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Sample records for coordinates endosomal sorting

  1. Physiology and Pathology of Endosome-to-Golgi Retrograde Sorting

    OpenAIRE

    Burd, Christopher G.

    2011-01-01

    Bi-directional traffic between the Golgi apparatus and the endosomal system sustains the functions of the trans Golgi network (TGN) in secretion and organelle biogenesis. Export of cargo from the TGN via the anterograde pathways depletes the organelle of sorting receptors, processing proteases, SNARE molecules, and other factors that are subsequently retrieved from endosomes via the retrograde pathway. Recent studies indicate that retrograde trafficking is vital to early metazoan development,...

  2. SNX-BAR proteins in phosphoinositide-mediated, tubular-based endosomal sorting.

    Science.gov (United States)

    van Weering, Jan R T; Verkade, Paul; Cullen, Peter J

    2010-06-01

    The endocytic network is morphologically characterized by a wide variety of membrane bound compartments that are able to undergo dynamic re-modeling through tubular and vesicular structures. The precise molecular mechanisms governing such re-modeling, and the events that co-ordinated this with the major role of endosomes, cargo sorting, remain unclear. That said, recent work on a protein family of sorting nexins (SNX) - especially a subfamily of SNX that contain a BAR domain (SNX-BARs) - has begun to shed some much needed light on these issues and in particular the process of tubular-based endosomal sorting. SNX-BARs are evolutionary conserved in endosomal protein complexes such as retromer, where they co-ordinate membrane deformation with cargo selection. Furthermore a central theme emerges of SNX-BARs linking the forming membrane carrier to cytoskeletal elements for transport through motor proteins such as dynein. By studying these SNX-BARs, we are gaining an increasingly detailed appreciation of the mechanistic basis of endosomal sorting and how this highly dynamic process functions in health and disease.

  3. Structural determinants allowing endolysosomal sorting and degradation of endosomal GTPases.

    Science.gov (United States)

    Valero, Ruth A; Oeste, Clara L; Stamatakis, Konstantinos; Ramos, Irene; Herrera, Mónica; Boya, Patricia; Pérez-Sala, Dolores

    2010-09-01

    Rapid control of protein degradation is usually achieved through the ubiquitin-proteasome pathway. We recently found that the short-lived GTPase RhoB is degraded in lysosomes. Moreover, the fusion of the RhoB C-terminal sequence CINCCKVL, containing the isoprenylation and palmitoylation sites, to other proteins directs their sorting into multivesicular bodies (MVBs) and rapid lysosomal degradation. Here, we show that this process is highly specific for RhoB. Alteration of late endosome lipid dynamics produced the accumulation of RhoB, but not of other endosomal GTPases, including Rab5, Rab7, Rab9 or Rab11, into enlarged MVB. Other isoprenylated and bipalmitoylated GTPases, such as H-Ras, Rap2A, Rap2B and TC10, were not accumulated into MVB and were stable. Remarkably, although TC10, which is highly homologous to RhoB, was stable, a sequence derived from its C-terminus (CINCCLIT) elicited MVB sorting and degradation of a green fluorescent protein (GFP)-chimeric protein. This led us to identify a cluster of basic amino acids (KKH) in the TC10 hypervariable region, constituting a secondary signal potentially involved in electrostatic interactions with membrane lipids. Mutation of this cluster allowed TC10 MVB sorting and degradation, whereas inserting it into RhoB hypervariable region rescued this protein from its lysosomal degradation pathway. These findings define a highly specific structural module for entering the MVB pathway and rapid lysosomal degradation.

  4. Evidence for a Golgi-to-endosome protein sorting pathway in Plasmodium falciparum.

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    Priscilla Krai

    Full Text Available During the asexual intraerythrocytic stage, the malaria parasite Plasmodium falciparum must traffic newly-synthesized proteins to a broad array of destinations within and beyond the parasite's plasma membrane. In this study, we have localized two well-conserved protein components of eukaryotic endosomes, the retromer complex and the small GTPase Rab7, to define a previously-undescribed endosomal compartment in P. falciparum. Retromer and Rab7 co-localized to a small number of punctate structures within parasites. These structures, which we refer to as endosomes, lie in close proximity to the Golgi apparatus and, like the Golgi apparatus, are inherited by daughter merozoites. However, the endosome is clearly distinct from the Golgi apparatus as neither retromer nor Rab7 redistributed to the endoplasmic reticulum upon brefeldin A treatment. Nascent rhoptries (specialized secretory organelles required for invasion developed adjacent to endosomes, an observation that suggests a role for the endosome in rhoptry biogenesis. A P. falciparum homolog of the sortilin family of protein sorting receptors (PfSortilin was localized to the Golgi apparatus. Together, these results elaborate a putative Golgi-to-endosome protein sorting pathway in asexual blood stage parasites and suggest that one role of retromer is to mediate the retrograde transport of PfSortilin from the endosome to the Golgi apparatus.

  5. PTP1B targets the endosomal sorting machinery

    DEFF Research Database (Denmark)

    Stuible, Matthew; Abella, Jasmine V; Feldhammer, Matthew;

    2010-01-01

    STAM2 specifically suppressed Akt activation, and a phosphorylation-deficient STAM2 mutant displayed prolonged localization on endosomes following EGF stimulation. These results reveal a novel link between the dephosphorylation and endocytic machinery and suggest that PTP1B can affect RTK signaling...

  6. Rab14 limits the sorting of Glut4 from endosomes into insulin-sensitive regulated secretory compartments in adipocytes.

    Science.gov (United States)

    Brewer, Paul Duffield; Habtemichael, Estifanos N; Romenskaia, Irina; Coster, Adelle C F; Mastick, Cynthia Corley

    2016-05-15

    Insulin increases glucose uptake by increasing the rate of exocytosis of the facilitative glucose transporter isoform 4 (Glut4) relative to its endocytosis. Insulin also releases Glut4 from highly insulin-regulated secretory compartments (GSVs or Glut4 storage vesicles) into constitutively cycling endosomes. Previously it was shown that both overexpression and knockdown of the small GTP-binding protein Rab14 decreased Glut4 translocation to the plasma membrane (PM). To determine the mechanism of this perturbation, we measured the effects of Rab14 knockdown on the trafficking kinetics of Glut4 relative to two proteins that partially co-localize with Glut4, the transferrin (Tf) receptor and low-density-lipoprotein-receptor-related protein 1 (LRP1). Our data support the hypothesis that Rab14 limits sorting of proteins from sorting (or 'early') endosomes into the specialized GSV pathway, possibly through regulation of endosomal maturation. This hypothesis is consistent with known Rab14 effectors. Interestingly, the insulin-sensitive Rab GTPase-activating protein Akt substrate of 160 kDa (AS160) affects both sorting into and exocytosis from GSVs. It has previously been shown that exocytosis of GSVs is rate-limited by Rab10, and both Rab10 and Rab14 are in vitro substrates of AS160. Regulation of both entry into and exit from GSVs by AS160 through sequential Rab substrates would provide a mechanism for the finely tuned 'quantal' increases in cycling Glut4 observed in response to increasing concentrations of insulin.

  7. The deubiquitinating enzyme USP8 promotes trafficking and degradation of the chemokine receptor 4 at the sorting endosome.

    Science.gov (United States)

    Berlin, Ilana; Higginbotham, Katherine M; Dise, Rebecca S; Sierra, Maria I; Nash, Piers D

    2010-11-26

    Reversible ubiquitination orchestrated by the opposition of ubiquitin ligases and deubiquitinating enzymes mediates endocytic trafficking of cell surface receptors for lysosomal degradation. Ubiquitin-specific protease 8 (USP8) has previously been implicated in endocytosis of several receptors by virtue of their deubiquitination. The present study explores an indirect role for USP8 in cargo trafficking through its regulation of the chemokine receptor 4 (CXCR4). Contrary to the effects of USP8 loss on enhanced green fluorescent protein, we find that USP8 depletion stabilizes CXCR4 on the cell surface and attenuates receptor degradation without affecting its ubiquitination status. In the presence of ligand, diminished CXCR4 turnover is accompanied by receptor accumulation on enlarged early endosomes and leads to enhancement of phospho-ERK signaling. Perturbation in CXCR4 trafficking, resulting from USP8 inactivation, occurs at the ESCRT-0 checkpoint, and catalytic mutation of USP8 specifically targeted to the ESCRT-0 complex impairs the spatial and temporal organization of the sorting endosome. USP8 functionally opposes the ubiquitin ligase AIP4 with respect to ESCRT-0 ubiquitination, thereby promoting trafficking of CXCR4. Collectively, our findings demonstrate a functional cooperation between USP8, AIP4, and the ESCRT-0 machinery at the early sorting phase of CXCR4 and underscore the versatility of USP8 in shaping trafficking events at the early-to-late endosome transition.

  8. Role of SKD1 Regulators LIP5 and IST1-LIKE1 in Endosomal Sorting and Plant Development1[OPEN

    Science.gov (United States)

    Paez-Valencia, Julio; Miller, Nathan D.; Goodman, Kaija

    2016-01-01

    SKD1 is a core component of the mechanism that degrades plasma membrane proteins via the Endosomal Sorting Complex Required for Transport (ESCRT) pathway. Its ATPase activity and endosomal recruitment are regulated by the ESCRT components LIP5 and IST1. How LIP5 and IST1 affect ESCRT-mediated endosomal trafficking and development in plants is not known. Here we use Arabidopsis mutants to demonstrate that LIP5 controls the constitutive degradation of plasma membrane proteins and the formation of endosomal intraluminal vesicles. Although lip5 mutants were able to polarize the auxin efflux facilitators PIN2 and PIN3, both proteins were mis-sorted to the tonoplast in lip5 root cells. In addition, lip5 root cells over-accumulated PIN2 at the plasma membrane. Consistently with the trafficking defects of PIN proteins, the lip5 roots showed abnormal gravitropism with an enhanced response within the first 4 h after gravistimulation. LIP5 physically interacts with IST1-LIKE1 (ISTL1), a protein predicted to be the Arabidopsis homolog of yeast IST1. However, we found that Arabidopsis contains 12 genes coding for predicted IST1-domain containing proteins (ISTL1–12). Within the ISTL1–6 group, ISTL1 showed the strongest interaction with LIP5, SKD1, and the ESCRT-III-related proteins CHMP1A in yeast two hybrid assays. Through the analysis of single and double mutants, we found that the synthetic interaction of LIP5 with ISTL1, but not with ISTL2, 3, or 6, is essential for normal plant growth, repression of spontaneous cell death, and post-embryonic lethality. PMID:26983994

  9. Molecular basis for SNX-BAR-mediated assembly of distinct endosomal sorting tubules

    DEFF Research Database (Denmark)

    van Weering, Jan R.T.; Sessions, Richard B.; Traer, Colin J.

    2012-01-01

    -BARs, we report that some, but not all, can elicit the formation of tubules with diameters that resemble sorting tubules observed in cells. We reveal that SNX-BARs display a restricted pattern of BAR domain-mediated dimerization, and by resolving a 2.8 Å structure of a SNX1-BAR domain homodimer, establish...... that dimerization is achieved in part through neutralization of charged residues in the hydrophobic BAR-dimerization interface. Membrane remodelling also requires functional amphipathic helices, predicted to be present in all SNX-BARs, and the formation of high order SNX-BAR oligomers through selective 'tip...

  10. The serotonin transporter undergoes constitutive internalization and is primarily sorted to late endosomes and lysosomal degradation

    DEFF Research Database (Denmark)

    Rahbek-Clemmensen, Troels; Bay, Tina; Eriksen, Jacob

    2014-01-01

    The serotonin transporter (SERT) plays a critical role in regulating serotonin signaling by mediating reuptake of serotonin from the extracellular space. The molecular and cellular mechanisms controlling SERT levels in the membrane remain poorly understood. To study trafficking of surface resident....... Furthermore, internalized SERT co-localized with the lysosomal marker LysoTracker and not with transferrin. The sorting pattern was further confirmed by visualizing internalization of SERT using the fluorescent cocaine analogue JHC1-64, and by reversible and pulse chase biotinylation assays showing evidence...

  11. CCC- and WASH-mediated endosomal sorting of LDLR is required for normal clearance of circulating LDL

    Science.gov (United States)

    Bartuzi, Paulina; Billadeau, Daniel D.; Favier, Robert; Rong, Shunxing; Dekker, Daphne; Fedoseienko, Alina; Fieten, Hille; Wijers, Melinde; Levels, Johannes H.; Huijkman, Nicolette; Kloosterhuis, Niels; van der Molen, Henk; Brufau, Gemma; Groen, Albert K.; Elliott, Alison M.; Kuivenhoven, Jan Albert; Plecko, Barbara; Grangl, Gernot; McGaughran, Julie; Horton, Jay D.; Burstein, Ezra; Hofker, Marten H.; van de Sluis, Bart

    2016-01-01

    The low-density lipoprotein receptor (LDLR) plays a pivotal role in clearing atherogenic circulating low-density lipoprotein (LDL) cholesterol. Here we show that the COMMD/CCDC22/CCDC93 (CCC) and the Wiskott–Aldrich syndrome protein and SCAR homologue (WASH) complexes are both crucial for endosomal sorting of LDLR and for its function. We find that patients with X-linked intellectual disability caused by mutations in CCDC22 are hypercholesterolaemic, and that COMMD1-deficient dogs and liver-specific Commd1 knockout mice have elevated plasma LDL cholesterol levels. Furthermore, Commd1 depletion results in mislocalization of LDLR, accompanied by decreased LDL uptake. Increased total plasma cholesterol levels are also seen in hepatic COMMD9-deficient mice. Inactivation of the CCC-associated WASH complex causes LDLR mislocalization, increased lysosomal degradation of LDLR and impaired LDL uptake. Furthermore, a mutation in the WASH component KIAA0196 (strumpellin) is associated with hypercholesterolaemia in humans. Altogether, this study provides valuable insights into the mechanisms regulating cholesterol homeostasis and LDLR trafficking. PMID:26965651

  12. Old world arenaviruses enter the host cell via the multivesicular body and depend on the endosomal sorting complex required for transport.

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    Giulia Pasqual

    2011-09-01

    Full Text Available The highly pathogenic Old World arenavirus Lassa virus (LASV and the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV use α-dystroglycan as a cellular receptor and enter the host cell by an unusual endocytotic pathway independent of clathrin, caveolin, dynamin, and actin. Upon internalization, the viruses are delivered to acidified endosomes in a Rab5-independent manner bypassing classical routes of incoming vesicular trafficking. Here we sought to identify cellular factors involved in the unusual and largely unknown entry pathway of LASV and LCMV. Cell entry of LASV and LCMV required microtubular transport to late endosomes, consistent with the low fusion pH of the viral envelope glycoproteins. Productive infection with recombinant LCMV expressing LASV envelope glycoprotein (rLCMV-LASVGP and LCMV depended on phosphatidyl inositol 3-kinase (PI3K as well as lysobisphosphatidic acid (LBPA, an unusual phospholipid that is involved in the formation of intraluminal vesicles (ILV of the multivesicular body (MVB of the late endosome. We provide evidence for a role of the endosomal sorting complex required for transport (ESCRT in LASV and LCMV cell entry, in particular the ESCRT components Hrs, Tsg101, Vps22, and Vps24, as well as the ESCRT-associated ATPase Vps4 involved in fission of ILV. Productive infection with rLCMV-LASVGP and LCMV also critically depended on the ESCRT-associated protein Alix, which is implicated in membrane dynamics of the MVB/late endosomes. Our study identifies crucial cellular factors implicated in Old World arenavirus cell entry and indicates that LASV and LCMV invade the host cell passing via the MVB/late endosome. Our data further suggest that the virus-receptor complexes undergo sorting into ILV of the MVB mediated by the ESCRT, possibly using a pathway that may be linked to the cellular trafficking and degradation of the cellular receptor.

  13. Old world arenaviruses enter the host cell via the multivesicular body and depend on the endosomal sorting complex required for transport.

    Science.gov (United States)

    Pasqual, Giulia; Rojek, Jillian M; Masin, Mark; Chatton, Jean-Yves; Kunz, Stefan

    2011-09-01

    The highly pathogenic Old World arenavirus Lassa virus (LASV) and the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) use α-dystroglycan as a cellular receptor and enter the host cell by an unusual endocytotic pathway independent of clathrin, caveolin, dynamin, and actin. Upon internalization, the viruses are delivered to acidified endosomes in a Rab5-independent manner bypassing classical routes of incoming vesicular trafficking. Here we sought to identify cellular factors involved in the unusual and largely unknown entry pathway of LASV and LCMV. Cell entry of LASV and LCMV required microtubular transport to late endosomes, consistent with the low fusion pH of the viral envelope glycoproteins. Productive infection with recombinant LCMV expressing LASV envelope glycoprotein (rLCMV-LASVGP) and LCMV depended on phosphatidyl inositol 3-kinase (PI3K) as well as lysobisphosphatidic acid (LBPA), an unusual phospholipid that is involved in the formation of intraluminal vesicles (ILV) of the multivesicular body (MVB) of the late endosome. We provide evidence for a role of the endosomal sorting complex required for transport (ESCRT) in LASV and LCMV cell entry, in particular the ESCRT components Hrs, Tsg101, Vps22, and Vps24, as well as the ESCRT-associated ATPase Vps4 involved in fission of ILV. Productive infection with rLCMV-LASVGP and LCMV also critically depended on the ESCRT-associated protein Alix, which is implicated in membrane dynamics of the MVB/late endosomes. Our study identifies crucial cellular factors implicated in Old World arenavirus cell entry and indicates that LASV and LCMV invade the host cell passing via the MVB/late endosome. Our data further suggest that the virus-receptor complexes undergo sorting into ILV of the MVB mediated by the ESCRT, possibly using a pathway that may be linked to the cellular trafficking and degradation of the cellular receptor.

  14. Novel regulation of Ski protein stability and endosomal sorting by actin cytoskeleton dynamics in hepatocytes.

    Science.gov (United States)

    Vázquez-Victorio, Genaro; Caligaris, Cassandre; Del Valle-Espinosa, Eugenio; Sosa-Garrocho, Marcela; González-Arenas, Nelly R; Reyes-Cruz, Guadalupe; Briones-Orta, Marco A; Macías-Silva, Marina

    2015-02-13

    TGF-β-induced antimitotic signals are highly regulated during cell proliferation under normal and pathological conditions, such as liver regeneration and cancer. Up-regulation of the transcriptional cofactors Ski and SnoN during liver regeneration may favor hepatocyte proliferation by inhibiting TGF-β signals. In this study, we found a novel mechanism that regulates Ski protein stability through TGF-β and G protein-coupled receptor (GPCR) signaling. Ski protein is distributed between the nucleus and cytoplasm of normal hepatocytes, and the molecular mechanisms controlling Ski protein stability involve the participation of actin cytoskeleton dynamics. Cytoplasmic Ski is partially associated with actin and localized in cholesterol-rich vesicles. Ski protein stability is decreased by TGF-β/Smads, GPCR/Rho signals, and actin polymerization, whereas GPCR/cAMP signals and actin depolymerization promote Ski protein stability. In conclusion, TGF-β and GPCR signals differentially regulate Ski protein stability and sorting in hepatocytes, and this cross-talk may occur during liver regeneration.

  15. Structure and Membrane Binding Properties of the Endosomal Tetratricopeptide Repeat (TPR) Domain-containing Sorting Nexins SNX20 and SNX21.

    Science.gov (United States)

    Clairfeuille, Thomas; Norwood, Suzanne J; Qi, Xiaying; Teasdale, Rohan D; Collins, Brett M

    2015-06-01

    Sorting nexins (SNX) orchestrate membrane trafficking and signaling events required for the proper distribution of proteins within the endosomal network. Their phox homology (PX) domain acts as a phosphoinositide (PI) recognition module that targets them to specific endocytic membrane domains. The modularity of SNX proteins confers a wide variety of functions from signaling to membrane deformation and cargo binding, and many SNXs are crucial modulators of endosome dynamics and are involved in a myriad of physiological and pathological processes such as neurodegenerative diseases, cancer, and inflammation. Here, we have studied the poorly characterized SNX20 and its paralogue SNX21, which contain an N-terminal PX domain and a C-terminal PX-associated B (PXB) domain of unknown function. The two proteins share similar PI-binding properties and are recruited to early endosomal compartments by their PX domain. The crystal structure of the SNX21 PXB domain reveals a tetratricopeptide repeat (TPR)-fold, a module that typically binds short peptide motifs, with three TPR α-helical repeats. However, the C-terminal capping helix adopts a highly unusual and potentially self-inhibitory topology. SAXS solution structures of SNX20 and SNX21 show that these proteins adopt a compact globular architecture, and membrane interaction analyses indicate the presence of overlapping PI-binding sites that may regulate their intracellular localization. This study provides the first structural analysis of this poorly characterized subfamily of SNX proteins, highlighting a likely role as endosome-associated scaffolds.

  16. The coordinating role of IQGAP1 in the regulation of local, endosome-specific actin networks

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    Edward B. Samson

    2017-06-01

    Full Text Available IQGAP1 is a large, multi-domain scaffold that helps orchestrate cell signaling and cytoskeletal mechanics by controlling interactions among a spectrum of receptors, signaling intermediates, and cytoskeletal proteins. While this coordination is known to impact cell morphology, motility, cell adhesion, and vesicular traffic, among other functions, the spatiotemporal properties and regulatory mechanisms of IQGAP1 have not been fully resolved. Herein, we describe a series of super-resolution and live-cell imaging analyses that identified a role for IQGAP1 in the regulation of an actin cytoskeletal shell surrounding a novel membranous compartment that localizes selectively to the basal cortex of polarized epithelial cells (MCF-10A. We also show that IQGAP1 appears to both stabilize the actin coating and constrain its growth. Loss of compartmental IQGAP1 initiates a disassembly mechanism involving rapid and unconstrained actin polymerization around the compartment and dispersal of its vesicle contents. Together, these findings suggest IQGAP1 achieves this control by harnessing both stabilizing and antagonistic interactions with actin. They also demonstrate the utility of these compartments for image-based investigations of the spatial and temporal dynamics of IQGAP1 within endosome-specific actin networks.

  17. Rab5 activity regulates GLUT4 sorting into insulin-responsive and non-insulin-responsive endosomal compartments: a potential mechanism for development of insulin resistance.

    Science.gov (United States)

    Tessneer, Kandice L; Jackson, Robert M; Griesel, Beth A; Olson, Ann Louise

    2014-09-01

    Glucose transporter isoform 4 (GLUT4) is the insulin-responsive glucose transporter mediating glucose uptake in adipose and skeletal muscle. Reduced GLUT4 translocation from intracellular storage compartments to the plasma membrane is a cause of peripheral insulin resistance. Using a chronic hyperinsulinemia (CHI)-induced cell model of insulin resistance and Rab5 mutant overexpression, we determined these manipulations altered endosomal sorting of GLUT4, thus contributing to the development of insulin resistance. We found that CHI induced insulin resistance in 3T3-L1 adipocytes by retaining GLUT4 in a Rab5-activity-dependent compartment that is unable to equilibrate with the cell surface in response to insulin. Furthermore, CHI-mediated retention of GLUT4 in this non-insulin-responsive compartment impaired filling of the transferrin receptor (TfR)-positive and TfR-negative insulin-responsive storage compartments. Our data suggest that hyperinsulinemia may inhibit GLUT4 by chronically maintaining GLUT4 in the Rab5 activity-dependent endosomal pathway and impairing formation of the TfR-negative and TfR-positive insulin-responsive GLUT4 pools. This model suggests that an early event in the development of insulin-resistant glucose transport in adipose tissue is to alter the intracellular localization of GLUT4 to a compartment that does not efficiently equilibrate with the cell surface when insulin levels are elevated for prolonged periods of time.

  18. Neuron specific Rab4 effector GRASP-1 coordinates membrane specialization and maturation of recycling endosomes

    NARCIS (Netherlands)

    C.C. Hoogenraad (Casper); I. Popa (Ioana); K. Futai (Kensuke); E. Sanchez-Martinez (Emma); P. Wulf (Phebe); T. van Vlijmen (Thijs); B.R. Dortland (Bjorn); V. Oorschot (Viola); R. Govers (Robert); M. Monti (Maria); A.J.R. Heck (Albert); M. Sheng (Morgan); J. Klumperman (Judith); H. Rehmann (Holger); D. Jaarsma (Dick); L.C. Kapitein (Lukas); P. van der Sluijs

    2010-01-01

    textabstractThe endosomal pathway in neuronal dendrites is essential for membrane receptor trafficking and proper synaptic function and plasticity. However, the molecular mechanisms that organize specific endocytic trafficking routes are poorly understood. Here, we identify GRIP-associated protein-1

  19. Wisconsin Card Sorting Test Performance in Children with Developmental Coordination Disorder

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    Wuang, Yee-Pay; Su, Chwen-Yng; Su, Jui-Hsing

    2011-01-01

    The primary purpose of this study was to investigate and compare the executive functions measured by the Wisconsin Card Sorting Test (WCST) between children with developmental coordination disorder (DCD) and age-matched normal controls. A second purpose was to examine the relations between executive functions and school functions in DCD children.…

  20. Detection of the Endosomal Sorting Complex Required for Transport in Entamoeba histolytica and Characterization of the EhVps4 Protein

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    Israel López-Reyes

    2010-01-01

    Full Text Available Eukaryotic endocytosis involves multivesicular bodies formation, which is driven by endosomal sorting complexes required for transport (ESCRT. Here, we showed the presence and expression of homologous ESCRT genes in Entamoeba histolytica. We cloned and expressed the Ehvps4 gene, an ESCRT member, to obtain the recombinant EhVps4 and generate specific antibodies, which immunodetected EhVps4 in cytoplasm of trophozoites. Bioinformatics and biochemical studies evidenced that rEhVps4 is an ATPase, whose activity depends on the conserved E211 residue. Next, we generated trophozoites overexpressing EhVps4 and mutant EhVps4-E211Q FLAG-tagged proteins. The EhVps4-FLAG was located in cytosol and at plasma membrane, whereas the EhVps4-E211Q-FLAG was detected as abundant cytoplasmic dots in trophozoites. Erythrophagocytosis, cytopathic activity, and hepatic damage in hamsters were not improved in trophozoites overexpressing EhVps4-FLAG. In contrast, EhVps4-E211Q-FLAG protein overexpression impaired these properties. The localization of EhVps4-FLAG around ingested erythrocytes, together with our previous results, strengthens the role for EhVps4 in E. histolytica phagocytosis and virulence.

  1. Detection of the Endosomal Sorting Complex Required for Transport in Entamoeba histolytica and Characterization of the EhVps4 Protein

    Science.gov (United States)

    López-Reyes, Israel; García-Rivera, Guillermina; Bañuelos, Cecilia; Herranz, Silvia; Vincent, Olivier; López-Camarillo, César; Marchat, Laurence A.; Orozco, Esther

    2010-01-01

    Eukaryotic endocytosis involves multivesicular bodies formation, which is driven by endosomal sorting complexes required for transport (ESCRT). Here, we showed the presence and expression of homologous ESCRT genes in Entamoeba histolytica. We cloned and expressed the Ehvps4 gene, an ESCRT member, to obtain the recombinant EhVps4 and generate specific antibodies, which immunodetected EhVps4 in cytoplasm of trophozoites. Bioinformatics and biochemical studies evidenced that rEhVps4 is an ATPase, whose activity depends on the conserved E211 residue. Next, we generated trophozoites overexpressing EhVps4 and mutant EhVps4-E211Q FLAG-tagged proteins. The EhVps4-FLAG was located in cytosol and at plasma membrane, whereas the EhVps4-E211Q-FLAG was detected as abundant cytoplasmic dots in trophozoites. Erythrophagocytosis, cytopathic activity, and hepatic damage in hamsters were not improved in trophozoites overexpressing EhVps4-FLAG. In contrast, EhVps4-E211Q-FLAG protein overexpression impaired these properties. The localization of EhVps4-FLAG around ingested erythrocytes, together with our previous results, strengthens the role for EhVps4 in E. histolytica phagocytosis and virulence. PMID:20508821

  2. Distinct mechanisms of recognizing endosomal sorting complex required for transport III (ESCRT-III) protein IST1 by different microtubule interacting and trafficking (MIT) domains.

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    Guo, Emily Z; Xu, Zhaohui

    2015-03-27

    The endosomal sorting complex required for transport (ESCRT) machinery is responsible for membrane remodeling in a number of biological processes including multivesicular body biogenesis, cytokinesis, and enveloped virus budding. In mammalian cells, efficient abscission during cytokinesis requires proper function of the ESCRT-III protein IST1, which binds to the microtubule interacting and trafficking (MIT) domains of VPS4, LIP5, and Spartin via its C-terminal MIT-interacting motif (MIM). Here, we studied the molecular interactions between IST1 and the three MIT domain-containing proteins to understand the structural basis that governs pairwise MIT-MIM interaction. Crystal structures of the three molecular complexes revealed that IST1 binds to the MIT domains of VPS4, LIP5, and Spartin using two different mechanisms (MIM1 mode versus MIM3 mode). Structural comparison revealed that structural features in both MIT and MIM contribute to determine the specific binding mechanism. Within the IST1 MIM sequence, two phenylalanine residues were shown to be important in discriminating MIM1 versus MIM3 binding. These observations enabled us to deduce a preliminary binding code, which we applied to provide CHMP2A, a protein that normally only binds the MIT domain in the MIM1 mode, the additional ability to bind the MIT domain of Spartin in the MIM3 mode. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Distinct Mechanisms of Recognizing Endosomal Sorting Complex Required for Transport III (ESCRT-III) Protein IST1 by Different Microtubule Interacting and Trafficking (MIT) Domains*

    Science.gov (United States)

    Guo, Emily Z.; Xu, Zhaohui

    2015-01-01

    The endosomal sorting complex required for transport (ESCRT) machinery is responsible for membrane remodeling in a number of biological processes including multivesicular body biogenesis, cytokinesis, and enveloped virus budding. In mammalian cells, efficient abscission during cytokinesis requires proper function of the ESCRT-III protein IST1, which binds to the microtubule interacting and trafficking (MIT) domains of VPS4, LIP5, and Spartin via its C-terminal MIT-interacting motif (MIM). Here, we studied the molecular interactions between IST1 and the three MIT domain-containing proteins to understand the structural basis that governs pairwise MIT-MIM interaction. Crystal structures of the three molecular complexes revealed that IST1 binds to the MIT domains of VPS4, LIP5, and Spartin using two different mechanisms (MIM1 mode versus MIM3 mode). Structural comparison revealed that structural features in both MIT and MIM contribute to determine the specific binding mechanism. Within the IST1 MIM sequence, two phenylalanine residues were shown to be important in discriminating MIM1 versus MIM3 binding. These observations enabled us to deduce a preliminary binding code, which we applied to provide CHMP2A, a protein that normally only binds the MIT domain in the MIM1 mode, the additional ability to bind the MIT domain of Spartin in the MIM3 mode. PMID:25657007

  4. Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT Machinery via Ubiquitination To Facilitate Viral Envelopment

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    Rina Barouch-Bentov

    2016-11-01

    Full Text Available Enveloped viruses commonly utilize late-domain motifs, sometimes cooperatively with ubiquitin, to hijack the endosomal sorting complex required for transport (ESCRT machinery for budding at the plasma membrane. However, the mechanisms underlying budding of viruses lacking defined late-domain motifs and budding into intracellular compartments are poorly characterized. Here, we map a network of hepatitis C virus (HCV protein interactions with the ESCRT machinery using a mammalian-cell-based protein interaction screen and reveal nine novel interactions. We identify HRS (hepatocyte growth factor-regulated tyrosine kinase substrate, an ESCRT-0 complex component, as an important entry point for HCV into the ESCRT pathway and validate its interactions with the HCV nonstructural (NS proteins NS2 and NS5A in HCV-infected cells. Infectivity assays indicate that HRS is an important factor for efficient HCV assembly. Specifically, by integrating capsid oligomerization assays, biophysical analysis of intracellular viral particles by continuous gradient centrifugations, proteolytic digestion protection, and RNase digestion protection assays, we show that HCV co-opts HRS to mediate a late assembly step, namely, envelopment. In the absence of defined late-domain motifs, K63-linked polyubiquitinated lysine residues in the HCV NS2 protein bind the HRS ubiquitin-interacting motif to facilitate assembly. Finally, ESCRT-III and VPS/VTA1 components are also recruited by HCV proteins to mediate assembly. These data uncover involvement of ESCRT proteins in intracellular budding of a virus lacking defined late-domain motifs and a novel mechanism by which HCV gains entry into the ESCRT network, with potential implications for other viruses.

  5. Negative regulation of phosphatidylinositol 3-phosphate levels in early-to-late endosome conversion.

    Science.gov (United States)

    Liu, Kai; Jian, Youli; Sun, Xiaojuan; Yang, Chengkui; Gao, Zhiyang; Zhang, Zhili; Liu, Xuezhao; Li, Yang; Xu, Jing; Jing, Yudong; Mitani, Shohei; He, Sudan; Yang, Chonglin

    2016-01-18

    Phosphatidylinositol 3-phosphate (PtdIns3P) plays a central role in endosome fusion, recycling, sorting, and early-to-late endosome conversion, but the mechanisms that determine how the correct endosomal PtdIns3P level is achieved remain largely elusive. Here we identify two new factors, SORF-1 and SORF-2, as essential PtdIns3P regulators in Caenorhabditis elegans. Loss of sorf-1 or sorf-2 leads to greatly elevated endosomal PtdIns3P, which drives excessive fusion of early endosomes. sorf-1 and sorf-2 function coordinately with Rab switching genes to inhibit synthesis of PtdIns3P, allowing its turnover for endosome conversion. SORF-1 and SORF-2 act in a complex with BEC-1/Beclin1, and their loss causes elevated activity of the phosphatidylinositol 3-kinase (PI3K) complex. In mammalian cells, inactivation of WDR91 and WDR81, the homologs of SORF-1 and SORF-2, induces Beclin1-dependent enlargement of PtdIns3P-enriched endosomes and defective degradation of epidermal growth factor receptor. WDR91 and WDR81 interact with Beclin1 and inhibit PI3K complex activity. These findings reveal a conserved mechanism that controls appropriate PtdIns3P levels in early-to-late endosome conversion.

  6. SNX12 role in endosome membrane transport.

    Directory of Open Access Journals (Sweden)

    Véronique Pons

    Full Text Available In this paper, we investigated the role of sorting nexin 12 (SNX12 in the endocytic pathway. SNX12 is a member of the PX domain-containing sorting nexin family and shares high homology with SNX3, which plays a central role in the formation of intralumenal vesicles within multivesicular endosomes. We found that SNX12 is expressed at very low levels compared to SNX3. SNX12 is primarily associated with early endosomes and this endosomal localization depends on the binding to 3-phosphoinositides. We find that overexpression of SNX12 prevents the detachment (or maturation of multivesicular endosomes from early endosomes. This in turn inhibits the degradative pathway from early to late endosomes/lysosomes, much like SNX3 overexpression, without affecting endocytosis, recycling and retrograde transport. In addition, while previous studies showed that Hrs knockdown prevents EGF receptor sorting into multivesicular endosomes, we find that overexpression of SNX12 restores the sorting process in an Hrs knockdown background. Altogether, our data show that despite lower expression level, SNX12 shares redundant functions with SNX3 in the biogenesis of multivesicular endosomes.

  7. Interplay of Endosomal pH and Ligand Occupancy in Integrin α5β1 Ubiquitination, Endocytic Sorting, and Cell Migration

    Directory of Open Access Journals (Sweden)

    Dmitri Kharitidi

    2015-10-01

    Full Text Available Membrane trafficking of integrins plays a pivotal role in cell proliferation and migration. How endocytosed integrins are targeted either for recycling or lysosomal delivery is not fully understood. Here, we show that fibronectin (FN binding to α5β1 integrin triggers ubiquitination and internalization of the receptor complex. Acidification facilitates FN dissociation from integrin α5β1 in vitro and in early endosomes, promoting receptor complex deubiquitination by the USP9x and recycling to the cell surface. Depending on residual ligand occupancy of receptors, some α5β1 integrins remain ubiquitinated and are captured by ESCRT-0/I, containing histidine domain-containing protein tyrosine phosphatase (HD-PTP and ubiquitin-associated protein 1 (UBAP1, and are directed for lysosomal proteolysis, limiting receptor downstream signaling and cell migration. Thus, HD-PTP or UBAP1 depletion confers a pro-invasive phenotype. Thus, pH-dependent FN-integrin dissociation and deubiquitination of the activated integrin α5β1 are required for receptor resensitization and cell migration, representing potential targets to modulate tumor invasiveness.

  8. The endosomal sorting complex required for transport (ESCRT) is required for the sensitivity of yeast cells to nickel ions in Saccharomyces cerevisiae.

    Science.gov (United States)

    Luo, Chong; Cao, Chunlei; Jiang, Linghuo

    2016-05-01

    Nickel is one of the toxic environment metal pollutants and is linked to various human diseases. In this study, through a functional genomics approach we have identified 16 nickel-sensitive and 22 nickel-tolerant diploid deletion mutants of budding yeast genes, many of which are novel players in the regulation of nickel homeostasis. The 16 nickel-sensitive mutants are of genes mainly involved in the protein folding, modification and destination and the cellular transport processes, while the 22 nickel-tolerant mutants are of genes encoding components of ESCRT complexes as well as protein factors involved in both the cell wall integrity maintenance and the vacuolar protein sorting process. In consistence with their phenotypes, most of these nickel-sensitive mutants show reduced intracellular nickel contents, while the majority of these nickel-tolerant mutants show elevated intracellular nickel contents, as compared to the wild type in response to nickel stress. Our data provides a basis for our understanding the regulation of nickel homeostasis and molecular mechanisms of nickel-induced human pathogenesis.

  9. Cell Sorting and Noise-Induced Cell Plasticity Coordinate to Sharpen Boundaries between Gene Expression Domains

    Science.gov (United States)

    2017-01-01

    A fundamental question in biology is how sharp boundaries of gene expression form precisely in spite of biological variation/noise. Numerous mechanisms position gene expression domains across fields of cells (e.g. morphogens), but how these domains are refined remains unclear. In some cases, domain boundaries sharpen through differential adhesion-mediated cell sorting. However, boundaries can also sharpen through cellular plasticity, with cell fate changes driven by up- or down-regulation of gene expression. In this context, we have argued that noise in gene expression can help cells transition to the correct fate. Here we investigate the efficacy of cell sorting, gene expression plasticity, and their combination in boundary sharpening using multi-scale, stochastic models. We focus on the formation of hindbrain segments (rhombomeres) in the developing zebrafish as an example, but the mechanisms investigated apply broadly to many tissues. Our results indicate that neither sorting nor plasticity is sufficient on its own to sharpen transition regions between different rhombomeres. Rather the two have complementary strengths and weaknesses, which synergize when combined to sharpen gene expression boundaries. PMID:28135279

  10. The structure and function of presynaptic endosomes

    Energy Technology Data Exchange (ETDEWEB)

    Jähne, Sebastian, E-mail: sebastian.jaehne1@stud.uni-goettingen.de [Department of Neuro- and Sensory Physiology, University of Göttingen Medical Center, Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain, Humboldtallee 23, 37073 Göttingen (Germany); International Max Planck Research School for Neurosciences, 37077 Göttingen (Germany); Rizzoli, Silvio O. [Department of Neuro- and Sensory Physiology, University of Göttingen Medical Center, Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain, Humboldtallee 23, 37073 Göttingen (Germany); Helm, Martin S., E-mail: martin.helm@med.uni-goettingen.de [Department of Neuro- and Sensory Physiology, University of Göttingen Medical Center, Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain, Humboldtallee 23, 37073 Göttingen (Germany); International Max Planck Research School for Molecular Biology, 37077 Göttingen (Germany)

    2015-07-15

    The function of endosomes and of endosome-like structures in the presynaptic compartment is still controversial. This is in part due to the absence of a consensus on definitions and markers for these compartments. Synaptic endosomes are sometimes seen as stable organelles, permanently present in the synapse. Alternatively, they are seen as short-lived intermediates in synaptic vesicle recycling, arising from the endocytosis of large vesicles from the plasma membrane, or from homotypic fusion of small vesicles. In addition, the potential function of the endosome is largely unknown in the synapse. Some groups have proposed that the endosome is involved in the sorting of synaptic vesicle proteins, albeit others have produced data that deny this possibility. In this review, we present the existing evidence for synaptic endosomes, we discuss their potential functions, and we highlight frequent technical pitfalls in the analysis of this elusive compartment. We also sketch a roadmap to definitely determine the role of synaptic endosomes for the synaptic vesicle cycle. Finally, we propose a common definition of synaptic endosome-like structures.

  11. Nanogold labeling of the yeast endosomal system for ultrastructural analyses

    NARCIS (Netherlands)

    Mari, Muriel; Griffith, Janice; Reggiori, Fulvio

    2014-01-01

    Endosomes are one of the major membrane sorting checkpoints in eukaryotic cells and they regulate recycling or destruction of proteins mostly from the plasma membrane and the Golgi. As a result the endosomal system plays a central role in maintaining cell homeostasis, and mutations in genes belongin

  12. Resolving sorting mechanisms into exosomes

    NARCIS (Netherlands)

    Stoorvogel, Willem

    2015-01-01

    The complexity of mechanisms driving protein sorting into exosomes is only beginning to emerge. In a paper recently published in Cell Research, Roucourt et al. report that trimming of heparan sulfate side chains of syndecans by endosomal heparanase facilitates sorting into exosomes by the formation

  13. Structure and function of endosomes in plant cells.

    Science.gov (United States)

    Contento, Anthony L; Bassham, Diane C

    2012-08-01

    Endosomes are a heterogeneous collection of organelles that function in the sorting and delivery of internalized material from the cell surface and the transport of materials from the Golgi to the lysosome or vacuole. Plant endosomes have some unique features, with an organization distinct from that of yeast or animal cells. Two clearly defined endosomal compartments have been studied in plant cells, the trans-Golgi network (equivalent to the early endosome) and the multivesicular body (equivalent to the late endosome), with additional endosome types (recycling endosome, late prevacuolar compartment) also a possibility. A model has been proposed in which the trans-Golgi network matures into a multivesicular body, which then fuses with the vacuole to release its cargo. In addition to basic trafficking functions, endosomes in plant cells are known to function in maintenance of cell polarity by polar localization of hormone transporters and in signaling pathways after internalization of ligand-bound receptors. These signaling functions are exemplified by the BRI1 brassinosteroid hormone receptor and by receptors for pathogen elicitors that activate defense responses. After endocytosis of these receptors from the plasma membrane, endosomes act as a signaling platform, thus playing an essential role in plant growth, development and defense responses. Here we describe the key features of plant endosomes and their differences from those of other organisms and discuss the role of these organelles in cell polarity and signaling pathways.

  14. Vps1 in the late endosome-to-vacuole traffic

    Indian Academy of Sciences (India)

    Jacob Hayden; Michelle Williams; Ann Granich; Hyoeun Ahn; Brandon Tenay; Joshua Lukehart; Chad Highfill; Sarah Dobard; Kyoungtae Kim

    2013-03-01

    Vacuolar protein sorting 1 (Vps1), the yeast homolog to human dynamin, is a GTP hydrolyzing protein, which plays an important role in protein sorting and targeting between the Golgi and late endosomal compartments. In this study, we assessed the functional significance of Vps1 in the membrane traffic towards the vacuole. We show here that vps1 cells accumulated FM4-64 to a greater extent than wild-type (WT) cells, suggesting slower endocytic degradation traffic toward the vacuole. In addition, we observed that two endosome-to-vacuole traffic markers, DsRed-FYVE and Ste2-GFP, were highly accumulated in Vps1-deficient cells, further supporting Vps1’s implication in efficient trafficking of endocytosed materials to the vacuole. Noteworthy, a simultaneous imaging analysis in conjunction with FM4-64 pulse-chase experiment further revealed that Vps1 plays a role in late endosome to the vacuole transport. Consistently, our subcellular localization analysis showed that Vps1 is present at the late endosome. The hyperaccumulation of endosomal intermediates in the vps1 mutant cells appears to be caused by the disruption of integrity of HOPS tethering complexes, manifested by mislocalization of Vps39 to the cytoplasm. Finally, we postulate that Vps1 functions together with the Endosomal Sorting Complex Required for Transport (ESCRT) complex at the late endosomal compartments, based on the observation that the double mutants, in which VPS1 along with singular ESCRT I, II and III genes have been disrupted, exhibited synthetic lethality. Together, we propose that Vps1 is required for correct and efficient trafficking from the late endosomal compartments to the vacuole.

  15. An Essential Role of Hrs/Vps27 in Endosomal Cholesterol Trafficking

    Directory of Open Access Journals (Sweden)

    Ximing Du

    2012-01-01

    Full Text Available The endosomal sorting complex required for transport (ESCRT plays a crucial role in the degradation of ubiquitinated endosomal membrane proteins. Here, we report that Hrs, a key protein of the ESCRT-0 complex, is required for the transport of low-density lipoprotein-derived cholesterol from endosomes to the endoplasmic reticulum. This function of Hrs in cholesterol transport is distinct from its previously defined role in lysosomal sorting and downregulation of membrane receptors via the ESCRT pathway. In line with this, knocking down other ESCRT proteins does not cause prominent endosomal cholesterol accumulation. Importantly, the localization and biochemical properties of key cholesterol-sorting proteins, NPC1 and NPC2, appear to be unchanged upon Hrs knockdown. Our data identify Hrs as a regulator of endosomal cholesterol trafficking and provide additional insights into the budding of intralumenal vesicles.

  16. Recycling endosomes in apical plasma membrane domain formation and epithelial cell polarity

    NARCIS (Netherlands)

    Golachowska, Magdalena R.; Hoekstra, Dick; van IJzendoorn, Sven C. D.

    2010-01-01

    Recycling endosomes have taken central stage in the intracellular sorting and polarized trafficking of apical and basolateral plasma membrane components. Molecular players in the underlying mechanisms are now emerging, including small GTPases, class V myosins and adaptor proteins. In particular,

  17. Structural basis for endosomal recruitment of ESCRT-I by ESCRT-0 in yeast

    Energy Technology Data Exchange (ETDEWEB)

    Ren, Xuefeng; Hurley, James H. (NIH)

    2011-10-28

    The ESCRT-0 and ESCRT-I complexes coordinate the clustering of ubiquitinated cargo with intralumenal budding of the endosomal membrane, two essential steps in vacuolar/lysosomal protein sorting from yeast to humans. The 1.85-{angstrom} crystal structure of interacting regions of the yeast ESCRT-0 and ESCRT-I complexes reveals that PSDP motifs of the Vps27 ESCRT-0 subunit bind to a novel electropositive N-terminal site on the UEV domain of the ESCRT-I subunit Vps23 centred on Trp16. This novel site is completely different from the C-terminal part of the human UEV domain that binds to P(S/T)AP motifs of human ESCRT-0 and HIV-1 Gag. Disruption of the novel PSDP-binding site eliminates the interaction in vitro and blocks enrichment of Vps23 in endosome-related class E compartments in yeast cells. However, this site is non-essential for sorting of the ESCRT cargo Cps1. Taken together, these results show how a conserved motif/domain pair can evolve to use strikingly different binding modes in different organisms.

  18. Use of a Card Sort Task to Assess Students' Ability to Coordinate Three Levels of Representation in Chemistry

    Science.gov (United States)

    Irby, Stefan M.; Phu, Andy L.; Borda, Emily J.; Haskell, Todd R.; Steed, Nicole; Meyer, Zachary

    2016-01-01

    There is much agreement among chemical education researchers that expertise in chemistry depends in part on the ability to coordinate understanding of phenomena on three levels: macroscopic (observable), sub-microscopic (atoms, molecules, and ions) and symbolic (chemical equations, graphs, etc.). We hypothesize this "level-coordination…

  19. Endosomal cholesterol trafficking: protein factors at a glance

    Institute of Scientific and Technical Information of China (English)

    Ximing Du; Hongyuan Yang

    2013-01-01

    The delivery of low-density lipoprotein-derived cholesterol (LDL-C) from endosomal compartments to the plasma membrane and the endoplasmic reticulum (ER) is an important yet poorly understood cellular process.NiemannPick C1 (NPC1),a multi-pass integral membrane protein on the limiting membranes of late endosomes (LE)/lysosomes (Ly),is known to insert lumenal LDL-C to the limiting membrane of LE/Ly.Recent progress has identified novel cytoplasmic proteins that regulate the exit of LDL-C from LE/Ly,such as ORP5,a member of the oxysterolbinding protein-related protein (ORPs) family,and Hrs/VPS27,a well-established regulator of the endosomal sorting complex required for transport pathway.Whereas ORP5/ORPs may serve as cytosolic cholesterol carriers and deliver cholesterol in a non-vesicular manner,how Hrs/VPS27 regulate endosomal cholesterol sorting remains enigmatic.We discuss the functional relationship between NPC1,Hrs,and ORP5,and formulate possible schemes on how LDL-C may be moved from endosomal compartments to other cellular organelles.

  20. A mechanism for retromer endosomal coat complex assembly with cargo.

    Science.gov (United States)

    Harrison, Megan S; Hung, Chia-Sui; Liu, Ting-ting; Christiano, Romain; Walther, Tobias C; Burd, Christopher G

    2014-01-07

    Retromer is an evolutionarily conserved protein complex composed of the VPS26, VPS29, and VPS35 proteins that selects and packages cargo proteins into transport carriers that export cargo from the endosome. The mechanisms by which retromer is recruited to the endosome and captures cargo are unknown. We show that membrane recruitment of retromer is mediated by bivalent recognition of an effector of PI3K, SNX3, and the RAB7A GTPase, by the VPS35 retromer subunit. These bivalent interactions prime retromer to capture integral membrane cargo, which enhances membrane association of retromer and initiates cargo sorting. The role of RAB7A is severely impaired by a mutation, K157N, that causes Charcot-Marie-Tooth neuropathy 2B. The results elucidate minimal requirements for retromer assembly on the endosome membrane and reveal how PI3K and RAB signaling are coupled to initiate retromer-mediated cargo export.

  1. Imaging and Quantitation Techniques for Tracking Cargo along Endosome-to-Golgi Transport Pathways

    Directory of Open Access Journals (Sweden)

    Paul A. Gleeson

    2013-02-01

    Full Text Available Recent improvements in the resolution of light microscopy, coupled with the development of a range of fluorescent-based probes, have provided new approaches to dissecting membrane domains and the regulation of membrane trafficking. Here, we review these advances, as well as highlight developments in quantitative image analysis and novel unbiased analytical approaches to quantitate protein localization. The application of these approaches to endosomal sorting and endosome-to-Golgi transport is discussed.

  2. Transport to late endosomes is required for efficient reovirus infection.

    Science.gov (United States)

    Mainou, Bernardo A; Dermody, Terence S

    2012-08-01

    Rab GTPases play an essential role in vesicular transport by coordinating the movement of various types of cargo from one cellular compartment to another. Individual Rab GTPases are distributed to specific organelles and thus serve as markers for discrete types of endocytic vesicles. Mammalian reovirus binds to cell surface glycans and junctional adhesion molecule-A (JAM-A) and enters cells by receptor-mediated endocytosis in a process dependent on β1 integrin. Within organelles of the endocytic compartment, reovirus undergoes stepwise disassembly catalyzed by cathepsin proteases, which allows the disassembly intermediate to penetrate endosomal membranes and release the transcriptionally active viral core into the cytoplasm. The pathway used by reovirus to traverse the endocytic compartment is largely unknown. In this study, we found that reovirus particles traffic through early, late, and recycling endosomes during cell entry. After attachment to the cell surface, reovirus particles and JAM-A codistribute into each of these compartments. Transfection of cells with constitutively active and dominant-negative Rab GTPases that affect early and late endosome biogenesis and maturation influenced reovirus infectivity. In contrast, reovirus infectivity was not altered in cells expressing mutant Rab GTPases that affect recycling endosomes. Thus, reovirus virions localize to early, late, and recycling endosomes during entry into host cells, but only those that traverse early and late endosomes yield a productive infection.

  3. Fission of SNX-BAR-coated endosomal retrograde transport carriers is promoted by the dynamin-related protein Vps1.

    Science.gov (United States)

    Chi, Richard J; Liu, Jingxuan; West, Matthew; Wang, Jing; Odorizzi, Greg; Burd, Christopher G

    2014-03-03

    Retromer is an endosomal sorting device that orchestrates capture and packaging of cargo into transport carriers coated with sorting nexin BAR domain proteins (SNX-BARs). We report that fission of retromer SNX-BAR-coated tubules from yeast endosomes is promoted by Vps1, a dynamin-related protein that localizes to endosomes decorated by retromer SNX-BARs and Mvp1, a SNX-BAR that is homologous to human SNX8. Mvp1 exhibits potent membrane remodeling activity in vitro, and it promotes association of Vps1 with the endosome in vivo. Retrograde transport carriers bud from the endosome coated by retromer and Mvp1, and cargo export is deficient in mvp1- and vps1-null cells, but with distinct endpoints; cargo export is delayed in mvp1-null cells, but cargo export completely fails in vps1-null cells. The results indicate that Mvp1 promotes Vps1-mediated fission of retromer- and Mvp1-coated tubules that bud from the endosome, revealing a functional link between the endosomal sorting and fission machineries to produce retrograde transport carriers.

  4. Transport of the cholera toxin B-subunit from recycling endosomes to the Golgi requires clathrin and AP-1.

    Science.gov (United States)

    Matsudaira, Tatsuyuki; Niki, Takahiro; Taguchi, Tomohiko; Arai, Hiroyuki

    2015-08-15

    The retrograde pathway is defined by the transport of proteins and lipids from the plasma membrane through endosomes to the Golgi complex, and is essential for a variety of cellular activities. Recycling endosomes are important sorting stations for some retrograde cargo. SMAP2, a GTPase-activating protein (GAP) for Arf1 with a putative clathrin-binding domain, has previously been shown to participate in the retrograde transport of the cholera toxin B-subunit (CTxB) from recycling endosomes. Here, we found that clathrin, a vesicle coat protein, and clathrin adaptor protein complex 1 (AP-1) were present at recycling endosomes and were needed for the retrograde transport of CTxB from recycling endosomes to the Golgi, but not from the plasma membrane to recycling endosomes. SMAP2 immunoprecipitated clathrin and AP-1 through a putative clathrin-binding domain and a CALM-binding domain, and SMAP2 mutants that did not interact with clathrin or AP-1 could not localize to recycling endosomes. Moreover, knockdown of Arf1 suppressed the retrograde transport of CTxB from recycling endosomes to the Golgi. These findings suggest that retrograde transport is mediated by clathrin-coated vesicles from recycling endosomes and that the role of the coat proteins is in the recruitment of Arf GAP to transport vesicles.

  5. Sorting of Clathrin-Independent Cargo Proteins Depends on Rab35 Delivered by Clathrin-Mediated Endocytosis.

    Science.gov (United States)

    Dutta, Dipannita; Donaldson, Julie G

    2015-09-01

    Clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE) co-exist in most cells but little is known about their communication and coordination. Here we show that when CME was inhibited, endocytosis by CIE continued but endosomal trafficking of CIE cargo proteins was altered. CIE cargo proteins that normally traffic directly into Arf6-associated tubules after internalization and avoid degradation (CD44, CD98 and CD147) now trafficked to lysosomes and were degraded. The endosomal tubules were also absent and Arf6-GTP levels were elevated. The altered trafficking, loss of the tubular endosomal network and elevated Arf6-GTP levels caused by inhibition of CME were rescued by expression of Rab35, a Rab associated with clathrin-coated vesicles, or its effector ACAPs, Arf6 GTPase activating proteins (GAP) that inactivate Arf6. Furthermore, siRNA knockdown of Rab35 recreated the phenotype of CME ablation on CIE cargo trafficking without altering endocytosis of transferrin. These observations suggest that Rab35 serves as a CME detector and that loss of CME, or Rab35 input, leads to elevated Arf6-GTP and shifts the sorting of CIE cargo proteins to lysosomes and degradation.

  6. Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7

    Science.gov (United States)

    Rojas, Raul; van Vlijmen, Thijs; Mardones, Gonzalo A.; Prabhu, Yogikala; Rojas, Adriana L.; Mohammed, Shabaz; Heck, Albert J.R.; Raposo, Graça; van der Sluijs, Peter; Bonifacino, Juan S.

    2008-01-01

    The retromer complex mediates retrograde transport of transmembrane cargo from endosomes to the trans-Golgi network (TGN). Mammalian retromer is composed of a sorting nexin (SNX) dimer that binds to phosphatidylinositol 3-phosphate–enriched endosomal membranes and a vacuolar protein sorting (Vps) 26/29/35 trimer that participates in cargo recognition. The mammalian SNX dimer is necessary but not sufficient for recruitment of the Vps26/29/35 trimer to membranes. In this study, we demonstrate that the guanosine triphosphatase Rab7 contributes to this recruitment. The Vps26/29/35 trimer specifically binds to Rab7–guanosine triphosphate (GTP) and localizes to Rab7-containing endosomal domains. Interference with Rab7 function causes dissociation of the Vps26/29/35 trimer but not the SNX dimer from membranes. This blocks retrieval of mannose 6-phosphate receptors to the TGN and impairs cathepsin D sorting. Rab5-GTP does not bind to the Vps26/29/35 trimer, but perturbation of Rab5 function causes dissociation of both the SNX and Vps26/29/35 components from membranes through inhibition of a pathway involving phosphatidylinositol 3-kinase. These findings demonstrate that Rab5 and Rab7 act in concert to regulate retromer recruitment to endosomes. PMID:18981234

  7. Antigen processing and remodeling of the endosomal pathway: requirements for antigen cross-presentation.

    Science.gov (United States)

    Compeer, Ewoud Bernardus; Flinsenberg, Thijs Willem Hendrik; van der Grein, Susanna Geertje; Boes, Marianne

    2012-01-01

    Cross-presentation of endocytosed antigen as peptide/class I major histocompatibility complex complexes plays a central role in the elicitation of CD8(+) T cell clones that mediate anti-viral and anti-tumor immune responses. While it has been clear that there are specific subsets of professional antigen presenting cells capable of antigen cross-presentation, identification of mechanisms involved is still ongoing. Especially amongst dendritic cells (DC), there are specialized subsets that are highly proficient at antigen cross-presentation. We here present a focused survey on the cell biological processes in the endosomal pathway that support antigen cross-presentation. This review highlights DC-intrinsic mechanisms that facilitate the cross-presentation of endocytosed antigen, including receptor-mediated uptake, maturation-induced endosomal sorting of membrane proteins, dynamic remodeling of endosomal structures and cell surface-directed endosomal trafficking. We will conclude with the description of pathogen-induced deviation of endosomal processing, and discuss how immune evasion strategies pertaining endosomal trafficking may preclude antigen cross-presentation.

  8. Sorting choanoflagellates

    Science.gov (United States)

    Marconi, Veronica I.; Miño, Gaston L.; Sparacino, Javier; Banchio, Adolfo J.; Condat, Carlos A.; Koehl, Mimi A. R.; King, Nicole; Stocker, Roman

    2015-03-01

    In freshwater environments, as well as in oceans, environmental conditions are in constant fluctuation. Some heterotrophic plankton must adapt their swimming behavior in order to survive under these conditions. In the case of the choanoflagellate, the closest animal ancestor, the ability to forage for food is given not only by its single flagellum, but also by its differentiation between fast and slow swimmers. The understanding of how these cells with different strategies to swim search for food can give us a better insight into how eukaryotes respond to different stimuli. In this work, we have designed a microfluidic device that sorts choanoflagellates by their speed. The optimal geometry was found by a numerical model using the experimentally determined motilities of each swimmer type.

  9. Hrs and SNX3 functions in sorting and membrane invagination within multivesicular bodies.

    Directory of Open Access Journals (Sweden)

    Véronique Pons

    2008-09-01

    Full Text Available After internalization, ubiquitinated signaling receptors are delivered to early endosomes. There, they are sorted and incorporated into the intralumenal invaginations of nascent multivesicular bodies, which function as transport intermediates to late endosomes. Receptor sorting is achieved by Hrs--an adaptor--like protein that binds membrane PtdIns3P via a FYVE motif-and then by ESCRT complexes, which presumably also mediate the invagination process. Eventually, intralumenal vesicles are delivered to lysosomes, leading to the notion that EGF receptor sorting into multivesicular bodies mediates lysosomal targeting. Here, we report that Hrs is essential for lysosomal targeting but dispensable for multivesicular body biogenesis and transport to late endosomes. By contrast, we find that the PtdIns3P-binding protein SNX3 is required for multivesicular body formation, but not for EGF receptor degradation. PtdIns3P thus controls the complementary functions of Hrs and SNX3 in sorting and multivesicular body biogenesis.

  10. Membrane tethering complexes in the endosomal system

    Directory of Open Access Journals (Sweden)

    Anne eSpang

    2016-05-01

    Full Text Available Vesicles that are generated by endocytic events at the plasma membrane are destined to early endosomes. A prerequisite for proper fusion is the tethering of two membrane entities. Tethering of vesicles to early endosomes is mediated by the CORVET complex, while fusion of late endosomes with lysosomes depends on the HOPS complex. Recycling through the TGN and to the plasma membrane is facilitated by the GARP and EARP complexes, respectively. However, there are other tethering functions in the endosomal system as there are multiple pathways through which proteins can be delivered from endosomes to either the TGN or the plasma membrane. Furthermore, complexes that may be part of novel tethering complexes have been recently identified. Thus it is likely that more tethering factors exist. In this review, I will provide an overview of different tethering complexes of the endosomal system and discuss how they may provide specificity in membrane traffic.

  11. Efficient sorting of Bessel beams [Conference paper

    CSIR Research Space (South Africa)

    Mhlanga, T

    2013-02-01

    Full Text Available A procedure to efficiently sort orbital angular momentum (OAM) states of light, by performing a Cartesian to log-polar coordinate transformation which translates helically phased beams into a transverse phase gradient, currently exists1. We...

  12. Identification of the ubiquitin ligase Triad1 as a regulator of endosomal transport

    Directory of Open Access Journals (Sweden)

    Gerco Hassink

    2012-05-01

    The ubiquitin system plays an important role in trafficking of signaling receptors from the plasma membrane to lysosomes. Triad1 is a ubiquitin ligase that catalyzes the formation of poly-ubiquitin chains linked via lysine-48 as well as lysine-63 residues. We show that depletion of Triad1 affects the sorting of both growth hormone and epidermal growth factor. Triad1-depleted cells accumulate both ligands in endosomes. While fluid phase transport to the lysosomes is reduced in the absence of Triad1, growth hormone receptor can recycle back to the plasma membrane together with transferrin. Using immune electron microscopy we show that Triad1 depletion results in enlarged endosomes with enlarged and irregular shaped intraluminal vesicles. The endosomes display prominent clathrin coats and show increased levels of growth hormone label. We conclude that Triad1 is required for the proper function of multivesicular bodies.

