development of nano drug delivery systems.

  9. Contributions of epsinR and gadkin to clathrin-mediated intracellular trafficking

    PubMed Central

    Hirst, Jennifer; Edgar, James R.; Borner, Georg H. H.; Li, Sam; Sahlender, Daniela A.; Antrobus, Robin; Robinson, Margaret S.

    2015-01-01

    The precise functions of most of the proteins that participate in clathrin-mediated intracellular trafficking are unknown. We investigated two such proteins, epsinR and gadkin, using the knocksideways method, which rapidly depletes proteins from the available pool by trapping them onto mitochondria. Although epsinR is known to be an N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE)-specific adaptor, the epsinR knocksideways blocked the production of the entire population of intracellular clathrin-coated vesicles (CCVs), suggesting a more global function. Using the epsinR knocksideways data, we were able to estimate the copy number of all major intracellular CCV proteins. Both sides of the vesicle are densely covered, indicating that CCVs sort their cargo by molecular crowding. Trapping of gadkin onto mitochondria also blocked the production of intracellular CCVs but by a different mechanism: vesicles became cross-linked to mitochondria and pulled out toward the cell periphery. Both phenotypes provide new insights into the regulation of intracellular CCV formation, which could not have been found using more conventional approaches. PMID:26179914

  10. Regulation of Epidermal Growth Factor Receptor Signaling by Endocytosis and Intracellular Trafficking

    SciTech Connect

    Burke, Patrick; Schooler, Kevin; Wiley, H S.

    2001-06-01

    Ligand activation of the epidermal growth factor receptor (EGFR) leads to its rapid internalization and eventual delivery to lysosomes. This process is thought to be a mechanism to attenuate signaling, but signals could potentially be generated following endocytosis. To directly evaluate EGFR signaling during receptor trafficking, we developed a technique to rapidly and selectively isolate internalized EGFR and associated molecules using reversibly-biotinylated anti-EGFR antibodies. In addition, we developed antibodies specific to tyrosine-phosphorylated EGFR. Using a combination of fluorescence imaging and affinity precipitation approaches, we evaluated the state of EGFR activation and substrate association during trafficking in epithelial cells. We found that following internalization, EGFR remained active in the early endosomes. However, receptors were inactivated prior to degradation, apparently due to ligand removal from endosomes. Adapter molecules, such as Shc, were associated with EGFR both at the cell surface and within endosomes. Some molecules, such as Grb2, were primarily found associated with surface EGFR, while others, such as Eps8, were only found with intracellular receptors. During the inactivation phase, c-Cbl became EGFR-associated, consistent with its postulated role in receptor attenuation. We conclude that the association of the EGFR with different proteins is compartment-specific . In addition, ligand loss is the proximal cause of EGFR inactivation. Thus, regulated trafficking could potentially influence the pattern as well as the duration of signal transduction.

  11. Intracellular trafficking of the pyridoxal cofactor. Implications for health and metabolic disease.

    PubMed

    Whittaker, James W

    2016-02-15

    The importance of the vitamin B6-derived pyridoxal cofactor for human health has been established through more than 70 years of intensive biochemical research, revealing its fundamental roles in metabolism. B6 deficiency, resulting from nutritional limitation or impaired uptake from dietary sources, is associated with epilepsy, neuromuscular disease and neurodegeneration. Hereditary disorders of B6 processing are also known, and genetic defects in pathways involved in transport of B6 into the cell and its transformation to the pyridoxal-5'-phosphate enzyme cofactor can contribute to cardiovascular disease by interfering with homocysteine metabolism and the biosynthesis of vasomodulatory polyamines. Compared to the processes involved in cellular uptake and processing of the B6 vitamers, trafficking of the PLP cofactor across intracellular membranes is very poorly understood, even though the availability of PLP within subcellular compartments (particularly the mitochondrion) may have important health implications. The aim of this review is to concisely summarize the state of current knowledge of intracellular trafficking of PLP and to identify key directions for future research.

  12. Intracellular trafficking by Star regulates cleavage of the Drosophila EGF receptor ligand Spitz

    PubMed Central

    Tsruya, Rachel; Schlesinger, Ayelet; Reich, Aderet; Gabay, Limor; Sapir, Amir; Shilo, Ben-Zion

    2002-01-01

    Spitz (Spi) is a TGFα homolog that is a cardinal ligand for the Drosophila EGF receptor throughout development. Cleavage of the ubiquitously expressed transmembrane form of Spi (mSpi) precedes EGF receptor activation. We show that the Star and Rhomboid (Rho) proteins are necessary for Spi cleavage in Drosophila cells. Complexes between the Spi and Star proteins, as well as between the Star and Rho proteins were identified, but no Spi–Star–Rho triple complex was detected. This observation suggests a sequential activity of Star and Rho in mSpi processing. The interactions between Spi and Star regulate the intracellular trafficking of Spi. The Spi precursor is retained in the periphery of the nucleus. Coexpression of Star promotes translocation of Spi to a compartment where Rho is present both in cells and in embryos. A Star deletion construct that maintains binding to Spi and Rho, but is unable to facilitate Spi translocation, lost biological activity. These results underscore the importance of regulated intracellular trafficking in processing of a TGFα family ligand. PMID:11799065

  13. Reactive oxygen species-mediated DJ-1 monomerization modulates intracellular trafficking involving karyopherin β2.

    PubMed

    Björkblom, Benny; Maple-Grødem, Jodi; Puno, Marc Rhyan; Odell, Mark; Larsen, Jan Petter; Møller, Simon Geir

    2014-08-01

    Mutations in DJ-1 are a cause of recessive, early-onset Parkinson's disease (PD). Although oxidative stress and mitochondrial integrity have been implicated in PD, it is largely unknown why neurons degenerate. DJ-1 is involved in oxidative stress-mediated responses and in mitochondrial maintenance; however, its specific function remains vague. Here we show that DJ-1 exhibits neuronal dynamic intracellular trafficking, with dimeric/monomeric cycling modulated by the oxidative environment. We demonstrate that oxidative stress enhances monomerization of wild-type cytosolic DJ-1, leading to nuclear recruitment. The pathogenic DJ-1/E163K variant is unable to homodimerize but is retained in the cytosol upon wild-type DJ-1 heterodimerization. We found that this wild-type/pathogenic heterodimer is disrupted by oxidative stress, leading to DJ-1/E163K mitochondrial translocation. We further demonstrated that endogenously expressed wild-type DJ-1 is imported into neuronal nuclei as a monomer and that nucleo-cytoplasmic transport is oxidative stress mediated. We identified a novel proline-tyrosine nuclear localization signal (PY-NLS) in DJ-1, and we found that nuclear monomeric DJ-1 import is mediated by an oxidative stress-dependent interaction with karyopherin β2. Our study provides evidence that oxidative stress-mediated intracellular trafficking of DJ-1, mediated by dynamic DJ-1 dimeric/monomeric cycling, is implicated in PD pathogenesis. PMID:24912681

  14. MUC1 intra-cellular trafficking is clathrin, dynamin, and rab5 dependent

    SciTech Connect

    Liu Xiaolong; Yuan Zhenglong; Chung, Maureen

    2008-11-28

    MUC1, a transmembrane glycoprotein, is abnormally over-expressed in most human adenocarcinomas. MUC1 association with cytoplasmic cell signal regulators and nuclear accumulation are important for its tumor related activities. Little is known about how MUC1 translocates from the cell membrane to the cytoplasm. In this study, live cell imaging was used to study MUC1 intracellular trafficking. The interaction between EGFR and MUC1 was mapped by FRET analysis and EGF stimulated MUC1 endocytosis was observed directly through live cell imaging. MUC1-CT endocytosis was clathrin and dynamin dependent. Rab5 over-expression resulted in decreased cell membrane localization of MUC1, with accumulation of MUC1 endocytic vesicles in the peri-nuclear region. Conversely, over-expression of a Rab5 dominant negative mutant (S34N) resulted in redistribution of MUC1 from the peri-nuclear region to the cytoplasm. Collectively, these results indicated that MUC1 intra-cellular trafficking occurs through a regulated process that was stimulated by direct EGFR and MUC1 interaction, mediated by clathrin coated pits that were dynamin dependent and regulated by Rab5.

  15. Live Cell Imaging of the Endocytosis and the Intracellular Trafficking of Multifunctional Lipid Nanoparticles

    SciTech Connect

    Zhang, Tieqiao; Danthi, S. N.; Xie, Jianwu; Hu, Dehong; Lu, H. Peter; Li, King H.

    2006-12-01

    Artificial lipid nanoparticles have drawn great attention due to their potential in medicine. Linked with targeting ligands, they can be used as probes and/or gene delivery vectors for specific types of target cells. Therefore, they are very promising agents in early detection, diagnosis and treatment of cancers and other genetic diseases. However, there are several barriers blocking the applications. Controlling the cellular uptake of the lipid nanoparticles is an important technical challenge to overcome. Understanding the mechanism of the endocytosis and the following intracellular trafficking is very important for improving the design and therefore the efficiency as a drug delivery system. By using fluorescence microscopy methods, we studied the endocytosis of lipid nanoparticles by live M21 cells. The movements of the nanoparticles inside the cell were quantitatively characterized and classified based on the diffusion behavior. The trajectories of nanoparticles movement over the cell membrane revealed hop-diffusion behavior prior to the endocytosis. Fast movement in large steps is observed in intracellular trafficking and is attributed to active movement along microtubule. These observations help to understand the mechanism of the endocytosis and the pathway of the particles in cells.

  16. Reactive oxygen species-mediated DJ-1 monomerization modulates intracellular trafficking involving karyopherin β2.

    PubMed

    Björkblom, Benny; Maple-Grødem, Jodi; Puno, Marc Rhyan; Odell, Mark; Larsen, Jan Petter; Møller, Simon Geir

    2014-08-01

    Mutations in DJ-1 are a cause of recessive, early-onset Parkinson's disease (PD). Although oxidative stress and mitochondrial integrity have been implicated in PD, it is largely unknown why neurons degenerate. DJ-1 is involved in oxidative stress-mediated responses and in mitochondrial maintenance; however, its specific function remains vague. Here we show that DJ-1 exhibits neuronal dynamic intracellular trafficking, with dimeric/monomeric cycling modulated by the oxidative environment. We demonstrate that oxidative stress enhances monomerization of wild-type cytosolic DJ-1, leading to nuclear recruitment. The pathogenic DJ-1/E163K variant is unable to homodimerize but is retained in the cytosol upon wild-type DJ-1 heterodimerization. We found that this wild-type/pathogenic heterodimer is disrupted by oxidative stress, leading to DJ-1/E163K mitochondrial translocation. We further demonstrated that endogenously expressed wild-type DJ-1 is imported into neuronal nuclei as a monomer and that nucleo-cytoplasmic transport is oxidative stress mediated. We identified a novel proline-tyrosine nuclear localization signal (PY-NLS) in DJ-1, and we found that nuclear monomeric DJ-1 import is mediated by an oxidative stress-dependent interaction with karyopherin β2. Our study provides evidence that oxidative stress-mediated intracellular trafficking of DJ-1, mediated by dynamic DJ-1 dimeric/monomeric cycling, is implicated in PD pathogenesis.

  17. Modulation of hydrogel nanoparticle intracellular trafficking by multivalent surface engineering with tumor targeting peptide

    NASA Astrophysics Data System (ADS)

    Karamchand, Leshern; Kim, Gwangseong; Wang, Shouyan; Hah, Hoe Jin; Ray, Aniruddha; Jiddou, Ruba; Koo Lee, Yong-Eun; Philbert, Martin A.; Kopelman, Raoul

    2013-10-01

    Surface engineering of a hydrogel nanoparticle (NP) with the tumor-targeting ligand, F3 peptide, enhances both the NP's binding affinity for, and internalization by, nucleolin overexpressing tumor cells. Remarkably, the F3-functionalized NPs consistently exhibited significantly lower trafficking to the degradative lysosomes than the non-functionalized NPs, in the tumor cells, after internalization. This is attributed to the non-functionalized NPs, but not the F3-functionalized NPs, being co-internalized with Lysosome-associated Membrane Protein-1 (LAMP1) from the surface of the tumor cells. Furthermore, it is shown that the intracellular trafficking of the F3-functionalized NPs differs significantly from that of the molecular F3 peptides (untethered to NPs). This has important implications for designing effective, chemically-responsive, controlled-release and multifunctional nanodrugs for multi-drug-resistant cancers.Surface engineering of a hydrogel nanoparticle (NP) with the tumor-targeting ligand, F3 peptide, enhances both the NP's binding affinity for, and internalization by, nucleolin overexpressing tumor cells. Remarkably, the F3-functionalized NPs consistently exhibited significantly lower trafficking to the degradative lysosomes than the non-functionalized NPs, in the tumor cells, after internalization. This is attributed to the non-functionalized NPs, but not the F3-functionalized NPs, being co-internalized with Lysosome-associated Membrane Protein-1 (LAMP1) from the surface of the tumor cells. Furthermore, it is shown that the intracellular trafficking of the F3-functionalized NPs differs significantly from that of the molecular F3 peptides (untethered to NPs). This has important implications for designing effective, chemically-responsive, controlled-release and multifunctional nanodrugs for multi-drug-resistant cancers. Electronic supplementary information (ESI) available: Effect of Potassium depletion on F3 peptide subcellular localization, MTT

  18. A Dual Role for the Nonreceptor Tyrosine Kinase Pyk2 during the Intracellular Trafficking of Human Papillomavirus 16

    PubMed Central

    Gottschalk, Elinor Y.

    2015-01-01

    ABSTRACT The infectious process of human papillomaviruses (HPVs) has been studied considerably, and many cellular components required for viral entry and trafficking continue to be revealed. In this study, we investigated the role of the nonreceptor tyrosine kinase Pyk2 during HPV16 pseudovirion infection of human keratinocytes. We found that Pyk2 is necessary for infection and appears to be involved in the intracellular trafficking of the virus. Small interfering RNA-mediated reduction of Pyk2 resulted in a significant decrease in infection but did not prevent viral entry at the plasma membrane. Pyk2 depletion resulted in altered endolysosomal trafficking of HPV16 and accelerated unfolding of the viral capsid. Furthermore, we observed retention of the HPV16 pseudogenome in the trans-Golgi network (TGN) in Pyk2-depleted cells, suggesting that the kinase could be required for the viral DNA to exit the TGN. While Pyk2 has previously been shown to function during the entry of enveloped viruses at the plasma membrane, the kinase has not yet been implicated in the intracellular trafficking of a nonenveloped virus such as HPV. Additionally, these data enrich the current literature on Pyk2's function in human keratinocytes. IMPORTANCE In this study, we investigated the role of the nonreceptor tyrosine kinase Pyk2 during human papillomavirus (HPV) infection of human skin cells. Infections with high-risk types of HPV such as HPV16 are the leading cause of cervical cancer and a major cause of genital and oropharyngeal cancer. As a nonenveloped virus, HPV enters cells by interacting with cellular receptors and established cellular trafficking routes to ensure that the viral DNA reaches the nucleus for productive infection. This study identified Pyk2 as a cellular component required for the intracellular trafficking of HPV16 during infection. Understanding the infectious pathways of HPVs is critical for developing additional preventive therapies. Furthermore, this study

  19. Intracellular trafficking of VP22 in bovine herpesvirus-1 infected cells

    SciTech Connect

    Lobanov, Vladislav A.; Babiuk, Lorne A.; Drunen Littel-van den Hurk, Sylvia van

    2010-01-20

    The intracellular trafficking of different VP22-enhanced yellow fluorescent protein (EYFP) fusion proteins expressed by bovine herpesvirus-1 (BHV-1) recombinants was examined by live-cell imaging. Our results demonstrate that (i) the fusion of EYFP to the C terminus of VP22 does not alter the trafficking of the protein in infected cells, (ii) VP22 expressed during BHV-1 infection translocates to the nucleus through three different pathways, namely early mitosis-dependent nuclear translocation, late massive nuclear translocation that follows a prolonged cytoplasmic stage of the protein in non-mitotic cells, and accumulation of a small subset of VP22 in discrete dot-like nuclear domains during its early cytoplasmic stage, (iii) the addition of the SV40 large-T-antigen nuclear localization signal (NLS) to VP22-EYFP abrogates its early cytoplasmic stage, and (iv) the VP22 {sup 131}PRPR{sup 134} NLS is not required for the late massive nuclear translocation of the protein, but this motif is essential for the targeting of VP22 to discrete dot-like nuclear domains during the early cytoplasmic stage. These results show that the amount of VP22 in the nucleus is precisely regulated at different stages of BHV-1 infection and suggest that the early pathways of VP22 nuclear accumulation may be more relevant to the infection process as the late massive nuclear influx starts when most of the viral progeny has already emerged from the cell.

  20. Intracellular trafficking of solid lipid nanoparticles and their distribution between cells through tunneling nanotubes.

    PubMed

    Kristl, Julijana; Plajnšek, Karmen Teskač; Kreft, Mateja Erdani; Janković, Biljana; Kocbek, Petra

    2013-09-27

    The intracellular fate of nanosized drug delivery systems is still not well understood. Various internalization pathways have been discovered, but knowledge of their intracellular trafficking is still incomplete. The aim of this study was to examine the internalization, pathways, and positioning taken by solid lipid nanoparticles (SLNs) in cells. SLNs were fluorescence labeled with a newly synthesized fluorescent probe, 14-DACA. The probe was strongly incorporated into the nanoparticle core under the influence of its long lipophilic chain, enabling superior visualization of SLNs under complex and dynamic intracellular conditions. The intracellular distribution of SLNs was studied qualitatively using a co-localization technique and quantitatively using fluorescence intensity profiles. SLNs were seen inside the cells as distinct bright blue dots that underwent dynamic movement and were finally positioned in the proximity of the nucleus. A few SLNs were shown to be present in mitochondria and between actin filaments, but none in the cell nucleus or lysosomes. SLNs are here reported to be present in tunneling nanotubes (TNTs), which could be a new route of SLN transfer between cells. More TNTs were observed in cells treated with SLNs. The presence of TNTs was additionally confirmed by atomic force microscopy analysis, which indicated that treated cells were more rough than control cells. Detailed investigation of the subcellular localization of SLNs and the evidence for their transfer and distribution via TNTs to the cells, which are not in direct contact with the source of SLNs, are important for understanding the mechanism of targeted drug delivery. Understanding the possible intercellular distribution of SLNs via TNTs can significantly influence approaches to treating organelle-specific diseases.

  1. Arf6-Dependent Intracellular Trafficking of Pasteurella multocida Toxin and pH-Dependent Translocation from Late Endosomes

    PubMed Central

    Repella, Tana L.; Ho, Mengfei; Chong, Tracy P. M.; Bannai, Yuka; Wilson, Brenda A.

    2011-01-01

    The potent mitogenic toxin from Pasteurella multocida (PMT) is the major virulence factor associated with a number of epizootic and zoonotic diseases caused by infection with this respiratory pathogen. PMT is a glutamine-specific protein deamidase that acts on its intracellular G-protein targets to increase intracellular calcium, cytoskeletal, and mitogenic signaling. PMT enters cells through receptor-mediated endocytosis and then translocates into the cytosol through a pH-dependent process that is inhibited by NH4Cl or bafilomycin A1. However, the detailed mechanisms that govern cellular entry, trafficking, and translocation of PMT remain unclear. Co-localization studies described herein revealed that while PMT shares an initial entry pathway with transferrin (Tfn) and cholera toxin (CT), the trafficking pathways of Tfn, CT, and PMT subsequently diverge, as Tfn is trafficked to recycling endosomes, CT is trafficked retrograde to the ER, and PMT is trafficked to late endosomes. Our studies implicate the small regulatory GTPase Arf6 in the endocytic trafficking of PMT. Translocation of PMT from the endocytic vesicle occurs through a pH-dependent process that is also dependent on both microtubule and actin dynamics, as evidenced by inhibition of PMT activity in our SRE-based reporter assay, with nocodazole and cytochalasin D, respectively, suggesting that membrane translocation and cytotoxicity of PMT is dependent on its transfer to late endosomal compartments. In contrast, disruption of Golgi-ER trafficking with brefeldin A increased PMT activity, suggesting that inhibiting PMT trafficking to non-productive compartments that do not lead to translocation, while promoting formation of an acidic tubulovesicle system more conducive to translocation, enhances PMT translocation and activity. PMID:22053287

  2. Cholera toxin subunit B-mediated intracellular trafficking of mesoporous silica nanoparticles toward the endoplasmic reticulum

    NASA Astrophysics Data System (ADS)

    Walker, William Andrew

    In recent decades, pharmaceutical research has led to the development of numerous treatments for human disease. Nanoscale delivery systems have the potential to maximize therapeutic outcomes by enabling target specific delivery of these therapeutics. The intracellular localization of many of these materials however, is poorly controlled, leading to sequestration in degradative cellular pathways and limiting the efficacy of their payloads. Numerous proteins, particularly bacterial toxins, have evolved mechanisms to subvert the degradative mechanisms of the cell. Here, we have investigated a possible strategy for shunting intracellular delivery of encapsulated cargoes from these pathways by modifying mesoporous silica nanoparticles (MSNs) with the well-characterized bacterial toxin Cholera toxin subunit B (CTxB). Using established optical imaging methods we investigated the internalization, trafficking, and subcellular localization of our modified MSNs in an in vitro animal cell model. We then attempted to demonstrate the practical utility of this approach by using CTxB-modified mesoporous silica nanoparticles to deliver propidium iodide, a membrane-impermeant fluorophore.

  3. Emerging roles of ARHGAP33 in intracellular trafficking of TrkB and pathophysiology of neuropsychiatric disorders

    PubMed Central

    Nakazawa, Takanobu; Hashimoto, Ryota; Sakoori, Kazuto; Sugaya, Yuki; Tanimura, Asami; Hashimotodani, Yuki; Ohi, Kazutaka; Yamamori, Hidenaga; Yasuda, Yuka; Umeda-Yano, Satomi; Kiyama, Yuji; Konno, Kohtarou; Inoue, Takeshi; Yokoyama, Kazumasa; Inoue, Takafumi; Numata, Shusuke; Ohnuma, Tohru; Iwata, Nakao; Ozaki, Norio; Hashimoto, Hitoshi; Watanabe, Masahiko; Manabe, Toshiya; Yamamoto, Tadashi; Takeda, Masatoshi; Kano, Masanobu

    2016-01-01

    Intracellular trafficking of receptor proteins is essential for neurons to detect various extracellular factors during the formation and refinement of neural circuits. However, the precise mechanisms underlying the trafficking of neurotrophin receptors to synapses remain elusive. Here, we demonstrate that a brain-enriched sorting nexin, ARHGAP33, is a new type of regulator for the intracellular trafficking of TrkB, a high-affinity receptor for brain-derived neurotrophic factor. ARHGAP33 knockout (KO) mice exhibit reduced expression of synaptic TrkB, impaired spine development and neuropsychiatric disorder-related behavioural abnormalities. These deficits are rescued by specific pharmacological enhancement of TrkB signalling in ARHGAP33 KO mice. Mechanistically, ARHGAP33 interacts with SORT1 to cooperatively regulate TrkB trafficking. Human ARHGAP33 is associated with brain phenotypes and reduced SORT1 expression is found in patients with schizophrenia. We propose that ARHGAP33/SORT1-mediated TrkB trafficking is essential for synapse development and that the dysfunction of this mechanism may be a new molecular pathology of neuropsychiatric disorders. PMID:26839058

  4. Intracellular trafficking of the human Wilson protein: the role of the six N-terminal metal-binding sites.

    PubMed Central

    Cater, Michael A; Forbes, John; La Fontaine, Sharon; Cox, Diane; Mercer, Julian F B

    2004-01-01

    The Wilson protein (ATP7B) is a copper-transporting CPx-type ATPase defective in the copper toxicity disorder Wilson disease. In hepatocytes, ATP7B delivers copper to apo-ceruloplasmin and mediates the excretion of excess copper into bile. These distinct functions require the protein to localize at two different subcellular compartments. At the trans-Golgi network, ATP7B transports copper for incorporation into apo-ceruloplasmin. When intracellular copper levels are increased, ATP7B traffics to post-Golgi vesicles in close proximity to the canalicular membrane to facilitate biliary copper excretion. In the present study, we investigated the role of the six N-terminal MBSs (metal-binding sites) in the trafficking process. Using site-directed mutagenesis, we mutated or deleted various combinations of the MBSs and assessed the effect of these changes on the localization and trafficking of ATP7B. Results show that the MBSs required for trafficking are the same as those previously found essential for the copper transport function. Either MBS 5 or MBS 6 alone was sufficient to support the redistribution of ATP7B to vesicular compartments. The first three N-terminal motifs were not required for copper-dependent intracellular trafficking and could not functionally replace sites 4-6 when placed in the same sequence position. Furthermore, the N-terminal region encompassing MBSs 1-5 (amino acids 64-540) was not essential for trafficking, with only one MBS close to the membrane channel, necessary and sufficient to support trafficking. Our findings were similar to those obtained for the closely related ATP7A protein, suggesting similar mechanisms for trafficking between copper-transporting CPx-type ATPases. PMID:14998371

  5. Intracellular Trafficking and Synaptic Function of APL-1 in Caenorhabditis elegans

    PubMed Central

    Wiese, Mary; Antebi, Adam; Zheng, Hui

    2010-01-01

    Background Alzheimer's disease (AD) is a neurodegenerative disorder primarily characterized by the deposition of β-amyloid plaques in the brain. Plaques are composed of the amyloid-β peptide derived from cleavage of the amyloid precursor protein (APP). Mutations in APP lead to the development of Familial Alzheimer's Disease (FAD), however, the normal function of this protein has proven elusive. The organism Caenorhabditis elegans is an attractive model as the amyloid precursor-like protein (APL-1) is the single ortholog of APP, and loss of apl-1 leads to a severe molting defect and early larval lethality. Methodology/Principal Findings We report here that lethality and molting can be rescued by full length APL-1, C-terminal mutations as well as a C-terminal truncation, suggesting that the extracellular region of the protein is essential for viability. RNAi knock-down of apl-1 followed by drug testing on the acetylcholinesterase inhibitor aldicarb showed that loss of apl-1 leads to aldicarb hypersensitivity, indicating a defect in synaptic function. The aldicarb hypersensitivity can be rescued by full length APL-1 in a dose dependent fashion. At the cellular level, kinesins UNC-104/KIF-1A and UNC-116/kinesin-1 are positive regulators of APL-1 expression in the neurons. Knock-down of the small GTPase rab-5 also leads to a dramatic decrease in the amount of apl-1 expression in neurons, suggesting that trafficking from the plasma membrane to the early endosome is important for apl-1 function. Loss of function of a different small GTPase, UNC-108, on the contrary, leads to the retention of APL-1 in the cell body. Conclusions/Significance Our results reveal novel insights into the intracellular trafficking of APL-1 and we report a functional role for APL-1 in synaptic transmission. PMID:20862215

  6. Role of Ostm1 Cytosolic Complex with Kinesin 5B in Intracellular Dispersion and Trafficking

    PubMed Central

    Pandruvada, Subramanya N. M.; Beauregard, Janie; Benjannet, Suzanne; Pata, Monica; Lazure, Claude; Seidah, Nabil G.

    2015-01-01

    In humans and in mice, mutations in the Ostm1 gene cause the most severe form of osteopetrosis, a major bone disease, and neuronal degeneration, both of which are associated with early death. To gain insight into Ostm1 function, we first investigated by sequence and biochemical analysis an immature 34-kDa type I transmembrane Ostm1 protein with a unique cytosolic tail. Mature Ostm1 is posttranslationally processed and highly N-glycosylated and has an apparent mass of ∼60 kDa. Analysis the subcellular localization of Ostm1 showed that it is within the endoplasmic reticulum, trans-Golgi network, and endosomes/lysosomes. By a wide protein screen under physiologic conditions, several novel cytosolic Ostm1 partners were identified and validated, for which a direct interaction with the kinesin 5B heavy chains was demonstrated. These results determined that Ostm1 is part of a cytosolic scaffolding multiprotein complex, imparting an adaptor function to Ostm1. Moreover, we uncovered a role for the Ostm1/KIF5B complex in intracellular trafficking and dispersion of cargos from the endoplasmic reticulum to late endosomal/lysosomal subcellular compartments. These Ostm1 molecular and cellular functions could elucidate all of the pathophysiologic mechanisms underlying the wide phenotypic spectrum of Ostm1-deficient mice. PMID:26598607

  7. Role of Ostm1 Cytosolic Complex with Kinesin 5B in Intracellular Dispersion and Trafficking.

    PubMed

    Pandruvada, Subramanya N M; Beauregard, Janie; Benjannet, Suzanne; Pata, Monica; Lazure, Claude; Seidah, Nabil G; Vacher, Jean

    2016-02-01

    In humans and in mice, mutations in the Ostm1 gene cause the most severe form of osteopetrosis, a major bone disease, and neuronal degeneration, both of which are associated with early death. To gain insight into Ostm1 function, we first investigated by sequence and biochemical analysis an immature 34-kDa type I transmembrane Ostm1 protein with a unique cytosolic tail. Mature Ostm1 is posttranslationally processed and highly N-glycosylated and has an apparent mass of ∼60 kDa. Analysis the subcellular localization of Ostm1 showed that it is within the endoplasmic reticulum, trans-Golgi network, and endosomes/lysosomes. By a wide protein screen under physiologic conditions, several novel cytosolic Ostm1 partners were identified and validated, for which a direct interaction with the kinesin 5B heavy chains was demonstrated. These results determined that Ostm1 is part of a cytosolic scaffolding multiprotein complex, imparting an adaptor function to Ostm1. Moreover, we uncovered a role for the Ostm1/KIF5B complex in intracellular trafficking and dispersion of cargos from the endoplasmic reticulum to late endosomal/lysosomal subcellular compartments. These Ostm1 molecular and cellular functions could elucidate all of the pathophysiologic mechanisms underlying the wide phenotypic spectrum of Ostm1-deficient mice. PMID:26598607

  8. Cellular uptake of a cystine-knot peptide and modulation of its intracellular trafficking

    PubMed Central

    Gao, Xinxin; Stanger, Karen; Kaluarachchi, Harini; Maurer, Till; Ciepla, Paulina; Chalouni, Cecile; Franke, Yvonne; Hannoush, Rami N.

    2016-01-01

    Cyclotides or cyclic cystine-knot peptides have emerged as a promising class of pharmacological ligands that modulate protein function. Interestingly, very few cyclotides have been shown to enter into cells. Yet, it remains unknown whether backbone cyclization is required for their cellular internalization. In this report, we studied the cellular behavior of EETI-II, a model acyclic cystine-knot peptide. Even though synthetic methods have been used to generate EETI-II, recombinant methods that allow efficient large scale biosynthesis of EETI-II have been lagging. Here, we describe a novel protocol for recombinant generation of folded EETI-II in high yields and to near homogeneity. We also uncover that EETI-II is efficiently uptaken via an active endocytic pathway to early endosomes in mammalian cells, eventually accumulating in late endosomes and lysosomes. Notably, co-incubation with a cell-penetrating peptide enhanced the cellular uptake and altered the trafficking of EETI-II, leading to its evasion of lysosomes. Our results demonstrate the feasibility of modulating the subcellular distribution and intracellular targeting of cystine-knot peptides, and hence enable future exploration of their utility in drug discovery and delivery. PMID:27734922

  9. Epitope Tags beside the N-Terminal Cytoplasmic Tail of Human BST-2 Alter Its Intracellular Trafficking and HIV-1 Restriction

    PubMed Central

    Zhang, Jingyao; Zhang, Biao; Wang, Xiaodan; Zhu, Yingzi; Zuo, Tao; Liu, Donglai; Li, Xiaojun; Wu, Jiaxin; Zhang, Haihong; Yu, Bin; Wu, Hui; Zhao, Xinghong; Kong, Wei; Yu, Xianghui

    2014-01-01

    BST-2 blocks the particle release of various enveloped viruses including HIV-1, and this antiviral activity is dependent on the topological arrangement of its four structural domains. Several functions of the cytoplasmic tail (CT) of BST-2 have been previously discussed, but the exact role of this domain remains to be clearly defined. In this study, we investigated the impact of truncation and commonly-used tags addition into the CT region of human BST-2 on its intracellular trafficking and signaling as well as its anti-HIV-1 function. The CT-truncated BST-2 exhibited potent inhibition on Vpu-defective HIV-1 and even wild-type HIV-1. However, the N-terminal HA-tagged CT-truncated BST-2 retained little antiviral activity and dramatically differed from its original protein in the cell surface level and intracellular localization. Further, we showed that the replacement of the CT domain with a hydrophobic tag altered BST-2 function possibly by preventing its normal vesicular trafficking. Notably, we demonstrated that a positive charged motif “KRXK” in the conjunctive region between the cytotail and the transmembrane domain which is conserved in primate BST-2 is important for the protein trafficking and the antiviral function. These results suggest that although the CT of BST-2 is not essential for its antiviral activity, the composition of residues in this region may play important roles in its normal trafficking which subsequently affected its function. These observations provide additional implications for the structure-function model of BST-2. PMID:25347789

  10. Epitope tags beside the N-terminal cytoplasmic tail of human BST-2 alter its intracellular trafficking and HIV-1 restriction.

    PubMed

    Lv, Mingyu; Wang, Jiawen; Zhang, Jingyao; Zhang, Biao; Wang, Xiaodan; Zhu, Yingzi; Zuo, Tao; Liu, Donglai; Li, Xiaojun; Wu, Jiaxin; Zhang, Haihong; Yu, Bin; Wu, Hui; Zhao, Xinghong; Kong, Wei; Yu, Xianghui

    2014-01-01

    BST-2 blocks the particle release of various enveloped viruses including HIV-1, and this antiviral activity is dependent on the topological arrangement of its four structural domains. Several functions of the cytoplasmic tail (CT) of BST-2 have been previously discussed, but the exact role of this domain remains to be clearly defined. In this study, we investigated the impact of truncation and commonly-used tags addition into the CT region of human BST-2 on its intracellular trafficking and signaling as well as its anti-HIV-1 function. The CT-truncated BST-2 exhibited potent inhibition on Vpu-defective HIV-1 and even wild-type HIV-1. However, the N-terminal HA-tagged CT-truncated BST-2 retained little antiviral activity and dramatically differed from its original protein in the cell surface level and intracellular localization. Further, we showed that the replacement of the CT domain with a hydrophobic tag altered BST-2 function possibly by preventing its normal vesicular trafficking. Notably, we demonstrated that a positive charged motif "KRXK" in the conjunctive region between the cytotail and the transmembrane domain which is conserved in primate BST-2 is important for the protein trafficking and the antiviral function. These results suggest that although the CT of BST-2 is not essential for its antiviral activity, the composition of residues in this region may play important roles in its normal trafficking which subsequently affected its function. These observations provide additional implications for the structure-function model of BST-2.

  11. Modular TRAPP complexes regulate intracellular protein trafficking through multiple Ypt/Rab GTPases in Saccharomyces cerevisiae.

    PubMed

    Zou, Shenshen; Liu, Yutao; Zhang, Xiu Qi; Chen, Yong; Ye, Min; Zhu, Xiaoping; Yang, Shu; Lipatova, Zhanna; Liang, Yongheng; Segev, Nava

    2012-06-01

    Ypt/Rab are key regulators of intracellular trafficking in all eukaryotic cells. In yeast, Ypt1 is essential for endoplasmic reticulum (ER)-to-Golgi transport, whereas Ypt31/32 regulate Golgi-to-plasma membrane and endosome-to-Golgi transport. TRAPP is a multisubunit complex that acts as an activator of Ypt/Rab GTPases. Trs85 and Trs130 are two subunits specific for TRAPP III and TRAPP II, respectively. Whereas TRAPP III was shown to acts as a Ypt1 activator, it is still controversial whether TRAPP II acts as a Ypt1 or Ypt31/32 activator. Here, we use GFP-Snc1 as a tool to study transport in Ypt and TRAPP mutant cells. First, we show that expression of GFP-Snc1 in trs85Δ mutant cells results in temperature sensitivity. Second, we suggest that in ypt1ts and trs85Δ, but not in ypt31Δ/32ts and trs130ts mutant cells, GFP-Snc1 accumulates in the ER. Third, we show that overexpression of Ypt1, but not Ypt31/32, can suppress both the growth and GFP-Snc1 accumulation phenotypes of trs85Δ mutant cells. In contrast, overexpression of Ypt31, but not Ypt1, suppresses the growth and GFP-Snc1 transport phenotypes of trs130ts mutant cells. These results provide genetic support for functional grouping of Ypt1 with Trs85-containing TRAPP III and Ypt31/32 with Trs130-containing TRAPP II.

  12. Prostaglandin E2 promotes Na1.8 trafficking via its intracellular RRR motif through the protein kinase A pathway.

    PubMed

    Liu, Chao; Li, Qian; Su, Yuanyuan; Bao, Lan

    2010-03-01

    Voltage-gated sodium channels (Na(v)) are essential for the initiation and propagation of action potentials in neurons. Na(v)1.8 activity is regulated by prostaglandin E(2) (PGE(2)). There is, however, no direct evidence showing the regulated trafficking of Na(v)1.8, and the molecular and cellular mechanism of PGE(2)-induced sodium channel trafficking is not clear. Here, we report that PGE(2) regulates the trafficking of Na(v)1.8 through the protein kinase A (PKA) signaling pathway, and an RRR motif in the first intracellular loop of Na(v)1.8 mediates this effect. In rat dorsal root ganglion (DRG) neurons, prolonged PGE(2) treatment enhanced Na(v)1.8 currents by increasing the channel density on the cell surface. Activation of PKA by forskolin had the same effect on DRG neurons and human embryonic kidney 293T cells expressing Na(v)1.8. Inhibition of PKA completely blocked the PGE(2)-promoted effect on Na(v)1.8. Mutation of five PKA phosphorylation sites or the RRR motif in the first intracellular loop of Na(v)1.8 abolished the PKA-promoted Na(v)1.8 surface expression. Furthermore, a membrane-tethered peptide containing the intracellular RRR motif disrupted the PGE(2)-induced promotion of the Na(v)1.8 current in DRG neurons. Our data indicate that PGE(2) promotes the surface expression of Na(v)1.8 via an intracellular RRR motif, and provide a novel mechanism for functional modulation of Na(v)1.8 by hyperalgesic agents. PMID:20028484

  13. Role of LIMP-2 in the intracellular trafficking of β-glucosidase in different human cellular models.

    PubMed

    Malini, Erika; Zampieri, Stefania; Deganuto, Marta; Romanello, Milena; Sechi, Annalisa; Bembi, Bruno; Dardis, Andrea

    2015-09-01

    Acid β-glucosidase (GCase), the enzyme deficient in Gaucher disease (GD), is transported to lysosomes by the lysosomal integral membrane protein (LIMP)-2. In humans, LIMP-2 deficiency leads to action myoclonus-renal failure (AMRF) syndrome. GD and AMRF syndrome share some clinical features. However, they are different from clinical and biochemical points of view, suggesting that the role of LIMP-2 in the targeting of GCase would be different in different tissues. Besides, the role of LIMP-2 in the uptake and trafficking of the human recombinant (hr)GCase used in the treatment of GD is unknown. Thus, we compared GCase activity and intracellular localization in immortalized lymphocytes, fibroblasts, and a neuronal model derived from multipotent adult stem cells, from a patient with AMRF syndrome, patients with GD, and control subjects. In fibroblasts and neuronlike cells, GCase targeting to the lysosomes is completely dependent on LIMP-2, whereas in blood cells, GCase is partially targeted to lysosomes by a LIMP-2-independent mechanism. Although hrGCase cellular uptake is independent of LIMP-2, its trafficking to the lysosomes is mediated by this receptor. These data provide new insights into the mechanisms involved in the intracellular trafficking of GCase and in the pathogeneses of GD and AMRF syndrome.

  14. Probe Intracellular Trafficking of a Polymeric DNA Delivery Vehicle by Functionalization with an Aggregation-Induced Emissive Tetraphenylethene Derivative.

    PubMed

    Han, Xiongqi; Chen, Qixian; Lu, Hongguang; Ma, Jianbiao; Gao, Hui

    2015-12-30

    Characteristic aggregation-induced quenching of π-fluorophores imposed substantial hindrance to their utilization in nanomedicine for insight into microscopic intracellular trafficking of therapeutic payload. To address this obstacle, we attempted to introduce a novel aggregation-induced emission (AIE) fluorophore into the cationic polymer, which was further used for formulation of a gene delivery carrier. Note that the selective restriction of the intramolecular rotation of the AIE fluorophore through its covalent bond to the polymer conduced to immense AIE. Furthermore, DNA payload labeled with the appropriate fluorophore as the Förster resonance energy transfer (FRET) acceptor verified a facile strategy to trace intracellular DNA releasing activity relying on the distance limitation requested by FRET (AIE fluorophore as FRET donor). Moreover, the hydrophobic nature of the AIE fluorophore appeared to promote colloidal stability of the constructed formulation. Together with other chemistry functionalization strategies (including endosome escape), the ultimate formulation exerted dramatic gene transfection efficiency. Hence, this report manifested a first nanomedicine platform combining AIE and FRET for microscopic insight into DNA intracellular trafficking activity. PMID:26634294

  15. Quantitative 3-D colocalization analysis as a tool to study the intracellular trafficking and dissociation of pDNA-chitosan polyplexes

    NASA Astrophysics Data System (ADS)

    Reitan, Nina Kristine; Sporsheim, Bjørnar; Bjørkøy, Astrid; Strand, Sabina; Davies, Catharina de Lange

    2012-02-01

    Multichannel microscopy is frequently used to study intermolecular interactions and spatial relationships between biomolecules and organelles or vesicles in cells. Based on multichannel images, quantitative colocalization analysis can provide valuable information about cellular internalization, vesicular transport, and the intracellular kinetics and location of biomolecules. However, such analyses should be performed carefully, because quantitative colocalization parameters have different interpretations and can be highly affected by image quality. We use quantitative three-dimensional colocalization analysis of deconvolved and chromatic-registered confocal images to study the dissociation of double-labeled pDNA-chitosan polyplexes in HeLa cells and their colocalization with early endosomes. Two chitosans that form polyplexes with highly different transfection efficacies are compared. Pearson's correlation coefficient, Manders' colocalization coefficients, and the intensity correlation quotient are estimated to determine the intracellular localization of polyplexes, free pDNA, and free chitosans. Differences are observed in the amount of uptake, and in the intracellular pathways and rates of dissociation for the two chitosans. The results support previous findings that polyplexes formed by self-branched, glycosylated chitosan oligomers are more favorable for cellular uptake and intracellular trafficking to the nucleus compared with polyplexes formed by linear chitosans.

  16. Cellular Internalization Mechanism and Intracellular Trafficking of Filamentous M13 Phages Displaying a Cell-Penetrating Transbody and TAT Peptide

    PubMed Central

    Shin, Seung-Min; Pham, Chuong D.; Choi, Dong-Ki; Kwon, Myung-Hee; Kim, Yong-Sung

    2012-01-01

    Cellular internalization of bacteriophage by surface-displayed cell penetrating peptides has been reported, though the underlying mechanism remains elusive. Here we describe in detail the internalization mechanism and intracellular trafficking and stability of filamentous M13 phages, the cellular entry of which is mediated by surface-displayed cell-penetrating light chain variable domain 3D8 VL transbody (3D8 VL-M13) or TAT peptide (TAT-M13). Recombinant 3D8 VL-M13 and TAT-M13 phages were efficiently internalized into living mammalian cells via physiologically relevant, energy-dependent endocytosis and were recovered from the cells in their infective form with the yield of 3D8 VL-M13 being higher (0.005∼0.01%) than that of TAT-M13 (0.001∼0.005%). Biochemical and genetic studies revealed that 3D8 VL-M13 was internalized principally by caveolae-mediated endocytosis via interaction with heparan sulfate proteoglycans as cell surface receptors, whereas TAT-M13 was internalized by clathrin- and caveolae-mediated endocytosis utilizing chondroitin sulfate proteoglycans as cell surface receptors, suggesting that phage internalization occurs by physiological endocytotic mechanism through specific cell surface receptors rather than non-specific transcytotic pathways. Internalized 3D8 VL-M13 phages routed to the cytosol and remained stable for more than 18 h without further trafficking to other subcellular compartments, whereas TAT-M13 phages routed to several subcellular compartments before being degraded in lysosomes even after 2 h of internalization. Our results suggest that the internalizing mechanism and intracellular trafficking of filamentous M13 bacteriophages largely follow the attributes of the displayed cell-penetrating moiety. Efficient internalization and cytosolic localization of 3D8 VL transbody-displayed phages will provide a useful tool for intracellular delivery of polar macromolecules such as proteins, peptides, and siRNAs. PMID:23251631

  17. The Interaction between the Fiber Knob Domain and the Cellular Attachment Receptor Determines the Intracellular Trafficking Route of Adenoviruses

    PubMed Central

    Shayakhmetov, Dmitry M.; Li, Zong-Yi; Ternovoi, Vladimir; Gaggar, Anuj; Gharwan, Helen; Lieber, André

    2003-01-01

    Most of the presently used adenovirus (Ad) vectors are based on serotype 5. However, the application of these vectors is limited by the native tropism of Ad5. To address this problem, a series of fiber chimeric vectors were produced to take advantage of the different cellular receptors used by Ad of different subgroups. In this study we utilize an Ad5-based chimeric vector containing sequences encoding the Ad35 fiber knob domain instead of the Ad5 knob (Ad5/35L) to analyze factors responsible for selection of intracellular trafficking routes by Ads. By competition analysis with recombinant Ad5 and Ad35 knobs we showed that the Ad5/35L vector infected cells through a receptor different from the Ad5 receptor. Intracellular trafficking of Ad5 and Ad5/35L viruses was analyzed in HeLa cells by tracking fluorophore-conjugated Ad particles, by immunostaining for capsid hexon protein, by electron microscopy, and by Southern blotting for viral DNA. These studies showed that the interaction with the Ad35 receptor(s) predestines Ad5/35L vector to intracellular trafficking pathways different from those of Ad5. Ad5 efficiently escaped from the endosomes early after infection. In contrast, Ad5/35L remained longer in late endosomal/lysosomal compartments and used them to achieve localization to the nucleus. However, a significant portion of Ad5/35L particles appeared to be recycled back to the cell surface. This phenomenon resulted in significantly less efficient Ad5/35L-mediated gene transfer compared to that of Ad5. We also demonstrated that the selection of intracellular trafficking routes was determined by the fiber knob domain and did not depend on the length of the fiber shaft. This study contributes to a better understanding of the mechanisms that govern the infection of retargeted, capsid-modified vectors which have potential application for hematopoietic stem cell and tumor gene therapy. PMID:12610146

  18. Mutations in the D1 domain of von Willebrand factor impair their propeptide-dependent multimerization, intracellular trafficking and secretion.

    PubMed

    Yin, Jie; Ma, Zhenni; Su, Jian; Wang, Jiong-Wei; Zhao, Xiaojuan; Ling, Jing; Bai, Xia; Ouyang, Wanyan; Wang, Zhaoyue; Yu, Ziqiang; Ruan, Changgeng

    2015-01-01

    We identified three novel mutations (p.Gly39Arg, p.Lys157Glu, p.Cys379Gly) and one previously known mutation (p.Asp141Asn) in the von Willebrand factor propeptide from three von Willebrand disease patients. All four mutations impaired multimerization of von Willebrand factor, due to reduced oxidoreductase activity of isomeric propeptide. These mutations resulted in the endothelial reticulum retention and impaired basal and stimulated secretions of von Willebrand factor. Our results support that the mutations in the D1 domain lead to defective multimerization, intracellular trafficking, and secretion of von Willebrand factor and result in bleeding of patients. PMID:26088471

  19. Phosphatidylinositol 3-kinase and 4-kinase have distinct roles in intracellular trafficking of cellulose synthase complexes in Arabidopsis thaliana.

    PubMed

    Fujimoto, Masaru; Suda, Yasuyuki; Vernhettes, Samantha; Nakano, Akihiko; Ueda, Takashi

    2015-02-01

    The oriented deposition of cellulose microfibrils in the plant cell wall plays a crucial role in various plant functions such as cell growth, organ formation and defense responses. Cellulose is synthesized by cellulose synthase complexes (CSCs) embedded in the plasma membrane (PM), which comprise the cellulose synthases (CESAs). The abundance and localization of CSCs at the PM should be strictly controlled for precise regulation of cellulose deposition, which strongly depends on the membrane trafficking system. However, the mechanism of the intracellular transport of CSCs is still poorly understood. In this study, we explored requirements for phosphoinositides (PIs) in CESA trafficking by analyzing the effects of inhibitors of PI synthesis in Arabidopsis thaliana expressing green fluorescent protein-tagged CESA3 (GFP-CESA3). We found that a shift to a sucrose-free condition accelerated re-localization of PM-localized GFP-CESA3 into the periphery of the Golgi apparatus via the clathrin-enriched trans-Golgi network (TGN). Treatment with wortmannin (Wm), an inhibitor of phosphatidylinositol 3- (PI3K) and 4- (PI4K) kinases, and phenylarsine oxide (PAO), a more specific inhibitor for PI4K, inhibited internalization of GFP-CESA3 from the PM. In contrast, treatment with LY294002, which impairs the PI3K activity, did not exert such an inhibitory effect on the sequestration of GFP-CESA3, but caused a predominant accumulation of GFP-CESA3 at the ring-shaped periphery of the Golgi apparatus, resulting in the removal of GFP-CESA3 from the PM. These results indicate that PIs are essential elements for localization and intracellular transport of CESA3 and that PI4K and PI3K are required for distinct steps in secretory and/or endocytic trafficking of CESA3. PMID:25516570

  20. Role of cellular FKBP52 protein in intracellular trafficking of recombinant adeno-associated virus 2 vectors

    SciTech Connect

    Zhao Weihong; Wu Jianqing ||; Zhong Li; Chen Linyuan; Weigel-Kelley, Kirsten A. |; Qing Keyun; Larsen, Steven H.; Shou Weinian; Warrington, Kenneth H. |; Srivastava, Arun |. E-mail: asrivastava@gtc.ufl.edu

    2006-09-30

    We have reported that tyrosine-phosphorylated forms of a cellular protein, FKBP52, inhibit the second-strand DNA synthesis of adeno-associated virus 2 (AAV), leading to inefficient transgene expression from recombinant AAV vectors. To further explore the role of FKBP52 in AAV-mediated transduction, we established murine embryo fibroblasts (MEFs) cultures from FKBP52 wild-type (WT), heterozygous (HE), and knockout (KO) mice. Conventional AAV vectors failed to transduce WT MEFs efficiently, and the transduction efficiency was not significantly increased in HE or KO MEFs. AAV vectors failed to traffic efficiently to the nucleus in these cells. Treatment with hydroxyurea (HU) increased the transduction efficiency of conventional AAV vectors by {approx}25-fold in WT MEFs, but only by {approx}4-fold in KO MEFs. The use of self-complementary AAV (scAAV) vectors, which bypass the requirement of viral second-strand DNA synthesis, revealed that HU treatment increased the transduction efficiency {approx}23-fold in WT MEFs, but only {approx}4-fold in KO MEFs, indicating that the lack of HU treatment-mediated increase in KO MEFs was not due to failure of AAV to undergo viral second-strand DNA synthesis. Following HU treatment, {approx}59% of AAV genomes were present in the nuclear fraction from WT MEFs, but only {approx}28% in KO MEFs, indicating that the pathway by which HU treatment mediates nuclear transport of AAV was impaired in KO MEFs. When KO MEFs were stably transfected with an FKBP52 expression plasmid, HU treatment-mediated increase in the transduction efficiency was restored in these cells, which correlated directly with improved intracellular trafficking. Intact AAV particles were also shown to interact with FKBP52 as well as with dynein, a known cellular protein involved in AAV trafficking. These studies suggest that FKBP52, being a cellular chaperone protein, facilitates intracellular trafficking of AAV, which has implications in the optimal use of recombinant

  1. Differential effects of Hsc70 and Hsp70 on the intracellular trafficking and functional expression of epithelial sodium channels.

    PubMed

    Goldfarb, Samuel B; Kashlan, Ossama B; Watkins, Jeffrey N; Suaud, Laurence; Yan, Wusheng; Kleyman, Thomas R; Rubenstein, Ronald C

    2006-04-11

    The members of the cytoplasmic 70-kDa heat shock protein family are involved in appropriate folding and trafficking of newly synthesized proteins in the cell. Hsc70, which is expressed constitutively, and Hsp70, the expression of which is stress- and heat shock-induced, are often considered to have similar cellular functions in this regard, but there are suggestions that the intracellular functions of these homologous but not identical proteins may differ. We tested the hypothesis that Hsc70 and Hsp70 would have differential effects on the expression of the epithelial sodium channel (ENaC). In Xenopus oocytes, overexpression of human Hsc70 decreased the functional (defined as amiloride-sensitive whole-oocyte current) and surface expression of murine ENaC (mENaC) in a concentration-dependent fashion. In contrast, coinjection of a moderate amount of Hsp70 cRNA (10 ng) increased the functional and surface expression of mENaC, whereas a higher amount of coinjected Hsp70 cRNA (30 ng) decreased mENaC functional and surface expression. The increase in mENaC functional expression with coinjection of 10 ng of Hsp70 cRNA was antagonized by the additional coinjection of Hsc70 cRNA in a concentration-dependent fashion. These data are consistent with Hsc70 and Hsp70 having differential and antagonistic effects with regard to the intracellular trafficking of mENaC in oocytes, which may have an impact on our understanding and potential treatment of diseases of aberrant ion channel trafficking. PMID:16585520

  2. Shedding PEG Palisade by Temporal Photostimulation and Intracellular Reducing Milieu for Facilitated Intracellular Trafficking and DNA Release.

    PubMed

    Wang, Tieyan; Chen, Qixian; Lu, Hongguang; Li, Wei; Li, Zaifen; Ma, Jianbiao; Gao, Hui

    2016-08-17

    The dilemma of poly(ethylene glycol) surface modification (PEGylation) inspired us to develop an intracellularly sheddable PEG palisade for synthetic delivery systems. Here, we attempted to conjugate PEG to polyethylenimine (PEI) through tandem linkages of disulfide-bridge susceptible to cytoplasmic reduction and an azobenzene/cyclodextrin inclusion complex responsive to external photoirradiation. The subsequent investigations revealed that facile PEG detachment could be achieved in endosomes upon photoirradiation, consequently engendering exposure of membrane-disruptive PEI for facilitated endosome escape. The liberated formulation in the cytosol was further subjected to complete PEG detachment relying on disulfide cleavage in the reductive cytosol, thus accelerating dissociation of electrostatically assembled PEI/DNA polyplex to release DNA by means of polyion exchange reaction with intracellularly charged species, ultimately contributing to efficient gene expression.

  3. Shedding PEG Palisade by Temporal Photostimulation and Intracellular Reducing Milieu for Facilitated Intracellular Trafficking and DNA Release.

    PubMed

    Wang, Tieyan; Chen, Qixian; Lu, Hongguang; Li, Wei; Li, Zaifen; Ma, Jianbiao; Gao, Hui

    2016-08-17

    The dilemma of poly(ethylene glycol) surface modification (PEGylation) inspired us to develop an intracellularly sheddable PEG palisade for synthetic delivery systems. Here, we attempted to conjugate PEG to polyethylenimine (PEI) through tandem linkages of disulfide-bridge susceptible to cytoplasmic reduction and an azobenzene/cyclodextrin inclusion complex responsive to external photoirradiation. The subsequent investigations revealed that facile PEG detachment could be achieved in endosomes upon photoirradiation, consequently engendering exposure of membrane-disruptive PEI for facilitated endosome escape. The liberated formulation in the cytosol was further subjected to complete PEG detachment relying on disulfide cleavage in the reductive cytosol, thus accelerating dissociation of electrostatically assembled PEI/DNA polyplex to release DNA by means of polyion exchange reaction with intracellularly charged species, ultimately contributing to efficient gene expression. PMID:27453033

  4. Sterol Carrier Protein-2, a Nonspecific Lipid-Transfer Protein, in Intracellular Cholesterol Trafficking in Testicular Leydig Cells.

    PubMed

    Li, Nancy C; Fan, Jinjiang; Papadopoulos, Vassilios

    2016-01-01

    Sterol carrier protein-2 (SCP2), also called nonspecific lipid-transfer protein, is thought to play a major role in intracellular lipid transport and metabolism, and it has been associated with diseases involving abnormalities in lipid trafficking, such as Zellweger syndrome. The Scp2 gene encodes the 58 kDa sterol carrier protein-x (SCPX) and 15 kDa pro-SCP2 proteins, both of which contain a 13 kDa SCP2 domain in their C-termini. We found that 22-NBD-cholesterol, a fluorescent analog of cholesterol and a preferred SCP2 ligands, was not localized in the peroxisomes. This raises questions about previous reports on the localization of the SCPX and SCP2 proteins and their relationship to peroxisomes and mitochondria in intracellular cholesterol transport. Immunofluorescent staining of cryosections of mouse testis and of MA-10 mouse tumor Leydig cells showed that SCPX and SCP2 are present in both mouse testicular interstitial tissue and in MA-10 cells. Fluorescent fusion proteins of SCPX and SCP2, as well as confocal live-cell imaging, were used to investigate the subcellular targeting of these proteins and the function of the putative mitochondrial targeting sequence. The results showed that SCPX and SCP2 are targeted to the peroxisomes by the C-terminal PTS1 domain, but the putative N-terminal mitochondrial targeting sequence alone is not potent enough to localize SCPX and SCP2 to the mitochondria. Homology modeling and molecular docking studies indicated that the SCP2 domain binds cholesterol, but lacks specificity of the binding and/or transport. These findings further our understanding of the role of SCPX and SCP2 in intracellular cholesterol transport, and present a new point of view on the role of these proteins in cholesterol trafficking.

  5. Sterol Carrier Protein-2, a Nonspecific Lipid-Transfer Protein, in Intracellular Cholesterol Trafficking in Testicular Leydig Cells

    PubMed Central

    Li, Nancy C.; Fan, Jinjiang; Papadopoulos, Vassilios

    2016-01-01

    Sterol carrier protein-2 (SCP2), also called nonspecific lipid-transfer protein, is thought to play a major role in intracellular lipid transport and metabolism, and it has been associated with diseases involving abnormalities in lipid trafficking, such as Zellweger syndrome. The Scp2 gene encodes the 58 kDa sterol carrier protein-x (SCPX) and 15 kDa pro-SCP2 proteins, both of which contain a 13 kDa SCP2 domain in their C-termini. We found that 22-NBD-cholesterol, a fluorescent analog of cholesterol and a preferred SCP2 ligands, was not localized in the peroxisomes. This raises questions about previous reports on the localization of the SCPX and SCP2 proteins and their relationship to peroxisomes and mitochondria in intracellular cholesterol transport. Immunofluorescent staining of cryosections of mouse testis and of MA-10 mouse tumor Leydig cells showed that SCPX and SCP2 are present in both mouse testicular interstitial tissue and in MA-10 cells. Fluorescent fusion proteins of SCPX and SCP2, as well as confocal live-cell imaging, were used to investigate the subcellular targeting of these proteins and the function of the putative mitochondrial targeting sequence. The results showed that SCPX and SCP2 are targeted to the peroxisomes by the C-terminal PTS1 domain, but the putative N-terminal mitochondrial targeting sequence alone is not potent enough to localize SCPX and SCP2 to the mitochondria. Homology modeling and molecular docking studies indicated that the SCP2 domain binds cholesterol, but lacks specificity of the binding and/or transport. These findings further our understanding of the role of SCPX and SCP2 in intracellular cholesterol transport, and present a new point of view on the role of these proteins in cholesterol trafficking. PMID:26901662

  6. Amino-terminal cysteine residues differentially influence RGS4 protein plasma membrane targeting, intracellular trafficking, and function.

    PubMed

    Bastin, Guillaume; Singh, Kevin; Dissanayake, Kaveesh; Mighiu, Alexandra S; Nurmohamed, Aliya; Heximer, Scott P

    2012-08-17

    Regulator of G-protein signaling (RGS) proteins are potent inhibitors of heterotrimeric G-protein signaling. RGS4 attenuates G-protein activity in several tissues. Previous work demonstrated that cysteine palmitoylation on residues in the amino-terminal (Cys-2 and Cys-12) and core domains (Cys-95) of RGS4 is important for protein stability, plasma membrane targeting, and GTPase activating function. To date Cys-2 has been the priority target for RGS4 regulation by palmitoylation based on its putative role in stabilizing the RGS4 protein. Here, we investigate differences in the contribution of Cys-2 and Cys-12 to the intracellular localization and function of RGS4. Inhibition of RGS4 palmitoylation with 2-bromopalmitate dramatically reduced its localization to the plasma membrane. Similarly, mutation of the RGS4 amphipathic helix (L23D) prevented membrane localization and its G(q) inhibitory function. Together, these data suggest that both RGS4 palmitoylation and the amphipathic helix domain are required for optimal plasma membrane targeting and function of RGS4. Mutation of Cys-12 decreased RGS4 membrane targeting to a similar extent as 2-bromopalmitate, resulting in complete loss of its G(q) inhibitory function. Mutation of Cys-2 did not impair plasma membrane targeting but did partially impair its function as a G(q) inhibitor. Comparison of the endosomal distribution pattern of wild type and mutant RGS4 proteins with TGN38 indicated that palmitoylation of these two cysteines contributes differentially to the intracellular trafficking of RGS4. These data show for the first time that Cys-2 and Cys-12 play markedly different roles in the regulation of RGS4 membrane localization, intracellular trafficking, and G(q) inhibitory function via mechanisms that are unrelated to RGS4 protein stabilization.

  7. Rab8 modulates metabotropic glutamate receptor subtype 1 intracellular trafficking and signaling in a protein kinase C-dependent manner.

    PubMed

    Esseltine, Jessica L; Ribeiro, Fabiola M; Ferguson, Stephen S G

    2012-11-21

    Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors (GPCRs) that are activated by glutamate, the primary excitatory neurotransmitter in the CNS. Alterations in glutamate receptor signaling are implicated in neuropathologies such as Alzheimer's disease, ischemia, and Huntington's disease among others. Group 1 mGluRs (mGluR1 and mGluR5) are primarily coupled to Gα(q/11) leading to the activation of phospholipase C and the formation of diacylglycerol and inositol 1,4,5-trisphosphate, which results in the release of intracellular calcium stores and protein kinase C (PKC) activation. Desensitization, endocytosis, and recycling are major mechanisms of GPCR regulation, and the intracellular trafficking of GPCRs is linked to the Rab family of small G proteins. Rab8 is a small GTPase that is specifically involved in the regulation of secretory/recycling vesicles, modulation of the actin cytoskeleton, and cell polarity. Rab8 has been shown to regulate the synaptic delivery of AMPA receptors during long-term potentiation and during constitutive receptor recycling. We show here that Rab8 interacts with the C-terminal tail of mGluR1a in an agonist-dependent manner and plays a role in regulating of mGluR1a signaling and intracellular trafficking in human embryonic kidney 293 cells. Specifically, Rab8 expression attenuates mGluR1a-mediated inositol phosphate formation and calcium release from mouse neurons in a PKC-dependent manner, while increasing cell surface mGluR1a expression via decreased receptor endocytosis. These experiments provide us with an understanding of the role Rabs play in coordinated regulation of mGluR1a and how this impacts mGluR1a signaling.

  8. Sterol Carrier Protein-2, a Nonspecific Lipid-Transfer Protein, in Intracellular Cholesterol Trafficking in Testicular Leydig Cells.

    PubMed

    Li, Nancy C; Fan, Jinjiang; Papadopoulos, Vassilios

    2016-01-01

    Sterol carrier protein-2 (SCP2), also called nonspecific lipid-transfer protein, is thought to play a major role in intracellular lipid transport and metabolism, and it has been associated with diseases involving abnormalities in lipid trafficking, such as Zellweger syndrome. The Scp2 gene encodes the 58 kDa sterol carrier protein-x (SCPX) and 15 kDa pro-SCP2 proteins, both of which contain a 13 kDa SCP2 domain in their C-termini. We found that 22-NBD-cholesterol, a fluorescent analog of cholesterol and a preferred SCP2 ligands, was not localized in the peroxisomes. This raises questions about previous reports on the localization of the SCPX and SCP2 proteins and their relationship to peroxisomes and mitochondria in intracellular cholesterol transport. Immunofluorescent staining of cryosections of mouse testis and of MA-10 mouse tumor Leydig cells showed that SCPX and SCP2 are present in both mouse testicular interstitial tissue and in MA-10 cells. Fluorescent fusion proteins of SCPX and SCP2, as well as confocal live-cell imaging, were used to investigate the subcellular targeting of these proteins and the function of the putative mitochondrial targeting sequence. The results showed that SCPX and SCP2 are targeted to the peroxisomes by the C-terminal PTS1 domain, but the putative N-terminal mitochondrial targeting sequence alone is not potent enough to localize SCPX and SCP2 to the mitochondria. Homology modeling and molecular docking studies indicated that the SCP2 domain binds cholesterol, but lacks specificity of the binding and/or transport. These findings further our understanding of the role of SCPX and SCP2 in intracellular cholesterol transport, and present a new point of view on the role of these proteins in cholesterol trafficking. PMID:26901662

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

    PubMed Central

    Callahan, Jon; Kopečková, Pavla; Kopeček, Jindřich

    2011-01-01

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

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

    PubMed

    Callahan, Jon; Kopečkov, Pavla; Kopeček, Jindřich

    2009-07-13

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

  11. Study of Mesoporous Silica Nanoparticles' (MSNs) intracellular trafficking and their application as drug delivery vehicles

    NASA Astrophysics Data System (ADS)

    Yanes, Rolando Eduardo

    Mesoporous silica nanoparticles (MSNs) are attractive drug delivery vehicle candidates due to their biocompatibility, stability, high surface area and efficient cellular uptake. In this dissertation, I discuss three aspects of MSNs' cellular behavior. First, MSNs are targeted to primary and metastatic cancer cell lines, then their exocytosis from cancer cells is studied, and finally they are used to recover intracellular proteins. Targeting of MSNs to primary cancer cells is achieved by conjugating transferrin on the surface of the mesoporous framework, which resulted in enhancement of nanoparticle uptake and drug delivery efficacy in cells that overexpress the transferrin receptor. Similarly, RGD peptides are used to target metastatic cancer cell lines that over-express integrin alphanubeta3. A circular RGD peptide is bound to the surface of MSNs and the endocytosis and cell killing efficacy of camptothecin loaded nanoparticles is significantly improved in cells that express the target receptor. Besides targeting, I studied the ultimate fate of phosphonate coated mesoporous silica nanoparticles inside cells. I discovered that the nanoparticles are exocytosed from cells through lysosomal exocytosis. The nanoparticles are exocytosed in intact form and the time that they remain inside the cells is affected by the surface properties of the nanoparticles and the type of cells. Cells that have a high rate of lysosomal exocytosis excrete the nanoparticles rapidly, which makes them more resistant to drug loaded nanoparticles because the amount of drug that is released inside the cell is limited. When the exocytosis of MSNs is inhibited, the cell killing efficacy of nanoparticles loaded with camptothecin is enhanced. The discovery that MSNs are exocytosed by cells led to a study to determine if proteins could be recovered from the exocytosed nanoparticles. The procedure to isolate exocytosed zinc-doped iron core MSNs and identify the proteins bound to them was developed

  12. Viral bimolecular fluorescence complementation: a novel tool to study intracellular vesicular trafficking pathways.

    PubMed

    Dirk, Brennan S; Jacob, Rajesh Abraham; Johnson, Aaron L; Pawlak, Emily N; Cavanagh, P Craig; Van Nynatten, Logan; Haeryfar, S M Mansour; Dikeakos, Jimmy D

    2015-01-01

    The Human Immunodeficiency Virus type 1 (HIV-1) accessory protein Nef interacts with a multitude of cellular proteins, manipulating the host membrane trafficking machinery to evade immune surveillance. Nef interactions have been analyzed using various in vitro assays, co-immunoprecipitation studies, and more recently mass spectrometry. However, these methods do not evaluate Nef interactions in the context of viral infection nor do they define the sub-cellular location of these interactions. In this report, we describe a novel bimolecular fluorescence complementation (BiFC) lentiviral expression tool, termed viral BiFC, to study Nef interactions with host cellular proteins in the context of viral infection. Using the F2A cleavage site from the foot and mouth disease virus we generated a viral BiFC expression vector capable of concurrent expression of Nef and host cellular proteins; PACS-1, MHC-I and SNX18. Our studies confirmed the interaction between Nef and PACS-1, a host membrane trafficking protein involved in Nef-mediated immune evasion, and demonstrated co-localization of this complex with LAMP-1 positive endolysosomal vesicles. Furthermore, we utilized viral BiFC to localize the Nef/MHC-I interaction to an AP-1 positive endosomal compartment. Finally, viral BiFC was observed between Nef and the membrane trafficking regulator SNX18. This novel demonstration of an association between Nef and SNX18 was localized to AP-1 positive vesicles. In summary, viral BiFC is a unique tool designed to analyze the interaction between Nef and host cellular proteins by mapping the sub-cellular locations of their interactions during viral infection. PMID:25915798

  13. Viral Bimolecular Fluorescence Complementation: A Novel Tool to Study Intracellular Vesicular Trafficking Pathways

    PubMed Central

    Johnson, Aaron L.; Pawlak, Emily N.; Cavanagh, P. Craig; Van Nynatten, Logan; Haeryfar, S. M. Mansour; Dikeakos, Jimmy D.

    2015-01-01

    The Human Immunodeficiency Virus type 1 (HIV-1) accessory protein Nef interacts with a multitude of cellular proteins, manipulating the host membrane trafficking machinery to evade immune surveillance. Nef interactions have been analyzed using various in vitro assays, co-immunoprecipitation studies, and more recently mass spectrometry. However, these methods do not evaluate Nef interactions in the context of viral infection nor do they define the sub-cellular location of these interactions. In this report, we describe a novel bimolecular fluorescence complementation (BiFC) lentiviral expression tool, termed viral BiFC, to study Nef interactions with host cellular proteins in the context of viral infection. Using the F2A cleavage site from the foot and mouth disease virus we generated a viral BiFC expression vector capable of concurrent expression of Nef and host cellular proteins; PACS-1, MHC-I and SNX18. Our studies confirmed the interaction between Nef and PACS-1, a host membrane trafficking protein involved in Nef-mediated immune evasion, and demonstrated co-localization of this complex with LAMP-1 positive endolysosomal vesicles. Furthermore, we utilized viral BiFC to localize the Nef/MHC-I interaction to an AP-1 positive endosomal compartment. Finally, viral BiFC was observed between Nef and the membrane trafficking regulator SNX18. This novel demonstration of an association between Nef and SNX18 was localized to AP-1 positive vesicles. In summary, viral BiFC is a unique tool designed to analyze the interaction between Nef and host cellular proteins by mapping the sub-cellular locations of their interactions during viral infection. PMID:25915798

  14. Protein-protein interactions involving voltage-gated sodium channels: Post-translational regulation, intracellular trafficking and functional expression.

    PubMed

    Shao, Dongmin; Okuse, Kenji; Djamgoz, Mustafa B A

    2009-07-01

    Voltage-gated sodium channels (VGSCs), classically known to play a central role in excitability and signalling in nerves and muscles, have also been found to be expressed in a range of 'non-excitable' cells, including lymphocytes, fibroblasts and endothelia. VGSC abnormalities are associated with various diseases including epilepsy, long-QT syndrome 3, Brugada syndrome, sudden infant death syndrome and, more recently, various human cancers. Given their pivotal role in a wide range of physiological and pathophysiological processes, regulation of functional VGSC expression has been the subject of intense study. An emerging theme is post-translational regulation and macro-molecular complexing by protein-protein interactions and intracellular trafficking, leading to changes in functional VGSC expression in plasma membrane. This partially involves endoplasmic reticulum associated degradation and ubiquitin-proteasome system. Several proteins have been shown to associate with VGSCs. Here, we review the interactions involving VGSCs and the following proteins: p11, ankyrin, syntrophin, beta-subunit of VGSC, papin, ERM and Nedd4 proteins. Protein kinases A and C, as well as Ca(2+)-calmodulin dependent kinase II that have also been shown to regulate intracellular trafficking of VGSCs by changing the balance of externalization vs. internalization, and an effort is made to separate these effects from the short-term phosphorylation of mature proteins in plasma membrane. Two further modulatory mechanisms are reciprocal interactions with the cytoskeleton and, late-stage, activity-dependent regulation. Thus, the review gives an updated account of the range of post-translational molecular mechanisms regulating functional VGSC expression. However, many details of VGSC subtype-specific regulation and pathophysiological aspects remain unknown and these are highlighted throughout for completeness. PMID:19401147

  15. Role of the HIV-1 Matrix Protein in Gag Intracellular Trafficking and Targeting to the Plasma Membrane for Virus Assembly

    PubMed Central

    Ghanam, Ruba H.; Samal, Alexandra B.; Fernandez, Timothy F.; Saad, Jamil S.

    2012-01-01

    Human immunodeficiency virus type-1 (HIV-1) encodes a polypeptide called Gag that is able to form virus-like particles in vitro in the absence of any cellular or viral constituents. During the late phase of the HIV-1 infection, Gag polyproteins are transported to the plasma membrane (PM) for assembly. In the past two decades, in vivo, in vitro, and structural studies have shown that Gag trafficking and targeting to the PM are orchestrated events that are dependent on multiple factors including cellular proteins and specific membrane lipids. The matrix (MA) domain of Gag has been the focus of these studies as it appears to be engaged in multiple intracellular interactions that are suggested to be critical for virus assembly and replication. The interaction between Gag and the PM is perhaps the most understood. It is now established that the ultimate localization of Gag on punctate sites on the PM is mediated by specific interactions between the MA domain of Gag and phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], a minor lipid localized on the inner leaflet of the PM. Structure-based studies revealed that binding of PI(4,5)P2 to MA induces minor conformational changes, leading to exposure of the myristyl (myr) group. Exposure of the myr group is also triggered by binding of calmodulin, enhanced by factors that promote protein self-association like the capsid domain of Gag, and is modulated by pH. Despite the steady progress in defining both the viral and cellular determinants of retroviral assembly and release, Gag’s intracellular interactions and trafficking to its assembly sites in the infected cell are poorly understood. In this review, we summarize the current understanding of the structural and functional role of MA in HIV replication. PMID:22363329

  16. Endocytosis of ABCG2 drug transporter caused by binding of 5D3 antibody: trafficking mechanisms and intracellular fate.

    PubMed

    Studzian, Maciej; Bartosz, Grzegorz; Pulaski, Lukasz

    2015-08-01

    ABCG2, a metabolite and xenobiotic transporter located at the plasma membrane (predominantly in barrier tissues and progenitor cells), undergoes a direct progressive endocytosis process from plasma membrane to intracellular compartments upon binding of 5D3 monoclonal antibody. This antibody is specific to an external epitope on the protein molecule and locks it in a discrete conformation within its activity cycle, presumably providing a structural trigger for the observed internalization phenomenon. Using routine and novel assays, we show that ABCG2 is endocytosed by a mixed mechanism: partially via a rapid, clathrin-dependent pathway and partially in a cholesterol-dependent, caveolin-independent manner. While the internalization process is entirely dynamin-dependent and converges initially at the early endosome, subsequent intracellular fate of ABCG2 is again twofold: endocytosis leads to only partial lysosomal degradation, while a significant fraction of the protein is retained in a post-endosomal compartment with the possibility of at least partial recycling back to the cell surface. This externally triggered, conformation-related trafficking pathway may serve as a general regulatory paradigm for membrane transporters, and its discovery was made possible thanks to consistent application of quantitative methods.

  17. Endocytosis of ABCG2 drug transporter caused by binding of 5D3 antibody: trafficking mechanisms and intracellular fate.

    PubMed

    Studzian, Maciej; Bartosz, Grzegorz; Pulaski, Lukasz

    2015-08-01

    ABCG2, a metabolite and xenobiotic transporter located at the plasma membrane (predominantly in barrier tissues and progenitor cells), undergoes a direct progressive endocytosis process from plasma membrane to intracellular compartments upon binding of 5D3 monoclonal antibody. This antibody is specific to an external epitope on the protein molecule and locks it in a discrete conformation within its activity cycle, presumably providing a structural trigger for the observed internalization phenomenon. Using routine and novel assays, we show that ABCG2 is endocytosed by a mixed mechanism: partially via a rapid, clathrin-dependent pathway and partially in a cholesterol-dependent, caveolin-independent manner. While the internalization process is entirely dynamin-dependent and converges initially at the early endosome, subsequent intracellular fate of ABCG2 is again twofold: endocytosis leads to only partial lysosomal degradation, while a significant fraction of the protein is retained in a post-endosomal compartment with the possibility of at least partial recycling back to the cell surface. This externally triggered, conformation-related trafficking pathway may serve as a general regulatory paradigm for membrane transporters, and its discovery was made possible thanks to consistent application of quantitative methods. PMID:25918011

  18. Effect of DNA/liposome mixing ratio on the physicochemical characteristics, cellular uptake and intracellular trafficking of plasmid DNA/cationic liposome complexes and subsequent gene expression.

    PubMed

    Sakurai, F; Inoue, R; Nishino, Y; Okuda, A; Matsumoto, O; Taga, T; Yamashita, F; Takakura, Y; Hashida, M

    2000-05-15

    In order to identify the important factors involved in cationic liposome-mediated gene transfer, in vitro transfection efficiencies by plasmid DNA complexed with DOTMA/DOPE liposomes at different DNA/liposome mixing ratios were evaluated using four types of cultured cells with respect to their physicochemical properties. Significant changes were observed in the particle size and zeta potential of the complexes as well as in their structures, assessed by atomic force microscopy, which depended on the mixing ratio. In transfection experiments, except for RAW 264.7 cells (mouse macrophages), efficient gene expression was obtained in MBT-2 cells (mouse bladder tumor), NLH3T3 cells (mouse fibroblasts) and HUVEC (human umbilical vein endothelial cells) at an optimal ratio of 1:5, 1:7.5 or 1:5, respectively. On the other hand, cellular uptake of the [32P]DNA/liposome complexes increased in all cell types with an increase in the mixing ratio, which was not reflected by the transfection efficiency. The cellular damage determined by MTT assay was minimal even at the highest DNA/liposome ratio (1:10), indicating that the lower gene expression level at the higher ratio was not due to cytotoxicity induced by the complex. An ethidium bromide intercalation assay showed that the release of plasmid DNA from the complex, following the addition of negatively charged liposomes, was restricted as the mixing ratio increased. Furthermore, confocal microscopic studies using HUVEC showed that the 1:5 complexes exhibited a dispersed distribution in the cytoplasm whereas a punctuate intracellular distribution was observed for the 1:10 complexes. This suggests that there was a significant difference in intracellular trafficking, probably release from the endosomes or lysosomes, of the plasmid DNA/cationic liposome complexes between these mixing ratios. Taken together, these findings suggest that the DNA/liposome mixing ratio significantly affects the intracellular trafficking of plasmid DNA

  19. Nuclear trafficking of the HIV-1 pre-integration complex depends on the ADAM10 intracellular domain

    SciTech Connect

    Endsley, Mark A.; Somasunderam, Anoma D.; Li, Guangyu; Oezguen, Numan; Thiviyanathan, Varatharasa; Murray, James L.; Rubin, Donald H.; Hodge, Thomas W.; and others

    2014-04-15

    Previously, we showed that ADAM10 is necessary for HIV-1 replication in primary human macrophages and immortalized cell lines. Silencing ADAM10 expression interrupted the HIV-1 life cycle prior to nuclear translocation of viral cDNA. Furthermore, our data indicated that HIV-1 replication depends on the expression of ADAM15 and γ-secretase, which proteolytically processes ADAM10. Silencing ADAM15 or γ-secretase expression inhibits HIV-1 replication between reverse transcription and nuclear entry. Here, we show that ADAM10 expression also supports replication in CD4{sup +} T lymphocytes. The intracellular domain (ICD) of ADAM10 associates with the HIV-1 pre-integration complex (PIC) in the cytoplasm and immunoprecipitates and co-localizes with HIV-1 integrase, a key component of PIC. Taken together, our data support a model whereby ADAM15/γ-secretase processing of ADAM10 releases the ICD, which then incorporates into HIV-1 PIC to facilitate nuclear trafficking. Thus, these studies suggest ADAM10 as a novel therapeutic target for inhibiting HIV-1 prior to nuclear entry. - Highlights: • Nuclear trafficking of the HIV-1 pre-integration complex depends on ADAM10. • ADAM10 associates with HIV-1 integrase in the pre-integration complex. • HIV-1 replication depends on the expression of ADAM15 and γ-secretase. • Silencing ADAM15 or γ-secretase expression inhibits nuclear import of viral cDNA. • ADAM10 is important for HIV-1 replication in human macrophages and CD4{sup +} T lymphocytes.

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

    NASA Astrophysics Data System (ADS)

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

    2008-09-01

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

  1. Common Pharmacophore of Structurally Distinct Small-Molecule Inhibitors of Intracellular Retrograde Trafficking of Ribosome Inactivating Proteins

    NASA Astrophysics Data System (ADS)

    Yu, Shichao; Park, Jewn Giew; Kahn, Jennifer Nielsen; Tumer, Nilgun E.; Pang, Yuan-Ping

    2013-12-01

    We reported previously (+/-)-2-(5-methylthiophen-2-yl)-3-phenyl-2,3-dihydroquinazolin-4(1H)-one [(+/-)-Retro-2cycl] as the chemical structure of Retro-2 that showed mouse protection against ricin, a notorious ribosome inactivating protein (RIP). Herein we report our chemical resolution of (+/-)-Retro-2cycl, analog synthesis, and cell-based evaluation showing that the two optically pure enantiomers and their achiral analog have nearly the same degree of cell protection against ricin as (+/-)-Retro-2cycl. We also report our computational studies explaining the lack of stereo preference and revealing a common pharmacophore of structurally distinct inhibitors of intracellular retrograde trafficking of RIPs. This pharmacophore comprises a central aromatic ring o-substituted by an aromatic ring and a moiety bearing an O or S atom attached to sp2 C atom(s). These results offer new insights into lead identification and optimization for RIP antidote development to minimize the global health threat caused by ribosome-inactivating proteins.

  2. Topogenesis and cell surface trafficking of GPR34 are facilitated by positive-inside rule that effects through a tri-basic motif in the first intracellular loop.

    PubMed

    Hasegawa, Haruki; Patel, Neha; Ettehadieh, Elham; Li, Peng; Lim, Ai Ching

    2016-07-01

    Protein folding, topogenesis and intracellular targeting of G protein-coupled receptors (GPCRs) must be precisely coordinated to ensure correct receptor localization. To elucidate how different steps of GPCR biosynthesis work together, we investigated the process of membrane topology determination and how it relates to the acquisition of cell surface trafficking competence in human GPR34. By monitoring a fused FLAG-tag and a conformation-sensitive native epitope during the expression of GPR34 mutant panel, a tri-basic motif in the first intracellular loop was identified as the key topogenic signal that dictates the orientation of transmembrane domain-1 (TM1). Charge disruption of the motif perturbed topogenic processes and resulted in the conformational epitope loss, post-translational processing alteration, and trafficking arrest in the Golgi. The placement of a cleavable N-terminal signal sequence as a surrogate topogenic determinant overcame the effects of tri-basic motif mutations and rectified the TM1 orientation; thereby restored the conformational epitope, post-translational modifications, and cell surface trafficking altogether. Progressive N-tail truncation and site-directed mutagenesis revealed that a proline-rich segment of the N-tail and all four cysteines individually located in the four separate extracellular regions must simultaneously reside in the ER lumen to muster the conformational epitope. Oxidation of all four cysteines was necessary for the epitope formation, but the cysteine residues themselves were not required for the trafficking event. The underlying biochemical properties of the conformational epitope was therefore the key to understand mechanistic processes propelled by positive-inside rule that simultaneously regulate the topogenesis and intracellular trafficking of GPR34. PMID:27086875

  3. Cellular uptake, intracellular trafficking, and antitumor efficacy of doxorubicin-loaded reduction-sensitive micelles.

    PubMed

    Cui, Can; Xue, Ya-Nan; Wu, Ming; Zhang, Yang; Yu, Ping; Liu, Lei; Zhuo, Ren-Xi; Huang, Shi-Wen

    2013-05-01

    Reduction-sensitive micelles were prepared from monomethoxy-poly(ethylene glycol)-S-S-hexadecyl (mPEG-S-S-C16), an amphiphilic poly(ethylene glycol) derivative containing a disulfide bond. The micelles were then used for the intracellular delivery of the anticancer drug doxorubicin (DOX) into tumor cells, and the cellular uptake mechanisms of the micelles were determined. To serve as a control, monomethoxy-poly(ethylene glycol)-C-C-hexadecyl (mPEG-C-C-C16) with an analogous structure but without a disulfide bond was also prepared. The polymer could self-assemble into micelles in an aqueous solution and be loaded with high-content DOX. In vitro release studies revealed that DOX-loaded mPEG-S-S-C16 micelles released DOX faster than DOX-loaded mPEG-C-C-C16 micelles in the presence of dithiothreitol (DTT), but showed similar release rates in the absence of DTT. MTT assay demonstrated significantly enhanced cytotoxicity of DOX-loaded mPEG-S-S-C16 micelles against the human cervical cancer cells (HeLa) compared with DOX-loaded mPEG-C-C-C16 micelles, but there was no significant difference in the cytotoxicity between the two DOX-loaded micelles against the african green monkey SV40-transformed kidney fibroblast cells (COS-7). Confocal laser scanning microscopy observation and flow cytometry analyses indicated that DOX-loaded mPEG-S-S-C16 micelles were efficiently internalized into HeLa cells, released DOX into the cytoplasm, and entered the nuclei. By contrast, in the case of DOX-loaded mPEG-C-C-C16 micelles, little DOX was found in the nuclei. Endocytosis inhibition results proved that both mPEG-S-S-C16 and mPEG-C-C-C16 micelles entered the HeLa cells mainly through the clathrin-mediated endocytosis pathway, and caveolae-mediated endocytosis was involved to a small extent. These results indicated that the different behaviors of cell uptake between reduction-sensitive and -insensitive micelles may occur after the micelles were internalized into the cells, but not during

  4. Association between Rare Variants in AP4E1, a Component of Intracellular Trafficking, and Persistent Stuttering.

    PubMed

    Raza, M Hashim; Mattera, Rafael; Morell, Robert; Sainz, Eduardo; Rahn, Rachel; Gutierrez, Joanne; Paris, Emily; Root, Jessica; Solomon, Beth; Brewer, Carmen; Basra, M Asim Raza; Khan, Shaheen; Riazuddin, Sheikh; Braun, Allen; Bonifacino, Juan S; Drayna, Dennis

    2015-11-01

    Stuttering is a common, highly heritable neurodevelopmental disorder characterized by deficits in the volitional control of speech. Whole-exome sequencing identified two heterozygous AP4E1 coding variants, c.1549G>A (p.Val517Ile) and c.2401G>A (p.Glu801Lys), that co-segregate with persistent developmental stuttering in a large Cameroonian family, and we observed the same two variants in unrelated Cameroonians with persistent stuttering. We found 23 other rare variants, including predicted loss-of-function variants, in AP4E1 in unrelated stuttering individuals in Cameroon, Pakistan, and North America. The rate of rare variants in AP4E1 was significantly higher in unrelated Pakistani and Cameroonian stuttering individuals than in population-matched control individuals, and coding variants in this gene are exceptionally rare in the general sub-Saharan West African, South Asian, and North American populations. Clinical examination of the Cameroonian family members failed to identify any symptoms previously reported in rare individuals carrying homozygous loss-of-function mutations in this gene. AP4E1 encodes the ε subunit of the heterotetrameric (ε-β4-μ4-σ4) AP-4 complex, involved in protein sorting at the trans-Golgi network. We found that the μ4 subunit of AP-4 interacts with NAGPA, an enzyme involved in the synthesis of the mannose 6-phosphate signal that targets acid hydrolases to the lysosome and the product of a gene previously associated with stuttering. These findings implicate deficits in intracellular trafficking in persistent stuttering. PMID:26544806

  5. Association between Rare Variants in AP4E1, a Component of Intracellular Trafficking, and Persistent Stuttering

    PubMed Central

    Raza, M. Hashim; Mattera, Rafael; Morell, Robert; Sainz, Eduardo; Rahn, Rachel; Gutierrez, Joanne; Paris, Emily; Root, Jessica; Solomon, Beth; Brewer, Carmen; Basra, M. Asim Raza; Khan, Shaheen; Riazuddin, Sheikh; Braun, Allen; Bonifacino, Juan S.; Drayna, Dennis

    2015-01-01

    Stuttering is a common, highly heritable neurodevelopmental disorder characterized by deficits in the volitional control of speech. Whole-exome sequencing identified two heterozygous AP4E1 coding variants, c.1549G>A (p.Val517Ile) and c.2401G>A (p.Glu801Lys), that co-segregate with persistent developmental stuttering in a large Cameroonian family, and we observed the same two variants in unrelated Cameroonians with persistent stuttering. We found 23 other rare variants, including predicted loss-of-function variants, in AP4E1 in unrelated stuttering individuals in Cameroon, Pakistan, and North America. The rate of rare variants in AP4E1 was significantly higher in unrelated Pakistani and Cameroonian stuttering individuals than in population-matched control individuals, and coding variants in this gene are exceptionally rare in the general sub-Saharan West African, South Asian, and North American populations. Clinical examination of the Cameroonian family members failed to identify any symptoms previously reported in rare individuals carrying homozygous loss-of-function mutations in this gene. AP4E1 encodes the ε subunit of the heterotetrameric (ε-β4-μ4-σ4) AP-4 complex, involved in protein sorting at the trans-Golgi network. We found that the μ4 subunit of AP-4 interacts with NAGPA, an enzyme involved in the synthesis of the mannose 6-phosphate signal that targets acid hydrolases to the lysosome and the product of a gene previously associated with stuttering. These findings implicate deficits in intracellular trafficking in persistent stuttering. PMID:26544806

  6. Association between Rare Variants in AP4E1, a Component of Intracellular Trafficking, and Persistent Stuttering.

    PubMed

    Raza, M Hashim; Mattera, Rafael; Morell, Robert; Sainz, Eduardo; Rahn, Rachel; Gutierrez, Joanne; Paris, Emily; Root, Jessica; Solomon, Beth; Brewer, Carmen; Basra, M Asim Raza; Khan, Shaheen; Riazuddin, Sheikh; Braun, Allen; Bonifacino, Juan S; Drayna, Dennis

    2015-11-01

    Stuttering is a common, highly heritable neurodevelopmental disorder characterized by deficits in the volitional control of speech. Whole-exome sequencing identified two heterozygous AP4E1 coding variants, c.1549G>A (p.Val517Ile) and c.2401G>A (p.Glu801Lys), that co-segregate with persistent developmental stuttering in a large Cameroonian family, and we observed the same two variants in unrelated Cameroonians with persistent stuttering. We found 23 other rare variants, including predicted loss-of-function variants, in AP4E1 in unrelated stuttering individuals in Cameroon, Pakistan, and North America. The rate of rare variants in AP4E1 was significantly higher in unrelated Pakistani and Cameroonian stuttering individuals than in population-matched control individuals, and coding variants in this gene are exceptionally rare in the general sub-Saharan West African, South Asian, and North American populations. Clinical examination of the Cameroonian family members failed to identify any symptoms previously reported in rare individuals carrying homozygous loss-of-function mutations in this gene. AP4E1 encodes the ε subunit of the heterotetrameric (ε-β4-μ4-σ4) AP-4 complex, involved in protein sorting at the trans-Golgi network. We found that the μ4 subunit of AP-4 interacts with NAGPA, an enzyme involved in the synthesis of the mannose 6-phosphate signal that targets acid hydrolases to the lysosome and the product of a gene previously associated with stuttering. These findings implicate deficits in intracellular trafficking in persistent stuttering.

  7. Preparation of HIV monoclonal antibody-conjugated pulchellin in order to study its intracellular trafficking pathway in HIV-infected cells by confocal microscopy

    NASA Astrophysics Data System (ADS)

    Sadraeian, M.; Tsutae, F. M.; Moreira, H. H. T.; Araujo, A. P. U.; Guimarães, F. E. G.; Pincus, S. H.

    2015-06-01

    Pulchellin is a type 2 of ribosome-inactivating proteins isolated from some seeds significantly growing in Brazil. It is a potent agent to inhibit the protein synthesis in cancer cells and also HIV-infected cells. Pulchellin can be conjugated to HIV monoclonal antibodies to specifically target the HIV-infected cells. To analyze the protein synthesis inhibition by Pulchellin, the intracellular localization of the immunoconjugate should be compared to Pulchellin. In this case, the intracellular trafficking of this protein in cells can be determined by confocal microscopy. In our study, we utilized Pulchellin to construct HIV monoclonal antibody-conjugated Pulchellin A chain in order to target HIV-infected lymphocyte cells. Afterward the conjugation was labeled with the superior Alexa Fluor 488 dye. As a subsequent step, we are interested in studying the intracellular trafficking pathway of this novel conjugation in HIV-infected cells by confocal microscopy. Moreover, possible quantitative methods for fluorescent labeling of the immunoconjugate during confocal microscopy will be investigated.

  8. The role of endocytosis in the uptake and intracellular trafficking of PepFect14-nucleic acid nanocomplexes via class A scavenger receptors.

    PubMed

    Juks, Carmen; Padari, Kärt; Margus, Helerin; Kriiska, Asko; Etverk, Indrek; Arukuusk, Piret; Koppel, Kaida; Ezzat, Kariem; Langel, Ülo; Pooga, Margus

    2015-12-01

    Cell penetrating peptides are efficient tools to deliver various bioactive cargos into cells, but their exact functioning mechanism is still debated. Recently, we showed that a delivery peptide PepFect14 condenses oligonucleotides (ON) into negatively charged nanocomplexes that are taken up by cells via class A scavenger receptors (SR-As). Here we unraveled the uptake mechanism and intracellular trafficking of PF14-ON nanocomplexes in HeLa cells. Macropinocytosis and caveolae-mediated endocytosis are responsible for the intracellular functionality of nucleic acids packed into nanocomplexes. However, only a negligible fraction of the complexes were trafficked to endoplasmic reticulum or Golgi apparatus - the common destinations of caveolar endocytosis. Neither were the PF14-SCO nanocomplexes routed to endo-lysosomal pathway, and they stayed in vesicles with slightly acidic pH, which were not marked with LysoSensor. "Naked" ON, in contrary, was rapidly targeted to acidic vesicles and lysosomes. The transmission electron microscopy analysis of interactions between SR-As and PF14-ON nanocomplexes on ultrastructural level revealed that nanocomplexes localized on the plasma membrane in close proximity to SR-As and their colocalization is retained in cells, suggesting that PF14-ON complexes associate with targeted receptors.

  9. Interplay between Clathrin and Rab5 Controls the Early Phagocytic Trafficking and Intracellular Survival of Brucella abortus within HeLa cells*

    PubMed Central

    Lee, Jin Ju; Kim, Dae Geun; Kim, Dong Hyeok; Simborio, Hannah Leah; Min, Wongi; Lee, Hu Jang; Her, Moon; Jung, Suk Chan; Watarai, Masahisa; Kim, Suk

    2013-01-01

    Lipid raft-associated clathrin is essential for host-pathogen interactions during infection. Brucella abortus is an intracellular pathogen that circumvents host defenses, but little is known about the precise infection mechanisms that involve interaction with lipid raft-associated mediators. The aim of this study was to elucidate the clathrin-mediated phagocytic mechanisms of B. abortus. The clathrin dependence of B. abortus infection in HeLa cells was investigated using an infection assay and immunofluorescence microscopy. The redistribution of clathrin in the membrane and in phagosomes was investigated using sucrose gradient fractionation of lipid rafts and the isolation of B. abortus-containing vacuoles, respectively. Clathrin and dynamin were concentrated into lipid rafts during B. abortus infection, and the entry and intracellular survival of B. abortus within HeLa cells were abrogated by clathrin inhibition. Clathrin disruption decreased actin polymerization and the colocalization of B. abortus-containing vacuoles with clathrin and Rab5 but not lysosome-associated membrane protein 1 (LAMP-1). Thus, our data demonstrate that clathrin plays a fundamental role in the entry and intracellular survival of B. abortus via interaction with lipid rafts and actin rearrangement. This process facilitates the early intracellular trafficking of B. abortus to safe replicative vacuoles. PMID:23940042

  10. Economics of human trafficking.

    PubMed

    Wheaton, Elizabeth M; Schauer, Edward J; Galli, Thomas V

    2010-01-01

    Because freedom of choice and economic gain are at the heart of productivity, human trafficking impedes national and international economic growth. Within the next 10 years, crime experts expect human trafficking to surpass drug and arms trafficking in its incidence, cost to human well-being, and profitability to criminals (Schauer and Wheaton, 2006: 164-165). The loss of agency from human trafficking as well as from modern slavery is the result of human vulnerability (Bales, 2000: 15). As people become vulnerable to exploitation and businesses continually seek the lowest-cost labour sources, trafficking human beings generates profit and a market for human trafficking is created. This paper presents an economic model of human trafficking that encompasses all known economic factors that affect human trafficking both across and within national borders. We envision human trafficking as a monopolistically competitive industry in which traffickers act as intermediaries between vulnerable individuals and employers by supplying differentiated products to employers. In the human trafficking market, the consumers are employers of trafficked labour and the products are human beings. Using a rational-choice framework of human trafficking we explain the social situations that shape relocation and working decisions of vulnerable populations leading to human trafficking, the impetus for being a trafficker, and the decisions by employers of trafficked individuals. The goal of this paper is to provide a common ground upon which policymakers and researchers can collaborate to decrease the incidence of trafficking in humans.

  11. Economics of human trafficking.

    PubMed

    Wheaton, Elizabeth M; Schauer, Edward J; Galli, Thomas V

    2010-01-01

    Because freedom of choice and economic gain are at the heart of productivity, human trafficking impedes national and international economic growth. Within the next 10 years, crime experts expect human trafficking to surpass drug and arms trafficking in its incidence, cost to human well-being, and profitability to criminals (Schauer and Wheaton, 2006: 164-165). The loss of agency from human trafficking as well as from modern slavery is the result of human vulnerability (Bales, 2000: 15). As people become vulnerable to exploitation and businesses continually seek the lowest-cost labour sources, trafficking human beings generates profit and a market for human trafficking is created. This paper presents an economic model of human trafficking that encompasses all known economic factors that affect human trafficking both across and within national borders. We envision human trafficking as a monopolistically competitive industry in which traffickers act as intermediaries between vulnerable individuals and employers by supplying differentiated products to employers. In the human trafficking market, the consumers are employers of trafficked labour and the products are human beings. Using a rational-choice framework of human trafficking we explain the social situations that shape relocation and working decisions of vulnerable populations leading to human trafficking, the impetus for being a trafficker, and the decisions by employers of trafficked individuals. The goal of this paper is to provide a common ground upon which policymakers and researchers can collaborate to decrease the incidence of trafficking in humans. PMID:20645472

  12. Mammalian farnesyltransferase α subunit regulates vacuolar protein sorting-associated protein 4A (Vps4A)--dependent intracellular trafficking through recycling endosomes.

    PubMed

    Kubala, Marta H; Norwood, Suzanne J; Gomez, Guillermo A; Jones, Alun; Johnston, Wayne; Yap, Alpha S; Mureev, Sergey; Alexandrov, Kirill

    2015-12-25

    The protein farnesyltransferase (FTase) mediates posttranslational modification of proteins with isoprenoid lipids. FTase is a heterodimer and although the β subunit harbors the active site, it requires the α subunit for its activity. Here we explore the other functions of the FTase α subunit in addition to its established role in protein prenylation. We found that in the absence of the β subunit, the α subunit of FTase forms a stable autonomous dimeric structure in solution. We identify interactors of FTase α using mass spectrometry, followed by rapid in vitro analysis using the Leishmania tarentolae cell - free system. Vps4A was validated for direct binding to the FTase α subunit both in vitro and in vivo. Analysis of the interaction with Vps4A in Hek 293 cells demonstrated that FTase α controls trafficking of transferrin receptor upstream of this protein. These results point to the existence of previously undetected biological functions of the FTase α subunit that includes control of intracellular membrane trafficking. PMID:26551458

  13. Tyrosine Binding Protein Sites Regulate the Intracellular Trafficking and Processing of Amyloid Precursor Protein through a Novel Lysosome-Directed Pathway

    PubMed Central

    Tam, Joshua H. K.; Cobb, M. Rebecca; Seah, Claudia; Pasternak, Stephen H.

    2016-01-01

    The amyloid hypothesis posits that the production of β-amyloid (Aβ) aggregates leads to neurodegeneration and cognitive decline associated with AD. Aβ is produced by sequential cleavage of the amyloid precursor protein (APP) by β- and γ-secretase. While nascent APP is well known to transit to the endosomal/ lysosomal system via the cell surface, we have recently shown that APP can also traffic to lysosomes intracellularly via its interaction with AP-3. Because AP-3 interacts with cargo protein via interaction with tyrosine motifs, we mutated the three tyrosines motif in the cytoplasmic tail of APP. Here, we show that the YTSI motif interacts with AP-3, and phosphorylation of the serine in this motif disrupts the interaction and decreases APP trafficking to lysosomes. Furthermore, we show that phosphorylation at this motif can decrease the production of neurotoxic Aβ 42. This demonstrates that reducing APP trafficking to lysosomes may be a strategy to reduce Aβ 42 in Alzheimer’s disease. PMID:27776132

  14. Sequential processing of the Toxoplasma apicoplast membrane protein FtsH1 in topologically distinct domains during intracellular trafficking.

    PubMed

    Karnataki, Anuradha; DeRocher, Amy E; Feagin, Jean E; Parsons, Marilyn

    2009-08-01

    FtsH proteins are hexameric transmembrane proteases found in chloroplasts, mitochondria and bacteria. In the protozoan Toxoplasma gondii, FtsH1 is localized to membranes of the apicoplast, a relict chloroplast present in many apicomplexan parasites. We have shown that although T. gondii FtsH1 lacks the typical bipartite targeting presequence seen on apicoplast luminal proteins, it is targeted to the apicoplast via the endoplasmic reticulum. In this report, we show that FtsH1 undergoes processing events to remove both the N- and C-termini, which are topologically separated by the membrane in which FtsH1 is embedded. Pulse-chase analysis showed that N-terminal cleavage precedes C-terminal cleavage. Unlike the processing of the N-terminal transit peptide of luminal proteins, which occurs in the apicoplast, analysis of ER-retained mutants showed that N-terminal processing of FtsH1 occurs in the endoplasmic reticulum. Two of four FtsH1 mutants bearing internal epitope tags accumulated in structures peripheral to the apicoplast, implying that FtsH1 trafficking is highly sensitive to changes in protein structure. These mutant proteins did not undergo C-terminal processing, suggesting that this processing step occurs after localization to the plastid. Mutation of the peptidase active site demonstrated that neither processing event occurs in cis. These data support a model in which multiple proteases act at different points of the trafficking pathway to form mature FtsH1, making its processing more complex than other FtsHs and unique among apicoplast proteins described thus far. PMID:19450729

  15. Doxorubicin as a molecular nanotheranostic agent: effect of doxorubicin encapsulation in micelles or nanoemulsions on the ultrasound-mediated intracellular delivery and nuclear trafficking

    PubMed Central

    Mohan, Praveena; Rapoport, Natalya

    2010-01-01

    Doxorubicin (DOX) is one of the most commonly used chemotherapeutic drugs and a popular research tool due to the inherent fluorescence of the DOX molecule. After DOX injection, fluorescence imaging of organs or cells can provide information on drug biodistribution. Therapeutic and imaging capabilities combined in a DOX molecule make it an excellent theranostic agent. However, DOX fluorescence depends on a number of factors that should be taken into consideration when interpreting results of DOX fluorescence measurements. Discussing these problems is the main thrust of the current paper. The sensitivity of DOX fluorescence intensity to DOX concentration, local microenvironment, and interaction with model cellular components is illustrated by fluorescence spectra of paired DOX/phosphilipid, DOX/histone, DOX/DNA, and triple DOX/histone/DNA and DOX/phospholipid/DNA systems. DOX fluorescence is dramatically quenched upon intercalation into the DNA; DOX fluorescence is also self-quenched at high concentrations of molecularly dissolved DOX; in contrast, DOX fluorescence is increased after binding to the histone or partitioning into the phospholipid phase of PEG-phospholipid micelles or hydrophobic cores of polymeric micelles. While flow cytometry is commonly used for characterization of DOX intracellular uptake, the above aspects of DOX fluorescence may significantly complicate interpretation of flow cytometry results. High cell fluorescence measured by flow cytometry may provide deceptive information on the actual intracellular DOX concentration and may not correlate with the therapeutic efficacy if DOX does not penetrate into the site of action in cell nuclei. These problems are illustrated in the experiments on the intracellular trafficking of DOX encapsulated in poly(ethylene oxide)-co-polycaprolactone (PEG-PCL) micelles or PEG-PCL stabilized perfluorocarbon nanodroplets, with and without the application of ultrasound used as an external trigger. For efficient

  16. Microtubule-targeting agents augment the toxicity of DNA-damaging agents by disrupting intracellular trafficking of DNA repair proteins.

    PubMed

    Poruchynsky, Marianne S; Komlodi-Pasztor, Edina; Trostel, Shana; Wilkerson, Julia; Regairaz, Marie; Pommier, Yves; Zhang, Xu; Kumar Maity, Tapan; Robey, Robert; Burotto, Mauricio; Sackett, Dan; Guha, Udayan; Fojo, Antonio Tito

    2015-02-01

    The paradigm that microtubule-targeting agents (MTAs) cause cell death via mitotic arrest applies to rapidly dividing cells but cannot explain MTA activity in slowly growing human cancers. Many preferred cancer regimens combine a MTA with a DNA-damaging agent (DDA). We hypothesized that MTAs synergize with DDAs by interfering with trafficking of DNA repair proteins on interphase microtubules. We investigated nine proteins involved in DNA repair: ATM, ATR, DNA-PK, Rad50, Mre11, p95/NBS1, p53, 53BP1, and p63. The proteins were sequestered in the cytoplasm by vincristine and paclitaxel but not by an aurora kinase inhibitor, colocalized with tubulin by confocal microscopy and coimmunoprecipitated with the microtubule motor dynein. Furthermore, adding MTAs to radiation, doxorubicin, or etoposide led to more sustained γ-H2AX levels. We conclude DNA damage-repair proteins traffic on microtubules and addition of MTAs sequesters them in the cytoplasm, explaining why MTA/DDA combinations are common anticancer regimens.

  17. Membrane targeting and intracellular trafficking of the human sodium-dependent multivitamin transporter in polarized epithelial cells.

    PubMed

    Subramanian, Veedamali S; Marchant, Jonathan S; Boulware, Michael J; Ma, Thomas Y; Said, Hamid M

    2009-04-01

    The human sodium-dependent multivitamin transporter (hSMVT) mediates sodium-dependent uptake of biotin in renal and intestinal epithelia. To date, however, there is nothing known about the structure-function relationship or targeting sequences in the hSMVT polypeptide that control its polarized expression within epithelia. Here, we focused on the role of the COOH-terminal tail of hSMVT in the targeting and functionality of this transporter. A full-length hSMVT-green fluorescent protein (GFP) fusion protein was functional and expressed at the apical membrane in renal and intestinal cell lines. Microtubule disrupting agents disrupted the mobility of trafficking vesicles and impaired cell surface delivery of hSMVT, which was also prevented in cells treated with dynamitin (p50), brefeldin, or monensin. Progressive truncation of the COOH-terminal tail impaired the functionality and targeting of the transporter. First, biotin transport decreased by approximately 20-30% on deletion of up to 15 COOH-terminal amino acids of hSMVT, a decrease mimicked solely by deletion of the terminal PDZ motif (TSL). Second, deletions into the COOH-terminal tail (between residues 584-612, containing a region of predicted high surface accessibility) resulted in a further drop in hSMVT transport (to approximately 40% of wild-type). Third, apical targeting was lost on deletion of a helical-prone region between amino acids 570-584. We conclude that the COOH tail of hSMVT contains several determinants important for polarized targeting and biotin transport.

  18. Human Trafficking

    MedlinePlus

    ... TRAFFICKING (English) Listen < Back to Search FACT SHEET: HUMAN TRAFFICKING (English) Published: August 2, 2012 Topics: Public Awareness , ... organizations that protect and serve trafficking victims. National Human Trafficking Resource Center at 1.888.373.7888 Last ...

  19. Binding of Host Factors Influences Internalization and Intracellular Trafficking of Streptococcus uberis in Bovine Mammary Epithelial Cells

    PubMed Central

    Almeida, Raul A.; Dunlap, John R.; Oliver, Stephen P.

    2010-01-01

    We showed that internalization of Streptococcus uberis into bovine mammary epithelial cells occurred through receptor- (RME) and caveolae-mediated endocytosis (CME). We reported also that treatment of S. uberis with host proteins including lactoferrin (LF) enhanced its internalization into host cells. Since the underlying mechanism(s) involved in such enhancement was unknown we investigated if preincubation of S. uberis with host proteins drives internalization of this pathogen into host cells through CME. Thus, experiments involving coculture of collagen-, fibronectin-, and LF-pretreated S. uberis with bovine mammary epithelial cells treated with RME and CME inhibitors were conducted. Results showed that internalization of host proteins-pretreated S. uberis into mammary epithelial cells treated with RME inhibitors was higher than that of untreated controls. These results suggest that pretreatment with selected host proteins commits S. uberis to CME, thus avoiding intracellular bactericidal mechanisms and allowing its persistence into bovine mammary epithelial cells. PMID:20614000

  20. Intracellular trafficking and metabolic turnover of yeast prepro-alpha-factor-SRIF precursors in GH3 cells.

    PubMed

    Lee, Myung Ae; Cheong, Kwang Ho; Shields, Dennis; Park, Sang Dai; Hong, Seung Hwan

    2002-09-30

    Chimeric genes coding for prepro region of yeast alpha-factor and anglerfish SRIF were expressed in rat GH3 cells to determine whether yeast signals could regulate hormone processing in mammalian cells. We report that nascent hybrid polypeptides were efficiently targeted to ER, where cleavage of signal peptides and core glycosylation occurred, and were localized mainly in Golgi. These data indicate that prepro region of yeast alpha-factor functions in sorting molecules to secretory pathway in mammalian cells. A hybrid construct with a mutated signal peptide underwent similar ER translocation, whereas such a mutation resulted in defective translocation in yeast (Cheong et al., 1997). This difference may be due to the differences in ER translocation between yeast and mammalian cells, i.e., posttranslational versus cotranslational translocation. Processing and secretion of metabolically labeled hybrid propeptides to mature SRIF peptides were assessed by HPLC. When pulse-labeled cells were chased for up to 2 h, intracellular propeptides disappeared with a half-life of approximately 25 min, showing that approximately 68% of initially synthesized propeptides were secreted constitutively. About 22% of SRIF-related products were proteolytically processed to mature SRIF, of which 38.7% were stored intracellularly with a half-life of approximately 2 h. In addition, immunocytochemical localization showed that a small proportion of SRIF molecules accumulated in secretory vesicles. All these results suggest that yeast prepropeptide could direct hybrid precursors to translocate into ER lumen and transit through secretory pathway to the distal elements of Golgi compartment, but could process and target it less efficiently to downstream in rat endocrine cells. PMID:12515394

  1. Scaling preferential flow processes in agricultural soils affected by tillage and trafficking at the field scale

    NASA Astrophysics Data System (ADS)

    Filipović, Vilim; Coquet, Yves

    2016-04-01

    There is an accumulation of experimental evidences that agricultural soils, at least the top horizons affected by tillage practices, are not homogeneous and present a structure that is strongly dependent on farming practices like tillage and trafficking. Soil tillage and trafficking can create compacted zones in the soil with hydraulic properties and porosity which are different from those of the non-compacted zones. This spatial variability can strongly influence transport processes and initiate preferential flow. Two or three dimensional models can be used to account for spatial variability created by agricultural practices, but such models need a detailed assessment of spatial heterogeneity which can be rather impractical to provide. This logically raises the question whether and how one dimensional model may be designed and used to account for the within-field spatial variability in soil structure created by agricultural practices. Preferential flow (dual-permeability) modelling performed with HYDRUS-1D will be confronted to classical modelling based on the Richards and convection-dispersion equations using HYDRUS-2D taking into account the various soil heterogeneities created by agricultural practices. Our goal is to derive one set of equivalent 1D soil hydraulic parameters from 2D simulations which accounts for soil heterogeneities created by agricultural operations. A field experiment was carried out in two phases: infiltration and redistribution on a plot by uniform sprinkle irrigation with water or bromide solution. Prior to the field experiment the soil structure of the tilled layer was determined along the face of a large trench perpendicular to the tillage direction (0.7 m depth and 3.1 m wide). Thirty TDR probes and tensiometers were installed in different soil structural zones (Δ compacted soil and Γ macroporous soil) which ensured soil water monitoring throughout the experiment. A map of bromide was constructed from small core samples (4 cm diam

  2. Identification of residues in ABCG2 affecting protein trafficking and drug transport, using co-evolutionary analysis of ABCG sequences

    PubMed Central

    Haider, Ameena J.; Cox, Megan H.; Jones, Natalie; Goode, Alice J.; Bridge, Katherine S.; Wong, Kelvin; Briggs, Deborah; Kerr, Ian D.

    2015-01-01

    ABCG2 is an ABC (ATP-binding cassette) transporter with a physiological role in urate transport in the kidney and is also implicated in multi-drug efflux from a number of organs in the body. The trafficking of the protein and the mechanism by which it recognizes and transports diverse drugs are important areas of research. In the current study, we have made a series of single amino acid mutations in ABCG2 on the basis of sequence analysis. Mutant isoforms were characterized for cell surface expression and function. One mutant (I573A) showed disrupted glycosylation and reduced trafficking kinetics. In contrast with many ABC transporter folding mutations which appear to be ‘rescued’ by chemical chaperones or low temperature incubation, the I573A mutation was not enriched at the cell surface by either treatment, with the majority of the protein being retained in the endoplasmic reticulum (ER). Two other mutations (P485A and M549A) showed distinct effects on transport of ABCG2 substrates reinforcing the role of TM helix 3 in drug recognition and transport and indicating the presence of intracellular coupling regions in ABCG2. PMID:26294421

  3. Intracellular Uptake and Trafficking of Difluoroboron Dibenzoylmethane-Poly(lactic acid) Nanoparticles in HeLa Cells

    PubMed Central

    Contreras, Janette; Xie, Jiansong; Chen, Yin Jie; Pei, Hua; Zhang, Guoqing; Fraser, Cassandra L.; Hamm-Alvarez, Sarah F.

    2010-01-01

    In this study, nanoparticles based on difluoroboron dibenzoylmethane-poly(lactic acid) (BF2dbmPLA) are prepared. Polylactic acid or polylactide is a commonly used degradable polymer, while the boron dye possesses a large extinction coefficient, high emission quantum yield, 2-photon absorption, and sensitivity to the surrounding environment. BF2dbmPLA exhibits molecular weight-dependent emission properties, and can be formulated as stable nanoparticles, suggesting that its unique optical properties may be useful in multiple contexts for probing intracellular environments. Here we show that BF2dbmPLA nanoparticles are internalized into cultured HeLa cells by endocytosis, and that within the cellular milieu they retain their fluorescence properties. BF2dbmPLA nanoparticles are photostable, resisting laser-induced photobleaching under conditions that destroy the fluorescence of a common photostable probe, LysoTracker™ blue. Their endocytosis is also lipid raft-dependent, as evidenced by their significant co-localization with cholera toxin B subunit in membrane compartments after uptake, and their sensitivity of uptake to methyl-β-cyclodextrin. Additionally, BF2dbmPLA nanoparticle endocytosis utilizes microtubules and actin filaments. Internalized BF2dbmPLA nanoparticles do not accumulate in acidic late endosomes and lysosomes, but within a perinuclear non-lysosomal compartment. These findings demonstrate the feasibility of using novel BF2dbmPLA nanoparticles exhibiting diverse emission properties for in situ, live cell imaging, and suggest that their endogenous uptake occurs through a lipid-raft dependent endocytosis mechanism. PMID:20420413

  4. The cytoskeletal inhibitors latrunculin A and blebbistatin exert antitumorigenic properties in human hepatocellular carcinoma cells by interfering with intracellular HuR trafficking

    SciTech Connect

    Doller, Anke; Badawi, Amel

    2015-01-01

    The impact of the RNA-binding protein HuR for the post-transcriptional deregulation of tumor-relevant genes is well established. Despite of elevations in HuR expression levels, an increase in cytoplasmic HuR abundance in many cases correlates with a high grade of malignancy. Here, we demonstrated that administration of the actin-depolymerizing macrolide latrunculin A, or blebbistatin, an inhibitor of myosin II ATPase activity, caused a dose- and time-dependent reduction in the high cytoplasmic HuR content of HepG2 and Huh7 hepatocellular carcinoma (HCC) cells. Subcellular fractionation revealed that in addition, both inhibitors strongly attenuated cytoskeletal and membrane-bound HuR abundance and conversely increased the HuR amount in nuclear cell fractions. Concomitant with changes in intracellular HuR localization, both cytoskeletal inhibitors markedly decreased the half-lives of cyclooxygenase-2 (COX-2), cyclin A and cyclin D{sub 1} encoding mRNAs resulting in a significant reduction in their expression levels in HepG2 cells. Importantly, a similar reduction in the expression of these HuR targets was achieved by a RNA interference (RNAi)-mediated knockdown of either HuR or nonmuscle myoin IIA. Using polysomal fractionation, we further demonstrate that the decrease in cytoplasmic HuR by latrunculin A or blebbistatin is accompanied by a marked change in the allocation of HuR and its mRNA cargo from polysomes to ribonucleoprotein (RNP) particles. Functionally, the basal migration and prostaglandin E{sub 2} synthesis are similarly impaired in inhibitor-treated and stable HuR-knockdown HepG2 cells. Our data demonstrate that interfering with the actomyosin-dependent HuR trafficking may comprise a valid therapeutic option for antagonizing pathologic posttranscriptional gene expression by HuR and furthermore emphasize the potential benefit of HuR inhibitory strategies for treatment of HCC. - Highlights: • We tested the effects of latrunculin A and blebbistatin on

  5. Trafficking regulation of proteins in Alzheimer’s disease

    PubMed Central

    2014-01-01

    The β-amyloid (Aβ) peptide has been postulated to be a key determinant in the pathogenesis of Alzheimer’s disease (AD). Aβ is produced through sequential cleavage of the β-amyloid precursor protein (APP) by β- and γ-secretases. APP and relevant secretases are transmembrane proteins and traffic through the secretory pathway in a highly regulated fashion. Perturbation of their intracellular trafficking may affect dynamic interactions among these proteins, thus altering Aβ generation and accelerating disease pathogenesis. Herein, we review recent progress elucidating the regulation of intracellular trafficking of these essential protein components in AD. PMID:24410826

  6. Defects in the COG complex and COG-related trafficking regulators affect neuronal Golgi function

    PubMed Central

    Climer, Leslie K.; Dobretsov, Maxim; Lupashin, Vladimir

    2015-01-01

    The Conserved Oligomeric Golgi (COG) complex is an evolutionarily conserved hetero-octameric protein complex that has been proposed to organize vesicle tethering at the Golgi apparatus. Defects in seven of the eight COG subunits are linked to Congenital Disorders of Glycosylation (CDG)-type II, a family of rare diseases involving misregulation of protein glycosylation, alterations in Golgi structure, variations in retrograde trafficking through the Golgi and system-wide clinical pathologies. A troublesome aspect of these diseases are the neurological pathologies such as low IQ, microcephaly, and cerebellar atrophy. The essential function of the COG complex is dependent upon interactions with other components of trafficking machinery, such as Rab-GTPases and SNAREs. COG-interacting Rabs and SNAREs have been implicated in neurodegenerative diseases like Alzheimer's disease and Parkinson's disease. Defects in Golgi maintenance disrupts trafficking and processing of essential proteins, frequently associated with and contributing to compromised neuron function and human disease. Despite the recent advances in molecular neuroscience, the subcellular bases for most neurodegenerative diseases are poorly understood. This article gives an overview of the potential contributions of the COG complex and its Rab and SNARE partners in the pathogenesis of different neurodegenerative disorders. PMID:26578865

  7. The cytoskeletal inhibitors latrunculin A and blebbistatin exert antitumorigenic properties in human hepatocellular carcinoma cells by interfering with intracellular HuR trafficking.

    PubMed

    Doller, Anke; Badawi, Amel; Schmid, Tobias; Brauss, Thilo; Pleli, Thomas; zu Heringdorf, Dagmar Meyer; Piiper, Albrecht; Pfeilschifter, Josef; Eberhardt, Wolfgang

    2015-01-01

    The impact of the RNA-binding protein HuR for the post-transcriptional deregulation of tumor-relevant genes is well established. Despite of elevations in HuR expression levels, an increase in cytoplasmic HuR abundance in many cases correlates with a high grade of malignancy. Here, we demonstrated that administration of the actin-depolymerizing macrolide latrunculin A, or blebbistatin, an inhibitor of myosin II ATPase activity, caused a dose- and time-dependent reduction in the high cytoplasmic HuR content of HepG2 and Huh7 hepatocellular carcinoma (HCC) cells. Subcellular fractionation revealed that in addition, both inhibitors strongly attenuated cytoskeletal and membrane-bound HuR abundance and conversely increased the HuR amount in nuclear cell fractions. Concomitant with changes in intracellular HuR localization, both cytoskeletal inhibitors markedly decreased the half-lives of cyclooxygenase-2 (COX-2), cyclin A and cyclin D1 encoding mRNAs resulting in a significant reduction in their expression levels in HepG2 cells. Importantly, a similar reduction in the expression of these HuR targets was achieved by a RNA interference (RNAi)-mediated knockdown of either HuR or nonmuscle myoin IIA. Using polysomal fractionation, we further demonstrate that the decrease in cytoplasmic HuR by latrunculin A or blebbistatin is accompanied by a marked change in the allocation of HuR and its mRNA cargo from polysomes to ribonucleoprotein (RNP) particles. Functionally, the basal migration and prostaglandin E2 synthesis are similarly impaired in inhibitor-treated and stable HuR-knockdown HepG2 cells. Our data demonstrate that interfering with the actomyosin-dependent HuR trafficking may comprise a valid therapeutic option for antagonizing pathologic posttranscriptional gene expression by HuR and furthermore emphasize the potential benefit of HuR inhibitory strategies for treatment of HCC.

  8. The intracellular domain of Dumbfounded affects myoblast fusion efficiency and interacts with Rolling pebbles and Loner.

    PubMed

    Bulchand, Sarada; Menon, Sree Devi; George, Simi Elizabeth; Chia, William

    2010-02-23

    Drosophila body wall muscles are multinucleated syncytia formed by successive fusions between a founder myoblast and several fusion competent myoblasts. Initial fusion gives rise to a bi/trinucleate precursor followed by more fusion cycles forming a mature muscle. This process requires the functions of various molecules including the transmembrane myoblast attractants Dumbfounded (Duf) and its paralogue Roughest (Rst), a scaffold protein Rolling pebbles (Rols) and a guanine nucleotide exchange factor Loner. Fusion completely fails in a duf, rst mutant, and is blocked at the bi/trinucleate stage in rols and loner single mutants. We analysed the transmembrane and intracellular domains of Duf, by mutating conserved putative signaling sites and serially deleting the intracellular domain. These were tested for their ability to translocate and interact with Rols and Loner and to rescue the fusion defect in duf, rst mutant embryos. Studying combinations of double mutants, further tested the function of Rols, Loner and other fusion molecules. Here we show that serial truncations of the Duf intracellular domain successively compromise its function to translocate and interact with Rols and Loner in addition to affecting myoblast fusion efficiency in embryos. Putative phosphorylation sites function additively while the extreme C terminus including a PDZ binding domain is dispensable for its function. We also show that fusion is completely blocked in a rols, loner double mutant and is compromised in other double mutants. These results suggest an additive function of the intracellular domain of Duf and an early function of Rols and Loner which is independent of Duf.

  9. Glucosylceramide synthesis inhibition affects cell cycle progression, membrane trafficking, and stage differentiation in Giardia lamblia.

    PubMed

    Stefanić, Sasa; Spycher, Cornelia; Morf, Laura; Fabriàs, Gemma; Casas, Josefina; Schraner, Elisabeth; Wild, Peter; Hehl, Adrian B; Sonda, Sabrina

    2010-09-01

    Synthesis of glucosylceramide via glucosylceramide synthase (GCS) is a crucial event in higher eukaryotes, both for the production of complex glycosphingolipids and for regulating cellular levels of ceramide, a potent antiproliferative second messenger. In this study, we explored the dependence of the early branching eukaryote Giardia lamblia on GCS activity. Biochemical analyses revealed that the parasite has a GCS located in endoplasmic reticulum (ER) membranes that is active in proliferating and encysting trophozoites. Pharmacological inhibition of GCS induced aberrant cell division, characterized by arrest of cytokinesis, incomplete cleavage furrow formation, and consequent block of replication. Importantly, we showed that increased ceramide levels were responsible for the cytokinesis arrest. In addition, GCS inhibition resulted in prominent ultrastructural abnormalities, including accumulation of cytosolic vesicles, enlarged lysosomes, and clathrin disorganization. Moreover, anterograde trafficking of the encystations-specific protein CWP1 was severely compromised and resulted in inhibition of stage differentiation. Our results reveal novel aspects of lipid metabolism in G. lamblia and specifically highlight the vital role of GCS in regulating cell cycle progression, membrane trafficking events, and stage differentiation in this parasite. In addition, we identified ceramide as a potent bioactive molecule, underscoring the universal conservation of ceramide signaling in eukaryotes. PMID:20335568

  10. Structural Features Affecting Trafficking, Processing, and Secretion of Trypanosoma cruzi Mucins*

    PubMed Central

    Cánepa, Gaspar E.; Mesías, Andrea C.; Yu, Hai; Chen, Xi; Buscaglia, Carlos A.

    2012-01-01

    Trypanosoma cruzi is wrapped by a dense coat of mucin-type molecules encoded by complex gene families termed TcSMUG and TcMUC, which are expressed in the insect- and mammal-dwelling forms of the parasite, respectively. Here, we dissect the contribution of distinct post-translational modifications on the trafficking of these glycoconjugates. In vivo tracing and characterization of tagged-variants expressed by transfected epimastigotes indicate that although the N-terminal signal peptide is responsible for targeting TcSMUG products to the endoplasmic reticulum (ER), the glycosyl phosphatidylinositol (GPI)-anchor likely functions as a forward transport signal for their timely progression along the secretory pathway. GPI-minus variants accumulate in the ER, with only a minor fraction being ultimately released to the medium as anchorless products. Secreted products, but not ER-accumulated ones, display several diagnostic features of mature mucin-type molecules including extensive O-type glycosylation, Galf-based epitopes recognized by monoclonal antibodies, and terminal Galp residues that become readily sialylated upon addition of parasite trans-sialidases. Processing of N-glycosylation site(s) is dispensable for the overall TcSMUG mucin-type maturation and secretion. Despite undergoing different O-glycosylation elaboration, TcMUC reporters yielded quite similar results, thus indicating that (i) molecular trafficking signals are structurally and functionally conserved between mucin families, and (ii) TcMUC and TcSMUG products are recognized and processed by a distinct repertoire of stage-specific glycosyltransferases. Thus, using the fidelity of a homologous expression system, we have defined some biosynthetic aspects of T. cruzi mucins, key molecules involved in parasite protection and virulence. PMID:22707724

  11. Identification of IAA transport inhibitors including compounds affecting cellular PIN trafficking by two chemical screening approaches using maize coleoptile systems.

    PubMed

    Nishimura, Takeshi; Matano, Naoyuki; Morishima, Taichi; Kakinuma, Chieko; Hayashi, Ken-Ichiro; Komano, Teruya; Kubo, Minoru; Hasebe, Mitsuyasu; Kasahara, Hiroyuki; Kamiya, Yuji; Koshiba, Tomokazu

    2012-10-01

    The monocot coleoptile tip region has been generally supposed to be the source of IAA to supply IAA to basal parts by the polar IAA transport system, which results in gravi- and phototropic curvature of coleoptiles. Based on this IAA transport system and gravitropism of maize coleoptiles, we have developed two screening methods to identify small molecules from a large chemical library that inhibit IAA transport. The methods detect molecules that affect (i) gravitropic curvature of coleoptiles; and (ii) the amount of IAA transported from the tip. From 10,000 chemicals, eight compounds were identified and categorized into two groups. Four chemicals in group A decreased IAA transport from the tip, and increased endogenous IAA levels in the tip. The structures of two compounds resembled that of 1-N-naphthylphthalamic acid (NPA), but those of the other two differed from structures of known IAA transport inhibitors. Four chemicals in group B strongly inhibited IAA transport from the tip, but IAA levels at the tip were only slightly affected. At higher concentrations, group B compounds inhibited germination of Arabidopsis, similarly to brefeldin A (BFA). Analysis of the cellular distribution of PIN2-green fluorescent protein (GFP) and PIN1-GFP in Arabidopsis revealed that one of the four chemicals in group B induced internalization of PIN1 and PIN2 proteins into vesicles smaller than BFA bodies, suggesting that this compound affects cellular vesicle trafficking systems related to PIN trafficking. The eight chemicals identified here will be a useful tool for understanding the mechanisms of IAA transport in plants. PMID:22875609

  12. Subversion of Retrograde Trafficking by Translocated Pathogen Effectors.

    PubMed

    Personnic, Nicolas; Bärlocher, Kevin; Finsel, Ivo; Hilbi, Hubert

    2016-06-01

    Intracellular bacterial pathogens subvert the endocytic bactericidal pathway to form specific replication-permissive compartments termed pathogen vacuoles or inclusions. To this end, the pathogens employ type III or type IV secretion systems, which translocate dozens, if not hundreds, of different effector proteins into their host cells, where they manipulate vesicle trafficking and signaling pathways in favor of the intruders. While the distinct cocktail of effectors defines the specific processes by which a pathogen vacuole is formed, the different pathogens commonly target certain vesicle trafficking routes, including the endocytic or secretory pathway. Recently, the retrograde transport pathway from endosomal compartments to the trans-Golgi network emerged as an important route affecting pathogen vacuole formation. Here, we review current insight into the host cell's retrograde trafficking pathway and how vacuolar pathogens of the genera Legionella, Coxiella, Salmonella, Chlamydia, and Simkania employ mechanistically distinct strategies to subvert this pathway, thus promoting intracellular survival and replication. PMID:26924068

  13. Reggies/flotillins regulate E-cadherin-mediated cell contact formation by affecting EGFR trafficking.

    PubMed

    Solis, Gonzalo P; Schrock, Yvonne; Hülsbusch, Nikola; Wiechers, Marianne; Plattner, Helmut; Stuermer, Claudia A O

    2012-05-01

    The reggie/flotillin proteins are implicated in membrane trafficking and, together with the cellular prion protein (PrP), in the recruitment of E-cadherin to cell contact sites. Here, we demonstrate that reggies, as well as PrP down-regulation, in epithelial A431 cells cause overlapping processes and abnormal formation of adherens junctions (AJs). This defect in cell adhesion results from reggie effects on Src tyrosine kinases and epidermal growth factor receptor (EGFR): loss of reggies reduces Src activation and EGFR phosphorylation at residues targeted by Src and c-cbl and leads to increased surface exposure of EGFR by blocking its internalization. The prolonged EGFR signaling at the plasma membrane enhances cell motility and macropinocytosis, by which junction-associated E-cadherin is internalized and recycled back to AJs. Accordingly, blockage of EGFR signaling or macropinocytosis in reggie-deficient cells restores normal AJ formation. Thus, by promoting EGFR internalization, reggies restrict the EGFR signaling involved in E-cadherin macropinocytosis and recycling and regulate AJ formation and dynamics and thereby cell adhesion.

  14. Leukocyte trafficking is not affected by multikinase inhibitors sunitinib or sorafenib in mice.

    PubMed

    Laurila, Juha P; Laukkanen, Mikko O; Joensuu, Heikki; Salmi, Marko; Jalkanen, Sirpa

    2016-11-15

    Sunitinib and sorafenib are broad-spectrum tyrosine kinase inhibitors (TKI) targeting, for example, VEGF1-3, PDGFRb, RET, FLT3, CD117 (c-KIT) and CSF-1R cell membrane receptors thus suppressing tumor angiogenesis and cancer cell growth. Recently it has been suggested that the kinases targeted by Sunitinib and/or Sorafenib regulate leukocyte transmigration, which might in part be responsible for the often-observed reduction in tumor-associated myeloid derived suppressor cells and regulatory T cells. The aim of the current study is to determine whether sunitinib or sorafenib inhibit leukocyte extravasation. Sunitinib, sorafenib, or vehicle treated animals did not show any difference in leukocyte trafficking either in peritonitis or in vivo homing experiments, although sunitinib treatment effectively inhibited growth of B16 melanoma tumors in WT, SCID and SCID beige mice. Inhibition of tumor growth was associated with an increased number of infiltrating CD11b+ cells in the tumor, while the numbers of CD8, Gr-1 and F4/80 expressing cells were unchanged. In conclusion, the findings suggest that despite multiple targets with a potential role in leukocyte extravasation, neither sunitinib nor sorafenib effectively inhibits this process in vivo. Thus, the observed specific effect on CD11b cells among tumor infiltrating leukocytes is most likely an indirect effect. PMID:27459381

  15. POPDC1S201F causes muscular dystrophy and arrhythmia by affecting protein trafficking

    PubMed Central

    Schindler, Roland F.R.; Scotton, Chiara; Zhang, Jianguo; Passarelli, Chiara; Ortiz-Bonnin, Beatriz; Simrick, Subreena; Schwerte, Thorsten; Poon, Kar-Lai; Fang, Mingyan; Rinné, Susanne; Froese, Alexander; Nikolaev, Viacheslav O.; Grunert, Christiane; Müller, Thomas; Tasca, Giorgio; Sarathchandra, Padmini; Drago, Fabrizio; Dallapiccola, Bruno; Rapezzi, Claudio; Arbustini, Eloisa; Di Raimo, Francesca Romana; Neri, Marcella; Selvatici, Rita; Gualandi, Francesca; Fattori, Fabiana; Pietrangelo, Antonello; Li, Wenyan; Jiang, Hui; Xu, Xun; Bertini, Enrico; Decher, Niels; Wang, Jun; Brand, Thomas; Ferlini, Alessandra

    2015-01-01

    The Popeye domain–containing 1 (POPDC1) gene encodes a plasma membrane–localized cAMP-binding protein that is abundantly expressed in striated muscle. In animal models, POPDC1 is an essential regulator of structure and function of cardiac and skeletal muscle; however, POPDC1 mutations have not been associated with human cardiac and muscular diseases. Here, we have described a homozygous missense variant (c.602C>T, p.S201F) in POPDC1, identified by whole-exome sequencing, in a family of 4 with cardiac arrhythmia and limb-girdle muscular dystrophy (LGMD). This allele was absent in known databases and segregated with the pathological phenotype in this family. We did not find the allele in a further screen of 104 patients with a similar phenotype, suggesting this mutation to be family specific. Compared with WT protein, POPDC1S201F displayed a 50% reduction in cAMP affinity, and in skeletal muscle from patients, both POPDC1S201F and WT POPDC2 displayed impaired membrane trafficking. Forced expression of POPDC1S201F in a murine cardiac muscle cell line (HL-1) increased hyperpolarization and upstroke velocity of the action potential. In zebrafish, expression of the homologous mutation (popdc1S191F) caused heart and skeletal muscle phenotypes that resembled those observed in patients. Our study therefore identifies POPDC1 as a disease gene causing a very rare autosomal recessive cardiac arrhythmia and LGMD, expanding the genetic causes of this heterogeneous group of inherited rare diseases. PMID:26642364

  16. A tyrosine-based motif and a casein kinase II phosphorylation site regulate the intracellular trafficking of the varicella-zoster virus glycoprotein I, a protein localized in the trans-Golgi network.

    PubMed Central

    Alconada, A; Bauer, U; Hoflack, B

    1996-01-01

    We have studied the intracellular trafficking of the envelope glycoprotein I (gpI) of the varicella-zoster virus, a human herpes virus whose assembly is believed to occur in the trans-Golgi network (TGN) and/or in endocytic compartments. When expressed in HeLa cells in the absence of additional virally encoded factors, this type-I membrane protein localizes to the TGN and cycles between this compartment and the cell surface. The expression of gpI promotes the recruitment of the AP-1 Golgi-specific assembly proteins onto TGN membranes, strongly suggesting that gpI, like the mannose 6-phosphate receptors, can leave the TGN in clathrin-coated vesicles for subsequent transport to endosomes. Its return from the cell surface to the TGN also occurs through endosomes. The transfer of the gpI cytoplasmic domain onto a reporter molecule shows that this domain is sufficient to confer TGN localization. Mutational analysis of this domain indicates that proper subcellular localization and cycling of gpI depend on two different determinants, a tyrosine-containing tetrapeptide related to endocytosis sorting signals and a cluster of acidic amino acids containing casein kinase II phosphorylatable residues. Thus, the VZV gpI and the mannose 6-phosphate receptors, albeit localized in different intracellular compartments at steady-state, follow similar trafficking pathways and share similar sorting mechanisms. Images PMID:8947032

  17. Biocompatibility, endocytosis, and intracellular trafficking of mesoporous silica and polystyrene nanoparticles in ovarian cancer cells: effects of size and surface charge groups

    PubMed Central

    Ekkapongpisit, Maneerat; Giovia, Antonino; Follo, Carlo; Caputo, Giuseppe; Isidoro, Ciro

    2012-01-01

    Background and methods Nanoparticles engineered to carry both a chemotherapeutic drug and a sensitive imaging probe are valid tools for early detection of cancer cells and to monitor the cytotoxic effects of anticancer treatment simultaneously. Here we report on the effect of size (10–30 nm versus 50 nm), type of material (mesoporous silica versus polystyrene), and surface charge functionalization (none, amine groups, or carboxyl groups) on biocompatibility, uptake, compartmentalization, and intracellular retention of fluorescently labeled nanoparticles in cultured human ovarian cancer cells. We also investigated the involvement of caveolae in the mechanism of uptake of nanoparticles. Results We found that mesoporous silica nanoparticles entered via caveolae-mediated endocytosis and reached the lysosomes; however, while the 50 nm nanoparticles permanently resided within these organelles, the 10 nm nanoparticles soon relocated in the cytoplasm. Naked 10 nm mesoporous silica nanoparticles showed the highest and 50 nm carboxyl-modified mesoporous silica nanoparticles the lowest uptake rates, respectively. Polystyrene nanoparticle uptake also occurred via a caveolae-independent pathway, and was negatively affected by serum. The 30 nm carboxyl-modified polystyrene nanoparticles did not localize in lysosomes and were not toxic, while the 50 nm amine-modified polystyrene nanoparticles accumulated within lysosomes and eventually caused cell death. Ovarian cancer cells expressing caveolin-1 were more likely to endocytose these nanoparticles. Conclusion These data highlight the importance of considering both the physicochemical characteristics (ie, material, size and surface charge on chemical groups) of nanoparticles and the biochemical composition of the cell membrane when choosing the most suitable nanotheranostics for targeting cancer cells. PMID:22904626

  18. Imp2, the PSTPIP homolog in fission yeast, affects sensitivity to the immunosuppressant FK506 and membrane trafficking in fission yeast

    SciTech Connect

    Kita, Ayako; Higa, Mari; Doi, Akira; Satoh, Ryosuke; Sugiura, Reiko

    2015-02-13

    Cytokinesis is a highly ordered process that divides one cell into two cells, which is functionally linked to the dynamic remodeling of the plasma membrane coordinately with various events such as membrane trafficking. Calcineurin is a highly conserved serine/threonine protein phosphatase, which regulates multiple biological functions, such as membrane trafficking and cytokinesis. Here, we isolated imp2-c3, a mutant allele of the imp2{sup +} gene, encoding a homolog of the mouse PSTPIP1 (proline-serine-threonine phosphatase interacting protein 1), using a genetic screen for mutations that are synthetically lethal with calcineurin deletion in fission yeast. The imp2-c3 mutants showed a defect in cytokinesis with multi-septated phenotypes, which was further enhanced upon treatment with the calcineurin inhibitor FK506. Notably, electron micrographs revealed that the imp2-c3 mutant cells accumulated aberrant multi-lamella Golgi structures and putative post-Golgi secretory vesicles, and exhibited fragmented vacuoles in addition to thickened septa. Consistently, imp2-c3 mutants showed a reduced secretion of acid phosphatase and defects in vacuole fusion. The imp2-c3 mutant cells exhibited a weakened cell wall, similar to the membrane trafficking mutants identified in the same genetic screen such as ypt3-i5. These findings implicate the PSTPIP1 homolog Imp2 in Golgi/vacuole function, thereby affecting various cellular processes, including cytokinesis and cell integrity. - Highlights: • We isolated imp2-c3, in a synthetic lethal screen with calcineurin in fission yeast. • The imp2{sup +} gene encodes a component of the actin contractile ring similar to Cdc15. • The imp2-c3 mutants showed defects in cytokinesis, which were exacerbated by FK506. • The imp2-c3 mutants were defective in membrane trafficking and cell wall integrity. • Our study revealed a novel role for Imp2 in the Golgi/vacuolar membrane trafficking.

  19. Intracellular trafficking of LET-756, a fibroblast growth factor of C. elegans, is controlled by a balance of export and nuclear signals

    SciTech Connect

    Popovici, Cornel; Fallet, Mathieu; Marguet, Didier; Birnbaum, Daniel; Roubin, Regine . E-mail: roubin@marseille.inserm.fr

    2006-05-15

    The superfamily of fibroblast growth factors (FGF), which counts 22 members in humans, exerts many functions during animal development and adult life. LET-756 is one of the two FGFs of the nematode C. elegans. Re-introduction of LET-756 in a null mutant strain restores viability, allowing the study of structural requirements for LET-756 trafficking and function. LET-756 protein has several regions and motifs, including a non-classical internal motif required for secretion. We show here that a main difference in the wild-type LET-756 molecule and a truncated molecule that mimics a partial loss-of-function mutant lies on subnuclear expression. Using Cos-1 cells and rescue activity we show that: (i) nuclear localization is due to various redundant NLS, one of them acting as a nucleolar localization signal; (ii) nuclear LET-756 is addressed to the speckles by a stretch of glutamine residues; (iii) nuclear LET-756 is trafficking between speckles and nucleoli; (iv) in the nucleolus, LET-756 is associated with proteins of the rRNA splicing compartment; (v) changing LET-756 secretion signal prevents its nuclear localization. We propose that LET-756 exerts its functions through a balance between secreted and nuclear forms due to two opposite addressing signals (i) synergy of several NLS and (ii) attenuated secretion signal.

  20. Effects of Membrane Trafficking on Signaling by Receptor Tyrosine Kinases

    PubMed Central

    Miaczynska, Marta

    2013-01-01

    The intracellular trafficking machinery contributes to the spatial and temporal control of signaling by receptor tyrosine kinases (RTKs). The primary role in this process is played by endocytic trafficking, which regulates the localization of RTKs and their downstream effectors, as well as the duration and the extent of their activity. The key regulatory points along the endocytic pathway are internalization of RTKs from the plasma membrane, their sorting to degradation or recycling, and their residence in various endosomal compartments. Here I will review factors and mechanisms that modulate RTK signaling by (1) affecting receptor internalization, (2) regulating the balance between degradation and recycling of RTK, and (3) compartmentalization of signals in endosomes and other organelles. Cumulatively, these mechanisms illustrate a multilayered control of RTK signaling exerted by the trafficking machinery. PMID:24186066

  1. Mutation of a C-Terminal Motif Affects Kaposi's Sarcoma-Associated Herpesvirus ORF57 RNA Binding, Nuclear Trafficking, and Multimerization ▿

    PubMed Central

    Taylor, Adam; Jackson, Brian R.; Noerenberg, Marko; Hughes, David J.; Boyne, James R.; Verow, Mark; Harris, Mark; Whitehouse, Adrian

    2011-01-01

    The Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein is essential for virus lytic replication. ORF57 regulates virus gene expression at multiple levels, enhancing transcription, stability, nuclear export, and translation of viral transcripts. To enhance the nuclear export of viral intronless transcripts, ORF57 (i) binds viral intronless mRNAs, (ii) shuttles between the nucleus, nucleolus, and the cytoplasm, and (iii) interacts with multiple cellular nuclear export proteins to access the TAP-mediated nuclear export pathway. We investigated the implications on the subcellular trafficking, cellular nuclear export factor recruitment, and ultimately nuclear mRNA export of an ORF57 protein unable to bind RNA. We observed that mutation of a carboxy-terminal RGG motif, which prevents RNA binding, affects the subcellular localization and nuclear trafficking of the ORF57 protein, suggesting that it forms subnuclear aggregates. Further analysis of the mutant shows that although it still retains the ability to interact with cellular nuclear export proteins, it is unable to export viral intronless mRNAs from the nucleus. Moreover, computational molecular modeling and biochemical studies suggest that, unlike the wild-type protein, this mutant is unable to self-associate. Therefore, these results suggest the mutation of a carboxy-terminal RGG motif affects ORF57 RNA binding, nuclear trafficking, and multimerization. PMID:21593148

  2. Human Trafficking

    ERIC Educational Resources Information Center

    Wilson, David McKay

    2011-01-01

    The shadowy, criminal nature of human trafficking makes evaluating its nature and scope difficult. The U.S. State Department and anti-trafficking groups estimate that worldwide some 27 million people are caught in a form of forced servitude today. Public awareness of modern-day slavery is gaining momentum thanks to new abolitionist efforts. Among…

  3. Strategic formulation of apigenin-loaded PLGA nanoparticles for intracellular trafficking, DNA targeting and improved therapeutic effects in skin melanoma in vitro.