  13. Human ClC-6 is a late endosomal glycoprotein that associates with detergent-resistant lipid domains.

    Directory of Open Access Journals (Sweden)

    Sofie Ignoul

    and ClC-7 when cotransfected in COS-1 cells. CONCLUSIONS: We conclude that human ClC-6 is an endosomal glycoprotein that partitions in detergent resistant lipid domains. The differential sorting of endogenous (late endosomal versus overexpressed (early and recycling endosomal ClC-6 is reminiscent of that of other late endosomal/lysosomal membrane proteins (e.g. LIMP II, and is consistent with a rate-limiting sorting step for ClC-6 between early endosomes and its final destination in late endosomes.

  14. Antigen processing and remodeling of the endosomal pathway: requirements for antigen cross-presentation.

    Directory of Open Access Journals (Sweden)

    Ewoud Bernardus Compeer

    2012-03-01

    Full Text Available The cross-presentation of endocytosed antigen as peptide/class I MHC complexes plays a central role in the elicitation of CD8+ T cell clones that mediate anti-viral and anti-tumor immune responses. While it has been clear that there are specific subsets of professional antigen presenting cells (APC capable of antigen cross-presentation, description of mechanisms involved is still ongoing. Especially amongst dendritic cells (DC, there are specialized subsets that are highly proficient at antigen cross-presentation. We here present a focused survey on the cell biological processes in the endosomal pathway that support antigen cross-presentation. This review highlight DC-intrinsic mechanisms that facilitate the cross-presentation of endocytosed antigen, including receptor-mediated uptake, recycling and maturation including the sorting of membrane proteins, dynamic remodeling of endosomal structures and cell-surface directed endosomal trafficking. We will conclude with description of pathogen-induced deviation of endosomal processing, and discuss how immune evasion strategies pertaining endosomal trafficking may preclude antigen cross-presentation.

  15. Endosome-lysosomes and neurodegeneration.

    Science.gov (United States)

    Mayer, R J; Tipler, C; Laszlo, L; Arnold, J; Lowe, J; Landon, M

    1994-01-01

    A number of the major human and animal neurodegenerative diseases, such as Alzheimer's disease and sheep scrapie, are characterised by deposits of amyloid, arising through incomplete breakdown of membrane proteins. Although our knowledge concerning these diseases is increasing, they remain largely untreatable. Recently, attention has focussed on the mechanisms of production of different types of amyloid and the likely involvement within cells of acid compartments called endosome-lysosomes. These organelles may be 'bioreactor' sites for the unfolding and partial degradation of membrane proteins to generate the amyloid materials. These subsequently become expelled from the cell, or are released from dead cells, and accumulate as pathological entities. Common features of the disease processes give new direction to therapeutic intervention.

  16. k -Bitonic sort

    Institute of Scientific and Technical Information of China (English)

    高庆狮; 胡玥; 刘志勇

    1999-01-01

    A k-bitonic sort which generalizes the bitonic sort is proposed. The theorem of the bitonic sort, which merges two monotonic sequences into one order sequence, is extended into the theorem of k-bitonic sort. The k-bitonic sort merges K (=2k or 2k-1) monotonic sequences into one order sequence in steps, where k=[K/2] is an integer and k≥1. The k-bitonic sort is the Batcher’s bitonic sort when k=1.

  17. Erythroid cell mitochondria receive endosomal iron by a "kiss-and-run" mechanism.

    Science.gov (United States)

    Hamdi, Amel; Roshan, Tariq M; Kahawita, Tanya M; Mason, Anne B; Sheftel, Alex D; Ponka, Prem

    2016-12-01

    In erythroid cells, more than 90% of transferrin-derived iron enters mitochondria where ferrochelatase inserts Fe(2+) into protoporphyrin IX. However, the path of iron from endosomes to mitochondrial ferrochelatase remains elusive. The prevailing opinion is that, after its export from endosomes, the redox-active metal spreads into the cytosol and mysteriously finds its way into mitochondria through passive diffusion. In contrast, this study supports the hypothesis that the highly efficient transport of iron toward ferrochelatase in erythroid cells requires a direct interaction between transferrin-endosomes and mitochondria (the "kiss-and-run" hypothesis). Using a novel method (flow sub-cytometry), we analyze lysates of reticulocytes after labeling these organelles with different fluorophores. We have identified a double-labeled population definitively representing endosomes interacting with mitochondria, as demonstrated by confocal microscopy. Moreover, we conclude that this endosome-mitochondrion association is reversible, since a "chase" with unlabeled holotransferrin causes a time-dependent decrease in the size of the double-labeled population. Importantly, the dissociation of endosomes from mitochondria does not occur in the absence of holotransferrin. Additionally, mutated recombinant holotransferrin, that cannot release iron, significantly decreases the uptake of (59)Fe by reticulocytes and diminishes (59)Fe incorporation into heme. This suggests that endosomes, which are unable to provide iron to mitochondria, cause a "traffic jam" leading to decreased endocytosis of holotransferrin. Altogether, our results suggest that a molecular mechanism exists to coordinate the iron status of endosomal transferrin with its trafficking. Besides its contribution to the field of iron metabolism, this study provides evidence for a new intracellular trafficking pathway of organelles. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Silencing by small RNAs is linked to endosomal trafficking.

    Science.gov (United States)

    Lee, Young Sik; Pressman, Sigal; Andress, Arlise P; Kim, Kevin; White, Jamie L; Cassidy, Justin J; Li, Xin; Lubell, Kim; Lim, Do Hwan; Cho, Ik Sang; Nakahara, Kenji; Preall, Jonathan B; Bellare, Priya; Sontheimer, Erik J; Carthew, Richard W

    2009-09-01

    Small RNAs direct RNA-induced silencing complexes (RISCs) to regulate stability and translation of mRNAs. RISCs associated with target mRNAs often accumulate in discrete cytoplasmic foci known as GW-bodies. However, RISC proteins can associate with membrane compartments such as the Golgi and endoplasmic reticulum. Here, we show that GW-bodies are associated with late endosomes (multivesicular bodies, MVBs). Blocking the maturation of MVBs into lysosomes by loss of the tethering factor HPS4 (ref. 5) enhances short interfering RNA (siRNA)- and micro RNA (miRNA)-mediated silencing in Drosophila melanogaster and humans. It also triggers over-accumulation of GW-bodies. Blocking MVB formation by ESCRT (endosomal sorting complex required for transport) depletion results in impaired miRNA silencing and loss of GW-bodies. These results indicate that active RISCs are physically and functionally coupled to MVBs. We further show that MVBs promote the competence of RISCs in loading small RNAs. We suggest that the recycling of RISCs is promoted by MVBs, resulting in RISCs more effectively engaging with small RNA effectors and possibly target RNAs. It may provide a means to enhance the dynamics of RNA silencing in the cytoplasm.

  19. What is a Sorting Function?

    DEFF Research Database (Denmark)

    Henglein, Fritz

    2009-01-01

    What is a sorting function—not a sorting function for a given ordering relation, but a sorting function with nothing given? Formulating four basic properties of sorting algorithms as defining requirements, we arrive at intrinsic notions of sorting and stable sorting: A function is a sorting...

  20. CIN85 associates with endosomal membrane and binds phosphatidic acid

    Institute of Scientific and Technical Information of China (English)

    Jing Zhang; Xiudan Zheng; Xiao Yang; Kan Liao

    2009-01-01

    CIN85 (Cbl-interacting protein of 85 kDa) is an important molecule involved in receptor tyrosine kinase endocy-tosis. Here we report that through its positively charged C-terminus, CIN85 associates with a fusogenic lipid - phos-phatidic acid. Its coiled-coil domain plays an important role in mediating this protein-lipid interaction. Deletion of the coiled-coil domain results in loss of membrane association, and reduced interaction with c-cbl, finally causing the blockage of epidermal growth factor receptor downregulation, In addition, a significant portion of CIN85 is located on the endosomai compartment and is related to endocytic cargo sorting, characterized by CIN85's localization on the "E class" compartment and EGF degradation blockage in CIN85 knockdown cells. Taken together, our results suggest that CIN85 may function as a scaffold molecule in both the internalization and endocytic cargo sorting pro-cesses through its association with the endosomal membrane.

  1. ATG12-ATG3 connects basal autophagy and late endosome function.

    Science.gov (United States)

    Murrow, Lyndsay; Debnath, Jayanta

    2015-01-01

    In addition to supporting cell survival in response to starvation or stress, autophagy promotes basal protein and organelle turnover. Compared to our understanding of stress-induced autophagy, little is known about how basal autophagy is regulated and how its activity is coordinated with other cellular processes. We recently identified a novel interaction between the ATG12-ATG3 conjugate and the ESCRT-associated protein PDCD6IP/Alix that promotes basal autophagy and endolysosomal trafficking. Moreover, ATG12-ATG3 is required for diverse PDCD6IP-mediated functions including late endosome distribution, exosome secretion, and viral budding. Our results highlight the importance of late endosomes for basal autophagic flux and reveal distinct roles for the core autophagy proteins ATG12 and ATG3 in controlling late endosome function.

  2. LUCID: A Quantitative Assay of ESCRT-Mediated Cargo Sorting into Multivesicular Bodies.

    Science.gov (United States)

    Nickerson, Daniel P; Merz, Alexey J

    2015-12-01

    Endosomes are transportation nodes, mediating selective transport of soluble and transmembrane cargos to and from the Golgi apparatus, plasma membrane and lysosomes. As endosomes mature to become multivesicular bodies (MVBs), Endosomal Sorting Complexes Required for Transport (ESCRTs) selectively incorporate transmembrane cargos into vesicles that bud into the endosome lumen. Luminal vesicles and their cargoes are targeted for destruction when MVBs fuse with lysosomes. Common assays of endosomal luminal targeting, including fluorescence microscopy and monitoring of proteolytic cargo maturation, possess significant limitations. We present a quantitative assay system called LUCID (LUCiferase reporter of Intraluminal Deposition) that monitors exposure of chimeric luciferase-cargo reporters to cytosol. Luciferase-chimera signal increases when sorting to the endosome lumen is disrupted, and silencing of signal from the chimera depends upon luminal delivery of the reporter rather than proteolytic degradation. The system presents several advantages, including rapidity, microscale operation and a high degree of reproducibility that enables detection of subtle phenotypic differences. Luciferase reporters provide linear signal over an extremely broad dynamic range, allowing analysis of reporter traffic even at anemic levels of expression. Furthermore, LUCID reports transport kinetics when applied to inducible trafficking reporters.

  3. Endosomal gene expression: a new indicator for prostate cancer patient prognosis?

    LENUS (Irish Health Repository)

    Johnson, Ian R D

    2015-11-10

    Prostate cancer continues to be a major cause of morbidity and mortality in men, but a method for accurate prognosis in these patients is yet to be developed. The recent discovery of altered endosomal biogenesis in prostate cancer has identified a fundamental change in the cell biology of this cancer, which holds great promise for the identification of novel biomarkers that can predict disease outcomes. Here we have identified significantly altered expression of endosomal genes in prostate cancer compared to non-malignant tissue in mRNA microarrays and confirmed these findings by qRT-PCR on fresh-frozen tissue. Importantly, we identified endosomal gene expression patterns that were predictive of patient outcomes. Two endosomal tri-gene signatures were identified from a previously published microarray cohort and had a significant capacity to stratify patient outcomes. The expression of APPL1, RAB5A, EEA1, PDCD6IP, NOX4 and SORT1 were altered in malignant patient tissue, when compared to indolent and normal prostate tissue. These findings support the initiation of a case-control study using larger cohorts of prostate tissue, with documented patient outcomes, to determine if different combinations of these new biomarkers can accurately predict disease status and clinical progression in prostate cancer patients.

  4. Late endosomal cholesterol accumulation leads to impaired intra-endosomal trafficking.

    Directory of Open Access Journals (Sweden)

    Komla Sobo

    Full Text Available BACKGROUND: Pathological accumulation of cholesterol in late endosomes is observed in lysosomal storage diseases such as Niemann-Pick type C. We here analyzed the effects of cholesterol accumulation in NPC cells, or as phenocopied by the drug U18666A, on late endosomes membrane organization and dynamics. METHODOLOGY/PRINCIPAL FINDINGS: Cholesterol accumulation did not lead to an increase in the raft to non-raft membrane ratio as anticipated. Strikingly, we observed a 2-3 fold increase in the size of the compartment. Most importantly, properties and dynamics of late endosomal intralumenal vesicles were altered as revealed by reduced late endosomal vacuolation induced by the mutant pore-forming toxin ASSP, reduced intoxication by the anthrax lethal toxin and inhibition of infection by the Vesicular Stomatitis Virus. CONCLUSIONS/SIGNIFICANCE: These results suggest that back fusion of intralumenal vesicles with the limiting membrane of late endosomes is dramatically perturbed upon cholesterol accumulation.

  5. A FYVE zinc finger domain protein specifically links mRNA transport to endosome trafficking.

    Science.gov (United States)

    Pohlmann, Thomas; Baumann, Sebastian; Haag, Carl; Albrecht, Mario; Feldbrügge, Michael

    2015-05-18

    An emerging theme in cellular logistics is the close connection between mRNA and membrane trafficking. A prominent example is the microtubule-dependent transport of mRNAs and associated ribosomes on endosomes. This coordinated process is crucial for correct septin filamentation and efficient growth of polarised cells, such as fungal hyphae. Despite detailed knowledge on the key RNA-binding protein and the molecular motors involved, it is unclear how mRNAs are connected to membranes during transport. Here, we identify a novel factor containing a FYVE zinc finger domain for interaction with endosomal lipids and a new PAM2-like domain required for interaction with the MLLE domain of the key RNA-binding protein. Consistently, loss of this FYVE domain protein leads to specific defects in mRNA, ribosome, and septin transport without affecting general functions of endosomes or their movement. Hence, this is the first endosomal component specific for mRNP trafficking uncovering a new mechanism to couple mRNPs to endosomes.

  6. Parallel sorting algorithms

    CERN Document Server

    Akl, Selim G

    1985-01-01

    Parallel Sorting Algorithms explains how to use parallel algorithms to sort a sequence of items on a variety of parallel computers. The book reviews the sorting problem, the parallel models of computation, parallel algorithms, and the lower bounds on the parallel sorting problems. The text also presents twenty different algorithms, such as linear arrays, mesh-connected computers, cube-connected computers. Another example where algorithm can be applied is on the shared-memory SIMD (single instruction stream multiple data stream) computers in which the whole sequence to be sorted can fit in the

  7. Parkinson Disease-linked Vps35 R524W Mutation Impairs the Endosomal Association of Retromer and Induces α-Synuclein Aggregation.

    Science.gov (United States)

    Follett, Jordan; Bugarcic, Andrea; Yang, Zhe; Ariotti, Nicholas; Norwood, Suzanne J; Collins, Brett M; Parton, Robert G; Teasdale, Rohan D

    2016-08-26

    Endosomal sorting is a highly orchestrated cellular process. Retromer is a heterotrimeric complex that associates with endosomal membranes and facilitates the retrograde sorting of multiple receptors, including the cation-independent mannose 6-phosphate receptor for lysosomal enzymes. The cycling of retromer on and off the endosomal membrane is regulated by a network of retromer-interacting proteins. Here, we find that Parkinson disease-associated Vps35 variant, R524W, but not P316S, is a loss-of-function mutation as marked by a reduced association with this regulatory network and dysregulation of endosomal receptor sorting. Expression of Vps35 R524W-containing retromer results in the accumulation of intracellular α-synuclein-positive aggregates, a hallmark of Parkinson disease. Overall, the Vps35 R524W-containing retromer has a decreased endosomal association, which can be partially rescued by R55, a small molecule previously shown to stabilize the retromer complex, supporting the potential for future targeting of the retromer complex in the treatment of Parkinson disease.

  8. A generation method for numerical specimen of granular materials by sort of coordinates%颗粒材料数值样本的坐标排序生成技术

    Institute of Scientific and Technical Information of China (English)

    楚锡华

    2011-01-01

    The mechanical behaviors of granular materials presented by discrete element method (DEM) based on discrete particle model are closely relative to its numerical specimen. The generation techniques for granular materials specimens has attracted comprehensive attentions with development of DEM in numerical simulation of granular materials. The object of this paper is how to generate a more dense granular assembly using random sequential analysis (RSA) model. For uniform grains, that is to say how to generate more number of grains in given region. Four modified RSA methods are suggested and discussed; numerical practices show that the more dense granular assembly will be generated by sort of coordinates based on RSA.%颗粒材料离散颗粒模型的数值模拟结果与颗粒材料的数值样本密切相关,随着离散单元在颗粒材料数值模拟领域的广泛应用,颗粒材料的数值样本生成技术日益受到重视.基于RSA模型研究如何使随机生成的颗粒材料更密实,对均匀颗粒而言亦即如何在指定区域内生成更多的颗粒,讨论了4类修正方案,并建议了一种基于坐标排序的样本生成技术.研究表明,在传统的颗粒体随机生成技术基础上,通过对随机生成的x坐标序列或y坐标序列进行排序,可使生成的颗粒材料数值样本更密实.

  9. Signal processing by the endosomal system.

    Science.gov (United States)

    Villaseñor, Roberto; Kalaidzidis, Yannis; Zerial, Marino

    2016-04-01

    Cells need to decode chemical or physical signals from their environment in order to make decisions on their fate. In the case of signalling receptors, ligand binding triggers a cascade of chemical reactions but also the internalization of the activated receptors in the endocytic pathway. Here, we highlight recent studies revealing a new role of the endosomal network in signal processing. The diversity of entry pathways and endosomal compartments is exploited to regulate the kinetics of receptor trafficking, and interactions with specific signalling adaptors and effectors. By governing the spatio-temporal distribution of signalling molecules, the endosomal system functions analogously to a digital-analogue computer that regulates the specificity and robustness of the signalling response.

  10. Proximity Labeling Reveals Molecular Determinants of FGFR4 Endosomal Transport.

    Science.gov (United States)

    Haugsten, Ellen Margrethe; Sørensen, Vigdis; Kunova Bosakova, Michaela; de Souza, Gustavo Antonio; Krejci, Pavel; Wiedlocha, Antoni; Wesche, Jørgen

    2016-10-07

    The fibroblast growth factor receptors (FGFRs) are important oncogenes promoting tumor progression in many types of cancer, such as breast, bladder, and lung cancer as well as multiple myeloma and rhabdomyosarcoma. However, little is known about how these receptors are internalized and down-regulated in cells. We have here applied proximity biotin labeling to identify proteins involved in FGFR4 signaling and trafficking. For this purpose we fused a mutated biotin ligase, BirA*, to the C-terminal tail of FGFR4 (FGFR4-BirA*) and the fusion protein was stably expressed in U2OS cells. Upon addition of biotin to these cells, proteins in proximity to the FGFR4-BirA* fusion protein became biotinylated and could be isolated and identified by quantitative mass spectrometry. We identified in total 291 proteins, including 80 proteins that were enriched in samples where the receptor was activated by the ligand (FGF1), among them several proteins previously found to be involved in FGFR signaling (e.g., FRS2, PLCγ, RSK2 and NCK2). Interestingly, many of the identified proteins were implicated in endosomal transport, and by precise annotation we were able to trace the intracellular pathways of activated FGFR4. Validating the data by confocal and three-dimensional structured illumination microscopy analysis, we concluded that FGFR4 uses clathrin-mediated endocytosis for internalization and is further sorted from early endosomes to the recycling compartment and the trans-Golgi network. Depletion of cells for clathrin heavy chain led to accumulation of FGFR4 at the cell surface and increased levels of active FGFR4 and PLCγ, while AKT and ERK signaling was diminished, demonstrating that functional clathrin-mediated endocytosis is required for proper FGFR4 signaling. Thus, this study reveals proteins and pathways involved in FGFR4 transport and signaling that provide possible targets and opportunities for therapeutic intervention in FGFR4 aberrant cancer.

  11. Membrane contacts between endosomes and ER provide sites for PTP1B-epidermal growth factor receptor interaction.

    Science.gov (United States)

    Eden, Emily R; White, Ian J; Tsapara, Anna; Futter, Clare E

    2010-03-01

    The epidermal growth factor receptor (EGFR) is a critical determinator of cell fate. Signalling from this receptor tyrosine kinase is spatially regulated by progression through the endocytic pathway, governing receptor half-life and accessibility to signalling proteins and phosphatases. Endocytosis of EGFR is required for interaction with the protein tyrosine phosphatase PTP1B (ref. 1), which localizes to the cytoplasmic face of the endoplasmic reticulum (ER), raising the question of how PTP1B comes into contact with endosomal EGFR. We show that EGFR-PTP1B interaction occurs by means of direct membrane contacts between the perimeter membrane of multivesicular bodies (MVBs) and the ER. The population of EGFR interacting with PTP1B is the same population that undergo ESCRT-mediated (endosomal sorting complex required for transport) sorting within MVBs, and PTP1B activity promotes the sequestration of EGFR on to MVB internal vesicles. Membrane contacts between endosomes and the ER form in both the presence and absence of stimulation by EGF. Thus membrane contacts between endosomes and the ER may represent a global mechanism for direct interaction between proteins on these two organelles.

  12. Sorting a distribution theory

    CERN Document Server

    Mahmoud, Hosam M

    2011-01-01

    A cutting-edge look at the emerging distributional theory of sorting Research on distributions associated with sorting algorithms has grown dramatically over the last few decades, spawning many exact and limiting distributions of complexity measures for many sorting algorithms. Yet much of this information has been scattered in disparate and highly specialized sources throughout the literature. In Sorting: A Distribution Theory, leading authority Hosam Mahmoud compiles, consolidates, and clarifies the large volume of available research, providing a much-needed, comprehensive treatment of the

  13. Individual cell sorting.

    Science.gov (United States)

    Stovel, R T; Sweet, R G

    1979-01-01

    Current cell sorting machines do not preserve the individual identity of processed cells; after analysis, the cells are assigned to a subpopulation where they are pooled with other similar cells. This paper reports progress on a system that sorts cells individually to precise locations on a microscope slide and preserves them for further observation with a light microscope while recording flow measurement data for each cell. Various electronic and mechanical modifications to an existing sorting machine are described that increase drop placement accuracy and permit individual cell sorting.

  14. Designing sorting networks

    CERN Document Server

    Baddar, Sherenaz W Al-Haj

    2012-01-01

    Designing Sorting Networks: A New Paradigm provides an in-depth guide to maximizing the efficiency of sorting networks, and uses 0/1 cases, partially ordered sets and Haase diagrams to closely analyze their behavior in an easy, intuitive manner. This book also outlines new ideas and techniques for designing faster sorting networks using Sortnet, and illustrates how these techniques were used to design faster 12-key and 18-key sorting networks through a series of case studies. Finally, it examines and explains the mysterious behavior exhibited by the fastest-known 9-step 16-key network. Designi

  15. Direct binding of retromer to human papillomavirus type 16 minor capsid protein L2 mediates endosome exit during viral infection.

    Directory of Open Access Journals (Sweden)

    Andreea Popa

    2015-02-01

    Full Text Available Trafficking of human papillomaviruses to the Golgi apparatus during virus entry requires retromer, an endosomal coat protein complex that mediates the vesicular transport of cellular transmembrane proteins from the endosome to the Golgi apparatus or the plasma membrane. Here we show that the HPV16 L2 minor capsid protein is a retromer cargo, even though L2 is not a transmembrane protein. We show that direct binding of retromer to a conserved sequence in the carboxy-terminus of L2 is required for exit of L2 from the early endosome and delivery to the trans-Golgi network during virus entry. This binding site is different from known retromer binding motifs and can be replaced by a sorting signal from a cellular retromer cargo. Thus, HPV16 is an unconventional particulate retromer cargo, and retromer binding initiates retrograde transport of viral components from the endosome to the trans-Golgi network during virus entry. We propose that the carboxy-terminal segment of L2 protein protrudes through the endosomal membrane and is accessed by retromer in the cytoplasm.

  16. Multiple Roles of VARP in Endosomal Trafficking: Rabs, Retromer Components and R-SNARE VAMP7 Meet on VARP.

    Science.gov (United States)

    Fukuda, Mitsunori

    2016-07-01

    VARP (VPS9-ankyrin-repeat protein, also known as ANKRD27) was originally identified as an N-terminal VPS9 (vacuolar protein sorting 9)-domain-containing protein that possesses guanine nucleotide exchange factor (GEF) activity toward small GTPase Rab21 and contains two ankyrin repeat (ANKR) domains in its central region. A number of VARP-interacting molecules have been identified during the past five years, and considerable attention is now being directed to the multiple roles of VARP in endosomal trafficking. More specifically, VARP is now known to interact with three different types of key membrane trafficking regulators, i.e. small GTPase Rabs (Rab32, Rab38 and Rab40C), the retromer complex (a sorting nexin dimer, VPS26, VPS29 and VPS35) and R-SNARE VAMP7. By binding to several of these molecules, VARP regulates endosomal trafficking, which underlies a variety of cellular events, including melanogenic enzyme trafficking to melanosomes, dendrite outgrowth of melanocytes, neurite outgrowth and retromer-mediated endosome-to-plasma membrane sorting of transmembrane proteins.

  17. Late Endosomal Cholesterol Accumulation Leads to Impaired Intra-Endosomal Trafficking

    OpenAIRE

    Komla Sobo; Isabelle Le Blanc; Pierre-Philippe Luyet; Marc Fivaz; Charles Ferguson; Parton, Robert G.; Jean Gruenberg; Gisou Van Der Goot, F.

    2007-01-01

    BACKGROUND: Pathological accumulation of cholesterol in late endosomes is observed in lysosomal storage diseases such as Niemann-Pick type C. We here analyzed the effects of cholesterol accumulation in NPC cells, or as phenocopied by the drug U18666A, on late endosomes membrane organization and dynamics. METHODOLOGY/PRINCIPAL FINDINGS: Cholesterol accumulation did not lead to an increase in the raft to non-raft membrane ratio as anticipated. Strikingly, we observed a 2-3 fold increase in the ...

  18. Sorting out Downside Beta

    NARCIS (Netherlands)

    G.T. Post (Thierry); P. van Vliet (Pim); S.D. Lansdorp (Simon)

    2009-01-01

    textabstractDownside risk, when properly defined and estimated, helps to explain the cross-section of US stock returns. Sorting stocks by a proper estimate of downside market beta leads to a substantially larger cross-sectional spread in average returns than sorting on regular market beta. This

  19. Cell sorting in development.

    Science.gov (United States)

    Krens, S F Gabby; Heisenberg, Carl-Philipp

    2011-01-01

    During the development of multicellular organisms, cell fate specification is followed by the sorting of different cell types into distinct domains from where the different tissues and organs are formed. Cell sorting involves both the segregation of a mixed population of cells with different fates and properties into distinct domains, and the active maintenance of their segregated state. Because of its biological importance and apparent resemblance to fluid segregation in physics, cell sorting was extensively studied by both biologists and physicists over the last decades. Different theories were developed that try to explain cell sorting on the basis of the physical properties of the constituent cells. However, only recently the molecular and cellular mechanisms that control the physical properties driving cell sorting, have begun to be unraveled. In this review, we will provide an overview of different cell-sorting processes in development and discuss how these processes can be explained by the different sorting theories, and how these theories in turn can be connected to the molecular and cellular mechanisms driving these processes.

  20. BCR and Endosomal TLR Signals Synergize to Increase AID Expression and Establish Central B Cell Tolerance.

    Science.gov (United States)

    Kuraoka, Masayuki; Snowden, Pilar B; Nojima, Takuya; Verkoczy, Laurent; Haynes, Barton F; Kitamura, Daisuke; Kelsoe, Garnett

    2017-02-14

    Activation-induced cytidine deaminase (AID) is required to purge autoreactive immature and transitional-1 (immature/T1) B cells at the first tolerance checkpoint, but how AID selectively removes self-reactive B cells is unclear. We now show that B cell antigen receptor (BCR) and endosomal Toll-like receptor (TLR) signals synergize to elicit high levels of AID expression in immature/T1 B cells. This synergy is restricted to ligands for endocytic TLR and requires phospholipase-D activation, endosomal acidification, and MyD88. The first checkpoint is significantly impaired in AID- or MyD88-deficient mice and in mice doubly heterozygous for AID and MyD88, suggesting interaction of these factors in central B cell tolerance. Moreover, administration of chloroquine, an inhibitor of endosomal acidification, results in a failure to remove autoreactive immature/T1 B cells in mice. We propose that a BCR/TLR pathway coordinately establishes central tolerance by hyper-activating AID in immature/T1 B cells that bind ligands for endosomal TLRs.

  1. BCR and Endosomal TLR Signals Synergize to Increase AID Expression and Establish Central B Cell Tolerance

    Directory of Open Access Journals (Sweden)

    Masayuki Kuraoka

    2017-02-01

    Full Text Available Activation-induced cytidine deaminase (AID is required to purge autoreactive immature and transitional-1 (immature/T1 B cells at the first tolerance checkpoint, but how AID selectively removes self-reactive B cells is unclear. We now show that B cell antigen receptor (BCR and endosomal Toll-like receptor (TLR signals synergize to elicit high levels of AID expression in immature/T1 B cells. This synergy is restricted to ligands for endocytic TLR and requires phospholipase-D activation, endosomal acidification, and MyD88. The first checkpoint is significantly impaired in AID- or MyD88-deficient mice and in mice doubly heterozygous for AID and MyD88, suggesting interaction of these factors in central B cell tolerance. Moreover, administration of chloroquine, an inhibitor of endosomal acidification, results in a failure to remove autoreactive immature/T1 B cells in mice. We propose that a BCR/TLR pathway coordinately establishes central tolerance by hyper-activating AID in immature/T1 B cells that bind ligands for endosomal TLRs.

  2. The novel endosomal membrane protein Ema interacts with the class C Vps-HOPS complex to promote endosomal maturation.

    Science.gov (United States)

    Kim, Sungsu; Wairkar, Yogesh P; Daniels, Richard W; DiAntonio, Aaron

    2010-03-08

    Endosomal maturation is critical for accurate and efficient cargo transport through endosomal compartments. Here we identify a mutation of the novel Drosophila gene, ema (endosomal maturation defective) in a screen for abnormal synaptic overgrowth and defective protein trafficking. Ema is an endosomal membrane protein required for trafficking of fluid-phase and receptor-mediated endocytic cargos. In the ema mutant, enlarged endosomal compartments accumulate as endosomal maturation fails, with early and late endosomes unable to progress into mature degradative late endosomes and lysosomes. Defective endosomal down-regulation of BMP signaling is responsible for the abnormal synaptic overgrowth. Ema binds to and genetically interacts with Vps16A, a component of the class C Vps-HOPS complex that promotes endosomal maturation. The human orthologue of ema, Clec16A, is a candidate susceptibility locus for autoimmune disorders, and its expression rescues the Drosophila mutant demonstrating conserved function. Characterizing this novel gene family identifies a new component of the endosomal pathway and provides insights into class C Vps-HOPS complex function.

  3. Chasing Ebola through the Endosomal Labyrinth

    Directory of Open Access Journals (Sweden)

    M. Javad Aman

    2016-03-01

    Full Text Available During virus entry, the surface glycoprotein of Ebola virus (EBOV undergoes a complex set of transformations within the endosomal network. Tools to study EBOV entry have been limited to static immunofluorescence or biochemical and functional analysis. In a recent article in mBio, Spence et al. reported a novel, live-cell-imaging method that tracks this transformational journey of EBOV in real time [J. S. Spence, T. B. Krause, E. Mittler, R. K. Jangra, and K. Chandran, mBio 7(1:e01857-15, 2016, http://dx.doi.org/10.1128/mBio.01857-15]. The assay validates known mechanisms of EBOV entry and sheds light on some novel intricacies. Direct evidence supports the hypothesis that fusion is a rare event that starts in maturing early endosomes, is completed in late endosomes, and occurs entirely in Niemann-Pick C1 (NPC1-positive (NPC1+ compartments. The study demonstrated that lipid mixing and productive fusion are temporally decoupled, with different energetic barriers and a protease-dependent step between the two events. Analysis of the mechanism of action of an important class of EBOV neutralizing antibodies, such as KZ52 and ZMapp, provides direct evidence that these antibodies act by inhibiting the membrane fusion.

  4. Conformational biosensors reveal GPCR signalling from endosomes.

    Science.gov (United States)

    Irannejad, Roshanak; Tomshine, Jin C; Tomshine, Jon R; Chevalier, Michael; Mahoney, Jacob P; Steyaert, Jan; Rasmussen, Søren G F; Sunahara, Roger K; El-Samad, Hana; Huang, Bo; von Zastrow, Mark

    2013-03-28

    A long-held tenet of molecular pharmacology is that canonical signal transduction mediated by G-protein-coupled receptor (GPCR) coupling to heterotrimeric G proteins is confined to the plasma membrane. Evidence supporting this traditional view is based on analytical methods that provide limited or no subcellular resolution. It has been subsequently proposed that signalling by internalized GPCRs is restricted to G-protein-independent mechanisms such as scaffolding by arrestins, or GPCR activation elicits a discrete form of persistent G protein signalling, or that internalized GPCRs can indeed contribute to the acute G-protein-mediated response. Evidence supporting these various latter hypotheses is indirect or subject to alternative interpretation, and it remains unknown if endosome-localized GPCRs are even present in an active form. Here we describe the application of conformation-specific single-domain antibodies (nanobodies) to directly probe activation of the β2-adrenoceptor, a prototypical GPCR, and its cognate G protein, Gs (ref. 12), in living mammalian cells. We show that the adrenergic agonist isoprenaline promotes receptor and G protein activation in the plasma membrane as expected, but also in the early endosome membrane, and that internalized receptors contribute to the overall cellular cyclic AMP response within several minutes after agonist application. These findings provide direct support for the hypothesis that canonical GPCR signalling occurs from endosomes as well as the plasma membrane, and suggest a versatile strategy for probing dynamic conformational change in vivo.

  5. ESCRT-II/Vps25 constrains digit number by endosome-mediated selective modulation of FGF-SHH signaling.

    Science.gov (United States)

    Handschuh, Karen; Feenstra, Jennifer; Koss, Matthew; Ferretti, Elisabetta; Risolino, Maurizio; Zewdu, Rediet; Sahai, Michelle A; Bénazet, Jean-Denis; Peng, Xiao P; Depew, Michael J; Quintana, Laura; Sharpe, James; Wang, Baolin; Alcorn, Heather; Rivi, Roberta; Butcher, Stephen; Manak, J Robert; Vaccari, Thomas; Weinstein, Harel; Anderson, Kathryn V; Lacy, Elizabeth; Selleri, Licia

    2014-10-23

    Sorting and degradation of receptors and associated signaling molecules maintain homeostasis of conserved signaling pathways during cell specification and tissue development. Yet, whether machineries that sort signaling proteins act preferentially on different receptors and ligands in different contexts remains mysterious. Here, we show that Vacuolar protein sorting 25, Vps25, a component of ESCRT-II (Endosomal Sorting Complex Required for Transport II), directs preferential endosome-mediated modulation of FGF signaling in limbs. By ENU-induced mutagenesis, we isolated a polydactylous mouse line carrying a hypomorphic mutation of Vps25 (Vps25(ENU)). Unlike Vps25-null embryos we generated, Vps25(ENU/ENU) mutants survive until late gestation. Their limbs display FGF signaling enhancement and consequent hyperactivation of the FGF-SHH feedback loop causing polydactyly, whereas WNT and BMP signaling remain unperturbed. Notably, Vps25(ENU/ENU) Mouse Embryonic Fibroblasts exhibit aberrant FGFR trafficking and degradation; however, SHH signaling is unperturbed. These studies establish that the ESCRT-II machinery selectively limits FGF signaling in vertebrate skeletal patterning.

  6. Effects of Endosomal Photodamage on Membrane Recycling and Endocytosis

    Science.gov (United States)

    Kessel, David; Santiago, Ann Marie; Andrzejak, Michelle

    2011-01-01

    The flux of receptor-independent endocytosis can be estimated by addition of wortmannin to cell cultures. Membrane influx is unaffected but traffic out of late endosomes is impaired, resulting in a substantial enlargement of these organelles. Using the 1c1c7 murine hepatoma, we investigated the effect of endosomal photodamage on this endocytic pathway. We previously reported that photodamage catalyzed by the lysosomal photosensitizer NPe6 prevented wortmannin-induced endosomal swelling, indicating an earlier block in the process. In this study, we show that endosomal photodamage, initiated by photodamage from an asymmetrically-substituted porphine or a phthalocyanine, also prevents wortmannin-induced endosomal swelling, even when the PDT dose is insufficient to cause endosomal disruption. As the PDT dose is increased, endosomal breakage occurs, as does apoptosis and cell death. Very high PDT doses result in necrosis. We propose that photodamage to endosomes results in alterations in the endosomal structure such that influx of new material is inhibited and receptor-independent endocytosis is prevented. In an additional series of studies, we found that the swollen late endosomes induced by wortmannin are unable to retain previously accumulated fluorescent probes or photosensitizers. PMID:21208213

  7. Endocytic sorting and recycling require membrane phosphatidylserine asymmetry maintained by TAT-1/CHAT-1.

    Science.gov (United States)

    Chen, Baohui; Jiang, Yue; Zeng, Sheng; Yan, Jiacong; Li, Xin; Zhang, Yan; Zou, Wei; Wang, Xiaochen

    2010-12-09

    Endocytic sorting is achieved through the formation of morphologically and functionally distinct sub-domains within early endosomes. Cargoes destined for recycling are sorted to and transported through newly-formed tubular membranes, but the processes that regulate membrane tubulation are poorly understood. Here, we identified a novel Caenorhabditis elegans Cdc50 family protein, CHAT-1, which acts as the chaperone of the TAT-1 P4-ATPase to regulate membrane phosphatidylserine (PS) asymmetry and endocytic transport. In chat-1 and tat-1 mutants, the endocytic sorting process is disrupted, leading to defects in both cargo recycling and degradation. TAT-1 and CHAT-1 colocalize to the tubular domain of the early endosome, the tubular endocytic recycling compartment (ERC), and the recycling endosome where PS is enriched on the cytosolic surface. Loss of tat-1 and chat-1 function disrupts membrane PS asymmetry and abrogates the tubular membrane structure. Our data suggest that CHAT-1 and TAT-1 maintain membrane phosphatidylserine asymmetry, thus promoting membrane tubulation and regulating endocytic sorting and recycling.

  8. Layers in sorting practices: Sorting out patients with potential cancer

    DEFF Research Database (Denmark)

    Møller, Naja Holten; Bjørn, Pernille

    2011-01-01

    mechanism, but is handled by informal sorting mechanisms. We identify two informal sorting mechanisms with large impact on the sorting practices, namely subtle categorizing and collective remembering. These informal sorting mechanisms have implications for the design of electronic booking systems because...

  9. Ligand recognition and domain structure of Vps10p, a vacuolar protein sorting receptor in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Jørgensen, M U; Emr, S D; Winther, Jakob R.

    1999-01-01

    Vp10p is a receptor that sorts several different vacuolar proteins by cycling between a late Golgi compartment and the endosome. The cytoplasmic tail of Vps10p is necessary for the recycling, whereas the lumenal domain is predicted to interact with the soluble ligands. We have studied ligand bind...

  10. LazySorted: A Lazily, Partially Sorted Python List

    Directory of Open Access Journals (Sweden)

    Naftali Harris

    2015-06-01

    Full Text Available LazySorted is a Python C extension implementing a partially and lazily sorted list data structure. It solves a common problem faced by programmers, in which they need just part of a sorted list, like its middle element (the median, but sort the entire list to get it. LazySorted presents them with the abstraction that they are working with a fully sorted list, while actually only sorting the list partially with quicksort partitions to return the requested sub-elements. This enables programmers to use naive "sort first" algorithms but nonetheless attain linear run-times when possible. LazySorted may serve as a drop-in replacement for the built-in sorted function in most cases, and can sometimes achieve run-times more than 7 times faster.

  11. The retromer component SNX6 interacts with dynactin p150~(Glued)and mediates endosome-to-TGN transport

    Institute of Scientific and Technical Information of China (English)

    Zhi Hong; Yanrui Yang; Cheng Zhang; Yang Niu; Ke Li; Xi Zhao; Jia-Jia Liu

    2009-01-01

    The retromer is a protein complex that mediates retrograde transport of transmembrane cargoes from endosomes to the fraws-Golgi network (TGN). It is comprised of a cargo-selection subcomplex of Vps26, Vps29 and Vps35 and a membrane-binding coat subcomplex of sorting nexins (SNXs). Previous studies identified SNX1/2 as one of the components of the SNX subcomplex, and SNX5/6 as candidates for the second SNX. How the retromer-associated cargoes are recognized and transported by molecular motors are largely unknown. In this study, we found that one of SNXl/2's dimerization partners, SNX6, interacts with the pl50~(Glued) subunit of the dynein/dynactin motor complex. We present evidence that SNX6 is a component of the retromer, and that recruitment of the motor complex to the membrane-associated retromer requires the SNX6-pl50~(Glued) interaction. Disruption of the SNX6-pl50~(Ghied) interaction causes failure in formation and detachment of the tubulovesicular sorting structures from endosomes and results in block of CI-MPR retrieval from endosomes to the TGN. These observations indicate that in addition to SNX 1/2, SNX6 in association with the dynein/dynactin complex drives the formation and movement of tubular retrograde intermediates.

  12. Sorting Plastic Waste in Hydrocyclone

    Directory of Open Access Journals (Sweden)

    Ernestas Šutinys

    2011-02-01

    Full Text Available The article presents material about sorting plastic waste in hydrocyclone. The tests on sorting plastic waste were carried out. Also, the findings received from the performed experiment on the technology of sorting plastic waste are interpreted applying an experimental model of the equipment used for sorting plastics of different density.Article in Lithuanian

  13. Ready, steady, SORT!

    CERN Multimedia

    Laëtitia Pedroso

    2010-01-01

    The selective or ecological sorting of waste is already second nature to many of us and concerns us all. As the GS Department's new awareness-raising campaign reminds us, everything we do to sort waste contributes to preserving the environment.    Placemats printed on recycled paper using vegetable-based ink will soon be distributed in Restaurant No.1.   Environmental protection is never far from the headlines, and CERN has a responsibility to ensure that the 3000 tonnes and more of waste it produces every year are correctly and selectively sorted. Materials can be given a second life through recycling and re-use, thereby avoiding pollution from landfill sites and incineration plants and saving on processing costs. The GS Department is launching a new poster campaign designed to raise awareness of the importance of waste sorting and recycling. "After conducting a survey to find out whether members of the personnel were prepared to make an effort to sort a...

  14. Endosomal sorting of Notch receptors through COMMD9-dependent pathways modulates Notch signaling

    NARCIS (Netherlands)

    Li, H.; Koo, Y.; Mao, X.; Sifuentes-Dominguez, L.; Morris, L.L.; Jia, D.; Miyata, N; Faulkner, R.A.; Deursen, J.M.A. van; Vooijs, M.; Billadeau, D.D.; Sluis, B. van de; Cleaver, O.; Burstein, E.

    2015-01-01

    Notch family members are transmembrane receptors that mediate essential developmental programs. Upon ligand binding, a proteolytic event releases the intracellular domain of Notch, which translocates to the nucleus to regulate gene transcription. In addition, Notch trafficking across the endolysosom

  15. Ubiquitin initiates sorting of Golgi and plasma membrane proteins into the vacuolar degradation pathway

    Directory of Open Access Journals (Sweden)

    Scheuring David

    2012-09-01

    Full Text Available Abstract Background In yeast and mammals, many plasma membrane (PM proteins destined for degradation are tagged with ubiquitin. These ubiquitinated proteins are internalized into clathrin-coated vesicles and are transported to early endosomal compartments. There, ubiquitinated proteins are sorted by the endosomal sorting complex required for transport (ESCRT machinery into the intraluminal vesicles of multivesicular endosomes. Degradation of these proteins occurs after endosomes fuse with lysosomes/lytic vacuoles to release their content into the lumen. In plants, some PM proteins, which cycle between the PM and endosomal compartments, have been found to be ubiquitinated, but it is unclear whether ubiquitin is sufficient to mediate internalization and thus acts as a primary sorting signal for the endocytic pathway. To test whether plants use ubiquitin as a signal for the degradation of membrane proteins, we have translationally fused ubiquitin to different fluorescent reporters for the plasma membrane and analyzed their transport. Results Ubiquitin-tagged PM reporters localized to endosomes and to the lumen of the lytic vacuole in tobacco mesophyll protoplasts and in tobacco epidermal cells. The internalization of these reporters was significantly reduced if clathrin-mediated endocytosis was inhibited by the coexpression of a mutant of the clathrin heavy chain, the clathrin hub. Surprisingly, a ubiquitin-tagged reporter for the Golgi was also transported into the lumen of the vacuole. Vacuolar delivery of the reporters was abolished upon inhibition of the ESCRT machinery, indicating that the vacuolar delivery of these reporters occurs via the endocytic transport route. Conclusions Ubiquitin acts as a sorting signal at different compartments in the endomembrane system to target membrane proteins into the vacuolar degradation pathway: If displayed at the PM, ubiquitin triggers internalization of PM reporters into the endocytic transport route

  16. Flow sorting in aquatic ecology

    OpenAIRE

    Marcus Reckermann

    2000-01-01

    Flow sorting can be a very helpful tool in revealing phytoplankton and bacterial community structure and elaborating specific physiological parameters of isolated species. Droplet sorting has been the most common technique. Despite the high optical and hydro-dynamic stress for the cells to be sorted, many species grow in culture subsequent to sorting. To date, flow sorting has been applied to post-incubation separation in natural water samples to account for group-specific physiological param...

  17. Distinct effects of endosomal escape and inhibition of endosomal trafficking on gene delivery via electrotransfection

    Science.gov (United States)

    Chang, Chun-Chi; Wang, Liangli; Yuan, Fan

    2017-01-01

    A recent theory suggests that endocytosis is involved in uptake and intracellular transport of electrotransfected plasmid DNA (pDNA). The goal of the current study was to understand if approaches used previously to improve endocytosis of gene delivery vectors could be applied to enhancing electrotransfection efficiency (eTE). Results from the study showed that photochemically induced endosomal escape, which could increase poly-L-lysine (PLL)-mediated gene delivery, decreased eTE. The decrease could not be blocked by treatment of cells with endonuclease inhibitors (aurintricarboxylic acid and zinc ion) or antioxidants (L-glutamine and ascorbic acid). Chemical treatment of cells with an endosomal trafficking inhibitor that blocks endosome progression, bafilomycin A1, resulted in a significant decrease in eTE. However, treatment of cells with lysosomotropic agents (chloroquine and ammonium chloride) had little effects on eTE. These data suggested that endosomes played important roles in protecting and intracellular trafficking of electrotransfected pDNA. PMID:28182739

  18. Sorting by Recursive Partitioning,

    Science.gov (United States)

    1983-12-01

    asymptotic time-complexity. This paper has the following main parts: First, a Pidgin -Algol version of the algorithm is presented and we discuss the main...those sorted subsets e) end "UsingBin*; end "AdaptSorting. 4 "Figure 1: A condensed Pidgin -Algol version of Adaptsort eiFor some conditions that we will...algorithm which have to be completed in either linear or constant times (these required critical times appear as comments in the Pidgin -Algol version

  19. Wage Sorting Trends

    DEFF Research Database (Denmark)

    Bagger, Jesper; Vejlin, Rune Majlund; Sørensen, Kenneth Lykke

    Using a population-wide Danish Matched Employer-Employee panel from 1980-2006, we document a strong trend towards more positive assortative wage sorting. The correlation between worker and firm fixed effects estimated from a log wage regression increases from -0.07 in 1981 to .14 in 2001. The non......Using a population-wide Danish Matched Employer-Employee panel from 1980-2006, we document a strong trend towards more positive assortative wage sorting. The correlation between worker and firm fixed effects estimated from a log wage regression increases from -0.07 in 1981 to .14 in 2001...

  20. Gender Differences in Sorting

    DEFF Research Database (Denmark)

    Merlino, Luca Paolo; Parrotta, Pierpaolo; Pozzoli, Dario

    In this paper, we investigate the sorting of workers in firms to understand gender gaps in labor market outcomes. Using Danish employer-employee matched data, we fiend strong evidence of glass ceilings in certain firms, especially after motherhood, preventing women from climbing the career ladder...

  1. Protein Sorting Prediction

    DEFF Research Database (Denmark)

    Nielsen, Henrik

    2017-01-01

    Many computational methods are available for predicting protein sorting in bacteria. When comparing them, it is important to know that they can be grouped into three fundamentally different approaches: signal-based, global-property-based and homology-based prediction. In this chapter, the strengths...

  2. Sorting and sustaining cooperation

    DEFF Research Database (Denmark)

    Vikander, Nick

    2013-01-01

    This paper looks at cooperation in teams where some people are selfish and others are conditional cooperators, and where lay-offs will occur at a fixed future date. I show that the best way to sustain cooperation prior to the lay-offs is often in a sorting equilibrium, where conditional cooperato...

  3. Det sorte USA

    DEFF Research Database (Denmark)

    Brøndal, Jørn

    Bogen gennemgår det sorte USAs historie fra 1776 til 2016, idet grundtemaet er spændingsforholdet mellem USAs grundlæggelsesidealer og den racemæssige praksis, et spændingsforhold som Gunnar Myrdal kaldte "det amerikanske dilemma." Bogen, der er opbygget som politisk, social og racemæssig historie...

  4. Online Sorted Range Reporting

    DEFF Research Database (Denmark)

    Brodal, Gerth Stølting; Fagerberg, Rolf; Greve, Mark

    2009-01-01

    We study the following one-dimensional range reporting problem: On an arrayA of n elements, support queries that given two indices i ≤ j and an integerk report the k smallest elements in the subarray A[i..j] in sorted order. We present a data structure in the RAM model supporting such queries in ...

  5. Molecular assemblies and membrane domains in multivesicular endosome dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Falguieres, Thomas; Luyet, Pierre-Philippe [Department of Biochemistry, University of Geneva, 30 quai Ernest Ansermet-1211 Geneva 4 (Switzerland); Gruenberg, Jean, E-mail: jean.gruenberg@biochem.unige.ch [Department of Biochemistry, University of Geneva, 30 quai Ernest Ansermet-1211 Geneva 4 (Switzerland)

    2009-05-15

    Along the degradation pathway, endosomes exhibit a characteristic multivesicular organization, resulting from the budding of vesicles into the endosomal lumen. After endocytosis and transport to early endosomes, activated signaling receptors are incorporated into these intralumenal vesicles through the action of the ESCRT machinery, a process that contributes to terminate signaling. Then, the vesicles and their protein cargo are further transported towards lysosomes for degradation. Evidence also shows that intralumenal vesicles can undergo 'back-fusion' with the late endosome limiting membrane, a route exploited by some pathogens and presumably followed by proteins and lipids that need to be recycled from within the endosomal lumen. This process depends on the late endosomal lipid lysobisphosphatidic acid and its putative effector Alix/AIP1, and is presumably coupled to the invagination of the endosomal limiting membrane at the molecular level via ESCRT proteins. In this review, we discuss the intra-endosomal transport routes in mammalian cells, and in particular the different mechanisms involved in membrane invagination, vesicle formation and fusion in a space inaccessible to proteins known to control intracellular membrane traffic.

  6. A Novel Type III Endosome Transmembrane Protein, TEMP

    Directory of Open Access Journals (Sweden)

    Rohan D. Teasdale

    2012-11-01

    Full Text Available As part of a high-throughput subcellular localisation project, the protein encoded by the RIKEN mouse cDNA 2610528J11 was expressed and identified to be associated with both endosomes and the plasma membrane. Based on this, we have assigned the name TEMP for Type III Endosome Membrane Protein. TEMP encodes a short protein of 111 amino acids with a single, alpha-helical transmembrane domain. Experimental analysis of its membrane topology demonstrated it is a Type III membrane protein with the amino-terminus in the lumenal, or extracellular region, and the carboxy-terminus in the cytoplasm. In addition to the plasma membrane TEMP was localized to Rab5 positive early endosomes, Rab5/Rab11 positive recycling endosomes but not Rab7 positive late endosomes. Video microscopy in living cells confirmed TEMP's plasma membrane localization and identified the intracellular endosome compartments to be tubulovesicular. Overexpression of TEMP resulted in the early/recycling endosomes clustering at the cell periphery that was dependent on the presence of intact microtubules. The cellular function of TEMP cannot be inferred based on bioinformatics comparison, but its cellular distribution between early/recycling endosomes and the plasma membrane suggests a role in membrane transport.

  7. K-sort: A new sorting algorithm that beats Heap sort for n <= 70 lakhs!

    CERN Document Server

    Sundararajan, Kiran Kumar; Chakraborty, Soubhik; Mahanti, N C

    2011-01-01

    Sundararajan and Chakraborty (2007) introduced a new version of Quick sort removing the interchanges. Khreisat (2007) found this algorithm to be competing well with some other versions of Quick sort. However, it uses an auxiliary array thereby increasing the space complexity. Here, we provide a second version of our new sort where we have removed the auxiliary array. This second improved version of the algorithm, which we call K-sort, is found to sort elements faster than Heap sort for an appreciably large array size (n <= 70,00,000) for uniform U[0, 1] inputs.

  8. Chip-based droplet sorting

    Energy Technology Data Exchange (ETDEWEB)

    Beer, Neil Reginald; Lee, Abraham; Hatch, Andrew

    2014-07-01

    A non-contact system for sorting monodisperse water-in-oil emulsion droplets in a microfluidic device based on the droplet's contents and their interaction with an applied electromagnetic field or by identification and sorting.

  9. Chip-based droplet sorting

    Science.gov (United States)

    Beer, Neil Reginald; Lee, Abraham; Hatch, Andrew

    2014-07-01

    A non-contact system for sorting monodisperse water-in-oil emulsion droplets in a microfluidic device based on the droplet's contents and their interaction with an applied electromagnetic field or by identification and sorting.

  10. Heideggers sorte arv

    DEFF Research Database (Denmark)

    Olesen, Søren Gosvig

    2015-01-01

    Martin Heidegger var antisemit, men er hans tænkning og intellektuelle arv det også? Søren Gosvig Olesen opsøger den store tyske tænkers arvinger og bindene fra 1938-48 i Heideggers efterladte ’Sorte hæfter’, hvor den lille mands meninger blander sig med en stor tænkers tanker......Martin Heidegger var antisemit, men er hans tænkning og intellektuelle arv det også? Søren Gosvig Olesen opsøger den store tyske tænkers arvinger og bindene fra 1938-48 i Heideggers efterladte ’Sorte hæfter’, hvor den lille mands meninger blander sig med en stor tænkers tanker...

  11. Sorting quantum systems efficiently

    Science.gov (United States)

    Ionicioiu, Radu

    2016-05-01

    Measuring the state of a quantum system is a fundamental process in quantum mechanics and plays an essential role in quantum information and quantum technologies. One method to measure a quantum observable is to sort the system in different spatial modes according to the measured value, followed by single-particle detectors on each mode. Examples of quantum sorters are polarizing beam-splitters (PBS) – which direct photons according to their polarization – and Stern-Gerlach devices. Here we propose a general scheme to sort a quantum system according to the value of any d-dimensional degree of freedom, such as spin, orbital angular momentum (OAM), wavelength etc. Our scheme is universal, works at the single-particle level and has a theoretical efficiency of 100%. As an application we design an efficient OAM sorter consisting of a single multi-path interferometer which is suitable for a photonic chip implementation.

  12. The R-SNARE endobrevin/VAMP-8 mediates homotypic fusion of early endosomes and late endosomes.

    Science.gov (United States)

    Antonin, W; Holroyd, C; Tikkanen, R; Höning, S; Jahn, R

    2000-10-01

    Endobrevin/VAMP-8 is an R-SNARE localized to endosomes, but it is unknown in which intracellular fusion step it operates. Using subcellular fractionation and quantitative immunogold electron microscopy, we found that endobrevin/VAMP-8 is present on all membranes known to communicate with early endosomes, including the plasma membrane, clathrin-coated pits, late endosomes, and membranes of the trans-Golgi network. Affinity-purified antibodies that block the ability of endobrevin/VAMP-8 to form SNARE core complexes potently inhibit homotypic fusion of both early and late endosomes in vitro. Fab fragments were as active as intact immunoglobulin Gs. Recombinant endobrevin/VAMP-8 inhibited both fusion reactions with similar potency. We conclude that endobrevin/VAMP-8 operates as an R-SNARE in the homotypic fusion of early and late endosomes.

  13. Non-26S Proteasome Proteolytic Role of Ubiquitin in Plant Endocytosis and Endosomal Trafficking

    Institute of Scientific and Technical Information of China (English)

    Miaomiao Tian; Qi Xie

    2013-01-01

    The 76 amino acid protein ubiquitin (Ub) is highly conserved in all eukaryotic species.It plays important roles in many cellular processes by covalently attaching to the target proteins.The best known function of Ub is marking substrate proteins for degradation by the 26S proteasome.In fact,other consequences of ubiquitination have been discovered in yeast and mammals,such as membrane trafficking,DNA repair,chromatin modification,and protein kinase activation.The common mechanism underlying these processes is that Ub serves as a signal to sort proteins to the vacuoles or lysosomes for degradation as opposed to 26S proteasome-dependent degradation.To date,several reports have indicated that a similar function of Ub also exists in plants.This review focuses on a summary and analysis of the recent research progress on Ub acting as a signal to mediate endocytosis and endosomal trafficking in plants.

  14. Actin-Sorting Nexin 27 (SNX27)-Retromer Complex Mediates Rapid Parathyroid Hormone Receptor Recycling.

    Science.gov (United States)

    McGarvey, Jennifer C; Xiao, Kunhong; Bowman, Shanna L; Mamonova, Tatyana; Zhang, Qiangmin; Bisello, Alessandro; Sneddon, W Bruce; Ardura, Juan A; Jean-Alphonse, Frederic; Vilardaga, Jean-Pierre; Puthenveedu, Manojkumar A; Friedman, Peter A

    2016-05-20

    The G protein-coupled parathyroid hormone receptor (PTHR) regulates mineral-ion homeostasis and bone remodeling. Upon parathyroid hormone (PTH) stimulation, the PTHR internalizes into early endosomes and subsequently traffics to the retromer complex, a sorting platform on early endosomes that promotes recycling of surface receptors. The C terminus of the PTHR contains a type I PDZ ligand that binds PDZ domain-containing proteins. Mass spectrometry identified sorting nexin 27 (SNX27) in isolated endosomes as a PTHR binding partner. PTH treatment enriched endosomal PTHR. SNX27 contains a PDZ domain and serves as a cargo selector for the retromer complex. VPS26, VPS29, and VPS35 retromer subunits were isolated with PTHR in endosomes from cells stimulated with PTH. Molecular dynamics and protein binding studies establish that PTHR and SNX27 interactions depend on the PDZ recognition motif in PTHR and the PDZ domain of SNX27. Depletion of either SNX27 or VPS35 or actin depolymerization decreased the rate of PTHR recycling following agonist stimulation. Mutating the PDZ ligand of PTHR abolished the interaction with SNX27 but did not affect the overall rate of recycling, suggesting that PTHR may directly engage the retromer complex. Coimmunoprecipitation and overlay experiments show that both intact and mutated PTHR bind retromer through the VPS26 protomer and sequentially assemble a ternary complex with PTHR and SNX27. SNX27-independent recycling may involve N-ethylmaleimide-sensitive factor, which binds both PDZ intact and mutant PTHRs. We conclude that PTHR recycles rapidly through at least two pathways, one involving the ASRT complex of actin, SNX27, and retromer and another possibly involving N-ethylmaleimide-sensitive factor.

  15. Actin-Sorting Nexin 27 (SNX27)-Retromer Complex Mediates Rapid Parathyroid Hormone Receptor Recycling*

    Science.gov (United States)

    McGarvey, Jennifer C.; Xiao, Kunhong; Bowman, Shanna L.; Mamonova, Tatyana; Zhang, Qiangmin; Bisello, Alessandro; Sneddon, W. Bruce; Ardura, Juan A.; Jean-Alphonse, Frederic; Vilardaga, Jean-Pierre; Puthenveedu, Manojkumar A.; Friedman, Peter A.

    2016-01-01

    The G protein-coupled parathyroid hormone receptor (PTHR) regulates mineral-ion homeostasis and bone remodeling. Upon parathyroid hormone (PTH) stimulation, the PTHR internalizes into early endosomes and subsequently traffics to the retromer complex, a sorting platform on early endosomes that promotes recycling of surface receptors. The C terminus of the PTHR contains a type I PDZ ligand that binds PDZ domain-containing proteins. Mass spectrometry identified sorting nexin 27 (SNX27) in isolated endosomes as a PTHR binding partner. PTH treatment enriched endosomal PTHR. SNX27 contains a PDZ domain and serves as a cargo selector for the retromer complex. VPS26, VPS29, and VPS35 retromer subunits were isolated with PTHR in endosomes from cells stimulated with PTH. Molecular dynamics and protein binding studies establish that PTHR and SNX27 interactions depend on the PDZ recognition motif in PTHR and the PDZ domain of SNX27. Depletion of either SNX27 or VPS35 or actin depolymerization decreased the rate of PTHR recycling following agonist stimulation. Mutating the PDZ ligand of PTHR abolished the interaction with SNX27 but did not affect the overall rate of recycling, suggesting that PTHR may directly engage the retromer complex. Coimmunoprecipitation and overlay experiments show that both intact and mutated PTHR bind retromer through the VPS26 protomer and sequentially assemble a ternary complex with PTHR and SNX27. SNX27-independent recycling may involve N-ethylmaleimide-sensitive factor, which binds both PDZ intact and mutant PTHRs. We conclude that PTHR recycles rapidly through at least two pathways, one involving the ASRT complex of actin, SNX27, and retromer and another possibly involving N-ethylmaleimide-sensitive factor. PMID:27008860

  16. Association with AflR in Endosomes Reveals New Functions for AflJ in Aflatoxin Biosynthesis

    Science.gov (United States)

    Ehrlich, Kenneth C.; Mack, Brian M.; Wei, Qijian; Li, Ping; Roze, Ludmila V.; Dazzo, Frank; Cary, Jeffrey W.; Bhatnagar, Deepak; Linz, John E.