    PubMed

    Das, Sreemanti; Das, Jayeeta; Samadder, Asmita; Paul, Avijit; Khuda-Bukhsh, Anisur Rahman

    2013-11-25

    The aim of the present study was the evaluation of anti-proliferative potentials of apigenin (Ap), (a dietary flavonoid) loaded in poly (lactic-co-glycolide) nanoparticles (NAp) in A375 cells in vitro. NAp was characterized for particle size, morphology, zeta potential, drug release and encapsulation. Cellular entry and intracellular localization of NAp were assessed by transmission electron and confocal microscopies. Circular dichroic spectral analysis and stability curve for Gibb's free energy of dsDNA of A375 cells were also analyzed. DNA fragmentation, intracellular ROS accumulation, superoxide-dismutase activity, intracellular glutathione-reductase content and mitochondrial functioning through relevant markers like mitochondrial transmembrane potential, ATPase activity, ATP/ADP ratio, volume changes/swelling, cytochrome-c release, expressions of Apaf-1, bax, bcl-2, caspase-9, 3, and PARP cleavage were analyzed. NAp produced better effects due to their smaller size, faster mobility and site-specific action. Photostability studies revealed that PLGA encapsulations were efficient at preserving apigenin ultraviolet-light mediated photodegradation. NAp readily entered cancer cells, could intercalate with dsDNA, inducing conformational change. Corresponding increase in ROS accumulation and depletion of the antioxidant enzyme activities exacerbated DNA damage, mediating apoptosis through mitochondrial dysfunction. Overall results indicate that therapeutic efficacy of NAp may be enhanced by PLGA nanoparticle formulations to have better ameliorative potentials in combating skin melanoma.

  4. Three-Dimensional Nanocomposites: Fluidics Driven Assembly of Metal Nanoparticles on Protein Nanostructures and Their Cell-Line-Dependent Intracellular Trafficking Pattern.

    PubMed

    Srikar, R; Suresh, Dhananjay; Saranathan, Sandhya; Zambre, Ajit; Kannan, Raghuraman

    2016-05-17

    Three-dimensional nanocomposites prepared using two different families of nanomaterials holds significant relevance pertaining to biological applications. However, integration of the two distinct nanomaterials with precision to control the overall compositional homogeneity of the resulting 3D nanocomposite is a synthetic challenge. Conventional reactions result in nanocomposites with heterogeneous composition and render useless. To address this challenge, we have developed a fluidics-mediated process for controlling the interaction of nanoparticles to yield a compositional uniform multidimensional nanoparticle; as an example, we demonstrated the integration of gold nanoparticles on gelatin nanoparticles. The composition of the nanocomposite is controlled by reacting predetermined number of gold nanoparticles to a known number of thiolated gelatin nanoparticles at any given time within a defined cross-sectional area. Using the fluidics process, we developed nanocomposites of different composition: [gelatin nanoparticles-(gold nanoparticles)x] where xaverage = 2, 12, or 25. The nanocomposites were further surface conjugated with organic molecules such as fluorescent dye or polyethylene glycol (PEG) molecules. To study the biological behavior of nanocomposite, we investigated the cellular internalization and trafficking characteristics of nanocomposites in two human cancer cell lines. The nanocomposites exhibited a three-stage cellular release mechanism that enables the translocation of gold nanoparticles within various cellular compartments. In summary, the three-dimensional nanocomposite serves as a novel platform for developing well-defined protein-metal nanocomposites for potential drug delivery, sensory, and molecular imaging applications. PMID:27088307

  5. Intracellular trafficking and cellular uptake mechanism of mPEG-PLGA-PLL and mPEG-PLGA-PLL-Gal nanoparticles for targeted delivery to hepatomas.

    PubMed

    Liu, Peifeng; Sun, Yanming; Wang, Qi; Sun, Ying; Li, He; Duan, Yourong

    2014-01-01

    The lysosomal escape of nanoparticles is crucial to enhancing their delivery and therapeutic efficiency. Here, we report the cellular uptake mechanism, lysosomal escape, and organelle morphology effect of monomethoxy (polyethylene glycol)-poly (D,L-lactide-co-glycolide)-poly (L-lysine) (mPEG-PLGA-PLL, PEAL) and 4-O-beta-D-Galactopyranosyl-D-gluconic acid (Gal)-modified PEAL (PEAL-Gal) for intracellular delivery to HepG2, Huh7, and PLC hepatoma cells. These results indicate that PEAL is taken up by clathrin-mediated endocytosis of HepG2, Huh7 and PLC cells. For PEAL-Gal, sialic acid receptor-mediated endocytosis and clathrin-mediated endocytosis are the primary uptake pathways in HepG2 cells, respectively, whereas PEAL-Gal is internalized by sag vesicle- and clathrin-mediated endocytosis in Huh7 cells. In the case of PLC cells, clathrin-mediated endocytosis and sialic acid receptor play a primary role in the uptake of PEAL-Gal. TEM results verify that PEAL and PEAL-Gal lead to a different influence on organelle morphology of HepG2, Huh7 and PLC cells. In addition, the results of intracellular distribution reveal that PEAL and PEAL-Gal are less entrapped in the lysosomes of HepG2 and Huh7 cells, demonstrating that they effectively escape from lysosomes and contribute to enhance the efficiency of intracellular delivery and tumor therapy. In vivo tumor targeting image results demonstrate that PEAL-Gal specifically delivers Rhodamine B (Rb) to the tumor tissue of mice with HepG2, Huh7, and PLC hepatomas and remains at a high concentration in tumor tissue until 48 h, properties that will greatly contribute to enhanced antitumor efficiency.

  6. Post-translational intracellular trafficking determines the type of immune response elicited by DNA vaccines expressing Gag antigen of Human Immunodeficiency Virus Type 1 (HIV-1)

    PubMed Central

    Wallace, Aaron; West, Kim; Rothman, Alan L; Ennis, Francis A; Lu, Shan; Wang, Shixia

    2013-01-01

    In the current study, immune responses induced by Gag DNA vaccines with different designs were evaluated in Balb/C mice. The results demonstrated that the DNA vaccine with the full length wild type gag gene (Wt-Gag) mainly produced Gag antigens intracellularly and induced a higher level of cell-mediated immune (CMI) responses, as measured by IFN-gamma ELISPOT, intracellular cytokine staining (ICS), and cytotoxic T lymphocytes (CTL) assays against a dominant CD8+ T cell epitope (AMQMLKETI). In contrast, the addition of a tissue plasminogen activator (tPA) leader sequence significantly improved overall Gag protein expression/secretion and Gag-specific antibody responses; however, Gag-specific CMI responses were decreased. The mutation of zinc-finger motif changed Gag protein expression patterns and reduced the ability to generate both CMI and antibody responses against Gag. These findings indicate that the structure and post-translational processing of antigens expressed by DNA vaccines play a critical role in eliciting optimal antibody or CMI responses. PMID:23941868

  7. Characterization of Helicobacter pylori VacA-containing vacuoles (VCVs), VacA intracellular trafficking and interference with calcium signalling in T lymphocytes.

    PubMed

    Kern, Beate; Jain, Utkarsh; Utsch, Ciara; Otto, Andreas; Busch, Benjamin; Jiménez-Soto, Luisa; Becher, Dörte; Haas, Rainer

    2015-12-01

    The human pathogen Helicobacter pylori colonizes half of the global population. Residing at the stomach epithelium, it contributes to the development of diseases such as gastritis, duodenal and gastric ulcers, and gastric cancer. A major factor is the secreted vacuolating toxin VacA, which forms anion-selective channels in the endosome membrane that cause the compartment to swell, but the composition and purpose of the resulting VacA-containing vacuoles (VCVs) are still unknown. VacA exerts influence on the host immune response in various ways, including inhibition of T-cell activation and proliferation and suppression of the host immune response. In this study, for the first time the composition of VCVs from T cells was comprehensively analysed to investigate VCV function. VCVs were successfully isolated via immunomagnetic separation, and the purified vacuoles were analysed by mass spectrometry. We detected a set of 122 VCV-specific proteins implicated among others in immune response, cell death and cellular signalling processes, all of which VacA is known to influence. One of the individual proteins studied further was stromal interaction molecule (STIM1), a calcium sensor residing in the endoplasmic reticulum (ER) that is important in store-operated calcium entry. Live cell imaging microscopy data demonstrated colocalization of VacA with STIM1 in the ER and indicated that VacA may interfere with the movement of STIM1 towards the plasma membrane-localized calcium release activated calcium channel protein ORAI1 in response to Ca(2+) store depletion. Furthermore, VacA inhibited the increase of cytosolic-free Ca(2+) in the Jurkat E6-1 T-cell line and human CD4(+) T cells. The presence of VacA in the ER and its trafficking to the Golgi apparatus was confirmed in HeLa cells, identifying these two cellular compartments as novel VacA target structures.

  8. New Verapamil Analogs Inhibit Intracellular Mycobacteria without Affecting the Functions of Mycobacterium-Specific T Cells

    PubMed Central

    Ruminiski, Peter G.; Kumar, Malkeet; Singh, Kawaljit; Hamzabegovic, Fahreta; Hoft, Daniel F.; Eickhoff, Christopher S.; Selimovic, Asmir; Campbell, Mary; Chibale, Kelly

    2015-01-01

    There is a growing interest in repurposing mycobacterial efflux pump inhibitors, such as verapamil, for tuberculosis (TB) treatment. To aid in the design of better analogs, we studied the effects of verapamil on macrophages and Mycobacterium tuberculosis-specific T cells. Macrophage activation was evaluated by measuring levels of nitric oxide, tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), and gamma interferon (IFN-γ). Since verapamil is a known autophagy inducer, the roles of autophagy induction in the antimycobacterial activities of verapamil and norverapamil were studied using bone marrow-derived macrophages from ATG5flox/flox (control) and ATG5flox/flox Lyz-Cre mice. Our results showed that despite the well-recognized effects of verapamil on calcium channels and autophagy, its action on intracellular M. tuberculosis does not involve macrophage activation or autophagy induction. Next, the effects of verapamil and norverapamil on M. tuberculosis-specific T cells were assessed using flow cytometry following the stimulation of peripheral blood mononuclear cells from TB-skin-test-positive donors with M. tuberculosis whole-cell lysate for 7 days in the presence or absence of drugs. We found that verapamil and norverapamil inhibit the expansion of M. tuberculosis-specific T cells. Additionally, three new verapamil analogs were found to inhibit intracellular Mycobacterium bovis BCG, and one of the three analogs (KSV21) inhibited intracellular M. tuberculosis replication at concentrations that did not inhibit M. tuberculosis-specific T cell expansion. KSV21 also inhibited mycobacterial efflux pumps to the same degree as verapamil. More interestingly, the new analog enhances the inhibitory activities of isoniazid and rifampin on intracellular M. tuberculosis. In conclusion, KSV21 is a promising verapamil analog on which to base structure-activity relationship studies aimed at identifying more effective analogs. PMID:26643325

  9. Adenovirus 5 and chimeric adenovirus 5/F35 employ distinct B-lymphocyte intracellular trafficking routes that are independent of their cognate cell surface receptor.

    PubMed

    Drouin, Mathieu; Cayer, Marie-Pierre; Jung, Daniel

    2010-06-01

    Gene transfer applications with adenovirus (Ad) type 5 are limited by its native tropism, hampering their use in several cell types. To address this limitation, several Ad vectors bearing chimeric fiber have been produced to take advantage of the different cellular receptors used by other subgroups of Ads. In this study, we have compared the transduction efficiency of Ad5 and the chimeric Ad5/F35 in primary human B lymphocytes and B-cell lines as a function of the developmental stage. We found that transduction efficiencies of the two Ads differ independently of their targeted cellular receptor but are related to the intracellular localization of the virus. In efficiently transduced cells, Ads were localized in early endosomes or cytosol, whereas in poorly transduced cells they were localized within late endosomes/lysosomes. Finally, we demonstrate that treatment of cells with phosphatase inhibitors known to redirect endocytosis towards caveolae, increased Ad5/F35 transduction efficiency.

  10. Sex trafficking and the exploitation of adolescents.

    PubMed

    McClain, Natalie M; Garrity, Stacy E

    2011-01-01

    Human trafficking affects a surprisingly large number of adolescents around the globe. Women and girls make up the majority of sex trafficking victims. Nurses must be aware of sex trafficking as a form of sexual violence in the adolescent population. Nurses can play a role in identifying, intervening, and advocating for victims of human trafficking as they currently do for patients that are the victims of other types of violent crimes. PMID:21284727

  11. Preliminary Validation of the Sex Trafficking Attitudes Scale.

    PubMed

    Houston-Kolnik, Jaclyn D; Todd, Nathan R; Wilson, Midge

    2016-09-01

    This study presents the Sex Trafficking Attitudes Scale (STAS), assessing cognitive, behavioral, and affective attitudes toward the sex trafficking of women and girls. Across two studies, exploratory and confirmatory factor analyses revealed and confirmed six subscales: (a) Knowledge About Sex Trafficking, (b) Awareness of Sex Trafficking, (c) Attitudes Toward Ability to Leave Sex Trafficking, (d) Attitudes Toward Helping Survivors, (e) Empathic Reactions Toward Sex Trafficking, and (f) Efficacy to Reduce Sex Trafficking. Results showed support for convergent validity as the subscales were associated with related measures. The STAS holds promise to expand research and inform efforts to support trafficking survivors.

  12. A truncated intracellular HER2/neu receptor produced by alternative RNA processing affects growth of human carcinoma cells.

    PubMed Central

    Scott, G K; Robles, R; Park, J W; Montgomery, P A; Daniel, J; Holmes, W E; Lee, J; Keller, G A; Li, W L; Fendly, B M

    1993-01-01

    Cloned sequences encoding a truncated form of the HER2 receptor were obtained from cDNA libraries derived from two HER2-overexpressing human breast cancer cell lines, BT-474 and SK-BR-3. The 5' 2.1 kb of the encoded transcript is identical to that of full-length 4.6-kb HER2 transcript and would be expected to produce a secreted form of HER2 receptor containing only the extracellular ligand binding domain (ECD). The 3' end of the truncated transcript diverges 61 nucleotides before the receptor's transmembrane region, reads through a consensus splice donor site containing an in-frame stop codon, and contains a poly(A) addition site, suggesting that the truncated transcript arises by alternative RNA processing. S1 nuclease protection assays show a 40-fold variation in the abundance of the truncated 2.3-kb transcript relative to full-length 4.6-kb transcript in a panel of eight HER2-expressing tumor cell lines of gastric, ovarian, and breast cancer origin. Expression of this truncated transcript in COS-1 cells produces both secreted and intracellular forms of HER2 ECD; however, immunofluorescent labeling of HER2 ECD protein in MKN7 tumor cells that natively overexpress the 2.3-kb transcript suggests that transcriptionally generated HER2 ECD is concentrated within the perinuclear cytoplasm. Metabolic labeling and endoglycosidase studies suggest that this HER2 ECD (100 kDa) undergoes differential trafficking between the endoplasmic reticulum and Golgi compartments compared with full-length (185-kDa) HER2 receptor. Transfection studies indicate that excess production of HER2 ECD in human tumor cells overexpressing full-length HER2 receptor can result in resistance to the growth-inhibiting effects of anti-HER2 monoclonal antibodies such as muMAb4D5. These findings demonstrate alternative processing of the HER2 transcript and implicate a potentially important growth regulatory role for intracellularly sequestered HER2 ECD in HER2-amplified human tumors. Images PMID:8096058

  13. Glucosylceramide synthesis inhibition affects cell cycle progression, membrane trafficking, and stage differentiation in Giardia lamblia[S

    PubMed Central

    Štefanić, Saša; Spycher, Cornelia; Morf, Laura; Fabriàs, Gemma; Casas, Josefina; Schraner, Elisabeth; Wild, Peter; Hehl, Adrian B.; Sonda, Sabrina

    2010-01-01

    Synthesis of glucosylceramide via glucosylceramide synthase (GCS) is a crucial event in higher eukaryotes, both for the production of complex glycosphingolipids and for regulating cellular levels of ceramide, a potent antiproliferative second messenger. In this study, we explored the dependence of the early branching eukaryote Giardia lamblia on GCS activity. Biochemical analyses revealed that the parasite has a GCS located in endoplasmic reticulum (ER) membranes that is active in proliferating and encysting trophozoites. Pharmacological inhibition of GCS induced aberrant cell division, characterized by arrest of cytokinesis, incomplete cleavage furrow formation, and consequent block of replication. Importantly, we showed that increased ceramide levels were responsible for the cytokinesis arrest. In addition, GCS inhibition resulted in prominent ultrastructural abnormalities, including accumulation of cytosolic vesicles, enlarged lysosomes, and clathrin disorganization. Moreover, anterograde trafficking of the encystations-specific protein CWP1 was severely compromised and resulted in inhibition of stage differentiation. Our results reveal novel aspects of lipid metabolism in G. lamblia and specifically highlight the vital role of GCS in regulating cell cycle progression, membrane trafficking events, and stage differentiation in this parasite. In addition, we identified ceramide as a potent bioactive molecule, underscoring the universal conservation of ceramide signaling in eukaryotes. PMID:20335568

  14. Intracellular calcium affects prestin's voltage operating point indirectly via turgor-induced membrane tension

    NASA Astrophysics Data System (ADS)

    Song, Lei; Santos-Sacchi, Joseph

    2015-12-01

    Recent identification of a calmodulin binding site within prestin's C-terminus indicates that calcium can significantly alter prestin's operating voltage range as gauged by the Boltzmann parameter Vh (Keller et al., J. Neuroscience, 2014). We reasoned that those experiments may have identified the molecular substrate for the protein's tension sensitivity. In an effort to understand how this may happen, we evaluated the effects of turgor pressure on such shifts produced by calcium. We find that the shifts are induced by calcium's ability to reduce turgor pressure during whole cell voltage clamp recording. Clamping turgor pressure to 1kPa, the cell's normal intracellular pressure, completely counters the calcium effect. Furthermore, following unrestrained shifts, collapsing the cells abolishes induced shifts. We conclude that calcium does not work by direct action on prestin's conformational state. The possibility remains that calcium interaction with prestin alters water movements within the cell, possibly via its anion transport function.

  15. Adaptor Protein-1 Complex Affects the Endocytic Trafficking and Function of Peptidylglycine α-Amidating Monooxygenase, a Luminal Cuproenzyme.

    PubMed

    Bonnemaison, Mathilde L; Bäck, Nils; Duffy, Megan E; Ralle, Martina; Mains, Richard E; Eipper, Betty A

    2015-08-28

    The adaptor protein-1 complex (AP-1), which transports cargo between the trans-Golgi network and endosomes, plays a role in the trafficking of Atp7a, a copper-transporting P-type ATPase, and peptidylglycine α-amidating monooxygenase (PAM), a copper-dependent membrane enzyme. Lack of any of the four AP-1 subunits impairs function, and patients with MEDNIK syndrome, a rare genetic disorder caused by lack of expression of the σ1A subunit, exhibit clinical and biochemical signs of impaired copper homeostasis. To explore the role of AP-1 in copper homeostasis in neuroendocrine cells, we used corticotrope tumor cells in which AP-1 function was diminished by reducing expression of its μ1A subunit. Copper levels were unchanged when AP-1 function was impaired, but cellular levels of Atp7a declined slightly. The ability of PAM to function was assessed by monitoring 18-kDa fragment-NH2 production from proopiomelanocortin. Reduced AP-1 function made 18-kDa fragment amidation more sensitive to inhibition by bathocuproine disulfonate, a cell-impermeant Cu(I) chelator. The endocytic trafficking of PAM was altered, and PAM-1 accumulated on the cell surface when AP-1 levels were reduced. Reduced AP-1 function increased the Atp7a presence in early/recycling endosomes but did not alter the ability of copper to stimulate its appearance on the plasma membrane. Co-immunoprecipitation of a small fraction of PAM and Atp7a supports the suggestion that copper can be transferred directly from Atp7a to PAM, a process that can occur only when both proteins are present in the same subcellular compartment. Altered luminal cuproenzyme function may contribute to deficits observed when the AP-1 function is compromised.

  16. Adaptor Protein-1 Complex Affects the Endocytic Trafficking and Function of Peptidylglycine α-Amidating Monooxygenase, a Luminal Cuproenzyme*

    PubMed Central

    Bonnemaison, Mathilde L.; Bäck, Nils; Duffy, Megan E.; Ralle, Martina; Mains, Richard E.; Eipper, Betty A.

    2015-01-01

    The adaptor protein-1 complex (AP-1), which transports cargo between the trans-Golgi network and endosomes, plays a role in the trafficking of Atp7a, a copper-transporting P-type ATPase, and peptidylglycine α-amidating monooxygenase (PAM), a copper-dependent membrane enzyme. Lack of any of the four AP-1 subunits impairs function, and patients with MEDNIK syndrome, a rare genetic disorder caused by lack of expression of the σ1A subunit, exhibit clinical and biochemical signs of impaired copper homeostasis. To explore the role of AP-1 in copper homeostasis in neuroendocrine cells, we used corticotrope tumor cells in which AP-1 function was diminished by reducing expression of its μ1A subunit. Copper levels were unchanged when AP-1 function was impaired, but cellular levels of Atp7a declined slightly. The ability of PAM to function was assessed by monitoring 18-kDa fragment-NH2 production from proopiomelanocortin. Reduced AP-1 function made 18-kDa fragment amidation more sensitive to inhibition by bathocuproine disulfonate, a cell-impermeant Cu(I) chelator. The endocytic trafficking of PAM was altered, and PAM-1 accumulated on the cell surface when AP-1 levels were reduced. Reduced AP-1 function increased the Atp7a presence in early/recycling endosomes but did not alter the ability of copper to stimulate its appearance on the plasma membrane. Co-immunoprecipitation of a small fraction of PAM and Atp7a supports the suggestion that copper can be transferred directly from Atp7a to PAM, a process that can occur only when both proteins are present in the same subcellular compartment. Altered luminal cuproenzyme function may contribute to deficits observed when the AP-1 function is compromised. PMID:26170456

  17. Small GTPases in vesicle trafficking.

    PubMed

    Molendijk, Arthur J; Ruperti, Benedetto; Palme, Klaus

    2004-12-01

    Plant small GTPases belonging to the Rop, Arf, and Rab families are regulators of vesicle trafficking. Rop GTPases regulate actin dynamics and modulate H(2)O(2) production in polar cell growth and pathogen defence. A candidate Rop GDP to Rop GTP exchange factor (RopGEF) SPIKE1 is involved in the morphogenesis of leaf epidermal cells. The ArfGEF GNOM regulates the endosomal recycling of the PIN proteins, which are involved in polar auxin transport. Intracellular localisation of small GTPases and functional studies using dominant mutant versions of Arf and Rab GTPases are defining novel plant-specific membrane compartments, especially those that participate in endosomal vesicle trafficking.

  18. Calcium influx affects intracellular transport and membrane repair following nanosecond pulsed electric field exposure

    NASA Astrophysics Data System (ADS)

    Thompson, Gary Lee; Roth, Caleb C.; Dalzell, Danielle R.; Kuipers, Marjorie; Ibey, Bennett L.

    2014-05-01

    The cellular response to subtle membrane damage following exposure to nanosecond pulsed electric fields (nsPEF) is not well understood. Recent work has shown that when cells are exposed to nsPEF, ion permeable nanopores (<2 nm) are created in the plasma membrane in contrast to larger diameter pores (>2 nm) created by longer micro- and millisecond duration pulses. Nanoporation of the plasma membrane by nsPEF has been shown to cause a transient increase in intracellular calcium concentration within milliseconds after exposure. Our research objective is to determine the impact of nsPEF on calcium-dependent structural and repair systems in mammalian cells. Chinese hamster ovary (CHO-K1) cells were exposed in the presence and absence of calcium ions in the outside buffer to either 1 or 20, 600-ns duration electrical pulses at 16.2 kV/cm, and pore size was determined using propidium iodide and calcium green. Membrane organization was observed with morphological changes and increases in FM1-43 fluorescence. Migration of lysosomes, implicated in membrane repair, was followed using confocal microscopy of red fluorescent protein-tagged LAMP1. Microtubule structure was imaged using mEmerald-tubulin. We found that at high 600-ns PEF dosage, calcium-induced membrane restructuring and microtubule depolymerization coincide with interruption of membrane repair via lysosomal exocytosis.

  19. Relationship between Microtubule Network Structure and Intracellular Transport in Cultured Endothelial Cells Affected by Shear Stress

    NASA Astrophysics Data System (ADS)

    Kudo, Susumu; Ikezawa, Kenji; Ikeda, Mariko; Tanishita, Kazuo

    Endothelial cells (ECs) that line the inner surface of blood vessels are barriers to the transport of various substances into or from vessel walls, and are continuously exposed to shear stress induced by blood flow in vivo. Shear stress affects the cytoskeleton (e.g., microtubules, microfilaments, intermediate filaments), and affects the transport of macromolecules. Here, the relationship between the microtubule network structure and this transport process for albumin uptake within cultured aortic endothelial cells affected by shear stress was studied. Based on fluorescent images of albumin uptake obtained by using confocal laser scanning microscopy (CLSM), both the microtubule network and albumin uptake in ECs were disrupted by colchicine and were affected by shear stress loading.

  20. Photoperiod affects the expression of sex and species differences in leukocyte number and leukocyte trafficking in congeneric hamsters.

    PubMed

    Bilbo, S D; Dhabhar, F S; Viswanathan, K; Saul, A; Nelson, R J

    2003-11-01

    Sex differences in immune function are well documented. These sex differences may be modulated by social and environmental factors. Individuals of polygynous species generally exhibit more pronounced sex differences in immune parameters than individuals of monogamous species, often displaying an energetic trade-off between enhanced immunity and high mating success. During winter, animals contend with environmental conditions (e.g. low temperatures and decreased food availability) that evoke energetic-stress responses; many mammals restrict reproduction in response to photoperiod as part of an annual winter coping strategy. To test the hypothesis that extant sex and species differences in immune surveillance may be modulated by photoperiod, we examined leukocyte numbers in males and females of two closely related hamster species (Phodopus). As predicted, uniparental P. sungorus exhibited a robust sex difference, with total white blood cells, total lymphocytes, T cells, and B cells higher in females than males, during long days when reproduction occurs, but not during short days when reproduction usually stops. In contrast, biparental male and female P. campbelli exhibited comparable leukocyte numbers during both long and short days. To study sex differences in stress responses, we also examined immune cell trafficking in response to an acute (2 h) restraint stressor. During stressful challenges, it appears beneficial for immune cells to exit the blood and move to primary immune defense areas such as the skin, in preparation for potential injury or infection. Acute stress moved lymphocytes and monocytes out of the blood in all animals. Blood cortisol concentrations were increased in P. sungorus females compared to males at baseline (52%) and in response to restraint stress (38%), but only in long days. P. campbelli males and females exhibited comparable blood cortisol and stress responses during both long and short days. Our results suggest that interactions among

  1. Early steps in steroidogenesis: intracellular cholesterol trafficking.

    PubMed

    Miller, Walter L; Bose, Himangshu S

    2011-12-01

    Steroid hormones are made from cholesterol, primarily derived from lipoproteins that enter cells via receptor-mediated endocytosis. In endo-lysosomes, cholesterol is released from cholesterol esters by lysosomal acid lipase (LAL; disordered in Wolman disease) and exported via Niemann-Pick type C (NPC) proteins (disordered in NPC disease). These diseases are characterized by accumulated cholesterol and cholesterol esters in most cell types. Mechanisms for trans-cytoplasmic cholesterol transport, membrane insertion, and retrieval from membranes are less clear. Cholesterol esters and "free" cholesterol are enzymatically interconverted in lipid droplets. Cholesterol transport to the cholesterol-poor outer mitochondrial membrane (OMM) appears to involve cholesterol transport proteins. Cytochrome P450scc (CYP11A1) then initiates steroidogenesis by converting cholesterol to pregnenolone on the inner mitochondrial membrane (IMM). Acute steroidogenic responses are regulated by cholesterol delivery from OMM to IMM, triggered by the steroidogenic acute regulatory protein (StAR). Chronic steroidogenic capacity is determined by CYP11A1 gene transcription. StAR mutations cause congenital lipoid adrenal hyperplasia, with absent steroidogenesis, potentially lethal salt loss, and 46,XY sex reversal. StAR mutations initially destroy most, but not all steroidogenesis; low levels of StAR-independent steroidogenesis are lost later due to cellular damage, explaining the clinical findings. Rare P450scc mutations cause a similar syndrome. This review addresses these early steps in steroid biosynthesis. PMID:21976778

  2. Early steps in steroidogenesis: intracellular cholesterol trafficking

    PubMed Central

    Miller, Walter L.; Bose, Himangshu S.

    2011-01-01

    Steroid hormones are made from cholesterol, primarily derived from lipoproteins that enter cells via receptor-mediated endocytosis. In endo-lysosomes, cholesterol is released from cholesterol esters by lysosomal acid lipase (LAL; disordered in Wolman disease) and exported via Niemann-Pick type C (NPC) proteins (disordered in NPC disease). These diseases are characterized by accumulated cholesterol and cholesterol esters in most cell types. Mechanisms for trans-cytoplasmic cholesterol transport, membrane insertion, and retrieval from membranes are less clear. Cholesterol esters and “free” cholesterol are enzymatically interconverted in lipid droplets. Cholesterol transport to the cholesterol-poor outer mitochondrial membrane (OMM) appears to involve cholesterol transport proteins. Cytochrome P450scc (CYP11A1) then initiates steroidogenesis by converting cholesterol to pregnenolone on the inner mitochondrial membrane (IMM). Acute steroidogenic responses are regulated by cholesterol delivery from OMM to IMM, triggered by the steroidogenic acute regulatory protein (StAR). Chronic steroidogenic capacity is determined by CYP11A1 gene transcription. StAR mutations cause congenital lipoid adrenal hyperplasia, with absent steroidogenesis, potentially lethal salt loss, and 46,XY sex reversal. StAR mutations initially destroy most, but not all steroidogenesis; low levels of StAR-independent steroidogenesis are lost later due to cellular damage, explaining the clinical findings. Rare P450scc mutations cause a similar syndrome. This review addresses these early steps in steroid biosynthesis. PMID:21976778

  3. Bacterial translocation affects intracellular neuroinflammatory pathways in a depression-like model in rats.

    PubMed

    Martín-Hernández, David; Caso, Javier R; Bris, Álvaro G; Maus, Sandra R; Madrigal, José L M; García-Bueno, Borja; MacDowell, Karina S; Alou, Luis; Gómez-Lus, Maria Luisa; Leza, Juan C

    2016-04-01

    Recent studies have suggested that depression is accompanied by an increased intestinal permeability which would be related to the inflammatory pathophysiology of the disease. This study aimed to evaluate whether experimental depression presents with bacterial translocation that in turn can lead to the TLR-4 in the brain affecting the mitogen-activated protein kinases (MAPK) and antioxidant pathways. Male Wistar rats were exposed to chronic mild stress (CMS) and the intestinal integrity, presence of bacteria in tissues and plasma lipopolysaccharide levels were analyzed. We also studied the expression in the prefrontal cortex of activated forms of MAPK and some of their activation controllers and the effects of CMS on the antioxidant Nrf2 pathway. Our results indicate that after exposure to a CMS protocol there is increased intestinal permeability and bacterial translocation. CMS also increases the expression of the activated form of the MAPK p38 while decreasing the expression of the antioxidant transcription factor Nrf2. The actions of antibiotic administration to prevent bacterial translocation on elements of the MAPK and Nrf2 pathways indicate that the translocated bacteria are playing a role in these effects. In effect, our results propose a role of the translocated bacteria in the pathophysiology of depression through the p38 MAPK pathway which could aggravate the neuroinflammation and the oxidative/nitrosative damage present in this pathology. Moreover, our results reveal that the antioxidant factor Nrf2 and its activators may be involved in the consequences of the CMS on the brain.

  4. Dendritic diameters affect the spatial variability of intracellular calcium dynamics in computer models

    PubMed Central

    Anwar, Haroon; Roome, Christopher J.; Nedelescu, Hermina; Chen, Weiliang; Kuhn, Bernd; De Schutter, Erik

    2014-01-01

    There is growing interest in understanding calcium dynamics in dendrites, both experimentally and computationally. Many processes influence these dynamics, but in dendrites there is a strong contribution of morphology because the peak calcium levels are strongly determined by the surface to volume ratio (SVR) of each branch, which is inversely related to branch diameter. In this study we explore the predicted variance of dendritic calcium concentrations due to local changes in dendrite diameter and how this is affected by the modeling approach used. We investigate this in a model of dendritic calcium spiking in different reconstructions of cerebellar Purkinje cells and in morphological analysis of neocortical and hippocampal pyramidal neurons. We report that many published models neglect diameter-dependent effects on calcium concentration and show how to implement this correctly in the NEURON simulator, both for phenomenological pool based models and for implementations using radial 1D diffusion. More detailed modeling requires simulation of 3D diffusion and we demonstrate that this does not dissipate the local concentration variance due to changes of dendritic diameter. In many cases 1D diffusion of models of calcium buffering give a good approximation provided an increased morphological resolution is implemented. PMID:25100945

  5. Factors affecting a recently purchased handgun's risk for use in crime under circumstances that suggest gun trafficking.

    PubMed

    Wright, Mona A; Wintemute, Garen J; Webster, Daniel W

    2010-05-01

    While many handguns are used in crime each year in the USA, most are not. We conducted this study to identify factors present at the time of a handgun's most recent retail sale that were associated with its subsequent use in crime under circumstances suggesting that the handgun had been trafficked--purchased with the intent of diverting it to criminal use. Handguns acquired in multiple-gun purchases were of particular interest. Using data for 180,321 handguns purchased from federally licensed retailers in California in 1996, we studied attributes of the handguns, the retailers selling them, the purchasers, and the sales transactions. Our outcome measure was a handgun's recovery by a police agency, followed by a gun ownership trace, conducted by the Bureau of Alcohol, Tobacco, Firearms and Explosives, that determined (a) that the recovery had occurred within 3 years of the handgun's most recent purchase from a licensed retailer and (b) that the person who possessed the gun when it was recovered by police was not its most recent purchaser. Altogether, 722 handguns were recovered and had trace results that met the additional criteria. Handguns acquired in multiple-gun, same-day transactions were more likely to be traced than were single-purchase handguns (odds ratio [OR] 1.33, 95% confidence intervals [CI] 1.08 to 1.63). This was not the case for multiple-purchase handguns defined more broadly as multiple handguns purchased by one individual over any 30-day period as used in "one-gun-a-month" laws. Bivariate regressions indicated increased risk of a handgun being traced when it sold new for $150 or less (OR 4.28, 95% CI 3.59 to 5.11) or had been purchased by a woman (OR 2.02, 95% CI 1.62 to 2.52). Handguns sold by retailers who also had a relatively high proportion (>or=2%) of purchases denied because the prospective purchasers were prohibited from owning firearms were more likely to be traced than were those sold by other retailers (OR 4.09, 95% CI 3.39 to 4

  6. Replacement of Val3 in Human Thymidylate Synthase Affects Its Kinetic Properties and Intracellular Stability

    SciTech Connect

    Huang, Xiao; Gibson, Lydia M.; Bell, Brittnaie J.; Lovelace, Leslie L.; Pea, Maria Marjorette O.; Berger, Franklin G.; Berger, Sondra H.; Lebioda, Lukasz

    2010-11-03

    Human and other mammalian thymidylate synthase (TS) enzymes have an N-terminal extension of {approx}27 amino acids that is not present in bacterial TSs. The extension, which is disordered in all reported crystal structures of TSs, has been considered to play a primary role in protein turnover but not in catalytic activity. In mammalian cells, the variant V3A has a half-life similar to that of wild-type human TS (wt hTS) while V3T is much more stable; V3L, V3F, and V3Y have half-lives approximately half of that for wt hTS. Catalytic turnover rates for most Val3 mutants are only slightly diminished, as expected. However, two mutants, V3L and V3F, have strongly compromised dUMP binding, with K{sub m,app} values increased by factors of 47 and 58, respectively. For V3L, this observation can be explained by stabilization of the inactive conformation of the loop of residues 181-197, which prevents substrate binding. In the crystal structure of V3L, electron density corresponding to a leucine residue is present in a position that stabilizes the loop of residues 181-197 in the inactive conformation. Since this density is not observed in other mutants and all other leucine residues are ordered in this structure, it is likely that this density represents Leu3. In the crystal structure of a V3F {center_dot} FdUMP binary complex, the nucleotide is bound in an alternative mode to that proposed for the catalytic complex, indicating that the high K{sub m,app} value is caused not by stabilization of the inactive conformer but by substrate binding in a nonproductive, inhibitory site. These observations show that the N-terminal extension affects the conformational state of the hTS catalytic region. Each of the mechanisms leading to the high K{sub m,app} values can be exploited to facilitate design of compounds acting as allosteric inhibitors of hTS.

  7. Replacement of Val3 in human thymidylate synthase affects its kinetic properties and intracellular stability .

    PubMed

    Huang, Xiao; Gibson, Lydia M; Bell, Brittnaie J; Lovelace, Leslie L; Peña, Maria Marjorette O; Berger, Franklin G; Berger, Sondra H; Lebioda, Lukasz

    2010-03-23

    Human and other mammalian thymidylate synthase (TS) enzymes have an N-terminal extension of approximately 27 amino acids that is not present in bacterial TSs. The extension, which is disordered in all reported crystal structures of TSs, has been considered to play a primary role in protein turnover but not in catalytic activity. In mammalian cells, the variant V3A has a half-life similar to that of wild-type human TS (wt hTS) while V3T is much more stable; V3L, V3F, and V3Y have half-lives approximately half of that for wt hTS. Catalytic turnover rates for most Val3 mutants are only slightly diminished, as expected. However, two mutants, V3L and V3F, have strongly compromised dUMP binding, with K(m,app) values increased by factors of 47 and 58, respectively. For V3L, this observation can be explained by stabilization of the inactive conformation of the loop of residues 181-197, which prevents substrate binding. In the crystal structure of V3L, electron density corresponding to a leucine residue is present in a position that stabilizes the loop of residues 181-197 in the inactive conformation. Since this density is not observed in other mutants and all other leucine residues are ordered in this structure, it is likely that this density represents Leu3. In the crystal structure of a V3F.FdUMP binary complex, the nucleotide is bound in an alternative mode to that proposed for the catalytic complex, indicating that the high K(m,app) value is caused not by stabilization of the inactive conformer but by substrate binding in a nonproductive, inhibitory site. These observations show that the N-terminal extension affects the conformational state of the hTS catalytic region. Each of the mechanisms leading to the high K(m,app) values can be exploited to facilitate design of compounds acting as allosteric inhibitors of hTS.

  8. DL-alpha-difluoromethylarginine inhibits intracellular Trypanosoma cruzi multiplication by affecting cell division but not trypomastigote-amastigote transformation.

    PubMed

    Yakubu, M A; Basso, B; Kierszenbaum, F

    1992-06-01

    DL-alpha-difluoromethylarginine (DFMA), a specific, irreversible inhibitor of arginine decarboxylase (ADC), decreases the capacity of Trypanosoma cruzi to invade and multiply within different types of mammalian host cells in vitro. In this work we found that inhibition of intracellular growth results from selective impairment of amastigote division without appreciable alteration of the capacity of the invading trypomastigotes to transform into the replicative amastigote form. Addition of agmatine, the product of arginine decarboxylation, reversed the inhibitory effect of DFMA. Inhibition of ornithine decarboxylase activity by DL-alpha-difluoromethylornithine present in the medium prior to and during infection did not affect trypomastigote transformation or amastigote replication and did not change the magnitude of the inhibitory effect of DFMA on parasite multiplication. Hence, neither polyamine synthesis via the ornithine decarboxylase pathway nor salvage of host cell polyamines by T. cruzi appeared to be a likely explanation for the normal rate of parasite transformation that was seen in the presence of DFMA. Two clones of T. cruzi, TMSU-1 and TMSU-2, were tested for their degrees of sensitivity to the inhibitory effects of DFMA. Both trypomastigote association with (i.e., binding to and penetration of) myoblasts, and intracellular amastigote multiplication by either clone were found to be significantly (P less than 0.05) but not completely inhibited by DFMA. Therefore, the partial inhibition of T. cruzi infectivity and replication caused by DFMA is unlikely to represent a composite of effects of the drug on DFMA-sensitive and insensitive clones.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. 'IntraCell' plugin for assessment of intracellular localization of nano-delivery systems and their targeting to the individual organelles.

    PubMed

    Sneh-Edri, Hadas; Stepensky, David

    2011-02-11

    Efficient intracellular targeting of drugs and drug delivery systems (DDSs) is a major challenge that should be overcome to enhance the therapeutic efficiency of biopharmaceuticals and other intracellularly-acting drugs. Studies that quantitatively assess the mechanisms, barriers, and efficiency of intracellular drug delivery are required to determine the therapeutic potential of intracellular targeting of nano-delivery systems. In this study we report development and application of a novel 'IntraCell' plugin for ImageJ that is useful for quantitative assessment of uptake and intracellular localization of the drug/DDS and estimation of targeting efficiency. The developed plugin is based on threshold-based identification of borders of cell and of the individual organelles on confocal images and pixel-by-pixel analysis of fluorescence intensities. We applied the developed 'IntraCell' plugin to investigate uptake and intracellular targeting of novel endoplasmic reticulum (ER)-targeted delivery system based on PLGA nanoparticles decorated with ER-targeting or control peptides and encapsulating antigenic peptide and fluorescent marker. Decoration of the nanoparticles with peptidic residues affected their uptake and intracellular trafficking in HeLa cells, indicating that the targeting peptide was identified as ER-targeting signal by the intracellular trafficking mechanisms in HeLa cells and that these mechanisms can handle nano-DDS of the size comparable to some intracellular vesicles (hundreds of nanometers in diameter). We conclude that decoration of nanoparticles with peptidic residues affects their intracellular localization and trafficking and can be potentially used for intracellularly-targeted drug delivery. 'IntraCell' plugin is an useful tool for quantitative assessment of efficiency of uptake and intracellular drug targeting. In combination with other experimental approaches, it will be useful for the development of intracellularly-targeted formulations with

  10. Human Trafficking in the Emergency Department

    PubMed Central

    Patel, Ronak B.; Ahn, Roy; Burke, Thomas F.

    2010-01-01

    Human trafficking continues to persist, affecting up to 200 million people worldwide. As clinicians in emergency departments commonly encounter victims of intimate partner violence, some of these encounters will be with trafficking victims. These encounters provide a rare opportunity for healthcare providers to intervene and help. This case report of a human trafficking patient from a teaching hospital illustrates the complexity in identifying these victims. Clinicians can better identify potential trafficking cases by increasing their awareness of this phenomenon, using qualified interpreters, isolating potential victims by providing privacy and using simple clear reassuring statements ensuring security. A multidisciplinary approach can then be mobilized to help these patients. PMID:21293753

  11. Human telomerase: biogenesis, trafficking, recruitment, and activation.

    PubMed

    Schmidt, Jens C; Cech, Thomas R

    2015-06-01

    Telomerase is the ribonucleoprotein enzyme that catalyzes the extension of telomeric DNA in eukaryotes. Recent work has begun to reveal key aspects of the assembly of the human telomerase complex, its intracellular trafficking involving Cajal bodies, and its recruitment to telomeres. Once telomerase has been recruited to the telomere, it appears to undergo a separate activation step, which may include an increase in its repeat addition processivity. This review covers human telomerase biogenesis, trafficking, and activation, comparing key aspects with the analogous events in other species.

  12. Does low intensity He-Ne laser radiation affect the intracellular pH of intact Escherichia coli cells?

    NASA Astrophysics Data System (ADS)

    Quickenden, Terence I.; Daniels, Lillian L. L.; Byrne, Lyndsay T.

    1995-05-01

    Claims that low levels of He-Ne laser light (cw, (lambda) equals 632.8 nm) can provide clinical benefits and can enhance in vitro cellular growth are still controversial (T.I. Quickenden and L.L. Daniels, 1993, Photochem. Photobiol. 57, 272-278; L.L. Daniels and T.I. Quickenden, 1994, Photochem. Photobiol., 60, 481-485). The present study tests the suggestion (T.I. Karu, 1988, Lasers life Sci. 2, 53-74; H. Friedmann, R. Lubart, I. Laulicht and S. Rochkink, 1991, J. Photochem. Photobiol. B: Biol. 11, 87-91) that red light stimulates mitosis by raising intracellular pH via absorption by chromophores in the respiratory chain. In order to search for photoinduced changes in intracellular pH, the effect of 5 mW He-Ne laser irradiation on cultures of E. coli was examined using a 300 MHz Nuclear Magnetic Resonance (NMR) spectrometer. The pH difference between the intracellular and extracellular fluid was monitored in the presence and absence of radiation by determining the difference in chemical shift for 31P resonances arising from the H2PO4- ⇔ HPO42- + H+ equilibrium in the two environments.

  13. Binding to Na(+) /H(+) exchanger regulatory factor 2 (NHERF2) affects trafficking and function of the enteropathogenic Escherichia coli type III secretion system effectors Map, EspI and NleH.

    PubMed

    Martinez, Eric; Schroeder, Gunnar N; Berger, Cedric N; Lee, Sau Fung; Robinson, Keith S; Badea, Luminita; Simpson, Nandi; Hall, Randy A; Hartland, Elizabeth L; Crepin, Valerie F; Frankel, Gad

    2010-12-01

    Enteropathogenic Escherichia coli (EPEC) strains are diarrhoeal pathogens that use a type III secretion system to translocate effector proteins into host cells in order to colonize and multiply in the human gut. Map, EspI and NleH1 are conserved EPEC effectors that possess a C-terminal class I PSD-95/Disc Large/ZO-1 (PDZ)-binding motif. Using a PDZ array screen we identified Na(+)/H(+) exchanger regulatory factor 2 (NHERF2), a scaffold protein involved in tethering and recycling ion channels in polarized epithelia that contains two PDZ domains, as a common target of Map, EspI and NleH1. Using recombinant proteins and co-immunoprecipitation we confirmed that NHERF2 binds each of the effectors. We generated a HeLa cell line stably expressing HA-tagged NHERF2 and found that Map, EspI and NleH1 colocalize and interact with intracellular NHERF2 via their C-terminal PDZ-binding motif. Overexpression of NHERF2 enhanced the formation and persistence of Map-induced filopodia, accelerated the trafficking of EspI to the Golgi and diminished the anti-apoptotic activity of NleH1. The binding of multiple T3SS effectors to a single scaffold protein is unique. Our data suggest that NHERF2 may act as a plasma membrane sorting site, providing a novel regulatory mechanism to control the intracellular spatial and temporal effector protein activity.

  14. Deletion of GPR40 Impairs Glucose-Induced Insulin Secretion In Vivo in Mice Without Affecting Intracellular Fuel Metabolism in Islets

    SciTech Connect

    Alquier, Thierry; Peyot, Marie-Line; Latour, M. G.; Kebede, Melkam; Sorensen, Christina M.; Gesta, Stephane; Kahn, C. R.; Smith, Richard D.; Jetton, Thomas L.; Metz, Thomas O.; Prentki, Marc; Poitout, Vincent J.

    2009-11-01

    The G protein-coupled receptor GPR40 mediates fatty-acid potentiation of glucose-stimulated insulin secretion, but its contribution to insulin secretion in vivo and mechanisms of action remain uncertain. This study was aimed to ascertain whether GPR40 controls insulin secretion in vivo and modulates intracellular fuel metabolism in islets. We observed that glucose- and arginine-stimulated insulin secretion, assessed by hyperglycemic clamps, was decreased by approximately 60% in GPR40 knock-out (KO) fasted and fed mice, without changes in insulin sensitivity assessed by hyperinsulinemic-euglycemic clamps. Glucose and palmitate metabolism were not affected by GPR40 deletion. Lipid profiling revealed a similar increase in triglyceride and decrease in lysophosphatidylethanolamine species in WT and KO islets in response to palmitate. These results demonstrate that GPR40 regulates insulin secretion in vivo not only in response to fatty acids but also to glucose and arginine, without altering intracellular fuel metabolism.

  15. Trafficking vesicles: pro or contra pathogens?

    PubMed

    Frei dit Frey, Nicolas; Robatzek, Silke

    2009-08-01

    Membrane compartmentalization and trafficking are pivotal for eukaryotic life and demand a higher order of coordination. Even in their resting state, most plant cells exhibit a polarized localization of membrane compartments, which is redirected when plant cells are attacked by microbes. Repositioning of organelles at pathogen penetration sites has been reported since more than a decade; however, only recently has targeted vesicle trafficking upon biotic stress emerged. It has become evident that vesicle secretion and endocytic pathways are engaged in the plant's immune system to actively defend against potential pathogens. By contrast, invasive pathogens have evolved means to utilize these trafficking pathways for the suppression of plant defenses and to the benefit of microbial proliferation. This review summarizes recent findings of host intracellular endomembrane adaptations in response to pathogens and how pathogens exploit them. PMID:19608452

  16. Glycosaminoglycans: Sorting determinants in intracellular protein traffic.

    PubMed

    Mihov, Deyan; Spiess, Martin

    2015-11-01

    Intracellular transport of proteins to their appropriate destinations is crucial for the maintenance of cellular integrity and function. Sorting information is contained either directly in the amino acid sequence or in a protein's post-translational modifications. Glycosaminoglycans (GAGs) are characteristic modifications of proteoglycans. GAGs are long unbranched polysaccharide chains with unique structural and functional properties also contributing to protein sorting in various ways. By deletion or insertion of GAG attachment sites it has been shown that GAGs affect polarized sorting in epithelial cells, targeting to and storage in secretory granules, and endocytosis. Most recently, the role of GAGs as signals for rapid trans-Golgi-to-cell surface transport, dominant over the cytosolic sorting motifs in the core protein, was demonstrated. Here, we provide an overview on existing data on the roles of GAGs on protein and proteoglycan trafficking.

  17. SAP97-mediated ADAM10 trafficking from Golgi outposts depends on PKC phosphorylation

    PubMed Central

    Saraceno, C; Marcello, E; Di Marino, D; Borroni, B; Claeysen, S; Perroy, J; Padovani, A; Tramontano, A; Gardoni, F; Di Luca, M

    2014-01-01

    A disintegrin and metalloproteinase 10 (ADAM10) is the major α-secretase that catalyzes the amyloid precursor protein (APP) ectodomain shedding in the brain and prevents amyloid formation. Its activity depends on correct intracellular trafficking and on synaptic membrane insertion. Here, we describe that in hippocampal neurons the synapse-associated protein-97 (SAP97), an excitatory synapse scaffolding element, governs ADAM10 trafficking from dendritic Golgi outposts to synaptic membranes. This process is mediated by a previously uncharacterized protein kinase C phosphosite in SAP97 SRC homology 3 domain that modulates SAP97 association with ADAM10. Such mechanism is essential for ADAM10 trafficking from the Golgi outposts to the synapse, but does not affect ADAM10 transport from the endoplasmic reticulum. Notably, this process is altered in Alzheimer's disease brains. These results help in understanding the mechanism responsible for the modulation of ADAM10 intracellular path, and can constitute an innovative therapeutic strategy to finely tune ADAM10 shedding activity towards APP. PMID:25429624

  18. A monoclonal antibody (PL/IM 430) to human platelet intracellular membranes which inhibits the uptake of Ca2+ without affecting the Ca2+ +Mg2+-ATPase.

    PubMed

    Hack, N; Wilkinson, J M; Crawford, N

    1988-03-01

    To probe the structure-function relationships of proteins present in the endoplasmic reticulum-like intracellular membranes of human blood platelets a panel of monoclonal antibodies have been raised, using as immunogen highly purified platelet intracellular membrane vesicles isolated by continuous flow electrophoresis [Menashi, Weintroub & Crawford (1981) J. Biol. Chem. 256, 4095-4101]. Four of these antibodies recognize a single 100 kDa polypeptide in the platelet membrane by immunoblotting. One antibody PL/IM 430 (of IgG1 subclass) inhibited (approximately 70%) the energy-dependent uptake of Ca2+ into the vesicles without affecting the Ca2+ +Mg2+-ATPase activity or the protein phosphorylation previously shown to proceed concomitantly with Ca2+ sequestration [Hack, Croset & Crawford (1986) Biochem. J. 233, 661-668]. The inhibition is independent of ATP concentration over a range 0-2 mM-ATP but shows dose-dependency for external [Ca2+] with maximum inhibition of Ca2+ translocation at concentrations of Ca2+ greater than 500 nM. This capacity of the antibody PL/IM 430 functionally to dislocate components of the intracellular membrane Ca2+ pump complex may have value in structural studies.

  19. 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.

  20. Monitoring receptor trafficking following retromer and WASH deregulation.

    PubMed

    Osborne, Douglas G; Phillips-Krawczak, Christine A; Billadeau, Daniel D

    2015-01-01

    Cell surface receptors that have been internalized and enter the endocytic pathway have multiple fates including entrance into the multivesicular body pathway on their way to lysosomal degradation, recycling back to the cell surface, or retrograde trafficking out of the endolysosomal system back to the Golgi apparatus. Two ubiquitously expressed protein complexes, WASH and the endosomal coat complex retromer, function together to play a central role in directing the fate of receptors into the latter two pathways. In this chapter, we describe fluorescent- and flow cytometry-based methods for analyzing the recycling and retrograde trafficking of two receptors, α5β1 and CI-M6PR, whose intracellular fates are regulated by WASH and retromer activity. The guidelines presented in this chapter can be applied to the analysis of any cell surface or intracellular membrane protein to determine the impact of WASH or retromer deregulation on its intracellular trafficking route. PMID:26360036

  1. Regulation of aquaporin-2 trafficking.

    PubMed

    Nedvetsky, Pavel I; Tamma, Grazia; Beulshausen, Sven; Valenti, Giovanna; Rosenthal, Walter; Klussmann, Enno

    2009-01-01

    Principal cells lining renal collecting ducts control the fine-tuning of body water homeostasis by regulating water reabsorption through the water channels aquaporin-2 (AQP2), aquaporin-3 (AQP3), and aquaporin-4 (AQP4). While the localization of AQP2 is subject to regulation by arginine-vasopressin (AVP), AQP3 and AQP4 are constitutively expressed in the basolateral plasma membrane. AVP adjusts the amount of AQP2 in the plasma membrane by triggering its redistribution from intracellular vesicles into the plasma membrane. This permits water entry into the cells and water exit through AQP3 and AQP4. The translocation of AQP2 is initiated by an increase in cAMP following V2R activation through AVP. The AVP-induced rise in cAMP activates protein kinase A (PKA), which in turn phosphorylates AQP2, and thereby triggers the redistribution of AQP2. Several proteins participating in the control of cAMP-dependent AQP2 trafficking have been identified; for example, A kinase anchoring proteins (AKAPs) tethering PKA to cellular compartments; phosphodiesterases (PDEs) regulating the local cAMP level; cytoskeletal components such as F-actin and microtubules; small GTPases of the Rho family controlling cytoskeletal dynamics; motor proteins transporting AQP2-bearing vesicles to and from the plasma membrane for exocytic insertion and endocytic retrieval; SNAREs inducing membrane fusions, hsc70, a chaperone, important for endocytic retrieval. In addition, cAMP-independent mechanisms of translocation mainly involving the F-actin cytoskeleton have been uncovered. Defects of AQP2 trafficking cause diseases such as nephrogenic diabetes insipidus (NDI), a disorder characterized by a massive loss of hypoosmotic urine.This review summarizes recent data elucidating molecular mechanisms underlying the trafficking of AQP2. In particular, we focus on proteins involved in the regulation of trafficking, and physiological and pathophysiological stimuli determining the cellular localization of AQP2

  2. Modulation of intracellular calcium levels by calcium lactate affects colon cancer cell motility through calcium-dependent calpain.

    PubMed

    Sundaramoorthy, Pasupathi; Sim, Jae Jun; Jang, Yeong-Su; Mishra, Siddhartha Kumar; Jeong, Keun-Yeong; Mander, Poonam; Chul, Oh Byung; Shim, Won-Sik; Oh, Seung Hyun; Nam, Ky-Youb; Kim, Hwan Mook

    2015-01-01

    Cancer cell motility is a key phenomenon regulating invasion and metastasis. Focal adhesion kinase (FAK) plays a major role in cellular adhesion and metastasis of various cancers. The relationship between dietary supplementation of calcium and colon cancer has been extensively investigated. However, the effect of calcium (Ca2+) supplementation on calpain-FAK-motility is not clearly understood. We sought to identify the mechanism of FAK cleavage through Ca2+ bound lactate (CaLa), its downstream signaling and role in the motility of human colon cancer cells. We found that treating HCT116 and HT-29 cells with CaLa immediately increased the intracellular Ca2+ (iCa2+) levels for a prolonged period of time. Ca2+ influx induced cleavage of FAK into an N-terminal FAK (FERM domain) in a dose-dependent manner. Phosphorylated FAK (p-FAK) was also cleaved in to its p-N-terminal FAK. CaLa increased colon cancer cells motility. Calpeptin, a calpain inhibitor, reversed the effects of CaLa on FAK and pFAK cleavage in both cancer cell lines. The cleaved FAK translocates into the nucleus and modulates p53 stability through MDM2-associated ubiquitination. CaLa-induced Ca2+ influx increased the motility of colon cancer cells was mediated by calpain activity through FAK and pFAK protein destabilization. In conclusion, these results suggest that careful consideration may be given in deciding dietary Ca2+ supplementation to patient undergoing treatment for metastatic cancer.

  3. The abcEDCBA-Encoded ABC Transporter and the virB Operon-Encoded Type IV Secretion System of Brucella ovis Are Critical for Intracellular Trafficking and Survival in Ovine Monocyte-Derived Macrophages.

    PubMed

    Macedo, Auricelio A; Silva, Ana P C; Mol, Juliana P S; Costa, Luciana F; Garcia, Luize N N; Araújo, Marcio S; Martins Filho, Olindo A; Paixão, Tatiane A; Santos, Renato L

    2015-01-01

    Brucella ovis infection is associated with epididymitis, orchitis and infertility in rams. Most of the information available on B. ovis and host cell interaction has been generated using murine macrophages or epithelial cell lines, but the interaction between B. ovis and primary ovine macrophages has not been studied. The aim of this study was to evaluate the role of the B. ovis abcEDCBA-encoded ABC transporter and the virB operon-encoded Type IV Secretion System (T4SS) during intracellular survival of B. ovis in ovine peripheral blood monocyte-derived macrophages. ΔabcBA and ΔvirB2 mutant strains were unable to survive in the intracellular environment when compared to the WT B. ovis at 48 hours post infection (hpi). In addition, these mutant strains cannot exclude the lysosomal marker LAMP1 from its vacuolar membrane, and their vacuoles do not acquire the endoplasmic reticulum marker calreticulin, which takes place in the WT B. ovis containing vacuole. Higher levels of nitric oxide production were observed in macrophages infected with WT B. ovis at 48 hpi when compared to macrophages infected with the ΔabcBA or ΔvirB2 mutant strains. Conversely, higher levels of reactive oxygen species were detected in macrophages infected with the ΔabcBA or ΔvirB2 mutant strains at 48 hpi when compared to macrophages infected with the WT strain. Our results demonstrate that B. ovis is able to persist and multiply in ovine macrophages, while ΔabcBA and ΔvirB2 mutations prevent intracellular multiplication, favor phagolysosome fusion, and impair maturation of the B. ovis vacuole towards an endoplasmic reticulum-derived compartment. PMID:26366863

  4. The abcEDCBA-Encoded ABC Transporter and the virB Operon-Encoded Type IV Secretion System of Brucella ovis Are Critical for Intracellular Trafficking and Survival in Ovine Monocyte-Derived Macrophages

    PubMed Central

    Macedo, Auricelio A.; Silva, Ana P. C.; Mol, Juliana P. S.; Costa, Luciana F.; Garcia, Luize N. N.; Araújo, Marcio S.; Martins Filho, Olindo A.; Paixão, Tatiane A.; Santos, Renato L.

    2015-01-01

    Brucella ovis infection is associated with epididymitis, orchitis and infertility in rams. Most of the information available on B. ovis and host cell interaction has been generated using murine macrophages or epithelial cell lines, but the interaction between B. ovis and primary ovine macrophages has not been studied. The aim of this study was to evaluate the role of the B. ovis abcEDCBA-encoded ABC transporter and the virB operon-encoded Type IV Secretion System (T4SS) during intracellular survival of B. ovis in ovine peripheral blood monocyte-derived macrophages. ΔabcBA and ΔvirB2 mutant strains were unable to survive in the intracellular environment when compared to the WT B. ovis at 48 hours post infection (hpi). In addition, these mutant strains cannot exclude the lysosomal marker LAMP1 from its vacuolar membrane, and their vacuoles do not acquire the endoplasmic reticulum marker calreticulin, which takes place in the WT B. ovis containing vacuole. Higher levels of nitric oxide production were observed in macrophages infected with WT B. ovis at 48 hpi when compared to macrophages infected with the ΔabcBA or ΔvirB2 mutant strains. Conversely, higher levels of reactive oxygen species were detected in macrophages infected with the ΔabcBA or ΔvirB2 mutant strains at 48 hpi when compared to macrophages infected with the WT strain. Our results demonstrate that B. ovis is able to persist and multiply in ovine macrophages, while ΔabcBA and ΔvirB2 mutations prevent intracellular multiplication, favor phagolysosome fusion, and impair maturation of the B. ovis vacuole towards an endoplasmic reticulum-derived compartment. PMID:26366863

  5. Amastin Knockdown in Leishmania braziliensis Affects Parasite-Macrophage Interaction and Results in Impaired Viability of Intracellular Amastigotes

    PubMed Central

    Nakagaki, Brenda Naemi; Mendonça-Neto, Rondon Pessoa; Canavaci, Adriana Monte Cassiano; Souza Melo, Normanda; Martinelli, Patrícia Massara; Fernandes, Ana Paula; daRocha, Wanderson Duarte; Teixeira, Santuza M. R.

    2015-01-01

    Leishmaniasis, a human parasitic disease with manifestations ranging from cutaneous ulcerations to fatal visceral infection, is caused by several Leishmania species. These protozoan parasites replicate as extracellular, flagellated promastigotes in the gut of a sandfly vector and as amastigotes inside the parasitophorous vacuole of vertebrate host macrophages. Amastins are surface glycoproteins encoded by large gene families present in the genomes of several trypanosomatids and highly expressed in the intracellular amastigote stages of Trypanosoma cruzi and Leishmania spp. Here, we showed that the genome of L. braziliensis contains 52 amastin genes belonging to all four previously described amastin subfamilies and that the expression of members of all subfamilies is upregulated in L. braziliensis amastigotes. Although primary sequence alignments showed no homology to any known protein sequence, homology searches based on secondary structure predictions indicate that amastins are related to claudins, a group of proteins that are components of eukaryotic tight junction complexes. By knocking-down the expression of δ-amastins in L. braziliensis, their essential role during infection became evident. δ-amastin knockdown parasites showed impaired growth after in vitro infection of mouse macrophages and completely failed to produce infection when inoculated in BALB/c mice, an attenuated phenotype that was reverted by the re-expression of an RNAi-resistant amastin gene. Further highlighting their essential role in host-parasite interactions, electron microscopy analyses of macrophages infected with amastin knockdown parasites showed significant alterations in the tight contact that is normally observed between the surface of wild type amastigotes and the membrane of the parasitophorous vacuole. PMID:26641088

  6. Modulation of Intracellular Calcium Levels by Calcium Lactate Affects Colon Cancer Cell Motility through Calcium-Dependent Calpain

    PubMed Central

    Sundaramoorthy, Pasupathi; Sim, Jae Jun; Jang, Yeong-Su; Mishra, Siddhartha Kumar; Jeong, Keun-Yeong; Mander, Poonam; Chul, Oh Byung; Shim, Won-Sik; Oh, Seung Hyun; Nam, Ky-Youb; Kim, Hwan Mook

    2015-01-01

    Cancer cell motility is a key phenomenon regulating invasion and metastasis. Focal adhesion kinase (FAK) plays a major role in cellular adhesion and metastasis of various cancers. The relationship between dietary supplementation of calcium and colon cancer has been extensively investigated. However, the effect of calcium (Ca2+) supplementation on calpain-FAK-motility is not clearly understood. We sought to identify the mechanism of FAK cleavage through Ca2+ bound lactate (CaLa), its downstream signaling and role in the motility of human colon cancer cells. We found that treating HCT116 and HT-29 cells with CaLa immediately increased the intracellular Ca2+ (iCa2+) levels for a prolonged period of time. Ca2+ influx induced cleavage of FAK into an N-terminal FAK (FERM domain) in a dose-dependent manner. Phosphorylated FAK (p-FAK) was also cleaved in to its p-N-terminal FAK. CaLa increased colon cancer cells motility. Calpeptin, a calpain inhibitor, reversed the effects of CaLa on FAK and pFAK cleavage in both cancer cell lines. The cleaved FAK translocates into the nucleus and modulates p53 stability through MDM2-associated ubiquitination. CaLa-induced Ca2+ influx increased the motility of colon cancer cells was mediated by calpain activity through FAK and pFAK protein destabilization. In conclusion, these results suggest that careful consideration may be given in deciding dietary Ca2+ supplementation to patient undergoing treatment for metastatic cancer. PMID:25629974

  7. Brucella suis Vaccine Strain 2 Induces Endoplasmic Reticulum Stress that Affects Intracellular Replication in Goat Trophoblast Cells In vitro

    PubMed Central

    Wang, Xiangguo; Lin, Pengfei; Li, Yang; Xiang, Caixia; Yin, Yanlong; Chen, Zhi; Du, Yue; Zhou, Dong; Jin, Yaping; Wang, Aihua

    2016-01-01

    Brucella has been reported to impair placental trophoblasts, a cellular target where Brucella efficiently replicates in association with the endoplasmic reticulum (ER), and ultimately trigger abortion in pregnant animals. However, the precise effects of Brucella on trophoblast cells remain unclear. Here, we describe the infection and replication of Brucella suis vaccine strain 2 (B.suis.S2) in goat trophoblast cells (GTCs) and the cellular and molecular responses induced in vitro. Our studies demonstrated that B.suis.S2 was able to infect and proliferate to high titers, hamper the proliferation of GTCs and induce apoptosis due to ER stress. Tunicamycin (Tm), a pharmacological chaperone that strongly mounts ER stress-induced apoptosis, inhibited B.suis.S2 replication in GTCs. In addition, 4 phenyl butyric acid (4-PBA), a pharmacological chaperone that alleviates ER stress-induced apoptosis, significantly enhanced B.suis.S2 replication in GTCs. The Unfolded Protein Response (UPR) chaperone molecule GRP78 also promoted B.suis.S2 proliferation in GTCs by inhibiting ER stress-induced apoptosis. We also discovered that the IRE1 pathway, but not the PERK or ATF6 pathway, was activated in the process. However, decreasing the expression of phosphoIRE1α and IRE1α proteins with Irestatin 9389 (IRE1 antagonist) in GTCs did not affect the proliferation of B.suis.S2. Although GTC implantation was not affected upon B.suis.S2 infection, progesterone secretion was suppressed, and prolactin and estrogen secretion increased; these effects were accompanied by changes in the expression of genes encoding key steroidogenic enzymes. This study systematically explored the mechanisms of abortion in Brucella infection from the viewpoint of pathogen invasion, ER stress and reproductive endocrinology. Our findings may provide new insight for understanding the mechanisms involved in goat abortions caused by Brucella infection. PMID:26904517

  8. Intracellular transport of nanocarriers across the intestinal epithelium.

    PubMed

    Fan, Weiwei; Xia, Dengning; Zhu, Quanlei; Hu, Lei; Gan, Yong

    2016-05-01

    The intestinal epithelium is the main barrier restricting the oral delivery of low-permeability drugs. Over recent years, numerous nanocarriers have been designed to improve the efficiency of oral drug delivery. However, the intracellular processes determining the transport of nanocarriers across the intestinal epithelium remain elusive, and only limited enhancement of the oral bioavailability of drugs has been achieved. Here, we review the processes involved in nanocarrier trafficking across the intestinal epithelium, including apical endocytosis, intracellular transport, and basolateral exocytosis. Understanding the complex intracellular processes of nanocarrier trafficking is particularly essential for the rational design of oral drug delivery systems. PMID:27094490

  9. Intracellular proteoglycans.

    PubMed Central

    Kolset, Svein Olav; Prydz, Kristian; Pejler, Gunnar

    2004-01-01

    Proteoglycans (PGs) are proteins with glycosaminoglycan chains, are ubiquitously expressed and have a wide range of functions. PGs in the extracellular matrix and on the cell surface have been the subject of extensive structural and functional studies. Less attention has so far been given to PGs located in intracellular compartments, although several reports suggest that these have biological functions in storage granules, the nucleus and other intracellular organelles. The purpose of this review is, therefore, to present some of these studies and to discuss possible functions linked to PGs located in different intracellular compartments. Reference will be made to publications relevant for the topics we present. It is beyond the scope of this review to cover all publications on PGs in intracellular locations. PMID:14759226

  10. Molecular dissection of Phaseolus vulgaris polygalacturonase-inhibiting protein 2 reveals the presence of hold/release domains affecting protein trafficking toward the cell wall

    PubMed Central

    De Caroli, Monica; Lenucci, Marcello S.; Manualdi, Francesca; Dalessandro, Giuseppe; De Lorenzo, Giulia; Piro, Gabriella

    2015-01-01

    The plant endomembrane system is massively involved in the synthesis, transport and secretion of cell wall polysaccharides and proteins; however, the molecular mechanisms underlying trafficking toward the apoplast are largely unknown. Besides constitutive, the existence of a regulated secretory pathway has been proposed. A polygalacturonase inhibitor protein (PGIP2), known to move as soluble cargo and reach the cell wall through a mechanism distinguishable from default, was dissected in its main functional domains (A, B, C, D), and C sub-fragments (C1–10), to identify signals essential for its regulated targeting. The secretion patterns of the fluorescent chimeras obtained by fusing different PGIP2 domains to the green fluorescent protein (GFP) were analyzed. PGIP2 N-terminal and leucine-rich repeat domains (B and C, respectively) seem to operate as holding/releasing signals, respectively, during PGIP2 transit through the Golgi. The B domain slows down PGIP2 secretion by transiently interacting with Golgi membranes. Its depletion leads, in fact, to the secretion via default (Sp2-susceptible) of the ACD-GFP chimera faster than PGIP2. Depending on its length (at least the first 5 leucine-rich repeats are required), the C domain modulates B interaction with Golgi membranes allowing the release of chimeras and their extracellular secretion through a Sp2 independent pathway. The addition of the vacuolar sorting determinant Chi to PGIP2 diverts the path of the protein from cell wall to vacuole, suggesting that C domain is a releasing rather than a cell wall sorting signal. PMID:26379688

  11. Molecular dissection of Phaseolus vulgaris polygalacturonase-inhibiting protein 2 reveals the presence of hold/release domains affecting protein trafficking toward the cell wall.