    2012-01-01

    Aflatoxins are the most potent naturally occurring carcinogens of fungal origin. Biosynthesis of aflatoxin involves the coordinated expression of more than 25 genes. The function of one gene in the aflatoxin gene cluster, aflJ, is not entirely understood but, because previous studies demonstrated a physical interaction between the Zn2Cys6 transcription factor AflR and AflJ, AflJ was proposed to act as a transcriptional co-activator. Image analysis revealed that, in the absence of aflJ in A. parasiticus, endosomes cluster within cells and near septa. AflJ fused to yellow fluorescent protein complemented the mutation in A. parasiticus ΔaflJ and localized mainly in endosomes. We found that AflJ co-localizes with AflR both in endosomes and in nuclei. Chromatin immunoprecipitation did not detect AflJ binding at known AflR DNA recognition sites suggesting that AflJ either does not bind to these sites or binds to them transiently. Based on these data, we hypothesize that AflJ assists in AflR transport to or from the nucleus, thus controlling the availability of AflR for transcriptional activation of aflatoxin biosynthesis cluster genes. AflJ may also assist in directing endosomes to the cytoplasmic membrane for aflatoxin export. PMID:23342682

  17. Association with AflR in Endosomes Reveals New Functions for AflJ in Aflatoxin Biosynthesis

    Directory of Open Access Journals (Sweden)

    John E. Linz

    2012-12-01

    Full Text Available Aflatoxins are the most potent naturally occurring carcinogens of fungal origin. Biosynthesis of aflatoxin involves the coordinated expression of more than 25 genes. The function of one gene in the aflatoxin gene cluster, aflJ, is not entirely understood but, because previous studies demonstrated a physical interaction between the Zn2Cys6 transcription factor AflR and AflJ, AflJ was proposed to act as a transcriptional co-activator. Image analysis revealed that, in the absence of aflJ in A. parasiticus, endosomes cluster within cells and near septa. AflJ fused to yellow fluorescent protein complemented the mutation in A. parasiticus ΔaflJ and localized mainly in endosomes. We found that AflJ co-localizes with AflR both in endosomes and in nuclei. Chromatin immunoprecipitation did not detect AflJ binding at known AflR DNA recognition sites suggesting that AflJ either does not bind to these sites or binds to them transiently. Based on these data, we hypothesize that AflJ assists in AflR transport to or from the nucleus, thus controlling the availability of AflR for transcriptional activation of aflatoxin biosynthesis cluster genes. AflJ may also assist in directing endosomes to the cytoplasmic membrane for aflatoxin export.

  18. Spin-the-bottle Sort and Annealing Sort: Oblivious Sorting via Round-robin Random Comparisons

    CERN Document Server

    Goodrich, Michael T

    2010-01-01

    We study sorting algorithms based on randomized round-robin comparisons. Specifically, we study Spin-the-bottle sort, where comparisons are unrestricted, and Annealing sort, where comparisons are restricted to a distance bounded by a \\emph{temperature} parameter. Both algorithms are simple, randomized, data-oblivious sorting algorithms, which are useful in privacy-preserving computations, but, as we show, Annealing sort is much more efficient. We show that there is an input permutation that causes Spin-the-bottle sort to require $\\Omega(n^2\\log n)$ expected time in order to succeed, and that in $O(n^2\\log n)$ time this algorithm succeeds with high probability for any input. We also show there is an implementation of Annealing sort that runs in $O(n\\log n)$ time and succeeds with very high probability.

  19. Spin-the-bottle Sort and Annealing Sort: Oblivious Sorting via Round-robin Random Comparisons.

    Science.gov (United States)

    Goodrich, Michael T

    2014-03-01

    We study sorting algorithms based on randomized round-robin comparisons. Specifically, we study Spin-the-bottle sort, where comparisons are unrestricted, and Annealing sort, where comparisons are restricted to a distance bounded by a temperature parameter. Both algorithms are simple, randomized, data-oblivious sorting algorithms, which are useful in privacy-preserving computations, but, as we show, Annealing sort is much more efficient. We show that there is an input permutation that causes Spin-the-bottle sort to require Ω(n(2) log n) expected time in order to succeed, and that in O(n(2) log n) time this algorithm succeeds with high probability for any input. We also show there is a specification of Annealing sort that runs in O(n log n) time and succeeds with very high probability.

  20. Deductive sort and climbing sort: new methods for non-dominated sorting.

    Science.gov (United States)

    McClymont, Kent; Keedwell, Ed

    2012-01-01

    In recent years an increasing number of real-world many-dimensional optimisation problems have been identified across the spectrum of research fields. Many popular evolutionary algorithms use non-dominance as a measure for selecting solutions for future generations. The process of sorting populations into non-dominated fronts is usually the controlling order of computational complexity and can be expensive for large populations or for a high number of objectives. This paper presents two novel methods for non-dominated sorting: deductive sort and climbing sort. The two new methods are compared to the fast non-dominated sort of NSGA-II and the non-dominated rank sort of the omni-optimizer. The results demonstrate the improved efficiencies of the deductive sort and the reductions in comparisons that can be made when applying inferred dominance relationships defined in this paper.

  1. Selective sorting of waste

    CERN Multimedia

    2007-01-01

    Not much effort needed, just willpower In order to keep the cost of disposing of waste materials as low as possible, CERN provides two types of recipient at the entrance to each building: a green plastic one for paper/cardboard and a metal one for general refuse. For some time now we have noticed, to our great regret, a growing negligence as far as selective sorting is concerned, with, for example, the green recipients being filled with a mixture of cardboard boxes full of polystyrene or protective wrappers, plastic bottles, empty yogurts pots, etc. …We have been able to ascertain, after careful checking, that this haphazard mixing of waste cannot be attributed to the cleaning staff but rather to members of the personnel who unscrupulously throw away their rubbish in a completely random manner. Non-sorted waste entails heavy costs for CERN. For information, once a non-compliant item is found in a green recipient, the entire contents are sent off for incineration rather than recycling… We are all concerned...

  2. Endocytic traffic: vesicle fusion cascade in the early endosomes.

    Science.gov (United States)

    Brenner, Michael P

    2012-08-01

    New research shows that vesicles in the early endosomal network coalesce according to a classical theoretical description of aggregation put forward by Smoluchowski more than 100 years ago. This gives a new tool for unraveling complexities of the endocytic pathways.

  3. Endosome-lysosomes, ubiquitin and neurodegeneration.

    Science.gov (United States)

    Mayer, R J; Tipler, C; Arnold, J; Laszlo, L; Al-Khedhairy, A; Lowe, J; Landon, M

    1996-01-01

    Before the advent of ubiquitin immunochemistry and immunogold electron microscopy, there was no known intracellular molecular commonality between neurodegenerative diseases. The application of antibodies which primarily detect ubiquitin protein conjugates has shown that all of the human and animal idiopathic and transmissible chronic neurodegenerative diseases, (including Alzheimer's disease (AD), Lewy body disease (LBD), amyotrophic lateral sclerosis (ALS), Creutzfeldt-Jakob disease (CJD) and scrapie) are related by some form of intraneuronal inclusion which contains ubiquitin protein conjugates. In addition, disorders such as Alzheimer's disease, CJD and sheep scrapie, are characterised by deposits of amyloid, arising through incomplete breakdown of membrane proteins which may be associated with cytoskeletal reorganisation. Although our knowledge about these diseases is increasing, they remain largely untreatable. Recently, attention has focused on the mechanisms of production of different types of amyloid and the likely involvement within cells of the endosome-lysosome system, organelles which are immuno-positive for ubiquitin protein conjugates. These organelles may be 'bioreactor' sites for the unfolding and partial degradation of membrane proteins to generate the amyloid materials or their precursors which subsequently become expelled from the cell, or are released from dead cells, and accumulate as pathological entities. Such common features of the disease processes give new direction to therapeutic intervention.

  4. Heterotrimeric G protein subunits are located on rat liver endosomes

    Directory of Open Access Journals (Sweden)

    Van Dyke Rebecca W

    2004-01-01

    Full Text Available Abstract Background Rat liver endosomes contain activated insulin receptors and downstream signal transduction molecules. We undertook these studies to determine whether endosomes also contain heterotrimeric G proteins that may be involved in signal transduction from G protein-coupled receptors. Results By Western blotting Gsα, Giα1,2, Giα3 and Gβ were enriched in both canalicular (CM and basolateral (BLM membranes but also readily detectable on three types of purified rat liver endosomes in the order recycling receptor compartment (RRC > compartment for uncoupling of receptor and ligand (CURL > multivesicular bodies (MVB >> purified secondary lysosomes. Western blotting with antibodies to Na, K-ATPase and to other proteins associated with plasma membranes and intracellular organelles indicated this was not due to contamination of endosome preparations by CM or BLM. Adenylate cyclase (AC was also identified on purified CM, BLM, RRC, CURL and MVB. Percoll gradient fractionation of liver postnuclear supernatants demonstrated co-occurrence of endosomes and heterotrimeric G protein subunits in fractions with little plasma membrane markers. By confocal microscopy, punctate staining for Gsα, Giα3 and Gβ corresponded to punctate areas of endocytosed Texas red-dextran in hepatocytes from control and cholera toxin-treated livers. Conclusion We conclude that heterotrimeric G protein subunits as well as AC likely traffic into hepatocytes on endosome membranes, possibly generating downstream signals spatially separate from signalling generated at the plasma membrane, analogous to the role(s of internalized insulin receptors.

  5. Biomechanics and thermodynamics of nanoparticle interactions with plasma and endosomal membrane lipids in cellular uptake and endosomal escape.

    Science.gov (United States)

    Peetla, Chiranjeevi; Jin, Shihua; Weimer, Jonathan; Elegbede, Adekunle; Labhasetwar, Vinod

    2014-07-01

    To be effective for cytoplasmic delivery of therapeutics, nanoparticles (NPs) taken up via endocytic pathways must efficiently transport across the cell membrane and subsequently escape from the secondary endosomes. We hypothesized that the biomechanical and thermodynamic interactions of NPs with plasma and endosomal membrane lipids are involved in these processes. Using model plasma and endosomal lipid membranes, we compared the interactions of cationic NPs composed of poly(D,L-lactide-co-glycolide) modified with the dichain surfactant didodecyldimethylammonium bromide (DMAB) or the single-chain surfactant cetyltrimethylammonium bromide (CTAB) vs anionic unmodified NPs of similar size. We validated our hypothesis in doxorubicin-sensitive (MCF-7, with relatively fluid membranes) and resistant breast cancer cells (MCF-7/ADR, with rigid membranes). Despite their cationic surface charges, DMAB- and CTAB-modified NPs showed different patterns of biophysical interaction: DMAB-modified NPs induced bending of the model plasma membrane, whereas CTAB-modified NPs condensed the membrane, thereby resisted bending. Unmodified NPs showed no effects on bending. DMAB-modified NPs also induced thermodynamic instability of the model endosomal membrane, whereas CTAB-modified and unmodified NPs had no effect. Since bending of the plasma membrane and destabilization of the endosomal membrane are critical biophysical processes in NP cellular uptake and endosomal escape, respectively, we tested these NPs for cellular uptake and drug efficacy. Confocal imaging showed that in both sensitive and resistant cells DMAB-modified NPs exhibited greater cellular uptake and escape from endosomes than CTAB-modified or unmodified NPs. Further, paclitaxel-loaded DMAB-modified NPs induced greater cytotoxicity even in resistant cells than CTAB-modified or unmodified NPs or drug in solution, demonstrating the potential of DMAB-modified NPs to overcome the transport barrier in resistant cells. In

  6. Endosome-mediated retrograde axonal transport of P2X3 receptor signals in primary sensory neurons

    Institute of Scientific and Technical Information of China (English)

    Xu-Qiao Chen; BinWang; Chengbiao Wu; Jin Pan; Bo Yuan; Yuan-Yuan Su; Xing-Yu Jiang; Xu Zhang; Lan Bao

    2012-01-01

    Neurotrophins and their receptors adopt signaling endosomes to transmit retrograde signals.However,the mechanisms of retrograde signaling for other ligand/receptor systems are poorly understood.Here,we report that the signals of the purinergic (P)2X3 receptor,an ATP-gated ion channel are retrogradely transported in dorsal root ganglion (DRG) neuron axons.We found that Rab5,a small GTPase,controls the early sorting of P2X3 receptors into endosomes,while Rab7 mediates the fast retrograde transport of P2X3 receptors.Intraplantar injection and axonal application into the microfluidic chamber of α,β-methylene-ATP (α,β-MeATP),a P2X selective agonist,enhanced the endocytosis and retrograde transport of P2X3 receptors.The α,β-MeATP-induced Ca2+ influx activated a pathway comprised of protein kinase C,rat sarcoma viral oncogene and extracellular signal-regulated protein kinase (ERK),which associated with endocytic P2X3 receptors to form signaling endosomes.Disruption of the lipid rafts abolished the α,β-MeATP-induced ERK phosphorylation,endocytosis and retrograde transport of P2X3 receptors.Furthermore,treatment of peripheral axons with α,β-MeATP increased the activation level of ERK and cAMP response element-binding protein in the cell bodies of DRG neurons and enhanced neuronal excitability.Impairment of either microtubule-based axonal transport in vivo or dynein function in vitro blocked α,β-MeATP-induced retrograde signals.These results indicate that P2X3 receptor-activated signals are transmitted via retrogradely transported endosomes in primary sensory neurons and provide a novel signaling mechanism for ligand-gated channels.

  7. Sorting nexin 27 couples PTHR trafficking to retromer for signal regulation in osteoblasts during bone growth.

    Science.gov (United States)

    Chan, Audrey S M; Clairfeuille, Thomas; Landao-Bassonga, Euphemie; Kinna, Genevieve; Ng, Pei Ying; Loo, Li Shen; Cheng, Tak Sum; Zheng, Minghao; Hong, Wanjin; Teasdale, Rohan D; Collins, Brett M; Pavlos, Nathan J

    2016-04-15

    The parathyroid hormone 1 receptor (PTHR) is central to the process of bone formation and remodeling. PTHR signaling requires receptor internalization into endosomes, which is then terminated by recycling or degradation. Here we show that sorting nexin 27 (SNX27) functions as an adaptor that couples PTHR to the retromer trafficking complex. SNX27 binds directly to the C-terminal PDZ-binding motif of PTHR, wiring it to retromer for endosomal sorting. The structure of SNX27 bound to the PTHR motif reveals a high-affinity interface involving conserved electrostatic interactions. Mechanistically, depletion of SNX27 or retromer augments intracellular PTHR signaling in endosomes. Osteoblasts genetically lacking SNX27 show similar disruptions in PTHR signaling and greatly reduced capacity for bone mineralization, contributing to profound skeletal deficits in SNX27-knockout mice. Taken together, our data support a critical role for SNX27-retromer mediated transport of PTHR in normal bone development. © 2016 Chan et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  8. Redistribution of Endosomal Membranes to the African Swine Fever Virus Replication Site

    Directory of Open Access Journals (Sweden)

    Miguel Ángel Cuesta-Geijo

    2017-06-01

    Full Text Available African swine fever virus (ASFV infection causes endosomal reorganization. Here, we show that the virus causes endosomal congregation close to the nucleus as the infection progresses, which is necessary to build a compact viral replication organelle. ASFV enters the cell by the endosomal pathway and reaches multivesicular late endosomes. Upon uncoating and fusion, the virus should exit to the cytosol to start replication. ASFV remodels endosomal traffic and redistributes endosomal membranes to the viral replication site. Virus replication also depends on endosomal membrane phosphoinositides (PtdIns synthesized by PIKfyve. Endosomes could act as platforms providing membranes and PtdIns, necessary for ASFV replication. Our study has revealed that ASFV reorganizes endosome dynamics, in order to ensure a productive infection.

  9. Enhancing endosomal escape for nanoparticle mediated siRNA delivery

    Science.gov (United States)

    Ma, Da

    2014-05-01

    Gene therapy with siRNA is a promising biotechnology to treat cancer and other diseases. To realize siRNA-based gene therapy, a safe and efficient delivery method is essential. Nanoparticle mediated siRNA delivery is of great importance to overcome biological barriers for systemic delivery in vivo. Based on recent discoveries, endosomal escape is a critical biological barrier to be overcome for siRNA delivery. This feature article focuses on endosomal escape strategies used for nanoparticle mediated siRNA delivery, including cationic polymers, pH sensitive polymers, calcium phosphate, and cell penetrating peptides. Work has been done to develop different endosomal escape strategies based on nanoparticle types, administration routes, and target organ/cell types. Also, enhancement of endosomal escape has been considered along with other aspects of siRNA delivery to ensure target specific accumulation, high cell uptake, and low toxicity. By enhancing endosomal escape and overcoming other biological barriers, great progress has been achieved in nanoparticle mediated siRNA delivery.

  10. Enhancing Endosomal Escape for Intracellular Delivery of Macromolecular Biologic Therapeutics.

    Science.gov (United States)

    Lönn, Peter; Kacsinta, Apollo D; Cui, Xian-Shu; Hamil, Alexander S; Kaulich, Manuel; Gogoi, Khirud; Dowdy, Steven F

    2016-09-08

    Bioactive macromolecular peptides and oligonucleotides have significant therapeutic potential. However, due to their size, they have no ability to enter the cytoplasm of cells. Peptide/Protein transduction domains (PTDs), also called cell-penetrating peptides (CPPs), can promote uptake of macromolecules via endocytosis. However, overcoming the rate-limiting step of endosomal escape into the cytoplasm remains a major challenge. Hydrophobic amino acid R groups are known to play a vital role in viral escape from endosomes. Here we utilize a real-time, quantitative live cell split-GFP fluorescence complementation phenotypic assay to systematically analyze and optimize a series of synthetic endosomal escape domains (EEDs). By conjugating EEDs to a TAT-PTD/CPP spilt-GFP peptide complementation assay, we were able to quantitatively measure endosomal escape into the cytoplasm of live cells via restoration of GFP fluorescence by intracellular molecular complementation. We found that EEDs containing two aromatic indole rings or one indole ring and two aromatic phenyl groups at a fixed distance of six polyethylene glycol (PEG) units from the TAT-PTD-cargo significantly enhanced cytoplasmic delivery in the absence of cytotoxicity. EEDs address the critical rate-limiting step of endosomal escape in delivery of macromolecular biologic peptide, protein and siRNA therapeutics into cells.

  11. The Role of the Clathrin Adaptor AP-1: Polarized Sorting and Beyond

    Directory of Open Access Journals (Sweden)

    Fubito Nakatsu

    2014-11-01

    Full Text Available The selective transport of proteins or lipids by vesicular transport is a fundamental process supporting cellular physiology. The budding process involves cargo sorting and vesicle formation at the donor membrane and constitutes an important process in vesicular transport. This process is particularly important for the polarized sorting in epithelial cells, in which the cargo molecules need to be selectively sorted and transported to two distinct destinations, the apical or basolateral plasma membrane. Adaptor protein (AP-1, a member of the AP complex family, which includes the ubiquitously expressed AP-1A and the epithelium-specific AP-1B, regulates polarized sorting at the trans-Golgi network and/or at the recycling endosomes. A growing body of evidence, especially from studies using model organisms and animals, demonstrates that the AP-1-mediated polarized sorting supports the development and physiology of multi-cellular units as functional organs and tissues (e.g., cell fate determination, inflammation and gut immune homeostasis. Furthermore, a possible involvement of AP-1B in the pathogenesis of human diseases, such as Crohn’s disease and cancer, is now becoming evident. These data highlight the significant contribution of AP-1 complexes to the physiology of multicellular organisms, as master regulators of polarized sorting in epithelial cells.

  12. The EARP Complex and Its Interactor EIPR-1 Are Required for Cargo Sorting to Dense-Core Vesicles.

    Science.gov (United States)

    Topalidou, Irini; Cattin-Ortolá, Jérôme; Pappas, Andrea L; Cooper, Kirsten; Merrihew, Gennifer E; MacCoss, Michael J; Ailion, Michael

    2016-05-01

    The dense-core vesicle is a secretory organelle that mediates the regulated release of peptide hormones, growth factors, and biogenic amines. Dense-core vesicles originate from the trans-Golgi of neurons and neuroendocrine cells, but it is unclear how this specialized organelle is formed and acquires its specific cargos. To identify proteins that act in dense-core vesicle biogenesis, we performed a forward genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We previously reported the identification of two conserved proteins that interact with the small GTPase RAB-2 to control normal dense-core vesicle cargo-sorting. Here we identify several additional conserved factors important for dense-core vesicle cargo sorting: the WD40 domain protein EIPR-1 and the endosome-associated recycling protein (EARP) complex. By assaying behavior and the trafficking of dense-core vesicle cargos, we show that mutants that lack EIPR-1 or EARP have defects in dense-core vesicle cargo-sorting similar to those of mutants in the RAB-2 pathway. Genetic epistasis data indicate that RAB-2, EIPR-1 and EARP function in a common pathway. In addition, using a proteomic approach in rat insulinoma cells, we show that EIPR-1 physically interacts with the EARP complex. Our data suggest that EIPR-1 is a new interactor of the EARP complex and that dense-core vesicle cargo sorting depends on the EARP-dependent trafficking of cargo through an endosomal sorting compartment.

  13. Flow sorting in aquatic ecology

    Directory of Open Access Journals (Sweden)

    Marcus Reckermann

    2000-06-01

    Full Text Available Flow sorting can be a very helpful tool in revealing phytoplankton and bacterial community structure and elaborating specific physiological parameters of isolated species. Droplet sorting has been the most common technique. Despite the high optical and hydro-dynamic stress for the cells to be sorted, many species grow in culture subsequent to sorting. To date, flow sorting has been applied to post-incubation separation in natural water samples to account for group-specific physiological parameters (radiotracer-uptake rates, to the production of clonal or non-clonal cultures from mixtures, to the isolaton of cell groups from natural assemblages for molecular analyses, and for taxonomic identification of sorted cells by microscopy. The application of cell sorting from natural water samples from the Wadden Sea, including different cryptophytes, cyanobacteria and diatoms, is shown, as well as the establishment of laboratory cultures from field samples. The optional use of a red laser to account for phycocyanine-rich cells is also discussed.

  14. Sara endosomes and the asymmetric division of intestinal stem cells.

    Science.gov (United States)

    Montagne, Chrystelle; Gonzalez-Gaitan, Marcos

    2014-05-01

    Tissue homeostasis is maintained by adult stem cells, which self-renew and give rise to differentiating cells. The generation of daughter cells with different fates is mediated by signalling molecules coming from an external niche or being asymmetrically dispatched between the two daughters upon stem cell mitosis. In the adult Drosophila midgut, the intestinal stem cell (ISC) divides to generate a new ISC and an enteroblast (EB) differentiating daughter. Notch signalling activity restricted to the EB regulates intestinal cell fate decision. Here, we show that ISCs divide asymmetrically, and Sara endosomes in ISCs are specifically dispatched to the presumptive EB. During ISC mitosis, Notch and Delta traffic through Sara endosomes, thereby contributing to Notch signalling bias, as revealed in Sara mutants: Sara itself contributes to the control of the ISC asymmetric division. Our data uncover an intrinsic endosomal mechanism during ISC mitosis, which participates in the maintenance of the adult intestinal lineage.

  15. Rapid endosomal escape of prickly nanodiamonds: implications for gene delivery

    Science.gov (United States)

    Chu, Zhiqin; Miu, Kaikei; Lung, Pingsai; Zhang, Silu; Zhao, Saisai; Chang, Huan-Cheng; Lin, Ge; Li, Quan

    2015-06-01

    The prickly nanodiamonds easily entered cells via endocytosis followed by unique intracellular translocation characteristics—quick endosomal escape followed by stable residence in cytoplasm. Endosomal membrane rupturing is identified as the major route of nanodiamonds’ escaping the vesicle confinement and to the cytoplasm. Little cytotoxicity is observed to associate with the nanodiamonds’ cytosolic release. Such features enable its application for gene delivery, which requires both effective cellular uptake and cytosolic release of the gene. Taking green fluorescent protein gene as an example, we demonstrate the successful cytosolic delivery and expression of such a gene using the prickly nanodiamonds as carrier.

  16. Molecular Characterization of Plant Prevacuolar and Endosomal Compartments

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Prevacuolar compartments (PVCs) and endosomal compartments are membrane-bound organelles mediating protein traffic to vacuoles in the secretory and endocytic pathways of plant cells. Over the years, great progress has been made towards our understanding in these two compartments in plant cells. In this review, we will summarize our contributions toward the identification and characterization of plant prevacuolar and endosomal compartments. Our studies will serve as important steps in future molecular characterization of PVC biogenesis and PVC-mediated protein trafficking in plant cells.

  17. Interchangeable but Essential Functions of SNX1 and SNX2 in the Association of Retromer with Endosomes and the Trafficking of Mannose 6-Phosphate Receptors▿ †

    Science.gov (United States)

    Rojas, Raul; Kametaka, Satoshi; Haft, Carol R.; Bonifacino, Juan S.

    2007-01-01

    The retromer is a cytosolic/peripheral membrane protein complex that mediates the retrieval of the cation-independent mannose 6-phosphate receptor from endosomes to the trans-Golgi network (TGN) in mammalian cells. Previous studies showed that the mammalian retromer comprises three proteins, named Vps26, Vps29, and Vps35, plus the sorting nexin, SNX1. There is conflicting evidence, however, as to whether a homologous sorting nexin, SNX2, is truly a component of the retromer. In addition, the nature of the subunit interactions and assembly of the mammalian retromer complex are poorly understood. We have addressed these issues by performing biochemical and functional analyses of endogenous retromers in the human cell line HeLa. We found that the mammalian retromer complex consists of two autonomously assembling subcomplexes, namely, a Vps26-Vps29-Vps35 obligate heterotrimer and a SNX1/2 alternative heterodimer or homodimer. The association of Vps26-Vps29-Vps35 with endosomes requires the presence of either SNX1 or SNX2, whereas SNX1/2 can be recruited to endosomes independently of Vps26-Vps29-Vps35. We also found that the presence of either SNX1 or SNX2 is essential for the retrieval of the cation-independent mannose 6-phosphate receptor to the TGN. These observations indicate that the mammalian retromer complex assembles by sequential association of SNX1/2 and Vps26-Vps29-Vps35 subcomplexes on endosomal membranes and that SNX1 and SNX2 play interchangeable but essential roles in retromer structure and function. PMID:17101778

  18. Rab coupling protein mediated endosomal recycling of N-cadherin influences cell motility.

    Science.gov (United States)

    Lindsay, Andrew J; McCaffrey, Mary W

    2016-07-09

    Rab coupling protein (RCP) is a Rab GTPase effector that functions in endosomal recycling. The RCP gene is frequently amplified in breast cancer, leading to increased cancer aggressiveness. Furthermore, RCP enhances the motility of ovarian cancer cells by coordinating the recycling of α5β1 integrin and EGF receptor to the leading edge of migrating cells. Here we report that RCP also influences the motility of lung adenocarcinoma cells. Knockdown of RCP inhibits the motility of A549 cells in 2D and 3D migration assays, while its overexpression enhances migration in these assays. Depletion of RCP leads to a reduction in N-cadherin protein levels, which could be restored with lysosomal inhibitors. Trafficking assays revealed that RCP knockdown inhibits the return of endocytosed N-cadherin to the cell surface. We propose that RCP regulates the endosomal recycling of N-cadherin, and in its absence N-cadherin is diverted to the degradative pathway. The increased aggressiveness of tumour cells that overexpress RCP may be due to biased recycling of N-cadherin in metastatic cancer cells.

  19. MICAL-L1-related and unrelated mechanisms underlying elongated tubular endosomal network (ETEN) in human dendritic cells

    NARCIS (Netherlands)

    Compeer, E.B.; Boes, Marianne

    2014-01-01

    The endosomal pathway constitutes a highly dynamic intracellular transport system, which is composed of vesicular and tubular compartments. Endosomal tubules enable geometry-based discrimination between membrane and luminal content. Extended tubular endosomes were suggested to deliver a steady strea

  20. The RCP-Rab11 complex regulates endocytic protein sorting.

    Science.gov (United States)

    Peden, Andrew A; Schonteich, Eric; Chun, John; Junutula, Jagath R; Scheller, Richard H; Prekeris, Rytis

    2004-08-01

    Rab 11 GTPase is an important regulator of endocytic membrane traffic. Recently, we and others have identified a novel family of Rab11 binding proteins, known as Rab11-family interacting proteins (FIPs). One of the family members, Rab coupling protein (RCP), was identified as a protein binding to both Rab4 and Rab11 GTPases. RCP was therefore suggested to serve a dual function as Rab4 and Rab11 binding protein. In this study, we characterized the cellular functions of RCP and mapped its interactions with Rab4 and Rab11. Our data show that RCP interacts only weakly with Rab4 in vitro and does not play the role of coupling Rab11 and Rab4 in vivo. Furthermore, our data indicate that the RCP-Rab11 complex regulates the sorting of transferrin receptors from the degradative to the recycling pathway. We therefore propose that RCP functions primarily as a Rab11 binding protein that regulates protein sorting in tubular endosomes.

  1. Sorting by the cytoplasmic domain of the amyloid precursor protein binding receptor SorLA

    DEFF Research Database (Denmark)

    Nielsen, Morten S; Gustafsen, Camilla; Madsen, Peder

    2007-01-01

    -formation with the amyloid precursor protein it downregulates generation of Alzheimer's disease-associated Abeta-peptide. The receptor is mainly located in vesicles, suggesting a function in protein sorting and transport. Here we examined SorLA's trafficking using full-length and chimeric receptors and find that its...... established that the AP-1 adaptor complex is essential to SorLA's transport between Golgi membranes and endosomes. Our results further implicate the GGA proteins in SorLA trafficking and provide evidence that SNX1 and Vps35, as parts of the retromer complex or possibly in a separate context, are engaged...

  2. Endosomal system genetics and autism spectrum disorders: A literature review.

    Science.gov (United States)

    Patak, Jameson; Zhang-James, Yanli; Faraone, Stephen V

    2016-06-01

    Autism spectrum disorders (ASDs) are a group of debilitating neurodevelopmental disorders thought to have genetic etiology, due to their high heritability. The endosomal system has become increasingly implicated in ASD pathophysiology. In an attempt to summarize the association between endosomal system genes and ASDs we performed a systematic review of the literature. We searched PubMed for relevant articles. Simons Foundation Autism Research Initiative (SFARI) gene database was used to exclude articles regarding genes with less than minimal evidence for association with ASDs. Our search retained 55 articles reviewed in two categories: genes that regulate and genes that are regulated by the endosomal system. Our review shows that the endosomal system is a novel pathway implicated in ASDs as well as other neuropsychiatric disorders. It plays a central role in aspects of cellular physiology on which neurons and glial cells are particularly reliant, due to their unique metabolic and functional demands. The system shows potential for biomarkers and pharmacological intervention and thus more research into this pathway is warranted.

  3. Nuclear targeting of an endosomal E3 ubiquitin ligase.

    Science.gov (United States)

    Bocock, Jeffrey P; Carmicle, Stephanie; Madamba, Egbert; Erickson, Ann H

    2010-06-01

    Ring finger protein 13 (RNF13) is an E3 ubiquitin ligase embedded in endosome membranes. The protein undergoes constitutive post-translational proteolysis, making its detection difficult unless cells are incubated with a proteasome inhibitor to allow biosynthetic forms to accumulate. When cells were treated with phorbol 12-myristate 13-acetate (PMA), RNF13 avoided proteolysis. A similar stabilization was seen on ionomycin treatment of cells. Drug treatment stabilized both the full-length protein and a membrane-embedded C-terminal fragment generated following ectodomain shedding. Immunofluorescence staining revealed that PMA treatment caused the protein to accumulate in recycling endosomes, where it colocalized with transferrin receptor, and on the inner nuclear membrane, where it colocalized with lamin B. Expression of dominant-negative Rab11 inhibited nuclear localization, suggesting RNF13 was targeted to the inner nuclear membrane through recycling endosomes. New protein synthesis was necessary for this targeting. Nuclear localization was confirmed by immunoelectron microscopy and by purification of the inner nuclear membrane. Stress-induced transport of an endosomal protein to the inner nuclear membrane is a novel mechanism for introduction of regulatory proteins to the DNA environment. RNF13, with its ubiquitin ligase-active RING domain, has the potential to turn over key nuclear proteins in response to signals received at the plasma membrane.

  4. Bacterial toxins and small molecules elucidate endosomal trafficking.

    Science.gov (United States)

    Slater, Louise H; Clatworthy, Anne E; Hung, Deborah T

    2014-02-01

    Bacterial toxins and small molecules are useful tools for studying eukaryotic cell biology. In a recent issue of PNAS, Gillespie and colleagues describe a novel small molecule inhibitor of bacterial toxins and virus trafficking through the endocytic pathway, 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA), that prevents transport from early to late endosomes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Marburg virus inclusions: A virus-induced microcompartment and interface to multivesicular bodies and the late endosomal compartment.

    Science.gov (United States)

    Dolnik, Olga; Stevermann, Lea; Kolesnikova, Larissa; Becker, Stephan

    2015-01-01

    Filovirus infection of target cells leads to the formation of virally induced cytoplasmic inclusions that contain viral nucleocapsids at different stages of maturation. While the role of the inclusions has been unclear since the identification of Marburg and Ebola viruses, it recently became clear that the inclusions are the sites of viral replication, nucleocapsid formation and maturation. Live cell imaging analyses revealed that mature nucleocapsids are transported from inclusions to the filopodia, which represent the major budding sites. Moreover, inclusions recruit cellular proteins that have been shown to support the transport of nucleocapsids. For example, the tumor susceptibility gene 101 protein (Tsg101) interacts with a late domain motif in the nucleocapsid protein NP and recruits the actin-nucleation factor IQGAP1. Complexes of nucleocapsids together with Tsg101 and IQGAP1 are then co-transported along actin filaments. We detected additional proteins (Alix, Nedd4 and the AAA-type ATPase VPS4) of the endosomal sorting complex required for transport (ESCRT) that are recruited into inclusions. Together, the results suggest that nucleocapsids recruit the machinery that enhances viral budding at the plasma membrane. Furthermore, we identified Lamp1 as a marker of the late endosomal compartment in inclusions, while ER, Golgi, TGN and early endosomal markers were absent. In addition, we observed that LC3, a marker of autophagosomal membranes, was present in inclusions. The 3D structures of inclusions show an intricate structure that seems to accommodate an intimate cooperation between cellular and viral components with the intention to support viral transport and budding.

  6. Sex-sorting sperm using flow cytometry/cell sorting.

    Science.gov (United States)

    Garner, Duane L; Evans, K Michael; Seidel, George E

    2013-01-01

    The sex of mammalian offspring can be predetermined by flow sorting relatively pure living populations of X- and Y-chromosome-bearing sperm. This method is based on precise staining of the DNA of sperm with the nucleic acid-specific fluorophore, Hoechst 33342, to differentiate between the subpopulations of X- and Y-sperm. The fluorescently stained sperm are then sex-sorted using a specialized high speed sorter, MoFlo(®) SX XDP, and collected into biologically supportive media prior to reconcentration and cryopreservation in numbers adequate for use with artificial insemination for some species or for in vitro fertilization. Sperm sorting can provide subpopulations of X- or Y-bearing bovine sperm at rates in the 8,000 sperm/s range while maintaining; a purity of 90% such that it has been applied to cattle on a commercial basis. The sex of offspring has been predetermined in a wide variety of mammalian species including cattle, swine, horses, sheep, goats, dogs, cats, deer, elk, dolphins, water buffalo as well as in humans using flow cytometric sorting of X- and Y-sperm.

  7. Automated Sorting of Transuranic Waste

    Energy Technology Data Exchange (ETDEWEB)

    Shurtliff, Rodney Marvin

    2001-03-01

    The HANDSS-55 Transuranic Waste Sorting Module is designed to sort out items found in 55-gallon drums of waste as determined by an operator. Innovative imaging techniques coupled with fast linear motor-based motion systems and a flexible end-effector system allow the operator to remove items from the waste stream by a touch of the finger. When all desired items are removed from the waste stream, the remaining objects are automatically moved to a repackaging port for removal from the glovebox/cell. The Transuranic Waste Sorting Module consists of 1) a high accuracy XYZ Stereo Measurement and Imaging system, 2) a vibrating/tilting sorting table, 3) an XY Deployment System, 4) a ZR Deployment System, 5) several user-selectable end-effectors, 6) a waste bag opening system, 7) control and instrumentation, 8) a noncompliant waste load-out area, and 9) a Human/Machine Interface (HMI). The system is modular in design to accommodate database management tools, additional load-out ports, and other enhancements. Manually sorting the contents of a 55-gallon drum takes about one day per drum. The HANDSS-55 Waste Sorting Module is designed to significantly increase the throughput of this sorting process by automating those functions that are strenuous and tiresome for an operator to perform. The Waste Sorting Module uses the inherent ability of an operator to identify the items that need to be segregated from the waste stream and then, under computer control, picks that item out of the waste and deposits it in the appropriate location. The operator identifies the object by locating the visual image on a large color display and touches the image on the display with his finger. The computer then determines the location of the object, and performing a highspeed image analysis determines its size and orientation, so that a robotic gripper can be deployed to pick it up. Following operator verification by voice or function key, the object is deposited into a specified location.

  8. Narcissistic self-sorting in self-assembled cages of rare Earth metals and rigid ligands.

    Science.gov (United States)

    Johnson, Amber M; Wiley, Calvin A; Young, Michael C; Zhang, Xing; Lyon, Yana; Julian, Ryan R; Hooley, Richard J

    2015-05-04

    Highly selective, narcissistic self-sorting can be achieved in the formation of self-assembled cages of rare earth metals with multianionic salicylhydrazone ligands. The assembly process is highly sensitive to the length of the ligand and the coordination geometry. Most surprisingly, high-fidelity sorting is possible between ligands of identical coordination angle and geometry, differing only in a single functional group on the ligand core, which is not involved in the coordination. Supramolecular effects allow discrimination between pendant functions as similar as carbonyl or methylene groups in a complex assembly process.

  9. Synaptic vesicle generation from central nerve terminal endosomes.

    Science.gov (United States)

    Kokotos, Alexandros C; Cousin, Michael A

    2015-03-01

    Central nerve terminals contain a small number of synaptic vesicles (SVs) that must sustain the fidelity of neurotransmission across a wide range of stimulation intensities. For this to be achieved, nerve terminals integrate a number of complementary endocytosis modes whose activation spans the breadth of these neuronal stimulation patterns. Two such modes are ultrafast endocytosis and activity-dependent bulk endocytosis, which are triggered by stimuli at either end of the physiological range. Both endocytosis modes generate endosomes directly from the nerve terminal plasma membrane, before the subsequent production of SVs from these structures. This review will discuss the current knowledge relating to the molecular mechanisms involved in the generation of SVs from nerve terminal endosomes, how this relates to other mechanisms of SV production and the functional role of such SVs.

  10. Promyelocytic leukemia bodies tether to early endosomes during mitosis.

    Science.gov (United States)

    Palibrk, Vuk; Lång, Emma; Lång, Anna; Schink, Kay Oliver; Rowe, Alexander D; Bøe, Stig Ove

    2014-01-01

    During mitosis the nuclear envelope breaks down, leading to potential interactions between cytoplasmic and nuclear components. PML bodies are nuclear structures with tumor suppressor and antiviral functions. Early endosomes, on the other hand, are cytoplasmic vesicles involved in transport and growth factor signaling. Here we demonstrate that PML bodies form stable interactions with early endosomes immediately following entry into mitosis. The 2 compartments remain stably associated throughout mitosis and dissociate in the cytoplasm of newly divided daughter cells. We also show that a minor subset of PML bodies becomes anchored to the mitotic spindle poles during cell division. The study demonstrates a stable mitosis-specific interaction between a cytoplasmic and a nuclear compartment.

  11. Sorting and selection in posets

    DEFF Research Database (Denmark)

    Daskalakis, Constantinos; Karp, Richard M.; Mossel, Elchanan

    2011-01-01

    Classical problems of sorting and searching assume an underlying linear ordering of the objects being compared. In this paper, we study these problems in the context of partially ordered sets, in which some pairs of objects are incomparable. This generalization is interesting from a combinatorial...

  12. Vamp-7 Mediates Vesicular Transport from Endosomes to Lysosomes

    Science.gov (United States)

    Advani, Raj J.; Yang, Bin; Prekeris, Rytis; Lee, Kelly C.; Klumperman, Judith; Scheller, Richard H.

    1999-01-01

    A more complete picture of the molecules that are critical for the organization of membrane compartments is beginning to emerge through the characterization of proteins in the vesicle-associated membrane protein (also called synaptobrevin) family of membrane trafficking proteins. To better understand the mechanisms of membrane trafficking within the endocytic pathway, we generated a series of monoclonal and polyclonal antibodies against the cytoplasmic domain of vesicle-associated membrane protein 7 (VAMP-7). The antibodies recognize a 25-kD membrane-associated protein in multiple tissues and cell lines. Immunohistochemical analysis reveals colocalization with a marker of late endosomes and lysosomes, lysosome-associated membrane protein 1 (LAMP-1), but not with other membrane markers, including p115 and transferrin receptor. Treatment with nocodozole or brefeldin A does not disrupt the colocalization of VAMP-7 and LAMP-1. Immunoelectron microscopy analysis shows that VAMP-7 is most concentrated in the trans-Golgi network region of the cell as well as late endosomes and transport vesicles that do not contain the mannose-6 phosphate receptor. In streptolysin- O–permeabilized cells, antibodies against VAMP-7 inhibit the breakdown of epidermal growth factor but not the recycling of transferrin. These data are consistent with a role for VAMP-7 in the vesicular transport of proteins from the early endosome to the lysosome. PMID:10459012

  13. Enhancing endosomal escape of transduced proteins by photochemical internalisation.

    Directory of Open Access Journals (Sweden)

    Kevin Mellert

    Full Text Available Induced internalisation of functional proteins into cultured cells has become an important aspect in a rising number of in vitro and in vivo assays. The endo-lysosomal entrapment of the transduced proteins remains the major problem in all transduction protocols. In this study we compared the efficiency, cytotoxicity and protein targeting of different commercially available transduction reagents by transducing a well-studied fluorescently labelled protein (Atto488-bovine serum albumin into cultured human sarcoma cells. The amount of internalised protein and toxicity differed between the different reagents, but the percentage of transduced cells was consistently high. Furthermore, in all protocols the signals of the transduced Atto488-BSA were predominantly punctual consistent with an endosomal localisation. To overcome the endosomal entrapment, the transduction protocols were combined with a photochemical internalisation (PCI treatment. Using this combination revealed that an endosomal disruption is highly effective in cell penetrating peptide (CPP mediated transduction, whereas lipid-mediated transductions lead to a lower signal spreading throughout the cytosol. No change in the signal distribution could be achieved in treatments using non-lipid polymers as a transduction reagent. Therefore, the combination of protein transduction protocols based on CPPs with the endosomolytic treatment PCI can facilitate protein transduction experiments in vitro.

  14. Development of a Kinetic Assay for Late Endosome Movement.

    Science.gov (United States)

    Esner, Milan; Meyenhofer, Felix; Kuhn, Michael; Thomas, Melissa; Kalaidzidis, Yannis; Bickle, Marc

    2014-08-01

    Automated imaging screens are performed mostly on fixed and stained samples to simplify the workflow and increase throughput. Some processes, such as the movement of cells and organelles or measuring membrane integrity and potential, can be measured only in living cells. Developing such assays to screen large compound or RNAi collections is challenging in many respects. Here, we develop a live-cell high-content assay for tracking endocytic organelles in medium throughput. We evaluate the added value of measuring kinetic parameters compared with measuring static parameters solely. We screened 2000 compounds in U-2 OS cells expressing Lamp1-GFP to label late endosomes. All hits have phenotypes in both static and kinetic parameters. However, we show that the kinetic parameters enable better discrimination of the mechanisms of action. Most of the compounds cause a decrease of motility of endosomes, but we identify several compounds that increase endosomal motility. In summary, we show that kinetic data help to better discriminate phenotypes and thereby obtain more subtle phenotypic clustering.

  15. Sorting nexin 17 regulates ApoER2 recycling and reelin signaling.

    Directory of Open Access Journals (Sweden)

    Pablo Sotelo

    Full Text Available ApoER2 is a member of the low density-lipoprotein receptor (LDL-R family. As a receptor for reelin, ApoER2 participates in neuronal migration during development as well as synaptic plasticity and survival in the adult brain. A previous yeast two-hybrid screen showed that ApoER2 is a binding partner of sorting nexin 17 (SNX17 - a cytosolic adaptor protein that regulates the trafficking of several membrane proteins in the endosomal pathway, including LRP1, P-selectin and integrins. However, no further studies have been performed to investigate the role of SNX17 in ApoER2 trafficking and function. In this study, we present evidence based on GST pull-down and inmunoprecipitation assays that the cytoplasmic NPxY endocytosis motif of ApoER2 interacts with the FERM domain of SNX17. SNX17 stimulates ApoER2 recycling in different cell lines including neurons without affecting its endocytic rate and also facilitates the transport of ApoER2 from the early endosomes to the recycling endosomes. The reduction of SNX17 was associated with accumulation of an ApoER2 carboxy-terminal fragment (CTF. In addition, in SNX17 knockdown cells, constitutive ApoER2 degradation was not modified, whereas reelin-induced ApoER2 degradation was increased, implying that SNX17 is a regulator of the receptor's half-life. Finally, in SNX17 silenced hippocampal and cortical neurons, we underscored a positive role of this endosomal protein in the development of the dendritic tree and reelin signaling. Overall, these results establish the role of SNX17 in ApoER2 trafficking and function and aid in identifying new links between endocytic trafficking and receptor signaling.

  16. Sorting nexin 17 regulates ApoER2 recycling and reelin signaling.

    Science.gov (United States)

    Sotelo, Pablo; Farfán, Pamela; Benitez, María Luisa; Bu, Guojun; Marzolo, María-Paz

    2014-01-01

    ApoER2 is a member of the low density-lipoprotein receptor (LDL-R) family. As a receptor for reelin, ApoER2 participates in neuronal migration during development as well as synaptic plasticity and survival in the adult brain. A previous yeast two-hybrid screen showed that ApoER2 is a binding partner of sorting nexin 17 (SNX17) - a cytosolic adaptor protein that regulates the trafficking of several membrane proteins in the endosomal pathway, including LRP1, P-selectin and integrins. However, no further studies have been performed to investigate the role of SNX17 in ApoER2 trafficking and function. In this study, we present evidence based on GST pull-down and inmunoprecipitation assays that the cytoplasmic NPxY endocytosis motif of ApoER2 interacts with the FERM domain of SNX17. SNX17 stimulates ApoER2 recycling in different cell lines including neurons without affecting its endocytic rate and also facilitates the transport of ApoER2 from the early endosomes to the recycling endosomes. The reduction of SNX17 was associated with accumulation of an ApoER2 carboxy-terminal fragment (CTF). In addition, in SNX17 knockdown cells, constitutive ApoER2 degradation was not modified, whereas reelin-induced ApoER2 degradation was increased, implying that SNX17 is a regulator of the receptor's half-life. Finally, in SNX17 silenced hippocampal and cortical neurons, we underscored a positive role of this endosomal protein in the development of the dendritic tree and reelin signaling. Overall, these results establish the role of SNX17 in ApoER2 trafficking and function and aid in identifying new links between endocytic trafficking and receptor signaling.

  17. Protein complexes and cholesterol in the control of late endosomal dynamicsCholesterol and multi-protein complexes in the control of late endosomal dynamics

    NARCIS (Netherlands)

    Kant, Rik Henricus Nicolaas van der

    2013-01-01

    Late endosomal transport is disrupted in several diseases such as Niemann-Pick type C, ARC syndrome and Alzheimer’s disease. This thesis describes the regulation of late endosomal dynamics by cholesterol and multi-protein complexes. We find that cholesterol acts as a cellular tomtom that steers the

  18. Retrieval of the Alzheimer's amyloid precursor protein from the endosome to the TGN is S655 phosphorylation state-dependent and retromer-mediated

    Directory of Open Access Journals (Sweden)

    Gandy Sam

    2010-10-01

    Full Text Available Abstract Background Retrograde transport of several transmembrane proteins from endosomes to the trans-Golgi network (TGN occurs via Rab 5-containing endosomes, mediated by clathrin and the recently characterized retromer complex. This complex and one of its putative sorting receptor components, SorLA, were reported to be associated to late onset Alzheimer's disease (AD. The pathogenesis of this neurodegenerative disorder is still elusive, although accumulation of amyloidogenic Abeta is a hallmark. This peptide is generated from the sucessive β- and γ- secretase proteolysis of the Alzheimer's amyloid precursor protein (APP, events which are associated with endocytic pathway compartments. Therefore, APP targeting and time of residence in endosomes would be predicted to modulate Abeta levels. However, the formation of an APP- and retromer-containing protein complex with potential functions in retrieval of APP from the endosome to the TGN had, to date, not been demonstrated directly. Further, the motif(s in APP that regulate its sorting to the TGN have not been characterized. Results Through the use of APP-GFP constructs, we show that APP containing endocytic vesicles targeted for the TGN, are also immunoreactive for clathrin-, Rab 5- and VPS35. Further, they frequently generate protruding tubules near the TGN, supporting an association with a retromer-mediated pathway. Importantly, we show for the first time, that mimicking APP phosphorylation at S655, within the APP 653YTSI656 basolateral motif, enhances APP retrieval via a retromer-mediated process. The phosphomimetic APP S655E displays decreased APP lysosomal targeting, enhanced mature half-life, and decreased tendency towards Abeta production. VPS35 downregulation impairs the phosphorylation dependent APP retrieval to the TGN, and decreases APP half-life. Conclusions We reported for the first time the importance of APP phosphorylation on S655 in regulating its retromer-mediated sorting to

  19. Fluorescent biosensors illuminate calcium levels within defined beta-cell endosome subpopulations.

    Science.gov (United States)

    Albrecht, Tobias; Zhao, Yongxin; Nguyen, Trang Hai; Campbell, Robert E; Johnson, James D

    2015-04-01

    Live cell imaging has revealed that calcium ions (Ca(2+)) pass in and out of many cellular organelles. However, technical hurdles have limited measurements of Ca(2+) in acidic organelles, such as endosomes. Although evidence hints that endosomes play a role in Ca(2+) signaling, direct measurements within endosomal lumina represent one of the final frontiers in organelle imaging. To measure Ca(2+) in a TiVAMP-positive endosome sub-population, the pH-resistant ratiometric Ca(2+) biosensor GEM-GECO1 and the ratiometric pH biosensor mKeima were used. A positive correlation between acidic endosomal pH and higher Ca(2+) was observed within these Rab5a- and Rab7-positive compartments. Ca(2+) concentration in most endosomes was estimated to be below 2μM, lower than Ca(2+) levels in several other intracellular stores, indicating that endosomes may take up Ca(2+) during physiological stimulation. Indeed, endosomes accumulated Ca(2+) during glucose-stimulation, a condition where endosomal pH did not change. Our biosensors permitted the first measurements revealing a role for endosomes in cellular Ca(2+) homeostasis during physiological stimulation.

  20. Endosome-mitochondria interactions are modulated by iron release from transferrin.

    Science.gov (United States)

    Das, Anupam; Nag, Sagarika; Mason, Anne B; Barroso, Margarida M

    2016-09-26

    Transient "kiss and run" interactions between endosomes containing iron-bound transferrin (Tf) and mitochondria have been shown to facilitate direct iron transfer in erythroid cells. In this study, we used superresolution three-dimensional (3D) direct stochastic optical reconstruction microscopy to show that Tf-containing endosomes directly interact with mitochondria in epithelial cells. We used live-cell time-lapse fluorescence microscopy, followed by 3D rendering, object tracking, and a distance transformation algorithm, to track Tf-endosomes and characterize the dynamics of their interactions with mitochondria. Quenching of iron sensor RDA-labeled mitochondria confirmed functional iron transfer by an interacting Tf-endosome. The motility of Tf-endosomes is significantly reduced upon interaction with mitochondria. To further assess the functional role of iron in the ability of Tf-endosomes to interact with mitochondria, we blocked endosomal iron release by using a Tf K206E/K534A mutant. Blocking intraendosomal iron release led to significantly increased motility of Tf-endosomes and increased duration of endosome-mitochondria interactions. Thus, intraendosomal iron regulates the kinetics of the interactions between Tf-containing endosomes and mitochondria in epithelial cells. © 2016 Das et al.

  1. GLUT4 traffic through an ESCRT-III-dependent sorting compartment in adipocytes.

    Directory of Open Access Journals (Sweden)

    Françoise Koumanov

    Full Text Available In insulin target tissues, GLUT4 is known to traffic through multiple compartments that may involve ubiquitin- and/or SUMO-dependent targeting. During these trafficking steps, GLUT4 is sorted into a storage reservoir compartment that is acutely released by insulin signalling processes that are downstream of PI 3-kinase associated changes in inositol phospholipids. As ESCRT components have recently been found to influence cellular sorting processes that are related to changes in both ubiquitination and inositol phospholipids, we have examined whether GLUT4 traffic is routed through ESCRT dependent sorting steps. Introduction of the dominant negative inhibitory constructs of the ESCRT-III components CHMP3 (CHMP3(1-179 and Vps4 (GFP-Vps4(E235Q into rat adipocytes leads to the accumulation of GLUT4 in large, coalesced and extended vesicles structures that co-localise with the inhibitory constructs over large parts of the extended structure. A new swollen hybrid and extensively ubiquitinated compartment is produced in which GLUT4 co-localises more extensively with the endosomal markers including EEA1 and transferrin receptors but also with the TGN marker syntaxin6. These perturbations are associated with failure of insulin action on GLUT4 traffic to the cell surface and suggest impairment in an ESCRT-dependent sorting step used for GLUT4 traffic to its specialised reservoir compartment.

  2. Sorting cells by their density

    Science.gov (United States)

    Norouzi, Nazila; Bhakta, Heran C.

    2017-01-01

    Sorting cells by their type is an important capability in biological research and medical diagnostics. However, most cell sorting techniques rely on labels or tags, which may have limited availability and specificity. Sorting different cell types by their different physical properties is an attractive alternative to labels because all cells intrinsically have these physical properties. But some physical properties, like cell size, vary significantly from cell to cell within a cell type; this makes it difficult to identify and sort cells based on their sizes alone. In this work we continuously sort different cells types by their density, a physical property with much lower cell-to-cell variation within a cell type (and therefore greater potential to discriminate different cell types) than other physical properties. We accomplish this using a 3D-printed microfluidic chip containing a horizontal flowing micron-scale density gradient. As cells flow through the chip, Earth’s gravity makes each cell move vertically to the point where the cell’s density matches the surrounding fluid’s density. When the horizontal channel then splits, cells with different densities are routed to different outlets. As a proof of concept, we use our density sorter chip to sort polymer microbeads by their material (polyethylene and polystyrene) and blood cells by their type (white blood cells and red blood cells). The chip enriches the fraction of white blood cells in a blood sample from 0.1% (in whole blood) to nearly 98% (in the output of the chip), a 1000x enrichment. Any researcher with access to a 3D printer can easily replicate our density sorter chip and use it in their own research using the design files provided as online Supporting Information. Additionally, researchers can simulate the performance of a density sorter chip in their own applications using the Python-based simulation software that accompanies this work. The simplicity, resolution, and throughput of this

  3. The RING-CH ligase K5 antagonizes restriction of KSHV and HIV-1 particle release by mediating ubiquitin-dependent endosomal degradation of tetherin.

    Directory of Open Access Journals (Sweden)

    Claire Pardieu

    2010-04-01

    Full Text Available Tetherin (CD317/BST2 is an interferon-induced membrane protein that inhibits the release of diverse enveloped viral particles. Several mammalian viruses have evolved countermeasures that inactivate tetherin, with the prototype being the HIV-1 Vpu protein. Here we show that the human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV is sensitive to tetherin restriction and its activity is counteracted by the KSHV encoded RING-CH E3 ubiquitin ligase K5. Tetherin expression in KSHV-infected cells inhibits viral particle release, as does depletion of K5 protein using RNA interference. K5 induces a species-specific downregulation of human tetherin from the cell surface followed by its endosomal degradation. We show that K5 targets a single lysine (K18 in the cytoplasmic tail of tetherin for ubiquitination, leading to relocalization of tetherin to CD63-positive endosomal compartments. Tetherin degradation is dependent on ESCRT-mediated endosomal sorting, but does not require a tyrosine-based sorting signal in the tetherin cytoplasmic tail. Importantly, we also show that the ability of K5 to substitute for Vpu in HIV-1 release is entirely dependent on K18 and the RING-CH domain of K5. By contrast, while Vpu induces ubiquitination of tetherin cytoplasmic tail lysine residues, mutation of these positions has no effect on its antagonism of tetherin function, and residual tetherin is associated with the trans-Golgi network (TGN in Vpu-expressing cells. Taken together our results demonstrate that K5 is a mechanistically distinct viral countermeasure to tetherin-mediated restriction, and that herpesvirus particle release is sensitive to this mode of antiviral inhibition.

  4. Swarm-Based Spatial Sorting

    CERN Document Server

    Amos, Martyn

    2008-01-01

    Purpose: To present an algorithm for spatially sorting objects into an annular structure. Design/Methodology/Approach: A swarm-based model that requires only stochastic agent behaviour coupled with a pheromone-inspired "attraction-repulsion" mechanism. Findings: The algorithm consistently generates high-quality annular structures, and is particularly powerful in situations where the initial configuration of objects is similar to those observed in nature. Research limitations/implications: Experimental evidence supports previous theoretical arguments about the nature and mechanism of spatial sorting by insects. Practical implications: The algorithm may find applications in distributed robotics. Originality/value: The model offers a powerful minimal algorithmic framework, and also sheds further light on the nature of attraction-repulsion algorithms and underlying natural processes.

  5. Sorting fluorescent nanocrystals with DNA

    Energy Technology Data Exchange (ETDEWEB)

    Gerion, Daniele; Parak, Wolfgang J.; Williams, Shara C.; Zanchet, Daniela; Micheel, Christine M.; Alivisatos, A. Paul

    2001-12-10

    Semiconductor nanocrystals with narrow and tunable fluorescence are covalently linked to oligonucleotides. These biocompounds retain the properties of both nanocrystals and DNA. Therefore, different sequences of DNA can be coded with nanocrystals and still preserve their ability to hybridize to their complements. We report the case where four different sequences of DNA are linked to four nanocrystal samples having different colors of emission in the range of 530-640 nm. When the DNA-nanocrystal conjugates are mixed together, it is possible to sort each type of nanoparticle using hybridization on a defined micrometer -size surface containing the complementary oligonucleotide. Detection of sorting requires only a single excitation source and an epifluorescence microscope. The possibility of directing fluorescent nanocrystals towards specific biological targets and detecting them, combined with their superior photo-stability compared to organic dyes, opens the way to improved biolabeling experiments, such as gene mapping on a nanometer scale or multicolor microarray analysis.

  6. Sorting Techniques for Plastics Recycling

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    This paper presents the basic principles of three different types of separating methods and a general guideline for choosing the most effective method for sorting plastic mixtures. It also presents the results of the tests carried out for separation of PVC, ABS and PET from different kinds of plastic mixtures in order to improve the grade of the raw input used in mechanical or feedstock recycling.

  7. Sorting of Sperm by Morphology

    Science.gov (United States)

    Koh, James; Marcos, Marcos

    2016-11-01

    Many studies have proven that the percentage of morphologically normal sperm is a significant factor in determining the success of assisted reproduction. The velocity of sperm in a microchannel with shear flow subjected to an external field will be explored theoretically. The difference in response between morphologically normal and abnormal sperm will be computed from a statistical approach, to study the feasibility and effectiveness of sorting by an external field to remove abnormal sperm. The full name of this author is Marcos.

  8. Unravelling the pivotal role of Alix in MVB sorting and silencing of the activated EGFR.

    Science.gov (United States)

    Sun, Sheng; Zhou, Xi; Zhang, Wei; Gallick, Gary E; Kuang, Jian

    2015-03-15

    Endosomal sorting complex required for transport (ESCRT)-III-mediated membrane invagination and scission are a critical step in multivesicular body (MVB) sorting of ubiquitinated membrane receptors, and generally thought to be required for degradation of these receptors in lysosomes. The adaptor protein Alix is critically involved in multiple ESCRT-III-mediated, membrane-remodelling processes in mammalian cells. However, Alix knockdown does not inhibit degradation of the activated epidermal growth factor receptor (EGFR) in mammalian cell lines, leading to a widely held notion that Alix is not critically involved in MVB sorting of ubiquitinated membrane receptors in mammalian cells. In the present study, we demonstrate that, despite its non-essential role in degradation of the activated EGFR, Alix plays a critical role in its MVB sorting and silencing Epidermal growth factor (EGF) stimulation of mammalian cell lines induces Alix's interaction with the ubiquitinated EGFR via the Alix V domain, and increases Alix's association with membrane-bound charged multivesicular body protein 4 (CHMP4) via the Alix Bro1 domain. Under both continuous and pulse-chase EGF stimulation conditions, inhibition of Alix's interaction with membrane-bound CHMP4, inhibition of Alix dimerization through the V domain or Alix knockdown dramatically inhibits MVB sorting of the activated EGFR and promotes sustained activation of extracellular-signal regulated kinase (ERK)1/2. Under the continuous EGF stimulation conditions, these cell treatments also retard degradation of the activated EGFR. These findings indicate that Alix is critically involved in MVB sorting of ubiquitinated membrane receptors in mammalian cells.

  9. Self-sorting molecular clips.

    Science.gov (United States)

    Ghosh, Soumyadip; Wu, Anxin; Fettinger, James C; Zavalij, Peter Y; Isaacs, Lyle

    2008-08-01

    We report the synthesis and characterization of 12 C-shaped methylene-bridged glycoluril dimers (1-12) bearing H-bonding groups on their aromatic rings. Compounds 1, 2, (+/-)-4a, (+/-)-5, (+/-)-7, and 8 form tightly associated homodimers in CDCl3, due to the combined driving force of pi-pi and H-bonding interactions. Compounds 2, (+/-)-5, and 8, having disparate spatial distribution of their H-bonding groups, display the ability to efficiently distinguish between self and nonself even within three-component mixtures in CDCl3. When the spatial distributions of the H-bonding groups of the molecular clips are similar (e.g., 1 and 2), a mixture of homodimers and heterodimers is formed. The effect of various structural modifications (e.g., chirality, side chain steric bulk, number and pattern of H-bonds) on the strength of self-assembly and the fidelity of self-sorting are presented. On the basis of these results we prepared self-sorting systems comprising three (e.g., 1, (+/-)-5, and (+/-)-7) and even four ( 2, (+/-)-5, 9, and 10) components. The potential of molecular clips 1-12 as robust, functionalizable, self-sorting modules to control the noncovalent interaction network in systems chemistry studies is described.

  10. Flow cytometry and cell sorting.

    Science.gov (United States)

    Ibrahim, Sherrif F; van den Engh, Ger

    2007-01-01

    Flow cytometry and cell sorting are well-established technologies in clinical diagnostics and biomedical research. Heterogeneous mixtures of cells are placed in suspension and passed single file across one or more laser interrogation points. Light signals emitted from the particles are collected and correlated to entities such as cell morphology, surface and intracellular protein expression, gene expression, and cellular physiology. Based on user-defined parameters, individual cells can then be diverted from the fluid stream and collected into viable, homogeneous fractions at exceptionally high speeds and a purity that approaches 100%. As such, the cell sorter becomes the launching point for numerous downstream studies. Flow cytometry is a cornerstone in clinical diagnostics, and cheaper, more versatile machines are finding their way into widespread and varied uses. In addition, advances in computing and optics have led to a new generation of flow cytometers capable of processing cells at orders of magnitudes faster than their predecessors, and with staggering degrees of complexity, making the cytometer a powerful discovery tool in biotechnology. This chapter will begin with a discussion of basic principles of flow cytometry and cell sorting, including a technical description of factors that contribute to the performance of these instruments. The remaining sections will then be divided into clinical- and research-based applications of flow cytometry and cell sorting, highlighting salient studies that illustrate the versatility of this indispensable technology.

  11. Word Sorts for General Music Classes

    Science.gov (United States)

    Cardany, Audrey Berger

    2015-01-01

    Word sorts are standard practice for aiding children in acquiring skills in English language arts. When included in the general music classroom, word sorts may aid students in acquiring a working knowledge of music vocabulary. The author shares a word sort activity drawn from vocabulary in John Lithgow's children's book "Never Play…

  12. How does the Shift-insertion sort behave when the sorting elements follow a Normal distribution?

    CERN Document Server

    Pal, Mita; Mahanti, N C

    2012-01-01

    The present paper examines the behavior of Shift-insertion sort (insertion sort with shifting) for normal distribution inputs and is in continuation of our earlier work on this new algorithm for discrete distribution inputs, namely, negative binomial. Shift insertion sort is found more sensitive for main effects but not for all interaction effects compared to conventional insertion sort.

  13. Recruitment of actin modifiers to TrkA endosomes governs retrograde NGF signaling and survival

    Science.gov (United States)

    Harrington, Anthony W.; Hillaire, Coryse St.; Zweifel, Larry S.; Glebova, Natalia O.; Philippidou, Polyxeni; Halegoua, Simon; Ginty, David D.

    2012-01-01

    Summary NGF and NT3 collaborate to support development of sympathetic neurons. Although both neurotrophins activate TrkA-dependent axonal extension, NGF is unique in its ability to promote retrograde transport of TrkA endosomes and retrograde survival. Here, we report that actin depolymerization is essential for initiation of NGF/TrkA endosome trafficking and that a Rac1–cofilin signaling module associated with TrkA early endosomes supports their maturation to retrograde transport-competent endosomes. Moreover, the actin-regulatory endosomal components are absent from NT3-formed TrkA endosomes, explaining the failure of NT3 to support retrograde TrkA transport and survival. The inability of NT3 to activate Rac1-GTP–cofilin signaling is likely due to the labile nature of NT3/TrkA complexes within the acidic environment of TrkA early endosomes. Thus, TrkA endosomes associate with actin-modulatory proteins to promote F-actin disassembly enabling their maturation into transport-competent signaling endosomes. Differential control of this process explains how NGF in final targets, but not NT3 from intermediate targets, supports retrograde survival of sympathetic neurons. PMID:21816277

  14. Membrane glycoprotein M6A promotes μ-opioid receptor endocytosis and facilitates receptor sorting into the recycling pathway

    Institute of Scientific and Technical Information of China (English)

    Ying-Jian Liang; Dai-Fei Wu; Ralf Stumm; Volker H(o)llt; Thomas Koch

    2008-01-01

    The interaction of μ-opioid receptor (MOPr) with the neuronal membrane glycoprotein M6a is known to facilitate MOPr endocytosis in human embryonic kidney 293 (HEK293) cells. To further study the role of M6a in the post-endocytotic sorting of MOPr, we investigated the agonist-induced co-internalization of MOPr and M6a and protein targeting after internalization in HEK293 cells that co-expressed HA-tagged MOPr and Myc-tagged M6a. We found that M6a, MOPr, and Rab 11, a marker for recycling endosomes, co-localized in endocytotic vesicles, indicating that MOPr and M6a are primarily targeted to recycling endosomes after endocytosis. Furthermore, co-expression of M6a augmented the post-endocytotic sorting of δ-opioid receptors into the recycling pathway, indicating that M6a might have a more general role in opioid receptor post-ndocytotic sorting. The enhanced post-endocytotic sorting of MOPr into the recycling pathway was accompanied by a decrease in agonist-induced receptor down-regulation of M6a in co-expressing cells. We tested the physiological relevance of these findings in primary cultures of cortical neurons and found that co-expression of M6a markedly increased the translocation of MOPrs from the plasma membrane to intracellular vesicles at steady state and significantly enhanced both constitutive and agonist-induced receptor endocytosis. In conclusion, our results strongly indicate that M6a modulates MOPr endocytosis and post-endocytotic sorting and has an important role in receptor regulation.

  15. Polarized sorting and trafficking in epithelial cells

    Institute of Scientific and Technical Information of China (English)

    Xinwang Cao; Michal A Surma; Kai Simons

    2012-01-01

    The polarized distribution of proteins and lipids at the surface membrane of epithelial cells results in the formation of an apical and a basolateral domain,which are separated by tight junctions.The generation and maintenance of epithelial polarity require elaborate mechanisms that guarantee correct sorting and vectorial delivery of cargo molecules.This dynamic process involves the interaction of sorting signals with sorting machineries and the formation of transport carriers.Here we review the recent advances in the field of polarized sorting in epithelial cells.We especially highlight the role of lipid rafts in apical sorting.

  16. Effect of diphtheria toxin T-domain on endosomal pH

    Directory of Open Access Journals (Sweden)

    A. J. Labyntsev

    2015-08-01

    Full Text Available A key step in the mode of cytotoxic action of diphtheria toxin (DT is the transfer of its catalytic domain (Cd from endosomes into the cytosol. The main activity in this process is performed by the transport domain (Td, but the molecular mechanism of its action remains unknown. We have previously shown that Td can have some influence on the endosomal transport of DT. The aim of this work was to study the effect of diphtheria toxin on the toxin compartmentalization in the intracellular transporting pathway and endosomal pH. We used recombinant fragments of DT, which differed only by the presence of Td in their structure, fused with fluorescent proteins. It was shown that the toxin fragment with Td moved slower by the pathway early-late endosomes-lysosomes, and had a slightly different pattern of colocalization with endosomal markers than DT fragment without Td. In addition, endosomes containing DT fragments with Td had a constant pH of about 6.5 from the 10th to 50th minute of observation, for the same time endosomes containing DT fragments without Td demons­trated a decrease in pH from 6.3 to 5.5. These results indicate that Td inhibits acidification of endosomal medium. One of possible explanations for this may be the effect of the ion channel formed by the T-domain on the process of the endosomal acidification. This property of Td may not only inhibit maturation of endosomes but also inhibit activation of endosomal pH-dependent proteases, and this promotes successful transport of Cd into the cell cytosol.

  17. Sorting and Selection in Posets

    CERN Document Server

    Daskalakis, Constantinos; Mossel, Elchanan; Riesenfeld, Samantha; Verbin, Elad

    2007-01-01

    Classical problems of sorting and searching assume an underlying linear ordering of the objects being compared. In this paper, we study a more general setting, in which some pairs of objects are incomparable. This generalization is relevant in applications related to rankings in sports, college admissions, or conference submissions. It also has potential applications in biology, such as comparing the evolutionary fitness of different strains of bacteria, or understanding input-output relations among a set of metabolic reactions or the causal influences among a set of interacting genes or proteins. Our results improve and extend results from two decades ago of Faigle and Tur\\'{a}n. A measure of complexity of a partially ordered set (poset) is its width. Our algorithms obtain information about a poset by queries that compare two elements. We present an algorithm that sorts, i.e. completely identifies, a width w poset of size n and has query complexity O(wn + nlog(n)), which is within a constant factor of the in...

  18. An Endosomal NAADP-Sensitive Two-Pore Ca2+ Channel Regulates ER-Endosome Membrane Contact Sites to Control Growth Factor Signaling

    Directory of Open Access Journals (Sweden)

    Bethan S. Kilpatrick

    2017-02-01

    Full Text Available Membrane contact sites are regions of close apposition between organelles that facilitate information transfer. Here, we reveal an essential role for Ca2+ derived from the endo-lysosomal system in maintaining contact between endosomes and the endoplasmic reticulum (ER. Antagonizing action of the Ca2+-mobilizing messenger NAADP, inhibiting its target endo-lysosomal ion channel, TPC1, and buffering local Ca2+ fluxes all clustered and enlarged late endosomes/lysosomes. We show that TPC1 localizes to ER-endosome contact sites and is required for their formation. Reducing NAADP-dependent contacts delayed EGF receptor de-phosphorylation consistent with close apposition of endocytosed receptors with the ER-localized phosphatase PTP1B. In accord, downstream MAP kinase activation and mobilization of ER Ca2+ stores by EGF were exaggerated upon NAADP blockade. Membrane contact sites between endosomes and the ER thus emerge as Ca2+-dependent hubs for signaling.

  19. Fixing the Sorting Algorithm for Android, Java and Python

    NARCIS (Netherlands)

    C.P.T. de Gouw (Stijn); F.S. de Boer (Frank)

    2015-01-01

    htmlabstractTim Peters developed the Timsort hybrid sorting algorithm in 2002. TimSort was first developed for Python, a popular programming language, but later ported to Java (where it appears as java.util.Collections.sort and java.util.Arrays.sort). TimSort is today used as the default sorting alg

  20. Fixing the Sorting Algorithm for Android, Java and Python

    NARCIS (Netherlands)

    Gouw, C.P.T. de; Boer, F.S. de

    2015-01-01

    Tim Peters developed the Timsort hybrid sorting algorithm in 2002. TimSort was first developed for Python, a popular programming language, but later ported to Java (where it appears as java.util.Collections.sort and java.util.Arrays.sort). TimSort is today used as the default sorting algorithm in Ja

  1. Fixing the Sorting Algorithm for Android, Java and Python

    NARCIS (Netherlands)

    C.P.T. de Gouw (Stijn); F.S. de Boer (Frank)

    2015-01-01

    htmlabstractTim Peters developed the Timsort hybrid sorting algorithm in 2002. TimSort was first developed for Python, a popular programming language, but later ported to Java (where it appears as java.util.Collections.sort and java.util.Arrays.sort). TimSort is today used as the default sorting

  2. Energy efficient data sorting using standard sorting algorithms

    KAUST Repository

    Bunse, Christian

    2011-01-01

    Protecting the environment by saving energy and thus reducing carbon dioxide emissions is one of todays hottest and most challenging topics. Although the perspective for reducing energy consumption, from ecological and business perspectives is clear, from a technological point of view, the realization especially for mobile systems still falls behind expectations. Novel strategies that allow (software) systems to dynamically adapt themselves at runtime can be effectively used to reduce energy consumption. This paper presents a case study that examines the impact of using an energy management component that dynamically selects and applies the "optimal" sorting algorithm, from an energy perspective, during multi-party mobile communication. Interestingly, the results indicate that algorithmic performance is not key and that dynamically switching algorithms at runtime does have a significant impact on energy consumption. © Springer-Verlag Berlin Heidelberg 2011.

  3. Regulation of endosomal motility and degradation by amyotrophic lateral sclerosis 2/alsin

    Directory of Open Access Journals (Sweden)

    Lai Chen

    2009-07-01

    Full Text Available Abstract Dysfunction of alsin, particularly its putative Rab5 guanine-nucleotide-exchange factor activity, has been linked to one form of juvenile onset recessive familial amyotrophic lateral sclerosis (ALS2. Multiple lines of alsin knockout (ALS2-/- mice have been generated to model this disease. However, it remains elusive whether the Rab5-dependent endocytosis is altered in ALS2-/- neurons. To directly examine the Rab5-mediated endosomal trafficking in ALS2-/- neurons, we introduced green fluorescent protein (GFP-tagged Rab5 into cultured hippocampal neurons to monitor the morphology and motility of Rab5-associated early endosomes. Here we report that Rab5-mediated endocytosis was severely altered in ALS2-/-neurons. Excessive accumulation of Rab5-positive vesicles was observed in ALS2-/- neurons, which correlated with a significant reduction in endosomal motility and augmentation in endosomal conversion to lysosomes. Consequently, a significant increase in endosome/lysosome-dependent degradation of internalized glutamate receptors was observed in ALS2-/- neurons. These phenotypes closely resembled the endosomal trafficking abnormalities induced by a constitutively active form of Rab5 in wild-type neurons. Therefore, our findings reveal a negatively regulatory mechanism of alsin in Rab5-mediated endosomal trafficking, suggesting that enhanced endosomal degradation in ALS2-/- neurons may underlie the pathogenesis of motor neuron degeneration in ALS2 and related motor neuron diseases.

  4. Endosomal SNARE proteins regulate CFTR activity and trafficking in epithelial cells.

    Science.gov (United States)

    Bilan, Frédéric; Nacfer, Magali; Fresquet, Fleur; Norez, Caroline; Melin, Patricia; Martin-Berge, Alice; Costa de Beauregard, Marie-Alyette; Becq, Frédéric; Kitzis, Alain; Thoreau, Vincent

    2008-07-01

    The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) protein is a chloride channel localized at the apical plasma membrane of epithelial cells. We previously described that syntaxin 8, an endosomal SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) protein, interacts with CFTR and regulates its trafficking to the plasma membrane and hence its channel activity. Syntaxin 8 belongs to the endosomal SNARE complex which also contains syntaxin 7, vti1b and VAMP8. Here, we report that these four endosomal SNARE proteins physically and functionally interact with CFTR. In LLC-PK1 cells transfected with CFTR and in Caco-2 cells endogenously expressing CFTR, we demonstrated that endosomal SNARE protein overexpression inhibits CFTR activity but not swelling- or calcium-activated iodide efflux, indicating a specific effect upon CFTR activity. Moreover, co-immunoprecipitation experiments in LLC-PK1-CFTR cells showed that CFTR and SNARE proteins belong to a same complex and pull-down assays showed that VAMP8 and vti1b preferentially interact with CFTR N-terminus tail. By cell surface biotinylation and immunofluorescence experiments, we evidenced that endosomal SNARE overexpression disturbs CFTR apical targeting. Finally, we found a colocalization of CFTR and endosomal SNARE proteins in Rab11-positive recycling endosomes, suggesting a new role for endosomal SNARE proteins in CFTR trafficking in epithelial cells.

  5. deep-orange and carnation define distinct stages in late endosomal biogenesis in Drosophila melanogaster.

    Science.gov (United States)

    Sriram, V; Krishnan, K S; Mayor, Satyajit

    2003-05-12

    Endosomal degradation is severely impaired in primary hemocytes from larvae of eye color mutants of Drosophila. Using high resolution imaging and immunofluorescence microscopy in these cells, products of eye color genes, deep-orange (dor) and carnation (car), are localized to large multivesicular Rab7-positive late endosomes containing Golgi-derived enzymes. These structures mature into small sized Dor-negative, Car-positive structures, which subsequently fuse to form tubular lysosomes. Defective endosomal degradation in mutant alleles of dor results from a failure of Golgi-derived vesicles to fuse with morphologically arrested Rab7-positive large sized endosomes, which are, however, normally acidified and mature with wild-type kinetics. This locates the site of Dor function to fusion of Golgi-derived vesicles with the large Rab7-positive endocytic compartments. In contrast, endosomal degradation is not considerably affected in car1 mutant; fusion of Golgi-derived vesicles and maturation of large sized endosomes is normal. However, removal of Dor from small sized Car-positive endosomes is slowed, and subsequent fusion with tubular lysosomes is abolished. Overexpression of Dor in car1 mutant aggravates this defect, implicating Car in the removal of Dor from endosomes. This suggests that, in addition to an independent role in fusion with tubular lysosomes, the Sec1p homologue, Car, regulates Dor function.

  6. Endosomal Trafficking of HIV-1 Gag and Genomic RNAs Regulates Viral Egress

    DEFF Research Database (Denmark)

    Molle, Dorothée; Segura-Morales, Carollna; Camus, Gregory

    2009-01-01

    HIV-1 Gag can assemble and generate virions at the plasma membrane, but it is also present in endosomes where its role remains incompletely characterized. Here, we show that HIV-1 RNAs and Gag are transported on endosomal vesicles positive for TiVamp, a v-SNARE involved in fusion events with the ...

  7. Potent Anti-HIV Chemokine Analogs Direct Post-Endocytic Sorting of CCR5.

    Directory of Open Access Journals (Sweden)

    Claudia Bönsch

    Full Text Available G protein-coupled receptors (GPCRs are desensitized and internalized following activation. They are then subjected to post-endocytic sorting (degradation, slow recycling or fast recycling. The majority of research on post-endocytic sorting has focused on the role of sequence-encoded address structures on receptors. This study focuses on trafficking of CCR5, a GPCR chemokine receptor and the principal entry coreceptor for HIV. Using Chinese Hamster Ovary cells stably expressing CCR5 we show that two different anti-HIV chemokine analogs, PSC-RANTES and 5P14-RANTES, direct receptor trafficking into two distinct subcellular compartments: the trans-Golgi network and the endosome recycling compartment, respectively. Our results indicate that a likely mechanism for ligand-directed sorting of CCR5 involves capacity of the chemokine analogs to elicit the formation of durable complexes of CCR5 and arrestin2 (beta-arrestin-1, with PSC-RANTES eliciting durable association in contrast to 5P14-RANTES, which elicits only transient association.

  8. Dengue virus ensures its fusion in late endosomes using compartment-specific lipids.

    Directory of Open Access Journals (Sweden)

    Elena Zaitseva

    Full Text Available Many enveloped viruses invade cells via endocytosis and use different environmental factors as triggers for virus-endosome fusion that delivers viral genome into cytosol. Intriguingly, dengue virus (DEN, the most prevalent mosquito-borne virus that infects up to 100 million people each year, fuses only in late endosomes, while activation of DEN protein fusogen glycoprotein E is triggered already at pH characteristic for early endosomes. Are there any cofactors that time DEN fusion to virion entry into late endosomes? Here we show that DEN utilizes bis(monoacylglycerophosphate, a lipid specific to late endosomes, as a co-factor for its endosomal acidification-dependent fusion machinery. Effective virus fusion to plasma- and intracellular- membranes, as well as to protein-free liposomes, requires the target membrane to contain anionic lipids such as bis(monoacylglycerophosphate and phosphatidylserine. Anionic lipids act downstream of low-pH-dependent fusion stages and promote the advance from the earliest hemifusion intermediates to the fusion pore opening. To reach anionic lipid-enriched late endosomes, DEN travels through acidified early endosomes, but we found that low pH-dependent loss of fusogenic properties of DEN is relatively slow in the presence of anionic lipid-free target membranes. We propose that anionic lipid-dependence of DEN fusion machinery protects it against premature irreversible restructuring and inactivation and ensures viral fusion in late endosomes, where the virus encounters anionic lipids for the first time during entry. Currently there are neither vaccines nor effective therapies for DEN, and the essential role of the newly identified DEN-bis(monoacylglycerophosphate interactions in viral genome escape from the endosome suggests a novel target for drug design.

  9. On the Construction of Sorted Reactive Systems

    DEFF Research Database (Denmark)

    Birkedal, Lars; Debois, Søren; Hildebrandt, Thomas

    2008-01-01

    We develop a theory of sorted bigraphical reactive systems. Every application of bigraphs in the literature has required an extension, a sorting, of pure bigraphs. In turn, every such application has required a redevelopment of the theory of pure bigraphical reactive systems for the sorting at hand...... bigraphs. Technically, we give our construction for ordinary reactive systems, then lift it to bigraphical reactive systems. As such, we give also a construction of sortings for ordinary reactive systems. This construction is an improvement over previous attempts in that it produces smaller and much more...

  10. Techniques to sort Bessel beams

    CSIR Research Space (South Africa)

    Dudley, Angela L

    2013-09-01

    Full Text Available -polar coordinate transformation, translating helically phased beams into a transverse phase gradient. By introducing two cylindrical lenses we can focus each of the azimuthal modes associated with each Bessel beam to a different lateral position in the Fourier...

  11. The GPRC6A Receptor displays Constitutive Internalization and Sorting to the Slow Recycling Pathway.

    Science.gov (United States)

    Jacobsen, Stine Engesgaard; Ammendrup-Johnsen, Ina; Jansen, Anna Mai; Gether, Ulrik; Madsen, Kenneth Lindegaard; Bräuner-Osborne, Hans

    2017-03-09

    The class C G protein-coupled receptor GPRC6A is a putative nutrient sensing receptor and represents a possible new drug target in metabolic disorders. However, the specific physiological role of this receptor has yet to be identified, and the mechanisms regulating its activity and cell surface availability also remain enigmatic. In the present study, we investigated the trafficking properties of GPRC6A by use of both a classical antibody feeding internalization assay in which cells were visualized using confocal microscopy and a novel internalization assay that is based on real-time measurements of fluorescence resonance energy transfer. Both assays revealed that GPRC6A predominantly undergoes constitutive internalization while the agonist-induced effects were imperceptible. Moreover, post-endocytic sorting was investigated by assessing the co-localization of internalized GPRC6A with selected Rab protein markers. Internalized GPRC6A was mainly co-localized with the early endosome marker Rab5 and the long loop recycling endosome marker Rab11 and to a much lesser extent with the late endosome marker Rab7. This suggests that upon agonist-independent internalization, GPRC6A is recycled via the Rab11-positive slow recycling pathway, which may be responsible for ensuring a persistent pool of GPRC6A receptors at the cell surface despite chronic agonist exposure. Distinct trafficking pathways have been reported for several of the class C receptors, and our results thus substantiate that non-canonical trafficking mechanisms are a common feature for the nutrient sensing class C family that ensure functional receptors in the cell membrane despite prolonged agonist exposure.

  12. Cytokinesis in plant and animal cells: endosomes 'shut the door'.

    Science.gov (United States)

    Baluska, Frantisek; Menzel, Diedrik; Barlow, Peter W

    2006-06-01

    For many years, cytokinesis in eukaryotic cells was considered to be a process that took a variety of forms. This is rather surprising in the face of an apparently conservative mitosis. Animal cytokinesis was described as a process based on an actomyosin-based contractile ring, assembling, and acting at the cell periphery. In contrast, cytokinesis of plant cells was viewed as the centrifugal generation of a new cell wall by fusion of Golgi apparatus-derived vesicles. However, recent advances in animal and plant cell biology have revealed that many features formerly considered as plant-specific are, in fact, valid also for cytokinetic animal cells. For example, vesicular trafficking has turned out to be important not only for plant but also for animal cytokinesis. Moreover, the terminal phase of animal cytokinesis based on midbody microtubule activity resembles plant cytokinesis in that interdigitating microtubules play a decisive role in the recruitment of cytokinetic vesicles and directing them towards the cytokinetic spaces which need to be plugged by fusing endosomes. Presently, we are approaching another turning point which brings cytokinesis in plant and animal cells even closer. As an unexpected twist, new studies reveal that both plant and animal cytokinesis is driven not so much by Golgi-derived vesicles but rather by homotypically and heterotypically fusing endosomes. These are generated from cytokinetic cortical sites defined by preprophase microtubules and contractile actomyosin ring, which induce local endocytosis of both the plasma membrane and cell wall material. Finally, plant and animal cytokinesis meet together at the physical separation of daughter cells despite obvious differences in their preparatory events.

  13. Design and realization of sort manipulator of crystal-angle sort machine

    Science.gov (United States)

    Wang, Ming-shun; Chen, Shu-ping; Guan, Shou-ping; Zhang, Yao-wei

    2005-12-01

    It is a current tendency of development in automation technology to replace manpower with manipulators in working places where dangerous, harmful, heavy or repetitive work is involved. The sort manipulator is installed in a crystal-angle sort machine to take the place of manpower, and engaged in unloading and sorting work. It is the outcome of combing together mechanism, electric transmission, and pneumatic element and micro-controller control. The step motor makes the sort manipulator operate precisely. The pneumatic elements make the sort manipulator be cleverer. Micro-controller's software bestows some simple artificial intelligence on the sort manipulator, so that it can precisely repeat its unloading and sorting work. The combination of manipulator's zero position and step motor counting control puts an end to accumulating error in long time operation. A sort manipulator's design in the practice engineering has been proved to be correct and reliable.

  14. Recent advances in flow cytometric cell sorting.

    Science.gov (United States)

    Osborne, Geoffrey W

    2011-01-01

    The classification and separation of one cell type or particle from others is a fundamental task in many areas of science. Numerous techniques are available to perform this task; however, electrostatic cell sorting has gained eminence over others because, when combined with the analysis capabilities of flow cytometry it provides flexible separations based on multiple parameters. Unlike competing technologies, such as gradient or magnetic separations that offer much larger total throughput, flow cytometric cell sorting permits selections based on various levels of fluorescent reporters, rather the complete presence or absence of the reporter. As such, this technology has found application in a huge range of fields. This chapter aims to describe the utility of single-cell sorting with particular emphasis given to index sorting. This is followed by two recently developed novel techniques of sorting cells or particles. The first of these is positional sorting which is useful in cell-based studies where sorting can proceed and produce meaningful results without being inherently dependant on prior knowledge of where gates should be set. Secondly, reflective plate sorting is introduced which positionally links multiwell sample and collection plates in a convenient assay format so that cells in the collection plate "reflect" those in the sample plate.

  15. Coronin-1 is a neurotrophin endosomal effector required for developmental competition for survival

    Science.gov (United States)

    Suo, Dong; Park, Juyeon; Harrington, Anthony W.; Zweifel, Larry S.; Mihalas, Stefan; Deppmann, Christopher D.

    2014-01-01

    Retrograde communication from axonal targets to neuronal cell bodies is critical for both development and function of the nervous system. Much progress has been made in recent years linking long-distance, retrograde signaling to a signaling endosome, yet the mechanisms governing the trafficking and signaling of these endosomes remain mainly uncharacterized. Here we report that in mouse sympathetic neurons the target-derived NGF-TrkA signaling endosome, upon arrival at the cell body, induces the expression and recruitment of a novel effector protein known as Coronin-1. In the absence of Coronin-1, the NGF-TrkA signaling endosome fuses to lysosomes 6–10 fold faster than when Coronin-1 is intact. We also define a novel Coronin-1-dependent trafficking event where signaling endosomes recycle and re-internalize upon arrival at the cell body. Beyond influencing endosomal trafficking, Coronin-1 is also required for several NGF-TrkA dependent-signaling events including calcium release, calcineurin activation, and CREB phosphorylation. These results establish Coronin-1 as an essential component of a novel feedback loop mediating NGF-TrkA endosome stability, recycling, and signaling as a critical mechanism governing developmental competition for survival. PMID:24270184

  16. ER network homeostasis is critical for plant endosome streaming and endocytosis

    Science.gov (United States)

    Stefano, Giovanni; Renna, Luciana; Lai, YaShiuan; Slabaugh, Erin; Mannino, Nicole; Buono, Rafael A; Otegui, Marisa S; Brandizzi, Federica

    2015-01-01

    Eukaryotic cells internalize cargo at the plasma membrane via endocytosis, a vital process that is accomplished through a complex network of endosomal organelles. In mammalian cells, the ER is in close association with endosomes and regulates their fission. Nonetheless, the physiological role of such interaction on endocytosis is yet unexplored. Here, we probed the existence of ER–endosome association in plant cells and assayed its physiological role in endocytosis. Through live-cell imaging and electron microscopy studies, we established that endosomes are extensively associated with the plant ER, supporting conservation of interaction between heterotypic organelles in evolutionarily distant kingdoms. Furthermore, by analyzing ER–endosome dynamics in genetic backgrounds with defects in ER structure and movement, we also established that the ER network integrity is necessary for homeostasis of the distribution and streaming of various endosome populations as well as for efficient endocytosis. These results support a novel model that endocytosis homeostasis depends on a spatiotemporal control of the endosome dynamics dictated by the ER membrane network. PMID:27462431

  17. The intact structural form of LLO in endosomes cannot protect against listeriosis

    Science.gov (United States)

    Rodriguez-Del Rio, Estela; Frande-Cabanes, Elisabet; Tobes, Raquel; Pareja, Eduardo; Lecea-Cuello, M. Jesús; Ruiz-Sáez, Marta; Carrasco-Marín, Eugenio; Alvarez-Dominguez, Carmen

    2011-01-01

    LLO is the major immuno-dominant antigen in listeriosis and is also required for protective immunity. Two forms of LLO can be observed in endosomal membranes, a LLO intact form and a Ctsd-processed LLO1-491 form. Endosomes obtained from resting macrophages contained only LLO intact forms, while endosomes obtained from IFN-activated macrophages contained both forms. Both types of endosomes elicited LLO90-91/CD8+ and LLO189-201/CD4+ specific immune responses. However, only endosomes containing the Ctsd-processed LLO1-491 form showed significant CD4+ and CD8+ T cell responses similar to LM infected bone marrow derived macrophages and characteristic of protective Listeria immunity. Moreover, endosomes with intact LLO could not confer protection as vaccine carriers against murine listeriosis. While endosomes with Ctsd-processed LLO1-491 form showed a moderate ability, slightly lower than high efficiency vaccine vectors as MØ infected with LM. These studies argue that all cell-free membrane vesicles might serve as valid vaccine carriers against infectious agents. Exclusively those cell-free vesicles MIIC competent for LLO processing are protective vaccines vectors since they recruit significant numbers of mature dendritic cells to the vaccination sites and contain a LLO1-491 form that might be accessible for MHC class I and class II antigen presentation. PMID:22003433

  18. Vacuolar Protein Sorting Genes in Parkinson's Disease: A Re-appraisal of Mutations Detection Rate and Neurobiology of Disease.

    Science.gov (United States)

    Gambardella, Stefano; Biagioni, Francesca; Ferese, Rosangela; Busceti, Carla L; Frati, Alessandro; Novelli, Giuseppe; Ruggieri, Stefano; Fornai, Francesco

    2016-01-01

    Mammalian retromers play a critical role in protein trans-membrane sorting from endosome to the trans-Golgi network (TGN). Recently, retromer alterations have been related to the onset of Parkinson's Disease (PD) since the variant p.Asp620Asn in VPS35 (Vacuolar Protein Sorting 35) was identified as a cause of late onset PD. This variant causes a primary defect in endosomal trafficking and retromers formation. Other mutations in VPS genes have been reported in both sporadic and familial PD. These mutations are less defined. Understanding the specific prevalence of all VPS gene mutations is key to understand the relevance of retromers impairment in the onset of PD. A number of PD-related mutations despite affecting different biochemical systems (autophagy, mitophagy, proteasome, endosomes, protein folding), all converge in producing an impairment in cell clearance. This may explain how genetic predispositions to PD may derive from slightly deleterious VPS mutations when combined with environmental agents overwhelming the clearance of the cell. This manuscript reviews genetic data produced in the last 5 years to re-define the actual prevalence of VPS gene mutations in the onset of PD. The prevalence of p.Asp620Asn mutation in VPS35 is 0.286 of familial PD. This increases up to 0.548 when considering mutations affecting all VPS genes. This configures mutations in VPS genes as the second most frequent autosomal dominant PD genotype. This high prevalence, joined with increased awareness of the role played by retromers in the neurobiology of PD, suggests environmentally-induced VPS alterations as crucial in the genesis of PD.

  19. Vacuolar Protein Sorting Genes in Parkinson's Disease: A Re-appraisal of Mutations Detection Rate and Neurobiology of Disease

    Science.gov (United States)

    Gambardella, Stefano; Biagioni, Francesca; Ferese, Rosangela; Busceti, Carla L.; Frati, Alessandro; Novelli, Giuseppe; Ruggieri, Stefano; Fornai, Francesco

    2016-01-01

    Mammalian retromers play a critical role in protein trans-membrane sorting from endosome to the trans-Golgi network (TGN). Recently, retromer alterations have been related to the onset of Parkinson's Disease (PD) since the variant p.Asp620Asn in VPS35 (Vacuolar Protein Sorting 35) was identified as a cause of late onset PD. This variant causes a primary defect in endosomal trafficking and retromers formation. Other mutations in VPS genes have been reported in both sporadic and familial PD. These mutations are less defined. Understanding the specific prevalence of all VPS gene mutations is key to understand the relevance of retromers impairment in the onset of PD. A number of PD-related mutations despite affecting different biochemical systems (autophagy, mitophagy, proteasome, endosomes, protein folding), all converge in producing an impairment in cell clearance. This may explain how genetic predispositions to PD may derive from slightly deleterious VPS mutations when combined with environmental agents overwhelming the clearance of the cell. This manuscript reviews genetic data produced in the last 5 years to re-define the actual prevalence of VPS gene mutations in the onset of PD. The prevalence of p.Asp620Asn mutation in VPS35 is 0.286 of familial PD. This increases up to 0.548 when considering mutations affecting all VPS genes. This configures mutations in VPS genes as the second most frequent autosomal dominant PD genotype. This high prevalence, joined with increased awareness of the role played by retromers in the neurobiology of PD, suggests environmentally-induced VPS alterations as crucial in the genesis of PD. PMID:27932943

  20. The MIT domain of UBPY constitutes a CHMP binding and endosomal localization signal required for efficient epidermal growth factor receptor degradation.

    Science.gov (United States)

    Row, Paula E; Liu, Han; Hayes, Sebastian; Welchman, Rebecca; Charalabous, Panagoula; Hofmann, Kay; Clague, Michael J; Sanderson, Christopher M; Urbé, Sylvie

    2007-10-19

    We have identified and characterized a Microtubule Interacting and Transport (MIT) domain at the N terminus of the deubiquitinating enzyme UBPY/USP8. In common with other MIT-containing proteins such as AMSH and VPS4, UBPY can interact with CHMP proteins, which are known to regulate endosomal sorting of ubiquitinated receptors. Comparison of binding preferences for the 11 members of the human CHMP family between the UBPY MIT domain and another ubiquitin isopeptidase, AMSH, reveals common interactions with CHMP1A and CHMP1B but a distinct selectivity of AMSH for CHMP3/VPS24, a core subunit of the ESCRT-III complex, and UBPY for CHMP7. We also show that in common with AMSH, UBPY deubiquitinating enzyme activity can be stimulated by STAM but is unresponsive to its cognate CHMPs. The UBPY MIT domain is dispensable for its catalytic activity but is essential for its localization to endosomes. This is functionally significant as an MIT-deleted UBPY mutant is unable to rescue its binding partner STAM from proteasomal degradation or reverse a block to epidermal growth factor receptor degradation imposed by small interfering RNA-mediated depletion of UBPY.

  1. Data parallel sorting for particle simulation

    Science.gov (United States)

    Dagum, Leonardo

    1992-01-01

    Sorting on a parallel architecture is a communications intensive event which can incur a high penalty in applications where it is required. In the case of particle simulation, only integer sorting is necessary, and sequential implementations easily attain the minimum performance bound of O (N) for N particles. Parallel implementations, however, have to cope with the parallel sorting problem which, in addition to incurring a heavy communications cost, can make the minimun performance bound difficult to attain. This paper demonstrates how the sorting problem in a particle simulation can be reduced to a merging problem, and describes an efficient data parallel algorithm to solve this merging problem in a particle simulation. The new algorithm is shown to be optimal under conditions usual for particle simulation, and its fieldwise implementation on the Connection Machine is analyzed in detail. The new algorithm is about four times faster than a fieldwise implementation of radix sort on the Connection Machine.

  2. Scalable, Multithreaded, Partially-in-Place Sorting

    Energy Technology Data Exchange (ETDEWEB)

    Haglin, David J.; Adolf, Robert D.; Mackey, Greg E.

    2013-05-20

    A recent trend in hardware development is producing computing systems that are stretching the number of cores and size of shared-memory beyond where most fundamental serial algorithms perform well. The expectation is that this trend will continue. So it makes sense to rethink our fundamental algorithms such as sorting. There are many situations where data that needs to be sorted will actually fit into the shared memory so applications could benefit from an efficient parallel sorting algorithm. When sorting large data (at least hundreds of Gigabytes) in a single shared memory, there are two factors that affect the algorithm choice. First, does the algorithm sort in-place? And second, does the algorithm scale well beyond tens of threads? Surprisingly, existing algorithms posses either one of these factors, but not both. We present an approach that gracefully degrades in performance as the amount of available working memory decreases relative to the size of the input.

  3. Data Sorting Using Graphics Processing Units

    Directory of Open Access Journals (Sweden)

    M. J. Mišić

    2012-06-01

    Full Text Available Graphics processing units (GPUs have been increasingly used for general-purpose computation in recent years. The GPU accelerated applications are found in both scientific and commercial domains. Sorting is considered as one of the very important operations in many applications, so its efficient implementation is essential for the overall application performance. This paper represents an effort to analyze and evaluate the implementations of the representative sorting algorithms on the graphics processing units. Three sorting algorithms (Quicksort, Merge sort, and Radix sort were evaluated on the Compute Unified Device Architecture (CUDA platform that is used to execute applications on NVIDIA graphics processing units. Algorithms were tested and evaluated using an automated test environment with input datasets of different characteristics. Finally, the results of this analysis are briefly discussed.

  4. Dmon1 controls recruitment of Rab7 to maturing endosomes in Drosophila.

    Science.gov (United States)

    Yousefian, Jahan; Troost, Tobias; Grawe, Ferdi; Sasamura, Takeshi; Fortini, Mark; Klein, Thomas

    2013-04-01

    The small GTPases Rab5 and Rab7 are important organisers of endosome formation and maturation. In addition, they orchestrate the trafficking of cargo through the endosomal pathway. A crucial event during maturation of endosomes is the replacement of the early organiser Rab5 with the late organiser Rab7 in a process called Rab conversion. Rab conversion is a prerequisite for late events, chief among them the fusion of matured endosomes with the lysosome. Recent work identifies members of the Sand1/Mon1 protein family as crucial factors during this process. Here, we present an analysis of the function of the Drosophila ortholog of mon1/sand1, Dmon1. We found that loss of function of Dmon1 results in an enlargement of maturing endosomes and loss of their association with Rab7. The enlarged endosomes contain Notch and other trans-membrane proteins as cargo. We report the first electron microscopy analysis of Dmon1 cells in a metazoan and extend the analysis of the endosomes in mutant cells. Our results suggest that the phenotype can be explained by the loss of function of Rab7. Moreover, the endosomes of Dmon1 cells mature normally in many aspects, despite the loss of association with Rab7. Surprisingly, we did not observe overactive or ectopic signalling through receptors such as Notch and RTKs in Dmon1 mutant cells, as would have been expected because of the accumulation of receptors in the maturing endosomes of these cells. This was the case even when receptor uptake into intraluminal vesicles was suppressed.

  5. Interactions between Rab and Arf GTPases regulate endosomal phosphatidylinositol-4,5-bisphosphate during endocytic recycling.

    Science.gov (United States)

    Shi, Anbing; Grant, Barth D

    2013-01-01

    After endocytosis, a selective endocytic recycling process returns many endocytosed molecules back to the plasma membrane. The RAB-10/Rab10 GTPase is known to be a key recycling regulator for specific cargo molecules. New evidence, focused on C. elegans RAB-10 in polarized epithelia, points to a key role of RAB-10 in the regulation of endosomal phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) levels. In turn, PI(4,5)P2 levels strongly influence the recruitment of many peripheral membrane proteins, including those important for vesicle budding through their membrane bending activities. Part of the effect of RAB-10 on endosomal PI(4,5)P2 is through its newly identified effector CNT-1, a predicted GTPase activating protein (GAP) of the small GTPase ARF-6/Arf6. In mammals PI(4,5)P2 generating enzymes are known Arf6 effectors. In C. elegans we found that RAB-10, CNT-1 and ARF-6 are present on the same endosomes, that RAB-10 recruits CNT-1 to endosomes, and that loss of CNT-1 or RAB-10 leads to overaccumulation of endosomal PI(4,5)P2, presumably via hyperactivation of endosomal ARF-6. In turn this leads to over-recruitment of PI(4,5)P2-dependent membrane-bending proteins RME-1/Ehd and SDPN-1/Syndapin/PACSIN. Conversely, in arf-6 mutants, endosomal PI(4,5)P2 levels were reduced and endosomal recruitment of RME-1 and SDPN-1 failed. This work makes an unexpected link between distinct classes of small GTPases that control endocytic recycling, and provides insight into how this interaction affects endosome function at the level of lipid phosphorylation.

  6. Sorting carbon nanotubes for electronics.

    Science.gov (United States)

    Martel, Richard

    2008-11-25

    Because of their unique structure and composition, single-wall carbon nanotubes (SWNTs) are at the interface between molecules and crystalline solids. They also present properties that are ideal for making lightweight, inexpensive, and flexible electronics. The raw material is composed of a heterogeneous mixture of SWNTs that differ in helicity and diameter and, therefore, requires purification and separation. In a series of groundbreaking experiments, a robust process serving this purpose was developed based on SWNTs encapsulated in surfactants and water. Ultracentrifugation in a density gradient combined with surfactant mixtures provided buoyant density differences, enabling enrichment for both diameter and electronic properties. A new paper in this issue explores further the process through the hydrodynamic properties of SWNT-surfactant complexes. The study reveals that we have just begun to uncover the dynamics and properties of nanotube-surfactant interactions and highlights the potential that could be gained from a better understanding of their chemistry. The time scale of integration of carbon nanotubes into electronics applications remains unclear, but the recent developments in sorting out SWNTs paves the way for improving on the properties of network-based SWNTs.

  7. Syntaxin 7 is localized to late endosome compartments, associates with Vamp 8, and Is required for late endosome-lysosome fusion.

    Science.gov (United States)

    Mullock, B M; Smith, C W; Ihrke, G; Bright, N A; Lindsay, M; Parkinson, E J; Brooks, D A; Parton, R G; James, D E; Luzio, J P; Piper, R C

    2000-09-01

    Protein traffic from the cell surface or the trans-Golgi network reaches the lysosome via a series of endosomal compartments. One of the last steps in the endocytic pathway is the fusion of late endosomes with lysosomes. This process has been reconstituted in vitro and has been shown to require NSF, alpha and gamma SNAP, and a Rab GTPase based on inhibition by Rab GDI. In Saccharomyces cerevisiae, fusion events to the lysosome-like vacuole are mediated by the syntaxin protein Vam3p, which is localized to the vacuolar membrane. In an effort to identify the molecular machinery that controls fusion events to the lysosome, we searched for mammalian homologues of Vam3p. One such candidate is syntaxin 7. Here we show that syntaxin 7 is concentrated in late endosomes and lysosomes. Coimmunoprecipitation experiments show that syntaxin 7 is associated with the endosomal v-SNARE Vamp 8, which partially colocalizes with syntaxin 7. Importantly, we show that syntaxin 7 is specifically required for the fusion of late endosomes with lysosomes in vitro, resulting in a hybrid organelle. Together, these data identify a SNARE complex that functions in the late endocytic system of animal cells.

  8. Enhancement of Selection, Bubble and Insertion Sorting Algorithm

    Directory of Open Access Journals (Sweden)

    Muhammad Farooq Umar

    2014-07-01

    Full Text Available In everyday life there is a large amount of data to arrange because sorting removes any ambiguities and make the data analysis and data processing very easy, efficient and provides with cost less effort. In this study a set of improved sorting algorithms are proposed which gives better performance and design idea. In this study five new sorting algorithms (Bi-directional Selection Sort, Bi-directional bubble sort, MIDBiDirectional Selection Sort, MIDBidirectional bubble sort and linear insertion sort are presented. Bi-directional Selection Sort and MIDBiDirectional Selection Sort are the enhancement on basic selection sort while Bidirectional bubble sort and MIDBidirectional bubble sort are the enhancement on basic bubble sort by changing the selection and swapping mechanism of data for sorting. Enhanced sorting algorithms reduced the iteration by half and quarter times respectively. Asymptotically complexities of these algorithms are reduced to O (n2/2 and O (n2/4 from O (n2. Linear insertion sort is the enhancement of insertion sort by changing the design of algorithm (convert two loops to one loop. So asymptotically this algorithm is converted to linear time complexity from quadratic complexity. These sorting algorithms are described using C. The proposed algorithms are analyzed using asymptotic analysis and also using machine-running time and compared with their basic sorting algorithms. In this study we also discuss how the performance and complexity can be improved by optimizing the code and design.

  9. An Unsupervised Online Spike-Sorting Framework.

    Science.gov (United States)

    Knieling, Simeon; Sridharan, Kousik S; Belardinelli, Paolo; Naros, Georgios; Weiss, Daniel; Mormann, Florian; Gharabaghi, Alireza

    2016-08-01

    Extracellular neuronal microelectrode recordings can include action potentials from multiple neurons. To separate spikes from different neurons, they can be sorted according to their shape, a procedure referred to as spike-sorting. Several algorithms have been reported to solve this task. However, when clustering outcomes are unsatisfactory, most of them are difficult to adjust to achieve the desired results. We present an online spike-sorting framework that uses feature normalization and weighting to maximize the distinctiveness between different spike shapes. Furthermore, multiple criteria are applied to either facilitate or prevent cluster fusion, thereby enabling experimenters to fine-tune the sorting process. We compare our method to established unsupervised offline (Wave_Clus (WC)) and online (OSort (OS)) algorithms by examining their performance in sorting various test datasets using two different scoring systems (AMI and the Adamos metric). Furthermore, we evaluate sorting capabilities on intra-operative recordings using established quality metrics. Compared to WC and OS, our algorithm achieved comparable or higher scores on average and produced more convincing sorting results for intra-operative datasets. Thus, the presented framework is suitable for both online and offline analysis and could substantially improve the quality of microelectrode-based data evaluation for research and clinical application.

  10. An improved infrared technique for sorting pecans

    Science.gov (United States)

    Graeve, Thorsten; Dereniak, Eustace L.; Lamonica, John A., Jr.

    1991-10-01

    This paper presents the results of a study of pecan spectral reflectances. It describes an experiment for measuring the contrast between several components of raw pecan product to be sorted. An analysis of the experimental data reveals high contrast ratios in the infrared spectrum, suggesting a potential improvement in sorting efficiency when separating pecan meat from shells. It is believed that this technique has the potential to dramatically improve the efficiency of current sorting machinery, and to reduce the cost of processing pecans for the consumer market.

  11. Minimal Model Semantics for Sorted Constraint Representation

    Institute of Scientific and Technical Information of China (English)

    廖乐健; 史忠植

    1995-01-01

    Sorted constraint representation is a very useful representation in AI which combines class hierarchies and constraint networks.For such sorted constraint representation,a problem is how to generalize the idea of default inheritance to constraint network,where the attributes in a class or between different classes interact with each other via the network.To give a formal account for the defeasible reasoning in such representation,a general sorted constraint logic is proposed,and a minimal-model semantics for the logic is presented.

  12. Semiconductor quantum dot/albumin complex is a long-life and highly photostable endosome marker.

    Science.gov (United States)

    Hanaki, Ken-ichi; Momo, Asami; Oku, Taisuke; Komoto, Atsushi; Maenosono, Shinya; Yamaguchi, Yukio; Yamamoto, Kenji

    2003-03-14

    For the purpose of selecting the efficient dispersion condition of hydrophilic semiconductor quantum dots (QDs) in biological buffers, the dispersion of the QDs mixed with a serum albumin from 9 different species or an ovalbumin was compared by a fluorescence intensity analysis. The QDs mixed with sheep serum albumin (SSA) showed the highest fluorescence of all when the mixtures were dissolved in Dulbecco's MEM. QD/SSA complexes were accumulated in the endosome/lysosome of Vero cells and the fluorescence could be detected over a 5-day post-incubation period. The photostability of QD/SSA complexes associated with the endosomes was detectable, at least, 30 times as long as that of fluorescein-labeled dextran involved in endosomes. QD/SSA complex, therefore, can be used as a long-life and highly photostable endosome marker.

  13. A measure of endosomal pH by flow cytometry in Dictyostelium

    Directory of Open Access Journals (Sweden)

    Cosson Pierre

    2009-01-01

    Full Text Available Abstract Background Dictyostelium amoebae are frequently used to study the organization and function of the endocytic pathway, and specific protocols are essential to measure the dynamics of endocytic compartments and their internal pH. Findings We have revisited these classical protocols to measure more accurately endosomal pH, making use of a fluorescent probe (Oregon green more adequate for very acidic pH values. This pH-sensitive probe was combined with a pH-insensitive marker, in order to visualize simultaneously endosome dynamics and pH changes. Finally, a flow cytometer was used to measure endosomal pH in individual cells. Conclusion Using these simple protocols the endosomal pH of endocytic compartments can be assessed accurately, revealing the extreme acidity of Dictyostelium lysosomes (pH

  14. Endosomal recycling controls plasma membrane area during mitosis.

    Science.gov (United States)

    Boucrot, Emmanuel; Kirchhausen, Tomas

    2007-05-08

    The shape and total surface of a cell and its daughters change during mitosis. Many cells round up during prophase and metaphase and reacquire their extended and flattened shape during cytokinesis. How does the total area of plasma membrane change to accommodate these morphological changes and by what mechanism is control of total membrane area achieved? Using single-cell imaging methods, we have found that the amount of plasma membrane in attached cells in culture decreases at the beginning of mitosis and recovers rapidly by the end. Clathrin-based endocytosis is normal throughout all phases of cell division, whereas recycling of internalized membranes back to the cell surface slows considerably during the rounding up period and resumes at the time at which recovery of cell membrane begins. Interference with either one of these processes by genetic or chemical means impairs cell division. The total cell-membrane area recovers even in the absence of a functional Golgi apparatus, which would be needed for export of newly synthesized membrane lipids and proteins. We propose a mechanism by which modulation of endosomal recycling controls cell area and surface expression of membrane-bound proteins during cell division.

  15. Mis-trafficking of endosomal urokinase proteins triggers drug-induced glioma nonapoptotic cell death.

    Science.gov (United States)

    Pasupuleti, Nagarekha; Grodzki, Ana Cristina; Gorin, Fredric

    2015-04-01

    5-Benzylglycinyl-amiloride (UCD38B) is the parent molecule of a class of anticancer small molecules that kill proliferative and nonproliferative high-grade glioma cells by programmed necrosis. UCD38B intracellularly triggers endocytosis, causing 40-50% of endosomes containing proteins of the urokinase plasminogen activator system (uPAS) to relocate to perinuclear mitochondrial regions. Endosomal "mis-trafficking" caused by UCD38B in human glioma cells corresponds to mitochondrial depolarization with the release and nuclear translocation of apoptotis-inducing factor (AIF) followed by irreversible caspase-independent cell demise. High-content quantification of immunocytochemical colocalization studies identified that UCD38B treatment increased endocytosis of the urokinase plasminogen activator (uPA), its receptor (uPAR), and plasminogen activator inhibitor-1 (PAI-1) into the early and late endosomes by 4- to 5-fold prior to AIF nuclear translocation and subsequent glioma demise. PAI-1 was found to comparably relocate with a subset of early and late endosomes in four different human glioma cell lines after UCD38B treatment, followed by caspase-independent, nonapoptotic cell death. Following UCD38B treatment, the receptor guidance protein LRP-1, which is required for endosomal recycling of the uPA receptor to the plasmalemma, remained abnormally associated with PAI-1 in early and late endosomes. The resultant aberrant endosomal recycling increased the total cellular content of the uPA-PAI-1 protein complex. Reversible inhibition of cellular endocytosis demonstrated that UCD38B bypasses the plasmalemmal uPAS complex and directly acts intracellularly to alter uPAS endocytotic trafficking. UCD38B represents a class of small molecules whose anticancer cytotoxicity is a consequence of causing the mis-trafficking of early and late endosomes containing uPAS cargo and leading to AIF-mediated necrotic cell death. Copyright © 2015 by The American Society for Pharmacology and

  16. Endosomal maturation, Rab7 GTPase and phosphoinositides in African swine fever virus entry.

    Directory of Open Access Journals (Sweden)

    Miguel A Cuesta-Geijo

    Full Text Available Here we analyzed the dependence of African swine fever virus (ASFV infection on the integrity of the endosomal pathway. Using confocal immunofluorescence with antibodies against viral capsid proteins, we found colocalization of incoming viral particles with early endosomes (EE during the first minutes of infection. Conversely, viral capsid protein was not detected in acidic late endosomal compartments, multivesicular bodies (MVBs, late endosomes (LEs or lysosomes (LY. Using an antibody against a viral inner core protein, we found colocalization of viral cores with late compartments from 30 to 60 minutes postinfection. The absence of capsid protein staining in LEs and LYs suggested that virus desencapsidation would take place at the acid pH of these organelles. In fact, inhibitors of intraluminal acidification of endosomes caused retention of viral capsid staining virions in Rab7 expressing endosomes and more importantly, severely impaired subsequent viral protein production. Endosomal acidification in the first hour after virus entry was essential for successful infection but not thereafter. In addition, altering the balance of phosphoinositides (PIs which are responsible of the maintenance of the endocytic pathway impaired ASFV infection. Early infection steps were dependent on the production of phosphatidylinositol 3-phosphate (PtdIns3P which is involved in EE maturation and multivesicular body (MVB biogenesis and on the interconversion of PtdIns3P to phosphatidylinositol 3, 5-biphosphate (PtdIns(3,5P(2. Likewise, GTPase Rab7 activity should remain intact, as well as processes related to LE compartment physiology, which are crucial during early infection. Our data demonstrate that the EE and LE compartments and the integrity of the endosomal maturation pathway orchestrated by Rab proteins and PIs play a central role during early stages of ASFV infection.

  17. deep-orange and carnation define distinct stages in late endosomal biogenesis in Drosophila melanogaster

    OpenAIRE

    Sriram, V.; Krishnan, K. S.; Mayor, Satyajit

    2003-01-01

    Endosomal degradation is severely impaired in primary hemocytes from larvae of eye color mutants of Drosophila. Using high resolution imaging and immunofluorescence microscopy in these cells, products of eye color genes, deep-orange (dor) and carnation (car), are localized to large multivesicular Rab7-positive late endosomes containing Golgi-derived enzymes. These structures mature into small sized Dor-negative, Car-positive structures, which subsequently fuse to form tubular lysosomes. Defec...

  18. Regulation of EGFR signal transduction by analogue-to-digital conversion in endosomes

    Science.gov (United States)

    Villaseñor, Roberto; Nonaka, Hidenori; Del Conte-Zerial, Perla; Kalaidzidis, Yannis; Zerial, Marino

    2015-01-01

    An outstanding question is how receptor tyrosine kinases (RTKs) determine different cell-fate decisions despite sharing the same signalling cascades. Here, we uncovered an unexpected mechanism of RTK trafficking in this process. By quantitative high-resolution FRET microscopy, we found that phosphorylated epidermal growth factor receptor (p-EGFR) is not randomly distributed but packaged at constant mean amounts in endosomes. Cells respond to higher EGF concentrations by increasing the number of endosomes but keeping the mean p-EGFR content per endosome almost constant. By mathematical modelling, we found that this mechanism confers both robustness and regulation to signalling output. Different growth factors caused specific changes in endosome number and size in various cell systems and changing the distribution of p-EGFR between endosomes was sufficient to reprogram cell-fate decision upon EGF stimulation. We propose that the packaging of p-RTKs in endosomes is a general mechanism to ensure the fidelity and specificity of the signalling response. DOI: http://dx.doi.org/10.7554/eLife.06156.001 PMID:25650738

  19. Enhanced Endosomal Escape by Light-Fueled Liquid-Metal Transformer.

    Science.gov (United States)

    Lu, Yue; Lin, Yiliang; Chen, Zhaowei; Hu, Quanyin; Liu, Yang; Yu, Shuangjiang; Gao, Wei; Dickey, Michael D; Gu, Zhen

    2017-04-12

    Effective endosomal escape remains as the "holy grail" for endocytosis-based intracellular drug delivery. To date, most of the endosomal escape strategies rely on small molecules, cationic polymers, or pore-forming proteins, which are often limited by the systemic toxicity and lack of specificity. We describe here a light-fueled liquid-metal transformer for effective endosomal escape-facilitated cargo delivery via a chemical-mechanical process. The nanoscale transformer can be prepared by a simple approach of sonicating a low-toxicity liquid-metal. When coated with graphene quantum dots (GQDs), the resulting nanospheres demonstrate the ability to absorb and convert photoenergy to drive the simultaneous phase separation and morphological transformation of the inner liquid-metal core. The morphological transformation from nanospheres to hollow nanorods with a remarkable change of aspect ratio can physically disrupt the endosomal membrane to promote endosomal escape of payloads. This metal-based nanotransformer equipped with GQDs provides a new strategy for facilitating effective endosomal escape to achieve spatiotemporally controlled drug delivery with enhanced efficacy.

  20. Regulation of EGFR signal transduction by analogue-to-digital conversion in endosomes.

    Science.gov (United States)

    Villaseñor, Roberto; Nonaka, Hidenori; Del Conte-Zerial, Perla; Kalaidzidis, Yannis; Zerial, Marino

    2015-02-04

    An outstanding question is how receptor tyrosine kinases (RTKs) determine different cell-fate decisions despite sharing the same signalling cascades. Here, we uncovered an unexpected mechanism of RTK trafficking in this process. By quantitative high-resolution FRET microscopy, we found that phosphorylated epidermal growth factor receptor (p-EGFR) is not randomly distributed but packaged at constant mean amounts in endosomes. Cells respond to higher EGF concentrations by increasing the number of endosomes but keeping the mean p-EGFR content per endosome almost constant. By mathematical modelling, we found that this mechanism confers both robustness and regulation to signalling output. Different growth factors caused specific changes in endosome number and size in various cell systems and changing the distribution of p-EGFR between endosomes was sufficient to reprogram cell-fate decision upon EGF stimulation. We propose that the packaging of p-RTKs in endosomes is a general mechanism to ensure the fidelity and specificity of the signalling response.

  1. The small GTP-binding protein rab4 is associated with early endosomes

    Energy Technology Data Exchange (ETDEWEB)

    van der Sluijs, P.; Hull, M.; Mellman, I. (Yale Univ. School of Medicine, New Haven, CT (United States)); Zahraoui, A.; Tavitian, A. (INSERM U 248, Paris (France)); Goud, B. (Unite de Genetique Somatique, Paris (France))

    1991-07-15

    Small GTP-binding proteins of the rab family have been implicated as playing important roles in controlling membrane traffic on the biosynthetic and endocytic pathways. The authors demonstrate that a distinct rab protein, rab4p, is associated with the population of early endosomes involved in transferrin-receptor recycling. An antibody to human rab4p was found to detect a doublet of {approx} 24-kDa proteins on immunoblots from various cell types. Seventy-five percent of these proteins were tightly membrane bound and could be released only by detergent treatment. Upon isolation of early endosomes, late endosomes, and lysosomes, by free-flow electrophoresis and Percoll density-gradient centrifugation, most (70%) of the rab4p was found to cofractionate with early endosomes and endocytic vesicles containing {sup 125}-labeled transferrin. The rab proteins previously localized to the endoplasmic reticulum and/or Golgi apparatus were not found in these fractions. They also localized rab4p to tansferrin-receptor-containing early endosomes by immunofluorescence after expression of rab4p cDNA. The association of rab4p with early endosomes and other vesicles involved in the intracellular tansport of transferrin receptor suggests that rab4p may play a role in regulating the pathway of receptor recycling.

  2. Poisson Coordinates.

    Science.gov (United States)

    Li, Xian-Ying; Hu, Shi-Min

    2013-02-01

    Harmonic functions are the critical points of a Dirichlet energy functional, the linear projections of conformal maps. They play an important role in computer graphics, particularly for gradient-domain image processing and shape-preserving geometric computation. We propose Poisson coordinates, a novel transfinite interpolation scheme based on the Poisson integral formula, as a rapid way to estimate a harmonic function on a certain domain with desired boundary values. Poisson coordinates are an extension of the Mean Value coordinates (MVCs) which inherit their linear precision, smoothness, and kernel positivity. We give explicit formulas for Poisson coordinates in both continuous and 2D discrete forms. Superior to MVCs, Poisson coordinates are proved to be pseudoharmonic (i.e., they reproduce harmonic functions on n-dimensional balls). Our experimental results show that Poisson coordinates have lower Dirichlet energies than MVCs on a number of typical 2D domains (particularly convex domains). As well as presenting a formula, our approach provides useful insights for further studies on coordinates-based interpolation and fast estimation of harmonic functions.

  3. Engineering a Cache-Oblivious Sorting Algorithm

    DEFF Research Database (Denmark)

    Brodal, Gerth Stølting; Fagerberg, Rolf; Vinther, Kristoffer

    2007-01-01

    This paper is an algorithmic engineering study of cache-oblivious sorting. We investigate by empirical methods a number of implementation issues and parameter choices for the cache-oblivious sorting algorithm Lazy Funnelsort, and compare the final algorithm with Quicksort, the established standard...... for comparison-based sorting, as well as with recent cache-aware proposals. The main result is a carefully implemented cache-oblivious sorting algorithm, which our experiments show can be faster than the best Quicksort implementation we are able to find, already for input sizes well within the limits of RAM....... It is also at least as fast as the recent cache-aware implementations included in the test. On disk the difference is even more pronounced regarding Quicksort and the cache-aware algorithms, whereas the algorithm is slower than a careful implementation of multiway Mergesort such as TPIE....

  4. Filter-less submicron hydrodynamic size sorting.

    Science.gov (United States)

    Fouet, M; Mader, M-A; Iraïn, S; Yanha, Z; Naillon, A; Cargou, S; Gué, A-M; Joseph, P

    2016-02-21

    We propose a simple microfluidic device able to separate submicron particles (critical size ∼0.1 μm) from a complex sample with no filter (minimum channel dimension being 5 μm) by hydrodynamic filtration. A model taking into account the actual velocity profile and hydrodynamic resistances enables prediction of the chip sorting properties for any geometry. Two design families are studied to obtain (i) small sizes within minutes (low-aspect ratio, two-level chip) and (ii) micron-sized sorting with a μL flow rate (3D architecture based on lamination). We obtain quantitative agreement of sorting performances both with experiments and with numerical solving, and determine the limits of the approach. We therefore demonstrate a passive, filter-less sub-micron size sorting with a simple, robust, and easy to fabricate design.

  5. Visual ergonomics interventions in mail sorting facilities.

    Science.gov (United States)

    Hemphälä, H; Hansson, G-Å; Dahlqvist, C; Eklund, J

    2012-01-01

    This study was performed between 2004 and 2011 at mail sorting facilities in Sweden. During this time, different interventions were performed. The first was a lighting intervention that had a positive impact on the postal workers, especially those with eyestrain. A new lighting system also improved the illuminance and gave better light distribution. The second intervention involved new personal spectacles for the postal workers who needed them and this had a positive effect on eyestrain. The third intervention involved a specific type of sorting spectacles for the postal workers who already used progressive lenses privately. The reading distances that the postal workers had while sorting the mail was inverted to the distances in their regular progressive lenses. The new sorting spectacles had a positive effect on head postures and on muscular activity.