    PubMed

    De Caroli, Monica; Lenucci, Marcello S; Manualdi, Francesca; Dalessandro, Giuseppe; De Lorenzo, Giulia; Piro, Gabriella

    2015-01-01

    The plant endomembrane system is massively involved in the synthesis, transport and secretion of cell wall polysaccharides and proteins; however, the molecular mechanisms underlying trafficking toward the apoplast are largely unknown. Besides constitutive, the existence of a regulated secretory pathway has been proposed. A polygalacturonase inhibitor protein (PGIP2), known to move as soluble cargo and reach the cell wall through a mechanism distinguishable from default, was dissected in its main functional domains (A, B, C, D), and C sub-fragments (C1-10), to identify signals essential for its regulated targeting. The secretion patterns of the fluorescent chimeras obtained by fusing different PGIP2 domains to the green fluorescent protein (GFP) were analyzed. PGIP2 N-terminal and leucine-rich repeat domains (B and C, respectively) seem to operate as holding/releasing signals, respectively, during PGIP2 transit through the Golgi. The B domain slows down PGIP2 secretion by transiently interacting with Golgi membranes. Its depletion leads, in fact, to the secretion via default (Sp2-susceptible) of the ACD-GFP chimera faster than PGIP2. Depending on its length (at least the first 5 leucine-rich repeats are required), the C domain modulates B interaction with Golgi membranes allowing the release of chimeras and their extracellular secretion through a Sp2 independent pathway. The addition of the vacuolar sorting determinant Chi to PGIP2 diverts the path of the protein from cell wall to vacuole, suggesting that C domain is a releasing rather than a cell wall sorting signal.

  12. TLR signals posttranscriptionally regulate the cytokine trafficking mediator sortilin

    PubMed Central

    Yabe-Wada, Toshiki; Matsuba, Shintaro; Takeda, Kazuya; Sato, Tetsuya; Suyama, Mikita; Ohkawa, Yasuyuki; Takai, Toshiyuki; Shi, Haifeng; Philpott, Caroline C.; Nakamura, Akira

    2016-01-01

    Regulating the transcription, translation and secretion of cytokines is crucial for controlling the appropriate balance of inflammation. Here we report that the sorting receptor sortilin plays a key role in cytokine production. We observed interactions of sortilin with multiple cytokines including IFN-α, and sortilin depletion in plasmacytoid dendritic cells (pDCs) led to a reduction of IFN-α secretion, suggesting a pivotal role of sortilin in the exocytic trafficking of IFN-α in pDCs. Moreover, sortilin mRNA was degraded posttranscriptionally upon stimulation with various TLR ligands. Poly-rC-binding protein 1 (PCBP1) recognized the C-rich element (CRE) in the 3′ UTR of sortilin mRNA, and depletion of PCBP1 enhanced the degradation of sortilin transcripts, suggesting that PCBP1 can act as a trans-acting factor to stabilize sortilin transcripts. The nucleotide-binding ability of PCBP1 was impaired by zinc ions and alterations of intracellular zinc affect sortilin expression. PCBP1 may therefore control the stability of sortilin transcripts by sensing intracellular zinc levels. Collectively, our findings provide insights into the posttranslational regulation of cytokine production through the posttranscriptional control of sortilin expression by TLR signals. PMID:27220277

  13. Stochastic models of intracellular transport

    NASA Astrophysics Data System (ADS)

    Bressloff, Paul C.; Newby, Jay M.

    2013-01-01

    The interior of a living cell is a crowded, heterogenuous, fluctuating environment. Hence, a major challenge in modeling intracellular transport is to analyze stochastic processes within complex environments. Broadly speaking, there are two basic mechanisms for intracellular transport: passive diffusion and motor-driven active transport. Diffusive transport can be formulated in terms of the motion of an overdamped Brownian particle. On the other hand, active transport requires chemical energy, usually in the form of adenosine triphosphate hydrolysis, and can be direction specific, allowing biomolecules to be transported long distances; this is particularly important in neurons due to their complex geometry. In this review a wide range of analytical methods and models of intracellular transport is presented. In the case of diffusive transport, narrow escape problems, diffusion to a small target, confined and single-file diffusion, homogenization theory, and fractional diffusion are considered. In the case of active transport, Brownian ratchets, random walk models, exclusion processes, random intermittent search processes, quasi-steady-state reduction methods, and mean-field approximations are considered. Applications include receptor trafficking, axonal transport, membrane diffusion, nuclear transport, protein-DNA interactions, virus trafficking, and the self-organization of subcellular structures.

  14. Protein trafficking during plant innate immunity.

    PubMed

    Wang, Wen-Ming; Liu, Peng-Qiang; Xu, Yong-Ju; Xiao, Shunyuan

    2016-04-01

    Plants have evolved a sophisticated immune system to fight against pathogenic microbes. Upon detection of pathogen invasion by immune receptors, the immune system is turned on, resulting in production of antimicrobial molecules including pathogenesis-related (PR) proteins. Conceivably, an efficient immune response depends on the capacity of the plant cell's protein/membrane trafficking network to deploy the right defense-associated molecules in the right place at the right time. Recent research in this area shows that while the abundance of cell surface immune receptors is regulated by endocytosis, many intracellular immune receptors, when activated, are partitioned between the cytoplasm and the nucleus for induction of defense genes and activation of programmed cell death, respectively. Vesicle transport is an essential process for secretion of PR proteins to the apoplastic space and targeting of defense-related proteins to the plasma membrane or other endomembrane compartments. In this review, we discuss the various aspects of protein trafficking during plant immunity, with a focus on the immunity proteins on the move and the major components of the trafficking machineries engaged. PMID:26345282

  15. Intracellular ice and cell survival in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum: an ultrastructural study of factors affecting cell and ice structures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cryogenic technologies are required to preserve embryonic axes of recalcitrant seeds. Formation of potentially lethal intracellular ice limits successful cryopreservation; thus, it is important to understand the relationships among cryo-exposure techniques, water content and survival. In this pap...

  16. Trafficking of Estrella lausannensis in human macrophages.

    PubMed

    Rusconi, Brigida; Kebbi-Beghdadi, Carole; Greub, Gilbert

    2015-07-01

    Estrella lausannensis is a new member of the Chlamydiales order. Like other Chlamydia-related bacteria, it is able to replicate in amoebae and in fish cell lines. A preliminary study investigating the pathogenic potential of Chlamydia-related bacteria found a correlation between antibody response to E. lausannensis and pneumonia in children. To further investigate the pathogenic potential of E. lausannensis, we determined its ability to grow in human macrophages and its intracellular trafficking. The replication in macrophages resulted in viable E. lausannensis; however, it caused a significant cytopathic effect. The intracellular trafficking of E. lausannensis was analyzed by determining the interaction of the Estrella-containing inclusions with various endocytic markers as well as host organelles. The E. lausannensis inclusion escaped the endocytic pathway rapidly avoiding maturation into phagolysosomes by preventing both EEA-1 and LAMP-1 accumulation. Compared to Waddlia chondrophila, another Chlamydia-related bacteria, the recruitment of mitochondria and endoplasmic reticulum was minimal for E. lausannensis inclusions. Estrella lausannensis appears to use a distinct source of nutrients and energy compared to other members of the Chlamydiales order. In conclusion, we hypothesize that E. lausannensis has a restricted growth in human macrophages, due to its reduced capacity to control programmed cell death. PMID:25857735

  17. Cationic Polymer Based Gene Delivery: Uptake and Intracellular Trafficking

    NASA Astrophysics Data System (ADS)

    Ho, Yoonkhei; Too, Heng-Phon

    2014-04-01

    To date, low transfection efficiency remains the major drawback of polymer based gene delivery. Many cell types including stem cells, fibroblast and neurons are known to be poorly transfected with polymer based gene carriers and the high toxicity severely restrict their utility in gene delivery. Continual efforts are made to identify cellular barriers to efficient transfection as these carriers have low immunogenicity, ease of manufacturing and scalability. Here, we summarize the current status of understanding on uptake mechanism of polymer-DNA complexes (polyplexes), their endosomal escape, cytosolic transport and nuclear entry of pDNA.

  18. Intracellular Assembly and Trafficking of MHC Class I Molecules

    PubMed Central

    Donaldson, Julie G.; Williams, David B.

    2009-01-01

    The presentation of antigenic peptides by class I molecules of the major histocompatibility complex begins in the endoplasmic reticulum where the coordinated action of molecular chaperones, folding enzymes and class I-specific factors ensure that class I molecules are loaded with high affinity peptide ligands that will survive prolonged display at the cell surface. Once assembled, class I molecules are released from the quality control machinery of the ER for export to the plasma membrane where they undergo dynamic endocytic cycling and turnover. We review recent progress in our understanding of class I assembly, anterograde transport and endocytosis and highlight some of the events targeted by viruses as a means to evade detection by cytotoxic T cells and natural killer cells. PMID:19761542

  19. Intracellular targeting of PLGA nanoparticles encapsulating antigenic peptide to the endoplasmic reticulum of dendritic cells and its effect on antigen cross-presentation in vitro.

    PubMed

    Sneh-Edri, Hadas; Likhtenshtein, Diana; Stepensky, David

    2011-08-01

    Intracellularly targeted delivery system based on PLGA nanoparticles decorated with endoplasmic reticulum (ER)-targeting or control peptides and encapsulating antigenic peptide and fluorescent marker, was developed and characterized. The cellular uptake by dendritic cells (murine DC2.4 cells), intracellular trafficking, and cross-presentation efficiency of this delivery system were studied in vitro. The prepared nanoparticles (an average diameter of ~350 nm) efficiently encapsulated antigenic peptide and fluorescent marker and gradually released them over several days. Yet, the nanoparticles' size was small enough to allow their efficient endocytosis by the antigen-presenting cells in vitro. Surface conjugation of the targeting or control peptides enhanced the endocytosis of the nanoparticles, affected their intracellular trafficking, and induced prolonged low-magnitude cross-presentation of the antigenic peptide. We demonstrated in vitro that the intracellular fate of nanoparticulate drug delivery systems can be altered by their surface decoration with peptidic targeting residues. More detailed investigation is required to determine the mechanisms and therapeutic potential of intracellular targeting of nanodelivery systems in vivo for the goal of an anticancer vaccine.

  20. Lysosomal Trafficking Regulator (LYST).

    PubMed

    Ji, Xiaojie; Chang, Bo; Naggert, Jürgen K; Nishina, Patsy M

    2016-01-01

    Regulation of vesicle trafficking to lysosomes and lysosome-related organelles (LROs) as well as regulation of the size of these organelles are critical to maintain their functions. Disruption of the lysosomal trafficking regulator (LYST) results in Chediak-Higashi syndrome (CHS), a rare autosomal recessive disorder characterized by oculocutaneous albinism, prolonged bleeding, severe immunodeficiency, recurrent bacterial infection, neurologic dysfunction and hemophagocytic lympohistiocytosis (HLH). The classic diagnostic feature of the syndrome is enlarged LROs in all cell types, including lysosomes, melanosomes, cytolytic granules and platelet dense bodies. The most striking CHS ocular pathology observed is an enlargement of melanosomes in the retinal pigment epithelium (RPE), which leads to aberrant distribution of eye pigmentation, and results in photophobia and decreased visual acuity. Understanding the molecular function of LYST and identification of its interacting partners may provide therapeutic targets for CHS and other diseases associated with the regulation of LRO size and/or vesicle trafficking, such as asthma, urticaria and Leishmania amazonensis infections. PMID:26427484

  1. Limited Efficiency of Drug Delivery to Specific Intracellular Organelles Using Subcellularly "Targeted" Drug Delivery Systems.

    PubMed

    Maity, Amit Ranjan; Stepensky, David

    2016-01-01

    Many drugs have been designed to act on intracellular targets and to affect intracellular processes inside target cells. For the desired effects to be exerted, these drugs should permeate target cells and reach specific intracellular organelles. This subcellular drug targeting approach has been proposed for enhancement of accumulation of these drugs in target organelles and improved efficiency. This approach is based on drug encapsulation in drug delivery systems (DDSs) and/or their decoration with specific targeting moieties that are intended to enhance the drug/DDS accumulation in the intracellular organelle of interest. During recent years, there has been a constant increase in interest in DDSs targeted to specific intracellular organelles, and many different approaches have been proposed for attaining efficient drug delivery to specific organelles of interest. However, it appears that in many studies insufficient efforts have been devoted to quantitative analysis of the major formulation parameters of the DDSs disposition (efficiency of DDS endocytosis and endosomal escape, intracellular trafficking, and efficiency of DDS delivery to the target organelle) and of the resulting pharmacological effects. Thus, in many cases, claims regarding efficient delivery of drug/DDS to a specific organelle and efficient subcellular targeting appear to be exaggerated. On the basis of the available experimental data, it appears that drugs/DDS decoration with specific targeting residues can affect their intracellular fate and result in preferential drug accumulation within an organelle of interest. However, it is not clear whether these approaches will be efficient in in vivo settings and be translated into preclinical and clinical applications. Studies that quantitatively assess the mechanisms, barriers, and efficiencies of subcellular drug delivery and of the associated toxic effects are required to determine the therapeutic potential of subcellular DDS targeting.

  2. Ipsen 5i is a Novel Potent Pharmacoperone for Intracellularly Retained Melanocortin-4 Receptor Mutants

    PubMed Central

    Tao, Ya-Xiong; Huang, Hui

    2014-01-01

    Inactivating mutations of the melanocortin-4 receptor (MC4R) cause early-onset severe obesity in humans. Comprehensive functional studies show that most of the inactivating mutants of the MC4R are retained intracellularly. In the present study, we investigated whether a small molecule inverse agonist of the MC4R, Ipsen 5i, could act as a pharmacoperone and correct the cell surface expression and function of intracellularly retained mutant MC4Rs using multiple cell lines, including HEK293 and two neuronal cell lines. We showed that Ipsen 5i rescued the cell surface expression of all 11 intracellularly retained mutant MC4Rs studied herein in at least one cell line. Ipsen 5i functionally rescued seven mutants in all cell lines used. One mutant (Y157S) was functionally rescued in HEK293 cells but not in the two neuronal cell lines. Ipsen 5i increased cell surface expression of three mutants (S58C, G98R, and F261S) but did not affect signaling. Ipsen 5i had no effect on mutant MC4Rs with other defects (Δ88-92, D90N, I102S) or no defect (N274S). It also did not affect trafficking of a misrouted MC3R mutant (I335S). Cell impermeable peptide ligands of the MC4R or cell permeable small molecule ligand of δ opioid receptor could not rescue misrouted mutant MC4R. In summary, we demonstrated that Ipsen 5i was a novel potent pharmacoperone of the MC4R, correcting trafficking and signaling of a significant portion (73%) of intracellularly retained mutants. Additional studies are needed to demonstrate its in vivo efficacy. PMID:25136332

  3. Health implications of human trafficking.

    PubMed

    Richards, Tiffany A

    2014-01-01

    Freedom is arguably the most cherished right in the United States. But each year, approximately 14,500 to 17,500 women, men and children are trafficked into the United States for the purposes of forced labor or sexual exploitation. Human trafficking has significant effects on both physical and mental health. This article describes the features of human trafficking, its physical and mental health effects and the vital role nurses can play in providing care to this vulnerable population. PMID:24750655

  4. Health implications of human trafficking.

    PubMed

    Richards, Tiffany A

    2014-01-01

    Freedom is arguably the most cherished right in the United States. But each year, approximately 14,500 to 17,500 women, men and children are trafficked into the United States for the purposes of forced labor or sexual exploitation. Human trafficking has significant effects on both physical and mental health. This article describes the features of human trafficking, its physical and mental health effects and the vital role nurses can play in providing care to this vulnerable population.

  5. Rab Family Proteins Regulate the Endosomal Trafficking and Function of RGS4*

    PubMed Central

    Bastin, Guillaume; Heximer, Scott P.

    2013-01-01

    RGS4, a heterotrimeric G-protein inhibitor, localizes to plasma membrane (PM) and endosomal compartments. Here, we examined Rab-mediated control of RGS4 internalization and recycling. Wild type and constitutively active Rab5 decreased RGS4 PM levels while increasing its endosomal targeting. Rab5, however, did not appreciably affect the PM localization or function of the M1 muscarinic receptor (M1R)/Gq signaling cascade. RGS4-containing endosomes co-localized with subsets of Rab5-, transferrin receptor-, and Lamp1/Lysotracker-marked compartments suggesting RGS4 traffics through PM recycling or acidified endosome pathways. Rab7 activity promoted TGN association, whereas Rab7(dominant negative) trapped RGS4 in late endosomes. Furthermore, RGS4 was found to co-localize with an endosomal pool marked by Rab11, the protein that mediates recycling/sorting of proteins to the PM. The Cys-12 residue in RGS4 appeared important for its Rab11-mediated trafficking to the PM. Rab11(dominant negative) decreased RGS4 PM levels and increased the number of RGS4-containing endosomes. Inhibition of Rab11 activity decreased RGS4 function as an inhibitor of M1R activity without affecting localization and function of the M1R/Gq signaling complex. Thus, both Rab5 activation and Rab11 inhibition decreased RGS4 function in a manner that is independent from their effects on the localization and function of the M1R/Gq signaling complex. This is the first study to implicate Rab GTPases in the intracellular trafficking of an RGS protein. Thus, Rab GTPases may be novel molecular targets for the selective regulation of M1R-mediated signaling via their specific effects on RGS4 trafficking and function. PMID:23733193

  6. Cortical development of AMPA receptor trafficking proteins

    PubMed Central

    Murphy, Kathryn M.; Tcharnaia, Lilia; Beshara, Simon P.; Jones, David G.

    2012-01-01

    AMPA-receptor trafficking plays a central role in excitatory plasticity, especially during development. Changes in the number of AMPA receptors and time spent at the synaptic surface are important factors of plasticity that directly affect long-term potentiation (LTP), long-term depression (LTD), synaptic scaling, and the excitatory-inhibitory (E/I) balance in the developing cortex. Experience-dependent changes in synaptic strength in visual cortex (V1) use a molecularly distinct AMPA trafficking pathway that includes the GluA2 subunit. We studied developmental changes in AMPA receptor trafficking proteins by quantifying expression of GluA2, pGluA2 (GluA2serine880), GRIP1, and PICK1 in rat visual and frontal cortex. We used Western Blot analysis of synaptoneurosome preparations of rat visual and frontal cortex from animals ranging in age from P0 to P105. GluA2 and pGluA2 followed different developmental trajectories in visual and frontal cortex, with a brief period of over expression in frontal cortex. The over expression of GluA2 and pGluA2 in immature frontal cortex raises the possibility that there may be a period of GluA2-dependent vulnerability in frontal cortex that is not found in V1. In contrast, GRIP1 and PICK1 had the same developmental trajectories and were expressed very early in development of both cortical areas. This suggests that the AMPA-interacting proteins are available to begin trafficking receptors as soon as GluA2-containing receptors are expressed. Finally, we used all four proteins to analyze the surface-to-internalization balance and found that this balance was roughly equal across both cortical regions, and throughout development. Our finding of an exquisite surface-to-internalization balance highlights that these AMPA receptor trafficking proteins function as a tightly controlled system in the developing cortex. PMID:22623912

  7. Depletion of Rab32 decreases intracellular lipid accumulation and induces lipolysis through enhancing ATGL expression in hepatocytes.

    PubMed

    Li, Qing; Wang, Jun; Wan, Ying; Chen, Dongfeng

    2016-03-18

    Nonalcoholic fatty liver disease (NAFLD) is a disease caused by the accumulation of lipids in hepatocytes. To date, however, the pathogenesis of NAFLD is still unclear. Recent studies have shown that Rab GTPases, a major protein family in vesicle trafficking, are associated with intracellular lipid accumulation. Here, we show that Rab32, the only Rab GTPase located in mitochondria, participates in hepatic steatosis. Ablation of Rab32 can decrease intracellular lipid accumulation in hepatocytes (HepG2, L02). Further studying the possible mechanism, we found that knockdown of Rab32 can enhance lipolysis instead of lipogenesis via inducing the expression of adipose triglyceride lipase (ATGL), a key enzyme on the surface of lipid droplets which has been proved to be significant in controlling intracellular lipid accumulation. Co-immunoprecipitation shows that Rab32 and ATGL are not directly associated. These findings suggest that knockdown of Rab32 indirectly affects lipolysis through increasing the expression of ATGL. Taken together, our study reveals that Rab32 can participate in regulating intracellular lipid accumulation and that knockdown of Rab32 can decrease intracellular lipid accumulation in hepatocytes. We also demonstrated that ablation of Rab32 can induce intracellular lipolysis by enhancing the expression of ATGL. PMID:26882978

  8. Intracellular ice and cell survival in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum: an ultrastructural study of factors affecting cell and ice structures

    PubMed Central

    Wesley-Smith, James; Berjak, Patricia; Pammenter, N. W.; Walters, Christina

    2014-01-01

    Background and Aims Cryopreservation is the only long-term conservation strategy available for germplasm of recalcitrant-seeded species. Efforts to cryopreserve this form of germplasm are hampered by potentially lethal intracellular freezing events; thus, it is important to understand the relationships among cryo-exposure techniques, water content, structure and survival. Methods Undried embryonic axes of Acer saccharinum and those rapidly dried to two different water contents were cooled at three rates and re-warmed at two rates. Ultrastructural observations were carried out on radicle and shoot tips prepared by freeze-fracture and freeze-substitution to assess immediate (i.e. pre-thaw) responses to cooling treatments. Survival of axes was assessed in vitro. Key Results Intracellular ice formation was not necessarily lethal. Embryo cells survived when crystal diameter was between 0·2 and 0·4 µm and fewer than 20 crystals were distributed per μm2 in the cytoplasm. Ice was not uniformly distributed within the cells. In fully hydrated axes cooled at an intermediate rate, the interiors of many organelles were apparently ice-free; this may have prevented the disruption of vital intracellular machinery. Intracytoplasmic ice formation did not apparently impact the integrity of the plasmalemma. The maximum number of ice crystals was far greater in shoot apices, which were more sensitive than radicles to cryo-exposure. Conclusions The findings challenge the accepted paradigm that intracellular ice formation is always lethal, as the results show that cells can survive intracellular ice if crystals are small and localized in the cytoplasm. Further understanding of the interactions among water content, cooling rate, cell structure and ice structure is required to optimize cryopreservation treatments without undue reliance on empirical approaches. PMID:24368198

  9. Would Controlled Substance Status Affect Steroid Trafficking?

    ERIC Educational Resources Information Center

    Cowart, Virginia S.

    1987-01-01

    Loss of control over the use of anabolic steriods had prompted the federal government to take steps to stem the black market manufacture and distribution of these drugs. However, these steps are likely to stop short of bestowing controlled substance status on steriods. (Author/CB)

  10. Intracellular calcium channels in protozoa.

    PubMed

    Docampo, Roberto; Moreno, Silvia N J; Plattner, Helmut

    2014-09-15

    Ca(2+)-signaling pathways and intracellular Ca(2+) channels are present in protozoa. Ancient origin of inositol 1,4,5-trisphosphate receptors (IP3Rs) and other intracellular channels predates the divergence of animals and fungi as evidenced by their presence in the choanoflagellate Monosiga brevicollis, the closest known relative to metazoans. The first protozoan IP3R cloned, from the ciliate Paramecium, displays strong sequence similarity to the rat type 3 IP3R. This ciliate has a large number of IP3- and ryanodine(Ry)-like receptors in six subfamilies suggesting the evolutionary adaptation to local requirements for an expanding diversification of vesicle trafficking. IP3Rs have also been functionally characterized in trypanosomatids, where they are essential for growth, differentiation, and establishment of infection. The presence of the mitochondrial calcium uniporter (MCU) in a number of protozoa indicates that mitochondrial regulation of Ca(2+) signaling is also an early appearance in evolution, and contributed to the discovery of the molecular nature of this channel in mammalian cells. There is only sequence evidence for the occurrence of two-pore channels (TPCs), transient receptor potential Ca(2+) channels (TRPCs) and intracellular mechanosensitive Ca(2+)-channels in Paramecium and in parasitic protozoa.

  11. Intracellular Calcium Channels in Protozoa

    PubMed Central

    Docampo, Roberto; Moreno, Silvia N.J.; Plattner, Helmut

    2014-01-01

    Ca2+-signaling pathways and intracellular Ca2+ channels are present in protozoa. Ancient origin of inositol 1,4,5-trisphosphate receptors (IP3Rs) and other intracellular channels predates the divergence of animals and fungi as evidenced by their presence in the choanoflagellate Monosiga brevicollis, the closest known relative to metazoans. The first protozoan IP3R cloned, from the ciliate Paramecium, displays strong sequence similarity to the rat type 3 IP3R. This ciliate has a large number of IP3- and ryanodine(Ry)-like receptors in 6 subfamilies suggesting the evolutionary adaptation to local requirements for an expanding diversification of vesicle trafficking. IP3Rs have also been functionally characterized in trypanosomatids, where they are essential for growth, differentiation, and establishment of infection. The presence of the mitochondrial calcium uniporter (MCU) in a number of protozoa indicates that mitochondrial regulation of Ca2+ signaling is also an early appearance in evolution, and contributed to the discovery of the molecular nature of this channel in mammalian cells. There is only sequence evidence for the occurrence of two-pore channels (TPCs), transient receptor potential Ca2+ channels (TRPCs) and intracellular mechanosensitive Ca2+-channels in Paramecium and in parasitic protozoa. PMID:24291099

  12. Effect of different chemical bonds in pegylation of zinc protoporphyrin that affects drug release, intracellular uptake, and therapeutic effect in the tumor.

    PubMed

    Tsukigawa, Kenji; Nakamura, Hideaki; Fang, Jun; Otagiri, Masaki; Maeda, Hiroshi

    2015-01-01

    Pegylated zinc protoporphyrin (PEG-ZnPP) is a water-soluble inhibitor of heme oxygenase-1. In this study, we prepared two types of PEG-ZnPP conjugates with different chemical bonds between PEG and ZnPP, i.e., ester bonds and ether bonds, where both conjugates also contain amide bonds. Cleavability of these bonds in vitro and in vivo, especially cancer tissue, and upon intracellular uptake, was investigated in parallel with biological activities of the conjugates. Each conjugate showed different cleavability by plasma esterases and tumor proteases, as revealed by HPLC analyses. PEG-ZnPP with ester bond (esPEG-ZnPP) was more sensitive than PEG-ZnPP with ether bond (etPEG-ZnPP) for cleavage of PEG chains. etPEG-ZnPP showed no cleavage of PEG chains and had lower intracellular uptake and antitumor activity than did esPEG-ZnPP. The degradation of esPEG-ZnPP appeared to be facilitated by both serine and cysteine proteases in tumor tissues, whereas it was significantly slower in normal organs except the liver. Depegylated products such as free ZnPP had higher intracellular uptake than did intact PEG-ZnPP. We also studied hydrolytic cleavage by blood plasma of different animal species; mouse plasma showed the fastest cleavage whereas human plasma showed the slowest. These results suggest that ester-linked conjugates manifest more efficient cleavage of PEG, and greater yield of the active principle from the conjugates in tumor tissues than in normal tissues. More efficient intracellular uptake and thus an improved therapeutic effect with ester-linked conjugates are thus anticipated with fain stability, particularly in human blood.

  13. Factors affecting the activation and inhibition of intracellular enzymes for degradation of 1,2 diamino benzene: kinetics and thermodynamic studies.

    PubMed

    P, Saranya; G, Sekaran

    2015-11-01

    Citrobacter freundii, the bacterium isolated from marine sediments was capable of degrading 1,2 diamino benzene (DAB), an endocrine disruptor. The mixed intracellular enzymes from C. freundii were extracted and purified. The mixed intracellular enzymes were used for the degradation of DAB and degree of degradation was evaluated in terms of pyruvic acid, the end product, formed. The variables such as effect of pH, temperature and metal ions on the degradation of DAB using mixed intracellular enzymes (MICE) were investigated. The maximum amount of pyruvic acid formed was found to be 569 ± 5 µg with 96% degradation efficiency at pH 7; temperature 25 °C; zinc nitrate 0.1 mM; and copper sulphate ions 0.15 mM. The stability of MICE at different temperatures and the interaction of MICE with metal ions were confirmed using FT-IR spectroscopy. The formation of pyruvic acid from degradation of DAB followed pseudo-second-order rate kinetics and it was a spontaneous, exothermic process. The activation energy of degradation of DAB by MICE was found to be 82.55 kJ/mol.

  14. Endocytic membrane trafficking and neurodegenerative disease.

    PubMed

    Schreij, Andrea M A; Fon, Edward A; McPherson, Peter S

    2016-04-01

    Neurodegenerative diseases are amongst the most devastating of human disorders. New technologies have led to a rapid increase in the identification of disease-related genes with an enhanced appreciation of the key roles played by genetics in the etiology of these disorders. Importantly, pinpointing the normal function of disease gene proteins leads to new understanding of the cellular machineries and pathways that are altered in the disease process. One such emerging pathway is membrane trafficking in the endosomal system. This key cellular process controls the localization and levels of a myriad of proteins and is thus critical for normal cell function. In this review we will focus on three neurodegenerative diseases; Parkinson disease, amyotrophic lateral sclerosis, and hereditary spastic paraplegias, for which a large number of newly discovered disease genes encode proteins that function in endosomal membrane trafficking. We will describe how alterations in these proteins affect endosomal function and speculate on the contributions of these disruptions to disease pathophysiology. PMID:26721251

  15. Role of PDZ Proteins in Regulating Trafficking, Signaling, and Function of GPCRs: Means, Motif, and Opportunity

    PubMed Central

    Romero, Guillermo; von Zastrow, Mark; Friedman, Peter A.

    2016-01-01

    PDZ proteins, named for the common structural domain shared by the postsynaptic density protein (PSD95), Drosophila disc large tumor suppressor (DlgA), and zonula occludens-1 protein (ZO-1), constitute a family of 200–300 recognized members. These cytoplasmic adapter proteins are capable of assembling a variety of membrane-associated proteins and signaling molecules in short-lived functional units. Here, we review PDZ proteins that participate in the regulation of signaling, trafficking, and function of G protein-coupled receptors. Salient structural features of PDZ proteins that allow them to recognize targeted GPCRs are considered. Scaffolding proteins harboring PDZ domains may contain single or multiple PDZ modules and may also include other protein–protein interaction modules. PDZ proteins may impact receptor signaling by diverse mechanisms that include retaining the receptor at the cell membrane, thereby increasing the duration of ligand binding, as well as importantly influencing GPCR internalization, trafficking, recycling, and intracellular sorting. PDZ proteins are also capable of modifying the assembled complex of accessory proteins such as β-arrestins that themselves regulate GPCR signaling. Additionally, PDZ proteins may modulate GPCR signaling by altering the G protein to which the receptor binds, or affect other regulatory proteins that impact GTPase activity, protein kinase A, phospholipase C, or modify downstream signaling events. Small molecules targeting the PDZ protein-GPCR interaction are being developed and may become important and selective drug candidates. PMID:21907913

  16. Mitochondrial trafficking in neurons and the role of the Miro family of GTPase proteins.

    PubMed

    Birsa, Nicol; Norkett, Rosalind; Higgs, Nathalie; Lopez-Domenech, Guillermo; Kittler, Josef T

    2013-12-01

    Correct mitochondrial dynamics are essential to neuronal function. These dynamics include mitochondrial trafficking and quality-control systems that maintain a precisely distributed and healthy mitochondrial network, so that local energy demands or Ca2+-buffering requirements within the intricate architecture of the neuron can be met. Mitochondria make use of molecular machinery that couples these organelles to microtubule-based transport via kinesin and dynein motors, facilitating the required long-range movements. These motors in turn are associated with a variety of adaptor proteins allowing additional regulation of the complex dynamics demonstrated by these organelles. Over recent years, a number of new motor and adaptor proteins have been added to a growing list of components implicated in mitochondrial trafficking and distribution. Yet, there are major questions that remain to be addressed about the regulation of mitochondrial transport complexes. One of the core components of this machinery, the mitochondrial Rho GTPases Miro1 (mitochondrial Rho 1) and Miro2 have received special attention due to their Ca2+-sensing and GTPase abilities, marking Miro an exceptional candidate for co-ordinating mitochondrial dynamics and intracellular signalling pathways. In the present paper, we discuss the wealth of literature regarding Miro-mediated mitochondrial transport in neurons and recently highlighted involvement of Miro proteins in mitochondrial turnover, emerging as a key process affected in neurodegeneration. PMID:24256248

  17. Defects in optineurin- and myosin VI-mediated cellular trafficking in amyotrophic lateral sclerosis.

    PubMed

    Sundaramoorthy, Vinod; Walker, Adam K; Tan, Vanessa; Fifita, Jennifer A; Mccann, Emily P; Williams, Kelly L; Blair, Ian P; Guillemin, Gilles J; Farg, Manal A; Atkin, Julie D

    2015-07-01

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder primarily affecting motor neurons. Mutations in optineurin cause a small proportion of familial ALS cases, and wild-type (WT) optineurin is misfolded and forms inclusions in sporadic ALS patient motor neurons. However, it is unknown how optineurin mutation or misfolding leads to ALS. Optineurin acts an adaptor protein connecting the molecular motor myosin VI to secretory vesicles and autophagosomes. Here, we demonstrate that ALS-linked mutations p.Q398X and p.E478G disrupt the association of optineurin with myosin VI, leading to an abnormal diffuse cytoplasmic distribution, inhibition of secretory protein trafficking, endoplasmic reticulum (ER) stress and Golgi fragmentation in motor neuron-like NSC-34 cells. We also provide further insight into the role of optineurin as an autophagy receptor. WT optineurin associated with lysosomes and promoted autophagosome fusion to lysosomes in neuronal cells, implying that it mediates trafficking of lysosomes during autophagy in association with myosin VI. However, either expression of ALS mutant optineurin or small interfering RNA-mediated knockdown of endogenous optineurin blocked lysosome fusion to autophagosomes, resulting in autophagosome accumulation. Together these results indicate that ALS-linked mutations in optineurin disrupt myosin VI-mediated intracellular trafficking processes. In addition, in control human patient tissues, optineurin displayed its normal vesicular localization, but in sporadic ALS patient tissues, vesicles were present in a significantly decreased proportion of motor neurons. Optineurin binding to myosin VI was also decreased in tissue lysates from sporadic ALS spinal cords. This study therefore links several previously described pathological mechanisms in ALS, including defects in autophagy, fragmentation of the Golgi and induction of ER stress, to disruption of optineurin function. These findings also indicate that

  18. Neutrophil elastase and proteinase 3 trafficking routes in myelomonocytic cells

    SciTech Connect

    Kaellquist, Linda; Rosen, Hanna; Nordenfelt, Pontus; Calafat, Jero; Janssen, Hans; Persson, Ann-Maj; Hansson, Markus; Olsson, Inge

    2010-11-15

    Neutrophil elastase (NE) and proteinase 3 (PR3) differ in intracellular localization, which may reflect different trafficking mechanisms of the precursor forms when synthesized at immature stages of neutrophils. To shed further light on these mechanisms, we compared the trafficking of precursor NE (proNE) and precursor PR3 (proPR3). Like proNE [1], proPR3 interacted with CD63 upon heterologous co-expression in COS cells but endogenous interaction was not detected although cell surface proNE/proPR3/CD63 were co-endocytosed in myelomonocytic cells. Cell surface proNE/proPR3 turned over more rapidly than cell surface CD63 consistent with processing/degradation of the pro-proteases but recycling of CD63. Colocalization of proNE/proPR3/CD63 with clathrin and Rab 7 suggested trafficking through coated vesicles and late endosomes. Partial caveolar trafficking of proNE/CD63 but not proPR3 was suggested by colocalization with caveolin-1. Blocking the C-terminus of proNE/proPR3 by creating a fusion with FK506 binding protein inhibited endosomal re-uptake of proNE but not proPR3 indicating 'pro{sub C}'-peptide-dependent structural/conformational requirements for proNE but not for proPR3 endocytosis. The NE aminoacid residue Y199 of a proposed NE sorting motif that interacts with AP-3 [2] was not required for proNE processing, sorting or endocytosis in rat basophilic leukemia (RBL) cells expressing heterologous Y199-deleted proNE; this suggests operation of another AP-3-link for proNE targeting. Our results show intracellular multi-step trafficking to be different between proNE and proPR3 consistent with their differential subcellular NE/PR3 localization in neutrophils.

  19. Ligand-directed trafficking of receptor stimulus.

    PubMed

    Chilmonczyk, Zdzisław; Bojarski, Andrzej J; Sylte, Ingebrigt

    2014-12-01

    GPCRs are seven transmembrane-spanning receptors that convey specific extracellular stimuli to intracellular signalling. They represent the largest family of cell surface proteins that are therapeutically targeted. According to the traditional two-state model of receptor theory, GPCRs were considered as operating in equilibrium between two functional conformations, an active (R*) and inactive (R) state. Thus, it was assumed that a GPCR can exist either in an "off" or "on" conformation causing either no activation or equal activation of all its signalling pathways. Over the past several years it has become evident that this model is too simple and that GPCR signalling is far more complex. Different studies have presented a multistate model of receptor activation in which ligand-specific receptor conformations are able to differentiate between distinct signalling partners. Recent data show that beside G proteins numerous other proteins, such as β-arrestins and kinases, may interact with GPCRs and activate intracellular signalling pathways. GPCR activation may therefore involve receptor desensitization, coupling to multiple G proteins, Gα or Gβγ signalling, and pathway activation that is independent of G proteins. This latter effect leads to agonist "functional selectivity" (also called ligand-directed receptor trafficking, stimulus trafficking, biased agonism, biased signalling), and agonist intervention with functional selectivity may improve the therapy. Many commercially available drugs with beneficial efficacy also show various undesirable side effects. Further studies of biased signalling might facilitate our understanding of the side effects of current drugs and take us to new avenues to efficiently design pathway-specific medications.

  20. Antibody-Fc receptor interactions in protection against intracellular pathogens.

    PubMed

    Joller, Nicole; Weber, Stefan S; Oxenius, Annette

    2011-04-01

    Intracellular pathogen-specific antibodies (Abs) can contribute to host protection by a number of different mechanisms. Ab opsonization of pathogens residing outside a host cell can prevent infection of target cells either via neutralization of the critical surface epitopes required for host cell entry, complement-mediated degradation, or via subsequent intracellular degradation. In the case of intracellular localization, Abs can bind to infected cells and thus mark them for destruction by Fc receptor (FcR)-bearing effector cells. This review focuses on the protective role of Abs against intracellular bacteria and parasites involving FcR interactions that modulate the intracellular trafficking of the pathogen, the ability of FcRs to interfere with the establishment of an intracellular replicative niche and the involvement of FcRs to modulate pathogen-specific T-cell responses. PMID:21413006

  1. Antibody-Fc receptor interactions in protection against intracellular pathogens.

    PubMed

    Joller, Nicole; Weber, Stefan S; Oxenius, Annette

    2011-04-01

    Intracellular pathogen-specific antibodies (Abs) can contribute to host protection by a number of different mechanisms. Ab opsonization of pathogens residing outside a host cell can prevent infection of target cells either via neutralization of the critical surface epitopes required for host cell entry, complement-mediated degradation, or via subsequent intracellular degradation. In the case of intracellular localization, Abs can bind to infected cells and thus mark them for destruction by Fc receptor (FcR)-bearing effector cells. This review focuses on the protective role of Abs against intracellular bacteria and parasites involving FcR interactions that modulate the intracellular trafficking of the pathogen, the ability of FcRs to interfere with the establishment of an intracellular replicative niche and the involvement of FcRs to modulate pathogen-specific T-cell responses.

  2. Lipid raft-dependent uptake, signaling, and intracellular fate of Porphyromonas gingivalis in mouse macrophages

    PubMed Central

    Wang, Min; Hajishengallis, George

    2009-01-01

    Summary Lipid rafts are cholesterol-enriched microdomains involved in cellular trafficking and implicated as portals for certain pathogens. We sought to determine whether the oral pathogen Porphyromonas gingivalis enters macrophages via lipid rafts, and if so, to examine the impact of raft entry on its intracellular fate. Using J774A.1 mouse macrophages, we found that P. gingivalis colocalizes with lipid rafts in a cholesterol-dependent way. Depletion of cellular cholesterol using methyl-β-cyclodextrin resulted in about 50% inhibition of P. gingivalis uptake, although this effect was reversed by cholesterol reconstitution. The intracellular survival of P. gingivalis was dramatically inhibited in cholesterol-depleted cells relative to untreated or cholesterol-reconstituted cells, even when infections were adjusted to allow equilibration of the initial intracellular bacterial load. P. gingivalis thus appeared to exploit raft-mediated uptake for promoting its survival. Consistent with this, lipid raft disruption enhanced the colocalization of internalized P. gingivalis with lysosomes. In contrast, raft disruption did not affect the expression of host receptors interacting with P. gingivalis, although it significantly inhibited signal transduction. In summary, P. gingivalis uses macrophage lipid rafts as signaling and entry platforms, which determine its intracellular fate to the pathogen’s own advantage. PMID:18547335

  3. 31 CFR 536.311 - Narcotics trafficking.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 31 Money and Finance:Treasury 3 2012-07-01 2012-07-01 false Narcotics trafficking. 536.311 Section... FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY NARCOTICS TRAFFICKING SANCTIONS REGULATIONS General Definitions § 536.311 Narcotics trafficking. The term narcotics trafficking means any activity...

  4. 31 CFR 536.311 - Narcotics trafficking.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 31 Money and Finance:Treasury 3 2013-07-01 2013-07-01 false Narcotics trafficking. 536.311 Section... FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY NARCOTICS TRAFFICKING SANCTIONS REGULATIONS General Definitions § 536.311 Narcotics trafficking. The term narcotics trafficking means any activity...

  5. 31 CFR 536.311 - Narcotics trafficking.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 31 Money and Finance:Treasury 3 2014-07-01 2014-07-01 false Narcotics trafficking. 536.311 Section... FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY NARCOTICS TRAFFICKING SANCTIONS REGULATIONS General Definitions § 536.311 Narcotics trafficking. The term narcotics trafficking means any activity...

  6. 31 CFR 536.311 - Narcotics trafficking.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 31 Money and Finance:Treasury 3 2011-07-01 2011-07-01 false Narcotics trafficking. 536.311 Section... FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY NARCOTICS TRAFFICKING SANCTIONS REGULATIONS General Definitions § 536.311 Narcotics trafficking. The term narcotics trafficking means any activity...

  7. 31 CFR 536.311 - Narcotics trafficking.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 31 Money and Finance: Treasury 3 2010-07-01 2010-07-01 false Narcotics trafficking. 536.311... OF FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY NARCOTICS TRAFFICKING SANCTIONS REGULATIONS General Definitions § 536.311 Narcotics trafficking. The term narcotics trafficking means any...

  8. Growth media simulating ileal and colonic environments affect the intracellular proteome and carbon fluxes of enterohemorrhagic Escherichia coli O157:H7 strain EDL933.

    PubMed

    Polzin, Sabrina; Huber, Claudia; Eylert, Eva; Elsenhans, Ines; Eisenreich, Wolfgang; Schmidt, Herbert

    2013-06-01

    In this study, the intracellular proteome of Escherichia coli O157:H7 strain EDL933 was analyzed by two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) spectrometry after growth in simulated ileal environment media (SIEM) and simulated colonic environment media (SCEM) under aerobic and microaerobic conditions. Differentially expressed intracellular proteins were identified and allocated to functional protein groups. Moreover, metabolic fluxes were analyzed by isotopologue profiling with [U-(13)C(6)]glucose as a tracer. The results of this study show that EDL933 responds with differential expression of a complex network of proteins and metabolic pathways, reflecting the high metabolic adaptability of the strain. Growth in SIEM and SCEM is obviously facilitated by the upregulation of nucleotide biosynthesis pathway proteins and could be impaired by exposition to 50 µM 6-mercaptopurine under aerobic conditions. Notably, various stress and virulence factors, including Shiga toxin, were expressed without having contact with a human host.

  9. Understanding the link between trafficking in persons and HIV and AIDS risk in Tanzania.

    PubMed

    Kamazima, Switbert R; Ezekiel, Mangi J; Kazaura, Method R; Fimbo, Benett

    2012-01-01

    The magnitude of trafficking in persons in Tanzania is unknown. Consequently, available information on health risks of persons trafficked for different forms of exploitation is extremely scanty. We conducted a baseline study in eight administrative regions of Tanzania using both qualitative and quantitative methods to generate data on the health conditions of trafficked persons to inform trafficking in persons control measures through HIV and AIDS interventions. Study participants included the national, regional and district community development officers, district medical officers, local government leaders, managers or representatives of non-governmental organizations involved in anti-trafficking in persons activities, members of the community and victims. Findings indicated that common forms of labour into which persons are trafficked include domestic services, agriculture (farming), construction, mining/quarrying, fishing, lumbering and manufacturing. Trafficked persons are reported to be exposed to risks like overcrowding, long working hours, psychological problems, physical injuries, impotence, breathing problems and sexually transmitted infections including HIV. It is concluded that the reported occupational hazards in industries where trafficked persons are forced into are not specific to trafficked persons as they affect all labourers. However, the underground nature of the trafficking in persons process increases health problems and risks, including the vulnerability to HIV infection. More tailored research is needed, especially to find means of how to reach out and provide services to this particular vulnerable population, validate labour forms of exploitation into which persons are trafficked to enable the integration or mainstreaming of HIV and AIDS and trafficking in persons at the policy and programmatic levels. In addition, findings would facilitate the understanding of the link between increased risk of IRV and trafficking in persons. PMID:26591750

  10. Understanding the link between trafficking in persons and HIV and AIDS risk in Tanzania.

    PubMed

    Kamazima, Switbert R; Ezekiel, Mangi J; Kazaura, Method R; Fimbo, Benett

    2012-01-01

    The magnitude of trafficking in persons in Tanzania is unknown. Consequently, available information on health risks of persons trafficked for different forms of exploitation is extremely scanty. We conducted a baseline study in eight administrative regions of Tanzania using both qualitative and quantitative methods to generate data on the health conditions of trafficked persons to inform trafficking in persons control measures through HIV and AIDS interventions. Study participants included the national, regional and district community development officers, district medical officers, local government leaders, managers or representatives of non-governmental organizations involved in anti-trafficking in persons activities, members of the community and victims. Findings indicated that common forms of labour into which persons are trafficked include domestic services, agriculture (farming), construction, mining/quarrying, fishing, lumbering and manufacturing. Trafficked persons are reported to be exposed to risks like overcrowding, long working hours, psychological problems, physical injuries, impotence, breathing problems and sexually transmitted infections including HIV. It is concluded that the reported occupational hazards in industries where trafficked persons are forced into are not specific to trafficked persons as they affect all labourers. However, the underground nature of the trafficking in persons process increases health problems and risks, including the vulnerability to HIV infection. More tailored research is needed, especially to find means of how to reach out and provide services to this particular vulnerable population, validate labour forms of exploitation into which persons are trafficked to enable the integration or mainstreaming of HIV and AIDS and trafficking in persons at the policy and programmatic levels. In addition, findings would facilitate the understanding of the link between increased risk of IRV and trafficking in persons.

  11. Illicit Trafficking of Natural Radionuclides

    SciTech Connect

    Friedrich, Steinhaeusler; Lyudmila, Zaitseva

    2008-08-07

    Natural radionuclides have been subject to trafficking worldwide, involving natural uranium ore (U 238), processed uranium (yellow cake), low enriched uranium (<20% U 235) or highly enriched uranium (>20% U 235), radium (Ra 226), polonium (Po 210), and natural thorium ore (Th 232). An important prerequisite to successful illicit trafficking activities is access to a suitable logistical infrastructure enabling an undercover shipment of radioactive materials and, in case of trafficking natural uranium or thorium ore, capable of transporting large volumes of material. Covert en route diversion of an authorised uranium transport, together with covert diversion of uranium concentrate from an operating or closed uranium mines or mills, are subject of case studies. Such cases, involving Israel, Iran, Pakistan and Libya, have been analyzed in terms of international actors involved and methods deployed. Using international incident data contained in the Database on Nuclear Smuggling, Theft and Orphan Radiation Sources (DSTO) and international experience gained from the fight against drug trafficking, a generic Trafficking Pathway Model (TPM) is developed for trafficking of natural radionuclides. The TPM covers the complete trafficking cycle, ranging from material diversion, covert material transport, material concealment, and all associated operational procedures. The model subdivides the trafficking cycle into five phases: (1) Material diversion by insider(s) or initiation by outsider(s); (2) Covert transport; (3) Material brokerage; (4) Material sale; (5) Material delivery. An Action Plan is recommended, addressing the strengthening of the national infrastructure for material protection and accounting, development of higher standards of good governance, and needs for improving the control system deployed by customs, border guards and security forces.

  12. Illicit Trafficking of Natural Radionuclides

    NASA Astrophysics Data System (ADS)

    Friedrich, Steinhäusler; Lyudmila, Zaitseva

    2008-08-01

    Natural radionuclides have been subject to trafficking worldwide, involving natural uranium ore (U 238), processed uranium (yellow cake), low enriched uranium (<20% U 235) or highly enriched uranium (>20% U 235), radium (Ra 226), polonium (Po 210), and natural thorium ore (Th 232). An important prerequisite to successful illicit trafficking activities is access to a suitable logistical infrastructure enabling an undercover shipment of radioactive materials and, in case of trafficking natural uranium or thorium ore, capable of transporting large volumes of material. Covert en route diversion of an authorised uranium transport, together with covert diversion of uranium concentrate from an operating or closed uranium mines or mills, are subject of case studies. Such cases, involving Israel, Iran, Pakistan and Libya, have been analyzed in terms of international actors involved and methods deployed. Using international incident data contained in the Database on Nuclear Smuggling, Theft and Orphan Radiation Sources (DSTO) and international experience gained from the fight against drug trafficking, a generic Trafficking Pathway Model (TPM) is developed for trafficking of natural radionuclides. The TPM covers the complete trafficking cycle, ranging from material diversion, covert material transport, material concealment, and all associated operational procedures. The model subdivides the trafficking cycle into five phases: (1) Material diversion by insider(s) or initiation by outsider(s); (2) Covert transport; (3) Material brokerage; (4) Material sale; (5) Material delivery. An Action Plan is recommended, addressing the strengthening of the national infrastructure for material protection and accounting, development of higher standards of good governance, and needs for improving the control system deployed by customs, border guards and security forces.

  13. Sex trafficking in South Asia.

    PubMed

    Huda, S

    2006-09-01

    Economic and social inequalities and political conflicts have led to the movement of persons within each country and across the borders in South Asia. Globalization has encouraged free mobility of capital, technology, experts and sex tourism. Illiteracy, dependency, violence, social stigma, cultural stereotypes, gender disparity and endemic poverty, among other factors, place women and children in powerless, non-negotiable situations that have contributed to the emergence and breeding of the cavernous problem of sex trafficking in the entire region. This alarming spread of sex trafficking has fuelled the spread of HIV infection in South Asia, posing a unique and serious threat to community health, poverty alleviation and other crucial aspects of human development. Although the SAARC (South Asian Association for Regional Cooperation) Convention on Trafficking in Women and Children has been an important breakthrough, most of the countries in the region do not have anti-trafficking legislation or means to protect the victims. Countries of the region should make a concerted effort to treat trafficking victims as "victims" of human rights violations in all anti-trafficking strategies and actions.

  14. Trafficking activity of myosin XXI is required in assembly of Leishmania flagellum.

    PubMed

    Katta, Santharam S; Tammana, Trinadh V Satish; Sahasrabuddhe, Amogh A; Bajpai, Virendra K; Gupta, Chhitar M

    2010-06-15

    Actin-based myosin motors have a pivotal role in intracellular trafficking in eukaryotic cells. The parasitic protozoan organism Leishmania expresses a novel class of myosin, myosin XXI (Myo21), which is preferentially localized at the proximal region of the flagellum. However, its function in this organism remains largely unknown. Here, we show that Myo21 interacts with actin, and its expression is dependent of the growth stage. We further reveal that depletion of Myo21 levels results in impairment of the flagellar assembly and intracellular trafficking. These defects are, however, reversed by episomal complementation. Additionally, it is shown that deletion of the Myo21 gene leads to generation of ploidy, suggesting an essential role of Myo21 in survival of Leishmania cells. Together, these results indicate that actin-dependent trafficking activity of Myo21 is essentially required during assembly of the Leishmania flagellum. PMID:20501700

  15. To discuss illicit nuclear trafficking

    SciTech Connect

    Balatsky, Galya I; Severe, William R; Wallace, Richard K

    2010-01-01

    The Illicit nuclear trafficking panel was conducted at the 4th Annual INMM workshop on Reducing the Risk from Radioactive and Nuclear Materials on February 2-3, 2010 in Washington DC. While the workshop occurred prior to the Nuclear Security Summit, April 12-13 2010 in Washington DC, some of the summit issues were raised during the workshop. The Communique of the Washington Nuclear Security Summit stated that 'Nuclear terrorism is one of the most challenging threats to international security, and strong nuclear security measures are the most effective means to prevent terrorists, criminals, or other unauthorized actors from acquiring nuclear materials.' The Illicit Trafficking panel is one means to strengthen nuclear security and cooperation at bilateral, regional and multilateral levels. Such a panel promotes nuclear security culture through technology development, human resources development, education and training. It is a tool which stresses the importance of international cooperation and coordination of assistance to improve efforts to prevent and respond to incidents of illicit nuclear trafficking. Illicit trafficking panel included representatives from US government, an international organization (IAEA), private industry and a non-governmental organization to discuss illicit nuclear trafficking issues. The focus of discussions was on best practices and challenges for addressing illicit nuclear trafficking. Terrorism connection. Workshop discussions pointed out the identification of terrorist connections with several trafficking incidents. Several trafficking cases involved real buyers (as opposed to undercover law enforcement agents) and there have been reports identifying individuals associated with terrorist organizations as prospective plutonium buyers. Some specific groups have been identified that consistently search for materials to buy on the black market, but no criminal groups were identified that specialize in nuclear materials or isotope smuggling

  16. Calcium signaling regulates trafficking of familial hypocalciuric hypercalcemia (FHH) mutants of the calcium sensing receptor.

    PubMed

    Grant, Michael P; Stepanchick, Ann; Breitwieser, Gerda E

    2012-12-01

    Calcium-sensing receptors (CaSRs) regulate systemic Ca(2+) homeostasis. Loss-of-function mutations cause familial benign hypocalciuric hypercalcemia (FHH) or neonatal severe hyperparathyroidism (NSHPT). FHH/NSHPT mutations can reduce trafficking of CaSRs to the plasma membrane. CaSR signaling is potentiated by agonist-driven anterograde CaSR trafficking, leading to a new steady state level of plasma membrane CaSR, which is maintained, with minimal functional desensitization, as long as extracellular Ca(2+) is elevated. This requirement for CaSR signaling to drive CaSR trafficking to the plasma membrane led us to reconsider the mechanism(s) contributing to dysregulated trafficking of FHH/NSHPT mutants. We simultaneously monitored dynamic changes in plasma membrane levels of CaSR and intracellular Ca(2+), using a chimeric CaSR construct, which allowed explicit tracking of plasma membrane levels of mutant or wild-type CaSRs in the presence of nonchimeric partners. Expression of mutants alone revealed severe defects in plasma membrane targeting and Ca(2+) signaling, which were substantially rescued by coexpression with wild-type CaSR. Biasing toward heterodimerization of wild-type and FHH/NSHPT mutants revealed that intracellular Ca(2+) oscillations were insufficient to rescue plasma membrane targeting. Coexpression of the nonfunctional mutant E297K with the truncation CaSRΔ868 robustly rescued trafficking and Ca(2+) signaling, whereas coexpression of distinct FHH/NSHPT mutants rescued neither trafficking nor signaling. Our study suggests that rescue of FHH/NSHPT mutants requires a steady state intracellular Ca(2+) response when extracellular Ca(2+) is elevated and argues that Ca(2+) signaling by wild-type CaSRs rescues FHH mutant trafficking to the plasma membrane.

  17. The Proteome of the Isolated Chlamydia trachomatis Containing Vacuole Reveals a Complex Trafficking Platform Enriched for Retromer Components.

    PubMed

    Aeberhard, Lukas; Banhart, Sebastian; Fischer, Martina; Jehmlich, Nico; Rose, Laura; Koch, Sophia; Laue, Michael; Renard, Bernhard Y; Schmidt, Frank; Heuer, Dagmar

    2015-06-01

    Chlamydia trachomatis is an important human pathogen that replicates inside the infected host cell in a unique vacuole, the inclusion. The formation of this intracellular bacterial niche is essential for productive Chlamydia infections. Despite its importance for Chlamydia biology, a holistic view on the protein composition of the inclusion, including its membrane, is currently missing. Here we describe the host cell-derived proteome of isolated C. trachomatis inclusions by quantitative proteomics. Computational analysis indicated that the inclusion is a complex intracellular trafficking platform that interacts with host cells' antero- and retrograde trafficking pathways. Furthermore, the inclusion is highly enriched for sorting nexins of the SNX-BAR retromer, a complex essential for retrograde trafficking. Functional studies showed that in particular, SNX5 controls the C. trachomatis infection and that retrograde trafficking is essential for infectious progeny formation. In summary, these findings suggest that C. trachomatis hijacks retrograde pathways for effective infection.

  18. 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.

  19. Identification of a new Pmp22 mouse mutant and trafficking analysis of a Pmp22 allelic series suggesting that protein aggregates may be protective in Pmp22-associated peripheral neuropathy.

    PubMed

    Isaacs, Adrian M; Jeans, Alexander; Oliver, Peter L; Vizor, Lucie; Brown, Steve D M; Hunter, A Jackie; Davies, Kay E

    2002-09-01

    We have identified and characterized a new peripheral myelin protein 22 (Pmp22) mouse mutant. The mutation results in a serine to threonine amino acid substitution at residue 72, which is a hot spot for mutation in human PMP22, leading to the peripheral neuropathy Dejerine-Sottas syndrome. We have previously described two other Pmp22 mutants, providing an allelic series for gene function analysis. Pmp22 mutations generally lead to abnormal intracellular trafficking of Pmp22, and we show that each mutant protein in the allelic series has a unique pattern of intracellular localization in transfected cell lines. The mutant protein from the less severely affected mutants occurs in large aggregates, while the mutant protein from the most severely affected mutant occurs in a diffuse perinuclear pattern that largely colocalizes with wild-type protein. This suggests that large Pmp22 aggregates may be protective in this form of peripheral neuropathy. PMID:12359155

  20. Rab GTPase mediated procollagen trafficking in ascorbic acid stimulated osteoblasts.

    PubMed

    Nabavi, Noushin; Pustylnik, Sofia; Harrison, Rene E

    2012-01-01

    Despite advances in investigating functional aspects of osteoblast (OB) differentiation, especially studies on how bone proteins are deposited and mineralized, there has been little research on the intracellular trafficking of bone proteins during OB differentiation. Collagen synthesis and secretion is the major function of OBs and is markedly up-regulated upon ascorbic acid (AA) stimulation, significantly more so than in fibroblast cells. Understanding the mechanism by which collagen is mobilized in specialized OB cells is important for both basic cell biology and diseases involving defects in bone protein secretion and deposition. Protein trafficking along the exocytic and endocytic pathways is aided by many molecules, with Rab GTPases being master regulators of vesicle targeting. In this study, we used microarray analysis to identify the Rab GTPases that are up-regulated during a 5-day AA differentiation of OBs, namely Rab1, Rab3d, and Rab27b. Further, we investigated the role of identified Rabs in regulating the trafficking of collagen from the site of synthesis in the ER to the Golgi and ultimately to the plasma membrane utilizing Rab dominant negative (DN) expression. We also observed that experimental halting of biosynthetic trafficking by these mutant Rabs initiated proteasome-mediated degradation of procollagen and ceased global protein translation. Acute expression of Rab1 and Rab3d DN constructs partially alleviated this negative feedback mechanism and resulted in impaired ER to Golgi trafficking of procollagen. Similar expression of Rab27b DN constructs resulted in dispersed collagen vesicles which may represent failed secretory vesicles sequestered in the cytosol. A significant and strong reduction in extracellular collagen levels was also observed implicating the functional importance of Rab1, Rab3d and Rab27b in these major collagen-producing cells.

  1. Role of H(+)-pyrophosphatase activity in the regulation of intracellular pH in a scuticociliate parasite of turbot: Physiological effects.

    PubMed

    Mallo, Natalia; Lamas, Jesús; de Felipe, Ana-Paula; Sueiro, Rosa-Ana; Fontenla, Francisco; Leiro, José-Manuel

    2016-10-01

    The scuticociliatosis is a very serious disease that affects the cultured turbot, and whose causal agent is the anphizoic and marine euryhaline ciliate Philasterides dicentrarchi. Several protozoans possess acidic organelles that contain high concentrations of pyrophosphate (PPi), Ca(2+) and other elements with essential roles in vesicular trafficking, pH homeostasis and osmoregulation. P. dicentrarchi possesses a pyrophosphatase (H(+)-PPase) that pumps H(+) through the membranes of vacuolar and alveolar sacs. These compartments share common features with the acidocalcisomes described in other parasitic protozoa (e.g. acid content and Ca(2+) storage). We evaluated the effects of Ca(2+) and ATP on H (+)-PPase activity in this ciliate and analyzed their role in maintaining intracellular pH homeostasis and osmoregulation, by the addition of PPi and inorganic molecules that affect osmolarity. Addition of PPi led to acidification of the intracellular compartments, while the addition of ATP, CaCl2 and bisphosphonates analogous of PPi and Ca(2+) metabolism regulators led to alkalinization and a decrease in H(+)-PPase expression in trophozoites. Addition of NaCl led to proton release, intracellular Ca(2+) accumulation and downregulation of H(+)-PPase expression. We conclude that the regulation of the acidification of intracellular compartments may be essential for maintaining the intracellular pH homeostasis necessary for survival of ciliates and their adaptation to salt stress, which they will presumably face during the endoparasitic phase, in which the salinity levels are lower than in their natural environment.

  2. Role of H(+)-pyrophosphatase activity in the regulation of intracellular pH in a scuticociliate parasite of turbot: Physiological effects.

    PubMed

    Mallo, Natalia; Lamas, Jesús; de Felipe, Ana-Paula; Sueiro, Rosa-Ana; Fontenla, Francisco; Leiro, José-Manuel

    2016-10-01

    The scuticociliatosis is a very serious disease that affects the cultured turbot, and whose causal agent is the anphizoic and marine euryhaline ciliate Philasterides dicentrarchi. Several protozoans possess acidic organelles that contain high concentrations of pyrophosphate (PPi), Ca(2+) and other elements with essential roles in vesicular trafficking, pH homeostasis and osmoregulation. P. dicentrarchi possesses a pyrophosphatase (H(+)-PPase) that pumps H(+) through the membranes of vacuolar and alveolar sacs. These compartments share common features with the acidocalcisomes described in other parasitic protozoa (e.g. acid content and Ca(2+) storage). We evaluated the effects of Ca(2+) and ATP on H (+)-PPase activity in this ciliate and analyzed their role in maintaining intracellular pH homeostasis and osmoregulation, by the addition of PPi and inorganic molecules that affect osmolarity. Addition of PPi led to acidification of the intracellular compartments, while the addition of ATP, CaCl2 and bisphosphonates analogous of PPi and Ca(2+) metabolism regulators led to alkalinization and a decrease in H(+)-PPase expression in trophozoites. Addition of NaCl led to proton release, intracellular Ca(2+) accumulation and downregulation of H(+)-PPase expression. We conclude that the regulation of the acidification of intracellular compartments may be essential for maintaining the intracellular pH homeostasis necessary for survival of ciliates and their adaptation to salt stress, which they will presumably face during the endoparasitic phase, in which the salinity levels are lower than in their natural environment. PMID:27480055

  3. Trafficking: a perspective from Asia.

    PubMed

    Skeldon, R

    2000-01-01

    The main theme of this article is market development and trafficking as a business. It touches upon most of the aspects of the phenomenon, which have been encountered elsewhere, and translates them into the relatively unfamiliar context of many of the Asian and South-East Asian economies. Equally, the literature cited is also probably unfamiliar. Themes touched upon include democratization, inter-state relations, human rights, and scale and perspectives, together with the problems of definitions, theory, and the reliability of data. The directions and characteristics of trafficking flows together with routes and border control are also considered. Coordinated official responses to criminality and criminal organizations, as well as to trafficked individuals, are beginning to emerge. There is a note of caution sounded that contextual and cultural perspectives, particularly on sex workers, must be viewed somewhat differently to those in Western societies. The article concludes that as long as countries in Asia maintain their policies of restrictive immigration, trafficking can be expected to continue and almost certainly increase. This is because accelerating development creates demand for labor at various skill levels and because even in times of recession migrants and brokers will seek to side-step attempts to expel immigrants and restrict access to labor markets. The elimination of trafficking is unlikely to be realistically achieved through legislation and declarations of intent but by improvements in the socioeconomic status of the population.

  4. Surface adsorption, intracellular accumulation and compartmentalization of Pb(II) in batch-operated lagoons with Salvinia minima as affected by environmental conditions, EDTA and nutrients.

    PubMed

    Olguín, Eugenia J; Sánchez-Galván, Gloria; Pérez-Pérez, Teresa; Pérez-Orozco, Arith

    2005-12-01

    The effects of environmental factors and nutrients on the various possible removal mechanisms (surface adsorption, intracellular accumulation and precipitation to sediments) and partitioning of lead among various compartments (plant biomass, water column and sediments) in Salvinia minima batch-operated lagoons, were evaluated. Surface adsorption was found to be the predominant mechanism for Pb(II) removal under all environmental conditions tested in the absence of nutrients (an average of 54.3%) and in a nutrient medium (modified Hutner 1/10 medium) free of EDTA and phosphates (54.41%) at "high" initial Pb(II) concentrations (in the range of 10.3+/-0.13 to 15.2+/-0.05 mg/L). Under these conditions, the bioconcentration factors (BCFs) were 2,431+/-276 and 2,065+/-35, respectively. Lead removal was very rapid during the first 4 h and reached 70% in the absence of nutrients at the "medium" light intensity and temperature (LIT) tested, 88% in nutrient medium free of EDTA and supplemented with synthetic wastewater (at the "lowest" LIT tested), and 85% in medium free of EDTA and phosphates. It was concluded that the mechanisms of lead removal by S. minima, and the compartmentalization of this metal in the microcosm of batch-operated lagoons, are primarily a function of the presence of certain nutrients and chelants, with secondary dependence on environmental conditions. In addition, the results indicate that the percentage of lead removed is only a gross parameter and that the complementary use of BCF and compartmentalization analysis is required to gain a full insight into the metal removal process.

  5. Trafficking in persons: a health concern?

    PubMed

    Zimmerman, Cathy; Kiss, Ligia; Houssain, Mazeda; Watts, Charlotte

    2009-01-01

    Human trafficking is a phenomenon that has now been documented in most regions in the world. Although trafficking of women and girls for sexual exploitation is the most commonly recognised form of trafficking, it is widely acknowledged that human trafficking also involves men, women and children who are trafficked for various forms of labour exploitation and into other abusive circumstances. Despite the violence and harm inherent in most trafficking situations, there remains extremely little evidence on the individual and public health implications of any form of human trafficking. The Brazilian government has recently launched a national plan to combat human trafficking. However, because the health risks associated with human trafficking have not been well-recognised or documented, there is extremely limited reliable data on the health needs of trafficked persons to inform policy and practices.. Brazilian policy-makers and service providers should be encouraged to learn about the likely range of health impacts of trafficking, and incorporate this into anti-trafficking protection and response strategies. As well as prevention activities, the government, international and local organisations should work together with the public health research community to study the health needs of trafficked persons and explore opportunities to provide safe and appropriate services to victims in need of care. PMID:19721944

  6. The Arabidopsis Intracellular Na+/H+ Antiporters NHX5 and NHX6 Are Endosome Associated and Necessary for Plant Growth and Development[w

    PubMed Central

    Bassil, Elias; Ohto, Masa-aki; Esumi, Tomoya; Tajima, Hiromi; Zhu, Zhu; Cagnac, Olivier; Belmonte, Mark; Peleg, Zvi; Yamaguchi, Toshio; Blumwald, Eduardo

    2011-01-01

    Intracellular Na+/H+ antiporters (NHXs) play important roles in cellular pH and Na+ and K+ homeostasis in all eukaryotes. Based on sequence similarity, the six intracellular Arabidopsis thaliana members are divided into two groups. Unlike the vacuolar NHX1-4, NHX5 and NHX6 are believed to be endosomal; however, little data exist to support either their function or localization. Using reverse genetics, we show that whereas single knockouts nhx5 or nhx6 did not differ from the wild type, the double knockout nhx5 nhx6 showed reduced growth, with smaller and fewer cells and increased sensitivity to salinity. Reduced growth of nhx5 nhx6 was due to slowed cell expansion. Transcriptome analysis indicated that nhx5, nhx6, and the wild type had similar gene expression profiles, whereas transcripts related to vesicular trafficking and abiotic stress were enriched in nhx5 nhx6. We show that unlike other intracellular NHX proteins, NHX5 and NHX6 are associated with punctate, motile cytosolic vesicles, sensitive to Brefeldin A, that colocalize to known Golgi and trans-Golgi network markers. We provide data to show that vacuolar trafficking is affected in nhx5 nhx6. Possible involvements of NHX5 and NHX6 in maintaining organelle pH and ion homeostasis with implications in endosomal sorting and cellular stress responses are discussed. PMID:21278129

  7. National Human Trafficking Initiatives: Dimensions of Policy Diffusion1

    PubMed Central

    Yoo, Eun-hye; Boyle, Elizabeth Heger

    2014-01-01

    The implementation of criminal law involves formal law enforcement, education and public outreach aimed at preventing criminal activity, and providing services for victims. Historically, quantitative research on global trends has tended to focus on a single policy dimension, potentially masking the unique factors that affect the diffusion of each policy dimension independently. Using an ordered-probit model to analyze new human trafficking policy data on national prosecution, prevention, and victim-protection efforts, we find that global ties and domestic interest groups matter more in areas where international law is less defined. While prosecution, officially mandated by the Trafficking Protocol, was relatively impervious to global ties and domestic interest groups, both trafficking prevention and victim protection were associated with these factors. Our findings also suggest that fear of repercussions is not a major driver of state actions to combat trafficking—neither ratification of the Trafficking Protocol nor levels of United States aid were associated with greater implementation of anti-trafficking measures. PMID:26538806

  8. Intracellular localization and subsequent redistribution of metal transporters in a rat choroid plexus model following exposure to manganese or iron

    SciTech Connect

    Wang Xueqian; Miller, David S.

    2008-07-15

    Confocal microscopy was used to investigate the effects of manganese (Mn) and iron (Fe) exposure on the subcellular distribution of metal transporting proteins, i.e., divalent metal transporter 1 (DMT1), metal transporter protein 1 (MTP1), and transferrin receptor (TfR), in the rat intact choroid plexus which comprises the blood-cerebrospinal fluid barrier. In control tissue, DMT1 was concentrated below the apical epithelial membrane, MTP1 was diffuse within the cytosol, and TfR was distributed in vesicles around nuclei. Following Mn or Fe treatment (1 and 10 {mu}M), the distribution of DMT1 was not affected. However, MTP1 and TfR moved markedly toward the apical pole of the cells. These shifts were abolished when microtubules were disrupted. Quantitative RT-PCR and Western blot analyses revealed a significant increase in mRNA and protein levels of TfR but not DMT1 and MTP1 after Mn exposure. These results suggest that early events in the tissue response to Mn or Fe exposure involve microtubule-dependent, intracellular trafficking of MTP1 and TfR. The intracellular trafficking of metal transporters in the choroid plexus following Mn exposure may partially contribute to Mn-induced disruption in Fe homeostasis in the cerebrospinal fluid (CSF) following Mn exposure.