  6. Quantum Database Search can do without Sorting

    CERN Document Server

    Patel, A

    2001-01-01

    Sorting is a fundamental computational process, which facilitates subsequent searching of a database. It can be thought of as factorisation of the search process. The location of a desired item in a sorted database can be found by classical queries that inspect one letter of the label at a time. For an unsorted database, no such classical quick search algorithm is available. If the database permits quantum queries, however, then mere digitisation is sufficient for efficient search. Sorting becomes redundant with the quantum superposition of states. A quantum algorithm is written down which locates the desired item in an unsorted database a factor of two faster than the best classical algorithm can in a sorted database. This algorithm has close resemblance to the assembly process in DNA replication.

  7. Card Sorts, State Tests, and Meaningful Mathematics

    Science.gov (United States)

    Chauvot, Jennifer B.; Benson, Sharon L. D.

    2008-01-01

    This article shares card-sorting activities that involve state-mandated test items to use with prospective and practicing mathematics teachers to teach about accountability measures while exploring reform-minded mathematics instruction recommendations. (Contains 2 figures.)

  8. Sorting it out: regulation of exosome loading.

    Science.gov (United States)

    Villarroya-Beltri, Carolina; Baixauli, Francesc; Gutiérrez-Vázquez, Cristina; Sánchez-Madrid, Francisco; Mittelbrunn, María

    2014-10-01

    Extracellular vesicles (EVs), a term that includes both exosomes of endocytic origin and vesicles derived from plasma membranes, are continuously secreted by cells to the extracellular environment, and represent a novel vehicle for cell-cell communication. Exosomes contain specific repertoires of proteins and RNAs, indicating the existence of mechanisms that control the sorting of molecules into them. Although the molecular mechanisms that regulate the loading of proteins into exosomes have been studied for years, the sorting of RNA has been elusive until recently. Here we review the molecular mechanisms that control the sorting of molecules into exosomes, with special attention to the sorting of RNA. We also discuss how the cellular context affects the composition of exosomes, and thus the outcome of the communication between the exosome-producer and recipient cells, with particular focus on the communication between tumor cells and with cells of the tumor microenvironment.

  9. Another Definition of Order—Sorted Algebra

    Institute of Scientific and Technical Information of China (English)

    何自强

    1998-01-01

    In this paper the definition of order-sorted algebra is generalized by introducing transformation functions between subtypes and supertypes.According to our definition,a type needn't be a subset of its supertype and a record model may form an order-sorted algebra.A new definition of equation is given.It has also been proved that equational theories and describing single inheritance have the initial model.

  10. A WASp-binding type II phosphatidylinositol 4-kinase required for actin polymerization-driven endosome motility.

    Science.gov (United States)

    Chang, Fanny S; Han, Gil-Soo; Carman, George M; Blumer, Kendall J

    2005-10-10

    Endosomes in yeast have been hypothesized to move through the cytoplasm by the momentum gained after actin polymerization has driven endosome abscision from the plasma membrane. Alternatively, after abscission, ongoing actin polymerization on endosomes could power transport. Here, we tested these hypotheses by showing that the Arp2/3 complex activation domain (WCA) of Las17 (Wiskott-Aldrich syndrome protein [WASp] homologue) fused to an endocytic cargo protein (Ste2) rescued endosome motility in las17DeltaWCA mutants, and that capping actin filament barbed ends inhibited endosome motility but not endocytic internalization. Motility therefore requires continual actin polymerization on endosomes. We also explored how Las17 is regulated. Endosome motility required the Las17-binding protein Lsb6, a type II phosphatidylinositol 4-kinase. Catalytically inactive Lsb6 interacted with Las17 and promoted endosome motility. Lsb6 therefore is a novel regulator of Las17 that mediates endosome motility independent of phosphatidylinositol 4-phosphate synthesis. Mammalian type II phosphatidylinositol 4-kinases may regulate WASp proteins and endosome motility.

  11. Modulation of RAB5A early endosome trafficking in response to KRas mediated macropinocytic fluxes in pancreatic cancer cells.

    Science.gov (United States)

    Teske, Christian; Schweitzer, Christine; Palamidessi, Andrea; Aust, Daniela E; Scita, Giorgio; Weitz, Jürgen; Welsch, Thilo

    2017-09-01

    KRAS is the key mutated gene in pancreatic ductal adenocarcinoma (PDAC). Emerging evidence indicates that KRas modulates endocytic uptake. The present study aimed to explore the fate of early endosomal trafficking under the control of KRas expression in PDAC. Surprisingly, PANC-1 cells lacking KRas exhibited significantly enlarged early and late endosomes containing internalized dextran and epidermal growth factor. Endosome enlargement was accompanied by reduced endosomal degradation. Both KRas silencing and lysosomal blockade caused an upregulation of the master regulator of early endosome biogenesis, RAB5A, which is likely responsible for the expansion of the early endosomal compartment, because simultaneous KRAS/RAB5A knockdown abolished endosome enlargement. In contrast, early endosome shrinkage was seen in MIA PaCa-2 cells despite RAB5A upregulation, indicating that distinct KRas-modulated responses operate in different metabolic subtypes of PDAC. In conclusion, mutant KRAS promotes endosomal degradation in PDAC cell lines, which is impaired by KRAS silencing. Moreover, KRAS silencing activates RAB5A upregulation and drives PDAC subtype-dependent modulation of endosome trafficking. Copyright © 2017. Published by Elsevier Inc.

  12. Automatic spike sorting using tuning information.

    Science.gov (United States)

    Ventura, Valérie

    2009-09-01

    Current spike sorting methods focus on clustering neurons' characteristic spike waveforms. The resulting spike-sorted data are typically used to estimate how covariates of interest modulate the firing rates of neurons. However, when these covariates do modulate the firing rates, they provide information about spikes' identities, which thus far have been ignored for the purpose of spike sorting. This letter describes a novel approach to spike sorting, which incorporates both waveform information and tuning information obtained from the modulation of firing rates. Because it efficiently uses all the available information, this spike sorter yields lower spike misclassification rates than traditional automatic spike sorters. This theoretical result is verified empirically on several examples. The proposed method does not require additional assumptions; only its implementation is different. It essentially consists of performing spike sorting and tuning estimation simultaneously rather than sequentially, as is currently done. We used an expectation-maximization maximum likelihood algorithm to implement the new spike sorter. We present the general form of this algorithm and provide a detailed implementable version under the assumptions that neurons are independent and spike according to Poisson processes. Finally, we uncover a systematic flaw of spike sorting based on waveform information only.

  13. The RCP–Rab11 Complex Regulates Endocytic Protein SortingD⃞

    Science.gov (United States)

    Peden, Andrew A.; Schonteich, Eric; Chun, John; Junutula, Jagath R.; Scheller, Richard H.; Prekeris, Rytis

    2004-01-01

    Rab 11 GTPase is an important regulator of endocytic membrane traffic. Recently, we and others have identified a novel family of Rab11 binding proteins, known as Rab11-family interacting proteins (FIPs). One of the family members, Rab coupling protein (RCP), was identified as a protein binding to both Rab4 and Rab11 GTPases. RCP was therefore suggested to serve a dual function as Rab4 and Rab11 binding protein. In this study, we characterized the cellular functions of RCP and mapped its interactions with Rab4 and Rab11. Our data show that RCP interacts only weakly with Rab4 in vitro and does not play the role of coupling Rab11 and Rab4 in vivo. Furthermore, our data indicate that the RCP–Rab11 complex regulates the sorting of transferrin receptors from the degradative to the recycling pathway. We therefore propose that RCP functions primarily as a Rab11 binding protein that regulates protein sorting in tubular endosomes. PMID:15181150

  14. Increased translocation of antigens to endosomes and TLR4 mediated endosomal recruitment of TAP contribute to nicotine augmented cross-presentation.

    Science.gov (United States)

    Wang, Yan Yan; Hu, Chun Fang; Li, Juan; You, Xiang; Gao, Feng Guang

    2016-06-21

    Cross-presentation by dendritic cells (DCs) requires surface molecules such as lectin, CD40, langerin, heat shock protein, mannose receptor, mediated endocytosis, the endosomal translocation of internalized antigen, and the relocation of transporter associated with antigen processing (TAP). Although the activation of α7 nicotinic acetylcholine receptor (α7 nAchR) up-regulate surface molecule expression, augment endocytosis, and enhance cross-presentation, the molecular mechanism of α7 nAchR activation-increased cross-presentation is still poorly understood. In this study, we investigated the role of mannose receptor in nicotine-increased cross-presentation and the mechanism that endotoxins orchestrating the recruitment of TAP toward endosomes. We demonstrated that nicotine increase the expressiones of mannose receptor and Toll-like receptor 4 (TLR4) via PI3K-Akt-mTOR-p70S6 pathway. Both endosomal translocation of mannose receptor-internalized antigens and TLR4 sig- naling are necessary for nicotine-augmented cross-presentation and cross-priming. Importantly, the recruitment of TAP toward endosomes via TLR4-MyD88-IRAK4 signaling contributes to nicotine-increased cross-presentation and cross-activation of T cells. Thus, these data suggest that increased recruitment of TAP to Ag-containing vesicles contributes to the superior cross-presentation efficacy of α7 nAchR activated DCs.

  15. Sensor module design and forward and inverse kinematics analysis of 6-DOF sorting transferring robot

    Science.gov (United States)

    Zhou, Huiying; Lin, Jiajian; Liu, Lei; Tao, Meng

    2017-09-01

    To meet the demand of high strength express sorting, it is significant to design a robot with multiple degrees of freedom that can sort and transfer. This paper uses infrared sensor, color sensor and pressure sensor to receive external information, combine the plan of motion path in advance and the feedback information from the sensors, then write relevant program. In accordance with these, we can design a 6-DOF robot that can realize multi-angle seizing. In order to obtain characteristics of forward and inverse kinematics, this paper describes the coordinate directions and pose estimation by the D-H parameter method and closed solution. On the basis of the solution of forward and inverse kinematics, geometric parameters of links and link parameters are optimized in terms of application requirements. In this way, this robot can identify route, sort and transfer.

  16. Laser ablation cell sorting in scanning cytometry

    Science.gov (United States)

    Shen, Feimo; Price, Jeffrey H.

    2001-05-01

    Flow cytometry has been an important tool for automated cells sorting. However, the lack of good sensitivity prevents it from being used for rare events sorting; furthermore, fragile cells, anchorage-dependent cells, and clump forming cells cannot be sorted this way. A fully automated, high-speed scanning cytometer with autofocus and image segmentation is capable of accurately locating contaminant cells in a monolayer cell population. A laser ablation system was incorporated into the cytometer to negatively sort out the unwanted cells by applying a focused, ultra-short laser pulse (sub-micron diameter, pulse duration = 4 nsec, wavelength - 500 nm) to each targeted cell. Due to the high power density (approximately 1010 W/cm2) that was present at the focal point, disruptive mechanical forces were generated and were responsible for the kill. Fluorescently stained NIH-3T3 fibroblast cells were used as a model contaminant target ells in an unstained NIH-3T3 population to determine the identification-kill effectiveness. The contaminant cells were stained with the fluorochrome CellTracker Blue CMAC, whereas the background cells were left intact. Ablation pulses were applied in frame-by-frame increment batches to the cell culture on the microscope. The negative sorting effectiveness was analyzed by automatically re-scanning the post-ablation cell culture in phase contrast and propidium iodide stained epi fluorescent fields to verify cell death.

  17. The p97 ATPase associates with EEA1 to regulate the size of early endosomes

    Institute of Scientific and Technical Information of China (English)

    Harish N Ramanathan; Yihong Ye

    2012-01-01

    The AAA ((A)TPase-(a)ssociated with various cellular (a)ctivities) ATPase p97 acts on diverse substrate proteins to partake in various cellular processes such as membrane fusion and endoplasmic reticulum-associated degradation (ERAD).In membrane fusion,p97 is thought to function in analogy to the related ATPase NSF (N-ethylmaleimidesensitive fusion protein),which promotes membrane fusion by disassembling a SNARE complex.In ERAD,p97 dislocates misfolded proteins from the ER membrane to facilitate their turnover by the proteasome.Here,we identify a novel function of p97 in endocytic trafficking by establishing the early endosomal autoantigen 1 (EEA1) as a new p97 substrate.We demonstrate that a fraction of p97 is localized to the early endosome membrane,where it binds EEA1 via the N-terminal C2H2 zinc finger domain.Inhibition of p97 either by siRNA or a pharmacological inhibitor results in clustering and enlargement of early endosomes,which is associated with an altered trafficking pattern for an endocytic cargo.Mechanistically,we show that p97 inhibition causes increased EEA1 self-association at the endosome membrane.We propose that p97 may regulate the size of early endosomes by governing the oligomeric state of EEA1.

  18. MiniCORVET is a Vps8-containing early endosomal tether in Drosophila

    Science.gov (United States)

    Lőrincz, Péter; Lakatos, Zsolt; Varga, Ágnes; Maruzs, Tamás; Simon-Vecsei, Zsófia; Darula, Zsuzsanna; Benkő, Péter; Csordás, Gábor; Lippai, Mónika; Andó, István; Hegedűs, Krisztina; Medzihradszky, Katalin F; Takáts, Szabolcs; Juhász, Gábor

    2016-01-01

    Yeast studies identified two heterohexameric tethering complexes, which consist of 4 shared (Vps11, Vps16, Vps18 and Vps33) and 2 specific subunits: Vps3 and Vps8 (CORVET) versus Vps39 and Vps41 (HOPS). CORVET is an early and HOPS is a late endosomal tether. The function of HOPS is well known in animal cells, while CORVET is poorly characterized. Here we show that Drosophila Vps8 is highly expressed in hemocytes and nephrocytes, and localizes to early endosomes despite the lack of a clear Vps3 homolog. We find that Vps8 forms a complex and acts together with Vps16A, Dor/Vps18 and Car/Vps33A, and loss of any of these proteins leads to fragmentation of endosomes. Surprisingly, Vps11 deletion causes enlargement of endosomes, similar to loss of the HOPS-specific subunits Vps39 and Lt/Vps41. We thus identify a 4 subunit-containing miniCORVET complex as an unconventional early endosomal tether in Drosophila. DOI: http://dx.doi.org/10.7554/eLife.14226.001 PMID:27253064

  19. Flow karyotyping and sorting of human chromosomes

    Energy Technology Data Exchange (ETDEWEB)

    Gray, J.W.; Lucas, J.; Peters, D.; Pinkel, D.; Trask, B.; van den Engh, G.; Van Dilla, M.A.

    1986-07-16

    Flow cytometry and sorting are becoming increasingly useful as tools for chromosome classfication and for the detection of numerical and structural chromosome aberrations. Chromosomes of a single type can be purified with these tools to facilitate gene mapping or production of chromosome specific recombinant DNA libraries. For analysis of chromosomes with flow cytometry, the chromosomes are extracted from mitotic cells, stained with one or more fluorescent dyes and classified one-by-one according to their dye content(s). Thus, the flow approach is fundamentally different than conventional karyotyping where chromosomes are classified within the context of a metaphase spread. Flow sorting allows purification of chromosomes that can be distinguished flow cytometrically. The authors describe the basic principles of flow cytometric chromosome classification i.e. flow karyotyping, and chromosome sorting and describe several applications. 30 refs., 8 figs.

  20. Applications of cell sorting in biotechnology

    Directory of Open Access Journals (Sweden)

    Mattanovich Diethard

    2006-03-01

    Full Text Available Abstract Due to its unique capability to analyze a large number of single cells for several parameters simultaneously, flow cytometry has changed our understanding of the behavior of cells in culture and of the population dynamics even of clonal populations. The potential of this method for biotechnological research, which is based on populations of living cells, was soon appreciated. Sorting applications, however, are still less frequent than one would expect with regard to their potential. This review highlights important contributions where flow cytometric cell sorting was used for physiological research, protein engineering, cell engineering, specifically emphasizing selection of overproducing cell lines. Finally conclusions are drawn concerning the impact of cell sorting on inverse metabolic engineering and systems biology.

  1. Differential Internalization Rates and Postendocytic Sorting of the Norepinephrine and Dopamine Transporters Are Controlled by Structural Elements in the N Termini

    DEFF Research Database (Denmark)

    Vuorenpää, Anne Elina; Jørgensen, Trine Nygaard; Newman, Amy H

    2016-01-01

    increased intracellular accumulation of JHC 1-64-labeled NET and caused a parallel reduction in uptake capacity. Internalized NET strongly colocalized with the “long loop” recycling marker Rab11, whereas less overlap was seen with the “short loop” recycling marker Rab4 and the late endosomal marker Rab7....... Moreover, mitigating Rab11 function by overexpression of dominant negative Rab11 impaired NET function. Sorting of NET to the Rab11 recycling compartment was further supported by confocal imaging and reversible biotinylation experiments in transfected differentiated CATH.a cells. In contrast to NET......, the dopamine transporter displayed markedly less constitutive internalization and limited sorting to the Rab11 recycling compartment in the differentiated CATH.a cells. Exchange of domains between the two homologous transporters revealed that this difference was determined by non-conserved structural elements...

  2. Optical cell sorting with multiple imaging modalities

    DEFF Research Database (Denmark)

    Banas, Andrew; Carrissemoux, Caro; Palima, Darwin

    2017-01-01

    techniques. Scattering forces from beams actuated via efficient phase-only efficient modulation has been adopted. This has lowered the required power for sorting cells to a tenth of our previous approach, and also makes the cell sorter safer for use in clinical settings. With the versatility of dynamically...... programmable phase spatial light modulators, a plurality of light shaping techniques, including hybrid approaches, can be utilized in cell sorting....... healthy cells. With the richness of visual information, a lot of microscopy techniques have been developed and have been crucial in biological studies. To utilize their complementary advantages we adopt both fluorescence and brightfield imaging in our optical cell sorter. Brightfield imaging has...

  3. Order-Sorted Parameterization and Induction

    Science.gov (United States)

    Meseguer, José

    Parameterization is one of the most powerful features to make specifications and declarative programs modular and reusable, and our best hope for scaling up formal verification efforts. This paper studies order-sorted parameterization at three different levels: (i) its mathematical semantics; (ii) its operational semantics by term rewriting; and (iii) the inductive reasoning principles that can soundly be used to prove properties about such specifications. It shows that achieving the desired properties at each of these three levels is a considerably subtler matter than for many-sorted specifications, but that such properties can be attained under reasonable conditions.

  4. MODELING WORK OF SORTING STATION USING UML

    Directory of Open Access Journals (Sweden)

    O. V. Gorbova

    2014-12-01

    Full Text Available Purpose. The purpose of this paper is the construction of methods and models for the graphical representation process of sorting station, using the unified modeling language (UML. Methodology. Methods of graph theory, finite automata and the representation theory of queuing systems were used as the methods of investigation. A graphical representation of the process was implemented with using the Unified Modeling Language UML. The sorting station process representation is implemented as a state diagram and actions through a set of IBM Rational Rose. Graphs can show parallel operation of sorting station, the parallel existence and influence of objects process and the transition from one state to another. The IBM Rational Rose complex allows developing a diagram of work sequence of varying degrees of detailing. Findings. The study has developed a graphical representation method of the process of sorting station of different kind of complexity. All graphical representations are made using the UML. They are represented as a directed graph with the states. It is clear enough in the study of the subject area. Applying the methodology of the representation process, it allows becoming friendly with the work of any automation object very fast, and exploring the process during algorithms construction of sorting stations and other railway facilities. This model is implemented with using the Unified Modeling Language (UML using a combination of IBM Rational Rose. Originality. The representation process of sorting station was developed by means of the Unified Modeling Language (UML use. Methodology of representation process allows creating the directed graphs based on the order of execution of the works chain, objects and performers of these works. The UML allows visualizing, specifying, constructing and documenting, formalizing the representation process of sorting station and developing sequence diagrams of works of varying degrees of detail. Practical

  5. Thermoresponsive pegylated bubble liposome nanovectors for efficient siRNA delivery via endosomal escape

    KAUST Repository

    Alamoudi, Kholod

    2017-05-19

    Improving the delivery of siRNA into cancer cells via bubble liposomes. Designing a thermoresponsive pegylated liposome through the introduction of ammonium bicarbonate salt into liposomes so as to control their endosomal escape for gene therapy.A sub-200 nm nanovector was fully characterized and examined for cellular uptake, cytotoxicity, endosomal escape and gene silencing.The siRNA-liposomes were internalized into cancer cells within 5 min and then released siRNAs in the cytosol prior to lysosomal degradation upon external temperature elevation. This was confirmed by confocal bioimaging and gene silencing reaching up to 90% and further demonstrated by the protein inhibition of both target genes.The thermoresponsiveness of ammonium bicarbonate containing liposomes enabled the rapid endosomal escape of the particles and resulted in an efficient gene silencing.

  6. Caenorhabditis elegans SAND-1 is essential for RAB-7 function in endosomal traffic.

    Science.gov (United States)

    Poteryaev, Dmitry; Fares, Hanna; Bowerman, Bruce; Spang, Anne

    2007-01-24

    The small rab-GTPase RAB-7 acts in endosome and endosome to lysosome traffic. We identified SAND-1 as a protein required for RAB-7 function based on similarities between SAND-1 and RAB-7 RNAi phenotypes. Although the initial uptake of yolk protein in oocytes, or of soluble secreted (ss) GFP in coelomocytes, appeared normal, further transport along the endocytic traffic route was delayed in the absence of SAND-1 function, and yolk proteins failed to reach yolk granules efficiently. Moreover, in coelomocytes, ssGFP and BSA-Texas-Red were endocytosed but not transported to lysosomes. We show that SAND-1 is essential for RAB-7 function at the transition from early to late endosomes, but not for RAB-7 function at lysosomes.

  7. Endosomal acidification and cathepsin L activity is required for calicivirus replication.

    Science.gov (United States)

    Shivanna, Vinay; Kim, Yunjeong; Chang, Kyeong-Ok

    2014-09-01

    The role of cellular proteases and endosome maturation in the entry of caliciviruses including porcine enteric calicivirus (PEC), murine norovirus (MNV)-1 and feline calicivirus (FCV) were investigated. Treatment with chloroquine or cathepsin L inhibitors, but not cathepsin B inhibitors, significantly reduced the replication of PEC, MNV and FCV. When concentrated PEC, MNV or FCV were incubated with recombinant cathepsin L, the minor capsid protein VP2 of PEC and the major capsid protein VP1 of MNV and FCV were cleaved by the protease based on the Western blot analysis. Confocal microscopy analysis of PEC and MNV-1 showed that viral capsid proteins were retained in the endosomes in the presence of a cathepsin L inhibitor or chloroquine during virus entry. The results of this study suggest the important role of endosome maturation and cathepsin L in the entry of caliciviruses, and cathepsin L as a potential therapeutic target for calicivirus infection. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Cell polarity and patterning by PIN trafficking through early endosomal compartments in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Hirokazu Tanaka

    2013-05-01

    Full Text Available PIN-FORMED (PIN proteins localize asymmetrically at the plasma membrane and mediate intercellular polar transport of the plant hormone auxin that is crucial for a multitude of developmental processes in plants. PIN localization is under extensive control by environmental or developmental cues, but mechanisms regulating PIN localization are not fully understood. Here we show that early endosomal components ARF GEF BEN1 and newly identified Sec1/Munc18 family protein BEN2 are involved in distinct steps of early endosomal trafficking. BEN1 and BEN2 are collectively required for polar PIN localization, for their dynamic repolarization, and consequently for auxin activity gradient formation and auxin-related developmental processes including embryonic patterning, organogenesis, and vasculature venation patterning. These results show that early endosomal trafficking is crucial for cell polarity and auxin-dependent regulation of plant architecture.

  9. Modeling the endosomal escape of cell-penetrating peptides using a transmembrane pH gradient.

    Science.gov (United States)

    Madani, Fatemeh; Abdo, Rania; Lindberg, Staffan; Hirose, Hisaaki; Futaki, Shiroh; Langel, Ulo; Gräslund, Astrid

    2013-04-01

    Cell-penetrating peptides (CPPs) can internalize into cells with covalently or non-covalently bound biologically active cargo molecules, which by themselves are not able to pass the cell membrane. Direct penetration and endocytosis are two main pathways suggested for the cellular uptake of CPPs. Cargo molecules which have entered the cell via an endocytotic pathway must be released from the endosome before degradation by enzymatic processes and endosomal acidification. Endosomal entrapment seems to be a major limitation in delivery of these molecules into the cytoplasm. Bacteriorhodopsin (BR) asymmetrically introduced into large unilamellar vesicles (LUVs) was used to induce a pH gradient across the lipid bilayer. By measuring pH outside the LUVs, we observed light-induced proton pumping mediated by BR from the outside to the inside of the LUVs, creating an acidic pH inside the LUVs, similar to the late endosomes in vivo. Here we studied the background mechanism(s) of endosomal escape. 20% negatively charged LUVs were used as model endosomes with incorporated BR into the membrane and fluorescein-labeled CPPs entrapped inside the LUVs, together with a fluorescence quencher. The translocation of different CPPs in the presence of a pH gradient across the membrane was studied. The results show that the light-induced pH gradient induced by BR facilitates vesicle membrane translocation, particularly for the intermediately hydrophobic CPPs, and much less for hydrophilic CPPs. The presence of chloroquine inside the LUVs or addition of pyrenebutyrate outside the LUVs destabilizes the vesicle membrane, resulting in significant changes of the pH gradient across the membrane.

  10. Syntaxin 7 and VAMP-7 are soluble N-ethylmaleimide-sensitive factor attachment protein receptors required for late endosome-lysosome and homotypic lysosome fusion in alveolar macrophages.

    Science.gov (United States)

    Ward, D M; Pevsner, J; Scullion, M A; Vaughn, M; Kaplan, J

    2000-07-01

    Endocytosis in alveolar macrophages can be reversibly inhibited, permitting the isolation of endocytic vesicles at defined stages of maturation. Using an in vitro fusion assay, we determined that each isolated endosome population was capable of homotypic fusion. All vesicle populations were also capable of heterotypic fusion in a temporally specific manner; early endosomes, isolated 4 min after internalization, could fuse with endosomes isolated 8 min after internalization but not with 12-min endosomes or lysosomes. Lysosomes fuse with 12-min endosomes but not with earlier endosomes. Using homogenous populations of endosomes, we have identified Syntaxin 7 as a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) required for late endosome-lysosome and homotypic lysosome fusion in vitro. A bacterially expressed human Syntaxin 7 lacking the transmembrane domain inhibited homotypic late endosome and lysosome fusion as well as heterotypic late endosome-lysosome fusion. Affinity-purified antibodies directed against Syntaxin 7 also inhibited lysosome fusion in vitro but had no affect on homotypic early endosome fusion. Previous work suggested that human VAMP-7 (vesicle-associated membrane protein-7) was a SNARE required for late endosome-lysosome fusion. A bacterially expressed human VAMP-7 lacking the transmembrane domain inhibited both late endosome-lysosome fusion and homotypic lysosome fusion in vitro. These studies indicate that: 1) fusion along the endocytic pathway is a highly regulated process, and 2) two SNARE molecules, Syntaxin 7 and human VAMP-7, are involved in fusion of vesicles in the late endocytic pathway in alveolar macrophages.

  11. Protein trafficking through the endosomal system prepares intracellular parasites for a home invasion.

    Science.gov (United States)

    Tomavo, Stanislas; Slomianny, Christian; Meissner, Markus; Carruthers, Vern B

    2013-10-01

    Toxoplasma (toxoplasmosis) and Plasmodium (malaria) use unique secretory organelles for migration, cell invasion, manipulation of host cell functions, and cell egress. In particular, the apical secretory micronemes and rhoptries of apicomplexan parasites are essential for successful host infection. New findings reveal that the contents of these organelles, which are transported through the endoplasmic reticulum (ER) and Golgi, also require the parasite endosome-like system to access their respective organelles. In this review, we discuss recent findings that demonstrate that these parasites reduced their endosomal system and modified classical regulators of this pathway for the biogenesis of apical organelles.

  12. Protein trafficking through the endosomal system prepares intracellular parasites for a home invasion.

    Directory of Open Access Journals (Sweden)

    Stanislas Tomavo

    2013-10-01

    Full Text Available Toxoplasma (toxoplasmosis and Plasmodium (malaria use unique secretory organelles for migration, cell invasion, manipulation of host cell functions, and cell egress. In particular, the apical secretory micronemes and rhoptries of apicomplexan parasites are essential for successful host infection. New findings reveal that the contents of these organelles, which are transported through the endoplasmic reticulum (ER and Golgi, also require the parasite endosome-like system to access their respective organelles. In this review, we discuss recent findings that demonstrate that these parasites reduced their endosomal system and modified classical regulators of this pathway for the biogenesis of apical organelles.

  13. SorLA Controls Neurotrophic Activity by Sorting of GDNF and Its Receptors GFRα1 and RET

    DEFF Research Database (Denmark)

    Glerup, Simon; Lume, Maria; Olsen, Ditte;

    2013-01-01

    Glial cell-line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor that has reached clinical trials for Parkinson's disease. GDNF binds to its coreceptor GFRα1 and signals through the transmembrane receptor tyrosine kinase RET, or RET independently through NCAM or syndecan-3....... Whereas the GDNF signaling cascades are well described, cellular turnover and trafficking of GDNF and its receptors remain poorly characterized. Here, we find that SorLA acts as sorting receptor for the GDNF/GFRα1 complex, directing it from the cell surface to endosomes. Through this mechanism, GDNF...... is targeted to lysosomes and degraded while GFRα1 recycles, creating an efficient GDNF clearance pathway. The SorLA/GFRα1 complex further targets RET for endocytosis but not for degradation, affecting GDNF-induced neurotrophic activities. SorLA-deficient mice display elevated GDNF levels, altered dopaminergic...

  14. System for optical sorting of microscopic objects

    DEFF Research Database (Denmark)

    2014-01-01

    The present invention relates to a system for optical sorting of microscopic objects and corresponding method. An optical detection system (52) is capable of determining the positions of said first and/or said second objects. One or more force transfer units (200, 205, 210, 215) are placed...

  15. Economics of Grading and Sorting Pallet Parts

    Science.gov (United States)

    Daniel L. Schmoldt; John A. McLeod; Philip A. Araman

    1993-01-01

    Before trying to develop an automated inspection system for pallet part grading we analyzed the economics of such a system. Our results suggest that higher quality pallets produced by grading and sorting pallet parts would be attractive to both manufacturers and their customers, who would have to pay increased prices for higher quality pallets. Reductions in cost-per-...

  16. Matlab Code for Sorted Real Schur Forms

    NARCIS (Netherlands)

    Brandts, J.H.

    2001-01-01

    In Matlab, there exists a standard command to generate a real Schur form, and another command transforms a real Schur form into a complex one. In Golub and Van Loan (1996), a Matlab-like routine is sketched that sorts a complex Schur form: given a target value ? in the complex plane, the diagonal el

  17. Wavelet adaptation for automatic voice disorders sorting.

    Science.gov (United States)

    Erfanian Saeedi, Nafise; Almasganj, Farshad

    2013-07-01

    Early diagnosis of voice disorders and abnormalities by means of digital speech processing is a subject of interest for many researchers. Various methods are introduced in the literature, some of which are able to extensively discriminate pathological voices from normal ones. Voice disorders sorting, on the other hand, has received less attention due to the complexity of the problem. Although, previous publications show satisfactory results in classifying one type of disordered voice from normal cases, or two different types of abnormalities from each other, no comprehensive approach for automatic sorting of vocal abnormalities has been offered yet. In this paper, a solution for this problem is suggested. We create a powerful wavelet feature extraction approach, in which, instead of standard wavelets, adaptive wavelets are generated and applied to the voice signals. Orthogonal wavelets are parameterized via lattice structure and then, the optimal parameters are investigated through an iterative process, using the genetic algorithm (GA). GA is guided by the classifier results. Based on the generated wavelet, a wavelet-filterbank is constructed and the voice signals are decomposed to compute eight energy-based features. A support vector machine (SVM) then classifies the signals using the extracted features. Experimental results show that six various types of vocal disorders: paralysis, nodules, polyps, edema, spasmodic dysphonia and keratosis are fully sorted via the proposed method. This could be a successful step toward sorting a larger number of abnormalities associated with the vocal system. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Credit Scores, Race, and Residential Sorting

    Science.gov (United States)

    Nelson, Ashlyn Aiko

    2010-01-01

    Credit scores have a profound impact on home purchasing power and mortgage pricing, yet little is known about how credit scores influence households' residential location decisions. This study estimates the effects of credit scores on residential sorting behavior using a novel mortgage industry data set combining household demographic, credit, and…

  19. Credit Scores, Race, and Residential Sorting

    Science.gov (United States)

    Nelson, Ashlyn Aiko

    2010-01-01

    Credit scores have a profound impact on home purchasing power and mortgage pricing, yet little is known about how credit scores influence households' residential location decisions. This study estimates the effects of credit scores on residential sorting behavior using a novel mortgage industry data set combining household demographic, credit, and…

  20. Integration through a Card-Sort Activity

    Science.gov (United States)

    Green, Kris; Ricca, Bernard P.

    2015-01-01

    Learning to compute integrals via the various techniques of integration (e.g., integration by parts, partial fractions, etc.) is difficult for many students. Here, we look at how students in a college level Calculus II course develop the ability to categorize integrals and the difficulties they encounter using a card sort-resort activity. Analysis…

  1. Integration through a Card-Sort Activity

    Science.gov (United States)

    Green, Kris; Ricca, Bernard P.

    2015-01-01

    Learning to compute integrals via the various techniques of integration (e.g., integration by parts, partial fractions, etc.) is difficult for many students. Here, we look at how students in a college level Calculus II course develop the ability to categorize integrals and the difficulties they encounter using a card sort-resort activity. Analysis…

  2. Irregular dunes, sediment sorting, and river morphodynamics

    NARCIS (Netherlands)

    Blom, Astrid; Weerts, H.J.T.; Ritsema, I.L; van Os, A.G.

    2006-01-01

    This research project focuses on modelling the large-scale morphodynamics of low-slope rivers dominated by mixed sediment, such as the Dutch part of the Rhine River. Usually we simply neglect the effects of sorting and variability in dune dimensions on the large-scale morphodynamics. This paper

  3. Sorting cells by their dynamical properties

    Science.gov (United States)

    Henry, Ewan; Holm, Stefan H.; Zhang, Zunmin; Beech, Jason P.; Tegenfeldt, Jonas O.; Fedosov, Dmitry A.; Gompper, Gerhard

    2016-10-01

    Recent advances in cell sorting aim at the development of novel methods that are sensitive to various mechanical properties of cells. Microfluidic technologies have a great potential for cell sorting; however, the design of many micro-devices is based on theories developed for rigid spherical particles with size as a separation parameter. Clearly, most bioparticles are non-spherical and deformable and therefore exhibit a much more intricate behavior in fluid flow than rigid spheres. Here, we demonstrate the use of cells’ mechanical and dynamical properties as biomarkers for separation by employing a combination of mesoscale hydrodynamic simulations and microfluidic experiments. The dynamic behavior of red blood cells (RBCs) within deterministic lateral displacement (DLD) devices is investigated for different device geometries and viscosity contrasts between the intra-cellular fluid and suspending medium. We find that the viscosity contrast and associated cell dynamics clearly determine the RBC trajectory through a DLD device. Simulation results compare well to experiments and provide new insights into the physical mechanisms which govern the sorting of non-spherical and deformable cells in DLD devices. Finally, we discuss the implications of cell dynamics for sorting schemes based on properties other than cell size, such as mechanics and morphology.

  4. Recruitment of Cbl-b to B cell antigen receptor couples antigen recognition to Toll-like receptor 9 activation in late endosomes.

    Directory of Open Access Journals (Sweden)

    Margaret Veselits

    Full Text Available Casitas B-lineage lymphoma-b (Cbl-b is a ubiquitin ligase (E3 that modulates signaling by tagging molecules for degradation. It is a complex protein with multiple domains and binding partners that are not involved in ubiquitinating substrates. Herein, we demonstrate that Cbl-b, but not c-Cbl, is recruited to the clustered B cell antigen receptor (BCR and that Cbl-b is required for entry of endocytosed BCRs into late endosomes. The E3 activity of Cbl-b is not necessary for BCR endocytic trafficking. Rather, the ubiquitin associated (UBA domain is required. Furthermore, the Cbl-b UBA domain is sufficient to confer the receptor trafficking functions of Cbl-b on c-Cbl. Cbl-b is also required for entry of the Toll-like receptor 9 (TLR9 into late endosomes and for the in vitro activation of TLR9 by BCR-captured ligands. These data indicate that Cbl-b acts as a scaffolding molecule to coordinate the delivery of the BCR and TLR9 into subcellular compartments required for productively delivering BCR-captured ligands to TLR9.

  5. TECHNICAL COORDINATION

    CERN Multimedia

    A. Ball

    Overview From a technical perspective, CMS has been in “beam operation” state since 6th November. The detector is fully closed with all components operational and the magnetic field is normally at the nominal 3.8T. The UXC cavern is normally closed with the radiation veto set. Access to UXC is now only possible during downtimes of LHC. Such accesses must be carefully planned, documented and carried out in agreement with CMS Technical Coordination, Experimental Area Management, LHC programme coordination and the CCC. Material flow in and out of UXC is now strictly controlled. Access to USC remains possible at any time, although, for safety reasons, it is necessary to register with the shift crew in the control room before going down.It is obligatory for all material leaving UXC to pass through the underground buffer zone for RP scanning, database entry and appropriate labeling for traceability. Technical coordination (notably Stephane Bally and Christoph Schaefer), the shift crew and run ...

  6. BECN1/Beclin 1 sorts cell-surface APP/amyloid β precursor protein for lysosomal degradation.

    Science.gov (United States)

    Swaminathan, Gayathri; Zhu, Wan; Plowey, Edward D

    2016-12-01

    The regulation of plasma membrane (PM)-localized transmembrane protein/receptor trafficking has critical implications for cell signaling, metabolism and survival. In this study, we investigated the role of BECN1 (Beclin 1) in the degradative trafficking of PM-associated APP (amyloid β precursor protein), whose metabolism to amyloid-β, an essential event in Alzheimer disease, is dependent on divergent PM trafficking pathways. We report a novel interaction between PM-associated APP and BECN1 that recruits macroautophagy/endosomal regulatory proteins PIK3C3 and UVRAG. We found that BECN1 promotes surface APP internalization and sorting predominantly to endosomes and endolysosomes. BECN1 also promotes the targeting of a smaller fraction of internalized APP to LC3-positive phagophores, suggesting a role for BECN1-dependent PM macroautophagy in APP degradation. Furthermore, BECN1 facilitates lysosomal degradation of surface APP and reduces the secretion of APP metabolites (soluble ectodomains, sAPP). The association between APP and BECN1 is dependent on the evolutionarily conserved domain (ECD) of BECN1 (amino acids 267-337). Deletion of a BECN1 ECD subregion (amino acids 285-299) did not impair BECN1- PIK3C3 interaction, PtdIns3K function or macroautophagy, but was sufficient to impair the APP-BECN1 interaction and BECN1's effects on surface APP internalization and degradation, resulting in increased secretion of sAPPs. Interestingly, both the BECN1-APP association and BECN1-dependent APP endocytosis and degradative trafficking were negatively regulated by active AKT. Our results further implicate phosphorylation of the BECN1 Ser295 residue in the inhibition of APP degradation by AKT. Our studies reveal a novel function for BECN1 in the sorting of a plasma membrane protein for endolysosomal and macroautophagic degradation.

  7. Vacuolar Protein Sorting genes in Parkinson’s Disease: a re-appraisal of mutations detection rate and neurobiology of disease

    Directory of Open Access Journals (Sweden)

    Stefano Gambardella

    2016-11-01

    Full Text Available Mammalian retromers play a critical role in protein trans-membrane sorting from endosome to the trans-Golgi network (TGN. Recently, retromers have been linked to Parkinson's Disease (PD since the identification of the variant p.Asp620Asn in VPS35 (Vacuolar Protein Sorting 35 as a cause of late onset PD. This variant causes a primary defect in endosomal trafficking and retromers formation, which represent critical steps in the molecular mechanisms of disease. Other slightly penetrant and mildly deleterious mutations in VPS genes have been reported in both sporadic and familial PD. Therefore, understanding the actual prevalence of the whole range of VPS gene mutations is key to understand the relevance of retromers impairment in PD. This scenario indicates a plethora of mutations occurring in different pathways (autophagy, mitophagy, proteasome, endosomes, protein misfolding all converging to cell clearing systems. This may explain how genetic predispositions to PD may derive from slightly deleterious mutations when combining with heterogeneous environmental factors. This manuscript is a re-appraisal of genetic data produced in the last five years redefining the prevalence of VPS mutations in PD. The prevalence of p.Asp620Asn in VPS35 is 0.286 of familial PD. This data increases up to 0.548 considering mutations affecting all VPS genes, thus representing the second most frequent autosomal dominant PD genotype. This high prevalence, joined with increased awareness of the key role of retromers alterations in PD, strongly candidate environmentally-induced VPS alterations as key molecular mechanisms in the genesis of PD. rations as key molecular mechanisms in the genesis of PD.

  8. Development of a Prototype Automated Sorting System for Plastic Recycling

    Directory of Open Access Journals (Sweden)

    D. A. Wahab

    2006-01-01

    Full Text Available Automated sorting for plastic recyclables has been seen as the way forward in the plastic recycling industry. Automated sorting provides significant improvements in terms of efficiency and consistency in the sorting process. In the case of macro sorting, which is the most common type of automated sorting, efficiency is determined by the mechanical details of the material handling system as well as the detection system. This paper provides a review on the state of-the-art technologies that have been deployed by some of the recycling facilities abroad. The design and development of a cost effective prototype automated system for sorting plastic recyclables is proposed and discussed.

  9. Multiple pathways for vacuolar sorting of yeast proteinase A

    DEFF Research Database (Denmark)

    Westphal, V; Marcusson, E G; Winther, Jakob R.

    1996-01-01

    The sorting of the yeast proteases proteinase A and carboxypeptidase Y to the vacuole is a saturable, receptor-mediated process. Information sufficient for vacuolar sorting of the normally secreted protein invertase has in fusion constructs previously been found to reside in the propeptide...... of proteinase A. We found that sorting of such a hybrid protein is dependent on the vacuolar protein-sorting receptor Vps10p. This was unexpected, as strains disrupted for VPS10 sort more than 85% of the proteinase A to the vacuole. Consistent with a role for Vps10p in sorting of proteinase A, we found that 1...

  10. PhySortR: a fast, flexible tool for sorting phylogenetic trees in R

    Directory of Open Access Journals (Sweden)

    Timothy G. Stephens

    2016-05-01

    Full Text Available A frequent bottleneck in interpreting phylogenomic output is the need to screen often thousands of trees for features of interest, particularly robust clades of specific taxa, as evidence of monophyletic relationship and/or reticulated evolution. Here we present PhySortR, a fast, flexible R package for classifying phylogenetic trees. Unlike existing utilities, PhySortR allows for identification of both exclusive and non-exclusive clades uniting the target taxa based on tip labels (i.e., leaves on a tree, with customisable options to assess clades within the context of the whole tree. Using simulated and empirical datasets, we demonstrate the potential and scalability of PhySortR in analysis of thousands of phylogenetic trees without a priori assumption of tree-rooting, and in yielding readily interpretable trees that unambiguously satisfy the query. PhySortR is a command-line tool that is freely available and easily automatable.

  11. Nbr1, a Receptor for ESCRT-Dependent Endosomal Microautophagy in Fission Yeast.

    Science.gov (United States)

    Mizushima, Noboru

    2015-09-17

    In this issue of Molecular Cell, Liu et al. (2015) report that fission yeast Nbr1, sharing a partial homology to the mammalian macroautophagy receptor NBR1, acts as a receptor for ESCRT-dependent endosomal microautophagy that delivers two hydrolytic enzymes from the cytosol to the vacuole.

  12. TLR2 ligands induce NF-κB activation from endosomal compartments of human monocytes.

    Directory of Open Access Journals (Sweden)

    Karim J Brandt

    Full Text Available Localization of Toll-like receptors (TLR in subcellular organelles is a major strategy to regulate innate immune responses. While TLR4, a cell-surface receptor, signals from both the plasma membrane and endosomal compartments, less is known about the functional role of endosomal trafficking upon TLR2 signaling. Here we show that the bacterial TLR2 ligands Pam3CSK4 and LTA activate NF-κB-dependent signaling from endosomal compartments in human monocytes and in a NF-κB sensitive reporter cell line, despite the expression of TLR2 at the cell surface. Further analyses indicate that TLR2-induced NF-κB activation is controlled by a clathrin/dynamin-dependent endocytosis mechanism, in which CD14 serves as an important upstream regulator. These findings establish that internalization of cell-surface TLR2 into endosomal compartments is required for NF-κB activation. These observations further demonstrate the need of endocytosis in the activation and regulation of TLR2-dependent signaling pathways.

  13. The cell outgrowth secretory endosome (COSE): a specialized compartment involved in neuronal morphogenesis.

    Science.gov (United States)

    Alberts, Philipp; Galli, Thierry

    2003-10-01

    The role of intracellular membrane trafficking in cellular morphogenesis is still unclear. We propose here a prominent function of a recently identified compartment that we propose to call the cell outgrowth secretory endosome (COSE), the exocytosis of which is controlled by the v-SNARE TIVAMP and by cell-cell adhesion.

  14. Directional Notch trafficking in Sara endosomes during asymmetric cell division in the spinal cord.

    Science.gov (United States)

    Kressmann, Sabine; Campos, Claudia; Castanon, Irinka; Fürthauer, Maximilian; González-Gaitán, Marcos

    2015-03-01

    Asymmetric division of neural precursor cells contributes to the generation of a variety of neuronal types. Asymmetric division is mediated by the asymmetric inheritance of fate determinants by the two daughter cells. In vertebrates, asymmetric fate determinants, such as Par3 and Mib, are only now starting to be identified. Here we show that, during mitosis of neural precursors in zebrafish, directional trafficking of Sara endosomes to one of the daughters can function as such a determinant. In asymmetric lineages, where one daughter cell becomes a neuron (n cell) whereas the other divides again to give rise to two neurons (p cell), we found that the daughter that inherits most of the Sara endosomes acquires the p fate. Sara endosomes carry an endocytosed pool of the Notch ligand DeltaD, which is thereby itself distributed asymmetrically. Sara and Notch are both essential for cell fate assignation within asymmetric lineages. Therefore, the Sara endosome system determines the fate decision between neuronal differentiation and mitosis in asymmetric lineages and thereby contributes to controlling the number of neural precursors and differentiated neurons during neurogenesis in a vertebrate.

  15. Endosome-based protein trafficking and Ca2+ homeostasis in the heart

    Science.gov (United States)

    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

  16. Increased neuronal Rab5 immunoreactive endosomes do not colocalize with TDP-43 in motor neuron disease.

    Science.gov (United States)

    Matej, Radoslav; Botond, Gergö; László, Lajos; Kopitar-Jerala, Natasa; Rusina, Robert; Budka, Herbert; Kovacs, Gabor G

    2010-09-01

    Sporadic motor neuron disease (MND) is characterized by progressive degeneration of motor neurons and intraneuronal cytoplasmic translocation and deposition of the nuclear protein TDP-43. There is a paucity of data on the subcellular mechanisms of the nuclear-cytoplasmic trafficking of TDP-43, particularly about the precise role of the endosomal-lysosomal system (ELS). In the present study, using a neuron-specific morphometric approach, we examined the expression of the early endosomal marker Rab5 and lysosomal cathepsins B, D, F, and L as well as PAS-stained structures in the anterior horn cells in 11 individuals affected by sporadic MND and 5 age-matched controls. This was compared with the expression of ubiquitin, p62 and TDP-43 and its phosphorylated form. The principal finding was the increased expression of the endosomal marker Rab5 and lysosomal cathepsin D, and of PAS-positive structures in motor neurons of MND cases. Furthermore, the area-portion of Rab5 immunoreactivity correlated well with the intracellular accumulation of ubiquitin, p62 and (phosphorylated) TDP-43. However, double immunolabelling and immunogold electron microscopy excluded colocalization of phosphorylated TDP-43 with the ELS. These data contrast with observations on neuronal cytopathology in Alzheimer's or prion diseases where the disease-specific proteins are processed within endosomes, and suggest a distinct role of the ELS in MND.

  17. Dynamin-dependent transferrin receptor recycling by endosome-derived clathrin-coated vesicles

    NARCIS (Netherlands)

    van Dam, EM; Stoorvogel, W

    2002-01-01

    Previously we described clathrin-coated buds on tubular early endosomes that are distinct from those at the plasma membrane and the traps-Golgi network. Here we show that these clathrin-coated buds, like plasma membrane clathrin-coated pits, contain endogenous dynamin-2. To study the itinerary that

  18. Cooperative endocytosis of the endosomal SNARE protein syntaxin-8 and the potassium channel TASK-1.

    Science.gov (United States)

    Renigunta, Vijay; Fischer, Thomas; Zuzarte, Marylou; Kling, Stefan; Zou, Xinle; Siebert, Kai; Limberg, Maren M; Rinné, Susanne; Decher, Niels; Schlichthörl, Günter; Daut, Jürgen

    2014-06-15

    The endosomal SNARE protein syntaxin-8 interacts with the acid-sensitive potassium channel TASK-1. The functional relevance of this interaction was studied by heterologous expression of these proteins (and mutants thereof) in Xenopus oocytes and in mammalian cell lines. Coexpression of syntaxin-8 caused a fourfold reduction in TASK-1 current, a corresponding reduction in the expression of TASK-1 at the cell surface, and a marked increase in the rate of endocytosis of the channel. TASK-1 and syntaxin-8 colocalized in the early endosomal compartment, as indicated by the endosomal markers 2xFYVE and rab5. The stimulatory effect of the SNARE protein on the endocytosis of the channel was abolished when both an endocytosis signal in TASK-1 and an endocytosis signal in syntaxin-8 were mutated. A syntaxin-8 mutant that cannot assemble with other SNARE proteins had virtually the same effect as wild-type syntaxin-8. Total internal reflection fluorescence microscopy showed formation and endocytosis of vesicles containing fluorescence-tagged clathrin, TASK-1, and/or syntaxin-8. Our results suggest that the unassembled form of syntaxin-8 and the potassium channel TASK-1 are internalized via clathrin-mediated endocytosis in a cooperative manner. This implies that syntaxin-8 regulates the endocytosis of TASK-1. Our study supports the idea that endosomal SNARE proteins can have functions unrelated to membrane fusion.

  19. Natural Modulators of Endosomal Toll-Like Receptor-Mediated Psoriatic Skin Inflammation

    Directory of Open Access Journals (Sweden)

    Chao-Yang Lai

    2017-01-01

    Full Text Available Psoriasis is a chronic inflammatory autoimmune disease that can be initiated by excessive activation of endosomal toll-like receptors (TLRs, particularly TLR7, TLR8, and TLR9. Therefore, inhibitors of endosomal TLR activation are being investigated for their ability to treat this disease. The currently approved biological drugs adalimumab, etanercept, infliximab, ustekinumab, ixekizumab, and secukizumab are antibodies against effector cytokines that participate in the initiation and development of psoriasis. Several immune modulatory oligonucleotides and small molecular weight compounds, including IMO-3100, IMO-8400, and CPG-52364, that block the interaction between endosomal TLRs and their ligands are under clinical investigation for their effectiveness in the treatment of psoriasis. In addition, several chemical compounds, including AS-2444697, PF-05387252, PF-05388169, PF-06650833, ML120B, and PHA-408, can inhibit TLR signaling. Although these compounds have demonstrated anti-inflammatory activity in animal models, their therapeutic potential for the treatment of psoriasis has not yet been tested. Recent studies demonstrated that natural compounds derived from plants, fungi, and bacteria, including mustard seed, Antrodia cinnamomea extract, curcumin, resveratrol, thiostrepton, azithromycin, and andrographolide, inhibited psoriasis-like inflammation induced by the TLR7 agonist imiquimod in animal models. These natural modulators employ different mechanisms to inhibit endosomal TLR activation and are administered via different routes. Therefore, they represent candidate psoriasis drugs and might lead to the development of new treatment options.

  20. RECYCLING PATHWAYS OF GLUCOSYLCERAMIDE IN BHK CELLS - DISTINCT INVOLVEMENT OF EARLY AND LATE ENDOSOMES

    NARCIS (Netherlands)

    KOK, JW; HOEKSTRA, K; ESKELINEN, S; HOEKSTRA, D

    1992-01-01

    Recycling pathways of the sphingolipid glucosylceramide were studied by employing a fluorescent analog of glucosylceramide, 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylglucosylsphingosine (C6-NBD-glucosylceramide). Direct recycling of the glycolipid from early endosomes to the plasma membra

  1. Investigation of endosome and lysosome biology by ultra pH-sensitive nanoprobes.

    Science.gov (United States)

    Wang, Chensu; Zhao, Tian; Li, Yang; Huang, Gang; White, Michael A; Gao, Jinming

    2016-09-06

    Endosomes and lysosomes play a critical role in various aspects of cell physiology such as nutrient sensing, receptor recycling, protein/lipid catabolism, and cell death. In drug delivery, endosomal release of therapeutic payloads from nanocarriers is also important in achieving efficient delivery of drugs to reach their intracellular targets. Recently, we invented a library of ultra pH-sensitive (UPS) nanoprobes with exquisite fluorescence response to subtle pH changes. The UPS nanoprobes also displayed strong pH-specific buffer effect over small molecular bases with broad pH responses (e.g., chloroquine and NH4Cl). Tunable pH transitions from 7.4 to 4.0 of UPS nanoprobes cover the entire physiological pH of endocytic organelles (e.g., early and late endosomes) and lysosomes. These unique physico-chemical properties of UPS nanoprobes allowed a 'detection and perturbation' strategy for the investigation of luminal pH in cell signaling and metabolism, which introduces a nanotechnology-enabled paradigm for the biological studies of endosomes and lysosomes.

  2. Role of receptor-mediated endocytosis, endosomal acidification and cathepsin D in cholera toxin cytotoxicity.

    Science.gov (United States)

    El Hage, Tatiana; Merlen, Clémence; Fabrega, Sylvie; Authier, François

    2007-05-01

    Using the in situ liver model system, we have recently shown that, after cholera toxin binding to hepatic cells, cholera toxin accumulates in a low-density endosomal compartment, and then undergoes endosomal proteolysis by the aspartic acid protease cathepsin-D [Merlen C, Fayol-Messaoudi D, Fabrega S, El Hage T, Servin A, Authier F (2005) FEBS J272, 4385-4397]. Here, we have used a subcellular fractionation approach to address the in vivo compartmentalization and cytotoxic action of cholera toxin in rat liver parenchyma. Following administration of a saturating dose of cholera toxin to rats, rapid endocytosis of both cholera toxin subunits was observed, coincident with massive internalization of both the 45 kDa and 47 kDa Gsalpha proteins. These events coincided with the endosomal recruitment of ADP-ribosylation factor proteins, especially ADP-ribosylation factor-6, with a time course identical to that of toxin and the A subunit of the stimulatory G protein (Gsalpha) translocation. After an initial lag phase of 30 min, these constituents were linked to NAD-dependent ADP-ribosylation of endogenous Gsalpha, with maximum accumulation observed at 30-60 min postinjection. Assessment of the subsequent postendosomal fate of internalized Gsalpha revealed sustained endolysosomal transfer of the two Gsalpha isoforms. Concomitantly, cholera toxin increased in vivo endosome acidification rates driven by the ATP-dependent H(+)-ATPase pump and in vitro vacuolar acidification in hepatoma HepG2 cells. The vacuolar H(+)-ATPase inhibitor bafilomycin and the cathepsin D inhibitor pepstatin A partially inhibited, both in vivo and in vitro, the cAMP response to cholera toxin. This cathepsin D-dependent action of cholera toxin under the control of endosomal acidity was confirmed using cellular systems in which modification of the expression levels of cathepsin D, either by transfection of the cathepsin D gene or small interfering RNA, was followed by parallel changes in the cytotoxic

  3. Colour based sorting station with Matlab simulation

    Directory of Open Access Journals (Sweden)

    Constantin Victor

    2017-01-01

    Full Text Available The paper presents the design process and manufacturing elements of a colour-based sorting station. The system is comprised of a gravitational storage, which also contains the colour sensor. Parts are extracted using a linear pneumatic motor and are fed onto an electrically driven conveyor belt. Extraction of the parts is done at 4 points, using two pneumatic motors and a geared DC motor, while the 4th position is at the end of the belt. The mechanical parts of the system are manufactured using 3D printer technology, allowing for easy modification and adaption to the geometry of different parts. The paper shows all of the stages needed to design, optimize, test and implement the proposed solution. System optimization was performed using a graphical Matlab interface which also allows for sorting algorithm optimization.

  4. Microtechnology for cell manipulation and sorting

    CERN Document Server

    Tseng, Peter; Carlo, Dino

    2017-01-01

    This book delves into the recent developments in the microscale and microfluidic technologies that allow manipulation at the single and cell aggregate level. Expert authors review the dominant mechanisms that manipulate and sort biological structures, making this a state-of-the-art overview of conventional cell sorting techniques, the principles of microfluidics, and of microfluidic devices. All chapters highlight the benefits and drawbacks of each technique they discuss, which include magnetic, electrical, optical, acoustic, gravity/sedimentation, inertial, deformability, and aqueous two-phase systems as the dominant mechanisms utilized by microfluidic devices to handle biological samples. Each chapter explains the physics of the mechanism at work, and reviews common geometries and devices to help readers decide the type of style of device required for various applications. This book is appropriate for graduate-level biomedical engineering and analytical chemistry students, as well as engineers and scientist...

  5. Machine-vision based optofluidic cell sorting

    DEFF Research Database (Denmark)

    Glückstad, Jesper; Bañas, Andrew

    In contemporary life science there is an increasing emphasis on sorting rare disease-indicating cells within small dilute quantities such as in the confines of optofluidic lab-on-chip devices. Our approach to this is based on the use of optical forces to isolate red blood cells detected by advanc...... the available light and creating 2D or 3D beam distributions aimed at the positions of the detected cells. Furthermore, the beam shaping freedom provided by GPC can allow optimizations in the beam’s propagation and its interaction with the laser catapulted and sorted cells....... machine vision1. This approach is gentler, less invasive and more economical compared to conventional FACS-systems. As cells are less responsive to plastic or glass objects commonly used in the optical manipulation literature2, and since laser safety would be an issue in clinical use, we develop efficient...

  6. A mower detector to judge soil sorting

    Energy Technology Data Exchange (ETDEWEB)

    Bramlitt, E.T.; Johnson, N.R. [Thermo Nuclear Services, Inc., Albuquerque, NM (United States)

    1995-12-31

    Thermo Nuclear Services (TNS) has developed a mower detector as an inexpensive and fast means for deciding potential value of soil sorting for cleanup. It is a shielded detector box on wheels pushed over the ground (as a person mows grass) at 30 ft/min with gamma-ray counts recorded every 0.25 sec. It mirror images detection by the TNS transportable sorter system which conveys soil at 30 ft/min and toggles a gate to send soil on separate paths based on counts. The mower detector shows if contamination is variable and suitable for sorting, and by unique calibration sources, it indicates detection sensitivity. The mower detector has been used to characterize some soil at Department of Energy sites in New Jersey and South Carolina.

  7. Efficient sorting using registers and caches

    DEFF Research Database (Denmark)

    Wickremesinghe, Rajiv; Arge, Lars Allan; Chase, Jeffrey S.;

    2002-01-01

    Modern computer systems have increasingly complex memory systems. Common machine models for algorithm analysis do not reflect many of the features of these systems, e.g., large register sets, lockup-free caches, cache hierarchies, associativity, cache line fetching, and streaming behavior...... on sorting performance. We introduce a new cache-conscious sorting algorithm, R-MERGE, which achieves better performance in practice over algorithms that are superior in the theoretical models. R-MERGE is designed to minimize memory stall cycles rather than cache misses by considering features common to many....... Inadequate models lead to poor algorithmic choices and an incomplete understanding of algorithm behavior on real machines.A key step toward developing better models is to quantify the performance effects of features not reflected in the models. This paper explores the effect of memory system features...

  8. TECHNICAL COORDINATION

    CERN Multimedia

    A. Ball

    2010-01-01

    Operational Experience At the end of the first full-year running period of LHC, CMS is established as a reliable, robust and mature experiment. In particular common systems and infrastructure faults accounted for <0.6 % CMS downtime during LHC pp physics. Technical operation throughout the entire year was rather smooth, the main faults requiring UXC access being sub-detector power systems and rack-cooling turbines. All such problems were corrected during scheduled technical stops, in the shadow of tunnel access needed by the LHC, or in negotiated accesses or access extensions. Nevertheless, the number of necessary accesses to the UXC averaged more than one per week and the technical stops were inevitably packed with work packages, typically 30 being executed within a few days, placing a high load on the coordination and area management teams. It is an appropriate moment for CMS Technical Coordination to thank all those in many CERN departments and in the Collaboration, who were involved in CMS techni...

  9. Sorting Network for Reversible Logic Synthesis

    CERN Document Server

    Islam, Md Saiful; Mahmud, Abdullah Al; karim, Muhammad Rezaul

    2010-01-01

    In this paper, we have introduced an algorithm to implement a sorting network for reversible logic synthesis based on swapping bit strings. The algorithm first constructs a network in terms of n*n Toffoli gates read from left to right. The number of gates in the circuit produced by our algorithm is then reduced by template matching and removing useless gates from the network. We have also compared the efficiency of the proposed method with the existing ones.

  10. The V-ATPase a2-subunit as a putative endosomal pH-sensor.

    Science.gov (United States)

    Marshansky, V

    2007-11-01

    V-ATPase (vesicular H(+)-ATPase)-driven intravesicular acidification is crucial for vesicular trafficking. Defects in vesicular acidification and trafficking have recently been recognized as essential determinants of various human diseases. An important role of endosomal acidification in receptor-ligand dissociation and in activation of lysosomal hydrolytic enzymes is well established. However, the molecular mechanisms by which luminal pH information is transmitted to the cytosolic small GTPases that control trafficking events such as budding, coat formation and fusion are unknown. Here, we discuss our recent discovery that endosomal V-ATPase is a pH-sensor regulating the degradative pathway. According to our model, V-ATPase is responsible for: (i) the generation of a pH gradient between vesicular membranes; (ii) sensing of intravesicular pH; and (iii) transmitting this information to the cytosolic side of the membrane. We also propose the hypothetical molecular mechanism involved in function of the V-ATPase a2-subunit as a putative pH-sensor. Based on extensive experimental evidence on the crucial role of histidine residues in the function of PSPs (pH-sensing proteins) in eukaryotic cells, we hypothesize that pH-sensitive histidine residues within the intra-endosomal loops and/or C-terminal luminal tail of the a2-subunit could also be involved in the pH-sensing function of V-ATPase. However, in order to identify putative pH-sensitive histidine residues and to test this hypothesis, it is absolutely essential that we increase our understanding of the folding and transmembrane topology of the a-subunit isoforms of V-ATPase. Thus the crucial role of intra-endosomal histidine residues in pH-dependent conformational changes of the V-ATPase a2-isoform, its interaction with cytosolic small GTPases and ultimately in its acidification-dependent regulation of the endosomal/lysosomal protein degradative pathway remain to be determined.