  9. The SNARE VAMP7 Regulates Exocytic Trafficking of Interleukin-12 in Dendritic Cells

    PubMed Central

    Chiaruttini, Giulia; Piperno, Giulia M.; Jouve, Mabel; De Nardi, Francesca; Larghi, Paola; Peden, Andrew A.; Baj, Gabriele; Müller, Sabina; Valitutti, Salvatore; Galli, Thierry; Benvenuti, Federica

    2016-01-01

    Summary Interleukin-12 (IL-12), produced by dendritic cells in response to activation, is central to pathogen eradication and tumor rejection. The trafficking pathways controlling spatial distribution and intracellular transport of IL-12 vesicles to the cell surface are still unknown. Here, we show that intracellular IL-12 localizes in late endocytic vesicles marked by the SNARE VAMP7. Dendritic cells (DCs) from VAMP7-deficient mice are partially impaired in the multidirectional release of IL-12. Upon encounter with antigen-specific T cells, IL-12-containing vesicles rapidly redistribute at the immune synapse and release IL-12 in a process entirely dependent on VAMP7 expression. Consistently, acquisition of effector functions is reduced in T cells stimulated by VAMP7-null DCs. These results provide insights into IL-12 intracellular trafficking pathways and show that VAMP7-mediated release of IL-12 at the immune synapse is a mechanism to transmit innate signals to T cells. PMID:26972013

  10. Rapid flow cytometric measurement of protein inclusions and nuclear trafficking

    PubMed Central

    Whiten, D. R.; San Gil, R.; McAlary, L.; Yerbury, J. J.; Ecroyd, H.; Wilson, M. R.

    2016-01-01

    Proteinaceous cytoplasmic inclusions are an indicator of dysfunction in normal cellular proteostasis and a hallmark of many neurodegenerative diseases. We describe a simple and rapid new flow cytometry-based method to enumerate, characterise and, if desired, physically recover protein inclusions from cells. This technique can analyse and resolve a broad variety of inclusions differing in both size and protein composition, making it applicable to essentially any model of intracellular protein aggregation. The method also allows rapid quantification of the nuclear trafficking of fluorescently labelled molecules. PMID:27516358

  11. Rapid flow cytometric measurement of protein inclusions and nuclear trafficking.

    PubMed

    Whiten, D R; San Gil, R; McAlary, L; Yerbury, J J; Ecroyd, H; Wilson, M R

    2016-01-01

    Proteinaceous cytoplasmic inclusions are an indicator of dysfunction in normal cellular proteostasis and a hallmark of many neurodegenerative diseases. We describe a simple and rapid new flow cytometry-based method to enumerate, characterise and, if desired, physically recover protein inclusions from cells. This technique can analyse and resolve a broad variety of inclusions differing in both size and protein composition, making it applicable to essentially any model of intracellular protein aggregation. The method also allows rapid quantification of the nuclear trafficking of fluorescently labelled molecules. PMID:27516358

  12. Understanding human trafficking in the United States.

    PubMed

    Logan, T K; Walker, Robert; Hunt, Gretchen

    2009-01-01

    The topic of modern-day slavery or human trafficking has received increased media and national attention. However, to date there has been limited research on the nature and scope of human trafficking in the United States. This article describes and synthesizes nine reports that assess the U.S. service organizations' legal representative knowledge of, and experience with, human trafficking cases, as well as information from actual cases and media reports. This article has five main goals: (a) to define what human trafficking is, and is not; (b) to describe factors identified as contributing to vulnerability to being trafficked and keeping a person entrapped in the situation; (c) to examine how the crime of human trafficking differs from other kinds of crimes in the United States; (d) to explore how human trafficking victims are identified; and, (e) to provide recommendations to better address human trafficking in the United States.

  13. Understanding human trafficking in the United States.

    PubMed

    Logan, T K; Walker, Robert; Hunt, Gretchen

    2009-01-01

    The topic of modern-day slavery or human trafficking has received increased media and national attention. However, to date there has been limited research on the nature and scope of human trafficking in the United States. This article describes and synthesizes nine reports that assess the U.S. service organizations' legal representative knowledge of, and experience with, human trafficking cases, as well as information from actual cases and media reports. This article has five main goals: (a) to define what human trafficking is, and is not; (b) to describe factors identified as contributing to vulnerability to being trafficked and keeping a person entrapped in the situation; (c) to examine how the crime of human trafficking differs from other kinds of crimes in the United States; (d) to explore how human trafficking victims are identified; and, (e) to provide recommendations to better address human trafficking in the United States. PMID:19056686

  14. Analysis of conventional and unconventional trafficking of CFTR and other membrane proteins.

    PubMed

    Gee, Heon Yung; Kim, Joo Young; Lee, Min Goo

    2015-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a polytopic transmembrane protein that functions as a cAMP-activated anion channel at the apical membrane of epithelial cells. Mutations in CFTR cause cystic fibrosis and are also associated with monosymptomatic diseases in the lung, pancreas, intestines, and vas deferens. Many disease-causing CFTR mutations, including the deletion of a single phenylalanine residue at position 508 (ΔF508-CFTR), result in protein misfolding and trafficking defects. Therefore, intracellular trafficking of wild-type and mutant CFTR has been studied extensively, and results from these studies significantly contribute to our general understanding of mechanisms involved in the cell-surface trafficking of membrane proteins. CFTR is a glycoprotein that undergoes complex N-glycosylation as it passes through Golgi-mediated conventional exocytosis. Interestingly, results from recent studies revealed that CFTR and other membrane proteins can reach the plasma membrane via an unconventional alternative route that bypasses Golgi in specific cellular conditions. Here, we describe methods that have been used to investigate the conventional and unconventional surface trafficking of CFTR. With appropriate modifications, the protocols described in this chapter can also be applied to studies investigating the intracellular trafficking of other plasma membrane proteins.

  15. 31 CFR 598.310 - Narcotics trafficking.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 31 Money and Finance:Treasury 3 2014-07-01 2014-07-01 false Narcotics trafficking. 598.310 Section... FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY FOREIGN NARCOTICS KINGPIN SANCTIONS REGULATIONS General Definitions § 598.310 Narcotics trafficking. The term narcotics trafficking means any illicit activity...

  16. 31 CFR 598.310 - Narcotics trafficking.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 31 Money and Finance:Treasury 3 2013-07-01 2013-07-01 false Narcotics trafficking. 598.310 Section... FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY FOREIGN NARCOTICS KINGPIN SANCTIONS REGULATIONS General Definitions § 598.310 Narcotics trafficking. The term narcotics trafficking means any illicit activity...

  17. 31 CFR 598.310 - Narcotics trafficking.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 31 Money and Finance:Treasury 3 2012-07-01 2012-07-01 false Narcotics trafficking. 598.310 Section... FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY FOREIGN NARCOTICS KINGPIN SANCTIONS REGULATIONS General Definitions § 598.310 Narcotics trafficking. The term narcotics trafficking means any illicit activity...

  18. 31 CFR 598.310 - Narcotics trafficking.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 31 Money and Finance: Treasury 3 2010-07-01 2010-07-01 false Narcotics trafficking. 598.310 Section 598.310 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE... General Definitions § 598.310 Narcotics trafficking. The term narcotics trafficking means any...

  19. 31 CFR 598.310 - Narcotics trafficking.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 31 Money and Finance:Treasury 3 2011-07-01 2011-07-01 false Narcotics trafficking. 598.310 Section 598.310 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) OFFICE OF... Definitions § 598.310 Narcotics trafficking. The term narcotics trafficking means any illicit activity...

  20. Endosome acidification and receptor trafficking: bafilomycin A1 slows receptor externalization by a mechanism involving the receptor's internalization motif.

    PubMed Central

    Johnson, L S; Dunn, K W; Pytowski, B; McGraw, T E

    1993-01-01

    To examine the relationship between endosome acidification and receptor trafficking, transferrin receptor trafficking was characterized in Chinese hamster ovary cells in which endosome acidification was blocked by treatment with the specific inhibitor of the vacuolar H(+)-ATPase, bafilomycin A1. Elevating endosome pH slowed the receptor externalization rate to approximately one-half of control but did not affect receptor internalization kinetics. The slowed receptor externalization required the receptor's cytoplasmic domain and was largely eliminated by substitutions replacing either of two aromatic amino acids within the receptor's cytoplasmic YTRF internalization motif. These results confirm, using a specific inhibitor of the vacuolar proton pump, that proper endosome acidification is necessary to maintain rapid recycling of intracellular receptors back to the plasma membrane. Moreover, receptor return to the plasma membrane is slowed in the absence of proper endosome acidification by a signal-dependent mechanism involving the receptor's cytoplasmic tyrosine-containing internalization motif. These results, in conjunction with results from other studies, suggest that the mechanism for clustering receptors in plasma membrane clathrin-coated pits may be an example of a more general mechanism that determines the dynamic distribution of membrane proteins among various compartments with luminal acidification playing a crucial role in this process. Images PMID:8167408

  1. Mucolipins: Intracellular TRPML1-3 channels.

    PubMed

    Cheng, Xiping; Shen, Dongbiao; Samie, Mohammad; Xu, Haoxing

    2010-05-17

    The mucolipin family of Transient Receptor Potential (TRPML) proteins is predicted to encode ion channels expressed in intracellular endosomes and lysosomes. Loss-of-function mutations of human TRPML1 cause type IV mucolipidosis (ML4), a childhood neurodegenerative disease. Meanwhile, gain-of-function mutations in the mouse TRPML3 result in the varitint-waddler (Va) phenotype with hearing and pigmentation defects. The broad spectrum phenotypes of ML4 and Va appear to result from certain aspects of endosomal/lysosomal dysfunction. Lysosomes, traditionally believed to be the terminal "recycling center" for biological "garbage", are now known to play indispensable roles in intracellular signal transduction and membrane trafficking. Studies employing animal models and cell lines in which TRPML genes have been genetically disrupted or depleted have uncovered roles of TRPMLs in multiple cellular functions including membrane trafficking, signal transduction, and organellar ion homeostasis. Physiological assays of mammalian cell lines in which TRPMLs are heterologously overexpressed have revealed the channel properties of TRPMLs in mediating cation (Ca(2+)/Fe(2+)) efflux from endosomes and lysosomes in response to unidentified cellular cues. This review aims to summarize these recent advances in the TRPML field and to correlate the channel properties of endolysosomal TRPMLs with their biological functions. We will also discuss the potential cellular mechanisms by which TRPML deficiency leads to neurodegeneration.

  2. Mucolipins: Intracellular TRPML1-3 Channels

    PubMed Central

    Cheng, Xiping; Shen, Dongbiao; Samie, Mohammad; Xu, Haoxing

    2010-01-01

    The mucolipin family of Transient Receptor Potential (TRPML) proteins is predicted to encode ion channels expressed in intracellular endosomes and lysosomes. Loss-of-function mutations of human TRPML1 cause type IV mucolipidosis (ML4), a childhood neurodegenerative disease. Meanwhile, gain-of-function mutations in the mouse TRPML3 result in the varitint-waddler (Va) phenotype with hearing and pigmentation defects. The broad spectrum phenotypes of ML4 and Va appear to result from certain aspects of endosomal/lysosomal dysfunction. Lysosomes, traditionally believed to be the terminal “recycling center” for biological “garbage”, are now known to play indispensable roles in intracellular signal transduction and membrane trafficking. Studies employing animal models and cell lines in which TRPML genes have been genetically disrupted or depleted have uncovered roles of TRPMLs in multiple cellular functions including membrane trafficking, signal transduction, and organellar ion homeostasis. Physiological assays of mammalian cell lines in which TRPMLs are heterologously over-expressed have revealed the channel properties of TRPMLs in mediating cation (Ca2+/Fe2+) efflux from endosomes and lysosomes in response to unidentified cellular cues. This review aims to summarize these recent advances in the TRPML field and to correlate the channel properties of endolysosomal TRPMLs with their biological functions. We will also discuss the potential cellular mechanisms by which TRPML deficiency leads to neurodegeneration. PMID:20074572

  3. Megalencephalic leukoencephalopathy with subcortical cysts protein-1 modulates endosomal pH and protein trafficking in astrocytes: relevance to MLC disease pathogenesis.

    PubMed

    Brignone, Maria S; Lanciotti, Angela; Visentin, Sergio; De Nuccio, Chiara; Molinari, Paola; Camerini, Serena; Diociaiuti, Marco; Petrini, Stefania; Minnone, Gaetana; Crescenzi, Marco; Laudiero, Luisa Bracci; Bertini, Enrico; Petrucci, Tamara C; Ambrosini, Elena

    2014-06-01

    Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare leukodystrophy caused by mutations in the gene encoding MLC1, a membrane protein mainly expressed in astrocytes in the central nervous system. Although MLC1 function is unknown, evidence is emerging that it may regulate ion fluxes. Using biochemical and proteomic approaches to identify MLC1 interactors and elucidate MLC1 function we found that MLC1 interacts with the vacuolar ATPase (V-ATPase), the proton pump that regulates endosomal acidity. Because we previously showed that in intracellular organelles MLC1 directly binds Na, K-ATPase, which controls endosomal pH, we studied MLC1 endosomal localization and trafficking and MLC1 effects on endosomal acidity and function using human astrocytoma cells overexpressing wild-type (WT) MLC1 or MLC1 carrying pathological mutations. We found that WT MLC1 is abundantly expressed in early (EEA1(+), Rab5(+)) and recycling (Rab11(+)) endosomes and uses the latter compartment to traffic to the plasma membrane during hyposmotic stress. We also showed that WT MLC1 limits early endosomal acidification and influences protein trafficking in astrocytoma cells by stimulating protein recycling, as revealed by FITC-dextran measurement of endosomal pH and transferrin protein recycling assay, respectively. WT MLC1 also favors recycling to the plasma-membrane of the TRPV4 cation channel which cooperates with MLC1 to activate calcium influx in astrocytes during hyposmotic stress. Although MLC disease-causing mutations differentially affect MLC1 localization and trafficking, all the mutated proteins fail to influence endosomal pH and protein recycling. This study demonstrates that MLC1 modulates endosomal pH and protein trafficking suggesting that alteration of these processes contributes to MLC pathogenesis.

  4. Functional rescue of a kidney anion exchanger 1 trafficking mutant in renal epithelial cells.

    PubMed

    Chu, Carmen Y S; King, Jennifer C; Berrini, Mattia; Alexander, R Todd; Cordat, Emmanuelle

    2013-01-01

    Mutations in the SLC4A1 gene encoding the anion exchanger 1 (AE1) can cause distal renal tubular acidosis (dRTA), a disease often due to mis-trafficking of the mutant protein. In this study, we investigated whether trafficking of a Golgi-retained dRTA mutant, G701D kAE1, or two dRTA mutants retained in the endoplasmic reticulum, C479W and R589H kAE1, could be functionally rescued to the plasma membrane of Madin-Darby Canine Kidney (MDCK) cells. Treatments with DMSO, glycerol, the corrector VX-809, or low temperature incubations restored the basolateral trafficking of G701D kAE1 mutant. These treatments had no significant rescuing effect on trafficking of the mis-folded C479W or R589H kAE1 mutants. DMSO was the only treatment that partially restored G701D kAE1 function in the plasma membrane of MDCK cells. Our experiments show that trafficking of intracellularly retained dRTA kAE1 mutants can be partially restored, and that one chemical treatment rescued both trafficking and function of a dRTA mutant. These studies provide an opportunity to develop alternative therapeutic solutions for dRTA patients. PMID:23460825

  5. LKB1/AMPK and PKA control ABCB11 trafficking and polarization in hepatocytes.

    PubMed

    Homolya, László; Fu, Dong; Sengupta, Prabuddha; Jarnik, Michal; Gillet, Jean-Pierre; Vitale-Cross, Lynn; Gutkind, J Silvio; Lippincott-Schwartz, Jennifer; Arias, Irwin M

    2014-01-01

    Polarization of hepatocytes is manifested by bile canalicular network formation and activation of LKB1 and AMPK, which control cellular energy metabolism. The bile acid, taurocholate, also regulates development of the canalicular network through activation of AMPK. In the present study, we used collagen sandwich hepatocyte cultures from control and liver-specific LKB1 knockout mice to examine the role of LKB1 in trafficking of ABCB11, the canalicular bile acid transporter. In polarized hepatocytes, ABCB11 traffics from Golgi to the apical plasma membrane and endogenously cycles through the rab 11a-myosin Vb recycling endosomal system. LKB1 knockout mice were jaundiced, lost weight and manifested impaired bile canalicular formation and intracellular trafficking of ABCB11, and died within three weeks. Using live cell imaging, fluorescence recovery after photobleaching (FRAP), particle tracking, and biochemistry, we found that LKB1 activity is required for microtubule-dependent trafficking of ABCB11 to the canalicular membrane. In control hepatocytes, ABCB11 trafficking was accelerated by taurocholate and cAMP; however, in LKB1 knockout hepatocytes, ABCB11 trafficking to the apical membrane was greatly reduced and restored only by cAMP, but not taurocholate. cAMP acted through a PKA-mediated pathway which did not activate AMPK. Our studies establish a regulatory role for LKB1 in ABCB11 trafficking to the canalicular membrane, hepatocyte polarization, and canalicular network formation. PMID:24643070

  6. Trafficking properties of the D5 dopamine receptor.

    PubMed

    Thompson, Dawn; Whistler, Jennifer L

    2011-05-01

    Dopamine receptors are important for diverse biological functions and are important pharmacological targets in human medicine. Signal transduction from the dopamine receptors is controlled at many levels, including by the process of receptor trafficking. Little is known regarding the endocytic and postendocytic trafficking properties of the D5 dopamine receptor. Here, we show that endocytosis of the D5 receptor can be achieved both homologously, through direct receptor activation by agonist, and also heterologously, due to independent activation of protein kinase C (PKC). In contrast, the D1 receptor is endocytosed only in response to agonist but not PKC activation. We have identified the residue in the third intracellular loop of the D5 receptor that is both necessary for PKC-mediated endocytosis of the D5 receptor and sufficient to induce PKC-mediated endocytosis when introduced to the D1 receptor. In addition, we show that endocytosis of D5 through both pathways is dependent on clathrin and dynamin but that only agonist-induced endocytosis engages β-arrestin 2. Together, these data show that the D5 receptor shows a trafficking profile distinct from that of any of the other dopamine receptors.

  7. Versatile roles of k63-linked ubiquitin chains in trafficking.

    PubMed

    Erpapazoglou, Zoi; Walker, Olivier; Haguenauer-Tsapis, Rosine

    2014-11-12

    Modification by Lys63-linked ubiquitin (UbK63) chains is the second most abundant form of ubiquitylation. In addition to their role in DNA repair or kinase activation, UbK63 chains interfere with multiple steps of intracellular trafficking. UbK63 chains decorate many plasma membrane proteins, providing a signal that is often, but not always, required for their internalization. In yeast, plants, worms and mammals, this same modification appears to be critical for efficient sorting to multivesicular bodies and subsequent lysosomal degradation. UbK63 chains are also one of the modifications involved in various forms of autophagy (mitophagy, xenophagy, or aggrephagy). Here, in the context of trafficking, we report recent structural studies investigating UbK63 chains assembly by various E2/E3 pairs, disassembly by deubiquitylases, and specifically recognition as sorting signals by receptors carrying Ub-binding domains, often acting in tandem. In addition, we address emerging and unanticipated roles of UbK63 chains in various recycling pathways that function by activating nucleators required for actin polymerization, as well as in the transient recruitment of signaling molecules at the plasma or ER membrane. In this review, we describe recent advances that converge to elucidate the mechanisms underlying the wealth of trafficking functions of UbK63 chains.

  8. Versatile Roles of K63-Linked Ubiquitin Chains in Trafficking

    PubMed Central

    Erpapazoglou, Zoi; Walker, Olivier; Haguenauer-Tsapis, Rosine

    2014-01-01

    Modification by Lys63-linked ubiquitin (UbK63) chains is the second most abundant form of ubiquitylation. In addition to their role in DNA repair or kinase activation, UbK63 chains interfere with multiple steps of intracellular trafficking. UbK63 chains decorate many plasma membrane proteins, providing a signal that is often, but not always, required for their internalization. In yeast, plants, worms and mammals, this same modification appears to be critical for efficient sorting to multivesicular bodies and subsequent lysosomal degradation. UbK63 chains are also one of the modifications involved in various forms of autophagy (mitophagy, xenophagy, or aggrephagy). Here, in the context of trafficking, we report recent structural studies investigating UbK63 chains assembly by various E2/E3 pairs, disassembly by deubiquitylases, and specifically recognition as sorting signals by receptors carrying Ub-binding domains, often acting in tandem. In addition, we address emerging and unanticipated roles of UbK63 chains in various recycling pathways that function by activating nucleators required for actin polymerization, as well as in the transient recruitment of signaling molecules at the plasma or ER membrane. In this review, we describe recent advances that converge to elucidate the mechanisms underlying the wealth of trafficking functions of UbK63 chains. PMID:25396681

  9. Human trafficking and the healthcare professional.

    PubMed

    Barrows, Jeffrey; Finger, Reginald

    2008-05-01

    Despite the legislation passed in the 19th century outlawing human slavery, it is more widespread today than at the conclusion of the civil war. Modern human slavery, termed human trafficking, comes in several forms. The most common type of human trafficking is sex trafficking, the sale of women and children into prostitution. Labor trafficking is the sale of men, women, and children into hard labor for which they receive little or no compensation. Other forms of trafficking include child soldiering, war brides, and organ removal. Healthcare professionals play a critical role in both finding victims of human trafficking while they are still in captivity, as well as caring for their mental and physical needs upon release. Those working in the healthcare profession need to be educated regarding how a trafficking victim may present, as well as their unique healthcare needs.

  10. Human trafficking and the healthcare professional.

    PubMed

    Barrows, Jeffrey; Finger, Reginald

    2008-05-01

    Despite the legislation passed in the 19th century outlawing human slavery, it is more widespread today than at the conclusion of the civil war. Modern human slavery, termed human trafficking, comes in several forms. The most common type of human trafficking is sex trafficking, the sale of women and children into prostitution. Labor trafficking is the sale of men, women, and children into hard labor for which they receive little or no compensation. Other forms of trafficking include child soldiering, war brides, and organ removal. Healthcare professionals play a critical role in both finding victims of human trafficking while they are still in captivity, as well as caring for their mental and physical needs upon release. Those working in the healthcare profession need to be educated regarding how a trafficking victim may present, as well as their unique healthcare needs. PMID:18414161

  11. The effect of pH dependence of antibody-antigen interactions on subcellular trafficking dynamics.

    PubMed

    Devanaboyina, Siva Charan; Lynch, Sandra M; Ober, Raimund J; Ram, Sripad; Kim, Dongyoung; Puig-Canto, Alberto; Breen, Shannon; Kasturirangan, Srinath; Fowler, Susan; Peng, Li; Zhong, Haihong; Jermutus, Lutz; Wu, Herren; Webster, Carl; Ward, E Sally; Gao, Changshou

    2013-01-01

    A drawback of targeting soluble antigens such as cytokines or toxins with long-lived antibodies is that such antibodies can prolong the half-life of the target antigen by a "buffering" effect. This has motivated the design of antibodies that bind to target with higher affinity at near neutral pH relative to acidic endosomal pH (~pH 6.0). Such antibodies are expected to release antigen within endosomes following uptake into cells, whereas antibody will be recycled and exocytosed in FcRn-expressing cells. To understand how the pH dependence of antibody-antigen interactions affects intracellular trafficking, we generated three antibodies that bind IL-6 with different pH dependencies in the range pH 6.0-7.4. The behavior of antigen in the presence of these antibodies has been characterized using a combination of fixed and live cell fluorescence microscopy. As the affinity of the antibody:IL-6 interaction at pH 6.0 decreases, an increasing amount of antigen dissociates from FcRn-bound antibody in early and late endosomes, and then enters lysosomes. Segregation of antibody and FcRn from endosomes in tubulovesicular transport carriers (TCs) into the recycling pathway can also be observed in live cells, and the extent of IL-6 association with TCs correlates with increasing affinity of the antibody:IL-6 interaction at acidic pH. These analyses result in an understanding, in spatiotemporal terms, of the effect of pH dependence of antibody-antigen interactions on subcellular trafficking and inform the design of antibodies with optimized binding properties for antigen elimination. PMID:24492341

  12. The effect of pH dependence of antibody-antigen interactions on subcellular trafficking dynamics

    PubMed Central

    Devanaboyina, Siva Charan; Lynch, Sandra M; Ober, Raimund J; Ram, Sripad; Kim, Dongyoung; Puig-Canto, Alberto; Breen, Shannon; Kasturirangan, Srinath; Fowler, Susan; Peng, Li; Zhong, Haihong; Jermutus, Lutz; Wu, Herren; Webster, Carl; Ward, E Sally; Gao, Changshou

    2013-01-01

    A drawback of targeting soluble antigens such as cytokines or toxins with long-lived antibodies is that such antibodies can prolong the half-life of the target antigen by a “buffering” effect. This has motivated the design of antibodies that bind to target with higher affinity at near neutral pH relative to acidic endosomal pH (~pH 6.0). Such antibodies are expected to release antigen within endosomes following uptake into cells, whereas antibody will be recycled and exocytosed in FcRn-expressing cells. To understand how the pH dependence of antibody-antigen interactions affects intracellular trafficking, we generated three antibodies that bind IL-6 with different pH dependencies in the range pH 6.0–7.4. The behavior of antigen in the presence of these antibodies has been characterized using a combination of fixed and live cell fluorescence microscopy. As the affinity of the antibody:IL-6 interaction at pH 6.0 decreases, an increasing amount of antigen dissociates from FcRn-bound antibody in early and late endosomes, and then enters lysosomes. Segregation of antibody and FcRn from endosomes in tubulovesicular transport carriers (TCs) into the recycling pathway can also be observed in live cells, and the extent of IL-6 association with TCs correlates with increasing affinity of the antibody:IL-6 interaction at acidic pH. These analyses result in an understanding, in spatiotemporal terms, of the effect of pH dependence of antibody-antigen interactions on subcellular trafficking and inform the design of antibodies with optimized binding properties for antigen elimination. PMID:24492341

  13. Trafficking and release of Leishmania metacyclic HASPB on macrophage invasion.

    PubMed

    Maclean, Lorna M; O'Toole, Peter J; Stark, Meg; Marrison, Jo; Seelenmeyer, Claudia; Nickel, Walter; Smith, Deborah F

    2012-05-01

    Proteins of the Leishmania hydrophilic acylated surface protein B (HASPB) family are only expressed in infective parasites (both extra- and intracellular stages) and, together with the peripheral membrane protein SHERP (small hydrophilic endoplasmic reticulum-associated protein), are essential for parasite differentiation (metacyclogenesis) in the sand fly vector. HASPB is a 'non-classically' secreted protein, requiring N-terminal acylation for trafficking to and exposure on the plasma membrane. Here, we use live cell imaging methods to further explore this pathway to the membrane and flagellum. Unlike HASPB trafficking in transfected mammalian cells, we find no evidence for a phosphorylation-regulated recycling pathway in metacyclic parasites. Once at the plasma membrane, HASPB18-GFP (green fluorescent protein) can undergo bidirectional movement within the inner leaflet of the membrane and on the flagellum. Transfer of fluorescent protein between the flagellum and the plasma membrane is compromised, however, suggesting the presence of a diffusion barrier at the base of the Leishmania flagellum. Full-length HASPB is released from the metacyclic parasite surface on to macrophages during phagocytosis but while expression is maintained in intracellular amastigotes, HASPB cannot be detected on the external surface in these cells. Thus HASPB may be a dual function protein that is shed by the infective metacyclic but retained internally once Leishmania are taken up by macrophages. PMID:22256896

  14. Multivesicular Bodies in Neurons: Distribution, Protein Content, and Trafficking Functions

    PubMed Central

    VON BARTHELD, CHRISTOPHER S.; ALTICK, AMY L.

    2011-01-01

    Summary Multivesicular bodies (MVBs) are intracellular endosomal organelles characterized by multiple internal vesicles that are enclosed within a single outer membrane. MVBs were initially regarded as purely prelysosomal structures along the degradative endosomal pathway of internalized proteins. MVBs are now known to be involved in numerous endocytic and trafficking functions, including protein sorting, recycling, transport, storage, and release. This review of neuronal MVBs summarizes their research history, morphology, distribution, accumulation of cargo and constitutive proteins, transport, and theories of functions of MVBs in neurons and glia. Due to their complex morphologies, neurons have expanded trafficking and signaling needs, beyond those of “geometrically simpler” cells, but it is not known whether neuronal MVBs perform additional transport and signaling functions. This review examines the concept of compartment-specific MVB functions in endosomal protein trafficking and signaling within synapses, axons, dendrites and cell bodies. We critically evaluate reports of the accumulation of neuronal MVBs based on evidence of stress-induced MVB formation. Furthermore, we discuss potential functions of neuronal and glial MVBs in development, in dystrophic neuritic syndromes, injury, disease, and aging. MVBs may play a role in Alzheimer’s, Huntington’s, and Niemann-Pick diseases, some types of frontotemporal dementia, prion and virus trafficking, as well as in adaptive responses of neurons to trauma and toxin or drug exposure. Functions of MVBs in neurons have been much neglected, and major gaps in knowledge currently exist. Developing truly MVB-specific markers would help to elucidate the roles of neuronal MVBs in intra- and intercellular signaling of normal and diseased neurons. PMID:21216273

  15. Genome-Wide Analysis of Sterol-Lipid Storage and Trafficking in Saccharomyces cerevisiae▿

    PubMed Central

    Fei, Weihua; Alfaro, Gabriel; Muthusamy, Baby-Periyanayaki; Klaassen, Zachary; Graham, Todd R.; Yang, Hongyuan; Beh, Christopher T.

    2008-01-01

    The pandemic of lipid-related disease necessitates a determination of how cholesterol and other lipids are transported and stored within cells. The first step in this determination is the identification of the genes involved in these transport and storage processes. Using genome-wide screens, we identified 56 yeast (Saccharomyces cerevisiae) genes involved in sterol-lipid biosynthesis, intracellular trafficking, and/or neutral-lipid storage. Direct biochemical and cytological examination of mutant cells revealed an unanticipated link between secretory protein glycosylation and triacylglycerol (TAG)/steryl ester (SE) synthesis for the storage of lipids. Together with the analysis of other deletion mutants, these results suggested at least two distinct events for the biogenesis of lipid storage particles: a step affecting neutral-lipid synthesis, generating the lipid core of storage particles, and another step for particle assembly. In addition to the lipid storage mutants, we identified mutations that affect the localization of unesterified sterols, which are normally concentrated in the plasma membrane. These findings implicated phospholipase C and the protein phosphatase Ptc1p in the regulation of sterol distribution within cells. This study identified novel sterol-related genes that define several distinct processes maintaining sterol homeostasis. PMID:18156287

  16. Trafficking of Vesicular Neurotransmitter Transporters

    PubMed Central

    Fei, Hao; Grygoruk, Anna; Brooks, Elizabeth S.; Chen, Audrey; Krantz, David E.

    2010-01-01

    Vesicular neurotransmitter transporters are required for the storage of all classical and amino acid neurotransmitters in secretory vesicles. Transporter expression can influence neurotransmitter storage and release, and trafficking targets the transporters to different types of secretory vesicles. Vesicular transporters traffic to synaptic vesicles as well as large dense core vesicles, and are recycled to synaptic vesicles at the nerve terminal. Some of the intrinsic signals for these trafficking events have been defined and include a dileucine motif present in multiple transporter subtypes, an acidic cluster in the neural isoform of the vesicular monoamine transporter (VMAT2) and a polyproline motif in the vesicular glutamate transporter VGLUT1. The sorting of VMAT2 and the vesicular acetylcholine transporter (VAChT) to secretory vesicles is regulated by phosphorylation. In addition, VGLUT1 uses alternative endocytic pathways for recycling back to synaptic vesicles following exocytosis. Regulation of these sorting events has the potential to influence synaptic transmission and behavior. PMID:18507811

  17. Intracellular Bacteria in Protozoa

    NASA Astrophysics Data System (ADS)

    Görtz, Hans-Dieter; Brigge, Theo

    Intracellular bacteria in humans are typically detrimental, and such infections are regarded by the patients as accidental and abnormal. In protozoa it seems obvious that many bacteria have coevolved with their hosts and are well adapted to the intracellular way of life. Manifold interactions between hosts and intracellular bacteria are found, and examples of antibacterial resistance of unknown mechanisms are observed. The wide diversity of intracellular bacteria in protozoa has become particularly obvious since they have begun to be classified by molecular techniques. Some of the bacteria are closely related to pathogens; others are responsible for the production of toxins.

  18. Viral subversion of nucleocytoplasmic trafficking.

    PubMed

    Yarbrough, Melanie L; Mata, Miguel A; Sakthivel, Ramanavelan; Fontoura, Beatriz M A

    2014-02-01

    Trafficking of proteins and RNA into and out of the nucleus occurs through the nuclear pore complex (NPC). Because of its critical function in many cellular processes, the NPC and transport factors are common targets of several viruses that disrupt key constituents of the machinery to facilitate viral replication. Many viruses such as poliovirus and severe acute respiratory syndrome (SARS) virus inhibit protein import into the nucleus, whereas viruses such as influenza A virus target and disrupt host mRNA nuclear export. Current evidence indicates that these viruses may employ such strategies to avert the host immune response. Conversely, many viruses co-opt nucleocytoplasmic trafficking to facilitate transport of viral RNAs. As viral proteins interact with key regulators of the host nuclear transport machinery, viruses have served as invaluable tools of discovery that led to the identification of novel constituents of nuclear transport pathways. This review explores the importance of nucleocytoplasmic trafficking to viral pathogenesis as these studies revealed new antiviral therapeutic strategies and exposed previously unknown cellular mechanisms. Further understanding of nuclear transport pathways will determine whether such therapeutics will be useful treatments for important human pathogens.

  19. Protein trafficking in kinetoplastid protozoa.

    PubMed Central

    Clayton, C; Häusler, T; Blattner, J

    1995-01-01

    The kinetoplastid protozoa infect hosts ranging from invertebrates to plants and mammals, causing diseases of medical and economic importance. They are the earliest-branching organisms in eucaryotic evolution to have either mitochondria or peroxisome-like microbodies. Investigation of their protein trafficking enables us to identify characteristics that have been conserved throughout eucaryotic evolution and also reveals how far variations, or alternative mechanisms, are possible. Protein trafficking in kinetoplastids is in many respects similar to that in higher eucaryotes, including mammals and yeasts. Differences in signal sequence specificities exist, however, for all subcellular locations so far examined in detail--microbodies, mitochondria, and endoplasmic reticulum--with signals being more degenerate, or shorter, than those of their higher eucaryotic counterparts. Some components of the normal array of trafficking mechanisms may be missing in most (if not all) kinetoplastids: examples are clathrin-coated vesicles, recycling receptors, and mannose 6-phosphate-mediated lysosomal targeting. Other aspects and structures are unique to the kinetoplastids or are as yet unexplained. Some of these peculiarities may eventually prove to be weak points that can be used as targets for chemotherapy; others may turn out to be much more widespread than currently suspected. PMID:7565409

  20. Viral Subversion of Nucleocytoplasmic Trafficking

    PubMed Central

    Yarbrough, Melanie L.; Mata, Miguel A.; Sakthivel, Ramanavelan; Fontoura, Beatriz M. A.

    2014-01-01

    Trafficking of proteins and RNA into and out of the nucleus occurs through the nuclear pore complex (NPC). Due to its critical function in many cellular processes, the NPC and transport factors are common targets of several viruses that disrupt key constituents of the machinery to facilitate viral replication. Many viruses such as poliovirus and severe acute respiratory syndrome (SARS) virus inhibit protein import into the nucleus, while viruses such as influenza A virus target and disrupt host mRNA nuclear export. Current evidence indicates that these viruses may employ such strategies to avert the host immune response. Conversely, many viruses co-opt nucleocytoplasmic trafficking to facilitate transport of viral RNAs. Since viral proteins interact with key regulators of the host nuclear transport machinery, viruses have served as invaluable tools of discovery that led to the identification of novel constituents of nuclear transport pathways. In addition, this review explores the importance of nucleocytoplasmic trafficking to viral pathogenesis as these studies revealed new antiviral therapeutic strategies and exposed previously unknown cellular mechanisms. Further understanding of nuclear transport pathways will determine whether such therapeutics will be useful treatments for important human pathogens. PMID:24289861

  1. Agonist-trafficking and hallucinogens.

    PubMed

    González-Maeso, Javier; Sealfon, Stuart C

    2009-01-01

    Seven transmembrane domain receptors, also termed G protein-coupled receptors (GPCRs), represent the most common molecular target for therapeutic drugs. The generally accepted pharmacological model for GPCR activation is the ternary complex model, in which GPCRs exist in a dynamic equilibrium between the active and inactive conformational states. However, the demonstration that different agonists sometimes elicit a different relative activation of two signaling pathways downstream of the same receptor has led to a revision of the ternary complex model. According to this agonist- trafficking model, agonists stabilize distinct activated receptor conformations that preferentially activate specific signaling pathways. Hallucinogenic drugs and non-hallucinogenic drugs represent an attractive experimental system with which to study agonist-trafficking of receptor signaling. Thus many of the behavioral responses induced by hallucinogenic drugs, such as lysergic acid diethylamide (LSD), psilocybin or mescaline, depend on activation of serotonin 5-HT(2A) receptors (5-HT2ARs). In contrast, this neuropsychological state in humans is not induced by closely related chemicals, such as lisuride or ergotamine, despite their similar in vitro activity at the 5-HT2AR. In this review, we summarize the current knowledge, as well as unresolved questions, regarding agonist-trafficking and the mechanism of action of hallucinogenic drugs.

  2. Plant vacuolar trafficking occurs through distinctly regulated pathways.

    PubMed

    Ebine, Kazuo; Inoue, Takeshi; Ito, Jun; Ito, Emi; Uemura, Tomohiro; Goh, Tatsuaki; Abe, Hiroshi; Sato, Ken; Nakano, Akihiko; Ueda, Takashi

    2014-06-16

    The multifunctional vacuole is the largest organelle in plant cells, and many proteins are transported to and stored in this organelle; thus, the vacuole has great physiological and agronomical importance. However, the molecular mechanism and regulation of plant vacuolar traffic remain largely unknown. In this study, we demonstrate that multiple vacuolar trafficking pathways operate in plants. RAB5 and RAB7 are evolutionarily conserved subfamilies of Rab GTPase, whose animal and yeast counterparts regulate vacuolar/endosomal trafficking in a sequential manner. Functional analyses of a putative activating complex for RAB7 indicated that this complex is responsible for maturation from RAB5- to RAB7-positive endosomes in plant cells. Moreover, these machinery components are recruited to a more complex trafficking network. Mutations in RAB5 and RAB7 conferred counteracting effects on the vti11 mutant. Furthermore, impairment of RAB5- and RAB7-dependent pathways differentially affected the transport of distinctive cargos. These results indicate that plants have developed a complex vacuolar transport system distinct from that of nonplant systems by assigning evolutionarily conserved machinery to unique trafficking pathways. These pathways provide a fundamental basis for plant development at the cellular and higher-ordered levels.

  3. Cellular membrane trafficking of mesoporous silica nanoparticles

    SciTech Connect

    Fang, I-Ju

    2012-01-01

    This dissertation mainly focuses on the investigation of the cellular membrane trafficking of mesoporous silica nanoparticles. We are interested in the study of endocytosis and exocytosis behaviors of mesoporous silica nanoparticles with desired surface functionality. The relationship between mesoporous silica nanoparticles and membrane trafficking of cells, either cancerous cells or normal cells was examined. Since mesoporous silica nanoparticles were applied in many drug delivery cases, the endocytotic efficiency of mesoporous silica nanoparticles needs to be investigated in more details in order to design the cellular drug delivery system in the controlled way. It is well known that cells can engulf some molecules outside of the cells through a receptor-ligand associated endocytosis. We are interested to determine if those biomolecules binding to cell surface receptors can be utilized on mesoporous silica nanoparticle materials to improve the uptake efficiency or govern the mechanism of endocytosis of mesoporous silica nanoparticles. Arginine-glycine-aspartate (RGD) is a small peptide recognized by cell integrin receptors and it was reported that avidin internalization was highly promoted by tumor lectin. Both RGD and avidin were linked to the surface of mesoporous silica nanoparticle materials to investigate the effect of receptor-associated biomolecule on cellular endocytosis efficiency. The effect of ligand types, ligand conformation and ligand density were discussed in Chapter 2 and 3. Furthermore, the exocytosis of mesoporous silica nanoparticles is very attractive for biological applications. The cellular protein sequestration study of mesoporous silica nanoparticles was examined for further information of the intracellular pathway of endocytosed mesoporous silica nanoparticle materials. The surface functionality of mesoporous silica nanoparticle materials demonstrated selectivity among the materials and cancer and normal cell lines. We aimed to determine

  4. Metabolic regulation of neutrophil spreading, membrane tubulovesicular extensions (cytonemes) formation and intracellular pH upon adhesion to fibronectin

    SciTech Connect

    Galkina, Svetlana I. . E-mail: galkina@genebee.msu.su; Sud'ina, Galina F.; Klein, Thomas

    2006-08-01

    Circulating leukocytes have a round cell shape and roll along vessel walls. However, metabolic disorders can lead them to adhere to the endothelium and spread (flatten). We studied the metabolic regulation of adhesion, spreading and intracellular pH (pHi) of neutrophils (polymorphonuclear leukocytes) upon adhesion to fibronectin-coated substrata. Resting neutrophils adhered and spread on fibronectin. An increase in pHi accompanied neutrophil spreading. Inhibition of oxidative phosphorylation or inhibition of P- and F-type ATPases affected neither neutrophil spreading nor pHi. Inhibition of glucose metabolism or V-ATPase impaired neutrophil spreading, blocked the increase in the pHi and induced extrusion of membrane tubulovesicular extensions (cytonemes), anchoring cells to substrata. Omission of extracellular Na{sup +} and inhibition of chloride channels caused a similar effect. We propose that these tubulovesicular extensions represent protrusions of exocytotic trafficking, supplying the plasma membrane of neutrophils with ion exchange mechanisms and additional membrane for spreading. Glucose metabolism and V-type ATPase could affect fusion of exocytotic trafficking with the plasma membrane, thus controlling neutrophil adhesive state and pHi. Cl{sup -} efflux through chloride channels and Na{sup +} influx seem to be involved in the regulation of the V-ATPase by carrying out charge compensation for the proton-pumping activity and through V-ATPase in regulation of neutrophil spreading and pHi.

  5. Regulation of cytoskeletal dynamics by redox signaling and oxidative stress: implications for neuronal development and trafficking

    PubMed Central

    Wilson, Carlos; González-Billault, Christian

    2015-01-01

    A proper balance between chemical reduction and oxidation (known as redox balance) is essential for normal cellular physiology. Deregulation in the production of oxidative species leads to DNA damage, lipid peroxidation and aberrant post-translational modification of proteins, which in most cases induces injury, cell death and disease. However, physiological concentrations of oxidative species are necessary to support important cell functions, such as chemotaxis, hormone synthesis, immune response, cytoskeletal remodeling, Ca2+ homeostasis and others. Recent evidence suggests that redox balance regulates actin and microtubule dynamics in both physiological and pathological contexts. Microtubules and actin microfilaments contain certain amino acid residues that are susceptible to oxidation, which reduces the ability of microtubules to polymerize and causes severing of actin microfilaments in neuronal and non-neuronal cells. In contrast, inhibited production of reactive oxygen species (ROS; e.g., due to NOXs) leads to aberrant actin polymerization, decreases neurite outgrowth and affects the normal development and polarization of neurons. In this review, we summarize emerging evidence suggesting that both general and specific enzymatic sources of redox species exert diverse effects on cytoskeletal dynamics. Considering the intimate relationship between cytoskeletal dynamics and trafficking, we also discuss the potential effects of redox balance on intracellular transport via regulation of the components of the microtubule and actin cytoskeleton as well as cytoskeleton-associated proteins, which may directly impact localization of proteins and vesicles across the soma, dendrites and axon of neurons. PMID:26483635

  6. Molybdenum Trafficking for Nitrogen Fixation†

    PubMed Central

    Hernandez, Jose A.; George, Simon J.; Rubio, Luis M.

    2009-01-01

    The molybdenum nitrogenase is responsible for most biological nitrogen fixation, a prokaryotic metabolic process that determines the global biogeochemical cycles of nitrogen and carbon. Here we describe the trafficking of molybdenum for nitrogen fixation in the model diazotrophic bacterium Azotobacter vinelandii. The genes and proteins involved in molybdenum uptake, homeostasis, storage, regulation, and nitrogenase cofactor biosynthesis are reviewed. Molybdenum biochemistry in A. vinelandii reveals unexpected mechanisms and a new role for iron-sulfur clusters in the sequestration and delivery of molybdenum. PMID:19772354

  7. Optical Control of Peroxisomal Trafficking.

    PubMed

    Spiltoir, Jessica I; Strickland, Devin; Glotzer, Michael; Tucker, Chandra L

    2016-07-15

    The blue-light-responsive LOV2 domain of Avena sativa phototropin1 (AsLOV2) has been used to regulate activity and binding of diverse protein targets with light. Here, we used AsLOV2 to photocage a peroxisomal targeting sequence, allowing light regulation of peroxisomal protein import. We generated a protein tag, LOV-PTS1, that can be appended to proteins of interest to direct their import to the peroxisome with light. This method provides a means to inducibly trigger peroxisomal protein trafficking in specific cells at user-defined times. PMID:26513473

  8. Examining the Risk of Nuclear Trafficking

    SciTech Connect

    Balatsky, Galya; Severe, William R; Schoeneck, Jeffery

    2009-01-01

    The need to stop illicit trafficking of nuclear and radioactive materials around the world is undeniable and urgent. This issue is particularly evident due to the highly dangerous consequences of the risks involved, the known interest of terrorist groups in acquiring such materials and the vulnerability of theft and diversion of such materials. Yet the phenomenon of nuclear trafficking remains a subject where the unknown dominates what is known on the subject. The trafficking panel at the Institute for Nuclear Materials Management (INMM) Workshop on Reducing the Risk of Radioactive and Nuclear Materials that took place in Albuquerque, New Mexico, March 10-11, 2009, dealt with some of the issues associated with nuclear trafficking. Different points of view on how to better address trafficking and thwart perpetrator efforts were discussed. This paper presents some of these views and addresses practical measures that should be considered to improve the situation.

  9. Retrograde trafficking of AB₅ toxins: mechanisms to therapeutics.

    PubMed

    Mukhopadhyay, Somshuvra; Linstedt, Adam D

    2013-10-01

    Bacterial AB5 toxins are a clinically relevant class of exotoxins that include several well-known members such as Shiga, cholera, and pertussis toxins. Infections with toxin-producing bacteria cause devastating human diseases that affect millions of individuals each year and have no definitive medical treatment. The molecular targets of AB5 toxins reside in the cytosol of infected cells, and the toxins reach the cytosol by trafficking through the retrograde membrane transport pathway that avoids degradative late endosomes and lysosomes. Focusing on Shiga toxin as the archetype member, we review recent advances in understanding the molecular mechanisms involved in the retrograde trafficking of AB5 toxins and highlight how these basic science advances are leading to the development of a promising new therapeutic approach based on inhibiting toxin transport.

  10. Gα13 and Rho mediate endosomal trafficking of CXCR4 into Rab11+ vesicles upon SDF-1 stimulation1

    PubMed Central

    Kumar, Ashok; Kremer, Kimberly N.; Dominguez, Daniel; Tadi, Madhavi; Hedin, Karen E.

    2011-01-01

    CXCR4, like other G protein coupled receptors (GPCRs), signals via heterotrimeric guanine nucleotide binding proteins (G proteins) to regulate gene transcription, migration, development, growth and transformation. We describe here a formerly-uncharacterized function of a G protein: a role in receptor trafficking. We previously showed that CXCR4 and the TCR physically associate and form a heterodimer upon SDF-1 stimulation in human T cells in order to prolong ERK activation, and thereby lead to gene-upregulation and cytokine secretion. The CXCR4-TCR heterodimers occur both on the cell surface and in an intracellular compartment in response to SDF-1. Neither the intracellular compartment to which the CXCR4-TCR heterodimers localize nor the mechanism for localization has been elucidated. Here, we characterize molecular mechanisms required for post-endocytic trafficking of CXCR4. Upon SDF-1 stimulation, CXCR4 localizes to Rab11+ vesicles, a recycling compartment, near the MTOC and Golgi apparatus. This trafficking requires the CXCR4 carboxyl-terminal tail domain but not the CXCR4 ubiquitination sites. The TCR also constitutively localizes to this Rab11+ compartment. Trafficking of CXCR4 into the Rab11+, TCR-containing endosomes requires actin polymerization. Furthermore, either inhibiting Rho activation or depleting Gα13 prevented trafficking of CXCR4 into the Rab11+ endosomes without hindering the ability of CXCR4 to endocytose. These results indicate that, upon SDF-1 treatment, Gα13 and Rho mediate the actin polymerization necessary for trafficking CXCR4 into the Rab11+, recycling endosomal compartment which also contains constitutively recycling TCR and thus CXCR4-TCR heterodimers. This is the first time that Gα13 has been described to mediate receptor trafficking. PMID:21148034

  11. Trafficking and function of the tetraspanin CD63

    SciTech Connect

    Pols, Maaike S.; Klumperman, Judith

    2009-05-15

    Tetraspanins comprise a large superfamily of cell surface-associated membrane proteins characterized by four transmembrane domains. They participate in a variety of cellular processes, like cell activation, adhesion, differentiation and tumour invasion. At the cell surface, tetraspanins form networks with a wide diversity of proteins called tetraspanin-enriched microdomains (TEMs). CD63 was the first characterized tetraspanin. In addition to its presence in TEMs, CD63 is also abundantly present in late endosomes and lysosomes. CD63 at the cell surface is endocytosed via a clathrin-dependent pathway, although recent studies suggest the involvement of other pathways as well and we here present evidence for a role of caveolae in CD63 endocytosis. In late endosomes, CD63 is enriched on the intraluminal vesicles, which by specialized cells are secreted as exosomes through fusion of endosomes with the plasma membrane. The complex localization pattern of CD63 suggests that its intracellular trafficking and distribution must be tightly regulated. In this review we discuss the latest insights in CD63 trafficking and its emerging function as a transport regulator of its interaction partners. Finally, the involvement of CD63 in cancer will be discussed.

  12. Cytomegalovirus immune evasion by perturbation of endosomal trafficking

    PubMed Central

    Lučin, Pero; Mahmutefendić, Hana; Blagojević Zagorac, Gordana; Ilić Tomaš, Maja

    2015-01-01

    Cytomegaloviruses (CMVs), members of the herpesvirus family, have evolved a variety of mechanisms to evade the immune response to survive in infected hosts and to establish latent infection. They effectively hide infected cells from the effector mechanisms of adaptive immunity by eliminating cellular proteins (major histocompatibility Class I and Class II molecules) from the cell surface that display viral antigens to CD8 and CD4 T lymphocytes. CMVs also successfully escape recognition and elimination of infected cells by natural killer (NK) cells, effector cells of innate immunity, either by mimicking NK cell inhibitory ligands or by downregulating NK cell-activating ligands. To accomplish these immunoevasion functions, CMVs encode several proteins that function in the biosynthetic pathway by inhibiting the assembly and trafficking of cellular proteins that participate in immune recognition and thereby, block their appearance at the cell surface. However, elimination of these proteins from the cell surface can also be achieved by perturbation of their endosomal route and subsequent relocation from the cell surface into intracellular compartments. Namely, the physiological route of every cellular protein, including immune recognition molecules, is characterized by specific features that determine its residence time at the cell surface. In this review, we summarize the current understanding of endocytic trafficking of immune recognition molecules and perturbations of the endosomal system during infection with CMVs and other members of the herpesvirus family that contribute to their immune evasion mechanisms. PMID:25263490

  13. Excess sphingomyelin disturbs ATG9A trafficking and autophagosome closure.

    PubMed

    Corcelle-Termeau, Elisabeth; Vindeløv, Signe Diness; Hämälistö, Saara; Mograbi, Baharia; Keldsbo, Anne; Bräsen, Jan Hinrich; Favaro, Elena; Adam, Dieter; Szyniarowski, Piotr; Hofman, Paul; Krautwald, Stefan; Farkas, Thomas; Petersen, Nikolaj H T; Rohde, Mikkel; Linkermann, Andreas; Jäättelä, Marja

    2016-05-01

    Sphingomyelin is an essential cellular lipid that traffics between plasma membrane and intracellular organelles until directed to lysosomes for SMPD1 (sphingomyelin phosphodiesterase 1)-mediated degradation. Inactivating mutations in the SMPD1 gene result in Niemann-Pick diseases type A and B characterized by sphingomyelin accumulation and severely disturbed tissue homeostasis. Here, we report that sphingomyelin overload disturbs the maturation and closure of autophagic membranes. Niemann-Pick type A patient fibroblasts and SMPD1-depleted cancer cells accumulate elongated and unclosed autophagic membranes as well as abnormally swollen autophagosomes in the absence of normal autophagosomes and autolysosomes. The immature autophagic membranes are rich in WIPI2, ATG16L1 and MAP1LC3B but display reduced association with ATG9A. Contrary to its normal trafficking between plasma membrane, intracellular organelles and autophagic membranes, ATG9A concentrates in transferrin receptor-positive juxtanuclear recycling endosomes in SMPD1-deficient cells. Supporting a causative role for ATG9A mistrafficking in the autophagy defect observed in SMPD1-deficient cells, ectopic ATG9A effectively reverts this phenotype. Exogenous C12-sphingomyelin induces a similar juxtanuclear accumulation of ATG9A and subsequent defect in the maturation of autophagic membranes in healthy cells while the main sphingomyelin metabolite, ceramide, fails to revert the autophagy defective phenotype in SMPD1-deficient cells. Juxtanuclear accumulation of ATG9A and defective autophagy are also evident in tissues of smpd1-deficient mice with a subsequent inability to cope with kidney ischemia-reperfusion stress. These data reveal sphingomyelin as an important regulator of ATG9A trafficking and maturation of early autophagic membranes. PMID:27070082

  14. Excess sphingomyelin disturbs ATG9A trafficking and autophagosome closure

    PubMed Central

    Corcelle-Termeau, Elisabeth; Vindeløv, Signe Diness; Hämälistö, Saara; Mograbi, Baharia; Keldsbo, Anne; Bräsen, Jan Hinrich; Favaro, Elena; Adam, Dieter; Szyniarowski, Piotr; Hofman, Paul; Krautwald, Stefan; Farkas, Thomas; Petersen, Nikolaj H.T.; Rohde, Mikkel; Linkermann, Andreas; Jäättelä, Marja

    2016-01-01

    ABSTRACT Sphingomyelin is an essential cellular lipid that traffics between plasma membrane and intracellular organelles until directed to lysosomes for SMPD1 (sphingomyelin phosphodiesterase 1)-mediated degradation. Inactivating mutations in the SMPD1 gene result in Niemann-Pick diseases type A and B characterized by sphingomyelin accumulation and severely disturbed tissue homeostasis. Here, we report that sphingomyelin overload disturbs the maturation and closure of autophagic membranes. Niemann-Pick type A patient fibroblasts and SMPD1-depleted cancer cells accumulate elongated and unclosed autophagic membranes as well as abnormally swollen autophagosomes in the absence of normal autophagosomes and autolysosomes. The immature autophagic membranes are rich in WIPI2, ATG16L1 and MAP1LC3B but display reduced association with ATG9A. Contrary to its normal trafficking between plasma membrane, intracellular organelles and autophagic membranes, ATG9A concentrates in transferrin receptor-positive juxtanuclear recycling endosomes in SMPD1-deficient cells. Supporting a causative role for ATG9A mistrafficking in the autophagy defect observed in SMPD1-deficient cells, ectopic ATG9A effectively reverts this phenotype. Exogenous C12-sphingomyelin induces a similar juxtanuclear accumulation of ATG9A and subsequent defect in the maturation of autophagic membranes in healthy cells while the main sphingomyelin metabolite, ceramide, fails to revert the autophagy defective phenotype in SMPD1-deficient cells. Juxtanuclear accumulation of ATG9A and defective autophagy are also evident in tissues of smpd1-deficient mice with a subsequent inability to cope with kidney ischemia-reperfusion stress. These data reveal sphingomyelin as an important regulator of ATG9A trafficking and maturation of early autophagic membranes. PMID:27070082

  15. Trafficking to the Ciliary Membrane

    PubMed Central

    Nachury, Maxence V.; Seeley, E. Scott; Jin, Hua

    2010-01-01

    The primary cilium organizes numerous signal transduction cascades and an understanding of signaling receptors trafficking to cilia is now emerging. A defining feature of cilia is the periciliary diffusion barrier that separates the ciliary and plasma membranes despite the topological continuity between these two membranes. Although lateral transport through this barrier may take place, polarized exocytosis to the base of the cilium has been the prevailing model for delivering membrane proteins to cilia. Key players for this polarized exocytosis model include the GTPases Rab8 and Rab11, the exocyst and possibly the intraflagellar tranport machinery. Sorting membrane proteins to cilia critically relies on the recognition of ciliary targeting signals by sorting machines such as the BBSome coat complex or the GTPase Arf4. Finally, signaling at the cilium entails the bidirectional movement of proteins between cytoplasm and cilia and ubiquitination may promote exit from cilia. PMID:19575670

  16. Heme in pathophysiology: a matter of scavenging, metabolism and trafficking across cell membranes.

    PubMed

    Chiabrando, Deborah; Vinchi, Francesca; Fiorito, Veronica; Mercurio, Sonia; Tolosano, Emanuela

    2014-01-01

    Heme (iron-protoporphyrin IX) is an essential co-factor involved in multiple biological processes: oxygen transport and storage, electron transfer, drug and steroid metabolism, signal transduction, and micro RNA processing. However, excess free-heme is highly toxic due to its ability to promote oxidative stress and lipid peroxidation, thus leading to membrane injury and, ultimately, apoptosis. Thus, heme metabolism needs to be finely regulated. Intracellular heme amount is controlled at multiple levels: synthesis, utilization by hemoproteins, degradation and both intracellular and intercellular trafficking. This review focuses on recent findings highlighting the importance of controlling intracellular heme levels to counteract heme-induced oxidative stress. The contributions of heme scavenging from the extracellular environment, heme synthesis and incorporation into hemoproteins, heme catabolism and heme transport in maintaining adequate intracellular heme content are discussed. Particular attention is put on the recently described mechanisms of heme trafficking through the plasma membrane mediated by specific heme importers and exporters. Finally, the involvement of genes orchestrating heme metabolism in several pathological conditions is illustrated and new therapeutic approaches aimed at controlling heme metabolism are discussed.

  17. Heme in pathophysiology: a matter of scavenging, metabolism and trafficking across cell membranes

    PubMed Central

    Chiabrando, Deborah; Vinchi, Francesca; Fiorito, Veronica; Mercurio, Sonia; Tolosano, Emanuela

    2014-01-01

    Heme (iron-protoporphyrin IX) is an essential co-factor involved in multiple biological processes: oxygen transport and storage, electron transfer, drug and steroid metabolism, signal transduction, and micro RNA processing. However, excess free-heme is highly toxic due to its ability to promote oxidative stress and lipid peroxidation, thus leading to membrane injury and, ultimately, apoptosis. Thus, heme metabolism needs to be finely regulated. Intracellular heme amount is controlled at multiple levels: synthesis, utilization by hemoproteins, degradation and both intracellular and intercellular trafficking. This review focuses on recent findings highlighting the importance of controlling intracellular heme levels to counteract heme-induced oxidative stress. The contributions of heme scavenging from the extracellular environment, heme synthesis and incorporation into hemoproteins, heme catabolism and heme transport in maintaining adequate intracellular heme content are discussed. Particular attention is put on the recently described mechanisms of heme trafficking through the plasma membrane mediated by specific heme importers and exporters. Finally, the involvement of genes orchestrating heme metabolism in several pathological conditions is illustrated and new therapeutic approaches aimed at controlling heme metabolism are discussed. PMID:24782769

  18. Syntabulin regulates the trafficking of PICK1-containing vesicles in neurons.

    PubMed

    Xu, Junyu; Wang, Na; Luo, Jian-Hong; Xia, Jun

    2016-01-01

    PICK1 (protein interacting with C-kinase 1) is a peripheral membrane protein that interacts with diverse membrane proteins. PICK1 has been shown to regulate the clustering and membrane localization of synaptic receptors such as AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors, metabotropic glutamate receptor 7, and ASICs (acid-sensing ion channels). Moreover, recent evidence suggests that PICK1 can mediate the trafficking of various vesicles out from the Golgi complex in several cell systems, including neurons. However, how PICK1 affects vesicle-trafficking dynamics remains unexplored. Here, we show that PICK1 mediates vesicle trafficking by interacting with syntabulin, a kinesin-binding protein that mediates the trafficking of both synaptic vesicles and mitochondria in axons. Syntabulin recruits PICK1 onto microtubule structures and mediates the trafficking of PICK1-containing vesicles along microtubules. In neurons, syntabulin alters PICK1 expression by recruiting PICK1 into axons and regulates the trafficking dynamics of PICK1-containing vesicles. Furthermore, we show that syntabulin forms a complex with PICK1 and ASICs, regulates ASIC protein expression in neurons, and participates in ASIC-induced acidotoxicity. PMID:26868290

  19. Child trafficking and commercial sexual exploitation: a review of promising prevention policies and programs.

    PubMed

    Rafferty, Yvonne

    2013-10-01

    Child trafficking, including commercial sexual exploitation (CSE), is one of the fastest growing and most lucrative criminal activities in the world. The global enslavement of children affects countless numbers of victims who are trafficked within their home countries or transported away from their homes and treated as commodities to be bought, sold, and resold for labor or sexual exploitation. All over the world, girls are particularly likely to be trafficked into the sex trade: Girls and women constitute 98% of those who are trafficked for CSE. Health and safety standards in exploitative settings are generally extremely low, and the degree of experienced violence has been linked with adverse physical, psychological, and social-emotional development. The human-rights-based approach to child trafficking provides a comprehensive conceptual framework whereby victim-focused and law enforcement responses can be developed, implemented, and evaluated. This article highlights promising policies and programs designed to prevent child trafficking and CSE by combating demand for sex with children, reducing supply, and strengthening communities. The literature reviewed includes academic publications as well as international and governmental and nongovernmental reports. Implications for social policy and future research are presented.

  20. Child trafficking and commercial sexual exploitation: a review of promising prevention policies and programs.

    PubMed

    Rafferty, Yvonne

    2013-10-01

    Child trafficking, including commercial sexual exploitation (CSE), is one of the fastest growing and most lucrative criminal activities in the world. The global enslavement of children affects countless numbers of victims who are trafficked within their home countries or transported away from their homes and treated as commodities to be bought, sold, and resold for labor or sexual exploitation. All over the world, girls are particularly likely to be trafficked into the sex trade: Girls and women constitute 98% of those who are trafficked for CSE. Health and safety standards in exploitative settings are generally extremely low, and the degree of experienced violence has been linked with adverse physical, psychological, and social-emotional development. The human-rights-based approach to child trafficking provides a comprehensive conceptual framework whereby victim-focused and law enforcement responses can be developed, implemented, and evaluated. This article highlights promising policies and programs designed to prevent child trafficking and CSE by combating demand for sex with children, reducing supply, and strengthening communities. The literature reviewed includes academic publications as well as international and governmental and nongovernmental reports. Implications for social policy and future research are presented. PMID:24164528

  1. Chloride Channels of Intracellular Membranes

    PubMed Central

    Edwards, John C.; Kahl, Christina R.

    2010-01-01

    Proteins implicated as intracellular chloride channels include the intracellular ClC proteins, the bestrophins, the cystic fibrosis transmembrane conductance regulator, the CLICs, and the recently described Golgi pH regulator. This paper examines current hypotheses regarding roles of intracellular chloride channels and reviews the evidence supporting a role in intracellular chloride transport for each of these proteins. PMID:20100480

  2. Vesicle trafficking plays a novel role in erythroblast enucleation.

    PubMed

    Keerthivasan, Ganesan; Small, Sara; Liu, Hui; Wickrema, Amittha; Crispino, John D

    2010-10-28

    Enucleation of mammalian erythroblasts is a process whose mechanism is largely undefined. The prevailing model suggests that nuclear extrusion occurs via asymmetric cytokinesis. To test this hypothesis, we treated primary erythroblasts with inhibitors of cytokinesis, including blebbistatin, hesperadin, and nocodazole, and then assayed for enucleation. Although these agents inhibited cell-cycle progression and subsequent enucleation when added early in culture, they failed to block enucleation proper when added to postmitotic cells. These results suggest that contraction of the actomyosin ring is not essential for nuclear expulsion. Next, by ultrastructural examination of primary erythroblasts, we observed an accumulation of vacuoles in the cytoplasm proximal to the extruding nucleus. This finding led us to hypothesize that vesicle trafficking contributes to erythroblast enucleation. Here, we show that chemical inhibitors of vesicle trafficking block enucleation of primary erythroblasts without affecting differentiation, cell division, or apoptosis. Moreover, knock-down of clathrin inhibited the enucleation of late erythroblasts. In contrast, vacuolin-1, a small molecule that induces vacuole formation, increased the percentage of enucleated cells. Together, these results illustrate that vesicle trafficking, specifically the formation, movement, and subsequent coalescence of vacuoles at the junction of the nucleus and the cytoplasm, is a critical component of mammalian erythroblast enucleation.

  3. Endosomal MR1 Trafficking Plays a Key Role in Presentation of Mycobacterium tuberculosis Ligands to MAIT Cells.

    PubMed

    Harriff, Melanie J; Karamooz, Elham; Burr, Ansen; Grant, Wilmon F; Canfield, Elizabeth T; Sorensen, Michelle L; Moita, Luis F; Lewinsohn, David M

    2016-03-01

    Mucosal-Associated Invariant T (MAIT) cells, present in high frequency in airway and other mucosal tissues, have Th1 effector capacity positioning them to play a critical role in the early immune response to intracellular pathogens, including Mycobacterium tuberculosis (Mtb). MR1 is a highly conserved Class I-like molecule that presents vitamin B metabolites to MAIT cells. The mechanisms for loading these ubiquitous small molecules are likely to be tightly regulated to prevent inappropriate MAIT cell activation. To define the intracellular localization of MR1, we analyzed the distribution of an MR1-GFP fusion protein in antigen presenting cells. We found that MR1 localized to endosomes and was translocated to the cell surface upon addition of 6-formyl pterin (6-FP). To understand the mechanisms by which MR1 antigens are presented, we used a lentiviral shRNA screen to identify trafficking molecules that are required for the presentation of Mtb antigen to HLA-diverse T cells. We identified Stx18, VAMP4, and Rab6 as trafficking molecules regulating MR1-dependent MAIT cell recognition of Mtb-infected cells. Stx18 but not VAMP4 or Rab6 knockdown also resulted in decreased 6-FP-dependent surface translocation of MR1 suggesting distinct pathways for loading of exogenous ligands and intracellular mycobacterially-derived ligands. We postulate that endosome-mediated trafficking of MR1 allows for selective sampling of the intracellular environment. PMID:27031111

  4. Endosomal MR1 Trafficking Plays a Key Role in Presentation of Mycobacterium tuberculosis Ligands to MAIT Cells

    PubMed Central

    Burr, Ansen; Grant, Wilmon F.; Canfield, Elizabeth T.; Sorensen, Michelle L.; Moita, Luis F.; Lewinsohn, David M.

    2016-01-01

    Mucosal-Associated Invariant T (MAIT) cells, present in high frequency in airway and other mucosal tissues, have Th1 effector capacity positioning them to play a critical role in the early immune response to intracellular pathogens, including Mycobacterium tuberculosis (Mtb). MR1 is a highly conserved Class I-like molecule that presents vitamin B metabolites to MAIT cells. The mechanisms for loading these ubiquitous small molecules are likely to be tightly regulated to prevent inappropriate MAIT cell activation. To define the intracellular localization of MR1, we analyzed the distribution of an MR1-GFP fusion protein in antigen presenting cells. We found that MR1 localized to endosomes and was translocated to the cell surface upon addition of 6-formyl pterin (6-FP). To understand the mechanisms by which MR1 antigens are presented, we used a lentiviral shRNA screen to identify trafficking molecules that are required for the presentation of Mtb antigen to HLA-diverse T cells. We identified Stx18, VAMP4, and Rab6 as trafficking molecules regulating MR1-dependent MAIT cell recognition of Mtb-infected cells. Stx18 but not VAMP4 or Rab6 knockdown also resulted in decreased 6-FP-dependent surface translocation of MR1 suggesting distinct pathways for loading of exogenous ligands and intracellular mycobacterially-derived ligands. We postulate that endosome-mediated trafficking of MR1 allows for selective sampling of the intracellular environment. PMID:27031111

  5. Female sex trafficking: conceptual issues, current debates, and future directions.

    PubMed

    Meshkovska, Biljana; Siegel, Melissa; Stutterheim, Sarah E; Bos, Arjan E R

    2015-01-01

    Female sex trafficking is a pressing concern. In this article, we provide a comprehensive overview of relevant issues regarding the concept of female sex trafficking and research in the field of human trafficking, drawing on a variety of disciplines, including economics, gender and sexuality studies, psychology, sociology, law, and social work. We discuss the debates surrounding the definition of human trafficking, compare and contrast it with human smuggling, and outline connections between female sex trafficking and the issue of sex work and prostitution. We further discuss the history and current estimations of female sex trafficking. We then outline the main actors in female sex trafficking, including trafficked persons, traffickers, clients, and service providers, and we overview the trafficking process from recruitment to identification, recovery, and (re)integration. Finally, we conclude with recommendations for future research that tie together the concepts of vulnerability, exploitation, and long-term recovery and (re)integration. PMID:25897567

  6. Female sex trafficking: conceptual issues, current debates, and future directions.

    PubMed

    Meshkovska, Biljana; Siegel, Melissa; Stutterheim, Sarah E; Bos, Arjan E R

    2015-01-01

    Female sex trafficking is a pressing concern. In this article, we provide a comprehensive overview of relevant issues regarding the concept of female sex trafficking and research in the field of human trafficking, drawing on a variety of disciplines, including economics, gender and sexuality studies, psychology, sociology, law, and social work. We discuss the debates surrounding the definition of human trafficking, compare and contrast it with human smuggling, and outline connections between female sex trafficking and the issue of sex work and prostitution. We further discuss the history and current estimations of female sex trafficking. We then outline the main actors in female sex trafficking, including trafficked persons, traffickers, clients, and service providers, and we overview the trafficking process from recruitment to identification, recovery, and (re)integration. Finally, we conclude with recommendations for future research that tie together the concepts of vulnerability, exploitation, and long-term recovery and (re)integration.

  7. Trafficking regulates the subcellular distribution of voltage-gated sodium channels in primary sensory neurons.

    PubMed

    Bao, Lan

    2015-01-01

    Voltage-gated sodium channels (Navs) comprise at least nine pore-forming α subunits. Of these, Nav1.6, Nav1.7, Nav1.8 and Nav1.9 are the most frequently studied in primary sensory neurons located in the dorsal root ganglion and are mainly localized to the cytoplasm. A large pool of intracellular Navs raises the possibility that changes in Nav trafficking could alter channel function. The molecular mediators of Nav trafficking mainly consist of signals within the Navs themselves, interacting proteins and extracellular factors. The surface expression of Navs is achieved by escape from the endoplasmic reticulum and proteasome degradation, forward trafficking and plasma membrane anchoring, and it is also regulated by channel phosphorylation and ubiquitination in primary sensory neurons. Axonal transport and localization of Navs in afferent fibers involves the motor protein KIF5B and scaffold proteins, including contactin and PDZ domain containing 2. Localization of Nav1.6 to the nodes of Ranvier in myelinated fibers of primary sensory neurons requires node formation and the submembrane cytoskeletal protein complex. These findings inform our understanding of the molecular and cellular mechanisms underlying Nav trafficking in primary sensory neurons. PMID:26423360

  8. Computational Model of VEGFR2 pathway to ERK activation and modulation through receptor trafficking

    PubMed Central

    Tan, Wan Hua; Popel, Aleksander S.; Mac Gabhann, Feilim

    2013-01-01

    Vascular Endothelial Growth Factor (VEGF) signal transduction is central to angiogenesis in development and in pathological conditions such as cancer, retinopathy and ischemic diseases. We constructed and validated a computational model of VEGFR2 trafficking and signaling, to study the role of receptor trafficking kinetics in modulating ERK phosphorylation in VEGF-stimulated endothelial cells. Trafficking parameters were optimized and validated against four previously published in vitro experiments. Based on these parameters, model simulations demonstrated interesting behaviors that may be highly relevant to understanding VEGF signaling in endothelial cells. First, at moderate VEGF doses, VEGFR2 phosphorylation and ERK phosphorylation are related in a log-linear fashion, with a stable duration of ERK activation; but with higher VEGF stimulation, phosphoERK becomes saturated, and its duration increases. Second, a large endosomal fraction of VEGFR2 makes the ERK activation reaction network less sensitive to perturbations in VEGF dosage. Third, extracellular-matrix-bound VEGF binds and activates VEGFR2, but by internalizing at a slower rate, matrix-bound VEGF-induced intracellular ERK phosphorylation is predicted to be greater in magnitude and more sustained, in agreement with experimental evidence. Fourth, different endothelial cell types appear to have different trafficking rates, which result in different levels of endosomal receptor localization and different ERK response profiles. PMID:23993967

  9. Human trafficking law and social structures.

    PubMed

    Wooditch, Alese

    2012-08-01

    Human trafficking has only recently emerged at the forefront of policy reform, even in developed nations. Yet, heightened awareness of the issue has not translated into effective policy as the majority of nations have ineffective antitrafficking practices; many countries have failed to criminalize human trafficking, whereas others do not actively enforce statutes in place. By applying Black's theory of law, this study offers a preliminary understanding into the variation of global prosecutorial efforts in human trafficking and adequacy of antitrafficking law. To isolate this relationship, the effects of trafficking markets are controlled. As with prior research, the study finds limited support for the theory. The article concludes with a discussion on the implications of the quantity of antitrafficking law and morphology association for policy development.

  10. Committee opinion no. 507: human trafficking.

    PubMed

    2011-09-01

    Human trafficking is a widespread problem with estimates ranging from 14,000 to 50,000 individuals trafficked into the United States annually. This hidden population involves the commercial sex industry, agriculture, factories, hotel and restaurant businesses, domestic workers, marriage brokers, and some adoption firms. Because 80% of trafficked individuals are women and girls, women’s health care providers may better serve their diverse patient population by increasing their awareness of this problem. The exploitation of people of any race, gender, sexual orientation, or ethnicity is unacceptable at any time, in any place. The members of the American College of Obstetricians and Gynecologists should be aware of this problem and strive to recognize and assist their patients who are victims or who have been victims of human trafficking. PMID:21860320

  11. Committee opinion no. 507: human trafficking.

    PubMed

    2011-09-01

    Human trafficking is a widespread problem with estimates ranging from 14,000 to 50,000 individuals trafficked into the United States annually. This hidden population involves the commercial sex industry, agriculture, factories, hotel and restaurant businesses, domestic workers, marriage brokers, and some adoption firms. Because 80% of trafficked individuals are women and girls, women’s health care providers may better serve their diverse patient population by increasing their awareness of this problem. The exploitation of people of any race, gender, sexual orientation, or ethnicity is unacceptable at any time, in any place. The members of the American College of Obstetricians and Gynecologists should be aware of this problem and strive to recognize and assist their patients who are victims or who have been victims of human trafficking.

  12. Sex trafficking of women and girls.

    PubMed

    Deshpande, Neha A; Nour, Nawal M

    2013-01-01

    Sex trafficking involves some form of forced or coerced sexual exploitation that is not limited to prostitution, and has become a significant and growing problem in both the United States and the larger global community. The costs to society include the degradation of human and women's rights, poor public health, disrupted communities, and diminished social development. Victims of sex trafficking acquire adverse physical and psychological health conditions and social disadvantages. Thus, sex trafficking is a critical health issue with broader social implications that requires both medical and legal attention. Healthcare professionals can work to improve the screening, identification, and assistance of victims of sex trafficking in a clinical setting and help these women and girls access legal and social services. PMID:23687554

  13. Ovarian Cystadenoma in a Trafficked Patient.

    PubMed

    Titchen, Kanani E; Katz, Douglas; Martinez, Kidian; White, Krishna

    2016-05-01

    The topic of child sex trafficking is receiving increased attention both in the lay press and in research articles. Recently, a number of physician organizations have issued policy statements calling for the education and involvement of physicians in combating this form of "modern-day slavery." Primary care and emergency medicine physicians have led these efforts, but a number of these victims may present to surgeons. Surgeons are in a unique position to identify trafficked patients; during the process of undraping, intubation, and surgical preparation, signs of trafficking such as tattoos, scars, dental injuries, and bruising may be evident. In addition, these patients may have specific needs in terms of anesthesia and postoperative care due to substance abuse. Here, we report the case of an 18-year-old girl with a history of sexual exploitation who presents for cystadenoma excision. To our knowledge, this is the first report of a sex-trafficked pediatric patient presenting for surgery. PMID:27244785

  14. Sex Trafficking of Women and Girls

    PubMed Central

    Deshpande, Neha A; Nour, Nawal M

    2013-01-01

    Sex trafficking involves some form of forced or coerced sexual exploitation that is not limited to prostitution, and has become a significant and growing problem in both the United States and the larger global community. The costs to society include the degradation of human and women’s rights, poor public health, disrupted communities, and diminished social development. Victims of sex trafficking acquire adverse physical and psychological health conditions and social disadvantages. Thus, sex trafficking is a critical health issue with broader social implications that requires both medical and legal attention. Healthcare professionals can work to improve the screening, identification, and assistance of victims of sex trafficking in a clinical setting and help these women and girls access legal and social services. PMID:23687554

  15. Intracellular chromium reduction.

    PubMed

    Arslan, P; Beltrame, M; Tomasi, A

    1987-10-22

    Two steps are involved in the uptake of Cr(VI): (1) the diffusion of the anion CrO4(2-) through a facilitated transport system, presumably the non-specific anion carrier and (2) the intracellular reduction of Cr(VI) to Cr(III). The intracellular reduction of Cr(VI), keeping the cytoplasmic concentration of Cr(VI) low, facilitates accumulation of chromate from extracellular medium into the cell. In the present paper, a direct demonstration of intracellular chromium reduction is provided by means of electron paramagnetic (spin) resonance (EPR) spectroscopy. Incubation of metabolically active rat thymocytes with chromate originates a signal which can be attributed to a paramagnetic species of chromium, Cr(V) or Cr(III). The EPR signal is originated by intracellular reduction of chromium since: (1) it is observed only when cells are incubated with chromate, (2) it is present even after extensive washings of the cells in a chromium-free medium; (3) it is abolished when cells are incubated with drugs able to reduce the glutathione pool, i.e., diethylmaleate or phorone; and (4) it is abolished when cells are incubated in the presence of a specific inhibitor of the anion carrier, 4-acetamido-4'-isothiocyanatostilbene-2-2'-disulfonic acid. PMID:2820507

  16. A systematic High-Content Screening microscopy approach reveals key roles for Rab33b, OATL1 and Myo6 in nanoparticle trafficking in HeLa cells

    PubMed Central

    Panarella, Angela; Bexiga, Mariana G.; Galea, George; O’ Neill, Elaine D.; Salvati, Anna; Dawson, Kenneth A.; Simpson, Jeremy C.

    2016-01-01

    Synthetic nanoparticles are promising tools for imaging and drug delivery; however the molecular details of cellular internalization and trafficking await full characterization. Current knowledge suggests that following endocytosis most nanoparticles pass from endosomes to lysosomes. In order to design effective drug delivery strategies that can use the endocytic pathway, or by-pass lysosomal accumulation, a comprehensive understanding of nanoparticle uptake and trafficking mechanisms is therefore fundamental. Here we describe and apply an RNA interference-based high-content screening microscopy strategy to assess the intracellular trafficking of fluorescently-labeled polystyrene nanoparticles in HeLa cells. We screened a total of 408 genes involved in cytoskeleton and membrane function, revealing roles for myosin VI, Rab33b and OATL1 in this process. This work provides the first systematic large-scale quantitative assessment of the proteins responsible for nanoparticle trafficking in cells, paving the way for subsequent genome-wide studies. PMID:27374232

  17. Trafficking of. cap alpha. -L-fucosidase in lymphoid cells

    SciTech Connect

    DiCioccio, R.A.; Brown, K.S.

    1987-05-01

    The quantity of ..cap alpha..-L-fucosidase in human serum is determined by heredity. The mechanism controlling levels of the enzyme in serum is unknown. To investigate this, lymphoid cell lines derived from individuals with either low, intermediate or high ..cap alpha..-L-fucosidase in serum were established. Steady state levels of extracellular ..cap alpha..-L-fucosidase protein and activity overlapped among the cell lines. Thus, in vivo serum phenotypes of ..cap alpha..-L-fucosidase are not adequately expressed in this system. ..cap alpha..-L-Fucosidase was also metabolically labelled with /sup 35/S-methionine, immunoprecipitated, and examined by SDS-PAGE. Cells pulse-labelled from 0.25-2 h had a major intracellular form of enzyme (Mr = 58,000). Cells pulsed for 1.5 h and chased for 21 h with unlabeled methionine had an intracellular form of Mr = 60,000 and an extracellular form of Mr = 62,000. Cells treated with chloroquine had only the 58,000-form both intra- and extra-cellularly. Moreover, chloroquine did not effect the quantitative distribution of ..cap alpha..-L-fucosidase between cells and medium. In fibroblasts, chloroquine enhanced the secretion of newly made lysosomal enzymes and blocked the processing of intercellular enzyme forms from a higher to a lower molecular mass. Thus, there are trafficking differences between ..cap alpha..-L-fucosidase in lymphoid cells and lysosomal enzymes in fibroblasts. This suggests that alternative targeting mechanisms for lysosomal enzymes exist in these cells.

  18. Cystic fibrosis transmembrane conductance regulator trafficking modulates the barrier function of airway epithelial cell monolayers.

    PubMed

    LeSimple, Pierre; Liao, Jie; Robert, Renaud; Gruenert, Dieter C; Hanrahan, John W

    2010-04-15

    The cystic fibrosis transmembrane conductance regulator (CFTR) is an integral membrane glycoprotein which functions as an anion channel and influences diverse cellular processes. We studied its role in the development of epithelial tightness by expressing wild-type (WT-CFTR) or mutant (Delta F508-CFTR) CFTR in human airway epithelial cell monolayers cultured at the air-liquid interface. Green fluorescent protein (GFP)-tagged WT or Delta F508 constructs were expressed in the CF bronchial cell line CFBE41o(-) using adenoviruses, and the results were compared with those obtained using CFBE41o(-) lines stably complemented with wild-type or mutant CFTR. As predicted, GFP-Delta WT-CFTR reached the apical membrane whereas GFP-F508-CFTR was only detected intracellularly. Although CFTR expression would be expected to reduce transepithelial resistance (TER), expressing GFP-CFTR significantly increased the TER of CFBE41o(-) monolayers whilst GFP-Delta F508-CFTR had no effect. Similar results were obtained with cell lines stably overexpressing Delta F508-CFTR or WT-CFTR. Preincubating Delta F508-CFTR monolayers at 29 degrees C reduced mannitol permeability and restored TER, and the effect on TER was reversible during temperature oscillations. Expression of GFP-Delta F508-CFTR or GFP-WT-CFTR in a cell line already containing endogenous WT-CFTR (Calu-3) did not alter TER. The CFTR- and temperature-dependence of TER were not affected by the CFTR inhibitor CFTR(inh)172 or low-chloride medium; therefore the effect of CFTR on barrier function was unrelated to its ion channel activity. Modulation of TER was blunted but not eliminated by genistein, implying the involvement of tyrosine phosphorylation and other mechanisms. Modulation of CFTR trafficking was correlated with an increase in tight junction depth. The results suggest that CFTR trafficking is required for the normal organisation and function of tight junctions. A reduction in barrier function caused by endoplasmic reticulum

  19. The hidden crime: human trafficking.

    PubMed

    Clause, Kristen J; Lawler, Kate Byrnes

    2013-01-01

    As the primary contact in the health care system, nurses can play a role in combating this crime and assisting the victims. Assessment for abuse, neglect, trauma, recurrent sexually transmitted infections (STIs) and fear of a controlling partner is critical. Following up on "red flags" and understanding methods of safe questioning can make the difference between slavery and recovery for victims. Nurses must also know the professional referrals in their areas once a potential victim has been identified. This may be a very dangerous undertaking and must be handled by experienced personnel. Referrals to forensic nurses or physicians, domestic violence professionals or law enforcement may be indicated. Initially, a nurse may want to consult with the agency social worker for guidance. Human trafficking is a human rights crime. Unfortunately, it is more prevalent in all types of communities than most people suspect. Nurses can be heroes to the victims through understanding of this crime and vigilance in the assessment and care of all people they encounter in their practices. PMID:24218718

  20. The hidden crime: human trafficking.

    PubMed

    Clause, Kristen J; Lawler, Kate Byrnes

    2013-01-01

    As the primary contact in the health care system, nurses can play a role in combating this crime and assisting the victims. Assessment for abuse, neglect, trauma, recurrent sexually transmitted infections (STIs) and fear of a controlling partner is critical. Following up on "red flags" and understanding methods of safe questioning can make the difference between slavery and recovery for victims. Nurses must also know the professional referrals in their areas once a potential victim has been identified. This may be a very dangerous undertaking and must be handled by experienced personnel. Referrals to forensic nurses or physicians, domestic violence professionals or law enforcement may be indicated. Initially, a nurse may want to consult with the agency social worker for guidance. Human trafficking is a human rights crime. Unfortunately, it is more prevalent in all types of communities than most people suspect. Nurses can be heroes to the victims through understanding of this crime and vigilance in the assessment and care of all people they encounter in their practices. To learn more or to help with this cause, visit the Somaly Mam Foundation at www.somaly.org or the U.S. Department of State at www. state.gov. PMID:23977773

  1. The hidden crime: human trafficking.

    PubMed

    Clause, Kristen J; Lawler, Kate Byrnes

    2013-01-01

    As the primary contact in the health care system, nurses can play a role in combating this crime and assisting the victims. Assessment for abuse, neglect, trauma, recurrent sexually transmitted infections (STIs) and fear of a controlling partner is critical. Following up on "red flags" and understanding methods of safe questioning can make the difference between slavery and recovery for victims. Nurses must also know the professional referrals in their areas once a potential victim has been identified. This may be a very dangerous undertaking and must be handled by experienced personnel. Referrals to forensic nurses or physicians, domestic violence professionals or law enforcement may be indicated. Initially, a nurse may want to consult with the agency social worker for guidance. Human trafficking is a human rights crime. Unfortunately, it is more prevalent in all types of communities than most people suspect. Nurses can be heroes to the victims through understanding of this crime and vigilance in the assessment and care of all people they encounter in their practices. To learn more or to help with this cause, visit the Somaly Mam Foundation at www.somaly.org or the U.S. Department of State at www. state.gov.

  2. The hidden crime: human trafficking.

    PubMed

    Clause, Kristen J; Lawler, Kate Byrnes

    2013-01-01

    As the primary contact in the health care system, nurses can play a role in combating this crime and assisting the victims. Assessment for abuse, neglect, trauma, recurrent sexually transmitted infections (STIs) and fear of a controlling partner is critical. Following up on "red flags" and understanding methods of safe questioning can make the difference between slavery and recovery for victims. Nurses must also know the professional referrals in their areas once a potential victim has been identified. This may be a very dangerous undertaking and must be handled by experienced personnel. Referrals to forensic nurses or physicians, domestic violence professionals or law enforcement may be indicated. Initially, a nurse may want to consult with the agency social worker for guidance. Human trafficking is a human rights crime. Unfortunately, it is more prevalent in all types of communities than most people suspect. Nurses can be heroes to the victims through understanding of this crime and vigilance in the assessment and care of all people they encounter in their practices.

  3. Lymphatic Regulation of Cellular Trafficking

    PubMed Central

    Jackson, David G.

    2016-01-01

    Lymphatic vessels play vital roles in immune surveillance and immune regulation by conveying antigen loaded dendritic cells, memory T cells, macrophages and neutrophils from the peripheral tissues to draining lymph nodes where they initiate as well as modify immune responses. Until relatively recently however, there was little understanding of how entry and migration through lymphatic vessels is organized or the specific molecular mechanisms that might be involved. Within the last decade, the situation has been transformed by an explosion of knowledge generated largely through the application of microscopic imaging, transgenic animals, specific markers and function blocking mAbs that is beginning to provide a rational conceptual framework. This article provides a critical review of the recent literature, highlighting seminal discoveries that have revealed the fascinating ultrastructure of leucocyte entry sites in lymphatic vessels, as well as generating controversies over the involvement of integrin adhesion, chemotactic and haptotactic mechanisms in DC entry under normal and inflamed conditions. It also discusses the major changes in lymphatic architecture that occur during inflammation and the different modes of leucocyte entry and trafficking within inflamed lymphatic vessels, as well as presenting a timely update on the likely role of hyaluronan and the major lymphatic endothelial hyaluronan receptor LYVE-1 in leucocyte transit.

  4. Salmonella Disrupts Host Endocytic Trafficking by SopD2-Mediated Inhibition of Rab7.

    PubMed

    D'Costa, Vanessa M; Braun, Virginie; Landekic, Marija; Shi, Rong; Proteau, Ariane; McDonald, Laura; Cygler, Miroslaw; Grinstein, Sergio; Brumell, John H

    2015-09-01

    Intracellular bacterial pathogens of a diverse nature share the ability to evade host immunity by impairing trafficking of endocytic cargo to lysosomes for degradation, a process that is poorly understood. Here, we show that the Salmonella enterica type 3 secreted effector SopD2 mediates this process by binding the host regulatory GTPase Rab7 and inhibiting its nucleotide exchange. Consequently, this limits Rab7 interaction with its dynein- and kinesin-binding effectors RILP and FYCO1 and thereby disrupts host-driven regulation of microtubule motors. Our study identifies a bacterial effector capable of directly binding and thereby modulating Rab7 activity and a mechanism of endocytic trafficking disruption that may provide insight into the pathogenesis of other bacteria. Additionally, we provide a powerful tool for the study of Rab7 function, and a potential therapeutic target. PMID:26299973

  5. Plasma membrane protein trafficking in plant–microbe interactions: a plant cell point of view

    PubMed Central

    Nathalie Leborgne-Castel; Bouhidel, Karim

    2014-01-01

    In order to ensure their physiological and cellular functions, plasma membrane (PM) proteins must be properly conveyed from their site of synthesis, i.e., the endoplasmic reticulum, to their final destination, the PM, through the secretory pathway. PM protein homeostasis also relies on recycling and/or degradation, two processes that are initiated by endocytosis. Vesicular membrane trafficking events to and from the PM have been shown to be altered when plant cells are exposed to mutualistic or pathogenic microbes. In this review, we will describe the fine-tune regulation of such alterations, and their consequence in PM protein activity. We will consider the formation of intracellular perimicrobial compartments, the PM protein trafficking machinery of the host, and the delivery or retrieval of signaling and transport proteins such as pattern-recognition receptors, producers of reactive oxygen species, and sugar transporters. PMID:25566303

  6. Salmonella Disrupts Host Endocytic Trafficking by SopD2-Mediated Inhibition of Rab7.

    PubMed

    D'Costa, Vanessa M; Braun, Virginie; Landekic, Marija; Shi, Rong; Proteau, Ariane; McDonald, Laura; Cygler, Miroslaw; Grinstein, Sergio; Brumell, John H

    2015-09-01

    Intracellular bacterial pathogens of a diverse nature share the ability to evade host immunity by impairing trafficking of endocytic cargo to lysosomes for degradation, a process that is poorly understood. Here, we show that the Salmonella enterica type 3 secreted effector SopD2 mediates this process by binding the host regulatory GTPase Rab7 and inhibiting its nucleotide exchange. Consequently, this limits Rab7 interaction with its dynein- and kinesin-binding effectors RILP and FYCO1 and thereby disrupts host-driven regulation of microtubule motors. Our study identifies a bacterial effector capable of directly binding and thereby modulating Rab7 activity and a mechanism of endocytic trafficking disruption that may provide insight into the pathogenesis of other bacteria. Additionally, we provide a powerful tool for the study of Rab7 function, and a potential therapeutic target.

  7. ESCRT-I Protein Tsg101 Plays a Role in the Post-macropinocytic Trafficking and Infection of Endothelial Cells by Kaposi’s Sarcoma-Associated Herpesvirus

    PubMed Central

    Kumar, Binod; Dutta, Dipanjan; Iqbal, Jawed; Ansari, Mairaj Ahmed; Roy, Arunava; Chikoti, Leela; Pisano, Gina; Veettil, Mohanan Valiya; Chandran, Bala

    2016-01-01

    Kaposi’s sarcoma-associated herpesvirus (KSHV) binding to the endothelial cell surface heparan sulfate is followed by sequential interactions with α3β1, αVβ3 and αVβ5 integrins and Ephrin A2 receptor tyrosine kinase (EphA2R). These interactions activate host cell pre-existing FAK, Src, PI3-K and RhoGTPase signaling cascades, c-Cbl mediated ubiquitination of receptors, recruitment of CIB1, p130Cas and Crk adaptor molecules, and membrane bleb formation leading to lipid raft dependent macropinocytosis of KSHV into human microvascular dermal endothelial (HMVEC-d) cells. The Endosomal Sorting Complexes Required for Transport (ESCRT) proteins, ESCRT-0, -I, -II, and–III, play a central role in clathrin-mediated internalized ubiquitinated receptor endosomal trafficking and sorting. ESCRT proteins have also been shown to play roles in viral egress. We have recently shown that ESCRT-0 component Hrs protein associates with the plasma membrane during macropinocytosis and mediates KSHV entry via ROCK1 mediated phosphorylation of NHE1 and local membrane pH change. Here, we demonstrate that the ESCRT-I complex Tsg101 protein also participates in the macropinocytosis of KSHV and plays a role in KSHV trafficking. Knockdown of Tsg101 did not affect virus entry in HMVEC-d and human umbilical vein endothelial (HUVEC) cells but significantly inhibited the KSHV genome entry into the nucleus and consequently viral gene expression in these cells. Double and triple immunofluorescence, proximity ligation immunofluorescence and co-immuoprecipitation studies revealed the association of Tsg101 with the KSHV containing macropinosomes, and increased levels of Tsg101 association/interactions with EphA2R, c-Cbl, p130Cas and Crk signal molecules, as well as with upstream and downstream ESCRT components such as Hrs (ESCRT-0), EAP45 (ESCRT-II), CHMP6 (ESCRT-III) and CHMP5 (ESCRT-III) in the KSHV infected cells. Tsg101 was also associated with early (Rab5) and late endosomal (Rab7) stages of

  8. Rha1, an Arabidopsis Rab5 homolog, plays a critical role in the vacuolar trafficking of soluble cargo proteins.

    PubMed

    Sohn, Eun Ju; Kim, Eol Sun; Zhao, Min; Kim, Soo Jin; Kim, Hyeran; Kim, Yong-Woo; Lee, Yong Jik; Hillmer, Stefan; Sohn, Uik; Jiang, Liwen; Hwang, Inhwan

    2003-05-01

    Rab proteins are members of the Ras superfamily of small GTP binding proteins and play important roles in various intracellular trafficking steps. We investigated the role of Rha1, an Arabidopsis Rab5 homolog, in intracellular trafficking in Arabidopsis protoplasts. In the presence of a dominant-negative mutant of Rha1, soluble vacuolar cargo proteins such as sporamin:green fluorescent protein (Spo:GFP) and Arabidopsis aleurain like protein:GFP are not delivered to the central vacuole; instead, they accumulate as a diffuse or punctate staining pattern within the cell. Spo:GFP at the punctate stains observed in the presence of hemagglutinin:Rha1[S24N] is colocalized with endogenous vacuolar sorting receptor (VSR(At-1)), which is known to localize primarily to the prevacuolar compartment, whereas Spo:GFP in the diffuse pattern is associated with tonoplasts. Furthermore, expression of Rha1[S24N] causes the secretion of a portion of the vacuolar proteins into medium. However, the inhibitory effect of Rha1[S24N] on vacuolar trafficking is relieved partially by coexpressed wild-type Rha1. Based on these results, we propose that Rha1 plays a critical role in the trafficking of soluble cargoes from the prevacuolar compartment to the central vacuole.

  9. Intracellular Distribution of Human T-Cell Leukemia Virus Type 1 Gag Proteins Is Independent of Interaction with Intracellular Membranes

    PubMed Central

    LeBlanc, Isabelle; Blot, Vincent; Bouchaert, Isabelle; Salamero, Jean; Goud, Bruno; Rosenberg, Arielle R.; Dokhélar, Marie-Christine

    2002-01-01

    Retrovirus Gag proteins are synthesized on free ribosomes, and are sufficient to govern the assembly and release of virus particles. Like type C retroviruses, human T-cell leukemia virus type 1 (HTLV-1) assembles and buds at the plasma membrane. After immunofluorescence staining, HTLV-1 Gag proteins appear as punctuated intracellular clusters, which suggests that they are associated either with intracellular membranes or with the plasma membrane. However, colocalization experiments using a panel of markers demonstrated that Gag proteins were not associated with the membranes involved in the secretory or endocytosis pathway. Small amounts of Gag proteins were detected at the plasma membrane and colocalized with the envelope glycoproteins. Moreover, Gag proteins were excluded from streptolysin-O permeabilized cells and in this respect behaved like cytoplasmic proteins. This suggests that the trafficking of HTLV-1 Gag proteins through the cytoplasm of the host cell is independent of any cell membrane system. PMID:11752179

  10. An international comparative public health analysis of sex trafficking of women and girls in eight cities: achieving a more effective health sector response.

    PubMed

    Macias Konstantopoulos, Wendy; Ahn, Roy; Alpert, Elaine J; Cafferty, Elizabeth; McGahan, Anita; Williams, Timothy P; Castor, Judith Palmer; Wolferstan, Nadya; Purcell, Genevieve; Burke, Thomas F

    2013-12-01

    Sex trafficking, trafficking for the purpose of forced sexual exploitation, is a widespread form of human trafficking that occurs in all regions of the world, affects mostly women and girls, and has far-reaching health implications. Studies suggest that up to 50 % of sex trafficking victims in the USA seek medical attention while in their trafficking situation, yet it is unclear how the healthcare system responds to the needs of victims of sex trafficking. To understand the intersection of sex trafficking and public health, we performed in-depth qualitative interviews among 277 antitrafficking stakeholders across eight metropolitan areas in five countries to examine the local context of sex trafficking. We sought to gain a new perspective on this form of gender-based violence from those who have a unique vantage point and intimate knowledge of push-and-pull factors, victim health needs, current available resources and practices in the health system, and barriers to care. Through comparative analysis across these contexts, we found that multiple sociocultural and economic factors facilitate sex trafficking, including child sexual abuse, the objectification of women and girls, and lack of income. Although there are numerous physical and psychological health problems associated with sex trafficking, health services for victims are patchy and poorly coordinated, particularly in the realm of mental health. Various factors function as barriers to a greater health response, including low awareness of sex trafficking and attitudinal biases among health workers. A more comprehensive and coordinated health system response to sex trafficking may help alleviate its devastating effects on vulnerable women and girls. There are numerous opportunities for local health systems to engage in antitrafficking efforts while partnering across sectors with relevant stakeholders. PMID:24151086

  11. An international comparative public health analysis of sex trafficking of women and girls in eight cities: achieving a more effective health sector response.

    PubMed

    Macias Konstantopoulos, Wendy; Ahn, Roy; Alpert, Elaine J; Cafferty, Elizabeth; McGahan, Anita; Williams, Timothy P; Castor, Judith Palmer; Wolferstan, Nadya; Purcell, Genevieve; Burke, Thomas F

    2013-12-01

    Sex trafficking, trafficking for the purpose of forced sexual exploitation, is a widespread form of human trafficking that occurs in all regions of the world, affects mostly women and girls, and has far-reaching health implications. Studies suggest that up to 50 % of sex trafficking victims in the USA seek medical attention while in their trafficking situation, yet it is unclear how the healthcare system responds to the needs of victims of sex trafficking. To understand the intersection of sex trafficking and public health, we performed in-depth qualitative interviews among 277 antitrafficking stakeholders across eight metropolitan areas in five countries to examine the local context of sex trafficking. We sought to gain a new perspective on this form of gender-based violence from those who have a unique vantage point and intimate knowledge of push-and-pull factors, victim health needs, current available resources and practices in the health system, and barriers to care. Through comparative analysis across these contexts, we found that multiple sociocultural and economic factors facilitate sex trafficking, including child sexual abuse, the objectification of women and girls, and lack of income. Although there are numerous physical and psychological health problems associated with sex trafficking, health services for victims are patchy and poorly coordinated, particularly in the realm of mental health. Various factors function as barriers to a greater health response, including low awareness of sex trafficking and attitudinal biases among health workers. A more comprehensive and coordinated health system response to sex trafficking may help alleviate its devastating effects on vulnerable women and girls. There are numerous opportunities for local health systems to engage in antitrafficking efforts while partnering across sectors with relevant stakeholders.

  12. Engineering an Anti-Transferrin Receptor ScFv for pH-Sensitive Binding Leads to Increased Intracellular Accumulation

    PubMed Central

    Tillotson, Benjamin J.; Goulatis, Loukas I.; Parenti, Isabelle; Duxbury, Elizabeth; Shusta, Eric V.

    2015-01-01

    The equilibrium binding affinity of receptor-ligand or antibody-antigen pairs may be modulated by protonation of histidine side-chains, and such pH-dependent mechanisms play important roles in biological systems, affecting molecular uptake and trafficking. Here, we aimed to manipulate cellular transport of single-chain antibodies (scFvs) against the transferrin receptor (TfR) by engineering pH-dependent antigen binding. An anti-TfR scFv was subjected to histidine saturation mutagenesis of a single CDR. By employing yeast surface display with a pH-dependent screening pressure, scFvs having markedly increased dissociation from TfR at pH 5.5 were identified. The pH-sensitivity generally resulted from a central cluster of histidine residues in CDRH1. When soluble, pH-sensitive, scFv clone M16 was dosed onto live cells, the internalized fraction was 2.6-fold greater than scFvs that lacked pH-sensitive binding and the increase was dependent on endosomal acidification. Differences in the intracellular distribution of M16 were also observed consistent with an intracellular decoupling of the scFv M16-TfR complex. Engineered pH-sensitive TfR binding could prove important for increasing the effectiveness of TfR-targeted antibodies seeking to exploit endocytosis or transcytosis for drug delivery purposes. PMID:26713870

  13. Engineering an Anti-Transferrin Receptor ScFv for pH-Sensitive Binding Leads to Increased Intracellular Accumulation.

    PubMed

    Tillotson, Benjamin J; Goulatis, Loukas I; Parenti, Isabelle; Duxbury, Elizabeth; Shusta, Eric V

    2015-01-01

    The equilibrium binding affinity of receptor-ligand or antibody-antigen pairs may be modulated by protonation of histidine side-chains, and such pH-dependent mechanisms play important roles in biological systems, affecting molecular uptake and trafficking. Here, we aimed to manipulate cellular transport of single-chain antibodies (scFvs) against the transferrin receptor (TfR) by engineering pH-dependent antigen binding. An anti-TfR scFv was subjected to histidine saturation mutagenesis of a single CDR. By employing yeast surface display with a pH-dependent screening pressure, scFvs having markedly increased dissociation from TfR at pH 5.5 were identified. The pH-sensitivity generally resulted from a central cluster of histidine residues in CDRH1. When soluble, pH-sensitive, scFv clone M16 was dosed onto live cells, the internalized fraction was 2.6-fold greater than scFvs that lacked pH-sensitive binding and the increase was dependent on endosomal acidification. Differences in the intracellular distribution of M16 were also observed consistent with an intracellular decoupling of the scFv M16-TfR complex. Engineered pH-sensitive TfR binding could prove important for increasing the effectiveness of TfR-targeted antibodies seeking to exploit endocytosis or transcytosis for drug delivery purposes. PMID:26713870

  14. [Progress in research on defective protein trafficking and functional restoration in HERG-associated long QT syndrome].

    PubMed

    Fang, Peiliang; Lian, Jiangfang

    2016-02-01

    The human ether-a-go-go related gene (HERG) encodes the α -subunit of the rapid component of the delayed rectifier K(+) channel, which is essential for the third repolarization of the action potential of human myocardial cells. Mutations of the HERG gene can cause type II hereditary long QT syndrome (LQT2), characterized by prolongation of the QT interval, abnormal T wave, torsade de pointes, syncope and sudden cardiac death. So far more than 300 HERG mutations have been identified, the majority of which can cause LQT2 due to HERG protein trafficking defect. It has been reported that certain drugs can induce acquired long QT syndrome through directly blocking the pore and/or affecting the HERG trafficking. The trafficking defects and K(+) currents can be restored with low temperature and certain drugs. However, the mechanisms underlying defective trafficking caused by HERG mutations and the inhibition/restoration of HERG trafficking by drugs are still unknown. This review summarizes the current understanding of the molecular mechanisms including HERG trafficking under physiological and pathological conditions, and the effects of drugs on the HERG trafficking, in order to provide theoretical evidence for the diagnosis and treatment of long QT syndrome.

  15. Differential Regulation of Endosomal GPCR/β-Arrestin Complexes and Trafficking by MAPK*

    PubMed Central

    Khoury, Etienne; Nikolajev, Ljiljana; Simaan, May; Namkung, Yoon; Laporte, Stéphane A.

    2014-01-01

    β-Arrestins are signaling adaptors that bind to agonist-occupied G protein-coupled receptors (GPCRs) and target them for endocytosis; however, the mechanisms regulating receptor/β-arrestin complexes and trafficking in endosomes, remain ill defined. Here we show, in live cells, differential dynamic regulation of endosomal bradykinin B2 receptor (B2R) complexes with either β-arrestin-1 or -2. We find a novel role for MAPK in the B2R/β-arrestin-2 complex formation, receptor trafficking and signaling mediated by an ERK1/2 regulatory motif in the hinge domain of the rat β-arrestin-2 (PET178P), but not rat β-arrestin-1 (PER177P). While the ERK1/2 regulatory motif is conserved between rat and mouse β-arrestin-2, it is surprisingly not conserved in human β-arrestin-2 (PEK178P). However, mutation of lysine 178 to threonine is sufficient to confer MAPK sensitivity to the human β-arrestin-2. Furthermore, substitution for a phosphomimetic residue in both the rat and the human β-arrestin-2 (T/K178D) significantly stabilizes B2R/β-arrestin complexes in endosomes, delays receptor recycling to the plasma membrane and maintains intracellular MAPK signaling. Similarly, the endosomal trafficking of β2-adrenergic, angiotensin II type 1 and vasopressin V2 receptors was altered by the β-arrestin-2 T178D mutant. Our findings unveil a novel subtype specific mode of MAPK-dependent regulation of β-arrestins in intracellular trafficking and signaling of GPCRs, and suggest differential endosomal receptor/β-arrestin-2 signaling roles among species. PMID:25016018

  16. Differential regulation of endosomal GPCR/β-arrestin complexes and trafficking by MAPK.

    PubMed

    Khoury, Etienne; Nikolajev, Ljiljana; Simaan, May; Namkung, Yoon; Laporte, Stéphane A

    2014-08-22

    β-Arrestins are signaling adaptors that bind to agonist-occupied G protein-coupled receptors (GPCRs) and target them for endocytosis; however, the mechanisms regulating receptor/β-arrestin complexes and trafficking in endosomes, remain ill defined. Here we show, in live cells, differential dynamic regulation of endosomal bradykinin B2 receptor (B2R) complexes with either β-arrestin-1 or -2. We find a novel role for MAPK in the B2R/β-arrestin-2 complex formation, receptor trafficking and signaling mediated by an ERK1/2 regulatory motif in the hinge domain of the rat β-arrestin-2 (PET(178)P), but not rat β-arrestin-1 (PER(177)P). While the ERK1/2 regulatory motif is conserved between rat and mouse β-arrestin-2, it is surprisingly not conserved in human β-arrestin-2 (PEK(178)P). However, mutation of lysine 178 to threonine is sufficient to confer MAPK sensitivity to the human β-arrestin-2. Furthermore, substitution for a phosphomimetic residue in both the rat and the human β-arrestin-2 (T/K178D) significantly stabilizes B2R/β-arrestin complexes in endosomes, delays receptor recycling to the plasma membrane and maintains intracellular MAPK signaling. Similarly, the endosomal trafficking of β2-adrenergic, angiotensin II type 1 and vasopressin V2 receptors was altered by the β-arrestin-2 T178D mutant. Our findings unveil a novel subtype specific mode of MAPK-dependent regulation of β-arrestins in intracellular trafficking and signaling of GPCRs, and suggest differential endosomal receptor/β-arrestin-2 signaling roles among species.

  17. Oxidative stress inhibits caveolin-1 palmitoylation and trafficking in endothelial cells

    NASA Technical Reports Server (NTRS)

    Parat, Marie-Odile; Stachowicz, Rafal Z.; Fox, Paul L.

    2002-01-01

    During normal and pathological conditions, endothelial cells (ECs) are subjected to locally generated reactive oxygen species, produced by themselves or by other vessel wall cells. In excess these molecules cause oxidative injury to the cell but at moderate levels they might modulate intracellular signalling pathways. We have investigated the effect of oxidative stress on the palmitoylation and trafficking of caveolin-1 in bovine aortic ECs. Exogenous H2O2 did not alter the intracellular localization of caveolin-1 in ECs. However, metabolic labelling experiments showed that H2O2 inhibited the trafficking of newly synthesized caveolin-1 to membrane raft domains. Several mechanisms potentially responsible for this inhibition were examined. Impairment of caveolin-1 synthesis by H2O2 was not responsible for diminished trafficking. Similarly, the inhibition was independent of H2O2-induced caveolin-1 phosphorylation as shown by the markedly different concentration dependences. We tested the effect of H2O2 on palmitoylation of caveolin-1 by the incorporation of [3H]palmitic acid. Exposure of ECs to H2O2 markedly inhibited the palmitoylation of caveolin-1. Comparable inhibition was observed after treatment of cells with H2O2 delivered either as a bolus or by continuous delivery with glucose and glucose oxidase. Kinetic studies showed that H2O2 did not alter the rate of caveolin-1 depalmitoylation but instead decreased the 'on-rate' of palmitoylation. Together these results show for the first time the modulation of protein palmitoylation by oxidative stress, and suggest a cellular mechanism by which stress might influence caveolin-1-dependent cell activities such as the concentration of signalling proteins and cholesterol trafficking.

  18. Gating characteristics control glutamate receptor distribution and trafficking in vivo.

    PubMed

    Petzoldt, Astrid G; Lee, Yü-Hien; Khorramshahi, Omid; Reynolds, Eric; Plested, Andrew J R; Herzel, Hanspeter; Sigrist, Stephan J

    2014-09-01

    Glutamate-releasing synapses dominate excitatory release in the brain. Mechanisms governing their assembly are of major importance for circuit development and long-term plasticity underlying learning and memory. AMPA/Kainate-type glutamate receptors (GluRs) are tetrameric ligand-gated ion channels that open their ion-conducting pores in response to binding of the neurotransmitter. Changes in subunit composition of postsynaptic GluRs are highly relevant for plasticity and development of glutamatergic synapses [1-4]. To date, posttranslational modifications, mostly operating via the intracellular C-terminal domains (CTDs) of GluRs, are presumed to be the major regulator of trafficking [5]. In recent years, structural and electrophysiological analyses have improved our understanding of GluR gating mechanism [6-11]. However, whether conformational changes subsequent to glutamate binding may per se be able to influence GluR trafficking has remained an unaddressed question. Using a Drosophila system allowing for extended visualization of GluR trafficking in vivo, we here provide evidence that mutations changing the gating behavior alter GluR distribution and trafficking. GluR mutants associated with reduced charge transfer segregated from coexpressed wild-type GluRs on the level of individual postsynaptic densities. Segregation was lost upon blocking of evoked glutamate release. Photobleaching experiments suggested increased mobility of mutants with reduced charge transfer, which accumulated prematurely during early steps of synapse assembly, but failed to further increase their level in accordance with assembly of the presynaptic scaffold. In summary, gating characteristics seem to be a new variable for the understanding of GluR trafficking relevant to both development and plasticity.

  19. Mutations in yeast ARV1 alter intracellular sterol distribution and are complemented by human ARV1.

    PubMed

    Tinkelenberg, A H; Liu, Y; Alcantara, F; Khan, S; Guo, Z; Bard, M; Sturley, S L

    2000-12-29

    Intracellular cholesterol redistribution between membranes and its subsequent esterification are critical aspects of lipid homeostasis that prevent free sterol toxicity. To identify genes that mediate sterol trafficking, we screened for yeast mutants that were inviable in the absence of sterol esterification. Mutations in the novel gene, ARV1, render cells dependent on sterol esterification for growth, nystatin-sensitive, temperature-sensitive, and anaerobically inviable. Cells lacking Arv1p display altered intracellular sterol distribution and are defective in sterol uptake, consistent with a role for Arv1p in trafficking sterol into the plasma membrane. Human ARV1, a predicted sequence ortholog of yeast ARV1, complements the defects associated with deletion of the yeast gene. The genes are predicted to encode transmembrane proteins with potential zinc-binding motifs. We propose that ARV1 is a novel mediator of eukaryotic sterol homeostasis.

  20. Imaging of Leukocyte Trafficking in Alzheimer’s Disease

    PubMed Central

    Pietronigro, Enrica; Zenaro, Elena; Constantin, Gabriela

    2016-01-01

    Alzheimer’s disease (AD) is the most common neurodegenerative disorder and is characterized by a progressive decline of cognitive functions. The neuropathological features of AD include amyloid beta (Aβ) deposition, intracellular neurofibrillary tangles derived from the cytoskeletal hyperphosphorylated tau protein, amyloid angiopathy, the loss of synapses, and neuronal degeneration. In the last decade, inflammation has emerged as a key feature of AD, but most studies have focused on the role of microglia-driven neuroinflammation mechanisms. A dysfunctional blood–brain barrier has also been implicated in the pathogenesis of AD, and several studies have demonstrated that the vascular deposition of Aβ induces the expression of adhesion molecules and alters the expression of tight junction proteins, potentially facilitating the transmigration of circulating leukocytes. Two-photon laser scanning microscopy (TPLSM) has become an indispensable tool to dissect the molecular mechanisms controlling leukocyte trafficking in the central nervous system (CNS). Recent TPLSM studies have shown that vascular deposition of Aβ in the CNS promotes intraluminal neutrophil adhesion and crawling on the brain endothelium and also that neutrophils extravasate in the parenchyma preferentially in areas with Aβ deposits. These studies have also highlighted a role for LFA-1 integrin in neutrophil accumulation in the CNS of AD-like disease models, revealing that LFA-1 inhibition reduces the corresponding cognitive deficit and AD neuropathology. In this article, we consider how current imaging techniques can help to unravel new inflammation mechanisms in the pathogenesis of AD and identify novel therapeutic strategies to treat the disease by interfering with leukocyte trafficking mechanisms. PMID:26913031

  1. Dynamic multibody protein interactions suggest versatile pathways for copper trafficking.

    PubMed

    Keller, Aaron M; Benítez, Jaime J; Klarin, Derek; Zhong, Linghao; Goldfogel, Matthew; Yang, Feng; Chen, Tai-Yen; Chen, Peng

    2012-05-30

    As part of intracellular copper trafficking pathways, the human copper chaperone Hah1 delivers Cu(+) to the Wilson's Disease Protein (WDP) via weak and dynamic protein-protein interactions. WDP contains six homologous metal binding domains (MBDs) connected by flexible linkers, and these MBDs all can receive Cu(+) from Hah1. The functional roles of the MBD multiplicity in Cu(+) trafficking are not well understood. Building on our previous study of the dynamic interactions between Hah1 and the isolated fourth MBD of WDP, here we study how Hah1 interacts with MBD34, a double-domain WDP construct, using single-molecule fluorescence resonance energy transfer (smFRET) combined with vesicle trapping. By alternating the positions of the smFRET donor and acceptor, we systematically probed Hah1-MBD3, Hah1-MBD4, and MBD3-MBD4 interaction dynamics within the multidomain system. We found that the two interconverting interaction geometries were conserved in both intermolecular Hah1-MBD and intramolecular MBD-MBD interactions. The Hah1-MBD interactions within MBD34 are stabilized by an order of magnitude relative to the isolated single-MBDs, and thermodynamic and kinetic evidence suggest that Hah1 can interact with both MBDs simultaneously. The enhanced interaction stability of Hah1 with the multi-MBD system, the dynamic intramolecular MBD-MBD interactions, and the ability of Hah1 to interact with multiple MBDs simultaneously suggest an efficient and versatile mechanism for the Hah1-to-WDP pathway to transport Cu(+).

  2. Rab GTPases regulate endothelial cell protein C receptor-mediated endocytosis and trafficking of factor VIIa.

    PubMed

    Nayak, Ramesh C; Keshava, Shiva; Esmon, Charles T; Pendurthi, Usha R; Rao, L Vijaya Mohan

    2013-01-01

    Recent studies have established that factor VIIa (FVIIa) binds to the endothelial cell protein C receptor (EPCR). FVIIa binding to EPCR may promote the endocytosis of this receptor/ligand complex. Rab GTPases are known to play a crucial role in the endocytic and exocytic pathways of receptors or receptor/ligand complexes. The present study was undertaken to investigate the role of Rab GTPases in the intracellular trafficking of EPCR and FVIIa. CHO-EPCR cells and human umbilical vein endothelial cells (HUVEC) were transduced with recombinant adenoviral vectors to express wild-type, constitutively active, or dominant negative mutant of various Rab GTPases. Cells were exposed to FVIIa conjugated with AF488 fluorescent probe (AF488-FVIIa), and intracellular trafficking of FVIIa, EPCR, and Rab proteins was evaluated by immunofluorescence confocal microscopy. In cells expressing wild-type or constitutively active Rab4A, internalized AF488-FVIIa accumulated in early/sorting endosomes and its entry into the recycling endosomal compartment (REC) was inhibited. Expression of constitutively active Rab5A induced large endosomal structures beneath the plasma membrane where EPCR and FVIIa accumulated. Dominant negative Rab5A inhibited the endocytosis of EPCR-FVIIa. Expression of constitutively active Rab11 resulted in retention of accumulated AF488-FVIIa in the REC, whereas expression of a dominant negative form of Rab11 led to accumulation of internalized FVIIa in the cytoplasm and prevented entry of internalized FVIIa into the REC. Expression of dominant negative Rab11 also inhibited the transport of FVIIa across the endothelium. Overall our data show that Rab GTPases regulate the internalization and intracellular trafficking of EPCR-FVIIa.

  3. Vesicle trafficking and cell surface membrane patchiness.

    PubMed

    Tang, Q; Edidin, M

    2001-07-01

    Membrane proteins and lipids often appear to be distributed in patches on the cell surface. These patches are often assumed to be membrane domains, arising from specific molecular associations. However, a computer simulation (Gheber and Edidin, 1999) shows that membrane patchiness may result from a combination of vesicle trafficking and dynamic barriers to lateral mobility. The simulation predicts that the steady-state patches of proteins and lipids seen on the cell surface will decay if vesicle trafficking is inhibited. To test this prediction, we compared the apparent sizes and intensities of patches of class I HLA molecules, integral membrane proteins, before and after inhibiting endocytic vesicle traffic from the cell surface, either by incubation in hypertonic medium or by expression of a dominant-negative mutant dynamin. As predicted by the simulation, the apparent sizes of HLA patches increased, whereas their intensities decreased after endocytosis and vesicle trafficking were inhibited. PMID:11423406

  4. Purinergic Signaling During Immune Cell Trafficking.

    PubMed

    Ferrari, Davide; McNamee, Eóin N; Idzko, Marco; Gambari, Roberto; Eltzschig, Holger K

    2016-06-01

    Migration and positioning of immune cells is fundamental for their differentiation and recruitment at sites of infection. Besides the fundamental role played by chemokines and their receptors, recent studies demonstrate that a complex network of purinergic signaling events plays a key role in these trafficking events. This process includes the release of nucleotides (such as ATP and ADP) and subsequent autocrine and paracrine signaling events through nucleotide receptors. At the same time, surface-expressed ectoapyrases and nucleotidases convert extracellular nucleotides to adenosine, and adenosine signaling events play additional functional roles in leucocyte trafficking. In this review we revisit classical paradigms of inflammatory cell trafficking in the context of recent studies implicating purinergic signaling events in this process. PMID:27142306

  5. Trafficking in persons and development: towards greater policy coherence.

    PubMed

    Danailova-Trainor, Gergana; Laczko, Frank

    2010-01-01

    Poverty is often regarded as the "root cause" of trafficking, but the linkages between poverty, a lack of development and trafficking are complex. For example, there is some evidence to suggest that victims of cross-border trafficking are more likely to originate from middle-income rather than lower-income countries. Trafficking and development have tended to be treated as very separate policy areas and the assessment of the development impact of counter-trafficking programmes is still at an early stage. This paper outlines a possible framework for a more evidence-based approach to understanding the linkages between trafficking, trafficking policy and human development. The paper argues that the human development gains from greater mobility could be significantly enhanced if there was greater coherence between policies to combat trafficking and policies to promote development. PMID:20645470

  6. 31 CFR 536.312 - Specially designated narcotics trafficker.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... designated narcotics trafficker means: (a) Persons listed in the annex to Executive Order 12978 (3 CFR, 1995... international narcotics trafficking centered in Colombia; or (2) Materially to assist in, or provide...

  7. Alternative Splicing in CaV2.2 Regulates Neuronal Trafficking via Adaptor Protein Complex-1 Adaptor Protein Motifs

    PubMed Central

    Macabuag, Natsuko

    2015-01-01

    N-type voltage-gated calcium (CaV2.2) channels are expressed in neurons and targeted to the plasma membrane of presynaptic terminals, facilitating neurotransmitter release. Here, we find that the adaptor protein complex-1 (AP-1) mediates trafficking of CaV2.2 from the trans-Golgi network to the cell surface. Examination of splice variants of CaV2.2, containing either exon 37a (selectively expressed in nociceptors) or 37b in the proximal C terminus, reveal that canonical AP-1 binding motifs, YxxΦ and [DE]xxxL[LI], present only in exon 37a, enhance intracellular trafficking of exon 37a-containing CaV2.2 to the axons and plasma membrane of rat DRG neurons. Finally, we identify differential effects of dopamine-2 receptor (D2R) and its agonist-induced activation on trafficking of CaV2.2 isoforms. D2R slowed the endocytosis of CaV2.2 containing exon 37b, but not exon 37a, and activation by the agonist quinpirole reversed the effect of the D2R. Our work thus reveals key mechanisms involved in the trafficking of N-type calcium channels. SIGNIFICANCE STATEMENT CaV2.2 channels are important for neurotransmitter release, but how they are trafficked is still poorly understood. Here, we describe a novel mechanism for trafficking of CaV2.2 from the trans-Golgi network to the cell surface which is mediated by the adaptor protein AP-1. Alternative splicing of exon 37 produces CaV2.2-exon 37a, selectively expressed in nociceptors, or CaV2.2-exon 37b, which is the major splice isoform. Our study reveals that canonical AP-1 binding motifs (YxxΦ and [DE]xxxL[LI]), present in exon 37a, but not 37b, enhance intracellular trafficking of exon 37a-containing CaV2.2 to axons and plasma membrane of DRG neurons. Interaction of APs with CaV2.2 channels may also be key underlying mechanisms for differential effects of the dopamine D2 receptor on trafficking of CaV2.2 splice variants. PMID:26511252

  8. Homeostatic plasticity and NMDA receptor trafficking.

    PubMed

    Pérez-Otaño, Isabel; Ehlers, Michael D

    2005-05-01

    Learning, memory and brain development are associated with long-lasting modifications of synapses that are guided by specific patterns of neuronal activity. Such modifications include classical Hebbian plasticities (such as long-term potentiation and long-term depression), which are rapid and synapse-specific, and others, such as synaptic scaling and metaplasticity, that work over longer timescales and are crucial for maintaining and orchestrating neuronal network function. The cellular mechanisms underlying Hebbian plasticity have been well studied and involve rapid changes in the trafficking of highly mobile AMPA receptors. An emerging concept is that activity-dependent alterations in NMDA receptor trafficking contribute to homeostatic plasticity at central glutamatergic synapses.

  9. Intracellular disassembly and localization of a new P123-PEI-R13/DNA complex.

    PubMed

    Zhu, Manman; Liu, Kehai; Zhu, Qing; Chen, Shunsheng; Lv, Hui; Zhao, Wenfang; Mao, Yuan; Hu, Jing

    2014-01-01

    The appropriate location and release of target gene is necessary for gene therapy. In our previous paper, a gene vector named P123-PEI-R13 has been successfully synthesized, and the physical characteristics and cellular trafficking of nanoparticle P123-PEI-R13/DNA has been explored explicitly, but little was known about its disassembly within cells. In order to investigate its intracellular disassembly, P123-PEI-R13/DNA complex was exposed to the different competitors (RNA, DNA, proteins) or different conditions of pH and osmolarity, DNA release was determined by gel electrophoresis. Meanwhile, confocal laser technology was used to locate the complex in cells. The results revealed that DNA, RNA and osmolarity could affect the stability of the complex obviously, especially RNA which exist in nucleus. In addition, the speed of DNA release decreased as the weight ratio of polymer increased. Images got by a confocal fluorescence microscope confirmed that after cell uptake, P123-PEI-R13 could translocate DNA into nucleus.

  10. Intracellular cotrafficking of factor VIII and von Willebrand factor type 2N variants to storage organelles.

    PubMed

    van den Biggelaar, Maartje; Meijer, Alexander B; Voorberg, Jan; Mertens, Koen

    2009-03-26

    Weibel-Palade bodies (WPBs) are the endothelial storage organelles that are formed upon von Willebrand factor (VWF) expression. Apart from VWF, WPBs contain a variety of hemostatic and inflammatory proteins. Some of these are thought to be targeted to WPBs by directly interacting with VWF in the secretory pathway. Previous studies have demonstrated that coexpression of factor VIII (FVIII) with VWF results in costorage of both proteins. However, whether cotrafficking is driven by intracellular FVIII-VWF assembly has remained unclear. We now have addressed this issue using recombinant VWF type 2N variants that are known to display reduced FVIII binding in the circulation. Binding studies using purified fluorescent FVIII and VWF type 2N variants revealed FVIII binding defects varying from moderate (Arg854Gln, Cys1060Arg) to severe (Arg763Gly, Thr791Met, Arg816Trp). Upon expression in HEK293 cells, all VWF variants induced formation of WPB-like organelles that were able to recruit P-selectin, as well as FVIII. WPBs containing FVIII did not display their typical elongated shape, suggesting that FVIII affects the organization of VWF tubules therein. The finding that VWF type 2N variants are still capable of cotargeting FVIII to storage granules implies that trafficking of WPB cargo proteins does not necessarily require high-affinity assembly with VWF. PMID:19088379

  11. Quantitative study of effects of free cationic chains on gene transfection in different intracellular stages.

    PubMed

    Cai, Jinge; Yue, Yanan; Wang, Yanjing; Jin, Zhenyu; Jin, Fan; Wu, Chi

    2016-09-28

    Previously, we revealed that in the application of using cationic polymer chains, polyethylenimine (PEI), to condense anionic plasmid DNA chains (pDNA) to form the DNA/polymer polyplexes, after all the pDNAs are complexed with PEI, further added PEIs exist individual chains and free in the solution mixture. It is those uncomplexed polycation chains that dramatically promote the gene transfection. In the current study, we studied how those free cationic chains with different lengths and topologies affect the intracellular trafficking of the polyplexes, the translocation of pDNA through the nuclear membrane, the transcription of pDNA to mRNA and the translocation of mRNA from nucleus to cytosol in HepG2 cells by using a combination of the three-dimensional confocal microscope and TaqMan real-time PCR. We found that free branched PEI chains with a molar mass of 25,000g/mol and a total concentration of 1.8×10(-6)g/mL promote the overall gene transfection efficiency by a factor of ~500 times. Our results quantitatively reveal that free chains help little in the cellular uptake, but clearly reduce the lysosomal entrapment of those internalized polyplexes (2-3 folds); assist the translocation of pDNA through nuclear membrane after it is released from the polyplexes in the cytosol (~5 folds); enhance the pDNA-to-mRNA transcription efficiency (~4 folds); and facilitate the nucleus-to-cytosol translocation of mRNA (7-8 folds). The total enhancement of those steps agrees well with the overall efficiency, demonstrating, for the first time, how free cationic polymer chains quantitatively promote the gene transfection in each step in the intracellular space. PMID:27448443

  12. Nanovehicular Intracellular Delivery Systems

    PubMed Central

    PROKOP, ALES; DAVIDSON, JEFFREY M.

    2013-01-01

    This article provides an overview of principles and barriers relevant to intracellular drug and gene transport, accumulation and retention (collectively called as drug delivery) by means of nanovehicles (NV). The aim is to deliver a cargo to a particular intracellular site, if possible, to exert a local action. Some of the principles discussed in this article apply to noncolloidal drugs that are not permeable to the plasma membrane or to the blood–brain barrier. NV are defined as a wide range of nanosized particles leading to colloidal objects which are capable of entering cells and tissues and delivering a cargo intracelullarly. Different localization and targeting means are discussed. Limited discussion on pharmacokinetics and pharmacodynamics is also presented. NVs are contrasted to micro-delivery and current nanotechnologies which are already in commercial use. Newer developments in NV technologies are outlined and future applications are stressed. We also briefly review the existing modeling tools and approaches to quantitatively describe the behavior of targeted NV within the vascular and tumor compartments, an area of particular importance. While we list “elementary” phenomena related to different level of complexity of delivery to cancer, we also stress importance of multi-scale modeling and bottom-up systems biology approach. PMID:18200527

  13. Intracellular events regulating cross-presentation

    PubMed Central

    Wagner, Claudia S.; Grotzke, Jeffrey E.; Cresswell, Peter

    2012-01-01

    Cross-presentation plays a fundamental role in the induction of CD8-T cell immunity. However, although more than three decades have passed since its discovery, surprisingly little is known about the exact mechanisms involved. Here we give an overview of the components involved at different stages of this process. First, antigens must be internalized into the cross-presenting cell. The involvement of different receptors, method of antigen uptake, and nature of the antigen can influence intracellular trafficking and access to the cross-presentation pathway. Once antigens access the endocytic system, different requirements for endosomal/phagosomal processing arise, such as proteolysis and reduction of disulfide bonds. The majority of cross-presented peptides are generated by proteasomal degradation. Therefore, antigens must cross a membrane barrier in a manner analogous to the fate of misfolded proteins in the endoplasmic reticulum (ER) that are retrotranslocated into the cytosol for degradation. Indeed, some components of the ER-associated degradation machinery have been implicated in cross-presentation. Further complicating the matter, endosomal and phagosomal compartments have been suggested as alternative sites to the ER for loading of peptides on major histocompatibility complex class I molecules. Finally, the antigen presenting cells involved, particularly dendritic cell subsets and their state of maturation, influence the efficiency of cross-presentation. PMID:22675326

  14. Leukocyte Trafficking to the Small Intestine and Colon.

    PubMed

    Habtezion, Aida; Nguyen, Linh P; Hadeiba, Husein; Butcher, Eugene C

    2016-02-01

    Leukocyte trafficking to the small and large intestines is tightly controlled to maintain intestinal immune homeostasis, mediate immune responses, and regulate inflammation. A wide array of chemoattractants, chemoattractant receptors, and adhesion molecules expressed by leukocytes, mucosal endothelium, epithelium, and stromal cells controls leukocyte recruitment and microenvironmental localization in intestine and in the gut-associated lymphoid tissues (GALTs). Naive lymphocytes traffic to the gut-draining mesenteric lymph nodes where they undergo antigen-induced activation and priming; these processes determine their memory/effector phenotypes and imprint them with the capacity to migrate via the lymph and blood to the intestines. Mechanisms of T-cell recruitment to GALT and of T cells and plasmablasts to the small intestine are well described. Recent advances include the discovery of an unexpected role for lectin CD22 as a B-cell homing receptor GALT, and identification of the orphan G-protein-coupled receptor 15 (GPR15) as a T-cell chemoattractant/trafficking receptor for the colon. GPR15 decorates distinct subsets of T cells in mice and humans, a difference in species that could affect translation of the results of mouse colitis models to humans. Clinical studies with antibodies to integrin α4β7 and its vascular ligand mucosal vascular addressin cell adhesion molecule 1 are proving the value of lymphocyte trafficking mechanisms as therapeutic targets for inflammatory bowel diseases. In contrast to lymphocytes, cells of the innate immune system express adhesion and chemoattractant receptors that allow them to migrate directly to effector tissue sites during inflammation. We review the mechanisms for innate and adaptive leukocyte localization to the intestinal tract and GALT, and discuss their relevance to human intestinal homeostasis and inflammation.

  15. Child organ trafficking: global reality and inadequate international response.

    PubMed

    Bagheri, Alireza

    2016-06-01

    In organ transplantation, the demand for human organs has grown far faster than the supply of organs. This has opened the door for illegal organ trade and trafficking including from children. Organized crime groups and individual organ brokers exploit the situation and, as a result, black markets are becoming more numerous and organized organ trafficking is expanding worldwide. While underprivileged and vulnerable men and women in developing countries are a major source of trafficked organs, and may themselves be trafficked for the purpose of illegal organ removal and trade, children are at especial risk of exploitation. With the confirmed cases of children being trafficked for their organs, child organ trafficking, which once called a "modern urban legend", is a sad reality in today's world. By presenting a global picture of child organ trafficking, this paper emphasizes that child organ trafficking is no longer a myth but a reality which has to be addressed. It argues that the international efforts against organ trafficking and trafficking in human beings for organ removal have failed to address child organ trafficking adequately. This chapter suggests that more orchestrated international collaboration as well as development of preventive measure and legally binding documents are needed to fight child organ trafficking and to support its victims. PMID:26612382

  16. 78 FR 70571 - Advisory Council on Wildlife Trafficking; Rescheduled Meeting

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-11-26

    ... Fish and Wildlife Service Advisory Council on Wildlife Trafficking; Rescheduled Meeting AGENCY: Fish... Service (Service), announce a public meeting of the Advisory Council on Wildlife Trafficking (Council... announce that the Advisory Council on Wildlife Trafficking (Council) will hold a meeting to...

  17. 48 CFR 52.222-50 - Combating Trafficking in Persons.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 2 2011-10-01 2011-10-01 false Combating Trafficking in....222-50 Combating Trafficking in Persons. As prescribed in 22.1705(a), insert the following clause: Combating Trafficking in Persons (FEB 2009) (a) Definitions. As used in this clause— Coercion means—...

  18. Child organ trafficking: global reality and inadequate international response.

    PubMed

    Bagheri, Alireza

    2016-06-01

    In organ transplantation, the demand for human organs has grown far faster than the supply of organs. This has opened the door for illegal organ trade and trafficking including from children. Organized crime groups and individual organ brokers exploit the situation and, as a result, black markets are becoming more numerous and organized organ trafficking is expanding worldwide. While underprivileged and vulnerable men and women in developing countries are a major source of trafficked organs, and may themselves be trafficked for the purpose of illegal organ removal and trade, children are at especial risk of exploitation. With the confirmed cases of children being trafficked for their organs, child organ trafficking, which once called a "modern urban legend", is a sad reality in today's world. By presenting a global picture of child organ trafficking, this paper emphasizes that child organ trafficking is no longer a myth but a reality which has to be addressed. It argues that the international efforts against organ trafficking and trafficking in human beings for organ removal have failed to address child organ trafficking adequately. This chapter suggests that more orchestrated international collaboration as well as development of preventive measure and legally binding documents are needed to fight child organ trafficking and to support its victims.

  19. Human Trafficking: A Review for Mental Health Professionals

    ERIC Educational Resources Information Center

    Yakushko, Oksana

    2009-01-01

    This article provides a review of current research on human trafficking for mental health practitioners and scholars. In addition to an overview of definitions, causes and processes of trafficking, the article highlights mental health consequences of trafficking along with suggestions for treatment of survivors. Directions for counseling services,…

  20. Trafficking of Children in Albania: Patterns of Recruitment and Reintegration

    ERIC Educational Resources Information Center

    Gjermeni, Eglantina; Van Hook, Mary P.; Gjipali, Saemira; Xhillari, Lindita; Lungu, Fatjon; Hazizi, Anila

    2008-01-01

    Problem: Many children in Albania and other countries of Eastern Europe are being trafficked as part of the global business of human trafficking. Objectives: The study sought to identify the patterns of child trafficking involving Albanian children, and especially children's views of the role of family issues and the nature of the trafficking…

  1. Miro1 Regulates Activity-Driven Positioning of Mitochondria within Astrocytic Processes Apposed to Synapses to Regulate Intracellular Calcium Signaling.

    PubMed

    Stephen, Terri-Leigh; Higgs, Nathalie F; Sheehan, David F; Al Awabdh, Sana; López-Doménech, Guillermo; Arancibia-Carcamo, I Lorena; Kittler, Josef T

    2015-12-01

    It is fast emerging that maintaining mitochondrial function is important for regulating astrocyte function, although the specific mechanisms that govern astrocyte mitochondrial trafficking and positioning remain poorly understood. The mitochondrial Rho-GTPase 1 protein (Miro1) regulates mitochondrial trafficking and detachment from the microtubule transport network to control activity-dependent mitochondrial positioning in neurons. However, whether Miro proteins are important for regulating signaling-dependent mitochondrial dynamics in astrocytic processes remains unclear. Using live-cell confocal microscopy of rat organotypic hippocampal slices, we find that enhancing neuronal activity induces transient mitochondrial remodeling in astrocytes, with a concomitant, transient reduction in mitochondrial trafficking, mediated by elevations in intracellular Ca(2+). Stimulating neuronal activity also induced mitochondrial confinement within astrocytic processes in close proximity to synapses. Furthermore, we show that the Ca(2+)-sensing EF-hand domains of Miro1 are important for regulating mitochondrial trafficking in astrocytes and required for activity-driven mitochondrial confinement near synapses. Additionally, activity-dependent mitochondrial positioning by Miro1 reciprocally regulates the levels of intracellular Ca(2+) in astrocytic processes. Thus, the regulation of intracellular Ca(2+) signaling, dependent on Miro1-mediated mitochondrial positioning, could have important consequences for astrocyte Ca(2+) wave propagation, gliotransmission, and ultimately neuronal function. PMID:26631479

  2. Intracellular potassium stabilizes human ether-à-go-go-related gene channels for export from endoplasmic reticulum.

    PubMed

    Wang, Lu; Dennis, Adrienne T; Trieu, Phan; Charron, Francois; Ethier, Natalie; Hebert, Terence E; Wan, Xiaoping; Ficker, Eckhard

    2009-04-01

    Several therapeutic compounds have been identified that prolong the QT interval on the electrocardiogram and cause torsade de pointes arrhythmias not by direct block of the cardiac potassium channel human ether-à-go-go-related gene (hERG) but via disruption of hERG trafficking to the cell surface membrane. One example of a clinically important compound class that potently inhibits hERG trafficking are cardiac glycosides. We have shown previously that inhibition of hERG trafficking by cardiac glycosides is initiated via direct block of Na(+)/K(+) pumps and not via off-target interactions with hERG or any other protein. However, it was not known how pump inhibition at the cell surface is coupled to hERG processing in the endoplasmic reticulum. Here, we show that depletion of intracellular K(+)-either indirectly after long-term exposure to cardiac glycosides or directly after exposure to gramicidin in low sodium media-is sufficient to disrupt hERG trafficking. In K(+)-depleted cells, hERG trafficking can be restored by permeating K(+) or Rb(+) ions, incubation at low temperature, exposure to the pharmacological chaperone astemizole, or specific mutations in the selectivity filter of hERG. Our data suggest a novel mechanism for drug-induced trafficking inhibition in which cardiac glycosides produce a [K(+)](i)-mediated conformational defect directly in the hERG channel protein. PMID:19139152

  3. Determination of intracellular nitrate.

    PubMed Central

    Romero, J M; Lara, C; Guerrero, M G

    1989-01-01

    A sensitive procedure has been developed for the determination of intracellular nitrate. The method includes: (i) preparation of cell lysates in 2 M-H3PO4 after separation of cells from the outer medium by rapid centrifugation through a layer of silicone oil, and (ii) subsequent nitrate analysis by ion-exchange h.p.l.c. with, as mobile phase, a solution containing 50 mM-H3PO4 and 2% (v/v) tetrahydrofuran, adjusted to pH 1.9 with NaOH. The determination of nitrate is subjected to interference by chloride and sulphate when present in the samples at high concentrations. Nitrite also interferes, but it is easily eliminated by treatment of the samples with sulphamic acid. The method has been successfully applied to the study of nitrate transport in the unicellular cyanobacterium Anacystis nidulans. PMID:2497740

  4. Intracellular trafficking pathways in silver nanoparticle uptake and toxicity in Caenorhabditis elegans.

    PubMed

    Maurer, Laura L; Yang, Xinyu; Schindler, Adam J; Taggart, Ross K; Jiang, Chuanjia; Hsu-Kim, Heileen; Sherwood, David R; Meyer, Joel N

    2016-09-01

    We used the nematode Caenorhabditis elegans to study the roles of endocytosis and lysosomal function in uptake and subsequent toxicity of silver nanoparticles (AgNP) in vivo. To focus on AgNP uptake and effects rather than silver ion (AgNO3) effects, we used a minimally dissolvable AgNP, citrate-coated AgNPs (CIT-AgNPs). We found that the clathrin-mediated endocytosis inhibitor chlorpromazine reduced the toxicity of CIT-AgNPs but not AgNO3. We also tested the sensitivity of three endocytosis-deficient mutants (rme-1, rme-6 and rme-8) and two lysosomal function deficient mutants (cup-5 and glo-1) as compared to wild-type (N2 strain). One of the endocytosis-deficient mutants (rme-6) took up less silver and was resistant to the acute toxicity of CIT-AgNPs compared to N2s. None of those mutants showed altered sensitivity to AgNO3. Lysosome and lysosome-related organelle mutants were more sensitive to the growth-inhibiting effects of both CIT-AgNPs and AgNO3. Our study provides mechanistic evidence suggesting that early endosome formation is necessary for AgNP-induced toxicity in vivo, as rme-6 mutants were less sensitive to the toxic effects of AgNPs than C. elegans with mutations involved in later steps in the endocytic process.

  5. Particle Tracking of Intracellular Trafficking of Octaarginine-modified Liposomes: A Comparative Study With Adenovirus

    PubMed Central

    Akita, Hidetaka; Enoto, Kaoru; Masuda, Tomoya; Mizuguchi, Hiroyuki; Tani, Tomomi; Harashima, Hideyoshi

    2010-01-01

    It is previously reported that octaarginine (R8)-modified liposome (R8-Lip) was taken up via macropinocytosis, and subsequently delivered to the nuclear periphery. In the present study, we investigated the mechanism for the cytoplasmic transport of R8-Lips, comparing with that for adenovirus. Treatment with microtubule-disruption reagent (nocodazole) inhibited the transfection activity of plasmid DNA (pDNA)-encapsulating R8-Lip more extensively than that of adenovirus. The directional transport of R8-Lips along green fluorescent protein (GFP)–tagged microtubules was observed; however, the velocity was slower than those for adenovirus or endosomes that were devoid of R8-Lips. These directional motions were abrogated in R8-Lips by nocodazole treatment, whereas adenovirus continued to undergo random motion. This finding suggests that the nuclear access of R8-Lip predominantly involves microtubule-dependent transport, whereas an apparent diffusive motion is also operative in nuclear access of adenovirus. Furthermore, quantum dot-labeled pDNA underwent directional motion concomitantly with rhodamine-labeled lipid envelopes, indicating that the R8-Lips were subject to microtubule-dependent transport in the intact form. Dual particle tracking of carriers and endosomes revealed that R8-Lip was directionally transported, associated with endosomes, whereas this occurs after endosomal escape in adenovirus. Collectively, the findings reported herein indicate that vesicular transport is a key factor in the cytoplasmic transport of R8-Lips. PMID:20216528

  6. Targeting and intracellular trafficking of clinically relevant hTHTR1 mutations in human cell lines.

    PubMed

    Subramanian, Veedamali S; Marchant, Jonathan S; Said, Hamid M

    2007-07-01

    The micronutrient thiamine is required for normal growth and development of human tissues, and is accumulated into cells through the activity of plasma membrane thiamine transporters, e.g. hTHTR1 (human thiamine transporter 1). Recent genetic evidence has linked mutations in hTHTR1 with the manifestation of TRMA (thiamine-responsive megaloblastic anaemia), a condition also associated with diabetes mellitus, sensorineural deafness and retinal disorders. To examine how mutations in hTHTR1 impair thiamine accumulation, we have investigated the targeting and functional properties of several different hTHTR1 mutants in human cell lines derived from epithelia relevant to thiamine absorption or tissues implicated in TRMA pathology. These constructs encompassed two newly identified point mutations (P51L and T158R) and two truncations of hTHTR1 identical with those found in TRMA kindreds (W358X and Delta383fs). Our results reveal a spectrum of mutant phenotypes, underlining that TRMA can result from decreased thiamine transport activity underpinned by changes in hTHTR1 expression levels, cellular targeting and/or protein transport activity.