  11. Human integrin α(3β(1 regulates TLR2 recognition of lipopeptides from endosomal compartments.

    Directory of Open Access Journals (Sweden)

    Meghan L Marre

    Full Text Available BACKGROUND: Toll-like receptor (TLR-2/TLR1 heterodimers recognize bacterial lipopeptides and initiate the production of inflammatory mediators. Adaptors and co-receptors that mediate this process, as well as the mechanisms by which these adaptors and co-receptors function, are still being discovered. METHODOLOGY/PRINCIPAL FINDINGS: Using shRNA, blocking antibodies, and fluorescent microscopy, we show that U937 macrophage responses to the TLR2/1 ligand, Pam(3CSK(4, are dependent upon an integrin, α(3β(1. The mechanism for integrin α(3β(1 involvement in TLR2/1 signaling is through its role in endocytosis of lipopeptides. Using inhibitors of endosomal acidification/maturation and physical tethering of the ligand, we show that the endocytosis of Pam(3CSK(4 is necessary for the complete TLR2/1-mediated pro-inflammatory cytokine response. We also show that TLR2/1 signaling from the endosome results in the induction of different inflammatory mediators than TLR2/1 signaling from the plasma membrane. CONCLUSION/SIGNIFICANCE: Here we identify integrin α(3β(1 as a novel regulator for the recognition of bacterial lipopeptides. We demonstrate that induction of a specific subset of cytokines is dependent upon integrin α(3β(1-mediated endocytosis of the ligand. In addition, we address an ongoing controversy regarding endosomal recognition of bacterial lipopeptides by demonstrating that TLR2/1 signals from within endosomal compartments as well as the plasma membrane, and that downstream responses may differ depending upon receptor localization. We propose that the regulation of endosomal TLR2/1 signaling by integrin α(3β(1 serves as a mechanism for modulating inflammatory responses.

  12. Evaluating Effects of Cell Sorting on Cellular Integrity

    OpenAIRE

    2014-01-01

    During the past year the Flow Cytometry Research Group has continued on its goal to establish best practice guidelines for cell sorting conditions that minimize cell stress, perturbation, or injury to the sorted cells. Towards this goal the group has followed up on an observation from our initial study that showed poor cell recovery when a clonal population of cells (Jurkat) was sorted aggressively under intentionally adverse sorting conditions (excessive pressure as well as undersized sortin...

  13. How Schwann Cells Sort Axons: New Concepts.

    Science.gov (United States)

    Feltri, M Laura; Poitelon, Yannick; Previtali, Stefano Carlo

    2016-06-01

    Peripheral nerves contain large myelinated and small unmyelinated (Remak) fibers that perform different functions. The choice to myelinate or not is dictated to Schwann cells by the axon itself, based on the amount of neuregulin I-type III exposed on its membrane. Peripheral axons are more important in determining the final myelination fate than central axons, and the implications for this difference in Schwann cells and oligodendrocytes are discussed. Interestingly, this choice is reversible during pathology, accounting for the remarkable plasticity of Schwann cells, and contributing to the regenerative potential of the peripheral nervous system. Radial sorting is the process by which Schwann cells choose larger axons to myelinate during development. This crucial morphogenetic step is a prerequisite for myelination and for differentiation of Remak fibers, and is arrested in human diseases due to mutations in genes coding for extracellular matrix and linkage molecules. In this review we will summarize progresses made in the last years by a flurry of reverse genetic experiments in mice and fish. This work revealed novel molecules that control radial sorting, and contributed unexpected ideas to our understanding of the cellular and molecular mechanisms that control radial sorting of axons.

  14. Chromosome analysis and sorting using flow cytometry.

    Science.gov (United States)

    Doležel, Jaroslav; Kubaláková, Marie; Cíhalíková, Jarmila; Suchánková, Pavla; Simková, Hana

    2011-01-01

    Chromosome analysis and sorting using flow cytometry (flow cytogenetics) is an attractive tool for fractionating plant genomes to small parts. The reduction of complexity greatly simplifies genetics and genomics in plant species with large genomes. However, as flow cytometry requires liquid suspensions of particles, the lack of suitable protocols for preparation of solutions of intact chromosomes delayed the application of flow cytogenetics in plants. This chapter outlines a high-yielding procedure for preparation of solutions of intact mitotic chromosomes from root tips of young seedlings and for their analysis using flow cytometry and sorting. Root tips accumulated at metaphase are mildly fixed with formaldehyde, and solutions of intact chromosomes are prepared by mechanical homogenization. The advantages of the present approach include the use of seedlings, which are easy to handle, and the karyological stability of root meristems, which can be induced to high degree of metaphase synchrony. Chromosomes isolated according to this protocol have well-preserved morphology, withstand shearing forces during sorting, and their DNA is intact and suitable for a range of applications.

  15. Generalized sorting profile of alluvial fans

    Science.gov (United States)

    Miller, Kimberly Litwin; Reitz, Meredith D.; Jerolmack, Douglas J.

    2014-10-01

    Alluvial rivers often exhibit self-similar gravel size distributions and abrupt gravel-sand transitions. Experiments suggest that these sorting patterns are established rapidly, but how—and how fast—this convergence occurs in the field is unknown. We examine the establishment of downstream sorting patterns in a kilometer-scale alluvial fan. The sharp transition from canyon to unconfined, channelized fan provides a well-defined boundary condition. The channel changes from deep and entrenched at the fan apex to shallow and depositional over a short distance, exhibiting nonequilibrium behavior. The resulting gravel-fining profile is not self-similar; the particle size distribution narrows until approximate equal mobility is achieved. Downfan, the gravel-sand transition appears to exhibit a self-similar form; field and laboratory data collapse when downstream distance is normalized by the location of the transition. Results suggest a generalized sorting profile for alluvial fans as a consequence of the threshold of motion and nonequilibrium channels.

  16. Categorizing Variations of Student-Implemented Sorting Algorithms

    Science.gov (United States)

    Taherkhani, Ahmad; Korhonen, Ari; Malmi, Lauri

    2012-01-01

    In this study, we examined freshmen students' sorting algorithm implementations in data structures and algorithms' course in two phases: at the beginning of the course before the students received any instruction on sorting algorithms, and after taking a lecture on sorting algorithms. The analysis revealed that many students have insufficient…

  17. Gender Sorting across K-12 Schools in the United States

    Science.gov (United States)

    Long, Mark C.; Conger, Dylan

    2013-01-01

    This article documents evidence of nonrandom gender sorting across K-12 schools in the United States. The sorting exists among coed schools and at all grade levels, and it is highest in the secondary school grades. We observe some gender sorting across school sectors and types: for instance, males are slightly underrepresented in private schools…

  18. Automatic Color Sorting of Hardwood Edge-Glued Panel Parts

    Science.gov (United States)

    D. Earl Kline; Richard Conners; Qiang Lu; Philip A. Araman

    1997-01-01

    This paper describes an automatic color sorting system for red oak edge-glued panel parts. The color sorting system simultaneously examines both faces of a panel part and then determines which face has the "best" color, and sorts the part into one of a number of color classes at plant production speeds. Initial test results show that the system generated over...

  19. Categorizing Variations of Student-Implemented Sorting Algorithms

    Science.gov (United States)

    Taherkhani, Ahmad; Korhonen, Ari; Malmi, Lauri

    2012-01-01

    In this study, we examined freshmen students' sorting algorithm implementations in data structures and algorithms' course in two phases: at the beginning of the course before the students received any instruction on sorting algorithms, and after taking a lecture on sorting algorithms. The analysis revealed that many students have insufficient…

  20. PI(3,5)P2 controls endosomal branched actin dynamics by regulating cortactin-actin interactions.

    Science.gov (United States)

    Hong, Nan Hyung; Qi, Aidong; Weaver, Alissa M

    2015-08-31

    Branched actin critically contributes to membrane trafficking by regulating membrane curvature, dynamics, fission, and transport. However, how actin dynamics are controlled at membranes is poorly understood. Here, we identify the branched actin regulator cortactin as a direct binding partner of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) and demonstrate that their interaction promotes turnover of late endosomal actin. In vitro biochemical studies indicated that cortactin binds PI(3,5)P2 via its actin filament-binding region. Furthermore, PI(3,5)P2 competed with actin filaments for binding to cortactin, thereby antagonizing cortactin activity. These findings suggest that PI(3,5)P2 formation on endosomes may remove cortactin from endosome-associated branched actin. Indeed, inhibition of PI(3,5)P2 production led to cortactin accumulation and actin stabilization on Rab7(+) endosomes. Conversely, inhibition of Arp2/3 complex activity greatly reduced cortactin localization to late endosomes. Knockdown of cortactin reversed PI(3,5)P2-inhibitor-induced actin accumulation and stabilization on endosomes. These data suggest a model in which PI(3,5)P2 binding removes cortactin from late endosomal branched actin networks and thereby promotes net actin turnover.

  1. Infection of XC cells by MLVs and Ebola virus is endosome-dependent but acidification-independent.

    Science.gov (United States)

    Kamiyama, Haruka; Kakoki, Katsura; Yoshii, Hiroaki; Iwao, Masatomo; Igawa, Tsukasa; Sakai, Hideki; Hayashi, Hideki; Matsuyama, Toshifumi; Yamamoto, Naoki; Kubo, Yoshinao

    2011-01-01

    Inhibitors of endosome acidification or cathepsin proteases attenuated infections mediated by envelope proteins of xenotropic murine leukemia virus-related virus (XMRV) and Ebola virus, as well as ecotropic, amphotropic, polytropic, and xenotropic murine leukemia viruses (MLVs), indicating that infections by these viruses occur through acidic endosomes and require cathepsin proteases in the susceptible cells such as TE671 cells. However, as previously shown, the endosome acidification inhibitors did not inhibit these viral infections in XC cells. It is generally accepted that the ecotropic MLV infection in XC cells occurs at the plasma membrane. Because cathepsin proteases are activated by low pH in acidic endosomes, the acidification inhibitors may inhibit the viral infections by suppressing cathepsin protease activation. The acidification inhibitors attenuated the activities of cathepsin proteases B and L in TE671 cells, but not in XC cells. Processing of cathepsin protease L was suppressed by the acidification inhibitor in NIH3T3 cells, but again not in XC cells. These results indicate that cathepsin proteases are activated without endosome acidification in XC cells. Treatment with an endocytosis inhibitor or knockdown of dynamin 2 expression by siRNAs suppressed MLV infections in all examined cells including XC cells. Furthermore, endosomal cathepsin proteases were required for these viral infections in XC cells as other susceptible cells. These results suggest that infections of XC cells by the MLVs and Ebola virus occur through endosomes and pH-independent cathepsin activation induces pH-independent infection in XC cells.

  2. Coordination Capacity

    CERN Document Server

    Cuff, Paul; Cover, Thomas

    2009-01-01

    We develop elements of a theory of cooperation and coordination in networks. Rather than considering a communication network as a means of distributing information, or of reconstructing random processes at remote nodes, we ask what dependence can be established among the nodes given the communication constraints. Specifically, in a network with communication rates between the nodes, we ask what is the set of all achievable joint distributions p(x1, ..., xm) of actions at the nodes on the network. Several networks are solved, including arbitrarily large cascade networks. Distributed cooperation can be the solution to many problems such as distributed games, distributed control, and establishing mutual information bounds on the influence of one part of a physical system on another.

  3. The late endosome/lysosome-anchored p18-mTORC1 pathway controls terminal maturation of lysosomes

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, Yusuke; Nada, Shigeyuki; Mori, Shunsuke; Soma-Nagae, Taeko; Oneyama, Chitose [Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871 (Japan); Okada, Masato, E-mail: okadam@biken.osaka-u.ac.jp [Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871 (Japan)

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer p18 is a membrane adaptor that anchors mTORC1 to late endosomes/lysosomes. Black-Right-Pointing-Pointer We examine the role of the p18-mTORC1 pathway in lysosome biogenesis. Black-Right-Pointing-Pointer The loss of p18 causes accumulation of intact late endosomes by arresting lysosome maturation. Black-Right-Pointing-Pointer Inhibition of mTORC1 activity with rapamycin phenocopies the defects of p18 loss. Black-Right-Pointing-Pointer The p18-mTORC1 pathway plays crucial roles in the terminal maturation of lysosomes. -- Abstract: The late endosome/lysosome membrane adaptor p18 (or LAMTOR1) serves as an anchor for the mammalian target of rapamycin complex 1 (mTORC1) and is required for its activation on lysosomes. The loss of p18 causes severe defects in cell growth as well as endosome dynamics, including membrane protein transport and lysosome biogenesis. However, the mechanisms underlying these effects on lysosome biogenesis remain unknown. Here, we show that the p18-mTORC1 pathway is crucial for terminal maturation of lysosomes. The loss of p18 causes aberrant intracellular distribution and abnormal sizes of late endosomes/lysosomes and an accumulation of late endosome specific components, including Rab7, RagC, and LAMP1; this suggests that intact late endosomes accumulate in the absence of p18. These defects are phenocopied by inhibiting mTORC1 activity with rapamycin. Loss of p18 also suppresses the integration of late endosomes and lysosomes, resulting in the defective degradation of tracer proteins. These results suggest that the p18-mTORC1 pathway plays crucial roles in the late stages of lysosomal maturation, potentially in late endosome-lysosome fusion, which is required for processing of various macromolecules.

  4. A lipid receptor sorts polyomavirus from the endolysosome to the endoplasmic reticulum to cause infection.

    Directory of Open Access Journals (Sweden)

    Mengding Qian

    2009-06-01

    Full Text Available The mechanisms by which receptors guide intracellular virus transport are poorly characterized. The murine polyomavirus (Py binds to the lipid receptor ganglioside GD1a and traffics to the endoplasmic reticulum (ER where it enters the cytosol and then the nucleus to initiate infection. How Py reaches the ER is unclear. We show that Py is transported initially to the endolysosome where the low pH imparts a conformational change that enhances its subsequent ER-to-cytosol membrane penetration. GD1a stimulates not viral binding or entry, but rather sorting of Py from late endosomes and/or lysosomes to the ER, suggesting that GD1a binding is responsible for ER targeting. Consistent with this, an artificial particle coated with a GD1a antibody is transported to the ER. Our results provide a rationale for transport of Py through the endolysosome, demonstrate a novel endolysosome-to-ER transport pathway that is regulated by a lipid, and implicate ganglioside binding as a general ER targeting mechanism.

  5. Cache-Aware and Cache-Oblivious Adaptive Sorting

    DEFF Research Database (Denmark)

    Brodal, Gerth Stølting; Fagerberg, Rolf; Moruz, Gabriel

    2005-01-01

    Two new adaptive sorting algorithms are introduced which perform an optimal number of comparisons with respect to the number of inversions in the input. The first algorithm is based on a new linear time reduction to (non-adaptive) sorting. The second algorithm is based on a new division protocol ...... for the GenericSort algorithm by Estivill-Castro and Wood. From both algorithms we derive I/O-optimal cache-aware and cache-oblivious adaptive sorting algorithms. These are the first I/O-optimal adaptive sorting algorithms....

  6. Nanoconjugation prolongs endosomal signaling of the epidermal growth factor receptor and enhances apoptosis

    Science.gov (United States)

    Wu, L.; Xu, F.; Reinhard, B. M.

    2016-07-01

    It is becoming increasingly clear that intracellular signaling can be subject to strict spatial control. As the covalent attachment of a signaling ligand to a nanoparticle (NP) impacts ligand-receptor binding, uptake, and trafficking, nanoconjugation provides new opportunities for manipulating intracellular signaling in a controlled fashion. To establish the effect of nanoconjugation on epidermal growth factor (EGF) mediated signaling, we investigate here the intracellular fate of nanoconjugated EGF (NP-EGF) and its bound receptor (EGFR) by quantitative correlated darkfield/fluorescence microscopy and density-based endosomal fractionation. We demonstrate that nanoconjugation prolongs the dwell time of phosphorylated receptors in the early endosomes and that the retention of activated EGFR in the early endosomes is accompanied by an EGF mediated apoptosis at effective concentrations that do not induce apoptosis in the case of free EGF. Overall, these findings indicate nanoconjugation as a rational strategy for modifying signaling that acts by modulating the temporo-spatial distribution of the activated EGF-EGFR ligand-receptor complex.It is becoming increasingly clear that intracellular signaling can be subject to strict spatial control. As the covalent attachment of a signaling ligand to a nanoparticle (NP) impacts ligand-receptor binding, uptake, and trafficking, nanoconjugation provides new opportunities for manipulating intracellular signaling in a controlled fashion. To establish the effect of nanoconjugation on epidermal growth factor (EGF) mediated signaling, we investigate here the intracellular fate of nanoconjugated EGF (NP-EGF) and its bound receptor (EGFR) by quantitative correlated darkfield/fluorescence microscopy and density-based endosomal fractionation. We demonstrate that nanoconjugation prolongs the dwell time of phosphorylated receptors in the early endosomes and that the retention of activated EGFR in the early endosomes is accompanied by an EGF

  7. α-Taxilin interacts with sorting nexin 4 and participates in the recycling pathway of transferrin receptor.

    Directory of Open Access Journals (Sweden)

    Hiroshi Sakane

    Full Text Available Membrane traffic plays a crucial role in delivering proteins and lipids to their intracellular destinations. We previously identified α-taxilin as a binding partner of the syntaxin family, which is involved in intracellular vesicle traffic. α-Taxilin is overexpressed in tumor tissues and interacts with polymerized tubulin, but the precise function of α-taxilin remains unclear. Receptor proteins on the plasma membrane are internalized, delivered to early endosomes and then either sorted to the lysosome for degradation or recycled back to the plasma membrane. In this study, we found that knockdown of α-taxilin induced the lysosomal degradation of transferrin receptor (TfnR, a well-known receptor which is generally recycled back to the plasma membrane after internalization, and impeded the recycling of transferrin. α-Taxilin was immunoprecipitated with sorting nexin 4 (SNX4, which is involved in the recycling of TfnR. Furthermore, knockdown of α-taxilin decreased the number and length of SNX4-positive tubular structures. We report for the first time that α-taxilin interacts with SNX4 and plays a role in the recycling pathway of TfnR.

  8. High Boron-induced Ubiquitination Regulates Vacuolar Sorting of the BOR1 Borate Transporter in Arabidopsis thaliana*

    Science.gov (United States)

    Kasai, Koji; Takano, Junpei; Miwa, Kyoko; Toyoda, Atsushi; Fujiwara, Toru

    2011-01-01

    Boron homeostasis is important for plants, as boron is essential but is toxic in excess. Under high boron conditions, the Arabidopsis thaliana borate transporter BOR1 is trafficked from the plasma membrane (PM) to the vacuole via the endocytic pathway for degradation to avoid excess boron transport. Here, we show that boron-induced ubiquitination is required for vacuolar sorting of BOR1. We found that a substitution of lysine 590 with alanine (K590A) in BOR1 blocked degradation. BOR1 was mono- or diubiquitinated within several minutes after applying a high concentration of boron, whereas the K590A mutant was not. The K590A mutation abolished vacuolar transport of BOR1 but did not apparently affect polar localization to the inner PM domains. Furthermore, brefeldin A and wortmannin treatment suggested that Lys-590 is required for BOR1 translocation from an early endosomal compartment to multivesicular bodies. Our results show that boron-induced ubiquitination of BOR1 is not required for endocytosis from the PM but is crucial for the sorting of internalized BOR1 to multivesicular bodies for subsequent degradation in vacuoles. PMID:21148314

  9. Starvation-Dependent Regulation of Golgi Quality Control Links the TOR Signaling and Vacuolar Protein Sorting Pathways

    Directory of Open Access Journals (Sweden)

    Niv Dobzinski

    2015-09-01

    Full Text Available Upon amino acid (AA starvation and TOR inactivation, plasma-membrane-localized permeases rapidly undergo ubiquitination and internalization via the vacuolar protein sorting/multivesicular body (VPS-MVB pathway and are degraded in the yeast vacuole. We now show that specific Golgi proteins are also directed to the vacuole under these conditions as part of a Golgi quality-control (GQC process. The degradation of GQC substrates is dependent upon ubiquitination by the defective-for-SREBP-cleavage (DSC complex, which was identified via genetic screening and includes the Tul1 E3 ligase. Using a model GQC substrate, GFP-tagged Yif1, we show that vacuolar targeting necessitates upregulation of the VPS pathway via proteasome-mediated degradation of the initial endosomal sorting complex required for transport, ESCRT-0, but not downstream ESCRT components. Thus, early cellular responses to starvation include the targeting of specific Golgi proteins for degradation, a phenomenon reminiscent of the inactivation of BTN1, the yeast Batten disease gene ortholog.

  10. Stochastic Models of Vesicular Sorting in Cellular Organelles

    CERN Document Server

    Vagne, Quentin

    2016-01-01

    The proper sorting of membrane components by regulated exchange between cellular organelles is crucial to intra-cellular organization. This process relies on the budding and fusion of transport vesicles, and should be strongly influenced by stochastic fluctuations considering the relatively small size of many organelles. We identify the perfect sorting of two membrane components initially mixed in a single compartment as a first passage process, and we show that the mean sorting time exhibits two distinct regimes as a function of the ratio of vesicle fusion to budding rates. Low ratio values leads to fast sorting, but results in a broad size distribution of sorted compartments dominated by small entities. High ratio values result in two well defined sorted compartments but is exponentially slow. Our results suggests an optimal balance between vesicle budding and fusion for the rapid and efficient sorting of membrane components, and highlight the importance of stochastic effects for the steady-state organizati...

  11. RUN COORDINATION

    CERN Multimedia

    C. Delaere

    2013-01-01

    Since the LHC ceased operations in February, a lot has been going on at Point 5, and Run Coordination continues to monitor closely the advance of maintenance and upgrade activities. In the last months, the Pixel detector was extracted and is now stored in the pixel lab in SX5; the beam pipe has been removed and ME1/1 removal has started. We regained access to the vactank and some work on the RBX of HB has started. Since mid-June, electricity and cooling are back in S1 and S2, allowing us to turn equipment back on, at least during the day. 24/7 shifts are not foreseen in the next weeks, and safety tours are mandatory to keep equipment on overnight, but re-commissioning activities are slowly being resumed. Given the (slight) delays accumulated in LS1, it was decided to merge the two global runs initially foreseen into a single exercise during the week of 4 November 2013. The aim of the global run is to check that we can run (parts of) CMS after several months switched off, with the new VME PCs installed, th...

  12. RUN COORDINATION

    CERN Multimedia

    Christophe Delaere

    2013-01-01

    The focus of Run Coordination during LS1 is to monitor closely the advance of maintenance and upgrade activities, to smooth interactions between subsystems and to ensure that all are ready in time to resume operations in 2015 with a fully calibrated and understood detector. After electricity and cooling were restored to all equipment, at about the time of the last CMS week, recommissioning activities were resumed for all subsystems. On 7 October, DCS shifts began 24/7 to allow subsystems to remain on to facilitate operations. That culminated with the Global Run in November (GriN), which   took place as scheduled during the week of 4 November. The GriN has been the first centrally managed operation since the beginning of LS1, and involved all subdetectors but the Pixel Tracker presently in a lab upstairs. All nights were therefore dedicated to long stable runs with as many subdetectors as possible. Among the many achievements in that week, three items may be highlighted. First, the Strip...

  13. Intracellular release of fluorescein anion from layered double hydroxide nanoparticles indicating endosomal escape

    Science.gov (United States)

    Tanaka, M.; Aisawa, S.; Hidetoshi, H.; Narita, E.; Dong, Q.; Yin, S.; Sato, T.

    2013-12-01

    In recent years, layered double hydroxide (LDH) has been attempted to be applied to a molecular container due to their anion exchange ability, low cytotoxicity and good biocompatibility. In this paper, we investigated the intracellular behaviour of LDH particles in mammalian cells after internalization. Nanoparticles of fluorescein (Fluo) intercalated LDH, Fluo/LDH, were prepared by the coprecipitation followed by subsequent hydrothermal treatment. As-prepared Fluo/LDH particles have the LDH structure and morphology of hexagonal sheet of 100 nm on the average. In addition, Fluo/LDH also exhibited high green fluorescence and low cytotoxicity. By a confocal laser scanning microscopy, the dim green fluorescence was observed throughout cells, including the nucleus. This result indicated that Fluo/LDH released guest anion (Fluo) from LDH structure inside cells. Furthermore, because the fluorescence was observed throughout the cell, Fluo was not retained within endosome structure, i.e., Fluo/LDH was dissolved to release Fluo from endosome.

  14. Endosomes derived from clathrin-independent endocytosis serve as precursors for endothelial lumen formation.

    Directory of Open Access Journals (Sweden)

    Natalie Porat-Shliom

    Full Text Available Clathrin-independent endocytosis (CIE is a form of bulk plasma membrane (PM endocytosis that allows cells to sample and evaluate PM composition. Once in endosomes, the internalized proteins and lipids can be recycled back to the PM or delivered to lysosomes for degradation. Endosomes arising from CIE contain lipid and signaling molecules suggesting that they might be involved in important biological processes. During vasculogenesis, new blood vessels are formed from precursor cells in a process involving internalization and accumulation of endocytic vesicles. Here, we found that CIE has a role in endothelial lumen formation. Specifically, we found that human vascular endothelial cells (HUVECs utilize CIE for internalization of distinct cargo molecules and that in three-dimensional cultures CIE membranes are delivered to the newly formed lumen.

  15. Involvement of the endosomal-lysosomal system correlates with regional pathology in Creutzfeldt-Jakob disease

    DEFF Research Database (Denmark)

    Kovács, Gábor G; Gelpi, Ellen; Ströbel, Thomas

    2007-01-01

    The endosomal-lysosomal system (ELS) has been suggested to play a role in the pathogenesis of prion diseases. The purpose of this study was to examine how experimental observations can be translated to human neuropathology and whether alterations of the ELS relate to neuropathologic changes....... Combined with stereologic techniques, we examined components of the ELS in human sporadic Creutzfeldt-Jakob disease brains. We immunostained for the early endosomal marker Rab5 and lysosomal enzymes cathepsin D and B. We determined neuron-specific changes in their expression and correlated......-immunoreactive lysosomes. The intraneuronal distribution of cathepsin D and B diverges between Purkinje cells and frontal cortical neurons in sporadic Creutzfeldt-Jakob disease brains. We demonstrated focal intra- and perineuronal colocalization of cathepsin D and PrP. Our results indicate that effects in the ELS...

  16. Endosome-based protein trafficking and Ca2+ homeostasis in the heart

    Directory of Open Access Journals (Sweden)

    Jerry eCurran

    2015-02-01

    Full Text Available 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 and cardiovascular system. In the heart, these systems are essential for the regulation of cardiac calcium, both at the level of the plasma membrane, but also at local domains of the endoplasmic reticulum, sarcoplasmic reticulum, mitochondria, nucleus, and nuclear envelope. One intracellular pathway often overlooked in relation to cardiovascular calcium 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. The vast majority of what is known has been derived from heterologous cell systems. However, recently, more appropriate cell and animal models been developed that have allowed researchers to begin to understand how this system functions within the intact physiological environment. All excitable cells, including cardiomyocytes, depend on the proper expression and organization of multiple ion channels, pumps, exchangers, and transporters within the plasma membrane. As the endosomal system acts to regulate the expression and localization of membrane proteins, understanding the in vivo function of this system in the heart is important. This review will focus on endosome-based protein trafficking in the heart in both health and disease. Special emphasis will be given to the role played by the family of endocytic regulatory proteins, C-terminal Eps15 homology domain -containing proteins (EHDs, as recent data demonstrates that this family of proteins is essential for the proper trafficking and localization and of key proteins involved in excitation-contraction coupling.

  17. Role of recycling endosomes and lysosomes in dynein-dependent entry of canine parvovirus.

    Science.gov (United States)

    Suikkanen, Sanna; Sääjärvi, Katja; Hirsimäki, Jonna; Välilehto, Outi; Reunanen, Hilkka; Vihinen-Ranta, Maija; Vuento, Matti

    2002-05-01

    Canine parvovirus (CPV) is a nonenveloped virus with a 5-kb single-stranded DNA genome. Lysosomotropic agents and low temperature are known to prevent CPV infection, indicating that the virus enters its host cells by endocytosis and requires an acidic intracellular compartment for penetration into the cytoplasm. After escape from the endocytotic vesicles, CPV is transported to the nucleus for replication. In the present study the intracellular entry pathway of the canine parvovirus in NLFK (Nordisk Laboratory feline kidney) cells was studied. After clustering in clathrin-coated pits and being taken up in coated vesicles, CPV colocalized with coendocytosed transferrin in endosomes resembling recycling endosomes. Later, CPV was found to enter, via late endosomes, a perinuclear vesicular compartment, where it colocalized with lysosomal markers. There was no indication of CPV entry into the trans-Golgi or the endoplasmic reticulum. Similar results were obtained both with full and with empty capsids. The data thus suggest that CPV or its DNA was released from the lysosomal compartment to the cytoplasm to be then transported to the nucleus. Electron microscopy analysis revealed endosomal vesicles containing CPV to be associated with microtubules. In the presence of nocodazole, a microtubule-disrupting drug, CPV entry was blocked and the virus was found in peripheral vesicles. Thus, some step(s) of the entry process were dependent on microtubules. Microinjection of antibodies to dynein caused CPV to remain in pericellular vesicles. This suggests an important role for the motor protein dynein in transporting vesicles containing CPV along the microtubule network.

  18. The p25 Subunit of the Dynactin Complex is Required for Dynein-Early Endosome Interaction

    Science.gov (United States)

    2011-01-01

    dynein–early endosome interaction in the filamen- tous fungus Aspergillus nidulans. In filamentous fungi , dynein and its regulators are important for...backbone of the dynactin complex, and its loss leads to a disruption of the whole complex. In Drosoph- ila and in filamentous fungi such as N. crassa...how the motor is targeted to these cargoes is still a topic under investigation. In filamentous fungi and higher eukaryotic cells such as neurons

  19. Protein trafficking through the endosomal system prepares intracellular parasites for a home invasion.

    OpenAIRE

    Tomavo, S; Slomianny, C; Meissner, M.; Carruthers, V B

    2013-01-01

    Toxoplasma (toxoplasmosis) and Plasmodium (malaria) use unique secretory organelles for migration, cell invasion, manipulation of host cell functions, and cell egress. In particular, the apical secretory micronemes and rhoptries of apicomplexan parasites are essential for successful host infection. New findings reveal that the contents of these organelles, which are transported through the endoplasmic reticulum (ER) and Golgi, also require the parasite endosome-like system to access their res...

  20. Imaging single retrovirus entry through alternative receptor isoforms and intermediates of virus-endosome fusion.

    Directory of Open Access Journals (Sweden)

    Naveen K Jha

    Full Text Available A large group of viruses rely on low pH to activate their fusion proteins that merge the viral envelope with an endosomal membrane, releasing the viral nucleocapsid. A critical barrier to understanding these events has been the lack of approaches to study virus-cell membrane fusion within acidic endosomes, the natural sites of virus nucleocapsid capsid entry into the cytosol. Here we have investigated these events using the highly tractable subgroup A avian sarcoma and leukosis virus envelope glycoprotein (EnvA-TVA receptor system. Through labeling EnvA pseudotyped viruses with a pH-sensitive fluorescent marker, we imaged their entry into mildly acidic compartments. We found that cells expressing the transmembrane receptor (TVA950 internalized the virus much faster than those expressing the GPI-anchored receptor isoform (TVA800. Surprisingly, TVA800 did not accelerate virus uptake compared to cells lacking the receptor. Subsequent steps of virus entry were visualized by incorporating a small viral content marker that was released into the cytosol as a result of fusion. EnvA-dependent fusion with TVA800-expressing cells occurred shortly after endocytosis and delivery into acidic endosomes, whereas fusion of viruses internalized through TVA950 was delayed. In the latter case, a relatively stable hemifusion-like intermediate preceded the fusion pore opening. The apparent size and stability of nascent fusion pores depended on the TVA isoforms and their expression levels, with TVA950 supporting more robust pores and a higher efficiency of infection compared to TVA800. These results demonstrate that surface receptor density and the intracellular trafficking pathway used are important determinants of efficient EnvA-mediated membrane fusion, and suggest that early fusion intermediates play a critical role in establishing low pH-dependent virus entry from within acidic endosomes.

  1. The Endosome-associated Deubiquitinating Enzyme USP8 Regulates BACE1 Enzyme Ubiquitination and Degradation.

    Science.gov (United States)

    Yeates, Eniola Funmilayo Aduke; Tesco, Giuseppina

    2016-07-22

    The β-site amyloid precursor protein-cleaving enzyme (BACE1) is the rate-limiting enzyme in the production of amyloid-β, the toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that that depletion of the trafficking molecule Golgi-localized γ-ear-containing ARF-binding protein 3 (GGA3) results in increased BACE1 levels and activity because of impaired lysosomal degradation. We also determined that GGA3 regulation of BACE1 levels requires its ability to bind ubiquitin. Accordingly, we reported that BACE1 is ubiquitinated at lysine 501 and that lack of ubiquitination at lysine 501 produces BACE1 stabilization. Ubiquitin conjugation is a reversible process mediated by deubiquitinating enzymes. The ubiquitin-specific peptidase 8 (USP8), an endosome-associated deubiquitinating enzyme, regulates the ubiquitination, trafficking, and lysosomal degradation of several plasma membrane proteins. Here, we report that RNAi-mediated depletion of USP8 reduced levels of both ectopically expressed and endogenous BACE1 in H4 human neuroglioma cells. Moreover, USP8 depletion increased BACE1 ubiquitination, promoted BACE1 accumulation in the early endosomes and late endosomes/lysosomes, and decreased levels of BACE1 in the recycling endosomes. We also found that decreased BACE1 protein levels were accompanied by a decrease in BACE1-mediated amyloid precursor protein cleavage and amyloid-β levels. Our findings demonstrate that USP8 plays a key role in the trafficking and degradation of BACE1 by deubiquitinating lysine 501. These studies suggest that therapies able to accelerate BACE1 degradation (e.g. by increasing BACE1 ubiquitination) may represent a potential treatment for Alzheimer disease.

  2. Sorting waste - A question of good will

    CERN Multimedia

    TS Department - FM Group

    2006-01-01

    In order to minimise waste-sorting costs, CERN provides two types of container at the entrance of buildings: a green plastic container for paper/cardboard and a metal container for household-type waste. We regret that recently there has been a significant decrease in the extent to which these types of waste are sorted, for example green containers have been found to hold assorted waste such as cardboard boxes filled with polystyrene, bubble-wrap or even plastic bottles, yoghurt pots, etc. Checks have shown that this 'non-compliant' waste does not come from the rubbish bins emptied by the cleaners but is deposited there directly by inconsiderate users. During the months of October and November alone, for example, only 15% of the waste from the paper/cardboard containers was recycled and the remaining 85% had to be incinerated, which entails a high cost for CERN. You should note that once an item of non-compliant waste is found in a green container its contents are immediately sent as waste to be incinerated ...

  3. Human-powered Sorts and Joins

    CERN Document Server

    Marcus, Adam; Karger, David; Madden, Samuel; Miller, Robert

    2011-01-01

    Crowdsourcing markets like Amazon's Mechanical Turk (MTurk) make it possible to task people with small jobs, such as labeling images or looking up phone numbers, via a programmatic interface. MTurk tasks for processing datasets with humans are currently designed with significant reimplementation of common workflows and ad-hoc selection of parameters such as price to pay per task. We describe how we have integrated crowds into a declarative workflow engine called Qurk to reduce the burden on workflow designers. In this paper, we focus on how to use humans to compare items for sorting and joining data, two of the most common operations in DBMSs. We describe our basic query interface and the user interface of the tasks we post to MTurk. We also propose a number of optimizations, including task batching, replacing pairwise comparisons with numerical ratings, and pre-filtering tables before joining them, which dramatically reduce the overall cost of running sorts and joins on the crowd. In an experiment joining tw...

  4. PACMan to Help Sort Hubble Proposals

    Science.gov (United States)

    Kohler, Susanna

    2017-04-01

    Every year, astronomers submit over a thousand proposals requesting time on the Hubble Space Telescope (HST). Currently, humans must sort through each of these proposals by hand before sending them off for review. Could this burden be shifted to computers?A Problem of VolumeAstronomer Molly Peeples gathered stats on the HST submissions sent in last week for the upcoming HST Cycle 25 (the deadline was Friday night), relative to previous years. This years proposal round broke the record, with over 1200 proposals submitted in total for Cycle 25. [Molly Peeples]Each proposal cycle for HST time attracts on the order of 1100 proposals accounting for far more HST time than is available. The proposals are therefore carefully reviewed by around 150 international members of the astronomy community during a six-month process to select those with the highest scientific merit.Ideally, each proposal will be read by reviewers that have scientific expertise relevant to the proposal topic: if a proposal requests HST time to study star formation, for instance, then the reviewers assigned to it should have research expertise in star formation.How does this matching of proposals to reviewers occur? The current method relies on self-reported categorization of the submitted proposals. This is unreliable, however; proposals are often mis-categorized by submitters due to misunderstanding or ambiguous cases.As a result, the Science Policies Group at the Space Telescope Science Institute (STScI) which oversees the review of HST proposals must go through each of the proposals by hand and re-categorize them. The proposals are then matched to reviewers with self-declared expertise in the same category.With the number of HST proposals on the rise and the expectation that the upcoming James Webb Space Telescope (JWST) will elicit even more proposals for time than Hubble scientists at STScI and NASA are now asking: could the human hours necessary for this task be spared? Could a computer program

  5. Quantum bounds for ordered searching and sorting

    CERN Document Server

    Hoyer, P; Shi, Y; Hoyer, Peter; Neerbek, Jan; Shi, Yaoyun

    2001-01-01

    We consider the quantum complexities of searching an ordered list and sorting an un-ordered list. For searching an ordered list of N elements, we prove a lower bound of \\frac{1}{\\pi}(\\ln(N)-1) on the number of oracle queries that access the list elements. This improves the previously best lower bound of ({1/12}\\log_2(N) - O(1)) due to Ambainis. For sorting N numbers, we prove a lower bound of \\frac{N}{2\\pi}(\\ln(N)-1) on the number of binary comparisons. The previously best lower bound is \\Omega(N). Our proofs are based on a weighted all-pairs inner product argument, and our results generalize to bounded error quantum algorithms. Both results are proven in the so-called quantum black box model, a quantum analogue of classical decision trees. In addition to our lower bound results, we give an exact quantum algorithm for ordered searching using (\\log_3(N) + O(1)) queries, which is roughly 0.631 \\log_2(N). Although our algorithm is worse than that of Farhi, Goldstone, Gutmann and Sipser, which makes 0.526 \\log_2(...

  6. Developing Automated Methods of Waste Sorting

    Energy Technology Data Exchange (ETDEWEB)

    Shurtliff, Rodney Marvin

    2002-08-01

    The U.S. Department of Energy (DOE) analyzed the need complex-wide for remote and automated technologies as they relate to the treatment and disposal of mixed wastes. This analysis revealed that several DOE sites need the capability to open drums containing waste, visually inspect and sort the contents, and finally repackage the containers that are acceptable at a waste disposal facility such as the Waste Isolation Pilot Plant (WIPP) in New Mexico. Conditioning contaminated waste so that it is compatible with the WIPP criteria for storage is an arduous task whether the waste is contact handled (waste having radioactivity levels below 200 mrem/hr) or remote handled. Currently, WIPP non-compliant items are removed from the waste stream manually, at a rate of about one 55-gallon drum per day. Issues relating to contamination-based health hazards as well as repetitive motion health hazards are steering industry towards a more user-friendly, method of conditioning or sorting waste.

  7. Corner Sort for Pareto-Based Many-Objective Optimization.

    Science.gov (United States)

    Wang, Handing; Yao, Xin

    2014-01-01

    Nondominated sorting plays an important role in Pareto-based multiobjective evolutionary algorithms (MOEAs). When faced with many-objective optimization problems multiobjective optimization problems (MOPs) with more than three objectives, the number of comparisons needed in nondominated sorting becomes very large. In view of this, a new corner sort is proposed in this paper. Corner sort first adopts a fast and simple method to obtain a nondominated solution from the corner solutions, and then uses the nondominated solution to ignore the solutions dominated by it to save comparisons. Obtaining the nondominated solutions requires much fewer objective comparisons in corner sort. In order to evaluate its performance, several state-of-the-art nondominated sorts are compared with our corner sort on three kinds of artificial solution sets of MOPs and the solution sets generated from MOEAs on benchmark problems. On one hand, the experiments on artificial solution sets show the performance on the solution sets with different distributions. On the other hand, the experiments on the solution sets generated from MOEAs show the influence that different sorts bring to MOEAs. The results show that corner sort performs well, especially on many-objective optimization problems. Corner sort uses fewer comparisons than others.

  8. In vivo determination of fluctuating forces during endosome trafficking using a combination of active and passive microrheology.

    Science.gov (United States)

    Robert, Damien; Nguyen, Thi-Hanh; Gallet, François; Wilhelm, Claire

    2010-04-06

    Regulation of intracellular trafficking is a central issue in cell biology. The forces acting on intracellular vesicles (endosomes) can be assessed in living cells by using a combination of active and passive microrheology. This dual approach is based on endosome labeling with magnetic nanoparticles. The resulting magnetic endosomes act both as probes that can be manipulated with external magnetic fields to infer the viscoelastic modulus of their surrounding microenvironment, and as biological vehicles that are trafficked along the microtubule network by means of forces generated by molecular motors. The intracellular viscoelastic modulus exhibits power law dependence with frequency, which is microtubule and actin-dependent. The mean square displacements of endosomes do not follow the predictions of the fluctuation-dissipation theorem, which offers evidence for active force generation. Microtubule disruption brings the intracellular medium closer to thermal equilibrium: active forces acting on the endosomes depend on microtubule-associated motors. The power spectra of these active forces, deduced through the use of a generalized Langevin equation, show a power law decrease with frequency and reveal an actin-dependent persistence of the force with time. Experimental spectra have been reproduced by a simple model consisting in a series of force steps power-law distributed in time. This model enlightens the role of the cytoskeleton dependent force exerted on endosomes to perform intracellular trafficking. In this work, the influence of cytoskeleton components and molecular motors on intracellular viscoelasticity and transport is addressed. The use of an original probe, the magnetic endosome, allows retrieving the power spectrum of active forces on these organelles thanks to interrelated active and passive measures. Finally a computational model gives estimates of the force itself and hence of the number of the motors pulling on endosomes.

  9. In vivo determination of fluctuating forces during endosome trafficking using a combination of active and passive microrheology.

    Directory of Open Access Journals (Sweden)

    Damien Robert

    Full Text Available BACKGROUND: Regulation of intracellular trafficking is a central issue in cell biology. The forces acting on intracellular vesicles (endosomes can be assessed in living cells by using a combination of active and passive microrheology. METHODOLOGY/PRINCIPAL FINDINGS: This dual approach is based on endosome labeling with magnetic nanoparticles. The resulting magnetic endosomes act both as probes that can be manipulated with external magnetic fields to infer the viscoelastic modulus of their surrounding microenvironment, and as biological vehicles that are trafficked along the microtubule network by means of forces generated by molecular motors. The intracellular viscoelastic modulus exhibits power law dependence with frequency, which is microtubule and actin-dependent. The mean square displacements of endosomes do not follow the predictions of the fluctuation-dissipation theorem, which offers evidence for active force generation. Microtubule disruption brings the intracellular medium closer to thermal equilibrium: active forces acting on the endosomes depend on microtubule-associated motors. The power spectra of these active forces, deduced through the use of a generalized Langevin equation, show a power law decrease with frequency and reveal an actin-dependent persistence of the force with time. Experimental spectra have been reproduced by a simple model consisting in a series of force steps power-law distributed in time. This model enlightens the role of the cytoskeleton dependent force exerted on endosomes to perform intracellular trafficking. CONCLUSIONS/SIGNIFICANCE: In this work, the influence of cytoskeleton components and molecular motors on intracellular viscoelasticity and transport is addressed. The use of an original probe, the magnetic endosome, allows retrieving the power spectrum of active forces on these organelles thanks to interrelated active and passive measures. Finally a computational model gives estimates of the force itself

  10. Cholesterol Regulates Syntaxin 6 Trafficking at trans-Golgi Network Endosomal Boundaries

    Directory of Open Access Journals (Sweden)

    Meritxell Reverter

    2014-05-01

    Full Text Available Inhibition of cholesterol export from late endosomes causes cellular cholesterol imbalance, including cholesterol depletion in the trans-Golgi network (TGN. Here, using Chinese hamster ovary (CHO Niemann-Pick type C1 (NPC1 mutant cell lines and human NPC1 mutant fibroblasts, we show that altered cholesterol levels at the TGN/endosome boundaries trigger Syntaxin 6 (Stx6 accumulation into VAMP3, transferrin, and Rab11-positive recycling endosomes (REs. This increases Stx6/VAMP3 interaction and interferes with the recycling of αVβ3 and α5β1 integrins and cell migration, possibly in a Stx6-dependent manner. In NPC1 mutant cells, restoration of cholesterol levels in the TGN, but not inhibition of VAMP3, restores the steady-state localization of Stx6 in the TGN. Furthermore, elevation of RE cholesterol is associated with increased amounts of Stx6 in RE. Hence, the fine-tuning of cholesterol levels at the TGN-RE boundaries together with a subset of cholesterol-sensitive SNARE proteins may play a regulatory role in cell migration and invasion.

  11. Human Metapneumovirus Is Capable of Entering Cells by Fusion with Endosomal Membranes.

    Directory of Open Access Journals (Sweden)

    Reagan G Cox

    2015-12-01

    Full Text Available Human metapneumovirus (HMPV, a member of the Paramyxoviridae family, is a leading cause of lower respiratory illness. Although receptor binding is thought to initiate fusion at the plasma membrane for paramyxoviruses, the entry mechanism for HMPV is largely uncharacterized. Here we sought to determine whether HMPV initiates fusion at the plasma membrane or following internalization. To study the HMPV entry process in human bronchial epithelial (BEAS-2B cells, we used fluorescence microscopy, an R18-dequenching fusion assay, and developed a quantitative, fluorescence microscopy assay to follow virus binding, internalization, membrane fusion, and visualize the cellular site of HMPV fusion. We found that HMPV particles are internalized into human bronchial epithelial cells before fusing with endosomes. Using chemical inhibitors and RNA interference, we determined that HMPV particles are internalized via clathrin-mediated endocytosis in a dynamin-dependent manner. HMPV fusion and productive infection are promoted by RGD-binding integrin engagement, internalization, actin polymerization, and dynamin. Further, HMPV fusion is pH-independent, although infection with rare strains is modestly inhibited by RNA interference or chemical inhibition of endosomal acidification. Thus, HMPV can enter via endocytosis, but the viral fusion machinery is not triggered by low pH. Together, our results indicate that HMPV is capable of entering host cells by multiple pathways, including membrane fusion from endosomal compartments.

  12. Augmenting the Efficacy of Immunotoxins and Other Targeted Protein Toxins by Endosomal Escape Enhancers

    Directory of Open Access Journals (Sweden)

    Hendrik Fuchs

    2016-07-01

    Full Text Available The toxic moiety of almost all protein-based targeted toxins must enter the cytosol of the target cell to mediate its fatal effect. Although more than 500 targeted toxins have been investigated in the past decades, no antibody-targeted protein toxin has been approved for tumor therapeutic applications by the authorities to date. Missing efficacy can be attributed in many cases to insufficient endosomal escape and therefore subsequent lysosomal degradation of the endocytosed toxins. To overcome this drawback, many strategies have been described to weaken the membrane integrity of endosomes. This comprises the use of lysosomotropic amines, carboxylic ionophores, calcium channel antagonists, various cell-penetrating peptides of viral, bacterial, plant, animal, human and synthetic origin, other organic molecules and light-induced techniques. Although the efficacy of the targeted toxins was typically augmented in cell culture hundred or thousand fold, in exceptional cases more than million fold, the combination of several substances harbors new problems including additional side effects, loss of target specificity, difficulties to determine the therapeutic window and cell type-dependent variations. This review critically scrutinizes the chances and challenges of endosomal escape enhancers and their potential role in future developments.

  13. Augmenting the Efficacy of Immunotoxins and Other Targeted Protein Toxins by Endosomal Escape Enhancers

    Science.gov (United States)

    Fuchs, Hendrik; Weng, Alexander; Gilabert-Oriol, Roger

    2016-01-01

    The toxic moiety of almost all protein-based targeted toxins must enter the cytosol of the target cell to mediate its fatal effect. Although more than 500 targeted toxins have been investigated in the past decades, no antibody-targeted protein toxin has been approved for tumor therapeutic applications by the authorities to date. Missing efficacy can be attributed in many cases to insufficient endosomal escape and therefore subsequent lysosomal degradation of the endocytosed toxins. To overcome this drawback, many strategies have been described to weaken the membrane integrity of endosomes. This comprises the use of lysosomotropic amines, carboxylic ionophores, calcium channel antagonists, various cell-penetrating peptides of viral, bacterial, plant, animal, human and synthetic origin, other organic molecules and light-induced techniques. Although the efficacy of the targeted toxins was typically augmented in cell culture hundred or thousand fold, in exceptional cases more than million fold, the combination of several substances harbors new problems including additional side effects, loss of target specificity, difficulties to determine the therapeutic window and cell type-dependent variations. This review critically scrutinizes the chances and challenges of endosomal escape enhancers and their potential role in future developments. PMID:27376327

  14. The nuclear protein Waharan is required for endosomal-lysosomal trafficking in Drosophila.

    Science.gov (United States)

    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.

  15. Vertical sorting and the morphodynamics of bed form-dominated rivers: a sorting evolution model

    NARCIS (Netherlands)

    Blom, Astrid; Ribberink, Jan S.; Parker, Gary

    2008-01-01

    Existing sediment continuity models for nonuniform sediment suffer from a number of shortcomings, as they fail to describe vertical sorting fluxes other than through net aggradation or degradation of the bed and are based on a discrete representation of the bed material interacting with the flow. We

  16. First-Generation Antipsychotic Haloperidol Alters the Functionality of the Late Endosomal/Lysosomal Compartment in Vitro

    Directory of Open Access Journals (Sweden)

    Alberto Canfrán-Duque

    2016-03-01

    Full Text Available First- and second-generation antipsychotics (FGAs and SGAs, respectively, have the ability to inhibit cholesterol biosynthesis and also to interrupt the intracellular cholesterol trafficking, interfering with low-density lipoprotein (LDL-derived cholesterol egress from late endosomes/lysosomes. In the present work, we examined the effects of FGA haloperidol on the functionality of late endosomes/lysosomes in vitro. In HepG2 hepatocarcinoma cells incubated in the presence of 1,1′-dioctadecyl-3,3,3,3′-tetramethylindocarbocyanineperchlorate (DiI-LDL, treatment with haloperidol caused the enlargement of organelles positive for late endosome markers lysosome-associated membrane protein 2 (LAMP-2 and LBPA (lysobisphosphatidic acid, which also showed increased content of both free-cholesterol and DiI derived from LDL. This indicates the accumulation of LDL-lipids in the late endosomal/lysosomal compartment caused by haloperidol. In contrast, LDL traffic through early endosomes and the Golgi apparatus appeared to be unaffected by the antipsychotic as the distribution of both early endosome antigen 1 (EEA1 and coatomer subunit β (β-COP were not perturbed. Notably, treatment with haloperidol significantly increased the lysosomal pH and decreased the activities of lysosomal protease and β-d-galactosidase in a dose-dependent manner. We conclude that the alkalinization of the lysosomes’ internal milieu induced by haloperidol affects lysosomal functionality.

  17. Ferlins Show Tissue-Specific Expression and Segregate as Plasma Membrane/Late Endosomal or Trans-Golgi/Recycling Ferlins.

    Science.gov (United States)

    Redpath, Gregory M I; Sophocleous, Reece A; Turnbull, Lynne; Whitchurch, Cynthia B; Cooper, Sandra T

    2016-03-01

    Ferlins are a family of transmembrane-anchored vesicle fusion proteins uniquely characterized by 5-7 tandem cytoplasmic C2 domains, Ca(2+)-regulated phospholipid-binding domains that regulate vesicle fusion in the synaptotagmin family. In humans, dysferlin mutations cause limb-girdle muscular dystrophy type 2B (LGMD2B) due to defective Ca(2+)-dependent, vesicle-mediated membrane repair and otoferlin mutations cause non-syndromic deafness due to defective Ca(2+)-triggered auditory neurotransmission. In this study, we describe the tissue-specific expression, subcellular localization and endocytic trafficking of the ferlin family. Studies of endosomal transit together with 3D-structured illumination microscopy reveals dysferlin and myoferlin are abundantly expressed at the PM and cycle to Rab7-positive late endosomes, supporting potential roles in the late-endosomal pathway. In contrast, Fer1L6 shows concentrated localization to a specific compartment of the trans-Golgi/recycling endosome, cycling rapidly between this compartment and the PM via Rab11 recycling endosomes. Otoferlin also shows trans-Golgi to PM cycling, with very low levels of PM otoferlin suggesting either brief PM residence, or rare incorporation of otoferlin molecules into the PM. Thus, type-I and type-II ferlins segregate as PM/late-endosomal or trans-Golgi/recycling ferlins, consistent with different ferlins mediating vesicle fusion events in specific subcellular locations.

  18. The Endosome Localized Arf-GAP AGAP1 Modulates Dendritic Spine Morphology Downstream of the Neurodevelopmental Disorder Factor Dysbindin

    Science.gov (United States)

    Arnold, Miranda; Cross, Rebecca; Singleton, Kaela S.; Zlatic, Stephanie; Chapleau, Christopher; Mullin, Ariana P.; Rolle, Isaiah; Moore, Carlene C.; Theibert, Anne; Pozzo-Miller, Lucas; Faundez, Victor; Larimore, Jennifer

    2016-01-01

    AGAP1 is an Arf1 GTPase activating protein that interacts with the vesicle-associated protein complexes adaptor protein 3 (AP-3) and Biogenesis of Lysosome Related Organelles Complex-1 (BLOC-1). Overexpression of AGAP1 in non-neuronal cells results in an accumulation of endosomal cargoes, which suggests a role in endosome-dependent traffic. In addition, AGAP1 is a candidate susceptibility gene for two neurodevelopmental disorders, autism spectrum disorder (ASD) and schizophrenia (SZ); yet its localization and function in neurons have not been described. Here, we describe that AGAP1 localizes to axons, dendrites, dendritic spines and synapses, colocalizing preferentially with markers of early and recycling endosomes. Functional studies reveal overexpression and down-regulation of AGAP1 affects both neuronal endosomal trafficking and dendritic spine morphology, supporting a role for AGAP1 in the recycling endosomal trafficking involved in their morphogenesis. Finally, we determined the sensitivity of AGAP1 expression to mutations in the DTNBP1 gene, which is associated with neurodevelopmental disorder, and found that AGAP1 mRNA and protein levels are selectively reduced in the null allele of the mouse ortholog of DTNBP1. We postulate that endosomal trafficking contributes to the pathogenesis of neurodevelopmental disorders affecting dendritic spine morphology, and thus excitatory synapse structure and function. PMID:27713690

  19. First-Generation Antipsychotic Haloperidol Alters the Functionality of the Late Endosomal/Lysosomal Compartment in Vitro.

    Science.gov (United States)

    Canfrán-Duque, Alberto; Barrio, Luis C; Lerma, Milagros; de la Peña, Gema; Serna, Jorge; Pastor, Oscar; Lasunción, Miguel A; Busto, Rebeca

    2016-03-18

    First- and second-generation antipsychotics (FGAs and SGAs, respectively), have the ability to inhibit cholesterol biosynthesis and also to interrupt the intracellular cholesterol trafficking, interfering with low-density lipoprotein (LDL)-derived cholesterol egress from late endosomes/lysosomes. In the present work, we examined the effects of FGA haloperidol on the functionality of late endosomes/lysosomes in vitro. In HepG2 hepatocarcinoma cells incubated in the presence of 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanineperchlorate (DiI)-LDL, treatment with haloperidol caused the enlargement of organelles positive for late endosome markers lysosome-associated membrane protein 2 (LAMP-2) and LBPA (lysobisphosphatidic acid), which also showed increased content of both free-cholesterol and DiI derived from LDL. This indicates the accumulation of LDL-lipids in the late endosomal/lysosomal compartment caused by haloperidol. In contrast, LDL traffic through early endosomes and the Golgi apparatus appeared to be unaffected by the antipsychotic as the distribution of both early endosome antigen 1 (EEA1) and coatomer subunit β (β-COP) were not perturbed. Notably, treatment with haloperidol significantly increased the lysosomal pH and decreased the activities of lysosomal protease and β-d-galactosidase in a dose-dependent manner. We conclude that the alkalinization of the lysosomes' internal milieu induced by haloperidol affects lysosomal functionality.

  20. The Endosome Localized Arf-GAP AGAP1 Modulates Dendritic Spine Morphology Downstream of the Neurodevelopmental Disorder Factor Dysbindin

    Directory of Open Access Journals (Sweden)

    Miranda Arnold

    2016-09-01

    Full Text Available AGAP1 is an Arf1 GTPase activating protein that interacts with the vesicle-associated protein complexes adaptor protein 3 (AP-3 and Biogenesis of Lysosome Related Organelles Complex-1 (BLOC-1. Overexpression of AGAP1 in non-neuronal cells results in an accumulation of endosomal cargoes, which suggests a role in endosome-dependent traffic. In addition, AGAP1 is a candidate susceptibility gene for two neurodevelopmental disorders, autism spectrum disorder (ASD and schizophrenia (SZ; yet its localization and function in neurons have not been described. Here, we describe that AGAP1 localizes to axons, dendrites, dendritic spines, and synapses, colocalizing preferentially with markers of early and recycling endosomes. Functional studies reveal overexpression and down-regulation of AGAP1 affects both neuronal endosomal trafficking and dendritic spine morphology, supporting a role for AGAP1 in the recycling endosomal trafficking involved in their morphogenesis. Finally, we determined the sensitivity of AGAP1 expression to mutations in the DTNBP1 gene, which is associated with neurodevelopmental disorder, and found that AGAP1 mRNA and protein levels are selectively reduced in the null allele of the mouse orthologue of DTNBP1. We postulate that endosomal trafficking contributes to the pathogenesis of neurodevelopmental disorders affecting dendritic spine morphology, and thus excitatory synapse structure and function.

  1. First-Generation Antipsychotic Haloperidol Alters the Functionality of the Late Endosomal/Lysosomal Compartment in Vitro

    Science.gov (United States)

    Canfrán-Duque, Alberto; Barrio, Luis C.; Lerma, Milagros; de la Peña, Gema; Serna, Jorge; Pastor, Oscar; Lasunción, Miguel A.; Busto, Rebeca

    2016-01-01

    First- and second-generation antipsychotics (FGAs and SGAs, respectively), have the ability to inhibit cholesterol biosynthesis and also to interrupt the intracellular cholesterol trafficking, interfering with low-density lipoprotein (LDL)-derived cholesterol egress from late endosomes/lysosomes. In the present work, we examined the effects of FGA haloperidol on the functionality of late endosomes/lysosomes in vitro. In HepG2 hepatocarcinoma cells incubated in the presence of 1,1′-dioctadecyl-3,3,3,3′-tetramethylindocarbocyanineperchlorate (DiI)-LDL, treatment with haloperidol caused the enlargement of organelles positive for late endosome markers lysosome-associated membrane protein 2 (LAMP-2) and LBPA (lysobisphosphatidic acid), which also showed increased content of both free-cholesterol and DiI derived from LDL. This indicates the accumulation of LDL-lipids in the late endosomal/lysosomal compartment caused by haloperidol. In contrast, LDL traffic through early endosomes and the Golgi apparatus appeared to be unaffected by the antipsychotic as the distribution of both early endosome antigen 1 (EEA1) and coatomer subunit β (β-COP) were not perturbed. Notably, treatment with haloperidol significantly increased the lysosomal pH and decreased the activities of lysosomal protease and β-d-galactosidase in a dose-dependent manner. We conclude that the alkalinization of the lysosomes’ internal milieu induced by haloperidol affects lysosomal functionality. PMID:26999125

  2. Motor coordination: a local hub for coordination.

    Science.gov (United States)

    Calabrese, Ronald L

    2014-03-31

    A local interneuron of a crayfish central pattern generator serves as a hub that integrates ascending and descending coordinating information and passes it on to a local oscillatory microcircuit to coordinate a series of segmental appendages known as swimmerets.

  3. Fluorescence activated cell sorting of plant protoplasts.

    Science.gov (United States)

    Bargmann, Bastiaan O R; Birnbaum, Kenneth D

    2010-02-18

    High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis. An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: P(SCR;)::GFP (endodermis and quiescent center) and P(WOX5;)::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution. FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time. This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce

  4. Receptor-mediated sorting of soluble vacuolar proteins: myths, facts, and a new model.

    Science.gov (United States)

    Robinson, David G; Neuhaus, Jean-Marc

    2016-08-01

    To prevent their being released to the cell exterior, acid hydrolases are recognized by receptors at some point in the secretory pathway and diverted towards the lytic compartment of the cell (lysosome or vacuole). In animal cells, the receptor is called the mannosyl 6-phosphate receptor (MPR) and it binds hydrolase ligands in the trans-Golgi network (TGN). These ligands are then sequestered into clathrin-coated vesicles (CCVs) because of motifs in the cytosolic tail of the MPR which interact first with monomeric adaptors (Golgi-localized, Gamma-ear-containing, ARF-binding proteins, GGAs) and then with tetrameric (adaptin) adaptor complexes. The CCVs then fuse with an early endosome, whose more acidic lumen causes the ligands to dissociate. The MPRs are then recycled back to the TGN via retromer-coated carriers. Plants have vacuolar sorting receptors (VSRs) which were originally identified in CCVs isolated from pea (Pisum sativum L.) cotyledons. It was therefore assumed that VSRs would have an analogous function in plants to MPRs in animals. Although this dogma has enjoyed wide support over the last 20 years there are many inconsistencies. Recently, results have been published which are quite contrary to it. It now emerges that VSRs and their ligands can interact very early in the secretory pathway, and dissociate in the TGN, which, in contrast to its mammalian counterpart, has a pH of 5.5. Multivesicular endosomes in plants lack proton pump complexes and consequently have an almost neutral internal pH, which discounts them as organelles of pH-dependent receptor-ligand dissociation. These data force a critical re-evaluation of the role of CCVs at the TGN, especially considering that vacuolar cargo ligands have never been identified in them. We propose that one population of TGN-derived CCVs participate in retrograde transport of VSRs from the TGN. We also present a new model to explain how secretory and vacuolar cargo proteins are effectively separated after

  5. Self-assembly of heteroleptic dinuclear metallosupramolecular kites from multivalent ligands via social self-sorting

    Directory of Open Access Journals (Sweden)

    Christian Benkhäuser

    2015-05-01

    Full Text Available A Tröger's base-derived racemic bis(1,10-phenanthroline ligand (rac-1 and a bis(2,2'-bipyridine ligand with a central 1,3-diethynylbenzene unit 2 were synthesized. Each of these ligands acts as a multivalent entity for the binding of two copper(I ions. Upon coordination to the metal ions these two ligands undergo selective self-assembly into heteroleptic dinuclear metallosupramolecular kites in a high-fidelity social self-sorting manner as evidenced by NMR spectroscopy and mass spectrometry.

  6. Passive chip-based droplet sorting

    Energy Technology Data Exchange (ETDEWEB)

    Beer, Neil Reginald; Lee, Abraham P; Hatch, Andrew C; Fisher, Jeffrey S

    2015-11-05

    An apparatus for passive sorting of microdroplets including a main flow channel, a flow stream of microdroplets in the main flow channel wherein the microdroplets have substantially the same diameter and wherein the flow stream of microdroplets includes first microdroplets having a first degree of stiffness and second microdroplets having a second degree of stiffness wherein the second degree of stiffness is different than the first degree of stiffness. A second flow channel is connected to the main flow channel for the second microdroplets having a second degree of stiffness. A separator separates the second microdroplets having a second degree of stiffness from the first microdroplets and directs the second microdroplets having a second degree of stiffness into the second flow channel.

  7. Phase sorting wave-particle correlator

    Science.gov (United States)

    Kletzing, C. A.; LaBelle, J.; Bounds, S. R.; Dolan, J.; Kaeppler, S. R.; Dombrowski, M.

    2017-02-01

    Wave-particle correlations, particularly of Langmuir waves and electrons, have been the subject of significant interest extending back to the 1970s. Often, these correlations have been simply observing modulation of the electrons at the plasma frequency with no phase resolution. The first phase-resolving correlators were developed at UC Berkeley in the late 1980s and reported by Ergun in the early 1990s. A design is presented which further improves on phase resolution in correlations of Langmuir waves and electrons with phase resolution of 22.5°. In this technique, a phase-locked loop (PLL) is used to lock onto the wave and subdivide the phase. Electrons are sorted on-the-fly as they arrive into the phase bins. Discussed are details of accurate timing, testing, and calibration of this system as well as results from rocket flights in which statistically significant phase correlations have been observed.

  8. Passive chip-based droplet sorting

    Energy Technology Data Exchange (ETDEWEB)

    Beer, Neil Reginald; Lee, Abraham P; Hatch, Andrew C; Fisher, Jeffrey S

    2015-03-03

    An apparatus for passive sorting of microdroplets including a main flow channel, a flow stream of microdroplets in the main flow channel wherein the microdroplets have substantially the same diameter and wherein the flow stream of microdroplets includes first microdroplets having a first degree of stiffness and second microdroplets having a second degree of stiffness wherein the second degree of stiffness is different than the first degree of stiffness. A second flow channel is connected to the main flow channel for the second microdroplets having a second degree of stiffness. A separator separates the second microdroplets having a second degree of stiffness from the first microdroplets and directs the second microdroplets having a second degree of stiffness into the second flow channel.

  9. Receptorligand sorting along the endocytic pathway

    CERN Document Server

    Linderman, Jennifer J

    1989-01-01

    This research monograph focuses on a biomolecular separation process that occurs within most cells. Two types of molecules, receptors and ligands, are separated and routed along different intracellular pathways; this is a critical step in the process of receptor-mediated endocytosis. The development of an understanding of the basic mechanisms of this separation process is presented, with an emphasis on discovering the fundamental and measurable parameters that influence the event. Mathematical models of sorting are evaluated to predict the range of possible outcomes. These are compared with a variety of experimental data on different receptor/ligand systems. In addition, the influence of the separation on overall receptor/ligand processing dynamics is discussed. The book is intended for both biomathematicians and biologists. It is not necessary to understand the details of the model equations and their solution in order to test the models experimentally. The analysis suggests experiments that might be done to...

  10. Carbon Nanotube–Purification and Sorting Protocols

    Directory of Open Access Journals (Sweden)

    Poornendu Chaturvedi

    2008-09-01

    Full Text Available Carbon nanotubes (CNTs have shown extraordinary mechanical, thermal, electrical, and electronic properties. Electronic properties of CNT are very sensitive to its diameter and chirality, making it metallicor semiconducting, depending upon its chiral vector. The extraordinary properties of CNTs have led to demonstration of several applications but commercial realisation of these devices require consistent qualityof CNTs, and these should be  free of any impurity. For development of electronic devices, CNTs should notjust be pure but also of similar length, diameter, and electronic behaviour. Such demanding requirements need development of elaborate purification and sorting protocols. In this paper,  a brief review of the existing technologies and the research done is presented.Defence Science Journal, 2008, 58(5, pp.591-599, DOI:http://dx.doi.org/10.14429/dsj.58.1694

  11. Improved method for pulse sorting based on PRI transform

    Science.gov (United States)

    Ren, Chunhui; Cao, Junqing; Fu, Yusheng; Barner, Kenneth E.

    2014-06-01

    To solve the problem of pulse sorting in complex electromagnetic environment, we propose an improved method for pulse sorting through in-depth analysis of the PRI transform algorithm principle and the advantages and disadvantages in this paper. The method is based on the traditional PRI transform algorithm, using spectral analysis of PRI transform spectrum to estimate the PRI centre value of jitter signal. Simulation results indicate that, the improved sorting method overcome the shortcomings of the traditional PRI jitter separation algorithm which cannot effectively sort jitter pulse sequence, in addition to the advantages of simple and accurate.

  12. Pattern matching based active optical sorting of colloids/cells

    Science.gov (United States)

    Verma, R. S.; Dasgupta, R.; Ahlawat, S.; Kumar, N.; Uppal, A.; Gupta, P. K.

    2013-08-01

    We report active optical sorting of colloids/cells by employing a cross correlation based pattern matching technique for selection of the desired objects and thereafter sorting using dynamically controllable holographic optical traps. The problem of possible collision between the different sets of objects during sorting was avoided by raising one set of particles to a different plane. We also present the results obtained on using this approach for some representative applications such as sorting of silica particles of two different sizes, of closely packed colloids and of white blood cells and red blood cells from a mixture of the two.

  13. A many-sorted calculus based on resolution and paramodulation

    CERN Document Server

    Walther, Christoph

    1987-01-01

    A Many-Sorted Calculus Based on Resolution and Paramodulation emphasizes the utilization of advantages and concepts of many-sorted logic for resolution and paramodulation based automated theorem proving.This book considers some first-order calculus that defines how theorems from given hypotheses by pure syntactic reasoning are obtained, shifting all the semantic and implicit argumentation to the syntactic and explicit level of formal first-order reasoning. This text discusses the efficiency of many-sorted reasoning, formal preliminaries for the RP- and ?RP-calculus, and many-sorted term rewrit

  14. Learning sorting algorithms through visualization construction

    Science.gov (United States)

    Cetin, Ibrahim; Andrews-Larson, Christine

    2016-01-01

    Recent increased interest in computational thinking poses an important question to researchers: What are the best ways to teach fundamental computing concepts to students? Visualization is suggested as one way of supporting student learning. This mixed-method study aimed to (i) examine the effect of instruction in which students constructed visualizations on students' programming achievement and students' attitudes toward computer programming, and (ii) explore how this kind of instruction supports students' learning according to their self-reported experiences in the course. The study was conducted with 58 pre-service teachers who were enrolled in their second programming class. They expect to teach information technology and computing-related courses at the primary and secondary levels. An embedded experimental model was utilized as a research design. Students in the experimental group were given instruction that required students to construct visualizations related to sorting, whereas students in the control group viewed pre-made visualizations. After the instructional intervention, eight students from each group were selected for semi-structured interviews. The results showed that the intervention based on visualization construction resulted in significantly better acquisition of sorting concepts. However, there was no significant difference between the groups with respect to students' attitudes toward computer programming. Qualitative data analysis indicated that students in the experimental group constructed necessary abstractions through their engagement in visualization construction activities. The authors of this study argue that the students' active engagement in the visualization construction activities explains only one side of students' success. The other side can be explained through the instructional approach, constructionism in this case, used to design instruction. The conclusions and implications of this study can be used by researchers and

  15. Intracellular mediators of transforming growth factor β superfamily signaling localize to endosomes in chicken embryo and mouse lenses in vivo

    Directory of Open Access Journals (Sweden)

    Ishii Shunsuke

    2007-06-01

    Full Text Available Abstract Background Endocytosis is a key regulator of growth factor signaling pathways. Recent studies showed that the localization to endosomes of intracellular mediators of growth factor signaling may be required for their function. Although there is substantial evidence linking endocytosis and growth factor signaling in cultured cells, there has been little study of the endosomal localization of signaling components in intact tissues or organs. Results Proteins that are downstream of the transforming growth factor-β superfamily signaling pathway were found on endosomes in chicken embryo and postnatal mouse lenses, which depend on signaling by members of the TGFβ superfamily for their normal development. Phosphorylated Smad1 (pSmad1, pSmad2, Smad4, Smad7, the transcriptional repressors c-Ski and TGIF and the adapter molecules Smad anchor for receptor activation (SARA and C184M, localized to EEA-1- and Rab5-positive vesicles in chicken embryo and/or postnatal mouse lenses. pSmad1 and pSmad2 also localized to Rab7-positive late endosomes. Smad7 was found associated with endosomes, but not caveolae. Bmpr1a conditional knock-out lenses showed decreased nuclear and endosomal localization of pSmad1. Many of the effectors in this pathway were distributed differently in vivo from their reported distribution in cultured cells. Conclusion Based on the findings reported here and data from other signaling systems, we suggest that the localization of activated intracellular mediators of the transforming growth factor-β superfamily to endosomes is important for the regulation of growth factor signaling.

  16. Seminal plasma affects sperm sex sorting in boars.

    Science.gov (United States)

    Alkmin, Diego V; Parrilla, Inmaculada; Tarantini, Tatiana; Del Olmo, David; Vazquez, Juan M; Martinez, Emilio A; Roca, Jordi

    2016-04-01

    Two experiments were conducted in boar semen samples to evaluate how both holding time (24h) and the presence of seminal plasma (SP) before sorting affect sperm sortability and the ability of sex-sorted spermatozoa to tolerate liquid storage. Whole ejaculate samples were divided into three aliquots immediately after collection: one was diluted (1:1, v/v) in Beltsville thawing solution (BTS; 50% SP); the SP of the other two aliquots was removed and the sperm pellets were diluted with BTS + 10% of their own SP (10% SP) or BTS alone (0% SP). The three aliquots of each ejaculate were divided into two portions, one that was processed immediately for sorting and a second that was sorted after 24h storage at 15-17°C. In the first experiment, the ability to exhibit well-defined X- and Y-chromosome-bearing sperm peaks (split) in the cytometry histogram and the subsequent sorting efficiency were assessed (20 ejaculates). In contrast with holding time, the SP proportion influenced the parameters examined, as evidenced by the higher number of ejaculates exhibiting split and better sorting efficiency (P<0.05) in semen samples with 0-10% SP compared with those with 50% SP. In a second experiment, the quality (viability, total and progressive motility) and functionality (plasma membrane fluidity and intracellular generation of reactive oxygen species) of sex-sorted spermatozoa were evaluated after 0, 72 and 120h storage at 15-17°C (10 ejaculates). Holding time and SP proportion did not influence the quality or functionality of stored sex-sorted spermatozoa. In conclusion, a holding time as long as 24h before sorting did not negatively affect sex sorting efficiency or the ability of sorted boar spermatozoa to tolerate long-term liquid storage. A high proportion of SP (50%) in the semen samples before sorting reduced the number of ejaculates to be sorted and negatively influenced the sorting efficiency, but did not affect the ability of sex-sorted spermatozoa to tolerate liquid

  17. Viable cell sorting of dinoflagellates by multiparametric flow cytometry.

    Science.gov (United States)

    Sinigalliano, Christopher D; Winshell, Jamie; Guerrero, Maria A; Scorzetti, Gloria; Fell, Jack W; Eaton, Richard W; Brand, Larry; Rein, Kathleen S

    2009-07-01

    Electronic cell sorting for isolation and culture of dinoflagellates and other marine eukaryotic phytoplankton was compared to the traditional method of manually picking cells using a micropipette. Trauma to electronically sorted cells was not a limiting factor, as fragile dinoflagellates, such as Karenia brevis (Dinophyceae), survived electronic cell sorting to yield viable cells. The rate of successful isolation of large-scale (> 4 litres) cultures was higher for manual picking than for electronic cell sorting (2% vs 0.5%, respectively). However, manual picking of cells is more labor intensive and time consuming. Most manually isolated cells required repicking, as the cultures were determined not to be unialgal after a single round of isolation; whereas, no cultures obtained in this study from electronic single-cell sorting required resorting. A broad flow cytometric gating logic was employed to enhance species diversity. The percentages of unique genotypes produced by manual picking or electronic cell sorting were similar (57% vs 54%, respectively), and each approach produced a variety of dinoflagellate or raphidophyte genera. Alternatively, a highly restrictive gating logic was successfully used to target K. brevis from a natural bloom sample. Direct electronic single-cell sorting was more successful than utilizing a pre-enrichment sort followed by electronic single-cell sorting. The appropriate recovery medium may enhance the rate of successful isolations. Seventy percent of isolated cells were recovered in a new medium (RE) reported here, which was optimized for axenic dinoflagellate cultures. The greatest limiting factor to the throughput of electronic cell sorting is the need for manual postsort culture maintenance and assessment of the large number of isolated cells. However, when combined with newly developed automated methods for growth screening, electronic single-cell sorting has the potential to accelerate the discovery of new algal strains.

  18. A Comparison of Card-sorting Analysis Methods

    DEFF Research Database (Denmark)

    Nawaz, Ather

    2012-01-01

    the recurrent patterns found and thus has consequences for the resulting website design. This paper draws an attention to the choice of card sorting analysis and techniques and shows how it impacts the results. The research focuses on how the same data for card sorting can lead to different website structures...

  19. Tradeoffs Between Branch Mispredictions and Comparisons for Sorting Algorithms

    DEFF Research Database (Denmark)

    Brodal, Gerth Stølting; Moruz, Gabriel

    2005-01-01

    Branch mispredictions is an important factor affecting the running time in practice. In this paper we consider tradeoffs between the number of branch mispredictions and the number of comparisons for sorting algorithms in the comparison model. We prove that a sorting algorithm using O(dnlog n...

  20. Real-Time Implementation of a Color Sorting System

    Science.gov (United States)

    Srikathyanyani Srikanteswara; Qiang Lu; William King; Thomas Drayer; Richard Conners; D. Earl Kline; Philip A. Araman

    1997-01-01

    Wood edge glued panels are used extensively in the furniture and cabinetry industries. They are used to make doors, tops, and sides of solid wood furniture and cabinets. Since lightly stained furniture and cabinets are gaining in popularity, there is an increasing demand to color sort the parts used to make these edge glued panels. The goal of the sorting processing is...

  1. Magnetic fluid equipment for sorting of secondary polyolefins from waste

    NARCIS (Netherlands)

    Rem, P.C.; Di Maio, F.; Hu, B.; Houzeaux, G...; Baltes, L.; Tierean, M.

    2012-01-01

    The paper presents the researches made on the FP7 project „Magnetic Sorting and Ultrasound Sensor Technologies for Production of High Purity Secondary Polyolefins from Waste” in order to develop a magnetic fluid equipment for sorting of polypropylene (PP) and polyethylene (PE) from polymers mixed

  2. Method and set-up for uranium ore sorting

    Energy Technology Data Exchange (ETDEWEB)

    Dragulescu, E.; Ivascu, M.; Popescu, D.; Semenescu, G. (Institutul de Fizica si Inginerie Nucleara, Bucharest (Romania)); Gherea, Gh. (Intreprinderea metalelor rare, Bucuresti (Romania)); Draga, Z.; Funaru, Gh. (Exploatarea miniera Oravita, Oravita (Romania))

    1981-01-01

    A method was studied for uranium ore sorting. After the discussion of the principle, some particular conditions of the sorting are pointed out. A radiometric assembly is described and some results obtained on the simulator and in industrial conditions are reported.

  3. Magnetic fluid equipment for sorting of secondary polyolefins from waste

    NARCIS (Netherlands)

    Rem, P.C.; Di Maio, F.; Hu, B.; Houzeaux, G...; Baltes, L.; Tierean, M.

    2012-01-01

    The paper presents the researches made on the FP7 project „Magnetic Sorting and Ultrasound Sensor Technologies for Production of High Purity Secondary Polyolefins from Waste” in order to develop a magnetic fluid equipment for sorting of polypropylene (PP) and polyethylene (PE) from polymers mixed wa

  4. Annexin A6 and Late Endosomal Cholesterol Modulate Integrin Recycling and Cell Migration*

    Science.gov (United States)

    García-Melero, Ana; Reverter, Meritxell; Hoque, Monira; Meneses-Salas, Elsa; Koese, Meryem; Conway, James R. W.; Johnsen, Camilla H.; Alvarez-Guaita, Anna; Morales-Paytuvi, Frederic; Elmaghrabi, Yasmin A.; Pol, Albert; Tebar, Francesc; Murray, Rachael Z.; Timpson, Paul; Enrich, Carlos; Grewal, Thomas; Rentero, Carles

    2016-01-01

    Annexins are a family of proteins that bind to phospholipids in a calcium-dependent manner. Earlier studies implicated annexin A6 (AnxA6) to inhibit secretion and participate in the organization of the extracellular matrix. We recently showed that elevated AnxA6 levels significantly reduced secretion of the extracellular matrix protein fibronectin (FN). Because FN is directly linked to the ability of cells to migrate, this prompted us to investigate the role of AnxA6 in cell migration. Up-regulation of AnxA6 in several cell models was associated with reduced cell migration in wound healing, individual cell tracking and three-dimensional migration/invasion assays. The reduced ability of AnxA6-expressing cells to migrate was associated with decreased cell surface expression of αVβ3 and α5β1 integrins, both FN receptors. Mechanistically, we found that elevated AnxA6 levels interfered with syntaxin-6 (Stx6)-dependent recycling of integrins to the cell surface. AnxA6 overexpression caused mislocalization and accumulation of Stx6 and integrins in recycling endosomes, whereas siRNA-mediated AnxA6 knockdown did not modify the trafficking of integrins. Given our recent findings that inhibition of cholesterol export from late endosomes (LEs) inhibits Stx6-dependent integrin recycling and that elevated AnxA6 levels cause LE cholesterol accumulation, we propose that AnxA6 and blockage of LE cholesterol transport are critical for endosomal function required for Stx6-mediated recycling of integrins in cell migration. PMID:26578516

  5. Structure and Function of Vps15 in the Endosomal G Protein Signaling Pathway

    Energy Technology Data Exchange (ETDEWEB)

    Heenan, Erin J.; Vanhooke, Janeen L.; Temple, Brenda R.; Betts, Laurie; Sondek, John E.; Dohlman, Henrik G.; (UNC)

    2009-09-11

    G protein-coupled receptors mediate cellular responses to a wide variety of stimuli, including taste, light, and neurotransmitters. In the yeast Saccharomyces cerevisiae, activation of the pheromone pathway triggers events leading to mating. The view had long been held that the G protein-mediated signal occurs principally at the plasma membrane. Recently, it has been shown that the G protein {alpha} subunit Gpa1 can promote signaling at endosomes and requires two components of the sole phosphatidylinositol-3-kinase in yeast, Vps15 and Vps34. Vps15 contains multiple WD repeats and also binds to Gpa1 preferentially in the GDP-bound state; these observations led us to hypothesize that Vps15 may function as a G protein {beta} subunit at the endosome. Here we show an X-ray crystal structure of the Vps15 WD domain that reveals a seven-bladed propeller resembling that of typical G{beta} subunits. We show further that the WD domain is sufficient to bind Gpa1 as well as to Atg14, a potential G{gamma} protein that exists in a complex with Vps15. The Vps15 kinase domain together with the intermediate domain (linking the kinase and WD domains) also contributes to Gpa1 binding and is necessary for Vps15 to sustain G protein signaling. These findings reveal that the Vps15 G{beta}-like domain serves as a scaffold to assemble Gpa1 and Atg14, whereas the kinase and intermediate domains are required for proper signaling at the endosome.

  6. Augmented cellular trafficking and endosomal escape of porous silicon nanoparticles via zwitterionic bilayer polymer surface engineering.

    Science.gov (United States)

    Shahbazi, Mohammad-Ali; Almeida, Patrick V; Mäkilä, Ermei M; Kaasalainen, Martti H; Salonen, Jarno J; Hirvonen, Jouni T; Santos, Hélder A

    2014-08-01

    The development of a stable vehicle with low toxicity, high cellular internalization, efficient endosomal escape, and optimal drug release profile is a key bottleneck in nanomedicine. To overcome all these problems, we have developed a successful layer-by-layer method to covalently conjugate polyethyleneimine (PEI) and poly(methyl vinyl ether-co-maleic acid) (PMVE-MA) copolymer on the surface of undecylenic acid functionalized thermally hydrocarbonized porous silicon nanoparticles (UnTHCPSi NPs), forming a bilayer zwitterionic nanocomposite containing free positive charge groups of hyper-branched PEI disguised by the PMVE-MA polymer. The surface smoothness, charge and hydrophilicity of the developed NPs considerably improved the colloidal and plasma stabilities via enhanced suspensibility and charge repulsion. Furthermore, despite the surface negative charge of the bilayer polymer-conjugated NPs, the cellular trafficking and endosomal escape were significantly increased in both MDA-MB-231 and MCF-7 breast cancer cells. Remarkably, we also showed that the conjugation of surface free amine groups of the highly toxic UnTHCPSi-PEI (Un-P) NPs to the carboxylic groups of PMVE-MA renders acceptable safety features to the system and preserves the endosomal escape properties via proton sponge mechanism of the free available amine groups located inside the hyper-branched PEI layer. Moreover, the double layer protection not only controlled the aggregation of the NPs and reduced the toxicity, but also sustained the drug release of an anticancer drug, methotrexate, with further improved cytotoxicity profile of the drug-loaded particles. These results provide a proof-of-concept evidence that such zwitterionic polymer-based PSi nanocomposites can be extensively used as a promising candidate for cytosolic drug delivery.

  7. Structural Studies of Adeno-Associated Virus Serotype 8 Capsid Transitions Associated with Endosomal Trafficking

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Hyun-Joo; Gurda, Brittney L.; McKenna, Robert; Potter, Mark; Byrne, Barry; Salganik, Maxim; Muzyczka, Nicholas; Agbandje-McKenna, Mavis (Florida)

    2012-09-17

    The single-stranded DNA (ssDNA) parvoviruses enter host cells through receptor-mediated endocytosis, and infection depends on processing in the early to late endosome as well as in the lysosome prior to nuclear entry for replication. However, the mechanisms of capsid endosomal processing, including the effects of low pH, are poorly understood. To gain insight into the structural transitions required for this essential step in infection, the crystal structures of empty and green fluorescent protein (GFP) gene-packaged adeno-associated virus serotype 8 (AAV8) have been determined at pH values of 6.0, 5.5, and 4.0 and then at pH 7.5 after incubation at pH 4.0, mimicking the conditions encountered during endocytic trafficking. While the capsid viral protein (VP) topologies of all the structures were similar, significant amino acid side chain conformational rearrangements were observed on (i) the interior surface of the capsid under the icosahedral 3-fold axis near ordered nucleic acid density that was lost concomitant with the conformational change as pH was reduced and (ii) the exterior capsid surface close to the icosahedral 2-fold depression. The 3-fold change is consistent with DNA release from an ordering interaction on the inside surface of the capsid at low pH values and suggests transitions that likely trigger the capsid for genome uncoating. The surface change results in disruption of VP-VP interface interactions and a decrease in buried surface area between VP monomers. This disruption points to capsid destabilization which may (i) release VP1 amino acids for its phospholipase A2 function for endosomal escape and nuclear localization signals for nuclear targeting and (ii) trigger genome uncoating.

  8. Structural studies of adeno-associated virus serotype 8 capsid transitions associated with endosomal trafficking.

    Science.gov (United States)

    Nam, Hyun-Joo; Gurda, Brittney L; McKenna, Robert; Potter, Mark; Byrne, Barry; Salganik, Maxim; Muzyczka, Nicholas; Agbandje-McKenna, Mavis

    2011-11-01

    The single-stranded DNA (ssDNA) parvoviruses enter host cells through receptor-mediated endocytosis, and infection depends on processing in the early to late endosome as well as in the lysosome prior to nuclear entry for replication. However, the mechanisms of capsid endosomal processing, including the effects of low pH, are poorly understood. To gain insight into the structural transitions required for this essential step in infection, the crystal structures of empty and green fluorescent protein (GFP) gene-packaged adeno-associated virus serotype 8 (AAV8) have been determined at pH values of 6.0, 5.5, and 4.0 and then at pH 7.5 after incubation at pH 4.0, mimicking the conditions encountered during endocytic trafficking. While the capsid viral protein (VP) topologies of all the structures were similar, significant amino acid side chain conformational rearrangements were observed on (i) the interior surface of the capsid under the icosahedral 3-fold axis near ordered nucleic acid density that was lost concomitant with the conformational change as pH was reduced and (ii) the exterior capsid surface close to the icosahedral 2-fold depression. The 3-fold change is consistent with DNA release from an ordering interaction on the inside surface of the capsid at low pH values and suggests transitions that likely trigger the capsid for genome uncoating. The surface change results in disruption of VP-VP interface interactions and a decrease in buried surface area between VP monomers. This disruption points to capsid destabilization which may (i) release VP1 amino acids for its phospholipase A2 function for endosomal escape and nuclear localization signals for nuclear targeting and (ii) trigger genome uncoating.

  9. Synaptic and endosomal localization of active gamma-secretase in rat brain.

    Directory of Open Access Journals (Sweden)

    Susanne Frykman

    Full Text Available BACKGROUND: A key player in the development of Alzheimer's disease (AD is the gamma-secretase complex consisting of at least four components: presenilin, nicastrin, Aph-1 and Pen-2. gamma-Secretase is crucial for the generation of the neurotoxic amyloid beta-peptide (Abeta but also takes part in the processing of many other substrates. In cell lines, active gamma-secretase has been found to localize primarily to the Golgi apparatus, endosomes and plasma membranes. However, no thorough studies have been performed to show the subcellular localization of the active gamma-secretase in the affected organ of AD, namely the brain. PRINCIPAL FINDINGS: We show by subcellular fractionation of rat brain that high gamma-secretase activity, as assessed by production of Abeta40, is present in an endosome- and plasma membrane-enriched fraction of an iodixanol gradient. We also prepared crude synaptic vesicles as well as synaptic membranes and both fractions showed high Abeta40 production and contained high amounts of the gamma-secretase components. Further purification of the synaptic vesicles verified the presence of the gamma-secretase components in these compartments. The localization of an active gamma-secretase in synapses and endosomes was confirmed in rat brain sections and neuronal cultures by using a biotinylated gamma-secretase inhibitor together with confocal microscopy. SIGNIFICANCE: The information about the subcellular localization of gamma-secretase in brain is important for the understanding of the molecular mechanisms of AD. Furthermore, the identified fractions can be used as sources for highly active gamma-secretase.

  10. Feed sorting in dairy cattle: Causes, consequences, and management.

    Science.gov (United States)

    Miller-Cushon, E K; DeVries, T J

    2016-12-29

    Dairy cattle commonly sort total mixed rations, a behavior that influences individual nutrient intake and reduces the nutritive value of the ration left in the bunk across the day. Typical patterns of feed sorting in lactating dairy cows, against longer forage particles, result in greater intake of highly-fermentable carbohydrates and lesser intake of effective fiber than intended, and are associated with reduced rumen pH and altered milk composition. To understand the reason for this behavior and reduce it on-farm, numerous studies have explored the influences of ration characteristics, feeding strategies, and management factors on the expression of feed sorting. In mature cows and young calves, feed sorting is influenced by forage inclusion rate, particle size, and dry matter content. Feeding strategies that increase the time available to manipulate feed-including decreased feeding frequency and increased feeding level-may result in increased feed sorting. The extent of feed sorting is also influenced by a variety of herd-level factors, but variability between individuals in the extent of feed sorting suggests that this behavior may be subject to additional factors, including previous experience and internal state. The development of feed sorting in young calves has been explored in several recent studies, suggesting that early opportunities to sort feed, as provided by access to mixed diets, may encourage the early onset of this behavior and help it persist beyond weaning. Evidence also supports the role of feedback mechanisms that influence this behavior at the individual level. In calves and adult cows, selective consumption of higher-energy ration components may be linked to energy demands, as influenced by the availability of supplemental feed or changing metabolic status. Further, considerable evidence suggests that cattle will adjust patterns of feed sorting in favor of physically effective fiber to attenuate low rumen pH, providing evidence for the role

  11. Degree of molecular self-sorting in multicomponent systems.

    Science.gov (United States)

    Saha, Manik Lal; Schmittel, Michael

    2012-06-28

    Self-sorting represents the spontaneous and high fidelity self and/or non-self-recognition of two or more related components within a complex mixture. While the effective management of self-sorting principles perceptibly requires some key expertise in molecular programming, at a higher stage of operation it is of supreme interest to guide the process to increasingly higher degrees of self-sorting. In this article, we present the emerging principles of how to guide several components toward formation of self-sorted multicomponent architectures. To provide further guidance in denominating such systems, we suggest to utilise a systematic classification as well as a formula to evaluate their degree of self-sorting (M).

  12. Standard practice for cell sorting in a BSL-3 facility.

    Science.gov (United States)

    Perfetto, Stephen P; Ambrozak, David R; Nguyen, Richard; Roederer, Mario; Koup, Richard A; Holmes, Kevin L

    2011-01-01

    Over the past decade, there has been a rapid growth in the number of BSL-3 and BSL-4 laboratories in the USA and an increase in demand for infectious cell sorting in BSL-3 laboratories. In 2007, the International Society for Advancement of Cytometry (ISAC) Biosafety Committee published standards for the sorting of unfixed cells and is an important resource for biosafety procedures when performing infectious cell sorting. Following a careful risk assessment, if it is determined that a cell sorter must be located within a BSL-3 laboratory, there are a variety of factors to be considered prior to the establishment of the laboratory. This chapter outlines procedures for infectious cell sorting in a BSL-3 environment to facilitate the establishment and safe operation of a BSL-3 cell sorting laboratory. Subjects covered include containment verification, remote operation, disinfection, personal protective equipment (PPE), and instrument-specific modifications for enhanced aerosol evacuation.

  13. Sorting Pairs of Points Based on Their Distances

    Directory of Open Access Journals (Sweden)

    Mohammad Farshi

    2016-02-01

    Full Text Available Sorting data is one of the main problems in computer science which studied vastly and used in several places. In several geometric problems, like problems on point sets or lines in the plane or Euclidean space with higher dimensions, the problem of sorting pairs of points based on the distance (between them is used. Using general sorting algorithms, sorting n 2 distances between n points can be done in O(n2 log n time. Ofcourse, sorting (n2 independent numbers does not have a faster solution, but since we have dependency between numbers in this case, finding a faster algorithm or showing that the problem in this case has O(n2 log n time complexity is interesting. In this paper, we try to answer this question.

  14. Presence of a lysosomal enzyme, arylsulfatase-A, in the prelysosome-endosome compartments of human cultured fibroblasts.

    Science.gov (United States)

    Kelly, B M; Yu, C Z; Chang, P L

    1989-02-01

    Although endosomes and lysosomes are associated with different subcellular functions, we present evidence that a lysosomal enzyme, arylsulfatase-A, is present in prelysosomal vesicles which constitute part of the endosomal compartment. When human cultured fibroblasts were subfractionated with Percoll gradients, arylsulfatase-A activity was enriched in three subcellular fractions: dense lysosomes, light lysosomes, and light membranous vesicles. Pulsing the cells for 1 to 10 min with the fluid-phase endocytic marker, horseradish peroxidase, showed that endosomes enriched with the marker were distributed partly in the light lysosome fraction but mainly in the light membranous fraction. By pulsing the fibroblasts for 10 min with horseradish peroxidase conjugated to colloidal gold and then staining the light membranous and light lysosomal fractions for arylsulfatase-A activity with a specific cytochemical technique, the endocytic marker was detected under the electron microscope in the same vesicles as the lysosomal enzyme. The origin of the lysosomal enzyme in this endosomal compartment was shown not to be acquired through mannose 6-phosphate receptor-mediated endocytosis of enzymes previously secreted from the cell. Together with our recent finding that the light membranous fraction contains prelysosomes distinct from bona fide lysosomes and was highly enriched with newly synthesized arylsulfatase-A molecules, these results demonstrate that prelysosomes also constitute part of the endosomal compartment to which intracellular lysosomal enzymes are targeted.

  15. The conjugation of diphtheria toxin T domain to poly(ethylenimine) based vectors for enhanced endosomal escape during gene transfection.

    Science.gov (United States)

    Kakimoto, Shinji; Hamada, Tsutomu; Komatsu, Yuuki; Takagi, Masahiro; Tanabe, Toshizumi; Azuma, Hideki; Shinkai, Seiji; Nagasaki, Takeshi

    2009-01-01

    The endosomal escape is a well-known serious obstacle for non-viral gene delivery. This is because of an acidic and enzymatic degradation of the contents of the endosome/lysosome. Therefore, the internalized gene needs to be efficient released into the cytosol to obtain the efficiently transfection efficiency. On the other hand, the diphtheria toxin T domain fuses with endosome membrane by pH decrease, then enhances the endosomal escape of the diphtheria toxin C fragment. In this study, we constructed diphtheria toxin T domain-conjugated poly(ethylenimine)s (PEI) polyplex for enhancing the endosomal escape of exogenous gene. The conjugation of diphtheria toxin T domain with PEI/pDNA polyplex leads to the significant enhancement of transfection efficiency when compared with plain PEI/pDNA polyplex. The pH-responsive increase in hydrophobicity of the diphtheria toxin T domain might not only trigger the perturbation of the endocytic vesicle membrane but might also increase the membrane permeability.

  16. Endosome-to-Plasma Membrane Recycling of VEGFR2 Receptor Tyrosine Kinase Regulates Endothelial Function and Blood Vessel Formation.

    Science.gov (United States)

    Jopling, Helen M; Odell, Adam F; Pellet-Many, Caroline; Latham, Antony M; Frankel, Paul; Sivaprasadarao, Asipu; Walker, John H; Zachary, Ian C; Ponnambalam, Sreenivasan

    2014-04-29

    Rab GTPases are implicated in endosome-to-plasma membrane recycling, but how such membrane traffic regulators control vascular endothelial growth factor receptor 2 (VEGFR2/KDR) dynamics and function are not well understood. Here, we evaluated two different recycling Rab GTPases, Rab4a and Rab11a, in regulating endothelial VEGFR2 trafficking and signalling with implications for endothelial cell migration, proliferation and angiogenesis. In primary endothelial cells, VEGFR2 displays co-localisation with Rab4a, but not Rab11a GTPase, on early endosomes. Expression of a guanosine diphosphate (GDP)-bound Rab4a S22N mutant caused increased VEGFR2 accumulation in endosomes. TfR and VEGFR2 exhibited differences in endosome-to-plasma membrane recycling in the presence of chloroquine. Depletion of Rab4a, but not Rab11a, levels stimulated VEGF-A-dependent intracellular signalling. However, depletion of either Rab4a or Rab11a levels inhibited VEGF-A-stimulated endothelial cell migration. Interestingly, depletion of Rab4a levels stimulated VEGF-A-regulated endothelial cell proliferation. Rab4a and Rab11a were also both required for endothelial tubulogenesis. Evaluation of a transgenic zebrafish model showed that both Rab4 and Rab11a are functionally required for blood vessel formation and animal viability. Rab-dependent endosome-to-plasma membrane recycling of VEGFR2 is important for intracellular signalling, cell migration and proliferation during angiogenesis.

  17. Adaptor protein complexes 1 and 3 are essential for generation of synaptic vesicles from activity-dependent bulk endosomes.

    Science.gov (United States)

    Cheung, Giselle; Cousin, Michael A

    2012-04-25

    Activity-dependent bulk endocytosis is the dominant synaptic vesicle retrieval mode during high intensity stimulation in central nerve terminals. A key event in this endocytosis mode is the generation of new vesicles from bulk endosomes, which replenish the reserve vesicle pool. We have identified an essential requirement for both adaptor protein complexes 1 and 3 in this process by employing morphological and optical tracking of bulk endosome-derived synaptic vesicles in rat primary neuronal cultures. We show that brefeldin A inhibits synaptic vesicle generation from bulk endosomes and that both brefeldin A knockdown and shRNA knockdown of either adaptor protein 1 or 3 subunits inhibit reserve pool replenishment from bulk endosomes. Conversely, no plasma membrane function was found for adaptor protein 1 or 3 in either bulk endosome formation or clathrin-mediated endocytosis. Simultaneous knockdown of both adaptor proteins 1 and 3 indicated that they generated the same population of synaptic vesicles. Thus, adaptor protein complexes 1 and 3 play an essential dual role in generation of synaptic vesicles during activity-dependent bulk endocytosis.

  18. Limitations of Radar Coordinates

    OpenAIRE

    Bini, Donato; Lusanna, Luca; Mashhoon, Bahram

    2004-01-01

    The construction of a radar coordinate system about the world line of an observer is discussed. Radar coordinates for a hyperbolic observer as well as a uniformly rotating observer are described in detail. The utility of the notion of radar distance and the admissibility of radar coordinates are investigated. Our results provide a critical assessment of the physical significance of radar coordinates.

  19. Help the planet by sorting your waste!

    CERN Multimedia

    2012-01-01

    Paper and cardboard waste comes in various forms, from newspapers to the toughest cardboard. Every year CERN dispatches about 200 tonnes of paper and cardboard to a recycling plant, but this is still too little when you take into consideration the tonnes of paper and cardboard that are still thrown out as part of ordinary rubbish or are incorrectly sorted into other rubbish skips.   Each office is equipped with a wastepaper bin, and a paper and cardboard container is available near every building. Cardboard boxes should be folded before they are placed in the containers in order to save space. Please note: Here are some sobering statistics: - 2 to 3 tonnes of wood pulp are required to manufacture 1 tonne of paper. - Each tonne of recycled paper means that we can save approximately 15 trees and substantial amounts of the water that is needed to extract cellulose (60 litres of water per kilo of paper). - A production of 100% recycled paper represents a 90% saving in water. - 5000 kWh of e...

  20. Cell sorting using efficient light shaping approaches

    Science.gov (United States)

    Bañas, Andrew; Palima, Darwin; Villangca, Mark; Glückstad, Jesper

    2016-03-01

    Early detection of diseases can save lives. Hence, there is emphasis in sorting rare disease-indicating cells within small dilute quantities such as in the confines of lab-on-a-chip devices. In our work, we use optical forces to isolate red blood cells detected by machine vision. This approach is gentler, less invasive and more economical compared to conventional FACS systems. As cells are less responsive to plastic or glass beads commonly used in the optical manipulation literature, and since laser safety would be an issue in clinical use, we develop efficient approaches in utilizing lasers and light modulation devices. The Generalized Phase Contrast (GPC) method that can be used for efficiently illuminating spatial light modulators or creating well-defined contiguous optical traps is supplemented by diffractive techniques capable of integrating the available light and creating 2D or 3D beam distributions aimed at the positions of the detected cells. Furthermore, the beam shaping freedom provided by GPC can allow optimizations in the beam's propagation and its interaction with the catapulted cells.

  1. Microfluidic-chip platform for cell sorting

    Science.gov (United States)

    Malik, Sarul; Balyan, Prerna; Akhtar, J.; Agarwal, Ajay

    2016-04-01

    Cell sorting and separation are considered to be very crucial preparatory steps for numerous clinical diagnostics and therapeutics applications in cell biology research arena. Label free cell separation techniques acceptance rate has been increased to multifold by various research groups. Size based cell separation method focuses on the intrinsic properties of the cell which not only avoids clogging issues associated with mechanical and centrifugation filtration methods but also reduces the overall cost for the process. Consequentially flow based cell separation method for continuous flow has attracted the attention of millions. Due to the realization of structures close to particle size in micro dimensions, the microfluidic devices offer precise and rapid particle manipulation which ultimately leads to an extraordinary cell separation results. The proposed microfluidic device is fabricated to separate polystyrene beads of size 1 µm, 5 µm, 10 µm and 20 µm. The actual dimensions of blood corpuscles were kept in mind while deciding the particle size of polystyrene beads which are used as a model particles for study.

  2. Defects in cellular sorting and retroviral assembly induced by GGA overexpression

    Directory of Open Access Journals (Sweden)

    Nagashima Kunio

    2009-09-01

    Full Text Available Abstract Background We previously demonstrated that overexpression of Golgi-localized, γ-ear containing, Arf-binding (GGA proteins inhibits retrovirus assembly and release by disrupting the function of endogenous ADP ribosylation factors (Arfs. GGA overexpression led to the formation of large, swollen vacuolar compartments, which in the case of GGA1 sequestered HIV-1 Gag. Results In the current study, we extend our previous findings to characterize in depth the GGA-induced compartments and the determinants for retroviral Gag sequestration in these structures. We find that GGA-induced structures are derived from the Golgi and contain aggresome markers. GGA overexpression leads to defects in trafficking of transferrin receptor and recycling of cation-dependent mannose 6-phosphate receptor. Additionally, we find that compartments induced by GGA overexpression sequester Tsg101, poly-ubiquitin, and, in the case of GGA3, Hrs. Interestingly, brefeldin A treatment, which leads to the dissociation of endogenous GGAs from membranes, does not dissociate the GGA-induced compartments. GGA mutants that are defective in Arf binding and hence association with membranes also induce the formation of GGA-induced structures. Overexpression of ubiquitin reverses the formation of GGA-induced structures and partially rescues HIV-1 particle production. We found that in addition to HIV-1 Gag, equine infectious anemia virus Gag is also sequestered in GGA1-induced structures. The determinants in Gag responsible for sequestration map to the matrix domain, and recruitment to these structures is dependent on Gag membrane binding. Conclusion These data provide insights into the composition of structures induced by GGA overexpression and their ability to disrupt endosomal sorting and retroviral particle production.

  3. Recognition of pathogen-associated nucleic acids by endosomal nucleic acid-sensing toll-like receptors

    Institute of Scientific and Technical Information of China (English)

    Xiaobing He; Huaijie Jia; Zhizhong Jing; Dingxiang Liu

    2013-01-01

    Foreign nucleic acids,the essential signature molecules of invading pathogens that act as danger signals for host cells,are detected by endosomal nucleic acid-sensing tolllike receptors (TLRs) 3,7,8,9,and 13.These TLRs have evolved to recognize ‘non-self' nucleic acids within endosomal compartments and rapidly initiate innate immune responses to ensure host protection through induction of type Ⅰ interferons,inflammatory cytokines,chemokines,and co-stimulatory molecules and maturation of immune cells.In this review,we highlight our understanding of the recognition of pathogen-associated nucleic acids and activation of corresponding signaling pathways through endosomal nucleic acid-sensing TLRs 3,7,8,9,and 13 for an enormous diversity of pathogens,with particular emphasis on their compartmentalization,intracellular trafficking,proteolytic cleavage,autophagy,and regulatory programs.

  4. Microfluidic EmbryoSort technology: towards in flow analysis, sorting and dispensing of individual vertebrate embryos

    Science.gov (United States)

    Fuad, Nurul M.; Wlodkowic, Donald

    2013-12-01

    The demand to reduce the numbers of laboratory animals has facilitated the emergence of surrogate models such as tests performed on zebrafish (Danio rerio) or African clawed frog's (Xenopus levis) eggs, embryos and larvae. Those two model organisms are becoming increasingly popular replacements to current adult animal testing in toxicology, ecotoxicology and also in drug discovery. Zebrafish eggs and embryos are particularly attractive for toxicological analysis due their size (diameter 1.6 mm), optical transparency, large numbers generated per fish and very straightforward husbandry. The current bottleneck in using zebrafish embryos for screening purposes is, however, a tedious manual evaluation to confirm the fertilization status and subsequent dispensing of single developing embryos to multitier plates to perform toxicity analysis. Manual procedures associated with sorting hundreds of embryos are very monotonous and as such prone to significant analytical errors due to operator's fatigue. In this work, we present a proofof- concept design of a continuous flow embryo sorter capable of analyzing, sorting and dispensing objects ranging in size from 1.5 - 2.5 mm. The prototypes were fabricated in polymethyl methacrylate (PMMA) transparent thermoplastic using infrared laser micromachining. The application of additive manufacturing processes to prototype Lab-on-a-Chip sorters using both fused deposition manufacturing (FDM) and stereolithography (SLA) were also explored. The operation of the device was based on a revolving receptacle capable of receiving, holding and positioning single fish embryos for both interrogation and subsequent sorting. The actuation of the revolving receptacle was performed using a DC motor and/or microservo motor. The system was designed to separate between fertilized (LIVE) and non-fertilized (DEAD) eggs, based on optical transparency using infrared (IR) emitters and receivers.

  5. Kinetics of iron release from transferrin bound to the transferrin receptor at endosomal pH.

    Science.gov (United States)

    Steere, Ashley N; Byrne, Shaina L; Chasteen, N Dennis; Mason, Anne B

    2012-03-01

    Human serum transferrin (hTF) is a bilobal glycoprotein that reversibly binds Fe(3+) and delivers it to cells by the process of receptor-mediated endocytosis. Despite decades of research, the precise events resulting in iron release from each lobe of hTF within the endosome have not been fully delineated. We provide an overview of the kinetics of iron release from hTF±the transferrin receptor (TFR) at endosomal pH (5.6). A critical evaluation of the array of biophysical techniques used to determine accurate rate constants is provided. Delivery of Fe(3+)to actively dividing cells by hTF is essential; too much or too little Fe(3+) directly impacts the well-being of an individual. Because the interaction of hTF with the TFR controls iron distribution in the body, an understanding of this process at the molecular level is essential. Not only does TFR direct the delivery of iron to the cell through the binding of hTF, kinetic data demonstrate that it also modulates iron release from the N- and C-lobes of hTF. Specifically, the TFR balances the rate of iron release from each lobe, resulting in efficient Fe(3+) release within a physiologically relevant time frame. This article is part of a Special Issue entitled Molecular Mechanisms of Iron Transport and Disorders. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. An inside job: how endosomal Na(+)/H(+) exchangers link to autism and neurological disease.

    Science.gov (United States)

    Kondapalli, Kalyan C; Prasad, Hari; Rao, Rajini

    2014-01-01

    Autism imposes a major impediment to childhood development and a huge emotional and financial burden on society. In recent years, there has been rapidly accumulating genetic evidence that links the eNHE, a subset of Na(+)/H(+) exchangers that localize to intracellular vesicles, to a variety of neurological conditions including autism, attention deficit hyperactivity disorder (ADHD), intellectual disability, and epilepsy. By providing a leak pathway for protons pumped by the V-ATPase, eNHE determine luminal pH and regulate cation (Na(+), K(+)) content in early and recycling endosomal compartments. Loss-of-function mutations in eNHE cause hyperacidification of endosomal lumen, as a result of imbalance in pump and leak pathways. Two isoforms, NHE6 and NHE9 are highly expressed in brain, including hippocampus and cortex. Here, we summarize evidence for the importance of luminal cation content and pH on processing, delivery and fate of cargo. Drawing upon insights from model organisms and mammalian cells we show how eNHE affect surface expression and function of membrane receptors and neurotransmitter transporters. These studies lead to cellular models of eNHE activity in pre- and post-synaptic neurons and astrocytes, where they could impact synapse development and plasticity. The study of eNHE has provided new insight on the mechanism of autism and other debilitating neurological disorders and opened up new possibilities for therapeutic intervention.

  7. Mitosis-coupled, microtubule-dependent clustering of endosomal vesicles around centrosomes.

    Science.gov (United States)

    Takatsu, Hiroyuki; Katoh, Yohei; Ueda, Tomoko; Waguri, Satoshi; Murayama, Takashi; Takahashi, Senye; Shin, Hye-Won; Nakayama, Kazuhisa

    2013-01-01

    Upon cell division, not only cells themselves but also their organelles undergo drastic shape changes, although the behaviors of organelles other than the Golgi apparatus remain poorly understood. We followed the spatiotemporal changes in the localization of transferrin receptor (TfnR) and other proteins. In early mitotic phases, a population of proteins cycling through the endocytic recycling compartment (ERC) exhibits a distinct spatiotemporal change from that of Golgi proteins. In prophase/prometaphase, when the cell surface-to-volume ratio is reaching its minimum, the ERC proteins are transiently assembled around the centrated centrosome in a microtubule- and dynein-dependent manner, and soon separated polewards into two clusters concomitant with separation of duplicated centrosomes. Electron microscopic analysis revealed that endosomal vesicles containing endocytosed transferrin cluster tightly around centrosomes without fusing with one another. As cytokinesis proceeds, the clusters gradually collapse, and the ERC proteins reassemble around the furrowing equatorial region. FRAP (fluorescence recovery after photobleaching) analyses of EGFP-TfnR-expressing cells revealed minimal membrane exchange between the endosomal clusters and other cellular compartments until anaphase/telophase, when membrane traffic resumes. Our observations indicate that ERC clustering around centrosomes plays a fundamental role in restricting membrane delivery to the plasma membrane during early mitotic phases, when the cell surface-to-volume ratio reaches its minimum.

  8. Early endosomal escape of a cyclic cell-penetrating peptide allows effective cytosolic cargo delivery.

    Science.gov (United States)

    Qian, Ziqing; LaRochelle, Jonathan R; Jiang, Bisheng; Lian, Wenlong; Hard, Ryan L; Selner, Nicholas G; Luechapanichkul, Rinrada; Barrios, Amy M; Pei, Dehua

    2014-06-24

    Cyclic heptapeptide cyclo(FΦRRRRQ) (cFΦR4, where Φ is l-2-naphthylalanine) was recently found to be efficiently internalized by mammalian cells. In this study, its mechanism of internalization was investigated by perturbing various endocytic events through the introduction of pharmacologic agents and genetic mutations. The results show that cFΦR4 binds directly to membrane phospholipids, is internalized into human cancer cells through endocytosis, and escapes from early endosomes into the cytoplasm. Its cargo capacity was examined with a wide variety of molecules, including small-molecule dyes, linear and cyclic peptides of various charged states, and proteins. Depending on the nature of the cargos, they may be delivered by endocyclic (insertion of cargo into the cFΦR4 ring), exocyclic (attachment of cargo to the Gln side chain), or bicyclic approaches (fusion of cFΦR4 and cyclic cargo rings). The overall delivery efficiency (i.e., delivery of cargo into the cytoplasm and nucleus) of cFΦR4 was 4-12-fold higher than those of nonaarginine, HIV Tat-derived peptide, or penetratin. The higher delivery efficiency, coupled with superior serum stability, minimal toxicity, and synthetic accessibility, renders cFΦR4 a useful transporter for intracellular cargo delivery and a suitable system for investigating the mechanism of endosomal escape.

  9. An Inside Job: How Endosomal Na+/H+ Exchangers Link to Autism and Neurological Disease

    Directory of Open Access Journals (Sweden)

    Kalyan C. Kondapalli

    2014-06-01

    Full Text Available Autism imposes a major impediment to childhood development and a huge emotional and financial burden on society. In recent years, there has been rapidly accumulating genetic evidence that links the eNHE, a subset of Na+/H+ exchangers that localize to intracellular vesicles, to a variety of neurological conditions including autism, attention deficit hyperactivity disorder, intellectual disability and epilepsy. By providing a leak pathway for protons pumped by the V-ATPase, eNHE determine luminal pH and regulate cation (Na+, K+ content in early and recycling endosomal compartments. Loss-of-function mutations in eNHE cause hyperacidification of endosomal lumen, as a result of imbalance in pump and leak pathways. Two isoforms, NHE6 and NHE9 are highly expressed in brain, including hippocampus and cortex. Here, we summarize evidence for the importance of luminal cation content and pH on processing, delivery and fate of cargo and on the surface expression and function of membrane receptors and neurotransmitter transporters, drawing upon insights from model organisms and mammalian cells. These studies lead to cellular models of eNHE activity in pre- and post-synaptic neurons and astrocytes, where they could impact synapse development and plasticity. The study of eNHE has provided new insight on the mechanism of autism and other debilitating neurological disorders and opened up new possibilities for therapeutic intervention.

  10. TDP-43 loss of function inhibits endosomal trafficking and alters trophic signaling in neurons.

    Science.gov (United States)

    Schwenk, Benjamin M; Hartmann, Hannelore; Serdaroglu, Alperen; Schludi, Martin H; Hornburg, Daniel; Meissner, Felix; Orozco, Denise; Colombo, Alessio; Tahirovic, Sabina; Michaelsen, Meike; Schreiber, Franziska; Haupt, Simone; Peitz, Michael; Brüstle, Oliver; Küpper, Clemens; Klopstock, Thomas; Otto, Markus; Ludolph, Albert C; Arzberger, Thomas; Kuhn, Peer-Hendrik; Edbauer, Dieter

    2016-11-02

    Nuclear clearance of TDP-43 into cytoplasmic aggregates is a key driver of neurodegeneration in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), but the mechanisms are unclear. Here, we show that TDP-43 knockdown specifically reduces the number and motility of RAB11-positive recycling endosomes in dendrites, while TDP-43 overexpression has the opposite effect. This is associated with delayed transferrin recycling in TDP-43-knockdown neurons and decreased β2-transferrin levels in patient CSF Whole proteome quantification identified the upregulation of the ESCRT component VPS4B upon TDP-43 knockdown in neurons. Luciferase reporter assays and chromatin immunoprecipitation suggest that TDP-43 represses VPS4B transcription. Preventing VPS4B upregulation or expression of its functional antagonist ALIX restores trafficking of recycling endosomes. Proteomic analysis revealed the broad reduction in surface expression of key receptors upon TDP-43 knockdown, including ErbB4, the neuregulin 1 receptor. TDP-43 knockdown delays the surface delivery of ErbB4. ErbB4 overexpression, but not neuregulin 1 stimulation, prevents dendrite loss upon TDP-43 knockdown. Thus, impaired recycling of ErbB4 and other receptors to the cell surface may contribute to TDP-43-induced neurodegeneration by blocking trophic signaling. © 2016 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

  11. Endosomal pH in Neuronal Signaling and Synaptic Transmission: Role of Na+/H+ Exchangers

    Directory of Open Access Journals (Sweden)

    Graham H Diering

    2014-01-01

    Full Text Available Neuronal precursor cells extend multiple neurites during development, one of which extends to form an axon whereas others develop into dendrites. Chemical stimulation of N-methyl D-aspartate (NMDA receptor in fully-differentiated neurons induces projection of dendritic spines, small spikes protruding from dendrites, thereby establishing another layer of polarity within the dendrite. Neuron-enriched Na+/H+ exchanger NHE5 contributes to both neurite growth and dendritic spine formation. In resting neurons and neuro-endocrine cells, neuron-enriched NHE5 is predominantly associated with recycling endosomes where it colocalizes with nerve growth factor (NGF receptor TrkA. NHE5 potently acidifies the lumen of TrkA-positive recycling endosomes and regulates cell-surface targeting of TrkA, whereas chemical stimulation of NMDA receptor rapidly recruits NHE5 to dendritic spines, alkalinizes dendrites and down-regulates the dendritic spine formation. Possible roles of NHE5 in neuronal signaling via proton movement in subcellular compartments are discussed.

  12. Sortilin regulates sorting and secretion of Sonic hedgehog.

    Science.gov (United States)

    Campbell, Charles; Beug, Shawn; Nickerson, Philip E B; Peng, Jimmy; Mazerolle, Chantal; Bassett, Erin A; Ringuette, Randy; Jama, Fadumo A; Morales, Carlos; Christ, Annabel; Wallace, Valerie A

    2016-10-15

    Sonic Hedgehog (Shh) is a secreted morphogen that is an essential regulator of patterning and growth. The Shh full-length protein undergoes autocleavage in the endoplasmic reticulum to generate the biologically active N-terminal fragment (ShhN), which is destined for secretion. We identified sortilin (Sort1), a member of the VPS10P-domain receptor family, as a new Shh trafficking receptor. We demonstrate that Sort-Shh interact by performing coimmunoprecipitation and proximity ligation assays in transfected cells and that they colocalize at the Golgi. Sort1 overexpression causes re-distribution of ShhN and, to a lesser extent, of full-length Shh to the Golgi and reduces Shh secretion. We show loss of Sort1 can partially rescue Hedgehog-associated patterning defects in a mouse model that is deficient in Shh processing, and we show that Sort1 levels negatively regulate anterograde Shh transport in axons in vitro and Hedgehog-dependent axon-glial interactions in vivo Taken together, we conclude that Shh and Sort1 can interact at the level of the Golgi and that Sort1 directs Shh away from the pathways that promote its secretion.

  13. Flow virometric sorting and analysis of HIV quasispecies from plasma

    Science.gov (United States)

    Jones, Jennifer C.; Keele, Brandon F.; Jenkins, Lisa M. Miller; Demberg, Thorsten

    2017-01-01

    Flow cytometry is utilized extensively for cellular analysis, but technical limitations have prevented its routine application for characterizing virus. The recent introduction of nanoscale fluorescence-activated cytometric cell sorting now allows analysis of individual virions. Here, we demonstrate staining and sorting of infectious HIV. Fluorescent antibodies specific for cellular molecules found on budding virions were used to label CCR5-tropic Bal HIV and CXCR4-tropic NL4.3 HIV Env-expressing pseudovirions made in THP-1 cells (monocyte/macrophage) and H9 cells (T cells), respectively. Using a flow cytometer, we resolved the stained virus beyond isotype staining and demonstrated purity and infectivity of sorted virus populations on cells with the appropriate coreceptors. We subsequently sorted infectious simian/human immunodeficiency virus from archived plasma. Recovery was approximately 0.5%, but virus present in plasma was already bound to viral-specific IgG generated in vivo, likely contributing to the low yield. Importantly, using two broadly neutralizing HIV antibodies, PG9 and VRC01, we also sorted virus from archived human plasma and analyzed the sorted populations genetically and by proteomics, identifying the quasispecies present. The ability to sort infectious HIV from clinically relevant samples provides material for detailed molecular, genetic, and proteomic analyses applicable to future design of vaccine antigens and potential development of personalized treatment regimens. PMID:28239654

  14. Microfluidic droplet sorting using integrated bilayer micro-valves

    Science.gov (United States)

    Chen, Yuncong; Tian, Yang; Xu, Zhen; Wang, Xinran; Yu, Sicong; Dong, Liang

    2016-10-01

    This paper reports on a microfluidic device capable of sorting microfluidic droplets utilizing conventional bilayer pneumatic micro-valves as sorting controllers. The device consists of two micro-valves placed symmetrically on two sides of a sorting area, each on top of a branching channel at an inclined angle with respect to the main channel. Changes in transmitted light intensity, induced by varying light absorbance by each droplet, are used to divert the droplet from the sorting area into one of the three outlet channels. When no valve is activated, the droplet flows into the outlet channel in the direction of the main channel. When one of the valves is triggered, the flexible membrane of valve will first be deflected. Once the droplet leaves the detection point, the deflected membrane will immediately return to its default flattened position, thereby exerting a drawing pressure on the droplet and deviating it from its original streamline to the outlet on the same side as the valve. This sorting method will be particularly suitable for numerous large-scale integrated microfluidic systems, where pneumatic micro-valves are already used. Only few structural modifications are needed to achieve droplet sorting capabilities in these systems. Due to the mechanical nature of diverting energy applied to droplets, the proposed sorting method may induce only minimal interference to biological species or microorganisms encapsulated inside the droplets that may accompany electrical, optical and magnetic-based techniques.

  15. IFITM3 restricts influenza A virus entry by blocking the formation of fusion pores following virus-endosome hemifusion.

    Directory of Open Access Journals (Sweden)

    Tanay M Desai

    2014-04-01

    Full Text Available Interferon-induced transmembrane proteins (IFITMs inhibit infection of diverse enveloped viruses, including the influenza A virus (IAV which is thought to enter from late endosomes. Recent evidence suggests that IFITMs block virus hemifusion (lipid mixing in the absence of viral content release by altering the properties of cell membranes. Consistent with this mechanism, excess cholesterol in late endosomes of IFITM-expressing cells has been reported to inhibit IAV entry. Here, we examined IAV restriction by IFITM3 protein using direct virus-cell fusion assay and single virus imaging in live cells. IFITM3 over-expression did not inhibit lipid mixing, but abrogated the release of viral content into the cytoplasm. Although late endosomes of IFITM3-expressing cells accumulated cholesterol, other interventions leading to aberrantly high levels of this lipid did not inhibit virus fusion. These results imply that excess cholesterol in late endosomes is not the mechanism by which IFITM3 inhibits the transition from hemifusion to full fusion. The IFITM3's ability to block fusion pore formation at a post-hemifusion stage shows that this protein stabilizes the cytoplasmic leaflet of endosomal membranes without adversely affecting the lumenal leaflet. We propose that IFITM3 interferes with pore formation either directly, through partitioning into the cytoplasmic leaflet of a hemifusion intermediate, or indirectly, by modulating the lipid/protein composition of this leaflet. Alternatively, IFITM3 may redirect IAV fusion to a non-productive pathway, perhaps by promoting fusion with intralumenal vesicles within multivesicular bodies/late endosomes.

  16. Role of endosomal trafficking dynamics on the regulation of hepatic insulin receptor activity: models for Fao cells.

    Science.gov (United States)

    Hori, Sharon S; Kurland, Irwin J; DiStefano, Joseph J

    2006-05-01

    Evidence indicates that endosomal insulin receptor (IR) trafficking plays a role in regulating insulin signal transduction. To evaluate its importance, we developed a series of biokinetic models for quantifying activated surface and endosomal IR dynamics from published experimental data. Starting with a published two-compartment Fao hepatoma model, a four-pool model was formulated that depicts IR autophosphorylation after receptor binding, IR endosomal internalization/trafficking, insulin dissociation from and dephosphorylation of internalized IR, and recycling of unliganded, dephosphorylated IR to the plasma membrane. Quantification required three additional data sets, two measured, but unmodeled by the same group. A five-pool model created to include endosomal trafficking of the nonphosphorylated insulin-IR complex was fitted using the same data sets, augmented with another published data set. Creation of a six-pool model added the physiologically relevant dissociation of insulin ligand from the activated endosomal IR. More importantly, all three models, validated against additional data not used in model fitting, predict that, mechanistically, internalization of activated IR is a rate-limiting step, at least under the receptor saturating conditions of the fitting data. This rate includes the transit time to a site where insulin dissociation from and/or dephosphorylation of the IR occurs by docking with protein-tyrosine phosphatases (PTPases), or where a sufficient conformational change occurs in the IR, perhaps due to insulin-IR dissociation, where associated PTPases may complete IR dephosphorylation. Our new models indicate that key events in endosomal IR trafficking have significance in mediating IR activity, possibly serving to regulate insulin signal transduction.