  7. Regulated phosphorylation and dephosphorylation of tau protein: effects on microtubule interaction, intracellular trafficking and neurodegeneration.

    PubMed Central

    Billingsley, M L; Kincaid, R L

    1997-01-01

    This review attempts to summarize what is known about tau phosphorylation in the context of both normal cellular function and dysfunction. However, conceptions of tau function continue to evolve, and it is likely that the regulation of tau distribution and metabolism is complex. The roles of microtubule-associated kinases and phosphatases have yet to be fully described, but may afford insight into how tau phosphorylation at the distal end of the axon regulates cytoskeletal-membrane interactions. Finally, lipid and glycosaminoglycan modification of tau structure affords yet more complexity for regulation and aggregation. Continued work will help to determine what is causal and what is coincidental in Alzheimer's disease, and may lead to identification of therapeutic targets for halting the progression of paired helical filament formation. PMID:9169588

  8. Regulation of polycystin-1 ciliary trafficking by motifs at its C-terminus and polycystin-2 but not by cleavage at the GPS site.

    PubMed

    Su, Xuefeng; Wu, Maoqing; Yao, Gang; El-Jouni, Wassim; Luo, Chong; Tabari, Azadeh; Zhou, Jing

    2015-11-15

    Failure to localize membrane proteins to the primary cilium causes a group of diseases collectively named ciliopathies. Polycystin-1 (PC1, also known as PKD1) is a large ciliary membrane protein defective in autosomal dominant polycystic kidney disease (ADPKD). Here, we developed a large set of PC1 expression constructs and identified multiple sequences, including a coiled-coil motif in the C-terminal tail of PC1, regulating full-length PC1 trafficking to the primary cilium. Ciliary trafficking of wild-type and mutant PC1 depends on the dose of polycystin-2 (PC2, also known as PKD2), and the formation of a PC1-PC2 complex. Modulation of the ciliary trafficking module mediated by the VxP ciliary-targeting sequence and Arf4 and Asap1 does not affect the ciliary localization of full-length PC1. PC1 also promotes PC2 ciliary trafficking. PC2 mutations truncating its C-terminal tail but not those changing the VxP sequence to AxA or impairing the pore of the channel, leading to a dead channel, affect PC1 ciliary trafficking. Cleavage at the GPCR proteolytic site (GPS) of PC1 is not required for PC1 trafficking to cilia. We propose a mutually dependent model for the ciliary trafficking of PC1 and PC2, and that PC1 ciliary trafficking is regulated by multiple cis-acting elements. As all pathogenic PC1 mutations tested here are defective in ciliary trafficking, ciliary trafficking might serve as a functional read-out for ADPKD.

  9. γ-SNAP stimulates disassembly of endosomal SNARE complexes and regulates endocytic trafficking pathways.

    PubMed

    Inoue, Hiroki; Matsuzaki, Yuka; Tanaka, Ayaka; Hosoi, Kaori; Ichimura, Kaoru; Arasaki, Kohei; Wakana, Yuichi; Asano, Kenichi; Tanaka, Masato; Okuzaki, Daisuke; Yamamoto, Akitsugu; Tani, Katsuko; Tagaya, Mitsuo

    2015-08-01

    Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) that reside in the target membranes and transport vesicles assemble into specific SNARE complexes to drive membrane fusion. N-ethylmaleimide-sensitive factor (NSF) and its attachment protein, α-SNAP (encoded by NAPA), catalyze disassembly of the SNARE complexes in the secretory and endocytic pathways to recycle them for the next round of fusion events. γ-SNAP (encoded by NAPG) is a SNAP isoform, but its function in SNARE-mediated membrane trafficking remains unknown. Here, we show that γ-SNAP regulates the endosomal trafficking of epidermal growth factor (EGF) receptor (EGFR) and transferrin. Immunoprecipitation and mass spectrometry analyses revealed that γ-SNAP interacts with a limited range of SNAREs, including endosomal ones. γ-SNAP, as well as α-SNAP, mediated the disassembly of endosomal syntaxin-7-containing SNARE complexes. Overexpression and small interfering (si)RNA-mediated depletion of γ-SNAP changed the morphologies and intracellular distributions of endosomes. Moreover, the depletion partially suppressed the exit of EGFR and transferrin from EEA1-positive early endosomes to delay their degradation and uptake. Taken together, our findings suggest that γ-SNAP is a unique SNAP that functions in a limited range of organelles - including endosomes - and their trafficking pathways.

  10. Characterization of a B₁₂trafficking chaperone protein from caenorhabditis elegans.

    PubMed

    Park, Jihyun; Kim, Jihoe

    2015-01-01

    The human B₁₂trafficking chaperone protein hCblC is responsible for escorted delivery and early processing of B₁₂in intracellular B12 metabolism. In this study, we characterized a putative B₁₂trafficking chaperone of Caenorhabditis elegans (cCblC), which shows 26% amino acid sequence identity with hCblC. cCblC was shown to bind B₁₂with a broad specificity for the upper axial ligand, as previously observed with other homologous proteins. In addition, cCblC catalyzed glutathione (GSH)-dependent elimination of alkyl and GSH upper axial ligands from alkylcobalamins and glutathionylcobalamin (GSCbl), respectively. Dealkylation of methylcobalamin (MeCbl) generated cob(II)alamin with S-methylglutathione. Cob(I)alamin was detected as the intermediate for cob(II)alamin generation, indicating that the reaction is a nucleophilic displacement using the thiolate of GSH. Deglutathionylation of GSCbl also generated cob(II)alamin, via cob(I)alamin intermediate, with glutathione disulfide, indicating the reaction is chemically analogous with dealkylation. Cob(II)alamin generated by dealkylation and deglutathionylation was bound to cCblC in the base-off state and stable under aerobic conditions, which would be favorable for subsequent enzyme cofactor synthesis. These results demonstrate that cCblC is a B₁₂trafficking chaperone of C. elegans catalyzing dealkylation and deglutathionylation via a nucleophilic displacement using the thiolate of GSH.

  11. Subcellular trafficking in rhabdovirus infection and immune evasion: a novel target for therapeutics.

    PubMed

    Oksayan, Sibil; Ito, Naoto; Moseley, Greg; Blondel, Danielle

    2012-02-01

    Vesicular stomatitis virus (VSV) and Rabies Virus (RABV) are the prototypic members of the rhabdovirus family. These viruses have a particularly broad host range, and despite the availability of vaccines, RABV still causes more than 50,000 human deaths a year. Trafficking of the virion or viral particles is important at several stages of the replicative life cycle, including cellular entry, localization into the cytoplasmic inclusion bodies which primarily house the transcription and replication of the viral genome, and migration to the plasma membrane from whence the virus is released by budding. Intriguingly, specific viral proteins, VSV M and RABV P have also been shown to undergo intracellular trafficking independent of the other viral apparatus. These proteins are multifunctional, and play roles in antagonism of host processes, namely the IFN system, and as such enable viral evasion of the innate cellular antiviral response. A body of recent research has been aimed at characterizing the mechanisms by which these proteins are able to shuttle between and localize to various subcellular sites, including the nucleus, which is not required during the cytoplasmic replicative life cycle of the virus. This work has indicated that trafficking of these proteins plays a significant role in determining the ability of the viruses to replicate and cause infection, and as such, represents a viable target for development of a new generation of vaccines and prophylactic therapeutics which are required to battle these pathogens of human and agricultural significance.

  12. Endosome-based protein trafficking and Ca2+ homeostasis in the heart

    PubMed Central

    Curran, Jerry; Makara, Michael A.; Mohler, Peter J.

    2015-01-01

    The ability to dynamically regulate, traffic, retain, and recycle proteins within the cell membrane is fundamental to life and central to the normal function of the heart. In the cardiomyocyte, these pathways are essential for the regulation of Ca2+, both at the level of the plasma membrane, but also in local cellular domains. One intracellular pathway often overlooked in relation to cardiovascular Ca2+ regulation and signaling is the endosome-based trafficking pathway. Highlighting its importance, this system and its molecular components are evolutionarily conserved across all metazoans. However, remarkably little is known of how endosome-based protein trafficking and recycling functions within mammalian cells systems, especially in the heart. As the endosomal system acts to regulate the expression and localization of membrane proteins central for cardiac Ca2+ regulation, understanding the in vivo function of this system in the heart is critical. This review will focus on endosome-based protein trafficking in the heart in both health and disease with special emphasis for the role of endocytic regulatory proteins, C-terminal Eps15 homology domain-containing proteins (EHDs). PMID:25709583

  13. The CD63-Syntenin-1 Complex Controls Post-Endocytic Trafficking of Oncogenic Human Papillomaviruses

    PubMed Central

    Gräßel, Linda; Fast, Laura Aline; Scheffer, Konstanze D.; Boukhallouk, Fatima; Spoden, Gilles A.; Tenzer, Stefan; Boller, Klaus; Bago, Ruzica; Rajesh, Sundaresan; Overduin, Michael; Berditchevski, Fedor; Florin, Luise

    2016-01-01

    Human papillomaviruses enter host cells via a clathrin-independent endocytic pathway involving tetraspanin proteins. However, post-endocytic trafficking required for virus capsid disassembly remains unclear. Here we demonstrate that the early trafficking pathway of internalised HPV particles involves tetraspanin CD63, syntenin-1 and ESCRT-associated adaptor protein ALIX. Following internalisation, viral particles are found in CD63-positive endosomes recruiting syntenin-1, a CD63-interacting adaptor protein. Electron microscopy and immunofluorescence experiments indicate that the CD63-syntenin-1 complex controls delivery of internalised viral particles to multivesicular endosomes. Accordingly, infectivity of high-risk HPV types 16, 18 and 31 as well as disassembly and post-uncoating processing of viral particles was markedly suppressed in CD63 or syntenin-1 depleted cells. Our analyses also present the syntenin-1 interacting protein ALIX as critical for HPV infection and CD63-syntenin-1-ALIX complex formation as a prerequisite for intracellular transport enabling viral capsid disassembly. Thus, our results identify the CD63-syntenin-1-ALIX complex as a key regulatory component in post-endocytic HPV trafficking. PMID:27578500

  14. The CD63-Syntenin-1 Complex Controls Post-Endocytic Trafficking of Oncogenic Human Papillomaviruses.

    PubMed

    Gräßel, Linda; Fast, Laura Aline; Scheffer, Konstanze D; Boukhallouk, Fatima; Spoden, Gilles A; Tenzer, Stefan; Boller, Klaus; Bago, Ruzica; Rajesh, Sundaresan; Overduin, Michael; Berditchevski, Fedor; Florin, Luise

    2016-08-31

    Human papillomaviruses enter host cells via a clathrin-independent endocytic pathway involving tetraspanin proteins. However, post-endocytic trafficking required for virus capsid disassembly remains unclear. Here we demonstrate that the early trafficking pathway of internalised HPV particles involves tetraspanin CD63, syntenin-1 and ESCRT-associated adaptor protein ALIX. Following internalisation, viral particles are found in CD63-positive endosomes recruiting syntenin-1, a CD63-interacting adaptor protein. Electron microscopy and immunofluorescence experiments indicate that the CD63-syntenin-1 complex controls delivery of internalised viral particles to multivesicular endosomes. Accordingly, infectivity of high-risk HPV types 16, 18 and 31 as well as disassembly and post-uncoating processing of viral particles was markedly suppressed in CD63 or syntenin-1 depleted cells. Our analyses also present the syntenin-1 interacting protein ALIX as critical for HPV infection and CD63-syntenin-1-ALIX complex formation as a prerequisite for intracellular transport enabling viral capsid disassembly. Thus, our results identify the CD63-syntenin-1-ALIX complex as a key regulatory component in post-endocytic HPV trafficking.

  15. The CD63-Syntenin-1 Complex Controls Post-Endocytic Trafficking of Oncogenic Human Papillomaviruses.

    PubMed

    Gräßel, Linda; Fast, Laura Aline; Scheffer, Konstanze D; Boukhallouk, Fatima; Spoden, Gilles A; Tenzer, Stefan; Boller, Klaus; Bago, Ruzica; Rajesh, Sundaresan; Overduin, Michael; Berditchevski, Fedor; Florin, Luise

    2016-01-01

    Human papillomaviruses enter host cells via a clathrin-independent endocytic pathway involving tetraspanin proteins. However, post-endocytic trafficking required for virus capsid disassembly remains unclear. Here we demonstrate that the early trafficking pathway of internalised HPV particles involves tetraspanin CD63, syntenin-1 and ESCRT-associated adaptor protein ALIX. Following internalisation, viral particles are found in CD63-positive endosomes recruiting syntenin-1, a CD63-interacting adaptor protein. Electron microscopy and immunofluorescence experiments indicate that the CD63-syntenin-1 complex controls delivery of internalised viral particles to multivesicular endosomes. Accordingly, infectivity of high-risk HPV types 16, 18 and 31 as well as disassembly and post-uncoating processing of viral particles was markedly suppressed in CD63 or syntenin-1 depleted cells. Our analyses also present the syntenin-1 interacting protein ALIX as critical for HPV infection and CD63-syntenin-1-ALIX complex formation as a prerequisite for intracellular transport enabling viral capsid disassembly. Thus, our results identify the CD63-syntenin-1-ALIX complex as a key regulatory component in post-endocytic HPV trafficking. PMID:27578500

  16. Dependence of PEI and PAMAM Gene Delivery on Clathrin- and Caveolin-Dependent Trafficking Pathways

    PubMed Central

    Hwang, Mark E.; Keswani, Rahul K.; Pack, Daniel W.

    2014-01-01

    Purpose Non-viral gene delivery vehicles such as polyethylenimine and polyamidoamine dendrimer effectively condense plasmid DNA, facilitate endocytosis, and deliver nucleic acid cargo to the nucleus in vitro. Better understanding of intracellular trafficking mechanisms involved in polymeric gene delivery is a prerequisite to clinical application. This study investigates the role of clathrin and caveolin endocytic pathways in cellular uptake and subsequent vector processing. Methods We formed 25-kD polyethylenimine (PEI) and generation 4 (G4) polyamidoamine (PAMAM) polyplexes at N/P 10 and evaluated internalization pathways and gene delivery in HeLa cells. Clathrin- and caveolin-dependent endocytosis inhibitors were used at varying concentrations to elucidate the roles of these important pathways. Results PEI and PAMAM polyplexes were internalized by both pathways. However, the amount of polyplex internalized poorly correlated with transgene expression. While the caveolin-dependent pathway generally led to effective gene delivery with both polymers, complete inhibition of the clathrin-dependent pathway was also deleterious to transfection with PEI polyplexes. Inhibition of one endocytic pathway may lead to an overall increase in uptake via unaffected pathways, suggesting the existence of compensatory endocytic mechanisms. Conclusions The well-studied clathrin- and caveolin-dependent endocytosis pathways are not necessarily independent, and perturbing one mechanism of trafficking influences the larger trafficking network. PMID:25511918

  17. Domestic minor sex trafficking: what the PNP needs to know.

    PubMed

    Hornor, Gail

    2015-01-01

    Human trafficking is a major global public health problem and represents a substantial human rights violation. Human trafficking has been receiving attention in both the lay media and professional literature. Human trafficking can include commercial sex, forced labor, child soldiers, and stealing of human organs. One form of human trafficking represents a significant American pediatric health problem: domestic minor sex trafficking (DMST). DMST is the commercial sexual abuse of children by selling, buying, or trading their sexual service. This continuing education article will define DMST and discuss it in terms of prevalence, risk factors, and practice implications for the pediatric nurse practitioner. PMID:25497135

  18. Domestic minor sex trafficking: what the PNP needs to know.

    PubMed

    Hornor, Gail

    2015-01-01

    Human trafficking is a major global public health problem and represents a substantial human rights violation. Human trafficking has been receiving attention in both the lay media and professional literature. Human trafficking can include commercial sex, forced labor, child soldiers, and stealing of human organs. One form of human trafficking represents a significant American pediatric health problem: domestic minor sex trafficking (DMST). DMST is the commercial sexual abuse of children by selling, buying, or trading their sexual service. This continuing education article will define DMST and discuss it in terms of prevalence, risk factors, and practice implications for the pediatric nurse practitioner.

  19. Targeting caspases in intracellular protozoan infections.

    PubMed

    Guillermo, Landi V C; Pereira, Wânia F; De Meis, Juliana; Ribeiro-Gomes, Flavia L; Silva, Elisabeth M; Kroll-Palhares, Karina; Takiya, Christina M; Lopes, Marcela F

    2009-06-01

    Caspases are cysteine aspartases acting either as initiators (caspases 8, 9, and 10) or executioners (caspases 3, 6, and 7) to induce programmed cell death by apoptosis. Parasite infections by certain intracellular protozoans increase host cell life span by targeting caspase activation. Conversely, caspase activation, followed by apoptosis of lymphocytes and other cells, prevents effective immune responses to chronic parasite infection. Here we discuss how pharmacological inhibition of caspases might affect the immunity to protozoan infections, by either blocking or delaying apoptosis.

  20. Agonist-promoted ubiquitination differentially regulates receptor trafficking of endothelin type A and type B receptors.

    PubMed

    Terada, Koji; Horinouchi, Takahiro; Fujioka, Yoichiro; Higashi, Tsunehito; Nepal, Prabha; Horiguchi, Mika; Karki, Sarita; Hatate, Chizuru; Hoshi, Akimasa; Harada, Takuya; Mai, Yosuke; Ohba, Yusuke; Miwa, Soichi

    2014-12-19

    Two types of G protein-coupled receptors for endothelin-1 (ET-1), ET type A receptor (ETAR) and ETBR, closely resemble each other, but upon ET-1 stimulation, they follow totally different intracellular trafficking pathways; ETAR is recycled back to plasma membrane, whereas ETBR is targeted to lysosome for degradation. However, the mechanisms for such different fates are unknown. Here we demonstrated that ETBR but not ETAR was ubiquitinated on the cell surface following ET-1 stimulation and that ETBR was internalized and degraded in lysosome more rapidly than ETAR. The mutant ETBR (designated "5KR mutant") in which 5 lysine residues in the C-tail were substituted to arginine was not ubiquitinated, and its rates of internalization and degradation after ET-1 stimulation became slower, being comparable with those of ETAR. Confocal microscopic study showed that following ET-1 stimulation, ETAR and 5KR mutant of ETBR were co-localized mainly with Rab11, a marker of recycling endosome, whereas ETBR was co-localized with Rab7, a marker of late endosome/lysosome. In the 5KR mutant, ET-1-induced ERK phosphorylation and an increase in the intracellular Ca(2+) concentration upon repetitive ET-1 stimulation were larger. A series of ETBR mutants (designated "4KR mutant"), in which either one of 5 arginine residues of the 5KR mutant was reverted to lysine, were normally ubiquitinated, internalized, and degraded, with ERK phosphorylation being normalized. These results demonstrate that agonist-induced ubiquitination at either lysine residue in the C-tail of ETBR but not ETAR switches intracellular trafficking from recycling to plasma membrane to targeting to lysosome, causing decreases in the cell surface level of ETBR and intracellular signaling.

  1. Emerging concepts in the pharmacogenomics of arrhythmias: ion channel trafficking

    PubMed Central

    Harkcom, William T; Abbott, Geoffrey W

    2010-01-01

    Continuous, rhythmic beating of the heart requires exquisite control of expression, localization and function of cardiac ion channels – the foundations of the cardiac myocyte action potential. Disruption of any of these processes can alter the shape of the action potential, predisposing to cardiac arrhythmias. These arrhythmias can manifest in a variety of ways depending on both the channels involved and the type of disruption (i.e., gain or loss of function). As much as 1% of the population of developed countries is affected by cardiac arrhythmia each year, and a detailed understanding of the mechanism of each arrhythmia is crucial to developing and prescribing the proper therapies. Many of the antiarrhythmic drugs currently on the market were developed before the underlying cause of the arrhythmia was known, and as a result lack specificity, causing side effects. The majority of the available drugs target the conductance of cardiac ion channels, either by blocking or enhancing current through the channel. In recent years, however, it has become apparent that specific targeting of ion channel conductance may not be the most effective means for treatment. Here we review increasing evidence that suggests defects in ion channel trafficking play an important role in the etiology of arrhythmias, and small molecule approaches to correct trafficking defects will likely play an important role in the future of arrhythmia treatment. PMID:20670193

  2. Trafficking and contract migrant workers in the Middle East.

    PubMed

    Jureidini, Ray

    2010-01-01

    The paper addresses a number of issues regarding the extent to which trafficking may be applied to migrant domestic workers who enter under the kafala system of sponsorship in the Middle East. Migrant domestic workers are the most numerous of those mentioned in reports on trafficking for labour exploitation in the region. The discussion seeks to determine whether "trafficking" can be ex post facto, rather than ex ante? In other words, can the label of trafficking be attributed only after the worker has arrived in the receiving country and is victimized according to the principles of trafficking protocols? In addition, must there be a proven intent to traffic by agents, or can employers who harm and/or exploit them be considered as traffickers alone? Should the harm done to workers on arrival at their place of work be classified (and assisted) as victims of trafficking, or as exploited workers?

  3. Cdk1-dependent control of membrane-trafficking dynamics

    PubMed Central

    McCusker, Derek; Royou, Anne; Velours, Christophe; Kellogg, Douglas

    2012-01-01

    Cyclin-dependent kinase 1 (Cdk1) is required for initiation and maintenance of polarized cell growth in budding yeast. Cdk1 activates Rho-family GTPases, which polarize the actin cytoskeleton for delivery of membrane to growth sites via the secretory pathway. Here we investigate whether Cdk1 plays additional roles in the initiation and maintenance of polarized cell growth. We find that inhibition of Cdk1 causes a cell surface growth defect that is as severe as that caused by actin depolymerization. However, unlike actin depolymerization, Cdk1 inhibition does not result in a massive accumulation of intracellular secretory vesicles or their cargoes. Analysis of post-Golgi vesicle dynamics after Cdk1 inhibition demonstrates that exocytic vesicles are rapidly mistargeted away from the growing bud, possibly to the endomembrane/vacuolar system. Inhibition of Cdk1 also causes defects in the organization of endocytic and exocytic zones at the site of growth. Cdk1 thus modulates membrane-trafficking dynamics, which is likely to play an important role in coordinating cell surface growth with cell cycle progression. PMID:22767578

  4. Active Trafficking of Alpha 1 Antitrypsin across the Lung Endothelium

    PubMed Central

    Lockett, Angelia D.; Brown, Mary Beth; Santos-Falcon, Nieves; Rush, Natalia I.; Oueini, Houssam; Oberle, Amber J.; Bolanis, Esther; Fragoso, Miryam A.; Petrusca, Daniela N.; Serban, Karina A.; Schweitzer, Kelly S.; Presson Jr., Robert G.

    2014-01-01

    The homeostatic lung protective effects of alpha-1 antitrypsin (A1AT) may require the transport of circulating proteinase inhibitor across an intact lung endothelial barrier. We hypothesized that uninjured pulmonary endothelial cells transport A1AT to lung epithelial cells. Purified human A1AT was rapidly taken up by confluent primary rat pulmonary endothelial cell monolayers, was secreted extracellularly, both apically and basolaterally, and was taken up by adjacent rat lung epithelial cells co-cultured on polarized transwells. Similarly, polarized primary human lung epithelial cells took up basolaterally-, but not apically-supplied A1AT, followed by apical secretion. Evidence of A1AT transcytosis across lung microcirculation was confirmed in vivo by two-photon intravital microscopy in mice. Time-lapse confocal microscopy indicated that A1AT co-localized with Golgi in the endothelium whilst inhibition of the classical secretory pathway with tunicamycin significantly increased intracellular retention of A1AT. However, inhibition of Golgi secretion promoted non-classical A1AT secretion, associated with microparticle release. Polymerized A1AT or A1AT supplied to endothelial cells exposed to soluble cigarette smoke extract had decreased transcytosis. These results suggest previously unappreciated pathways of A1AT bidirectional uptake and secretion from lung endothelial cells towards the alveolar epithelium and airspaces. A1AT trafficking may determine its functional bioavailablity in the lung, which could be impaired in individuals exposed to smoking or in those with A1AT deficiency. PMID:24743137

  5. The retrovirus RNA trafficking granule: from birth to maturity

    PubMed Central

    Cochrane, Alan W; McNally, Mark T; Mouland, Andrew J

    2006-01-01

    Post-transcriptional events in the life of an RNA including RNA processing, transport, translation and metabolism are characterized by the regulated assembly of multiple ribonucleoprotein (RNP) complexes. At each of these steps, there is the engagement and disengagement of RNA-binding proteins until the RNA reaches its final destination. For retroviral genomic RNA, the final destination is the capsid. Numerous studies have provided crucial information about these processes and serve as the basis for studies on the intracellular fate of retroviral RNA. Retroviral RNAs are like cellular mRNAs but their processing is more tightly regulated by multiple cis-acting sequences and the activities of many trans-acting proteins. This review describes the viral and cellular partners that retroviral RNA encounters during its maturation that begins in the nucleus, focusing on important events including splicing, 3' end-processing, RNA trafficking from the nucleus to the cytoplasm and finally, mechanisms that lead to its compartmentalization into progeny virions. PMID:16545126

  6. Quantitative analysis of virus and plasmid trafficking in cells

    NASA Astrophysics Data System (ADS)

    Lagache, Thibault; Dauty, Emmanuel; Holcman, David

    2009-01-01

    Intracellular transport of DNA carriers is a fundamental step of gene delivery. By combining both theoretical and numerical approaches we study here single and several viruses and DNA particles trafficking in the cell cytoplasm to a small nuclear pore. We present a physical model to account for certain aspects of cellular organization, starting with the observation that a viral trajectory consists of epochs of pure diffusion and epochs of active transport along microtubules. We define a general degradation rate to describe the limitations of the delivery of plasmid or viral particles to a nuclear pore imposed by various types of direct and indirect hydrolysis activity inside the cytoplasm. By replacing the switching dynamics by a single steady state stochastic description, we obtain estimates for the probability and the mean time for the first one of many particles to go from the cell membrane to a small nuclear pore. Computational simulations confirm that our model can be used to analyze and interpret viral trajectories and estimate quantitatively the success of nuclear delivery.

  7. Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

    PubMed Central

    El Ayadi, Amina; Stieren, Emily S.; Barral, José M.; Oberhauser, Andres F.; Boehning, Darren

    2012-01-01

    Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed α, β, and γ-secretases1. Sequential proteolytic cleavage of APP with β and γ-secretases leads to the production of a small proteolytic peptide, termed Aβ, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes2,3. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro5,6, however it obtains stable secondary structure when bound to Fe657. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells4. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis8, to modulate signaling pathways9, and to regulate calcium signaling10. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology11, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques12. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a

  8. TAG1 regulates the endocytic trafficking and signalling of the Semaphorin3A receptor complex

    PubMed Central

    Dang, Puneet; Smythe, Elizabeth; Furley, Andrew J. W.

    2012-01-01

    Endocytic trafficking of membrane proteins is essential for neuronal structure and function. We show that Transient Axonal Glycoprotein1 (TAG1 or CNTN2), a contactin-related adhesion molecule, plays a central role in the differential trafficking of components of the semaphorin3A receptor complex into distinct endosomal compartments in murine spinal sensory neuron growth cones. The semaphorin3A receptor is composed of Neuropilin1 (NRP1), PlexinA4 and L1, with NRP1 being the ligand-binding component. TAG1 interacts with NRP1 causing a change in its association with L1 in the Sema3A response, such that L1 is lost from the complex following Sema3A binding. Initially, however, L1 and NRP1 endocytose together and only become separated intracellularly, NRP1 becoming associated with endosomes enriched in lipid rafts and co-localising with TAG1 and PlexinA4. When TAG1 is missing, NRP1 and L1 fail to separate and NRP1 does not become raft-associated; co-localisation with PlexinA4 is reduced and Plexin signalling is not initiated. These observations identify a novel role for TAG1 in modulating the intracellular sorting of signalling receptor complexes. PMID:22836270

  9. Subcellular localization and mechanisms of nucleocytoplasmic trafficking of steroid receptor coactivator-1.

    PubMed

    Amazit, Larbi; Alj, Youssef; Tyagi, Rakesh Kumar; Chauchereau, Anne; Loosfelt, Hugues; Pichon, Christophe; Pantel, Jacques; Foulon-Guinchard, Emmanuelle; Leclerc, Philippe; Milgrom, Edwin; Guiochon-Mantel, Anne

    2003-08-22

    Steroid hormone receptors are ligand-stimulated transcription factors that modulate gene transcription by recruiting coregulators to gene promoters. Subcellular localization and dynamic movements of transcription factors have been shown to be one of the major means of regulating their transcriptional activity. In the present report we describe the subcellular localization and the dynamics of intracellular trafficking of steroid receptor coactivator 1 (SRC-1). After its synthesis in the cytoplasm, SRC-1 is imported into the nucleus, where it activates transcription and is subsequently exported back to the cytoplasm. In both the nucleus and cytoplasm, SRC-1 is localized in speckles. The characterization of SRC-1 nuclear localization sequence reveals that it is a classic bipartite signal localized in the N-terminal region of the protein, between amino acids 18 and 36. This sequence is highly conserved within the other members of the p160 family. Additionally, SRC-1 nuclear export is inhibited by leptomycin B. The region involved in its nuclear export is localized between amino acids 990 and 1038. It is an unusually large domain differing from the classic leucine-rich NES sequences. Thus SRC-1 nuclear export involves either an alternate type of NES or is dependent on the interaction of SRC-1 with a protein, which is exported through the crm1/exportin pathway. Overall, the intracellular trafficking of SRC-1 might be a mechanism to regulate the termination of hormone action, the interaction with other signaling pathways in the cytoplasm and its degradation. PMID:12791702

  10. The nicotine metabolite, cotinine, alters the assembly and trafficking of a subset of nicotinic acetylcholine receptors.

    PubMed

    Fox, Ashley M; Moonschi, Faruk H; Richards, Christopher I

    2015-10-01

    Exposure to nicotine alters the trafficking and assembly of nicotinic receptors (nAChRs), leading to their up-regulation on the plasma membrane. Although the mechanism is not fully understood, nicotine-induced up-regulation is believed to contribute to nicotine addiction. The effect of cotinine, the primary metabolite of nicotine, on nAChR trafficking and assembly has not been extensively investigated. We utilize a pH-sensitive variant of GFP, super ecliptic pHluorin, to differentiate between intracellular nAChRs and those expressed on the plasma membrane to quantify changes resulting from cotinine and nicotine exposure. Similar to nicotine, exposure to cotinine increases the number of α4β2 receptors on the plasma membrane and causes a redistribution of intracellular receptors. In contrast to this, cotinine exposure down-regulates α6β2β3 receptors. We also used single molecule fluorescence studies to show that cotinine and nicotine both alter the assembly of α4β2 receptors to favor the high sensitivity (α4)2(β2)3 stoichiometry.

  11. Surface chemistry dependent "switch" regulates the trafficking and therapeutic performance of drug-loaded carbon nanotubes.

    PubMed

    Das, Manasmita; Singh, Raman Preet; Datir, Satyajit R; Jain, Sanyog

    2013-04-17

    The present study explores the possibility of exploiting surface functionality as one of the key regulators for modulating the intracellular trafficking and therapeutic performance of drug loaded carbon nanotubes (CNTs). In line with that approach, a series of biofunctionalized multiwalled carbon nanotubes (f-CNTs 1-6) decorated with various functional molecules including antifouling polymer (PEG), tumor recognition modules (folic acid/hyaluronic acid/estradiol), and fluorophores (rhodamine B isothiocyanate/Alexa Fluor) were synthesized. By loading different anticancer agents (methotrexate (MTX), doxorubicin (DOX), and paclitaxel (PTX)) onto each functionalized CNT preparation, we tried to elucidate how the surface functional molecules associated with each f-CNT influence their therapeutic potential. We observed that antiproliferative or apoptotic activity of drug-loaded CNTs critically depends on their mechanistic pathway of cellular internalization and intracellular trafficking, which in turn had an intimate rapport with their surface chemistry. To our knowledge, for the first time, we have embarked on the possibility of using a surface chemistry dependent "switch" to remote-control the second and third order targeting of chemotherapeutic agents supramolecularly complexed/adsorbed on CNTs, which in turn is expected to benefit the development of futuristic nanobots for cancer theranostics.

  12. The Nicotine Metabolite, Cotinine, Alters the Assembly and Trafficking of a Subset of Nicotinic Acetylcholine Receptors*

    PubMed Central

    Fox, Ashley M.; Moonschi, Faruk H.; Richards, Christopher I.

    2015-01-01

    Exposure to nicotine alters the trafficking and assembly of nicotinic receptors (nAChRs), leading to their up-regulation on the plasma membrane. Although the mechanism is not fully understood, nicotine-induced up-regulation is believed to contribute to nicotine addiction. The effect of cotinine, the primary metabolite of nicotine, on nAChR trafficking and assembly has not been extensively investigated. We utilize a pH-sensitive variant of GFP, super ecliptic pHluorin, to differentiate between intracellular nAChRs and those expressed on the plasma membrane to quantify changes resulting from cotinine and nicotine exposure. Similar to nicotine, exposure to cotinine increases the number of α4β2 receptors on the plasma membrane and causes a redistribution of intracellular receptors. In contrast to this, cotinine exposure down-regulates α6β2β3 receptors. We also used single molecule fluorescence studies to show that cotinine and nicotine both alter the assembly of α4β2 receptors to favor the high sensitivity (α4)2(β2)3 stoichiometry. PMID:26269589

  13. Regulation of GPCR activity, trafficking and localization by GPCR-interacting proteins

    PubMed Central

    Magalhaes, Ana C; Dunn, Henry; Ferguson, Stephen SG

    2012-01-01

    GPCRs represent the largest family of integral membrane proteins and were first identified as receptor proteins that couple via heterotrimeric G-proteins to regulate a vast variety of effector proteins to modulate cellular function. It is now recognized that GPCRs interact with a myriad of proteins that not only function to attenuate their signalling but also function to couple these receptors to heterotrimeric G-protein-independent signalling pathways. In addition, intracellular and transmembrane proteins associate with GPCRs and regulate their processing in the endoplasmic reticulum, trafficking to the cell surface, compartmentalization to plasma membrane microdomains, endocytosis and trafficking between intracellular membrane compartments. The present review will overview the functional consequence of β-arrestin, receptor activity-modifying proteins (RAMPS), regulators of G-protein signalling (RGS), GPCR-associated sorting proteins (GASPs), Homer, small GTPases, PSD95/Disc Large/Zona Occludens (PDZ), spinophilin, protein phosphatases, calmodulin, optineurin and Src homology 3 (SH3) containing protein interactions with GPCRs. LINKED ARTICLES This article is part of a themed section on the Molecular Pharmacology of G Protein-Coupled Receptors (GPCRs). To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-6. To view the 2010 themed section on the same topic visit http://onlinelibrary.wiley.com/doi/10.1111/bph.2010.159.issue-5/issuetoc PMID:21699508

  14. Physical health symptoms reported by trafficked women receiving post-trafficking support in Moldova: prevalence, severity and associated factors

    PubMed Central

    2012-01-01

    Background Many trafficked people suffer high levels of physical, sexual and psychological abuse. Yet, there has been limited research on the physical health problems associated with human trafficking or how the health needs of women in post-trafficking support settings vary according to socio-demographic or trafficking characteristics. Methods We analysed the prevalence and severity of 15 health symptoms reported by 120 trafficked women who had returned to Moldova between December 2007 and December 2008 and were registered with the International Organisation for Migration Assistance and Protection Programme. Women had returned to Moldova an average of 5.9 months prior to interview (range 2-12 months). Results Headaches (61.7%), stomach pain (60.9%), memory problems (44.2%), back pain (42.5%), loss of appetite (35%), and tooth pain (35%) were amongst the most commonly reported symptoms amongst both women trafficked for sexual exploitation and women trafficked for labour exploitation. The prevalence of headache and memory problems was strongly associated with duration of exploitation. Conclusions Trafficked women who register for post-trafficking support services after returning to their country of origin are likely to have long-term physical and dental health needs and should be provided with access to comprehensive medical services. Health problems among women who register for post-trafficking support services after returning to their country of origin are not limited to women trafficked for sexual exploitation but are also experienced by victims of labour exploitation. PMID:22834807

  15. Three-dimensional imaging of nucleolin trafficking in normal cells, transfectants, and heterokaryons

    NASA Astrophysics Data System (ADS)

    Ballou, Byron T.; Fisher, Gregory W.; Deng, Jau-Shyong; Hakala, Thomas R.; Srivastava, Meera; Farkas, Daniel L.

    1996-04-01

    The study of intracellular trafficking using labeled molecules has been aided by the development of the cyanine fluorochromes, which are easily coupled, very soluble, resist photobleaching, and fluoresce at far-red wavelengths where background fluorescence is minimal. We have used Cy3-, Cy5-, and Cy5.5-labeled antibodies, antigen-binding fragments, and specifically binding single-stranded oligonucleotides to follow expression and trafficking of nucleolin, the most abundant protein of the nucleolus. Nucleolin shuttles between the nucleolus and the cytoplasm, and is also expressed on the cell surface, allowing us to test our techniques at all three cellular sites. Differentially cyanine-labeled non-specific antibodies were used to control for non-specific binding. Similarly, the differentially labeled non-binding strand of the cloned oligonucleotide served as a control. The multimode microscope allowed us to follow both rapid and slow redistributions of labeled ligands in the same study. We also performed 3-D reconstructions of nucleolin distribution in cells using rapid acquisition and deconvolution. Microinjection of labeled ligands was used to follow intracellular distribution, while incubation of whole cells with antibody and antigen-binding fragments was used to study uptake. To unambiguously define trafficking, and eliminate the possibility of interference by cross-reactive proteins, we transfected mouse renal cell carcinoma cells that express cell surface nucleolin with human nucleolin. We used microinjection and cell surface staining with Cy3- or Cy5- labeled monoclonal antibody D3 (specific for human nucleolin) to assess the cellular distribution of the human protein. Several clones expressed human nucleolin on their surfaces and showed high levels of transport of the human protein into the mouse nucleus and nucleolus. This distribution roughly parallels that of mouse nucleolin as determined by labeled polyclonal antibody. We have used these engineered

  16. Axons provide the secretory machinery for trafficking of voltage-gated sodium channels in peripheral nerve.

    PubMed

    González, Carolina; Cánovas, José; Fresno, Javiera; Couve, Eduardo; Court, Felipe A; Couve, Andrés

    2016-02-16

    The regulation of the axonal proteome is key to generate and maintain neural function. Fast and slow axoplasmic waves have been known for decades, but alternative mechanisms to control the abundance of axonal proteins based on local synthesis have also been identified. The presence of the endoplasmic reticulum has been documented in peripheral axons, but it is still unknown whether this localized organelle participates in the delivery of axonal membrane proteins. Voltage-gated sodium channels are responsible for action potentials and are mostly concentrated in the axon initial segment and nodes of Ranvier. Despite their fundamental role, little is known about the intracellular trafficking mechanisms that govern their availability in mature axons. Here we describe the secretory machinery in axons and its contribution to plasma membrane delivery of sodium channels. The distribution of axonal secretory components was evaluated in axons of the sciatic nerve and in spinal nerve axons after in vivo electroporation. Intracellular protein trafficking was pharmacologically blocked in vivo and in vitro. Axonal voltage-gated sodium channel mRNA and local trafficking were examined by RT-PCR and a retention-release methodology. We demonstrate that mature axons contain components of the endoplasmic reticulum and other biosynthetic organelles. Axonal organelles and sodium channel localization are sensitive to local blockade of the endoplasmic reticulum to Golgi transport. More importantly, secretory organelles are capable of delivering sodium channels to the plasma membrane in isolated axons, demonstrating an intrinsic capacity of the axonal biosynthetic route in regulating the axonal proteome in mammalian axons. PMID:26839409

  17. Sorting Motifs Involved in the Trafficking and Localization of the PIN1 Auxin Efflux Carrier.

    PubMed

    Sancho-Andrés, Gloria; Soriano-Ortega, Esther; Gao, Caiji; Bernabé-Orts, Joan Miquel; Narasimhan, Madhumitha; Müller, Anna Ophelia; Tejos, Ricardo; Jiang, Liwen; Friml, Jiří; Aniento, Fernando; Marcote, María Jesús

    2016-07-01

    In contrast with the wealth of recent reports about the function of μ-adaptins and clathrin adaptor protein (AP) complexes, there is very little information about the motifs that determine the sorting of membrane proteins within clathrin-coated vesicles in plants. Here, we investigated putative sorting signals in the large cytosolic loop of the Arabidopsis (Arabidopsis thaliana) PIN-FORMED1 (PIN1) auxin transporter, which are involved in binding μ-adaptins and thus in PIN1 trafficking and localization. We found that Phe-165 and Tyr-280, Tyr-328, and Tyr-394 are involved in the binding of different μ-adaptins in vitro. However, only Phe-165, which binds μA(μ2)- and μD(μ3)-adaptin, was found to be essential for PIN1 trafficking and localization in vivo. The PIN1:GFP-F165A mutant showed reduced endocytosis but also localized to intracellular structures containing several layers of membranes and endoplasmic reticulum (ER) markers, suggesting that they correspond to ER or ER-derived membranes. While PIN1:GFP localized normally in a μA (μ2)-adaptin mutant, it accumulated in big intracellular structures containing LysoTracker in a μD (μ3)-adaptin mutant, consistent with previous results obtained with mutants of other subunits of the AP-3 complex. Our data suggest that Phe-165, through the binding of μA (μ2)- and μD (μ3)-adaptin, is important for PIN1 endocytosis and for PIN1 trafficking along the secretory pathway, respectively. PMID:27208248

  18. Cellular trafficking and degradation of erythropoietin and novel erythropoiesis stimulating protein (NESP).

    PubMed

    Gross, Alec W; Lodish, Harvey F

    2006-01-27

    Erythropoietin (Epo) is essential for the production of mature red blood cells, and recombinant Epo is commonly used to treat anemia, but how Epo is degraded and cleared from the body is not understood. Glycosylation of Epo is required for its in vivo bioactivity, although not for in vitro receptor binding or stimulation of Epo-dependent cell lines; Epo glycosylation actually reduces the affinity of Epo for the Epo receptor (EpoR). Interestingly, a hyperglycosylated analog of Epo, called novel erythropoiesis-stimulating protein (NESP), has a lower affinity than Epo for the EpoR but has greater in vivo activity and a longer serum half-life than Epo. We hypothesize that a major mechanism for degradation of Epo in the body occurs in cells expressing the Epo receptor, through receptor-mediated endocytosis of Epo followed by degradation in lysosomes, and therefore investigated the trafficking and degradation of Epo and NESP by EpoR-expressing cells. We show that Epo and NESP are degraded only by cultured cells that express the EpoR, and their receptor binding, dissociation, and trafficking properties determine their rates of intracellular degradation. Epo binds surface EpoR faster than NESP (k(on) = 5.0 x 10(8) m(-1) min(-1) versus 1.1 x 10(8) m(-1) min(-1)) but dissociates slower (k(off) = 0.029 min(-1) versus 0.042 min(-1)). Surface-bound Epo and NESP are internalized at the same rate (k(in) = 0.06 min(-1)), and after internalization 60% of each ligand is resecreted intact and 40% degraded. Our kinetic model of Epo and NESP receptor binding, intracellular trafficking, and degradation explains why Epo is degraded faster than NESP at the cellular level.

  19. Axons provide the secretory machinery for trafficking of voltage-gated sodium channels in peripheral nerve

    PubMed Central

    González, Carolina; Cánovas, José; Fresno, Javiera; Couve, Eduardo; Court, Felipe A.; Couve, Andrés

    2016-01-01

    The regulation of the axonal proteome is key to generate and maintain neural function. Fast and slow axoplasmic waves have been known for decades, but alternative mechanisms to control the abundance of axonal proteins based on local synthesis have also been identified. The presence of the endoplasmic reticulum has been documented in peripheral axons, but it is still unknown whether this localized organelle participates in the delivery of axonal membrane proteins. Voltage-gated sodium channels are responsible for action potentials and are mostly concentrated in the axon initial segment and nodes of Ranvier. Despite their fundamental role, little is known about the intracellular trafficking mechanisms that govern their availability in mature axons. Here we describe the secretory machinery in axons and its contribution to plasma membrane delivery of sodium channels. The distribution of axonal secretory components was evaluated in axons of the sciatic nerve and in spinal nerve axons after in vivo electroporation. Intracellular protein trafficking was pharmacologically blocked in vivo and in vitro. Axonal voltage-gated sodium channel mRNA and local trafficking were examined by RT-PCR and a retention-release methodology. We demonstrate that mature axons contain components of the endoplasmic reticulum and other biosynthetic organelles. Axonal organelles and sodium channel localization are sensitive to local blockade of the endoplasmic reticulum to Golgi transport. More importantly, secretory organelles are capable of delivering sodium channels to the plasma membrane in isolated axons, demonstrating an intrinsic capacity of the axonal biosynthetic route in regulating the axonal proteome in mammalian axons. PMID:26839409

  20. Intracellular Uropathogenic E. coli Exploits Host Rab35 for Iron Acquisition and Survival within Urinary Bladder Cells.

    PubMed

    Dikshit, Neha; Bist, Pradeep; Fenlon, Shannon N; Pulloor, Niyas Kudukkil; Chua, Christelle En Lin; Scidmore, Marci A; Carlyon, Jason A; Tang, Bor Luen; Chen, Swaine L; Sukumaran, Bindu

    2015-08-01

    Recurrent urinary tract infections (UTIs) caused by uropathogenic E. coli (UPEC) are common and morbid infections with limited therapeutic options. Previous studies have demonstrated that persistent intracellular infection of bladder epithelial cells (BEC) by UPEC contributes to recurrent UTI in mouse models of infection. However, the mechanisms employed by UPEC to survive within BEC are incompletely understood. In this study we aimed to understand the role of host vesicular trafficking proteins in the intracellular survival of UPEC. Using a cell culture model of intracellular UPEC infection, we found that the small GTPase Rab35 facilitates UPEC survival in UPEC-containing vacuoles (UCV) within BEC. Rab35 plays a role in endosomal recycling of transferrin receptor (TfR), the key protein responsible for transferrin-mediated cellular iron uptake. UPEC enhance the expression of both Rab35 and TfR and recruit these proteins to the UCV, thereby supplying UPEC with the essential nutrient iron. Accordingly, Rab35 or TfR depleted cells showed significantly lower intracellular iron levels and reduced ability to support UPEC survival. In the absence of Rab35, UPEC are preferentially trafficked to degradative lysosomes and killed. Furthermore, in an in vivo murine model of persistent intracellular infection, Rab35 also colocalizes with intracellular UPEC. We propose a model in which UPEC subverts two different vesicular trafficking pathways (endosomal recycling and degradative lysosomal fusion) by modulating Rab35, thereby simultaneously enhancing iron acquisition and avoiding lysosomal degradation of the UCV within bladder epithelial cells. Our findings reveal a novel survival mechanism of intracellular UPEC and suggest a potential avenue for therapeutic intervention against recurrent UTI.

  1. A calcineurin-dependent switch controls the trafficking function of α-arrestin Aly1/Art6.

    PubMed

    O'Donnell, Allyson F; Huang, Laiqiang; Thorner, Jeremy; Cyert, Martha S

    2013-08-16

    Proper regulation of plasma membrane protein endocytosis by external stimuli is required for cell growth and survival. In yeast, excess levels of certain nutrients induce endocytosis of the cognate permeases to prevent toxic accumulation of metabolites. The α-arrestins, a family of trafficking adaptors, stimulate ubiquitin-dependent and clathrin-mediated endocytosis by interacting with both a client permease and the ubiquitin ligase Rsp5. However, the molecular mechanisms that control α-arrestin function are not well understood. Here, we show that α-arrestin Aly1/Art6 is a phosphoprotein that specifically interacts with and is dephosphorylated by the Ca(2+)- and calmodulin-dependent phosphoprotein phosphatase calcineurin/PP2B. Dephosphorylation of Aly1 by calcineurin at a subset of phospho-sites is required for Aly1-mediated trafficking of the aspartic acid and glutamic acid transporter Dip5 to the vacuole, but it does not alter Rsp5 binding, ubiquitinylation, or stability of Aly1. In addition, dephosphorylation of Aly1 by calcineurin does not regulate the ability of Aly1 to promote the intracellular sorting of the general amino acid permease Gap1. These results suggest that phosphorylation of Aly1 inhibits its vacuolar trafficking function and, conversely, that dephosphorylation of Aly1 by calcineurin serves as a regulatory switch to promote Aly1-mediated trafficking to the vacuole.

  2. Monitoring G protein-coupled receptor and β-arrestin trafficking in live cells using enhanced bystander BRET.

    PubMed

    Namkung, Yoon; Le Gouill, Christian; Lukashova, Viktoria; Kobayashi, Hiroyuki; Hogue, Mireille; Khoury, Etienne; Song, Mideum; Bouvier, Michel; Laporte, Stéphane A

    2016-01-01

    Endocytosis and intracellular trafficking of receptors are pivotal to maintain physiological functions and drug action; however, robust quantitative approaches are lacking to study such processes in live cells. Here we present new bioluminescence resonance energy transfer (BRET) sensors to quantitatively monitor G protein-coupled receptors (GPCRs) and β-arrestin trafficking. These sensors are based on bystander BRET and use the naturally interacting chromophores luciferase (RLuc) and green fluorescent protein (rGFP) from Renilla. The versatility and robustness of this approach are exemplified by anchoring rGFP at the plasma membrane or in endosomes to generate high dynamic spectrometric BRET signals on ligand-promoted recruitment or sequestration of RLuc-tagged proteins to, or from, specific cell compartments, as well as sensitive subcellular BRET imaging for protein translocation visualization. These sensors are scalable to high-throughput formats and allow quantitative pharmacological studies of GPCR trafficking in real time, in live cells, revealing ligand-dependent biased trafficking of receptor/β-arrestin complexes. PMID:27397672

  3. Monitoring G protein-coupled receptor and β-arrestin trafficking in live cells using enhanced bystander BRET

    PubMed Central

    Namkung, Yoon; Le Gouill, Christian; Lukashova, Viktoria; Kobayashi, Hiroyuki; Hogue, Mireille; Khoury, Etienne; Song, Mideum; Bouvier, Michel; Laporte, Stéphane A.

    2016-01-01

    Endocytosis and intracellular trafficking of receptors are pivotal to maintain physiological functions and drug action; however, robust quantitative approaches are lacking to study such processes in live cells. Here we present new bioluminescence resonance energy transfer (BRET) sensors to quantitatively monitor G protein-coupled receptors (GPCRs) and β-arrestin trafficking. These sensors are based on bystander BRET and use the naturally interacting chromophores luciferase (RLuc) and green fluorescent protein (rGFP) from Renilla. The versatility and robustness of this approach are exemplified by anchoring rGFP at the plasma membrane or in endosomes to generate high dynamic spectrometric BRET signals on ligand-promoted recruitment or sequestration of RLuc-tagged proteins to, or from, specific cell compartments, as well as sensitive subcellular BRET imaging for protein translocation visualization. These sensors are scalable to high-throughput formats and allow quantitative pharmacological studies of GPCR trafficking in real time, in live cells, revealing ligand-dependent biased trafficking of receptor/β-arrestin complexes. PMID:27397672

  4. Mechanisms of intracellular ice formation.

    PubMed Central

    Muldrew, K; McGann, L E

    1990-01-01

    The phenomenon of intracellular freezing in cells was investigated by designing experiments with cultured mouse fibroblasts on a cryomicroscope to critically assess the current hypotheses describing the genesis of intracellular ice: (a) intracellular freezing is a result of critical undercooling; (b) the cytoplasm is nucleated through aqueous pores in the plasma membrane; and (c) intracellular freezing is a result of membrane damage caused by electrical transients at the ice interface. The experimental data did not support any of these theories, but was consistent with the hypothesis that the plasma membrane is damaged at a critical gradient in osmotic pressure across the membrane, and intracellular freezing occurs as a result of this damage. An implication of this hypothesis is that mathematical models can be used to design protocols to avoid damaging gradients in osmotic pressure, allowing new approaches to the preservation of cells, tissues, and organs by rapid cooling. PMID:2306499

  5. Vaccinia virus kelch protein A55 is a 64 kDa intracellular factor that affects virus-induced cytopathic effect and the outcome of infection in a murine intradermal model.

    PubMed

    Beard, Philippa M; Froggatt, Graham C; Smith, Geoffrey L

    2006-06-01

    The vaccinia virus (VACV) protein A55 is a BTB/kelch protein with a broad-complex, tramtrack and bric-a-brac (BTB) domain in the N-terminal region and five kelch repeats in the C-terminal half. The BTB/kelch subgroup of the kelch superfamily of proteins has been associated with a wide variety of functions including regulation of the cytoskeleton. VACV contains three genes predicted to encode BTB/kelch proteins: A55R, F3L and C2L. The A55R gene product has been identified as an intracellular protein of 64 kDa that is expressed late in infection. A VACV strain lacking 93.6% of the A55R open reading frame (vdeltaA55) was constructed and found to have an unaltered growth rate in vivo but a different plaque morphology and cytopathic effect, as well as reduced development of VACV-induced Ca2+-independent cell/extracellular matrix adhesion. In a murine intradermal model of VACV infection, a virus lacking the A55R gene induced larger lesions than wild-type and revertant control viruses.

  6. Sex Trafficking: Policies, Programs, and Services.

    PubMed

    Orme, Julie; Ross-Sheriff, Fariyal

    2015-10-01

    Sex trafficking (ST), a contemporary form of female slavery, is a human rights issue of critical concern to social work. The global response to ST has been substantial, and 166 countries have adopted anti-ST legislation. Despite considerable efforts to combat ST, the magnitude is increasing. To date, the majority of anti-ST efforts have focused on criminalization policies that target traffickers or purchasers of sexual services, who are predominantly male; prevention programming and services for predominantly female victims have received less support. Therapeutic services to assist pornography addicts and purchasers of sexual services are also necessary. In this article, authors examine current anti-ST policies, programs, and services, both domestically and globally, and present an innovative paradigm that addresses social inequities and emphasizes prevention programming. They conclude with a discussion of the paradigm's implications for social work policies, practices, and services. PMID:26489349

  7. Technosocial Predictive Analytics for Illicit Nuclear Trafficking

    SciTech Connect

    Sanfilippo, Antonio P.; Butner, R. Scott; Cowell, Andrew J.; Dalton, Angela C.; Haack, Jereme N.; Kreyling, Sean J.; Riensche, Roderick M.; White, Amanda M.; Whitney, Paul D.

    2011-03-29

    Illicit nuclear trafficking networks are a national security threat. These networks can directly lead to nuclear proliferation, as state or non-state actors attempt to identify and acquire nuclear weapons-related expertise, technologies, components, and materials. The ability to characterize and anticipate the key nodes, transit routes, and exchange mechanisms associated with these networks is essential to influence, disrupt, interdict or destroy the function of the networks and their processes. The complexities inherent to the characterization and anticipation of illicit nuclear trafficking networks requires that a variety of modeling and knowledge technologies be jointly harnessed to construct an effective analytical and decision making workflow in which specific case studies can be built in reasonable time and with realistic effort. In this paper, we explore a solution to this challenge that integrates evidentiary and dynamic modeling with knowledge management and analytical gaming, and demonstrate its application to a geopolitical region at risk.

  8. Maritime drug trafficking: an underrated problem.

    PubMed

    Aune, B R

    1990-01-01

    Seizure data indicate that a substantial proportion of the total quantity of drugs seized is confiscated from maritime modes of conveyance or has been transported by sea. The trafficking of narcotic drugs by sea has virtually become an industry comprised of many individual enterprises of varying size and organization. The maritime medium is one of the main ways by which drugs may enter some countries. In response to the problem, various sophisticated anti-trafficking offensives and strategies have been established or contemplated in certain geographical areas. The shipment of drugs to the primary consuming countries has not been curbed, however, and there is every indication that the overall movement of drugs is still unimpeded.

  9. Regulatory T-cell compartmentalization and trafficking

    PubMed Central

    Wei, Shuang; Kryczek, Ilona; Zou, Weiping

    2006-01-01

    CD4+CD25+FOXP3+ regulatory T cells (CD4+ Treg cells) are thought to differentiate in the thymus and immigrate from the thymus to the periphery. Treg cells can regulate both acquired and innate immunity through multiple modes of suppression. The cross-talk between Treg cells and targeted cells, such as antigen-presenting cells (APCs) and T cells, is crucial for ensuring suppression by Treg cells in the appropriate microenvironment. Emerging evidence suggests that Treg compartmentalization and trafficking may be tissue or/and organ specific and that distinct chemokine receptor and integrin expression may contribute to selective retention and trafficking of Treg cells at sites where regulation is required. In this review, the cellular and molecular signals that control specialized migration and retention of Treg cells are discussed. PMID:16537800

  10. Sex Trafficking: Policies, Programs, and Services.

    PubMed

    Orme, Julie; Ross-Sheriff, Fariyal

    2015-10-01

    Sex trafficking (ST), a contemporary form of female slavery, is a human rights issue of critical concern to social work. The global response to ST has been substantial, and 166 countries have adopted anti-ST legislation. Despite considerable efforts to combat ST, the magnitude is increasing. To date, the majority of anti-ST efforts have focused on criminalization policies that target traffickers or purchasers of sexual services, who are predominantly male; prevention programming and services for predominantly female victims have received less support. Therapeutic services to assist pornography addicts and purchasers of sexual services are also necessary. In this article, authors examine current anti-ST policies, programs, and services, both domestically and globally, and present an innovative paradigm that addresses social inequities and emphasizes prevention programming. They conclude with a discussion of the paradigm's implications for social work policies, practices, and services.

  11. Key Role for Intracellular K+ and Protein Kinases Sat4/Hal4 and Hal5 in the Plasma Membrane Stabilization of Yeast Nutrient Transporters▿

    PubMed Central

    Pérez-Valle, Jorge; Jenkins, Huw; Merchan, Stephanie; Montiel, Vera; Ramos, José; Sharma, Sukesh; Serrano, Ramón; Yenush, Lynne

    2007-01-01

    K+ transport in living cells must be tightly controlled because it affects basic physiological parameters such as turgor, membrane potential, ionic strength, and pH. In yeast, the major high-affinity K+ transporter, Trk1, is inhibited by high intracellular K+ levels and positively regulated by two redundant “halotolerance” protein kinases, Sat4/Hal4 and Hal5. Here we show that these kinases are not required for Trk1 activity; rather, they stabilize the transporter at the plasma membrane under low K+ conditions, preventing its endocytosis and vacuolar degradation. High concentrations (0.2 M) of K+, but not Na+ or sorbitol, transported by undefined low-affinity systems, maintain Trk1 at the plasma membrane in the hal4 hal5 mutant. Other nutrient transporters, such as Can1 (arginine permease), Fur4 (uracil permease), and Hxt1 (low-affinity glucose permease), are also destabilized in the hal4 hal5 mutant under low K+ conditions and, in the case of Can1, are stabilized by high K+ concentrations. Other plasma membrane proteins such as Pma1 (H+-pumping ATPase) and Sur7 (an eisosomal protein) are not regulated by halotolerance kinases or by high K+ levels. This novel regulatory mechanism of nutrient transporters may participate in the quiescence/growth transition and could result from effects of intracellular K+ and halotolerance kinases on membrane trafficking and/or on the transporters themselves. PMID:17548466

  12. The nuclear protein Waharan is required for endosomal-lysosomal trafficking in Drosophila.

    PubMed

    Lone, Mohiddin; Kungl, Theresa; Koper, Andre; Bottenberg, Wolfgang; Kammerer, Richard; Klein, Melanie; Sweeney, Sean T; Auburn, Richard P; O'Kane, Cahir J; Prokop, Andreas

    2010-07-15

    Here we report Drosophila Waharan (Wah), a 170-kD predominantly nuclear protein with two potential human homologues, as a newly identified regulator of endosomal trafficking. Wah is required for neuromuscular-junction development and muscle integrity. In muscles, knockdown of Wah caused novel accumulations of tightly packed electron-dense tubules, which we termed 'sausage bodies'. Our data suggest that sausage bodies coincide with sites at which ubiquitylated proteins and a number of endosomal and lysosomal markers co-accumulate. Furthermore, loss of Wah function generated loss of the acidic LysoTracker compartment. Together with data demonstrating that Wah acts earlier in the trafficking pathway than the Escrt-III component Drosophila Shrb (snf7 in Schizosaccharomyces pombe), our results indicate that Wah is essential for endocytic trafficking at the late endosome. Highly unexpected phenotypes result from Wah knockdown, in that the distribution of ubiquitylated cargos and endolysosomal morphologies are affected despite Wah being a predominant nuclear protein. This finding suggests the existence of a relationship between nuclear functions and endolysosomal trafficking. Future studies of Wah function will give us insights into this interesting phenomenon.

  13. Following intracellular cholesterol transport by linear and non-linear optical microscopy of intrinsically fluorescent sterols.

    PubMed

    Wüstner, Daniel

    2012-02-01

    Elucidation of intracellular cholesterol transport is important for understanding the molecular basis of several metabolic and neuronal diseases, like atheroclerosis or lysosomal storage disorders. Progress in this field depends crucially on the development of new technical approaches to follow the cellular movement of this essential lipid molecule. In this article, a survey of the various methods being used for analysis of sterol trafficking is given. Various classical biochemical methods are presented and their suitability for analysis of sterol trafficking is assessed. Special emphasis is on recent developments in imaging technology to follow the intracellular fate of intrinsically fluorescent sterols as faithful cholesterol markers. In particular, UV-sensitive wide field and multiphoton microscopy of the sterol dehydroergosterol, DHE, is explained and new methods of quantitative image analysis like pixel-wise bleach rate fitting and multiphoton image correlation spectroscopy are introduced. Several applications of the new technology including observation of vectorial sterol trafficking in polarized human hepatoma cells for investigation of reverse cholesterol transport are presented. PMID:21470123

  14. Role of adaptor proteins and clathrin in the trafficking of human kidney anion exchanger 1 (kAE1) to the cell surface.

    PubMed

    Junking, Mutita; Sawasdee, Nunghathai; Duangtum, Natapol; Cheunsuchon, Boonyarit; Limjindaporn, Thawornchai; Yenchitsomanus, Pa-thai

    2014-07-01

    Kidney anion exchanger 1 (kAE1) plays an important role in acid-base homeostasis by mediating chloride/bicarbornate (Cl-/HCO3-) exchange at the basolateral membrane of α-intercalated cells in the distal nephron. Impaired intracellular trafficking of kAE1 caused by mutations of SLC4A1 encoding kAE1 results in kidney disease - distal renal tubular acidosis (dRTA). However, it is not known how the intracellular sorting and trafficking of kAE1 from trans-Golgi network (TGN) to the basolateral membrane occurs. Here, we studied the role of basolateral-related sorting proteins, including the mu1 subunit of adaptor protein (AP) complexes, clathrin and protein kinase D, on kAE1 trafficking in polarized and non-polarized kidney cells. By using RNA interference, co-immunoprecipitation, yellow fluorescent protein-based protein fragment complementation assays and immunofluorescence staining, we demonstrated that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin (but not AP-1 mu1B, PKD1 or PKD2) play crucial roles in intracellular sorting and trafficking of kAE1. We also demonstrated colocalization of kAE1 and basolateral-related sorting proteins in human kidney tissues by double immunofluorescence staining. These findings indicate that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin are required for kAE1 sorting and trafficking from TGN to the basolateral membrane of acid-secreting α-intercalated cells. PMID:24698155

  15. Role of adaptor proteins and clathrin in the trafficking of human kidney anion exchanger 1 (kAE1) to the cell surface.

    PubMed

    Junking, Mutita; Sawasdee, Nunghathai; Duangtum, Natapol; Cheunsuchon, Boonyarit; Limjindaporn, Thawornchai; Yenchitsomanus, Pa-thai

    2014-07-01

    Kidney anion exchanger 1 (kAE1) plays an important role in acid-base homeostasis by mediating chloride/bicarbornate (Cl-/HCO3-) exchange at the basolateral membrane of α-intercalated cells in the distal nephron. Impaired intracellular trafficking of kAE1 caused by mutations of SLC4A1 encoding kAE1 results in kidney disease - distal renal tubular acidosis (dRTA). However, it is not known how the intracellular sorting and trafficking of kAE1 from trans-Golgi network (TGN) to the basolateral membrane occurs. Here, we studied the role of basolateral-related sorting proteins, including the mu1 subunit of adaptor protein (AP) complexes, clathrin and protein kinase D, on kAE1 trafficking in polarized and non-polarized kidney cells. By using RNA interference, co-immunoprecipitation, yellow fluorescent protein-based protein fragment complementation assays and immunofluorescence staining, we demonstrated that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin (but not AP-1 mu1B, PKD1 or PKD2) play crucial roles in intracellular sorting and trafficking of kAE1. We also demonstrated colocalization of kAE1 and basolateral-related sorting proteins in human kidney tissues by double immunofluorescence staining. These findings indicate that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin are required for kAE1 sorting and trafficking from TGN to the basolateral membrane of acid-secreting α-intercalated cells.

  16. Forensic medical examination of victims of trafficking in human beings.

    PubMed

    Alempijevic, Djordie; Jecmenica, Dragan; Pavlekic, Snezana; Savic, Slobodan; Aleksandric, Branimir

    2007-01-01

    Trafficking in human beigns (THB) is recognized as a global public health issue as well as a violation of human rights. Trafficking has been identified to be associated with several health risks including psychological trauma, injuries from violence, and substance misuse. Public and media reports suggest that the morbidity and mortality associated with trafficking are substantial. The need of medico-legal healthcare for THB victims is being neglected. Forensic medical examination, as specific intervention, is a highly desirable element of ermegency health care provided for victims of tracking. Acting in such a way, the investigation should establish the facts related to the allegatation of trafficking, thereby assisting in identifying those responsible, but also contributing to the procedures designed to obtain redress for the victims. Local anti-trafficking policies and interventions, however, have not acknowledged these needs. Therefore, the agenda of anti-trafficking policies needs to be redrawn to include forensic medical assessment of victims for legal purposes.

  17. Multiple ecto-nucleoside triphosphate diphosphohydrolases facilitate intracellular replication of Legionella pneumophila.

    PubMed

    Riedmaier, Patrice; Sansom, Fiona M; Sofian, Trifina; Beddoe, Travis; Schuelein, Ralf; Newton, Hayley J; Hartland, Elizabeth L

    2014-09-01

    Legionella pneumophila is an opportunistic pathogen that replicates within alveolar macrophages resulting in the onset of severe atypical pneumonia. Previously we have identified Lpg1905, a eukaryotic-type ecto-NTPDase (nucleoside triphosphate diphosphohydrolase) from L. pneumophila that was required for optimal intracellular replication and virulence in a mouse lung infection model. In the present study, we characterized the activity of a second eukaryotic-type NTPDase, Lpg0971, from L. pneumophila. We observed that recombinant Lpg0971 hydrolysed only ATP and exhibited divalent cation preference for manganese (II) ions. Similar to lpg1905, an lpg0971 mutant carrying the plasmid pMIP was attenuated in a mouse lung infection model and impaired for replication in human macrophages and amoebae. Increased trafficking of the LCV (Legionella-containing vacuole) to a LAMP-1 (lysosome-associated membrane protein-1)-positive compartment was observed for both the lpg1905 and lpg0971 mutants carrying pMIP. Complementation with either lpg1905 or lpg0971 restored intracellular replication, suggesting that a minimum level of ATPase activity was required for this function. A double lpg1905/0971 mutant was not more impaired for intracellular replication than the single mutants and complementation of the double mutant with lpg0971, but not lpg1905, restored intracellular replication. This suggested that although the NTPDases have overlapping activities they have distinct functions. Unlike many eukaryotic-type proteins from L. pneumophila, neither Lpg1905 nor Lpg0971 were translocated into the host cell by the Dot/Icm (defective in organelle trafficking/intracellular multiplication) type IV secretion system. Overall our data suggest that the ability of L. pneumophila to replicate in eukaryotic cells relies in part on the ability of the pathogen to hydrolyse ATP within an intracellular compartment.

  18. Epithelial Cell Gene Expression Induced by Intracellular Staphylococcus aureus

    PubMed Central

    Li, Xianglu; Fusco, William G.; Seo, Keun S.; Bayles, Kenneth W.; Mosley, Erin E.; McGuire, Mark A.; Bohach, Gregory A.

    2009-01-01

    HEp-2 cell monolayers were cocultured with intracellular Staphylococcus aureus, and changes in gene expression were profiled using DNA microarrays. Intracellular S. aureus affected genes involved in cellular stress responses, signal transduction, inflammation, apoptosis, fibrosis, and cholesterol biosynthesis. Transcription of stress response and signal transduction-related genes including atf3, sgk, map2k1, map2k3, arhb, and arhe was increased. In addition, elevated transcription of proinflammatory genes was observed for tnfa, il1b, il6, il8, cxcl1, ccl20, cox2, and pai1. Genes involved in proapoptosis and fibrosis were also affected at transcriptional level by intracellular S. aureus. Notably, intracellular S. aureus induced strong transcriptional down-regulation of several cholesterol biosynthesis genes. These results suggest that epithelial cells respond to intracellular S. aureus by inducing genes affecting immunity and in repairing damage caused by the organism, and are consistent with the possibility that the organism exploits an intracellular environment to subvert host immunity and promote colonization. PMID:20016671

  19. Topography of the Human Papillomavirus Minor Capsid Protein L2 during Vesicular Trafficking of Infectious Entry

    PubMed Central

    DiGiuseppe, Stephen; Keiffer, Timothy R.; Bienkowska-Haba, Malgorzata; Luszczek, Wioleta; Guion, Lucile G. M.; Müller, Martin

    2015-01-01

    ABSTRACT The human papillomavirus (HPV) capsid is composed of the major capsid protein L1 and the minor capsid protein L2. During entry, the HPV capsid undergoes numerous conformational changes that result in endosomal uptake and subsequent trafficking of the L2 protein in complex with the viral DNA to the trans-Golgi network. To facilitate this transport, the L2 protein harbors a number of putative motifs that, if capable of direct interaction, would interact with cytosolic host cell factors. These data imply that a portion of L2 becomes cytosolic during infection. Using a low concentration of digitonin to selectively permeabilize the plasma membrane of infected cells, we mapped the topography of the L2 protein during infection. We observed that epitopes within amino acid residues 64 to 81 and 163 to 170 and a C-terminal tag of HPV16 L2 are exposed on the cytosolic side of intracellular membranes, whereas an epitope within residues 20 to 38, which are upstream of a putative transmembrane region, is luminal. Corroborating these findings, we also found that L2 protein is sensitive to trypsin digestion during infection. These data demonstrate that the majority of the L2 protein becomes accessible on the cytosolic side of intracellular membranes in order to interact with cytosolic factors to facilitate vesicular trafficking. IMPORTANCE In order to complete infectious entry, nonenveloped viruses have to pass cellular membranes. This is often achieved through the viral capsid protein associating with or integrating into intracellular membrane. Here, we determine the topography of HPV L2 protein in the endocytic vesicular compartment, suggesting that L2 becomes a transmembrane protein with a short luminal portion and with the majority facing the cytosolic side for interaction with host cell transport factors. PMID:26246568

  20. NAD+-Glycohydrolase Promotes Intracellular Survival of Group A Streptococcus

    PubMed Central

    Sharma, Onkar; O’Seaghdha, Maghnus; Velarde, Jorge J.; Wessels, Michael R.

    2016-01-01

    A global increase in invasive infections due to group A Streptococcus (S. pyogenes or GAS) has been observed since the 1980s, associated with emergence of a clonal group of strains of the M1T1 serotype. Among other virulence attributes, the M1T1 clone secretes NAD+-glycohydrolase (NADase). When GAS binds to epithelial cells in vitro, NADase is translocated into the cytosol in a process mediated by streptolysin O (SLO), and expression of these two toxins is associated with enhanced GAS intracellular survival. Because SLO is required for NADase translocation, it has been difficult to distinguish pathogenic effects of NADase from those of SLO. To resolve the effects of the two proteins, we made use of anthrax toxin as an alternative means to deliver NADase to host cells, independently of SLO. We developed a novel method for purification of enzymatically active NADase fused to an amino-terminal fragment of anthrax toxin lethal factor (LFn-NADase) that exploits the avid, reversible binding of NADase to its endogenous inhibitor. LFn-NADase was translocated across a synthetic lipid bilayer in vitro in the presence of anthrax toxin protective antigen in a pH-dependent manner. Exposure of human oropharyngeal keratinocytes to LFn-NADase in the presence of protective antigen resulted in cytosolic delivery of NADase activity, inhibition of protein synthesis, and cell death, whereas a similar construct of an enzymatically inactive point mutant had no effect. Anthrax toxin-mediated delivery of NADase in an amount comparable to that observed during in vitro infection with live GAS rescued the defective intracellular survival of NADase-deficient GAS and increased the survival of SLO-deficient GAS. Confocal microscopy demonstrated that delivery of LFn-NADase prevented intracellular trafficking of NADase-deficient GAS to lysosomes. We conclude that NADase mediates cytotoxicity and promotes intracellular survival of GAS in host cells. PMID:26938870

  1. AP-3 mediated trafficking of TLR2 ligands controls specificity of inflammatory responses but not adaptor complex assembly

    PubMed Central

    Petnicki-Ocwieja, Tanja; Kern, Aurelie; Killpack, Tess L.; Bunnell, Stephen C.; Hu, Linden T.

    2015-01-01

    Innate immune engagement results in the activation of host defenses that produce microbe-specific inflammatory responses. A long-standing interest in the field of innate immunity is to understand how varied host responses are generated through the signaling of just a limited number of receptors. Recently, intracellular trafficking and compartmental partitioning have been identified as mechanism that provide signaling specificity for receptors by regulating signaling platform assembly. We show that cytokine activation as a result of Toll-like receptor 2 (TLR2) stimulation occurs at different intracellular locations and is mediated by the phagosomal trafficking molecule AP-3. AP-3 is required for trafficking TLR2 purified ligands or the Lyme disease causing bacterium, Borrelia burgdorferi, to LAMP-1 lysosomal compartments. The presence of AP-3 is necessary for the activation of cytokines such as IL-6 but not TNF-α or type I interferons, suggesting induction of these cytokines occurs from a different compartment. Lack of AP-3 does not interfere with the recruitment of TLR signaling adaptors TRAM and MyD88 to the phagosome, indicating that the TLR-MyD88 signaling complex is assembled at a pre-lysosomal stage and that IL-6 activation depends on proper localization of signaling molecules downstream of MyD88. Finally, infection of AP-3 deficient mice with B. burgdorferi resulted in altered joint inflammation during murine Lyme arthritis. Our studies further elucidate the effects of phagosomal trafficking on tailoring immune responses in vitro and in vivo. PMID:26423153

  2. INTRACELLULAR SIGNALING AND DEVELOPMENTAL NEUROTOXICITY.

    EPA Science Inventory

    A book chapter in ?Molecular Toxicology: Transcriptional Targets? reviewed the role of intracellular signaling in the developmental neurotoxicity of environmental chemicals. This chapter covered a number of aspects including the development of the nervous system, role of intrace...

  3. Domestic minor sex trafficking in the United States.

    PubMed

    Kotrla, Kimberly

    2010-04-01

    By now, most social workers are familiar with the issue of human trafficking. However, many are likely unfamiliar with research indicating that youths constitute the most vulnerable group in the United States for becoming victims of sex trafficking and that most women in prostitution actually entered as minors. Some experts are now referring to the sex trafficking of U.S. children and youths as "domestic minor sex trafficking," or DMST. This article seeks to acquaint readers with what is currently known regarding the extent of DMST, who is at risk for becoming a victim, and implications for the social work profession in addressing this tragedy.

  4. Domestic minor sex trafficking in the United States.

    PubMed

    Kotrla, Kimberly

    2010-04-01

    By now, most social workers are familiar with the issue of human trafficking. However, many are likely unfamiliar with research indicating that youths constitute the most vulnerable group in the United States for becoming victims of sex trafficking and that most women in prostitution actually entered as minors. Some experts are now referring to the sex trafficking of U.S. children and youths as "domestic minor sex trafficking," or DMST. This article seeks to acquaint readers with what is currently known regarding the extent of DMST, who is at risk for becoming a victim, and implications for the social work profession in addressing this tragedy. PMID:20408359

  5. Expression of Nucleolin Affects Microtubule Dynamics.

    PubMed

    Gaume, Xavier; Place, Christophe; Delage, Helene; Mongelard, Fabien; Monier, Karine; Bouvet, Philippe

    2016-01-01

    Nucleolin is present in diverse cellular compartments and is involved in a variety of cellular processes from nucleolar structure and function to intracellular trafficking, cell adhesion and migration. Recently, nucleolin has been localized at the mature centriole where it is involved in microtubule nucleation and anchoring. Although this new function of nucleolin linked to microtubule regulation has been identified, the global effects of nucleolin on microtubule dynamics have not been addressed yet. In the present study, we analyzed the roles of nucleolin protein levels on global microtubule dynamics by tracking the EB3 microtubule plus end binding protein in live cells. We have found that during microtubule growth phases, nucleolin affects both the speed and life time of polymerization and by analyzing catastrophe events, we showed that nucleolin reduces catastrophe frequency. This new property of nucleolin was then confirmed in a cold induced microtubule depolymerization experiment in which we have found that cold resistant microtubules were totally destabilized in nucleolin depleted cells. Altogether, our data demonstrate a new function of nucleolin on microtubule stabilization, thus bringing novel insights into understanding the multifunctional properties of nucleolin in healthy and cancer cells. PMID:27309529

  6. Expression of Nucleolin Affects Microtubule Dynamics

    PubMed Central

    Gaume, Xavier; Place, Christophe; Delage, Helene; Mongelard, Fabien; Monier, Karine; Bouvet, Philippe

    2016-01-01

    Nucleolin is present in diverse cellular compartments and is involved in a variety of cellular processes from nucleolar structure and function to intracellular trafficking, cell adhesion and migration. Recently, nucleolin has been localized at the mature centriole where it is involved in microtubule nucleation and anchoring. Although this new function of nucleolin linked to microtubule regulation has been identified, the global effects of nucleolin on microtubule dynamics have not been addressed yet. In the present study, we analyzed the roles of nucleolin protein levels on global microtubule dynamics by tracking the EB3 microtubule plus end binding protein in live cells. We have found that during microtubule growth phases, nucleolin affects both the speed and life time of polymerization and by analyzing catastrophe events, we showed that nucleolin reduces catastrophe frequency. This new property of nucleolin was then confirmed in a cold induced microtubule depolymerization experiment in which we have found that cold resistant microtubules were totally destabilized in nucleolin depleted cells. Altogether, our data demonstrate a new function of nucleolin on microtubule stabilization, thus bringing novel insights into understanding the multifunctional properties of nucleolin in healthy and cancer cells. PMID:27309529

  7. Mapping the H(+) (V)-ATPase interactome: identification of proteins involved in trafficking, folding, assembly and phosphorylation.

    PubMed

    Merkulova, Maria; Păunescu, Teodor G; Azroyan, Anie; Marshansky, Vladimir; Breton, Sylvie; Brown, Dennis

    2015-01-01

    V-ATPases (H(+) ATPases) are multisubunit, ATP-dependent proton pumps that regulate pH homeostasis in virtually all eukaryotes. They are involved in key cell biological processes including vesicle trafficking, endosomal pH sensing, membrane fusion and intracellular signaling. They also have critical systemic roles in renal acid excretion and blood pH balance, male fertility, bone remodeling, synaptic transmission, olfaction and hearing. Furthermore, V-ATPase dysfunction either results in or aggravates various other diseases, but little is known about the complex protein interactions that regulate these varied V-ATPase functions. Therefore, we performed a proteomic analysis to identify V-ATPase associated proteins and construct a V-ATPase interactome. Our analysis using kidney tissue revealed V-ATPase-associated protein clusters involved in protein quality control, complex assembly and intracellular trafficking. ARHGEF7, DMXL1, EZR, NCOA7, OXR1, RPS6KA3, SNX27 and 9 subunits of the chaperonin containing TCP1 complex (CCT) were found to interact with V-ATPase for the first time in this study. Knockdown of two interacting proteins, DMXL1 and WDR7, inhibited V-ATPase-mediated intracellular vesicle acidification in a kidney cell line, providing validation for the utility of our interactome as a screen for functionally important novel V-ATPase-regulating proteins. Our data, therefore, provide new insights and directions for the analysis of V-ATPase cell biology and (patho)physiology. PMID:26442671

  8. Mapping the H+ (V)-ATPase interactome: identification of proteins involved in trafficking, folding, assembly and phosphorylation

    PubMed Central

    Merkulova, Maria; Păunescu, Teodor G.; Azroyan, Anie; Marshansky, Vladimir; Breton, Sylvie; Brown, Dennis

    2015-01-01

    V-ATPases (H+ ATPases) are multisubunit, ATP-dependent proton pumps that regulate pH homeostasis in virtually all eukaryotes. They are involved in key cell biological processes including vesicle trafficking, endosomal pH sensing, membrane fusion and intracellular signaling. They also have critical systemic roles in renal acid excretion and blood pH balance, male fertility, bone remodeling, synaptic transmission, olfaction and hearing. Furthermore, V-ATPase dysfunction either results in or aggravates various other diseases, but little is known about the complex protein interactions that regulate these varied V-ATPase functions. Therefore, we performed a proteomic analysis to identify V-ATPase associated proteins and construct a V-ATPase interactome. Our analysis using kidney tissue revealed V-ATPase-associated protein clusters involved in protein quality control, complex assembly and intracellular trafficking. ARHGEF7, DMXL1, EZR, NCOA7, OXR1, RPS6KA3, SNX27 and 9 subunits of the chaperonin containing TCP1 complex (CCT) were found to interact with V-ATPase for the first time in this study. Knockdown of two interacting proteins, DMXL1 and WDR7, inhibited V-ATPase-mediated intracellular vesicle acidification in a kidney cell line, providing validation for the utility of our interactome as a screen for functionally important novel V-ATPase-regulating proteins. Our data, therefore, provide new insights and directions for the analysis of V-ATPase cell biology and (patho)physiology. PMID:26442671

  9. Studies on the Roles of Clathrin-Mediated Membrane Trafficking and Zinc Transporter Cis4 in the Transport of GPI-Anchored Proteins in Fission Yeast

    PubMed Central

    Ma, Yan; Sugiura, Reiko; Kuno, Takayoshi

    2012-01-01

    We previously identified Cis4, a zinc transporter belonging to the cation diffusion facilitator protein family, and we demonstrated that Cis4 is implicated in Golgi membrane trafficking in fission yeast. Here, we identified three glycosylphosphatidylinositol (GPI)-anchored proteins, namely Ecm33, Aah3, and Gaz2, as multicopy suppressors of the MgCl2-sensitive phenotype of cis4-1 mutant. The phenotypes of ecm33, aah3 and gaz2 deletion cells were distinct from each other, and Cis4 overexpression suppressed Δecm33 phenotypes but did not suppress Δaah3 defects. Notably, green fluorescent protein-tagged Ecm33, which was observed at the cell surface in wild-type cells, mostly localized as intracellular dots that are presumed to be the Golgi and endosomes in membrane-trafficking mutants, including Δapm1, ypt3-i5, and chc1-1 mutants. Interestingly, all these membrane-trafficking mutants showed hypersensitivity to BE49385A, an inhibitor of Its8 that is involved in GPI-anchored protein synthesis. Taken together, these results suggest that GPI-anchored proteins are transported through a clathrin-mediated post-Golgi membrane trafficking pathway and that zinc transporter Cis4 may play roles in membrane trafficking of GPI-anchored proteins in fission yeast. PMID:22848669

  10. Mutational analysis of the intramembranous H10 loop of yeast Nhx1 reveals a critical role in ion homoeostasis and vesicle trafficking.

    PubMed

    Mukherjee, Sanchita; Kallay, Laura; Brett, Christopher L; Rao, Rajini

    2006-08-15

    Yeast Nhx1 [Na+(K+)/H+ exchanger 1] is an intracellular Na+(K+)/H+ exchanger, localizing to the late endosome where it is important for ion homoeostasis and vesicle trafficking. Phylogenetic analysis of NHE (Na+/H+ exchanger) sequences has identified orthologous proteins, including HsNHE6 (human NHE6), HsNHE7 and HsNHE9 of unknown physiological role. These appear distinct from well-studied mammalian plasma membrane isoforms (NHE1-NHE5). To explore the differences between plasma membrane and intracellular NHEs and understand the link between ion homoeostasis and vesicle trafficking, we examined the consequence of replacing residues in the intramembranous H10 loop of Nhx1 between transmembrane segments 9 and 10. The critical role for the carboxy group of Glu355 in ion transport is consistent with the invariance of this residue in all NHEs. Surprisingly, residues specifically conserved in the intracellular isoforms (such as Phe357 and Tyr361) could not be replaced with closely similar residues (leucine and phenylalanine) found in the plasma membrane isoforms without loss of function, revealing unexpected side chain specificity. The trafficking phenotypes of all Nhx1 mutants, including hygromycin-sensitivity and missorting of carboxypeptidase Y, were found to directly correlate with pH homoeostasis defects and could be proportionately corrected by titration with weak base. The present study demonstrates the importance of the H10 loop of the NHE family, highlights the differences between plasma membrane and intracellular isoforms and shows that trafficking defects are tightly coupled with pH homoeostasis.

  11. 3 CFR - Continuation of the National Emergency With Respect to Significant Narcotics Traffickers Centered...

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... to Significant Narcotics Traffickers Centered in Colombia Presidential Documents Other Presidential... Narcotics Traffickers Centered in Colombia On October 21, 1995, by Executive Order 12978, the President... economy of the United States constituted by the actions of significant narcotics traffickers centered...

  12. 3 CFR - Continuation of the National Emergency With Respect to Significant Narcotics Traffickers Centered...

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... to Significant Narcotics Traffickers Centered in Colombia Presidential Documents Other Presidential... Narcotics Traffickers Centered in Colombia On October 21, 1995, by Executive Order 12978, the President... economy of the United States constituted by the actions of significant narcotics traffickers centered...

  13. 3 CFR - Continuation of the National Emergency With Respect to Significant Narcotics Traffickers Centered...

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... to Significant Narcotics Traffickers Centered in Colombia Presidential Documents Other Presidential... Narcotics Traffickers Centered in Colombia On October 21, 1995, by Executive Order 12978, the President declared a national emergency with respect to significant narcotics traffickers centered in...

  14. Extracellular Matrix Stiffness and Architecture Govern Intracellular Rheology in Cancer

    PubMed Central

    Baker, Erin L.; Bonnecaze, Roger T.; Zaman, Muhammad H.

    2009-01-01

    Abstract Little is known about the complex interplay between the extracellular mechanical environment and the mechanical properties that characterize the dynamic intracellular environment. To elucidate this relationship in cancer, we probe the intracellular environment using particle-tracking microrheology. In three-dimensional (3D) matrices, intracellular effective creep compliance of prostate cancer cells is shown to increase with increasing extracellular matrix (ECM) stiffness, whereas modulating ECM stiffness does not significantly affect the intracellular mechanical state when cells are attached to two-dimensional (2D) matrices. Switching from 2D to 3D matrices induces an order-of-magnitude shift in intracellular effective creep compliance and apparent elastic modulus. However, for a given matrix stiffness, partial blocking of β1 integrins mitigates the shift in intracellular mechanical state that is invoked by switching from a 2D to 3D matrix architecture. This finding suggests that the increased cell-matrix engagement inherent to a 3D matrix architecture may contribute to differences observed in viscoelastic properties between cells attached to 2D matrices and cells embedded within 3D matrices. In total, our observations show that ECM stiffness and architecture can strongly influence the intracellular mechanical state of cancer cells. PMID:19686648

  15. Assisting victims of human trafficking: strategies to facilitate identification, exit from trafficking, and the restoration of wellness.

    PubMed

    Hodge, David R

    2014-04-01

    Human trafficking is a pressing social justice concern. Social work is uniquely situated to address this problem. However, despite the profession's commitment to social justice, the scholarship to equip social workers to address this issue has been largely absent from professional discourse. To address this gap, this article helps social work practitioners to assist victims of human trafficking. After orienting readers to the scope and process of human trafficking, the topics of victim identification, exit from trafficking, and the restoration of psychological wellness are discussed. By equipping themselves in these three areas, practitioners can advance social justice on behalf of some of the most exploited people in the world. PMID:24855860

  16. Assisting victims of human trafficking: strategies to facilitate identification, exit from trafficking, and the restoration of wellness.

    PubMed

    Hodge, David R

    2014-04-01

    Human trafficking is a pressing social justice concern. Social work is uniquely situated to address this problem. However, despite the profession's commitment to social justice, the scholarship to equip social workers to address this issue has been largely absent from professional discourse. To address this gap, this article helps social work practitioners to assist victims of human trafficking. After orienting readers to the scope and process of human trafficking, the topics of victim identification, exit from trafficking, and the restoration of psychological wellness are discussed. By equipping themselves in these three areas, practitioners can advance social justice on behalf of some of the most exploited people in the world.

  17. Brain Region-Specific Effects of cGMP-Dependent Kinase II Knockout on AMPA Receptor Trafficking and Animal Behavior

    ERIC Educational Resources Information Center

    Kim, Seonil; Pick, Joseph E.; Abera, Sinedu; Khatri, Latika; Ferreira, Danielle D. P.; Sathler, Matheus F.; Morison, Sage L.; Hofmann, Franz; Ziff, Edward B.

    2016-01-01

    Phosphorylation of GluA1, a subunit of AMPA receptors (AMPARs), is critical for AMPAR synaptic trafficking and control of synaptic transmission. cGMP-dependent protein kinase II (cGKII) mediates this phosphorylation, and cGKII knockout (KO) affects GluA1 phosphorylation and alters animal behavior. Notably, GluA1 phosphorylation in the KO…

  18. Nitrogen Monoxide (NO) Storage and Transport by Dinitrosyl-Dithiol-Iron Complexes: Long-lived NO That Is Trafficked by Interacting Proteins*

    PubMed Central

    Suryo Rahmanto, Yohan; Kalinowski, Danuta S.; Lane, Darius J. R.; Lok, Hiu Chuen; Richardson, Vera; Richardson, Des R.

    2012-01-01

    Nitrogen monoxide (NO) markedly affects intracellular iron metabolism, and recent studies have shown that molecules traditionally involved in drug resistance, namely GST and MRP1 (multidrug resistance-associated protein 1), are critical molecular players in this process. This is mediated by interaction of these proteins with dinitrosyl-dithiol-iron complexes (Watts, R. N., Hawkins, C., Ponka, P., and Richardson, D. R. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 7670–7675; Lok, H. C., Suryo Rahmanto, Y., Hawkins, C. L., Kalinowski, D. S., Morrow, C. S., Townsend, A. J., Ponka, P., and Richardson, D. R. (2012) J. Biol. Chem. 287, 607–618). These complexes are bioavailable, have a markedly longer half-life compared with free NO, and form in cells after an interaction between iron, NO, and glutathione. The generation of dinitrosyl-dithiol-iron complexes acts as a common currency for NO transport and storage by MRP1 and GST P1-1, respectively. Understanding the biological trafficking mechanisms involved in the metabolism of NO is vital for elucidating its many roles in cellular signaling and cytotoxicity and for development of new therapeutic targets. PMID:22262835

  19. Ganglioside Regulation of AMPA Receptor Trafficking

    PubMed Central

    Prendergast, Jillian; Umanah, George K.E.; Yoo, Seung-Wan; Lagerlöf, Olof; Motari, Mary G.; Cole, Robert N.; Huganir, Richard L.; Dawson, Ted M.; Dawson, Valina L.

    2014-01-01

    Gangliosides are major cell-surface determinants on all vertebrate neurons. Human congenital disorders of ganglioside biosynthesis invariably result in intellectual disability and are often associated with intractable seizures. To probe the mechanisms of ganglioside functions, affinity-captured ganglioside-binding proteins from rat cerebellar granule neurons were identified by quantitative proteomic mass spectrometry. Of the six proteins that bound selectively to the major brain ganglioside GT1b (GT1b:GM1 > 4; p < 10−4), three regulate neurotransmitter receptor trafficking: Thorase (ATPase family AAA domain-containing protein 1), soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (γ-SNAP), and the transmembrane protein Nicalin. Thorase facilitates endocytosis of GluR2 subunit-containing AMPA-type glutamate receptors (AMPARs) in an ATPase-dependent manner; its deletion in mice results in learning and memory deficits (J. Zhang et al., 2011b). GluR2-containing AMPARs did not bind GT1b, but bound specifically to another ganglioside, GM1. Addition of noncleavable ATP (ATPγS) significantly disrupted ganglioside binding, whereas it enhanced AMPAR association with Thorase, NSF, and Nicalin. Mutant mice lacking GT1b expressed markedly higher brain Thorase, whereas Thorase-null mice expressed higher GT1b. Treatment of cultured hippocampal neurons with sialidase, which cleaves GT1b (and other sialoglycans), resulted in a significant reduction in the size of surface GluR2 puncta. These data support a model in which GM1-bound GluR2-containing AMPARs are functionally segregated from GT1b-bound AMPAR-trafficking complexes. Release of ganglioside binding may enhance GluR2-containing AMPAR association with its trafficking complexes, increasing endocytosis. Disrupting ganglioside biosynthesis may result in reduced synaptic expression of GluR2-contianing AMPARs resulting in intellectual deficits and seizure susceptibility in mice and humans. PMID:25253868

  20. Toward Intracellular Targeted Delivery of Cancer Therapeutics

    PubMed Central

    Pandya, Hetal; Debinski, Waldemar

    2013-01-01

    A number of anti-cancer drugs have their targets localized to particular intracellular compartments. These drugs reach the targets mainly through diffusion, dependent on biophysical and biochemical forces that allow cell penetration. This means that both cancer cells and normal cells will be subjected to such diffusion; hence many of these drugs, like chemotherapeutics, are potentially toxic and the concentration achieved at the site of their action is often suboptimal. The same relates to radiation that indiscriminately affects normal and diseased cells. However, nature-designed systems enable compounds present in the extracellular environment to end up inside the cell and even travel to more specific intracellular compartments. For example, viruses and bacterial toxins can more or less specifically recognize eukaryotic cells, enter these cells, and direct some protein portions to designated intracellular areas. These phenomena have led to creative thinking, such as employing viruses or bacterial toxins for cargo delivery to cells and, more specifically, to cancer cells. Proteins can be genetically engineered in order to not only mimic what viruses and bacterial toxins can do, but also to add new functions, extending or changing the intracellular routes. It is possible to make conjugates or, more preferably, single-chain proteins that recognize cancer cells and deliver cargo inside the cells, even to the desired subcellular compartment. These findings offer new opportunities to deliver drugs/labels only to cancer cells and only to their site of action within the cells. The development of such dual-specificity vectors for targeting cancer cells is an attractive and potentially safer and more efficacious way of delivering drugs. We provide examples of this approach for delivering brain cancer therapeutics, using a specific biomarker on glioblastoma tumor cells. PMID:22671766

  1. Plant vacuole morphology and vacuolar trafficking

    PubMed Central

    Zhang, Chunhua; Hicks, Glenn R.; Raikhel, Natasha V.

    2014-01-01

    Plant vacuoles are essential organelles for plant growth and development, and have multiple functions. Vacuoles are highly dynamic and pleiomorphic, and their size varies depending on the cell type and growth conditions. Vacuoles compartmentalize different cellular components such as proteins, sugars, ions and other secondary metabolites and play critical roles in plants response to different biotic/abiotic signaling pathways. In this review, we will summarize the patterns of changes in vacuole morphology in certain cell types, our understanding of the mechanisms of plant vacuole biogenesis, and the role of SNAREs and Rab GTPases in vacuolar trafficking. PMID:25309565

  2. Sequence and structure-specific elements of HERG mRNA determine channel synthesis and trafficking efficiency

    PubMed Central

    Sroubek, Jakub; Krishnan, Yamini; McDonald, Thomas V.

    2013-01-01

    Human ether-á-gogo-related gene (HERG) encodes a potassium channel that is highly susceptible to deleterious mutations resulting in susceptibility to fatal cardiac arrhythmias. Most mutations adversely affect HERG channel assembly and trafficking. Why the channel is so vulnerable to missense mutations is not well understood. Since nothing is known of how mRNA structural elements factor in channel processing, we synthesized a codon-modified HERG cDNA (HERG-CM) where the codons were synonymously changed to reduce GC content, secondary structure, and rare codon usage. HERG-CM produced typical IKr-like currents; however, channel synthesis and processing were markedly different. Translation efficiency was reduced for HERG-CM, as determined by heterologous expression, in vitro translation, and polysomal profiling. Trafficking efficiency to the cell surface was greatly enhanced, as assayed by immunofluorescence, subcellular fractionation, and surface labeling. Chimeras of HERG-NT/CM indicated that trafficking efficiency was largely dependent on 5′ sequences, while translation efficiency involved multiple areas. These results suggest that HERG translation and trafficking rates are independently governed by noncoding information in various regions of the mRNA molecule. Noncoding information embedded within the mRNA may play a role in the pathogenesis of hereditary arrhythmia syndromes and could provide an avenue for targeted therapeutics.—Sroubek, J., Krishnan, Y., McDonald, T V. Sequence- and structure-specific elements of HERG mRNA determine channel synthesis and trafficking efficiency. PMID:23608144

  3. TRPM6 kinase activity regulates TRPM7 trafficking and inhibits cellular growth under hypomagnesic conditions.

    PubMed

    Brandao, Katherine; Deason-Towne, Francina; Zhao, Xiaoyun; Perraud, Anne-Laure; Schmitz, Carsten

    2014-12-01

    The channel kinases TRPM6 and TRPM7 are both members of the melastatin-related transient receptor potential (TRPM) subfamily of ion channels and the only known fusions of an ion channel pore with a kinase domain. TRPM6 and TRPM7 form functional, tetrameric channel complexes at the plasma membrane by heteromerization. TRPM6 was previously shown to cross-phosphorylate TRPM7 on threonine residues, but not vice versa. Genetic studies demonstrated that TRPM6 and TRPM7 fulfill non-redundant functions and that each channel contributes uniquely to the regulation of Mg(2+) homeostasis. Although there are indications that TRPM6 and TRPM7 can influence each other's cellular distribution and activity, little is known about the functional relationship between these two channel-kinases. In the present study, we examined how TRPM6 kinase activity influences TRPM7 serine phosphorylation, intracellular trafficking, and cell surface expression of TRPM7, as well as Mg(2+)-dependent cellular growth. We found TRPM7 serine phosphorylation via the TRPM6 kinase, but no TRPM6 serine phosphorylation via the TRPM7 kinase. Intracellular trafficking of TRPM7 was altered in HEK-293 epithelial kidney cells and DT40 B cells in the presence of TRPM6 with intact kinase activity, independently of the availability of extracellular Mg(2+), but TRPM6/7 surface labeling experiments indicate comparable levels of the TRPM6/7 channels at the plasma membrane. Furthermore, using a complementation approach in TRPM7-deficient DT40 B-cells, we demonstrated that wild-type TRPM6 inhibited cell growth under hypomagnesic cell culture conditions in cells co-expressing TRPM6 and TRPM7; however, co-expression of a TRPM6 kinase dead mutant had no effect-a similar phenotype was also observed in TRPM6/7 co-expressing HEK-293 cells. Our results provide first clues about how heteromer formation between TRPM6 and TRPM7 influences the biological activity of these ion channels. We show that TRPM6 regulates TRPM7 intracellular

  4. Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

    PubMed Central

    McIntosh, Avery L.; Atshaves, Barbara P.; Huang, Huan; Gallegos, Adalberto M.; Kier, Ann B.; Schroeder, Friedhelm

    2008-01-01

    Cholesterol itself has very few structural/chemical features suitable for real-time imaging in living cells. Thus, the advent of dehydroergosterol [ergosta-5,7,9(11),22-tetraen-3β-ol, DHE] the fluorescent sterol most structurally and functionally similar to cholesterol to date, has proven to be a major asset for real-time probing/elucidating the sterol environment and intracellular sterol trafficking in living organisms. DHE is a naturally-occurring, fluorescent sterol analog that faithfully mimics many of the properties of cholesterol. Because these properties are very sensitive to sterol structure and degradation, such studies require the use of extremely pure (>98%) quantities of fluorescent sterol. DHE is readily bound by cholesterol-binding proteins, is incorporated into lipoproteins (from the diet of animals or by exchange in vitro), and for real-time imaging studies is easily incorporated into cultured cells where it co-distributes with endogenous sterol. Incorporation from an ethanolic stock solution to cell culture media is effective, but this process forms an aqueous dispersion of dehydroergosterol crystals which can result in endocytic cellular uptake and distribution into lysosomes which is problematic in imaging DHE at the plasma membrane of living cells. In contrast, monomeric DHE can be incorporated from unilamellar vesicles by exchange/fusion with the plasma membrane or from DHE-methyl-β-cyclodextrin (DHE-MβCD) complexes by exchange with the plasma membrane. Both of the latter techniques can deliver large quantities of monomeric dehydroergosterol with significant distribution into the plasma membrane. The properties and behavior of DHE in protein-binding, lipoproteins, model membranes, biological membranes, lipid rafts/caveolae, and real-time imaging in living cells indicate that this naturally-occurring fluorescent sterol is a useful mimic for probing the properties of cholesterol in these systems. PMID:18536950

  5. Cell type-specific response to high intracellular loading of polyacrylic acid-coated magnetic nanoparticles

    PubMed Central

    Lojk, Jasna; Bregar, Vladimir B; Rajh, Maruša; Miš, Katarina; Kreft, Mateja Erdani; Pirkmajer, Sergej; Veranič, Peter; Pavlin, Mojca

    2015-01-01

    Magnetic nanoparticles (NPs) are a special type of NP with a ferromagnetic, electron-dense core that enables several applications such as cell tracking, hyperthermia, and magnetic separation, as well as multimodality. So far, superparamagnetic iron oxide NPs (SPIONs) are the only clinically approved type of metal oxide NPs, but cobalt ferrite NPs have properties suitable for biomedical applications as well. In this study, we analyzed the cellular responses to magnetic cobalt ferrite NPs coated with polyacrylic acid (PAA) in three cell types: Chinese Hamster Ovary (CHO), mouse melanoma (B16) cell line, and primary human myoblasts (MYO). We compared the internalization pathway, intracellular trafficking, and intracellular fate of our NPs using fluorescence and transmission electron microscopy (TEM) as well as quantified NP uptake and analyzed uptake dynamics. We determined cell viability after 24 or 96 hours’ exposure to increasing concentrations of NPs, and quantified the generation of reactive oxygen species (ROS) upon 24 and 48 hours’ exposure. Our NPs have been shown to readily enter and accumulate in cells in high quantities using the same two endocytic pathways; mostly by macropinocytosis and partially by clathrin-mediated endocytosis. The cell types differed in their uptake rate, the dynamics of intracellular trafficking, and the uptake capacity, as well as in their response to higher concentrations of internalized NPs. The observed differences in cell responses stress the importance of evaluation of NP–cell interactions on several different cell types for better prediction of possible toxic effects on different cell and tissue types in vivo. PMID:25733835

  6. Coat as a Dagger: The Use of Capsid Proteins to Perforate Membranes during Non-Enveloped DNA Viruses Trafficking

    PubMed Central

    Bilkova, Eva; Forstova, Jitka; Abrahamyan, Levon

    2014-01-01

    To get access to the replication site, small non-enveloped DNA viruses have to cross the cell membrane using a limited number of capsid proteins, which also protect the viral genome in the extracellular environment. Most of DNA viruses have to reach the nucleus to replicate. The capsid proteins involved in transmembrane penetration are exposed or released during endosomal trafficking of the virus. Subsequently, the conserved domains of capsid proteins interact with cellular membranes and ensure their efficient permeabilization. This review summarizes our current knowledge concerning the role of capsid proteins of small non-enveloped DNA viruses in intracellular membrane perturbation in the early stages of infection. PMID:25055856

  7. Determining the role of Rab7 in constitutive and ligand-mediated epidermal growth factor receptor endocytic trafficking using single cell assays.

    PubMed

    Ceresa, Brian P

    2015-01-01

    RAB proteins are essential for the proper membrane trafficking of a number of proteins. Each of the 60+ RABs that have been identified has a discrete role in coordinating the movement from one subcellular compartment to another. Early attempts at deciphering the roles of individual RAB proteins relied heavily on the use of activating and/or dominant negative mutants (Ceresa, Histol Histopathol 21:987-993, 2006). However, overexpression of mutant proteins can lead to misleading information; high levels of expression can drive low affinity (and possibly, nonphysiological) interactions as well as cause mislocalization. The use of RNAi for transient protein knock down will reveal which membrane trafficking steps absolutely require the attenuated RAB. When determining the role of RAB protein in epidermal growth factor receptor (EGFR) membrane trafficking, there are special considerations. The EGFR undergoes constitutive and ligand-mediated endocytic trafficking. Both affect receptor signaling, but via different mechanisms. Here, we discuss how to experimentally dissect those two processes.

  8. Alternative routes to the cell surface underpin insulin-regulated membrane trafficking of GLUT4

    PubMed Central

    Kioumourtzoglou, Dimitrios; Pryor, Paul R.; Gould, Gwyn W.; Bryant, Nia J.

    2015-01-01

    ABSTRACT Insulin-stimulated delivery of glucose transporters (GLUT4, also known as SLC2A4) from specialized intracellular GLUT4 storage vesicles (GSVs) to the surface of fat and muscle cells is central to whole-body glucose regulation. This translocation and subsequent internalization of GLUT4 back into intracellular stores transits through numerous small membrane-bound compartments (internal GLUT4-containing vesicles; IGVs) including GSVs, but the function of these different compartments is not clear. Cellugyrin (also known as synaptogyrin-2) and sortilin define distinct populations of IGV; sortilin-positive IGVs represent GSVs, but the function of cellugyrin-containing IGVs is unknown. Here, we demonstrate a role for cellugyrin in intracellular sequestration of GLUT4 in HeLa cells and have used a proximity ligation assay to follow changes in pairwise associations between cellugyrin, sortilin, GLUT4 and membrane trafficking machinery following insulin-stimulation of 3T3-L1 adipoctyes. Our data suggest that insulin stimulates traffic from cellugyrin-containing to sortilin-containing membranes, and that cellugyrin-containing IGVs provide an insulin-sensitive reservoir to replenish GSVs following insulin-stimulated exocytosis of GLUT4. Furthermore, our data support the existence of a pathway from cellugyrin-containing membranes to the surface of 3T3-L1 adipocytes that bypasses GSVs under basal conditions, and that insulin diverts traffic away from this into GSVs. PMID:26071524

  9. Intracellular Dynamics of Synucleins: "Here, There and Everywhere".

    PubMed

    Surguchov, Andrei

    2015-01-01

    Synucleins are small, soluble proteins expressed primarily in neural tissue and in certain tumors. The synuclein family consists of three members: α-, β-, and γ-synucleins present only in vertebrates. Members of the synuclein family have high sequence identity, especially in the N-terminal regions. The synuclein gene family came into the spotlight, when one of its members, α-synuclein, was found to be associated with Parkinson's disease and other neurodegenerative disorders, whereas γ-synuclein was linked to several forms of cancer. There are a lot of controversy and exciting debates concerning members of the synuclein family, including their normal functions, toxicity, role in pathology, transmission between cells and intracellular localization. Important findings which remain undisputable for many years are synuclein localization in synapses and their role in the regulation of synaptic vesicle trafficking, whereas their presence and function in mitochondria and nucleus is a debated topic. In this review, we present the data on the localization of synucleins in two intracellular organelles: the nucleus and mitochondria. PMID:26614873

  10. 31 CFR 536.312 - Specially designated narcotics trafficker.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... designated narcotics trafficker means: (a) Persons listed in the annex to Executive Order 12978 (3 CFR, 1995... 31 Money and Finance:Treasury 3 2014-07-01 2014-07-01 false Specially designated narcotics... (Continued) OFFICE OF FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY NARCOTICS TRAFFICKING...

  11. 31 CFR 536.312 - Specially designated narcotics trafficker.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... designated narcotics trafficker means: (a) Persons listed in the annex to Executive Order 12978 (3 CFR, 1995... 31 Money and Finance:Treasury 3 2013-07-01 2013-07-01 false Specially designated narcotics... (Continued) OFFICE OF FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY NARCOTICS TRAFFICKING...

  12. 31 CFR 536.312 - Specially designated narcotics trafficker.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... designated narcotics trafficker means: (a) Persons listed in the annex to Executive Order 12978 (3 CFR, 1995... 31 Money and Finance:Treasury 3 2011-07-01 2011-07-01 false Specially designated narcotics... (Continued) OFFICE OF FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY NARCOTICS TRAFFICKING...

  13. 31 CFR 536.312 - Specially designated narcotics trafficker.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... designated narcotics trafficker means: (a) Persons listed in the annex to Executive Order 12978 (3 CFR, 1995... 31 Money and Finance:Treasury 3 2012-07-01 2012-07-01 false Specially designated narcotics... (Continued) OFFICE OF FOREIGN ASSETS CONTROL, DEPARTMENT OF THE TREASURY NARCOTICS TRAFFICKING...

  14. Adolescent Black Males' Drug Trafficking and Addiction: Three Theoretical Perspectives.

    ERIC Educational Resources Information Center

    Moore, Sharon E.

    1995-01-01

    Explains the incidence and nature of drug trafficking and chemical dependency among adolescent black males. The paper also discusses the social science theories of Emile Durkheim, Karl Marx, and Molefi Asante to better understand the behaviors, and the consequences of those behaviors, of young black males who participate in drug trafficking. (GR)

  15. 78 FR 59317 - Federal Acquisition Regulation; Ending Trafficking in Persons

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-09-26

    ... INFORMATION: I. Background The United States has long had a zero-tolerance policy regarding Government... subjection to involuntary servitude, peonage, debt bondage, or slavery, and sex trafficking. As the largest... (77 FR 60029, October 2, 2012), and Title XVII, entitled ``Ending Trafficking in...

  16. Protein kinesis: The dynamics of protein trafficking and stability

    SciTech Connect

    1995-12-31

    The purpose of this conference is to provide a multidisciplinary forum for exchange of state-of-the-art information on protein kinesis. This volume contains abstracts of papers in the following areas: protein folding and modification in the endoplasmic reticulum; protein trafficking; protein translocation and folding; protein degradation; polarity; nuclear trafficking; membrane dynamics; and protein import into organelles.

  17. Aggression in Sexually Abused Trafficked Girls and Efficacy of Intervention

    ERIC Educational Resources Information Center

    Deb, Sibnath; Mukherjee, Aparna; Mathews, Ben

    2011-01-01

    The broad objective of this study was to understand the incidence and severity of aggression among sexually abused girls who were trafficked and who were then further used for commercial sexual exploitation (referred to subsequently as sexually abused trafficked girls). In addition, the impact of counseling for minimizing aggression in these girls…

  18. Domestic Minor Sex Trafficking in the United States

    ERIC Educational Resources Information Center

    Kotrla, Kimberly

    2010-01-01

    By now, most social workers are familiar with the issue of human trafficking. However, many are likely unfamiliar with research indicating that youths constitute the most vulnerable group in the United States for becoming victims of sex trafficking and that most women in prostitution actually entered as minors. Some experts are now referring to…

  19. Teaching about Trafficking: Opportunities and Challenges for Critical Engagement

    ERIC Educational Resources Information Center

    Dragiewicz, Molly

    2008-01-01

    The author came to know about trafficking by accident, when she was hired as a research assistant at The Protection Project (TPP) in 1999. As a feminist teacher, the author was very aware of the divisions among feminists on the subject of trafficking, and was interested in communicating these differences to students who were not well versed in the…

  20. Vesicle trafficking in plant immune responses.

    PubMed

    Robatzek, Silke

    2007-01-01

    In plants, perception of pathogen-associated molecular patterns at the surface is the first line of defence in cellular immunity. This review summarizes recent evidence of the involvement of vesicle trafficking in the plant's immune response against pathogens. I first discuss aspects of ligand-stimulated receptor endocytosis. The best-characterized pattern-recognition receptor (PRR), FLS2, is a transmembrane leucine-rich repeat receptor kinase that recognizes bacterial flagellin. FLS2 was recently shown to undergo internalization upon activation with its cognate ligand. An animal PRR, TLR4 that mediates perception of bacterial-derived lipopolysaccharides, similarly exhibits ligand-stimulated endocytosis. The second focus is N-ethylmaleimide-sensitive factor adaptor protein receptor (SNARE)-mediated immunity involving syntaxins and their cognate partners. One of the genes involved in basal immunity in Arabidopsis, PEN1, encodes a syntaxin that focally accumulates at fungal penetration sites, raising the possibility that induced exocytosis is important for active defence. Pathogen-triggered endocytic and exocytic processes have to be balanced to ensure host cell homeostasis. Thus, understanding how phytopathogens have evolved strategies to exploit host cell vesicle trafficking to manipulate immune responses is currently an area of intense study. PMID:17081192

  1. Secretory protein trafficking in Giardia intestinalis.

    PubMed

    Hehl, Adrian B; Marti, Matthias

    2004-07-01

    Early diverged extant organisms, which may serve as convenient laboratory models to look for and study evolutionary ancient features of eukaryotic cell biology, are rare. The diplomonad Giardia intestinalis, a protozoan parasite known to cause diarrhoeal disease, has become an increasingly popular object of basic research in cell biology, not least because of a genome sequencing project nearing completion. Commensurate with its phylogenetic status, the Giardia trophozoite has a very basic secretory system and even lacks hallmark structures such as a morphologically identifiable Golgi apparatus. The cell's capacity for protein sorting is nevertheless unimpeded, exemplified by its ability to cope with massive amounts of newly synthesized cyst wall proteins and glycans, which are sorted to dedicated Golgi-like compartments termed encystation-specific vesicles (ESVs) generated from endoplasmic reticulum (ER)-derived transport intermediates. This soluble bulk cargo is kept strictly separate from constitutively transported variant surface proteins during export, a function that is dependent on the stage-specific recognition of trafficking signals. Encysting Giardia therefore provide a unique system for the study of unconventional, Golgi-independent protein trafficking mechanisms in the broader context of eukaryotic endomembrane organization and evolution. PMID:15225300

  2. Entrapment and Enmeshment Schemes Used by Sex Traffickers.

    PubMed

    Reid, Joan A

    2016-09-01

    Emerging research suggests that sex traffickers/pimps control the majority of trafficked girls in the United States. The youthfulness of these victims and their lack of psychosocial maturity severely diminish their ability to detect exploitative motives or withstand manipulation of traffickers. A review of 43 cases of sexually exploited girls involving non-relative traffickers and 10 semi-structured interviews with social service providers revealed numerous scripts and schemes used by sex traffickers to entrap and entangle victims including boyfriend/lover scripts, ruses involving debt bondage, friendship or faux-family scripts, threats of forced abortion or to take away children, and coerced co-offending. These findings inform potential prevention efforts and highlight the need for multi-systemic, victim-centered approaches to intervention. PMID:25079777

  3. Entrapment and Enmeshment Schemes Used by Sex Traffickers.

    PubMed

    Reid, Joan A

    2016-09-01

    Emerging research suggests that sex traffickers/pimps control the majority of trafficked girls in the United States. The youthfulness of these victims and their lack of psychosocial maturity severely diminish their ability to detect exploitative motives or withstand manipulation of traffickers. A review of 43 cases of sexually exploited girls involving non-relative traffickers and 10 semi-structured interviews with social service providers revealed numerous scripts and schemes used by sex traffickers to entrap and entangle victims including boyfriend/lover scripts, ruses involving debt bondage, friendship or faux-family scripts, threats of forced abortion or to take away children, and coerced co-offending. These findings inform potential prevention efforts and highlight the need for multi-systemic, victim-centered approaches to intervention.

  4. The role of the nurse in combating human trafficking.

    PubMed

    Sabella, Donna

    2011-02-01

    Human trafficking, also called modern slavery, happens worldwide--and the United States is no exception. Within our borders, thousands of foreign nationals and U.S. citizens, many of them children, are forced or coerced into sex work or various forms of labor every year. Nurses and other health care providers who encounter victims of trafficking often don't realize it, and opportunities to intervene are lost. Although no one sign can demonstrate with certainty when someone is being trafficked, there are several indicators that clinicians should know. This article provides an overview of human trafficking, describes how to recognize signs that a person is being trafficked and how to safely intervene, and offers an extensive resource list.

  5. The role of the nurse in combating human trafficking.

    PubMed

    Sabella, Donna

    2011-02-01

    Human trafficking, also called modern slavery, happens worldwide--and the United States is no exception. Within our borders, thousands of foreign nationals and U.S. citizens, many of them children, are forced or coerced into sex work or various forms of labor every year. Nurses and other health care providers who encounter victims of trafficking often don't realize it, and opportunities to intervene are lost. Although no one sign can demonstrate with certainty when someone is being trafficked, there are several indicators that clinicians should know. This article provides an overview of human trafficking, describes how to recognize signs that a person is being trafficked and how to safely intervene, and offers an extensive resource list. PMID:21270581

  6. Human trafficking: the role of the health care provider.

    PubMed

    Dovydaitis, Tiffany

    2010-01-01

    Human trafficking is a major public health problem, both domestically and internationally. Health care providers are often the only professionals to interact with trafficking victims who are still in captivity. The expert assessment and interview skills of providers contribute to their readiness to identify victims of trafficking. The purpose of this article is to provide clinicians with knowledge on trafficking and give specific tools that they may use to assist victims in the clinical setting. Definitions, statistics, and common health care problems of trafficking victims are reviewed. The role of the health care provider is outlined through a case study and clinical practice tools are provided. Suggestions for future research are also briefly addressed. PMID:20732668

  7. Psychological Coercion in Human Trafficking: An Application of Biderman's Framework.

    PubMed

    Baldwin, Susie B; Fehrenbacher, Anne E; Eisenman, David P

    2015-09-01

    This study examined coercive conditions experienced by trafficked persons in the context of Biderman's theory of coercion. We conducted semi-structured interviews with 12 adult women trafficked into Los Angeles County, from 10 countries, for domestic work and/or sex work. Participants described health problems they experienced in relation to their trafficking experience and their perceptions of conditions that caused health problems. Utilizing a framework analysis approach, we analyzed themes using Biderman's framework. Participants reported experiencing the range of nonphysical coercive tactics outlined by Biderman, including isolation, monopolization of perception, induced debility or exhaustion, threats, occasional indulgences, demonstration of omnipotence, degradation, and enforcement of trivial demands. Our analysis demonstrates how these coercion tactics reinforced the submission of trafficked persons to their traffickers even in the absence of physical force or restraints. Such psychological abuse creates extreme stress that can lead to acute and chronic, physical and mental health problems. PMID:25371382

  8. Human Trafficking: The Role of the Health Care Provider

    PubMed Central

    Dovydaitis, Tiffany

    2011-01-01

    Human trafficking is a major public health problem, both domestically and internationally. Health care providers are often the only professionals to interact with trafficking victims who are still in captivity. The expert assessment and interview skills of providers contribute to their readiness to identify victims of trafficking. The purpose of this article is to provide clinicians with knowledge on trafficking and give specific tools that they may use to assist victims in the clinical setting. Definitions, statistics, and common health care problems of trafficking victims are reviewed. The role of the health care provider is outlined through a case study and clinical practice tools are provided. Suggestions for future research are also briefly addressed. PMID:20732668

  9. New Insights into How Trafficking Regulates T Cell Receptor Signaling

    PubMed Central

    Lou, Jieqiong; Rossy, Jérémie; Deng, Qiji; Pageon, Sophie V.; Gaus, Katharina

    2016-01-01

    There is emerging evidence that exocytosis plays an important role in regulating T cell receptor (TCR) signaling. The trafficking molecules involved in lytic granule (LG) secretion in cytotoxic T lymphocytes (CTL) have been well-studied due to the immune disorder known as familial hemophagocytic lymphohistiocytosis (FHLH). However, the knowledge of trafficking machineries regulating the exocytosis of receptors and signaling molecules remains quite limited. In this review, we summarize the reported trafficking molecules involved in the transport of the TCR and downstream signaling molecules to the cell surface. By combining this information with the known knowledge of LG exocytosis and general exocytic trafficking machinery, we attempt to draw a more complete picture of how the TCR signaling network and exocytic trafficking matrix are interconnected to facilitate T cell activation. This also highlights how membrane compartmentalization facilitates the spatiotemporal organization of cellular responses that are essential for immune functions. PMID:27508206

  10. A vesicle-trafficking protein commandeers Kv channel voltage sensors for voltage-dependent secretion.

    PubMed

    Grefen, Christopher; Karnik, Rucha; Larson, Emily; Lefoulon, Cécile; Wang, Yizhou; Waghmare, Sakharam; Zhang, Ben; Hills, Adrian; Blatt, Michael R

    2015-01-01

    Growth in plants depends on ion transport for osmotic solute uptake and secretory membrane trafficking to deliver material for wall remodelling and cell expansion. The coordination of these processes lies at the heart of the question, unresolved for more than a century, of how plants regulate cell volume and turgor. Here we report that the SNARE protein SYP121 (SYR1/PEN1), which mediates vesicle fusion at the Arabidopsis plasma membrane, binds the voltage sensor domains (VSDs) of K(+) channels to confer a voltage dependence on secretory traffic in parallel with K(+) uptake. VSD binding enhances secretion in vivo subject to voltage, and mutations affecting VSD conformation alter binding and secretion in parallel with channel gating, net K(+) concentration, osmotic content and growth. These results demonstrate a new and unexpected mechanism for secretory control, in which a subset of plant SNAREs commandeer K(+) channel VSDs to coordinate membrane trafficking with K(+) uptake for growth.

  11. Golgi-localized STELLO proteins regulate the assembly and trafficking of cellulose synthase complexes in Arabidopsis.

    PubMed

    Zhang, Yi; Nikolovski, Nino; Sorieul, Mathias; Vellosillo, Tamara; McFarlane, Heather E; Dupree, Ray; Kesten, Christopher; Schneider, René; Driemeier, Carlos; Lathe, Rahul; Lampugnani, Edwin; Yu, Xiaolan; Ivakov, Alexander; Doblin, Monika S; Mortimer, Jenny C; Brown, Steven P; Persson, Staffan; Dupree, Paul

    2016-01-01

    As the most abundant biopolymer on Earth, cellulose is a key structural component of the plant cell wall. Cellulose is produced at the plasma membrane by cellulose synthase (CesA) complexes (CSCs), which are assembled in the endomembrane system and trafficked to the plasma membrane. While several proteins that affect CesA activity have been identified, components that regulate CSC assembly and trafficking remain unknown. Here we show that STELLO1 and 2 are Golgi-localized proteins that can interact with CesAs and control cellulose quantity. In the absence of STELLO function, the spatial distribution within the Golgi, secretion and activity of the CSCs are impaired indicating a central role of the STELLO proteins in CSC assembly. Point mutations in the predicted catalytic domains of the STELLO proteins indicate that they are glycosyltransferases facing the Golgi lumen. Hence, we have uncovered proteins that regulate CSC assembly in the plant Golgi apparatus. PMID:27277162

  12. Multiple roles for membrane-associated protein trafficking and signaling in gravitropism

    PubMed Central

    Strohm, Allison K.; Baldwin, Katherine L.; Masson, Patrick H.

    2012-01-01

    Gravitropism is a process that allows plant organs to guide their growth relative to the gravity vector. It requires them to sense changes in their orientation and generate a biochemical signal that they transmit to the tissues that drive organ curvature. Trafficking between the plasma membrane and endosomal compartments is important for all of these phases of the gravitropic response. The sedimentation of starch-filled organelles called amyloplasts plays a key role in sensing reorientation, and vacuolar integrity is required for amyloplast sedimentation in shoots. Other proteins associated with the vesicle trafficking pathway contribute to early gravity signal transduction independently of amyloplast sedimentation in both roots and hypocotyls. Phosphatidylinositol signaling, which starts at the plasma membrane and later affects the localization of auxin efflux facilitators, is a likely second messenger in the signal transduction phase of gravitropism. Finally, membrane-localized auxin influx and efflux facilitators contribute to a differential auxin gradient across the gravistimulated organs, which directs root curvature. PMID:23248632

  13. Golgi-localized STELLO proteins regulate the assembly and trafficking of cellulose synthase complexes in Arabidopsis

    PubMed Central

    Zhang, Yi; Nikolovski, Nino; Sorieul, Mathias; Vellosillo, Tamara; McFarlane, Heather E.; Dupree, Ray; Kesten, Christopher; Schneider, René; Driemeier, Carlos; Lathe, Rahul; Lampugnani, Edwin; Yu, Xiaolan; Ivakov, Alexander; Doblin, Monika S.; Mortimer, Jenny C.; Brown, Steven P.; Persson, Staffan; Dupree, Paul

    2016-01-01

    As the most abundant biopolymer on Earth, cellulose is a key structural component of the plant cell wall. Cellulose is produced at the plasma membrane by cellulose synthase (CesA) complexes (CSCs), which are assembled in the endomembrane system and trafficked to the plasma membrane. While several proteins that affect CesA activity have been identified, components that regulate CSC assembly and trafficking remain unknown. Here we show that STELLO1 and 2 are Golgi-localized proteins that can interact with CesAs and control cellulose quantity. In the absence of STELLO function, the spatial distribution within the Golgi, secretion and activity of the CSCs are impaired indicating a central role of the STELLO proteins in CSC assembly. Point mutations in the predicted catalytic domains of the STELLO proteins indicate that they are glycosyltransferases facing the Golgi lumen. Hence, we have uncovered proteins that regulate CSC assembly in the plant Golgi apparatus. PMID:27277162

  14. Rab11-FIP1A regulates early trafficking into the recycling endosomes.

    PubMed

    Schafer, Jenny C; McRae, Rebecca E; Manning, Elizabeth H; Lapierre, Lynne A; Goldenring, James R

    2016-01-15

    The Rab11 family of small GTPases, along with the Rab11-family interacting proteins (Rab11-FIPs), are critical regulators of intracellular vesicle trafficking and recycling. We have identified a point mutation of Threonine-197 site to an Alanine in Rab11-FIP1A, which causes a dramatic dominant negative phenotype when expressed in HeLa cells. The normally perinuclear distribution of GFP-Rab11-FIP1A was condensed into a membranous cisternum with almost no GFP-Rab11-FIP1A(T197A) remaining outside of this central locus. Also, this condensed GFP-FIP1A(T197A) altered the distribution of proteins in the Rab11a recycling pathway including endogenous Rab11a, Rab11-FIP1C, and transferrin receptor (CD71). Furthermore, this condensed GFP-FIP1A(T197A)-containing structure exhibited little movement in live HeLa cells. Expression of GFP-FIP1A(T197A) caused a strong blockade of transferrin recycling. Treatment of cells expressing GFP-FIP1A(T197A) with nocodazole did not disperse the Rab11a-containing recycling system. We also found that Rab5 and EEA1 were accumulated in membranes by GFP-Rab11-FIP1A but Rab4 was unaffected, suggesting that a direct pathway may exist from early endosomes into the Rab11a-containing recycling system. Our study of a potent inhibitory trafficking mutation in Rab11-FIP1A shows that Rab11-FIP1A associates with and regulates trafficking at an early step in the process of membrane recycling.

  15. The tyrosine phosphatase PTPN14 (Pez) inhibits metastasis by altering protein trafficking.

    PubMed

    Belle, Leila; Ali, Naveid; Lonic, Ana; Li, Xiaochun; Paltridge, James L; Roslan, Suraya; Herrmann, David; Conway, James R W; Gehling, Freya K; Bert, Andrew G; Crocker, Lesley A; Tsykin, Anna; Farshid, Gelareh; Goodall, Gregory J; Timpson, Paul; Daly, Roger J; Khew-Goodall, Yeesim

    2015-02-17

    Factors secreted by tumor cells shape the local microenvironment to promote invasion and metastasis, as well as condition the premetastatic niche to enable secondary-site colonization and growth. In addition to this secretome, tumor cells have increased abundance of growth-promoting receptors at the cell surface. We found that the tyrosine phosphatase PTPN14 (also called Pez, which is mutated in various cancers) suppressed metastasis by reducing intracellular protein trafficking through the secretory pathway. Knocking down PTPN14 in tumor cells or injecting the peritoneum of mice with conditioned medium from PTPN14-deficient cell cultures promoted the growth and metastasis of breast cancer xenografts. Loss of catalytically functional PTPN14 increased the secretion of growth factors and cytokines, such as IL-8 (interleukin-8), and increased the abundance of EGFR (epidermal growth factor receptor) at the cell surface of breast cancer cells and of FLT4 (vascular endothelial growth factor receptor 3) at the cell surface of primary lymphatic endothelial cells. We identified RIN1 (Ras and Rab interactor 1) and PRKCD (protein kinase C-δ) as binding partners and substrates of PTPN14. Similar to cells overexpressing PTPN14, receptor trafficking to the cell surface was inhibited in cells that lacked PRKCD or RIN1 or expressed a nonphosphorylatable RIN1 mutant, and cytokine secretion was decreased in cells treated with PRKCD inhibitors. Invasive breast cancer tissue had decreased expression of PTPN14, and patient survival was worse when tumors had increased expression of the genes encoding RIN1 or PRKCD. Thus, PTPN14 prevents metastasis by restricting the trafficking of both soluble and membrane-bound proteins. PMID:25690013

  16. Urothelial cultures support intracellular bacterial community formation by uropathogenic Escherichia coli.

    PubMed

    Berry, Ruth E; Klumpp, David J; Schaeffer, Anthony J

    2009-07-01

    Uropathogenic Escherichia coli (UPEC) causes most community-acquired and nosocomial urinary tract infections (UTI). In a mouse model of UTI, UPEC invades superficial bladder cells and proliferates rapidly, forming biofilm-like structures called intracellular bacterial communities (IBCs). Using a gentamicin protection assay and fluorescence microscopy, we developed an in vitro model for studying UPEC proliferation within immortalized human urothelial cells. By pharmacologic manipulation of urothelial cells with the cholesterol-sequestering drug filipin, numbers of intracellular UPEC CFU increased 8 h and 24 h postinfection relative to untreated cultures. Enhanced UPEC intracellular proliferation required that the urothelial cells, but not the bacteria, be filipin treated prior to infection. However, neither UPEC frequency of invasion nor early intracellular trafficking events to a Lamp1-positive compartment were modulated by filipin. Upon inspection by fluorescence microscopy, cultures with enhanced UPEC intracellular proliferation exhibited large, dense bacterial aggregates within cells that resembled IBCs but were contained with Lamp1-positive vacuoles. While an isogenic fimH mutant was capable of forming these IBC-like structures, the mutant formed significantly fewer than wild-type UPEC. Similar to IBCs, expression of E. coli iron acquisition systems was upregulated by intracellular UPEC. Expression of other putative virulence factors, including hlyA, cnf1, fliC, kpsD, and the biofilm adhesin yfaL also increased, while expression of fimA decreased and that of flu did not change. These results indicate that UPEC differentially regulates virulence factors in the intracellular environment. Thus, immortalized urothelial cultures that recapitulate IBC formation in vitro represent a novel system for the molecular and biochemical characterization of the UPEC intracellular life cycle.

  17. Illicit trafficking of radiological & nuclear materials : modeling and analysis of trafficking trends and risks.

    SciTech Connect

    York, David L.; Love, Tracia L.; Rochau, Gary Eugene

    2005-01-01

    Concerns over the illicit trafficking of radiological and nuclear materials were focused originally on the lack of security and accountability of such material throughout the former Soviet states. This is primarily attributed to the frequency of events that have occurred involving the theft and trafficking of critical material components that could be used to construct a Radiological Dispersal Device (RDD) or even a rudimentary nuclear device. However, with the continued expansion of nuclear technology and the deployment of a global nuclear fuel cycle these materials have become increasingly prevalent, affording a more diverse inventory of dangerous materials and dual-use items. To further complicate the matter, the list of nuclear consumers has grown to include: (1) Nation-states that have gone beyond the IAEA agreed framework and additional protocols concerning multiple nuclear fuel cycles and processes that reuse the fuel through reprocessing to exploit technologies previously confined to the more industrialized world; (2) Terrorist organizations seeking to acquire nuclear and radiological material due to the potential devastation and psychological effect of their use; (3) Organized crime, which has discovered a lucrative market in trafficking of illicit material to international actors and/or countries; and (4) Amateur smugglers trying to feed their families in a post-Soviet era. An initial look at trafficking trends of this type seems scattered and erratic, localized primarily to a select group of countries. This is not necessarily the case. The success with which other contraband has been smuggled throughout the world suggests that nuclear trafficking may be carried out with relative ease along the same routes by the same criminals or criminal organizations. Because of the inordinately high threat posed by terrorist or extremist groups acquiring the ingredients for unconventional weapons, it is necessary that illicit trafficking of these materials be better

  18. Surface functionalized mesoporous silica nanoparticles for intracellular drug delivery

    NASA Astrophysics Data System (ADS)

    Vivero-Escoto, Juan Luis

    Mesoporous silica nanoparticles (MSNs) are a highly promising platform for intracellular controlled release of drugs and biomolecules. Despite that the application of MSNs in the field of intracellular drug delivery is still at its infancy very exciting breakthroughs have been achieved in the last years. A general review of the most recent progress in this area of research is presented, including a description of the latest findings on the pathways of entry into live mammalian cells together with the intracellular trafficking, a summary on the contribution of MSNs to the development of site-specific drug delivery systems, a report on the biocompatibility of this material in vitro andin vivo, and a discussion on the most recent breakthroughs in the synthesis and application of stimuli-responsive mesoporous silica-based delivery vehicles. A gold nanoparticles (AuNPs)-capped MSNs-based intracellular photoinduced drug delivery system (PR-AuNPs-MSNs) for the controlled release of anticancer drug inside of human fibroblast and liver cells was synthesized and characterized. We found that the mesoporous channels of MSNs could be efficiently capped by the photoresponsive AuNPs without leaking the toxic drug, paclitaxel, inside of human cells. Furthermore, we demonstrated that the cargo-release property of this PR-AuNPs-MSNs system could be easily photo-controlled under mild and biocompatible conditions in vitro. In collaboration with Renato Mortera (a visiting student from Italy), a MSNs based intracellular delivery system for controlled release of cell membrane impermeable cysteine was developed. A large amount of cysteine molecules were covalently attached to the silica surface of MSNs through cleavable disulfide linkers. These cysteine-containing nanoparticles were efficiently endocytosed by human cervical cancer cells HeLa. These materials exhibit 450 times higher cell growth inhibition capability than that of the conventional N-acetylcysteine prodrug. The ability to

  19. Functional analysis of host factors that mediate the intracellular lifestyle of Cryptococcus neoformans.

    PubMed

    Qin, Qing-Ming; Luo, Jijing; Lin, Xiaorong; Pei, Jianwu; Li, Lei; Ficht, Thomas A; de Figueiredo, Paul

    2011-06-01

    Cryptococcus neoformans (Cn), the major causative agent of human fungal meningoencephalitis, replicates within phagolysosomes of infected host cells. Despite more than a half-century of investigation into host-Cn interactions, host factors that mediate infection by this fungal pathogen remain obscure. Here, we describe the development of a system that employs Drosophila S2 cells and RNA interference (RNAi) to define and characterize Cn host factors. The system recapitulated salient aspects of fungal interactions with mammalian cells, including phagocytosis, intracellular trafficking, replication, cell-to-cell spread and escape of the pathogen from host cells. Fifty-seven evolutionarily conserved host factors were identified using this system, including 29 factors that had not been previously implicated in mediating fungal pathogenesis. Subsequent analysis indicated that Cn exploits host actin cytoskeletal elements, cell surface signaling molecules, and vesicle-mediated transport proteins to establish a replicative niche. Several host molecules known to be associated with autophagy (Atg), including Atg2, Atg5, Atg9 and Pi3K59F (a class III PI3-kinase) were also uncovered in our screen. Small interfering RNA (siRNA) mediated depletion of these autophagy proteins in murine RAW264.7 macrophages demonstrated their requirement during Cn infection, thereby validating findings obtained using the Drosophila S2 cell system. Immunofluorescence confocal microscopy analyses demonstrated that Atg5, LC3, Atg9a were recruited to the vicinity of Cn containing vacuoles (CnCvs) in the early stages of Cn infection. Pharmacological inhibition of autophagy and/or PI3-kinase activity further demonstrated a requirement for autophagy associated host proteins in supporting infection of mammalian cells by Cn. Finally, systematic trafficking studies indicated that CnCVs associated with Atg proteins, including Atg5, Atg9a and LC3, during trafficking to a terminal intracellular compartment that

  20. Miro1 Regulates Activity-Driven Positioning of Mitochondria within Astrocytic Processes Apposed to Synapses to Regulate Intracellular Calcium Signaling

    PubMed Central

    Stephen, Terri-Leigh; Higgs, Nathalie F.; Sheehan, David F.; Al Awabdh, Sana; López-Doménech, Guillermo; Arancibia-Carcamo, I. Lorena

    2015-01-01

    It is fast emerging that maintaining mitochondrial function is important for regulating astrocyte function, although the specific mechanisms that govern astrocyte mitochondrial trafficking and positioning remain poorly understood. The mitochondrial Rho-GTPase 1 protein (Miro1) regulates mitochondrial trafficking and detachment from the microtubule transport network to control activity-dependent mitochondrial positioning in neurons. However, whether Miro proteins are important for regulating signaling-dependent mitochondrial dynamics in astrocytic processes remains unclear. Using live-cell confocal microscopy of rat organotypic hippocampal slices, we find that enhancing neuronal activity induces transient mitochondrial remodeling in astrocytes, with a concomitant, transient reduction in mitochondrial trafficking, mediated by elevations in intracellular Ca2+. Stimulating neuronal activity also induced mitochondrial confinement within astrocytic processes in close proximity to synapses. Furthermore, we show that the Ca2+-sensing EF-hand domains of Miro1 are important for regulating mitochondrial trafficking in astrocytes and required for activity-driven mitochondrial confinement near synapses. Additionally, activity-dependent mitochondrial positioning by Miro1 reciprocally regulates the levels of intracellular Ca2+ in astrocytic processes. Thus, the regulation of intracellular Ca2+ signaling, dependent on Miro1-mediated mitochondrial positioning, could have important consequences for astrocyte Ca2+ wave propagation, gliotransmission, and ultimately neuronal function. SIGNIFICANCE STATEMENT Mitochondria are key cellular organelles that play important roles in providing cellular energy and buffering intracellular calcium ions. The mechanisms that control mitochondrial distribution within the processes of glial cells called astrocytes and the impact this may have on calcium signaling remains unclear. We show that activation of glutamate receptors or increased neuronal

  1. Cellular trafficking and anticancer activity of Garcinia mangostana extract-encapsulated polymeric nanoparticles.

    PubMed

    Pan-In, Porntip; Wanichwecharungruang, Supason; Hanes, Justin; Kim, Anthony J

    2014-01-01

    Garcinia mangostana Linn extract (GME) is a natural product that has received considerable attention in cancer therapy, and has the potential to reduce side effects of chemotherapeutics and improve efficacy. We formulated GME-encapsulated ethyl cellulose (GME-EC) and a polymer blend of ethyl cellulose and methyl cellulose (GME-EC/MC) nanoparticles. We achieved high drug-loading and encapsulation efficiency using a solvent-displacement method with particle sizes around 250 nm. Cellular uptake and accumulation of GME was higher for GME-encapsulated nanoparticles compared to free GME. In vitro cytotoxicity analysis showed effective anticancer activity of GME-EC and GME-EC/MC nanoparticles in HeLa cells in a dose-dependent manner. GME-EC/MC nanoparticles showed approximately twofold-higher anticancer activity compared to GME-EC nanoparticles, likely due to their enhanced bioavailability. GME-encapsulated nanoparticles primarily entered HeLa cells by clathrin-mediated endocytosis and trafficked through the endolysosomal pathway. As far as we know, this is the first report on the cellular uptake and intracellular trafficking mechanism of drug-loaded cellulose-based nanoparticles. In summary, encapsulation of GME using cellulose-derivative nanoparticles - GME-EC and GME-EC/MC nanoparticles - successfully improved the bioavailability of GME in aqueous solution, enhanced cellular uptake, and displayed effective anticancer activity. PMID:25125977

  2. Regulation of Cell Migration and β1 Integrin Trafficking by the Endosomal Adaptor GGA3.

    PubMed

    Ratcliffe, Colin D H; Sahgal, Pranshu; Parachoniak, Christine A; Ivaska, Johanna; Park, Morag

    2016-06-01

    The integrin family of cell adhesion receptors link extracellular matrices to intracellular signaling pathways and the actin cytoskeleton; and regulate cell migration, proliferation and survival in normal and diseased tissues. The subcellular location of integrin receptors is critical for their function and deregulated trafficking is implicated in various human diseases. Here we identify a role for Golgi-localized gamma-ear containing Arf-binding protein 3 (GGA3), in regulating trafficking of β1 integrin. GGA3 knockdown reduces cell surface and total levels of α2, α5 and β1 integrin subunits, inhibits cell spreading, reduces focal adhesion number, as well as cell migration. In the absence of GGA3, integrins are increasingly retained inside the cell, traffic toward the perinuclear lysosomal compartment and their degradation is enhanced. Integrin traffic and maintenance of integrin levels are dependent on the integrity of the Arf binding site of GGA3. Furthermore, sorting nexin 17 (SNX17), a critical regulator of integrin recycling, becomes mislocalized to enlarged late endosomes upon GGA3 depletion. These data support a model whereby GGA3, through its ability to regulate SNX17 endosomal localization and through interaction with Arf6 diverts integrins from the degradative pathway supporting cell migration. PMID:26935970

  3. Cellular trafficking and anticancer activity of Garcinia mangostana extract-encapsulated polymeric nanoparticles

    PubMed Central

    Pan-In, Porntip; Wanichwecharungruang, Supason; Hanes, Justin; Kim, Anthony J

    2014-01-01

    Garcinia mangostana Linn extract (GME) is a natural product that has received considerable attention in cancer therapy, and has the potential to reduce side effects of chemotherapeutics and improve efficacy. We formulated GME-encapsulated ethyl cellulose (GME-EC) and a polymer blend of ethyl cellulose and methyl cellulose (GME-EC/MC) nanoparticles. We achieved high drug-loading and encapsulation efficiency using a solvent-displacement method with particle sizes around 250 nm. Cellular uptake and accumulation of GME was higher for GME-encapsulated nanoparticles compared to free GME. In vitro cytotoxicity analysis showed effective anticancer activity of GME-EC and GME-EC/MC nanoparticles in HeLa cells in a dose-dependent manner. GME-EC/MC nanoparticles showed approximately twofold-higher anticancer activity compared to GME-EC nanoparticles, likely due to their enhanced bioavailability. GME-encapsulated nanoparticles primarily entered HeLa cells by clathrin-mediated endocytosis and trafficked through the endolysosomal pathway. As far as we know, this is the first report on the cellular uptake and intracellular trafficking mechanism of drug-loaded cellulose-based nanoparticles. In summary, encapsulation of GME using cellulose-derivative nanoparticles – GME-EC and GME-EC/MC nanoparticles – successfully improved the bioavailability of GME in aqueous solution, enhanced cellular uptake, and displayed effective anticancer activity. PMID:25125977

  4. LIN-12/Notch trafficking and regulation of DSL ligand activity during vulval induction in Caenorhabditis elegans.

    PubMed

    Shaye, Daniel D; Greenwald, Iva

    2005-11-01

    A novel mode of crosstalk between the EGFR-Ras-MAPK and LIN-12/Notch pathways occurs during the patterning of a row of vulval precursor cells (VPCs) in Caenorhabditis elegans: activation of the EGFR-Ras-MAPK pathway in the central VPC promotes endocytosis and degradation of LIN-12 protein. LIN-12 downregulation in the central VPC is a prerequisite for the activity of the lateral signal, which activates LIN-12 in neighboring VPCs. Here we characterize cis-acting targeting sequences in the LIN-12 intracellular domain and find that in addition to a di-leucine motif, serine/threonine residues are important for internalization and lysine residues are important for post-internalization trafficking and degradation. We also identify two trans-acting factors that are required for post-internalization trafficking and degradation: ALX-1, a homolog of yeast Bro1p and mammalian Alix and the WWP-1/Su(dx)/Itch ubiquitin ligase. By examining the effects of mutated forms of LIN-12 and reduced wwp-1 or alx-1 activity on subcellular localization and activity of LIN-12, we provide evidence that the lateral signal-inhibit