  17. Automatic gear sorting system based on monocular vision

    Directory of Open Access Journals (Sweden)

    Wenqi Wu

    2015-11-01

    Full Text Available An automatic gear sorting system based on monocular vision is proposed in this paper. A CCD camera fixed on the top of the sorting system is used to obtain the images of the gears on the conveyor belt. The gears׳ features including number of holes, number of teeth and color are extracted, which is used to categorize the gears. Photoelectric sensors are used to locate the gears׳ position and produce the trigger signals for pneumatic cylinders. The automatic gear sorting is achieved by using pneumatic actuators to push different gears into their corresponding storage boxes. The experimental results verify the validity and reliability of the proposed method and system.

  18. A Novel Auto-Sorting System for Chinese Cabbage Seeds.

    Science.gov (United States)

    Huang, Kuo-Yi; Cheng, Jian-Feng

    2017-04-18

    This paper presents a novel machine vision-based auto-sorting system for Chinese cabbage seeds. The system comprises an inlet-outlet mechanism, machine vision hardware and software, and control system for sorting seed quality. The proposed method can estimate the shape, color, and textural features of seeds that are provided as input neurons of neural networks in order to classify seeds as "good" and "not good" (NG). The results show the accuracies of classification to be 91.53% and 88.95% for good and NG seeds, respectively. The experimental results indicate that Chinese cabbage seeds can be sorted efficiently using the developed system.

  19. Efficient Sorting of Free Electron Orbital Angular Momentum

    CERN Document Server

    McMorran, Benjamin J; Lavery, Martin P J

    2016-01-01

    We propose a method for sorting electrons by orbital angular momentum (OAM). Several methods now exist to prepare electron wavefunctions in OAM states, but no technique has been developed for efficient, parallel measurement of pure and mixed electron OAM states. The proposed technique draws inspiration from the recent demonstration of the sorting of OAM through modal transformation. We show that the same transformation can be performed with electrostatic electron optical elements. Specifically, we show that a charged needle and an array of electrodes perform the transformation and phase correction necessary to sort orbital angular momentum states. This device may enable the analysis of the spatial mode distribution of inelastically scattered electrons.

  20. Movement and Coordination

    Science.gov (United States)

    ... Español Text Size Email Print Share Movement and Coordination Page Content Article Body At this age, your ... level will strengthen his body and develop his coordination. In the months ahead, your child’s running will ...

  1. Developmental coordination disorder

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/001533.htm Developmental coordination disorder To use the sharing features on this page, please enable JavaScript. Developmental coordination disorder is a childhood disorder. It leads to ...

  2. Running worms: C. elegans self-sorting by electrotaxis.

    Directory of Open Access Journals (Sweden)

    Xavier Manière

    Full Text Available The nematode C. elegans displays complex dynamical behaviors that are commonly used to identify relevant phenotypes. Although its maintenance is straightforward, sorting large populations of worms when looking for a behavioral phenotype is difficult, time consuming and hardly quantitative when done manually. Interestingly, when submitted to a moderate electric field, worms move steadily along straight trajectories. Here, we report an inexpensive method to measure worms crawling velocities and sort them within a few minutes by taking advantage of their electrotactic skills. This method allows to quantitatively measure the effect of mutations and aging on worm's crawling velocity. We also show that worms with different locomotory phenotypes can be spatially sorted, fast worms traveling away from slow ones. Group of nematodes with comparable locomotory fitness could then be isolated for further analysis. C. elegans is a growing model for neurodegenerative diseases and using electrotaxis for self-sorting can improve the high-throughput search of therapeutic bio-molecules.

  3. Sorting and quantifying orbital angular momentum of laser beams

    CSIR Research Space (South Africa)

    Schulze, C

    2013-10-01

    Full Text Available We present a novel tool for sorting the orbital angular momentum and to determine the orbital angular momentum density of laser beams, which is based on the use of correlation filters....

  4. Natural Selection Is a Sorting Process: What Does that Mean?

    Science.gov (United States)

    Price, Rebecca M.

    2013-01-01

    To learn why natural selection acts only on existing variation, students categorize processes as either creative or sorting. This activity helps students confront the misconception that adaptations evolve because species need them.

  5. FPGA-based Accelerators for Parallel Data Sort

    Directory of Open Access Journals (Sweden)

    Sklyarov Valery

    2014-12-01

    Full Text Available The paper is dedicated to parallel data sort based on sorting networks. The proposed methods and circuits have the following characteristics: 1 using two-level parallel comparators in even-odd transition networks with feedback to a register keeping input/intermediate data; 2 parallel merging of many sorted sequences; 3 using even-odd transition networks built from other sorting networks; 4 rational reuse of comparators in different types of networks, namely even-odd transition and for discovering maximum/minimum values. The experiments in FPGA, which were done for up to 16×220 32-bit data items, demonstrate very good results (as fast as 3-5 ns per data item.

  6. Unsupervised Spike Sorting Based on Discriminative Subspace Learning

    CERN Document Server

    Keshtkaran, Mohammad Reza

    2014-01-01

    Spike sorting is a fundamental preprocessing step for many neuroscience studies which rely on the analysis of spike trains. In this paper, we present two unsupervised spike sorting algorithms based on discriminative subspace learning. The first algorithm simultaneously learns the discriminative feature subspace and performs clustering. It uses histogram of features in the most discriminative projection to detect the number of neurons. The second algorithm performs hierarchical divisive clustering that learns a discriminative 1-dimensional subspace for clustering in each level of the hierarchy until achieving almost unimodal distribution in the subspace. The algorithms are tested on synthetic and in-vivo data, and are compared against two widely used spike sorting methods. The comparative results demonstrate that our spike sorting methods can achieve substantially higher accuracy in lower dimensional feature space, and they are highly robust to noise. Moreover, they provide significantly better cluster separab...

  7. Combination of the Sorting Line Priority Polling Control Mechanism

    Directory of Open Access Journals (Sweden)

    Wenxue Ran

    2013-10-01

    Full Text Available The paper proposed the priority polling control mechanism of the unit material combinations sorting lines, and sorters operating process is understood to be the arrival process of orders, service process of each sorter sorts orders and the conversion process between sorting machines. Control process, sorters of the combined sorting lines are divided into the priority sorters and ordinary sorters, priority sorters use full service control, ordinary sorters use limited service (k = 1, applies the polling service system theory, through the embedded Markov chain and probability generating function to establish the mathematical model of the system, the exact solution of the control mechanism of the polling system model and the first and second characteristic parameters, combined with the actual production priority parameters of orders picking for simulation analysis

  8. Using Sorting Networks for Skill Building and Reasoning

    Science.gov (United States)

    Andre, Robert; Wiest, Lynda R.

    2007-01-01

    Sorting networks, used in graph theory, have instructional value as a skill- building tool as well as an interesting exploration in discrete mathematics. Students can practice mathematics facts and develop reasoning and logic skills with this topic. (Contains 4 figures.)

  9. Optical sorting and photo-transfection of mammalian cells

    CSIR Research Space (South Africa)

    Mthunzi, P

    2010-02-01

    Full Text Available Recently, laser light sources of different regimes have emerged as an essential tool in the biophotonics research area. Classic applications include, for example: manipulating single cells and their subcellular organelles, sorting cells...

  10. Using Sorting Networks for Skill Building and Reasoning

    Science.gov (United States)

    Andre, Robert; Wiest, Lynda R.

    2007-01-01

    Sorting networks, used in graph theory, have instructional value as a skill- building tool as well as an interesting exploration in discrete mathematics. Students can practice mathematics facts and develop reasoning and logic skills with this topic. (Contains 4 figures.)

  11. SorLA Controls Neurotrophic Activity by Sorting of GDNF and Its Receptors GFRα1 and RET

    Directory of Open Access Journals (Sweden)

    Simon Glerup

    2013-01-01

    Full Text Available Glial cell-line-derived neurotrophic factor (GDNF is a potent neurotrophic factor that has reached clinical trials for Parkinson’s disease. GDNF binds to its coreceptor GFRα1 and signals through the transmembrane receptor tyrosine kinase RET, or RET independently through NCAM or syndecan-3. Whereas the GDNF signaling cascades are well described, cellular turnover and trafficking of GDNF and its receptors remain poorly characterized. Here, we find that SorLA acts as sorting receptor for the GDNF/GFRα1 complex, directing it from the cell surface to endosomes. Through this mechanism, GDNF is targeted to lysosomes and degraded while GFRα1 recycles, creating an efficient GDNF clearance pathway. The SorLA/GFRα1 complex further targets RET for endocytosis but not for degradation, affecting GDNF-induced neurotrophic activities. SorLA-deficient mice display elevated GDNF levels, altered dopaminergic function, marked hyperactivity, and reduced anxiety, all of which are phenotypes related to abnormal GDNF activity. Taken together, these findings establish SorLA as a critical regulator of GDNF activity in the CNS.

  12. SEPT8 modulates β-amyloidogenic processing of APP by affecting the sorting and accumulation of BACE1.

    Science.gov (United States)

    Kurkinen, Kaisa M A; Marttinen, Mikael; Turner, Laura; Natunen, Teemu; Mäkinen, Petra; Haapalinna, Fanni; Sarajärvi, Timo; Gabbouj, Sami; Kurki, Mitja; Paananen, Jussi; Koivisto, Anne M; Rauramaa, Tuomas; Leinonen, Ville; Tanila, Heikki; Soininen, Hilkka; Lucas, Fiona R; Haapasalo, Annakaisa; Hiltunen, Mikko

    2016-06-01

    Dysfunction and loss of synapses are early pathogenic events in Alzheimer's disease. A central step in the generation of toxic amyloid-β (Aβ) peptides is the cleavage of amyloid precursor protein (APP) by β-site APP-cleaving enzyme (BACE1). Here, we have elucidated whether downregulation of septin (SEPT) protein family members, which are implicated in synaptic plasticity and vesicular trafficking, affects APP processing and Aβ generation. SEPT8 was found to reduce soluble APPβ and Aβ levels in neuronal cells through a post-translational mechanism leading to decreased levels of BACE1 protein. In the human temporal cortex, we identified alterations in the expression of specific SEPT8 transcript variants in a manner that correlated with Alzheimer's-disease-related neurofibrillary pathology. These changes were associated with altered β-secretase activity. We also discovered that the overexpression of a specific Alzheimer's-disease-associated SEPT8 transcript variant increased the levels of BACE1 and Aβ peptides in neuronal cells. These changes were related to an increased half-life of BACE1 and the localization of BACE1 in recycling endosomes. These data suggest that SEPT8 modulates β-amyloidogenic processing of APP through a mechanism affecting the intracellular sorting and accumulation of BACE1.

  13. Coordination and Cooperation

    OpenAIRE

    Janssen, Maarten

    2003-01-01

    textabstractThis comment makes four related points. First, explaining coordination is different from explaining cooperation. Second, solving the coordination problem is more important for the theory of games than solving the cooperation problem. Third, a version of the Principle of Coordination can be rationalized on individualistic grounds. Finally, psychological game theory should consider how players perceive their gaming situation. ---------------------------------------------------------...

  14. Processing Coordination Ambiguity

    Science.gov (United States)

    Engelhardt, Paul E.; Ferreira, Fernanda

    2010-01-01

    We examined temporarily ambiguous coordination structures such as "put the butter in the bowl and the pan on the towel." Minimal Attachment predicts that the ambiguous noun phrase "the pan" will be interpreted as a noun-phrase coordination structure because it is syntactically simpler than clausal coordination. Constraint-based theories assume…

  15. Processing Coordination Ambiguity

    Science.gov (United States)

    Engelhardt, Paul E.; Ferreira, Fernanda

    2010-01-01

    We examined temporarily ambiguous coordination structures such as "put the butter in the bowl and the pan on the towel." Minimal Attachment predicts that the ambiguous noun phrase "the pan" will be interpreted as a noun-phrase coordination structure because it is syntactically simpler than clausal coordination. Constraint-based…

  16. Coordination and Cooperation

    NARCIS (Netherlands)

    M.C.W. Janssen (Maarten)

    2003-01-01

    textabstractThis comment makes four related points. First, explaining coordination is different from explaining cooperation. Second, solving the coordination problem is more important for the theory of games than solving the cooperation problem. Third, a version of the Principle of Coordination can

  17. Coordination and Cooperation

    NARCIS (Netherlands)

    M.C.W. Janssen (Maarten)

    2003-01-01

    textabstractThis comment makes four related points. First, explaining coordination is different from explaining cooperation. Second, solving the coordination problem is more important for the theory of games than solving the cooperation problem. Third, a version of the Principle of Coordination can

  18. Molecular characterization of flow-sorted mammalian centromeres

    Energy Technology Data Exchange (ETDEWEB)

    Hamkalo, B.A.; Henschen, A.; Parseghian, M.H. [Univ. of Calfornia, Irvine, CA (United States). Dept. of Molecular Biology and Biochemistry] [and others

    1998-12-31

    This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). The project involved experiments directed towards developing a molecular characterization of the centromere region of mammalian chromosomes. Attempts to purify this essential chromosomal locus by conventional methods have thus far been unsuccessful. However, preliminary data obtained in collaboration with the National Flow Cytometry Resource (NFCR) showed that it is possible to purify a chromosome fragment that is present in certain cultured mouse cell lines and has all the properties expected of an intact centromere region. To begin sorting this minichromosome for the identification of proteins preferentially associated with centromere regions, standard buffers utilized in chromosome sorting were evaluated for potential effects on maintenance of chromosomal proteins during sorting. The data indicate that the presence of several buffer constituents results in the extraction of all but a few chromosomal proteins. The subsequent use of a magnesium sulfate buffer resulted in the sorting of mouse chromosomes that do not suffer a significant loss of proteins. Several DNA stains were also evaluated for causing protein dissociation, but no significant losses were observed. Although flow-sorted chromosomes have been used extensively for DNA analysis and cloning, this is a pioneering effort by the NFCR, and its collaborators, to exploit chromosome sorting capabilities for the analysis of chromosomal proteins.

  19. Automatic particle detection and sorting in an electrokinetic microfluidic chip.

    Science.gov (United States)

    Song, Yongxin; Peng, Ran; Wang, Junsheng; Pan, Xinxiang; Sun, Yeqing; Li, Dongqing

    2013-03-01

    This paper reports a lab-on-a-chip device that can automatically detect and sort particles based on their size differences with a high resolution. The PDMS-glass microfluidic chip is made by soft-lithography technique. A differential resistive pulse sensor is employed to electrically detect the sizes of the particles in EOF generated by applying DC voltages across channels. The detected resistive pulse sensor signals, whose amplitudes are proportional to particles' sizes, will automatically trigger the sorting process that is controlled by applying a voltage pulse (36 V) whenever a target particle is detected. This method was applied to automatically detect and sort polystyrene particles and microalgae in aqueous solutions. Sorting 5 μm polymer particle from a mixture of 4- and 5-μm polystyrene particles in aqueous solution, i.e. 1 μm sorting resolution, was demonstrated. The device described in this paper is simple, automatic, and label-free with high sorting resolution. It has wide applications in sample pretreatment and target particles detection. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Molecular characterization of flow-sorted mammalian centromeres

    Energy Technology Data Exchange (ETDEWEB)

    Hamkalo, B.A.; Henschen, A.; Parseghian, M.H. [Univ. of Calfornia, Irvine, CA (United States). Dept. of Molecular Biology and Biochemistry] [and others

    1998-12-31

    This is the final report of a three-year, Laboratory Directed Research and Development (LDRD) project at the Los Alamos National Laboratory (LANL). The project involved experiments directed towards developing a molecular characterization of the centromere region of mammalian chromosomes. Attempts to purify this essential chromosomal locus by conventional methods have thus far been unsuccessful. However, preliminary data obtained in collaboration with the National Flow Cytometry Resource (NFCR) showed that it is possible to purify a chromosome fragment that is present in certain cultured mouse cell lines and has all the properties expected of an intact centromere region. To begin sorting this minichromosome for the identification of proteins preferentially associated with centromere regions, standard buffers utilized in chromosome sorting were evaluated for potential effects on maintenance of chromosomal proteins during sorting. The data indicate that the presence of several buffer constituents results in the extraction of all but a few chromosomal proteins. The subsequent use of a magnesium sulfate buffer resulted in the sorting of mouse chromosomes that do not suffer a significant loss of proteins. Several DNA stains were also evaluated for causing protein dissociation, but no significant losses were observed. Although flow-sorted chromosomes have been used extensively for DNA analysis and cloning, this is a pioneering effort by the NFCR, and its collaborators, to exploit chromosome sorting capabilities for the analysis of chromosomal proteins.

  1. NOVEL RADAR SIGNAL SORTING METHOD BASED ON GEOMETRIC COVERING

    Institute of Scientific and Technical Information of China (English)

    万建; 国强; 宋文明

    2013-01-01

    With the increase of complexity of electromagnetic environment and continuous appearance of advanced system radars ,signals received by radar reconnaissance receivers become even more intensive and complex .There-fore ,traditional radar sorting methods based on neural network algorithms and support vector machine (SVM ) cannot process them effectively .Aiming at solving this problem ,a novel radar signal sorting method based on the cloud model theory and the geometric covering algorithm is proposed .By applying the geometric covering algo-rithm to divide input signals into different covering domains based on their distribution characteristics ,the method can overcome a typical problem that it is easy for traditional sorting algorithms to fall into the local extrema due to the use of complex nonlinear equation to describe input signals .The method uses the cloud model to describe the membership degree between signals to be sorted and their covering domains ,thus it avoids the disadvantage that traditional sorting methods based on hard clustering cannot deinterleave the signal samples with overlapped param-eters .Experimental results show that the presented method can effectively sort advanced system radar signals with overlapped parameters in complex electromagnetic environment .

  2. Random walk models of worker sorting in ant colonies.

    Science.gov (United States)

    Sendova-Franks, Ana B; Van Lent, Jan

    2002-07-21

    Sorting can be an important mechanism for the transfer of information from one level of biological organization to another. Here we study the algorithm underlying worker sorting in Leptothorax ant colonies. Worker sorting is related to task allocation and therefore to the adaptive advantages associated with an efficient system for the division of labour in ant colonies. We considered four spatially explicit individual-based models founded on two-dimensional correlated random walk. Our aim was to establish whether sorting at the level of the worker population could occur with minimal assumptions about the behavioural algorithm of individual workers. The behaviour of an individual worker in the models could be summarized by the rule "move if you can, turn always". We assume that the turning angle of a worker is individually specific and negatively dependent on the magnitude of an internal parameter micro which could be regarded as a measure of individual experience or task specialization. All four models attained a level of worker sortedness that was compatible with results from experiments onLeptothorax ant colonies. We found that the presence of a sorting pivot, such as the nest wall or an attraction force towards the centre of the worker population, was crucial for sorting. We make a distinction between such pivots and templates and discuss the biological implications of their difference.

  3. An Evaluation of the Critical Factors Affecting the Efficiency of Some Sorting Techniques

    Directory of Open Access Journals (Sweden)

    Olabiyisi S.O.

    2013-02-01

    Full Text Available Sorting allows information or data to be put into a meaningful order. As efficiency is a major concern of computing, data are sorted in order to gain the efficiency in retrieving or searching tasks. The factors affecting the efficiency of shell, Heap, Bubble, Quick and Merge sorting techniques in terms of running time, memory usage and the number of exchanges were investigated. Experiment was conducted for the decision variables generated from algorithms implemented in Java programming and factor analysis by principal components of the obtained experimental data was carried out in order to estimate the contribution of each factor to the success of the sorting algorithms. Further statistical analysis was carried out to generate eigenvalue of the extracted factor and hence, a system of linear equations which was used to estimate the assessment of each factor of the sorting techniques was proposed. The study revealed that the main factor affecting these sorting techniques was time taken to sort. It contributed 97.842%, 97.693%, 89.351%, 98.336% and 90.480% for Bubble sort, Heap sort, Merge sort, Quick sort and Shell sort respectively. The number of swap came second contributing 1.587% for Bubble sort, 2.305% for Heap sort, 10.63% for Merge sort, 1.643% for Quick sort and 9.514% for Shell sort. The memory used was the least of the factors contributing negligible percentage for the five sorting techniques. It contributed 0.571% for Bubble sort, 0.002% for Heap sort, 0.011% for Merge sort, 0.021% for Quick sort and 0.006% for Shell sort.

  4. FIP1/RCP binding to Golgin-97 regulates retrograde transport from recycling endosomes to the trans-Golgi network.

    Science.gov (United States)

    Jing, Jian; Junutula, Jagath R; Wu, Christine; Burden, Jemima; Matern, Hugo; Peden, Andrew A; Prekeris, Rytis

    2010-09-01

    Many proteins are retrieved to the trans-Golgi Network (TGN) from the endosomal system through several retrograde transport pathways to maintain the composition and function of the TGN. However, the molecular mechanisms involved in these distinct retrograde pathways remain to be fully understood. Here we have used fluorescence and electron microscopy as well as various functional transport assays to show that Rab11a/b and its binding protein FIP1/RCP are both required for the retrograde delivery of TGN38 and Shiga toxin from early/recycling endosomes to the TGN, but not for the retrieval of mannose-6-phosphate receptor from late endosomes. Furthermore, by proteomic analysis we identified Golgin-97 as a FIP1/RCP-binding protein. The FIP1/RCP-binding domain maps to the C-terminus of Golgin-97, adjacent to its GRIP domain. Binding of FIP1/RCP to Golgin-97 does not affect Golgin-97 recruitment to the TGN, but appears to regulate the targeting of retrograde transport vesicles to the TGN. Thus, we propose that FIP1/RCP binding to Golgin-97 is required for tethering and fusion of recycling endosome-derived retrograde transport vesicles to the TGN.

  5. Cationic Polymer Intercalation into the Lipid Membrane Enables Intact Polyplex DNA Escape from Endosomes for Gene Delivery.

    Science.gov (United States)

    Vaidyanathan, Sriram; Chen, Junjie; Orr, Bradford G; Banaszak Holl, Mark M

    2016-06-06

    Developing improved cationic polymer-DNA polyplexes for gene delivery requires improved understanding of DNA transport from endosomes into the nucleus. Using a FRET-capable oligonucleotide molecular beacon (OMB), we monitored the transport of intact DNA to cell organelles. We observed that for effective (jetPEI) and ineffective (G5 PAMAM) vectors, the fraction of cells displaying intact OMB in the cytosol (jetPEI ≫ G5 PAMAM) quantitatively predicted the fraction expressing transgene (jetPEI ≫ G5 PAMAM). Intact OMB delivered with PAMAM and confined to endosomes could be released to the cytosol by the subsequent addition of L-PEI, with a corresponding 10-fold increase in transgene expression. These results suggest that future vector development should optimize vectors for intercalation into, and destabilization of, the endosomal membrane. Finally, the study highlights a two-step strategy in which the pDNA is loaded in cells using one vector and endosomal release is mediated by a second agent.

  6. RhoB and the mammalian Diaphanous-related formin mDia2 in endosome trafficking.

    Science.gov (United States)

    Wallar, Bradley J; Deward, Aaron D; Resau, James H; Alberts, Arthur S

    2007-02-01

    Rho GTPases and the dynamic assembly and disassembly of actin filaments have been shown to have critical roles in both the internalization and trafficking of growth factor receptors. While all three mammalian Diaphanous-related (mDia1/2/3) formin GTPase effector proteins have been localized on endosomes, a role for their actin nucleation, filament elongation, and/or bundling remains poorly understood in the context of intracellular trafficking. In a study of a functional relationship between RhoB, a GTPase known to associate with both early- and late-endosomes, and the formin mDia2, we show that 1) RhoB and mDia2 interact on endosomes; 2) GTPase activity-the ability to hydrolyze GTP to GDP-is required for the ability of RhoB to govern endosome dynamics; and 3) the actin dynamics controlled by RhoB and mDia2 is necessary for vesicle trafficking. These studies further suggest that Rho GTPases significantly influence the activity of mDia family formins in driving cellular membrane remodeling through the regulation of actin dynamics.

  7. Kinesin-3 and dynein cooperate in long-range retrograde endosome motility along a nonuniform microtubule array

    NARCIS (Netherlands)

    Schuster, M.; Kilaru, S.; Fink, G.; Collemare, J.A.R.; Roger, Y.; Steinberg, G.

    2011-01-01

    The polarity of microtubules (MTs) determines the motors for intracellular motility, with kinesins moving to plus ends and dynein to minus ends. In elongated cells of Ustilago maydis, dynein is thought to move early endosomes (EEs) toward the septum (retrograde), whereas kinesin-3 transports them to

  8. Characterization of PEBBLEs as a Tool for Real-Time Measurement of Dictyostelium discoideum Endosomal pH

    Directory of Open Access Journals (Sweden)

    Everett Moding

    2009-01-01

    Full Text Available The measurement of intracellular ion concentration change is important for understanding the cellular mechanisms for communication. Recently developed nanosensors, (Photonic Explorers for Biomedical use with Biologically Localized Embedding PEBBLEs, have a number of advantages for measuring ions in cells over established methods using microelectrodes, unbound fluorescent dyes, or NMR. PEBBLE sensors have been shown to work in principle for measuring dynamic ion changes, but few in vivo applications have been demonstrated. We modified the protocol for the fabrication of pH sensing PEBBLEs and developed a protocol for the utilization of these sensors for the monitoring of dynamic pH changes in the endosomes of slime mold Dictyostelium discoideum (D. discoideum. Oregon Green 514-CdSe Quantum Dot PEBBLEs were used to measure real-time pH inside D. discoideum endosomes during cAMP stimulation. Endosomal pH was shown to decrease during cAMP signaling, demonstrating a movement of protons into the endosomes of D. discoideum amoebae.

  9. The protein transportation pathway from Golgi to vacuoles via endosomes plays a role in enhancement of methylmercury toxicity

    Science.gov (United States)

    Hwang, Gi-Wook; Murai, Yasutaka; Takahashi, Tsutomu; Naganuma, Akira

    2014-07-01

    Methylmercury causes serious damage to the central nervous system, but the molecular mechanisms of methylmercury toxicity are only marginally understood. In this study, we used a gene-deletion mutant library of budding yeast to conduct genome-wide screening for gene knockouts affecting the sensitivity of methylmercury toxicity. We successfully identified 31 genes whose deletions confer resistance to methylmercury in yeast, and 18 genes whose deletions confer hypersensitivity to methylmercury. Yeast genes whose deletions conferred resistance to methylmercury included many gene encoding factors involved in protein transport to vacuoles. Detailed examination of the relationship between the factors involved in this transport system and methylmercury toxicity revealed that mutants with loss of the factors involved in the transportation pathway from the trans-Golgi network (TGN) to the endosome, protein uptake into the endosome, and endosome-vacuole fusion showed higher methylmercury resistance than did wild-type yeast. The results of our genetic engineering study suggest that this vesicle transport system (proteins moving from the TGN to vacuole via endosome) is responsible for enhancing methylmercury toxicity due to the interrelationship between the pathways. There is a possibility that there may be proteins in the cell that enhance methylmercury toxicity through the protein transport system.

  10. Kinesin-3 and dynein cooperate in long-range retrograde endosome motility along a nonuniform microtubule array

    NARCIS (Netherlands)

    Schuster, M.; Kilaru, S.; Fink, G.; Collemare, J.A.R.; Roger, Y.; Steinberg, G.

    2011-01-01

    The polarity of microtubules (MTs) determines the motors for intracellular motility, with kinesins moving to plus ends and dynein to minus ends. In elongated cells of Ustilago maydis, dynein is thought to move early endosomes (EEs) toward the septum (retrograde), whereas kinesin-3 transports them to

  11. COMMD1 is linked to the WASH complex and regulates endosomal trafficking of the copper transporter ATP7A.

    Science.gov (United States)

    Phillips-Krawczak, Christine A; Singla, Amika; Starokadomskyy, Petro; Deng, Zhihui; Osborne, Douglas G; Li, Haiying; Dick, Christopher J; Gomez, Timothy S; Koenecke, Megan; Zhang, Jin-San; Dai, Haiming; Sifuentes-Dominguez, Luis F; Geng, Linda N; Kaufmann, Scott H; Hein, Marco Y; Wallis, Mathew; McGaughran, Julie; Gecz, Jozef; Sluis, Bart van de; Billadeau, Daniel D; Burstein, Ezra

    2015-01-01

    COMMD1 deficiency results in defective copper homeostasis, but the mechanism for this has remained elusive. Here we report that COMMD1 is directly linked to early endosomes through its interaction with a protein complex containing CCDC22, CCDC93, and C16orf62. This COMMD/CCDC22/CCDC93 (CCC) complex interacts with the multisubunit WASH complex, an evolutionarily conserved system, which is required for endosomal deposition of F-actin and cargo trafficking in conjunction with the retromer. Interactions between the WASH complex subunit FAM21, and the carboxyl-terminal ends of CCDC22 and CCDC93 are responsible for CCC complex recruitment to endosomes. We show that depletion of CCC complex components leads to lack of copper-dependent movement of the copper transporter ATP7A from endosomes, resulting in intracellular copper accumulation and modest alterations in copper homeostasis in humans with CCDC22 mutations. This work provides a mechanistic explanation for the role of COMMD1 in copper homeostasis and uncovers additional genes involved in the regulation of copper transporter recycling.

  12. COMMD1 is linked to the WASH complex and regulates endosomal trafficking of the copper transporter ATP7A

    NARCIS (Netherlands)

    Phillips-Krawczak, Christine A.; Singla, Amika; Starokadomskyy, Petro; Deng, Zhihui; Osborne, Douglas G.; Li, Haiying; Dick, Christopher J.; Gomez, Timothy S.; Koenecke, Megan; Zhang, Jin-San; Dai, Haiming; Sifuentes-Dominguez, Luis F.; Geng, Linda N.; Kaufmann, Scott H.; Hein, Marco Y.; Wallis, Mathew; McGaughran, Julie; Gecz, Jozef; De Sluis, Bart van; Billadeau, Daniel D.; Burstein, Ezra

    2015-01-01

    COMMD1 deficiency results in defective copper homeostasis, but the mechanism for this has remained elusive. Here we report that COMMD1 is directly linked to early endosomes through its interaction with a protein complex containing CCDC22, CCDC93, and C16orf62. This COMMD/CCDC22/CCDC93 (CCC) complex

  13. Dendronized Mesoporous Silica Nanoparticles Provide an Internal Endosomal Escape Mechanism for Successful Cytosolic Drug Release

    CERN Document Server

    Weiss, Veronika; Torrano, Adriano A; Strobel, Claudia; Mackowiak, Stephan A; Gatzenmeier, Tim; Hilger, Ingrid; Braeuchle, Christoph; Bein, Thomas

    2015-01-01

    Mesoporous silica nanoparticles (MSNs) attract increasing interest in the field of gene and drug delivery due to their versatile features as a multifunctional drug delivery platform. Here, we describe poly(amidoamine) (PAMAM) dendron-functionalized MSNs that fulfill key prerequisites for a controllable intracellular drug release. In addition to high loading capacity, they offer 1) low cytotoxicity, showing no impact on the metabolism of endothelial cells, 2) specific cancer cell targeting due to receptor-mediated cell uptake, 3) a redox-driven cleavage of disulfide bridges allowing for stimuli-responsive cargo release, and most importantly, 4) a specific internal trigger based on the high buffering capacity of PAMAM dendrons to provide endosomal escape.

  14. The late endosomal adaptor p14 is a macrophage host-defense factor against Salmonella infection.

    Science.gov (United States)

    Taub, Nicole; Nairz, Manfred; Hilber, Diana; Hess, Michael W; Weiss, Günter; Huber, Lukas A

    2012-06-01

    The outcome of an infection depends on the balance between host resistance and bacterial virulence. Here, we show that the late endosomal adaptor p14 (also known as LAMTOR2) is one of the components for cellular host defense against the intracellular pathogen Salmonella enterica serovar Typhimurium. During Salmonella infection, the complex of p14 and MP1 is required for the accurately timed transport of Salmonella through the endolysosomal system. Loss of p14 opens a time window that allows Salmonella to populate a replication niche, in which early and late antimicrobial effector systems, comprising NADPH phagocytic oxidase and inducible nitric oxide synthase, respectively, are inappropriately activated. Thus, p14 supports the accurate transport of Salmonella through the endolysosomal system, thereby limiting bacterial replication in both, professional phagocytes and in non-phagocytic cells in vitro, and helps mice to successfully battle Salmonella infection in vivo.

  15. Clathrin and AP1B: Key roles in basolateral trafficking through trans-endosomal routes

    Science.gov (United States)

    Gonzalez, Alfonso; Rodriguez-Boulan, Enrique

    2013-01-01

    Research following introduction of the MDCK model system to study epithelial polarity (1978) led to an initial paradigm that posited independent roles of the trans Golgi network (TGN) and recycling endosomes (RE) in the generation of, respectively, biosynthetic and recycling routes of plasma membrane (PM) proteins to apical and basolateral PM domains. This model dominated the field for 20 years. However, studies over the past decade and the discovery of the involvement of clathrin and clathrin adaptors in protein trafficking to the basolateral PM has led to a new paradigm. TGN and RE are now believed to cooperate closely in both biosynthetic and recycling trafficking routes. Here, we critically review these recent advances and the questions that remain unanswered. PMID:19854182

  16. Atg8 is involved in endosomal and phagosomal acidification in the parasitic protist Entamoeba histolytica.

    Science.gov (United States)

    Picazarri, Karina; Nakada-Tsukui, Kumiko; Tsuboi, Kumiko; Miyamoto, Eri; Watanabe, Naoko; Kawakami, Eiryo; Nozaki, Tomoyoshi

    2015-10-01

    Autophagy is one of two major bulk protein degradation systems and is conserved throughout eukaryotes. The protozoan Entamoeba histolytica, which is a human intestinal parasite, possesses a restricted set of autophagy-related (Atg) proteins compared with other eukaryotes and thus represents a suitable model organism for studying the minimal essential components and ancestral functions of autophagy. E. histolytica possesses two conjugation systems: Atg8 and Atg5/12, although a gene encoding Atg12 is missing in the genome. Atg8 is considered to be the central and authentic marker of autophagosomes, but recent studies have demonstrated that Atg8 is not exclusively involved in autophagy per se, but other fundamental mechanisms of vesicular traffic. To investigate this question in E. histolytica, we studied on Atg8 during the proliferative stage. Atg8 was constitutively expressed in both laboratory-maintained and recently established clinical isolates and appeared to be lipid-modified in logarithmic growth phase, suggesting a role of Atg8 in non-stress and proliferative conditions. These findings are in contrast to those for Entamoeba invadens, in which autophagy is markedly induced during an early phase of differentiation from the trophozoite into the cyst. The repression of Atg8 gene expression in En. histolytica by antisense small RNA-mediated transcriptional gene silencing resulted in growth retardation, delayed endocytosis and reduced acidification of endosomes and phagosomes. Taken together, these results suggest that Atg8 and the Atg8 conjugation pathway have some roles in the biogenesis of endosomes and phagosomes in this primitive eukaryote.

  17. Presenilin 1 regulates epidermal growth factor receptor turnover and signaling in the endosomal-lysosomal pathway.

    Science.gov (United States)

    Repetto, Emanuela; Yoon, Il-Sang; Zheng, Hui; Kang, David E

    2007-10-26

    Mutations in the gene encoding presenilin 1 (PS1) cause the most aggressive form of early-onset familial Alzheimer disease. In addition to its well established role in Abeta production and Notch proteolysis, PS1 has been shown to mediate other physiological activities, such as regulation of the Wnt/beta-catenin signaling pathway, modulation of phosphatidylinositol 3-kinase/Akt and MEK/ERK signaling, and trafficking of select membrane proteins and/or intracellular vesicles. In this study, we present evidence that PS1 is a critical regulator of a key signaling receptor tyrosine kinase, epidermal growth factor receptor (EGFR). Specifically, EGFR levels were robustly increased in fibroblasts deficient in both PS1 and PS2 (PS(-/-)) due to delayed turnover of EGFR protein. Stable transfection of wild-type PS1 but not PS2 corrected EGFR to levels comparable to PS(+/+) cells, while FAD PS1 mutations showed partial loss of activity. The C-terminal fragment of PS1 was sufficient to fully reduce EGFR levels. In addition, the rapid ligand-induced degradation of EGFR was markedly delayed in PS(-/-) cells, resulting in prolonged signal activation. Despite the defective turnover of EGFR, ligand-induced autophosphorylation, ubiquitination, and endocytosis of EGFR were not affected by the lack of PS1. Instead, the trafficking of EGFR from early endosomes to lysosomes was severely delayed by PS1 deficiency. Elevation of EGFR was also seen in brains of adult mice conditionally ablated in PS1 and in skin tumors associated with the loss of PS1. These findings demonstrate a critical role of PS1 in the trafficking and turnover of EGFR and suggest potential pathogenic effects of elevated EGFR as well as perturbed endosomal-lysosomal trafficking in cell cycle control and Alzheimer disease.

  18. Targeted in vivo delivery of siRNA and an endosome-releasing agent to hepatocytes.

    Science.gov (United States)

    Sebestyén, Magdolna G; Wong, So C; Trubetskoy, Vladimir; Lewis, David L; Wooddell, Christine I

    2015-01-01

    The discoveries of RNA interference (RNAi) and short interfering RNAs (siRNAs) have provided the opportunity to treat diseases in a fundamentally new way: by co-opting a natural process to inhibit gene expression at the mRNA level. Given that siRNAs must interact with the cells' natural RNAi machinery in order to exert their silencing effect, one of the most fundamental requirements for their use is efficient delivery to the desired cell type and, specifically, into the cytoplasm of those cells. Numerous research efforts involving the testing of a large number of delivery approaches using various carrier molecules and inventing several distinct formulation technologies during the past decade illustrate the difficulty and complexity of this task. We have developed synthetic polymer formulations for in vivo siRNA delivery named Dynamic PolyConjugates™ (DPCs) that are designed to mimic the features viruses possess for efficient delivery of their nucleic acids. These include small size, long half-life in circulation, capability of displaying distinct host cell tropism, efficient receptor binding and cell entry, disassembly in the endosome and subsequent release of the nucleic acid cargo to the cytoplasm. Here we present an example of this delivery platform composed of a hepatocyte-targeted endosome-releasing agent and a cholesterol-conjugated siRNA (chol-siRNA). This delivery platform forms the basis of ARC-520, an siRNA-based therapeutic for the treatment of chronic hepatitis B virus (HBV) infection. In this chapter, we provide a general overview of the steps in developing ARC-520 and detailed protocols for two critical stages of the discovery process: (1) verifying targeted in vivo delivery to hepatocytes and (2) evaluating in vivo drug efficacy using a mouse model of chronic HBV infection.

  19. Microfluidic train station: highly robust and multiplexable sorting of droplets on electric rails.

    Science.gov (United States)

    Frenzel, Daniel; Merten, Christoph A

    2017-02-24

    Fluorescence-activated droplet sorting (FADS) has become a widely used technique for high-throughput screening applications. However, existing methods are very sensitive to fluctuating flow rates at the sorting junction, which can be caused by the pulsing effects of mechanical pumps, droplet aggregates or the accumulation of precipitates during lengthy biological screening applications. Furthermore, existing sorting devices allow only 2-way sorting. We present here a dielectrophoretic sorting system in which the droplets are sorted along multiple electrode pairs that run parallel to the channels. This enables highly reliable sorting (no errors were detected for more than 2000 sorting events) even when inverting the relative flow rates at a 2-way sorting junction from 80 : 20 to 20 : 80. Furthermore, our toolbox is scalable: we demonstrate on the example of a triple-colour sorting experiment with a total of four decoupled electrodes that multi-way sorting is feasible.

  20. Conserved V-ATPase c subunit plays a role in plant growth by influencing V-ATPase-dependent endosomal trafficking.

    Science.gov (United States)

    Zhou, Aimin; Bu, Yuanyuan; Takano, Tetsuo; Zhang, Xinxin; Liu, Shenkui

    2016-01-01

    In plant cells, the vacuolar-type H(+)-ATPases (V-ATPase) are localized in the tonoplast, Golgi, trans-Golgi network and endosome. However, little is known about how V-ATPase influences plant growth, particularly with regard to the V-ATPase c subunit (VHA-c). Here, we characterized the function of a VHA-c gene from Puccinellia tenuiflora (PutVHA-c) in plant growth. Compared to the wild-type, transgenic plants overexpressing PutVHA-c in Arabidopsis thaliana exhibit better growth phenotypes in root length, fresh weight, plant height and silique number under the normal and salt stress conditions due to noticeably higher V-ATPase activity. Consistently, the Arabidopsis atvha-c5 mutant shows reduced V-ATPase activity and retarded plant growth. Furthermore, confocal and immunogold electron microscopy assays demonstrate that PutVHA-c is mainly localized to endosomal compartments. The treatment of concanamycin A (ConcA), a specific inhibitor of V-ATPases, leads to obvious aggregation of the endosomal compartments labelled with PutVHA-c-GFP. Moreover, ConcA treatment results in the abnormal localization of two plasma membrane (PM) marker proteins Pinformed 1 (AtPIN1) and regulator of G protein signalling-1 (AtRGS1). These findings suggest that the decrease in V-ATPase activity blocks endosomal trafficking. Taken together, our results strongly suggest that the PutVHA-c plays an important role in plant growth by influencing V-ATPase-dependent endosomal trafficking.

  1. pH regulation in early endosomes and interferon-inducible transmembrane proteins control avian retrovirus fusion.

    Science.gov (United States)

    Desai, Tanay M; Marin, Mariana; Mason, Caleb; Melikyan, Gregory B

    2017-05-12

    Enveloped viruses infect host cells by fusing their membranes with those of the host cell, a process mediated by viral glycoproteins upon binding to cognate host receptors or entering into acidic intracellular compartments. Whereas the effect of receptor density on viral infection has been well studied, the role of cell type-specific factors/processes, such as pH regulation, has not been characterized in sufficient detail. Here, we examined the effects of cell-extrinsic factors (buffer environment) and cell-intrinsic factors (interferon-inducible transmembrane proteins, IFITMs), on the pH regulation in early endosomes and on the efficiency of acid-dependent fusion of the avian sarcoma and leukosis virus (ASLV), with endosomes. First, we found that a modest elevation of external pH can raise the pH in early endosomes in a cell type-dependent manner and thereby delay the acid-induced fusion of endocytosed ASLV. Second, we observed a cell type-dependent delay between the low pH-dependent and temperature-dependent steps of viral fusion, consistent with the delayed enlargement of the fusion pore. Third, ectopic expression of IFITMs, known to potently block influenza virus fusion with late compartments, was found to only partially inhibit ASLV fusion with early endosomes. Interestingly, IFITM expression promoted virus uptake and the acidification of endosomal compartments, resulting in an accelerated fusion rate when driven by the glycosylphosphatidylinositol-anchored, but not by the transmembrane isoform of the ASLV receptor. Collectively, these results highlight the role of cell-extrinsic and cell-intrinsic factors in regulating the efficiency and kinetics of virus entry and fusion with target cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Brucella abortus Cell Cycle and Infection Are Coordinated.

    Science.gov (United States)

    De Bolle, Xavier; Crosson, Sean; Matroule, Jean-Yves; Letesson, Jean-Jacques

    2015-12-01

    Brucellae are facultative intracellular pathogens. The recent development of methods and genetically engineered strains allowed the description of cell-cycle progression of Brucella abortus, including unipolar growth and the ordered initiation of chromosomal replication. B. abortus cell-cycle progression is coordinated with intracellular trafficking in the endosomal compartments. Bacteria are first blocked at the G1 stage, growth and chromosome replication being resumed shortly before reaching the intracellular proliferation compartment. The control mechanisms of cell cycle are similar to those reported for the bacterium Caulobacter crescentus, and they are crucial for survival in the host cell. The development of single-cell analyses could also be applied to other bacterial pathogens to investigate their cell-cycle progression during infection.

  3. Neuronal spike sorting based on radial basis function neural networks

    Directory of Open Access Journals (Sweden)

    Taghavi Kani M

    2011-02-01

    Full Text Available "nBackground: Studying the behavior of a society of neurons, extracting the communication mechanisms of brain with other tissues, finding treatment for some nervous system diseases and designing neuroprosthetic devices, require an algorithm to sort neuralspikes automatically. However, sorting neural spikes is a challenging task because of the low signal to noise ratio (SNR of the spikes. The main purpose of this study was to design an automatic algorithm for classifying neuronal spikes that are emitted from a specific region of the nervous system."n "nMethods: The spike sorting process usually consists of three stages: detection, feature extraction and sorting. We initially used signal statistics to detect neural spikes. Then, we chose a limited number of typical spikes as features and finally used them to train a radial basis function (RBF neural network to sort the spikes. In most spike sorting devices, these signals are not linearly discriminative. In order to solve this problem, the aforesaid RBF neural network was used."n "nResults: After the learning process, our proposed algorithm classified any arbitrary spike. The obtained results showed that even though the proposed Radial Basis Spike Sorter (RBSS reached to the same error as the previous methods, however, the computational costs were much lower compared to other algorithms. Moreover, the competitive points of the proposed algorithm were its good speed and low computational complexity."n "nConclusion: Regarding the results of this study, the proposed algorithm seems to serve the purpose of procedures that require real-time processing and spike sorting.

  4. Spike sorting for polytrodes: a divide and conquer approach

    Directory of Open Access Journals (Sweden)

    Nicholas V. Swindale

    2014-02-01

    Full Text Available In order to determine patterns of neural activity, spike signals recorded by extracellular electrodes have to be clustered (sorted with the aim of ensuring that each cluster represents all the spikes generated by an individual neuron. Many methods for spike sorting have been proposed but few are easily applicable to recordings from polytrodes which may have 16 or more recording sites. As with tetrodes, these are spaced sufficiently closely that signals from single neurons will usually be recorded on several adjacent sites. Although this offers a better chance of distinguishing neurons with similarly shaped spikes, sorting is difficult in such cases because of the high dimensionality of the space in which the signals must be classified. This report details a method for spike sorting based on a divide and conquer approach. Clusters are initially formed by assigning each event to the channel on which it is largest. Each channel-based cluster is then sub-divided into as many distinct clusters as possible. These are then recombined on the basis of pairwise tests into a final set of clusters. Pairwise tests are also performed to establish how distinct each cluster is from the others. A modified gradient ascent clustering (GAC algorithm is used to do the clustering. The method can sort spikes with minimal user input in times comparable to real time for recordings lasting up to 45 minutes. Our results illustrate some of the difficulties inherent in spike sorting, including changes in spike shape over time. We show that some physiologically distinct units may have very similar spike shapes. We show that RMS measures of spike shape similarity are not sensitive enough to discriminate clusters that can otherwise be separated by principal components analysis. Hence spike sorting based on least-squares matching to templates may be unreliable. Our methods should be applicable to tetrodes and scaleable to larger multi-electrode arrays (MEAs.

  5. COMPRESSIBLE VIRTUAL WINDOW ALGORITHM IN PICKING PROCESS CONTROL OF AUTOMATED SORTING SYSTEM

    Institute of Scientific and Technical Information of China (English)

    WU Yaohua; ZHANG Yigong; WU Yingying

    2008-01-01

    Compared to fixed virtual window algorithm (FVWA), the dynamic virtual window algorithm (DVWA) determines the length of each virtual container according to the sizes of goods of each order, which saves space of virtual containers and improves the picking efficiency. However, the interval of consecutive goods caused by dispensers on conveyor can not be eliminated by DVWA, which limits a further improvement of picking efficiency. In order to solve this problem, a compressible virtual window algorithm (CVWA) is presented. It not only inherits the merit of DVWA but also compresses the length of virtual containers without congestion of order accumulation by advancing the beginning time of order picking and reasonably coordinating the pace of order accumulation. The simulation result proves that the picking efficiency of automated sorting system is greatly improved by CVWA.

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

    Science.gov (United States)

    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. Copyright © 2014. Published by Elsevier Inc.

  7. An Exploratory Study of Critical Factors Affecting the Efficiency of Sorting Techniques (Shell, Heap and Treap)

    CERN Document Server

    Folorunso, Olusegun; Salako, Oluwatimilehin

    2012-01-01

    The efficiency of sorting techniques has a significant impact on the overall efficiency of a program. The efficiency of Shell, Heap and Treap sorting techniques in terms of both running time and memory usage was studied, experiments conducted and results subjected to factor analysis by SPSS. The study revealed the main factor affecting these sorting techniques was time taken to sort.

  8. Rac1-Rab11-FIP3 regulatory hub coordinates vesicle traffic with actin remodeling and T-cell activation.

    Science.gov (United States)

    Bouchet, Jérôme; Del Río-Iñiguez, Iratxe; Lasserre, Rémi; Agüera-Gonzalez, Sonia; Cuche, Céline; Danckaert, Anne; McCaffrey, Mary W; Di Bartolo, Vincenzo; Alcover, Andrés

    2016-06-01

    The immunological synapse generation and function is the result of a T-cell polarization process that depends on the orchestrated action of the actin and microtubule cytoskeleton and of intracellular vesicle traffic. However, how these events are coordinated is ill defined. Since Rab and Rho families of GTPases control intracellular vesicle traffic and cytoskeleton reorganization, respectively, we investigated their possible interplay. We show here that a significant fraction of Rac1 is associated with Rab11-positive recycling endosomes. Moreover, the Rab11 effector FIP3 controls Rac1 intracellular localization and Rac1 targeting to the immunological synapse. FIP3 regulates, in a Rac1-dependent manner, key morphological events, like T-cell spreading and synapse symmetry. Finally, Rab11-/FIP3-mediated regulation is necessary for T-cell activation leading to cytokine production. Therefore, Rac1 endosomal traffic is key to regulate T-cell activation.

  9. Stigmergy, self-organization, and sorting in collective robotics.

    Science.gov (United States)

    Holland, O; Melhuish, C

    1999-01-01

    Many structures built by social insects are the outcome of a process of self-organization, in which the repeated actions of the insects interact over time with the changing physical environment to produce a characteristic end state. A major mediating factor is stigmergy, the elicitation of specific environment-changing behaviors by the sensory effects of local environmental changes produced by previous behavior. A typical task involving stigmergic self-organization is brood sorting: Many ant species sort their brood so that items at similar stages of development are grouped together and separated from items at different stages of development. This article examines the operation of stigmergy and self-organization in a homogeneous group of physical robots, in the context of the task of clustering and sorting Frisbees of two different types. Using a behavioral rule set simpler than any yet proposed for ant sorting, and having no capacity for spatial orientation or memory, the robots are able to achieve effective clustering and sorting showing all the signs of self-organization. It is argued that the success of this demonstration is crucially dependent on the exploitation of real-world physics, and that the use of simulation alone to investigate stigmergy may fail to reveal its power as an evolutionary option for collective life forms.

  10. A Sort-Last Rendering System over an Optical Backplane

    Directory of Open Access Journals (Sweden)

    Yasuhiro Kirihata

    2005-06-01

    Full Text Available Sort-Last is a computer graphics technique for rendering extremely large data sets on clusters of computers. Sort-Last works by dividing the data set into even-sized chunks for parallel rendering and then composing the images to form the final result. Since sort-last rendering requires the movement of large amounts of image data among cluster nodes, the network interconnecting the nodes becomes a major bottleneck. In this paper, we describe a sort-last rendering system implemented on a cluster of computers whose nodes are connected by an all-optical switch. The rendering system introduces the notion of the Photonic Computing Engine, a computing system built dynamically by using the optical switch to create dedicated network connections among cluster nodes. The sort-last volume rendering algorithm was implemented on the Photonic Computing Engine, and its performance is evaluated. Prelimi- nary experiments show that performance is affected by the image composition time and average payload size. In an attempt to stabilize the performance of the system, we have designed a flow control mechanism that uses feedback messages to dynamically adjust the data flow rate within the computing engine.

  11. Mechanically robust microfluidics and bulk wave acoustics to sort microparticles

    Science.gov (United States)

    Dauson, Erin R.; Gregory, Kelvin B.; Greve, David W.; Healy, Gregory P.; Oppenheim, Irving J.

    2016-04-01

    Sorting microparticles (or cells, or bacteria) is significant for scientific, medical and industrial purposes. Research groups have used lithium niobate SAW devices to produce standing waves, and then to align microparticles at the node lines in polydimethylsiloxane (PDMS, silicone) microfluidic channels. The "tilted angle" (skewed) configuration is a recent breakthrough producing particle trajectories that cross multiple node lines, making it practical to sort particles. However, lithium niobate wafers and PDMS microfluidic channels are not mechanically robust. We demonstrate "tilted angle" microparticle sorting in novel devices that are robust, rapidly prototyped, and manufacturable. We form our microfluidic system in a rigid polymethyl methacrylate (PMMA, acrylic) prism, sandwiched by lead-zirconium-titanate (PZT) wafers, operating in through-thickness mode with inertial backing, that produce standing bulk waves. The overall configuration is compact and mechanically robust, and actuating PZT wafers in through-thickness mode is highly efficient. Moving to this novel configuration introduced new acoustics questions involving internal reflections, but we show experimental images confirming the intended nodal geometry. Microparticles in "tilted angle" devices display undulating trajectories, where deviation from the straight path increases with particle diameter and with excitation voltage to create the mechanism by which particles are sorted. We show a simplified analytical model by which a "phase space" is constructed to characterize effective particle sorting, and we compare our experimental data to the predictions from that simplified model; precise correlation is not expected and is not observed, but the important physical trends from the model are paralleled in the measured particle trajectories.

  12. Enhanced time overcurrent coordination

    Energy Technology Data Exchange (ETDEWEB)

    Enriquez, Arturo Conde; Martinez, Ernesto Vazquez [Universidad Autonoma de Nuevo Leon, Facultad de Ingenieria Mecanica y Electrica, Apdo. Postal 114-F, Ciudad Universitaria, CP 66450 San Nicolas de los Garza, Nuevo Leon (Mexico)

    2006-04-15

    In this paper, we recommend a new coordination system for time overcurrent relays. The purpose of the coordination process is to find a time element function that allows it to operate using a constant back-up time delay, for any fault current. In this article, we describe the implementation and coordination results of time overcurrent relays, fuses and reclosers. Experiments were carried out in a laboratory test situation using signals of a power electrical system physics simulator. (author)

  13. A microfluidic device to sort capsules by deformability

    CERN Document Server

    Zhu, L; Mitra, Dhrubaditya; Brandt, Luca

    2014-01-01

    Guided by extensive numerical simulations, we propose a microfluidic device that can sort elastic capsules by their deformability. The device consists of a duct embedded with a semi-cylindrical obstacle, and a diffuser which further enhances the sorting capability. We demonstrate that the device can operate reasonably well under changes in the initial position of the the capsule. The efficiency of the device remains essentially unaltered under small changes of the obstacle shape (from semi-circular to semi-elliptic cross-section). Confinement along the direction perpendicular to the plane of the device increases its efficiency. This work is the first numerical study of cell sorting by a realistic microfluidic device.

  14. System for sorting microscopic objects using electromagnetic radiation

    DEFF Research Database (Denmark)

    2013-01-01

    There is presented a system 10,100 for sorting microscopic objects 76, 78, 80, where the system comprises a fluid channel 66 with an inlet 68 and an outlet 70, where the fluid channel is arranged for allowing the fluid flow to be laminar. The system furthermore comprises a detection system 52 which...... enables detecting microscopic objects in the fluid channel and furthermore enables determining their position. The system furthermore comprises a controller 67, such as a computer, which receives the positions and accordingly controls a source of light beams so as to "shoot" light beams towards selected...... microscopic objects so as to "push" them into a new position. The system thereby enables sorting the selected microscopic objects. In more specific embodiments, the detection system furthermore assigns different categories to different microscopic objects, so as to enable sorting based on multiple categories....

  15. Efficient searching and sorting applications using an associative array processor

    Science.gov (United States)

    Pace, W.; Quinn, M. J.

    1978-01-01

    The purpose of this paper is to describe a method of searching and sorting data by using some of the unique capabilities of an associative array processor. To understand the application, the associative array processor is described in detail. In particular, the content addressable memory and flip network are discussed because these two unique elements give the associative array processor the power to rapidly sort and search. A simple alphanumeric sorting example is explained in hardware and software terms. The hardware used to explain the application is the STARAN (Goodyear Aerospace Corporation) associative array processor. The software used is the APPLE (Array Processor Programming Language) programming language. Some applications of the array processor are discussed. This summary tries to differentiate between the techniques of the sequential machine and the associative array processor.

  16. A Novel Auto-Sorting System for Chinese Cabbage Seeds

    Directory of Open Access Journals (Sweden)

    Kuo-Yi Huang

    2017-04-01

    Full Text Available This paper presents a novel machine vision-based auto-sorting system for Chinese cabbage seeds. The system comprises an inlet-outlet mechanism, machine vision hardware and software, and control system for sorting seed quality. The proposed method can estimate the shape, color, and textural features of seeds that are provided as input neurons of neural networks in order to classify seeds as “good” and “not good” (NG. The results show the accuracies of classification to be 91.53% and 88.95% for good and NG seeds, respectively. The experimental results indicate that Chinese cabbage seeds can be sorted efficiently using the developed system.

  17. Particle migration and sorting in microbubble streaming flows

    Science.gov (United States)

    Thameem, Raqeeb; Hilgenfeldt, Sascha

    2016-01-01

    Ultrasonic driving of semicylindrical microbubbles generates strong streaming flows that are robust over a wide range of driving frequencies. We show that in microchannels, these streaming flow patterns can be combined with Poiseuille flows to achieve two distinctive, highly tunable methods for size-sensitive sorting and trapping of particles much smaller than the bubble itself. This method allows higher throughput than typical passive sorting techniques, since it does not require the inclusion of device features on the order of the particle size. We propose a simple mechanism, based on channel and flow geometry, which reliably describes and predicts the sorting behavior observed in experiment. It is also shown that an asymptotic theory that incorporates the device geometry and superimposed channel flow accurately models key flow features such as peak speeds and particle trajectories, provided it is appropriately modified to account for 3D effects caused by the axial confinement of the bubble. PMID:26958103

  18. Sorting, Searching, and Simulation in the MapReduce Framework

    DEFF Research Database (Denmark)

    Goodrich, Michael T.; Sitchinava, Nodari; Zhang, Qin

    2011-01-01

    usefulness of our approach by designing and analyzing efficient MapReduce algorithms for fundamental sorting, searching, and simulation problems. This study is motivated by a goal of ultimately putting the MapReduce framework on an equal theoretical footing with the well-known PRAM and BSP parallel...... models, which would benefit both the theory and practice of MapReduce algorithms. We describe efficient MapReduce algorithms for sorting, multi-searching, and simulations of parallel algorithms specified in the BSP and CRCW PRAM models. We also provide some applications of these results to problems...... in parallel computational geometry for the MapReduce framework, which result in efficient MapReduce algorithms for sorting, 2- and 3-dimensional convex hulls, and fixed-dimensional linear programming. For the case when mappers and reducers have a memory/message-I/O size of M = (N), for a small constant > 0...

  19. Sorting of bending magnets for the SSRF booster

    Institute of Scientific and Technical Information of China (English)

    HOU Jie; LIU Gui-Min; LI Hao-Hu; ZHANG Man-Zhou

    2008-01-01

    The Shanghai Synchrotron Radiation Facility(SSRF)booster ring,a full energy injector for the storage ring,is deigned to accelerate the electron beam energy from 150MeV to 3.5GeV that demands high extraction efficiency at the extraction energy with low beam loss rate when electrons are ramping.Closed orbit distortion(COD)caused by bending magnet field uniformity errors which affects the machine performance harmfully could be effectively reduced by bending magnet location sorting.Considering the affections of random errors in measurement,both ideal sorting and realistic sorting are studied based on measured bending magnet field uniformity errors and one reasonable combination of bending magnets which can reduce the horizontal COD by a factor of 5is given as the final installation sequence of the booster bending magnets in this paper.

  20. Sorting of bending magnets for the SSRF booster

    Science.gov (United States)

    Hou, Jie; Liu, Gui-Min; Li, Hao-Hu; Zhang, Man-Zhou

    2008-04-01

    The Shanghai Synchrotron Radiation Facility (SSRF)booster ring, a full energy injector for the storage ring, is deigned to accelerate the electron beam energy from 150 MeV to 3.5 GeV that demands high extraction efficiency at the extraction energy with low beam loss rate when electrons are ramping. Closed orbit distortion (COD) caused by bending magnet field uniformity errors which affects the machine performance harmfully could be effectively reduced by bending magnet location sorting. Considering the affections of random errors in measurement, both ideal sorting and realistic sorting are studied based on measured bending magnet field uniformity errors and one reasonable combination of bending magnets which can reduce the horizontal COD by a factor of 5 is given as the final installation sequence of the booster bending magnets in this paper. Supported by SSRF Project

  1. Scalable orbital-angular-momentum sorting without destroying photon states

    CERN Document Server

    Wang, Fang-Xiang; Yin, Zhen-Qiang; Wang, Shuang; Guo, Guang-Can; Han, Zheng-Fu

    2016-01-01

    Single photons with orbital angular momentum (OAM) have attracted substantial attention from researchers. A single photon can carry infinite OAM values theoretically. Thus, OAM photon states have been widely used in quantum information and fundamental quantum mechanics. Although there have been many methods for sorting quantum states with different OAM values, the nondestructive and efficient sorter of high-dimensional OAM remains a fundamental challenge. Here, we propose a scalable OAM sorter which can categorize different OAM states simultaneously, meanwhile, preserving both OAM and spin angular momentum. Fundamental elements of the sorter are composed of symmetric multiport beam splitters (BSs) and Dove prisms with cascading structure, which in principle can be flexibly and effectively combined to sort arbitrarily high-dimensional OAM photons. The scalable structures proposed here greatly reduce the number of BSs required for sorting high-dimensional OAMstates. In view of the nondestructive and extensible ...

  2. Cholesterol-induced protein sorting: an analysis of energetic feasibility

    DEFF Research Database (Denmark)

    Lundbaek, J A; Andersen, O S; Werge, T;

    2003-01-01

    transmembrane domain (TMD). M. S. Bretscher and S. Munro (SCIENCE: 261:1280-1281, 1993) therefore proposed a physical sorting mechanism based on the hydrophobic match between the proteins' TMD and the bilayer thickness, in which cholesterol would regulate protein sorting by increasing the lipid bilayer...... thickness. In this model, Golgi proteins with short TMDs would be excluded from cholesterol-enriched domains (lipid rafts) that are incorporated into transport vesicles destined for the plasma membrane. Although attractive, this model remains unproven. We therefore evaluated the energetic feasibility...... thickness per se, however, have only a modest effect on sorting; the major effect arises because cholesterol changes also the bilayer material properties, which augments the energetic penalty for incorporating short TMDs into cholesterol-enriched domains. We conclude that cholesterol-induced changes...

  3. Cognitive impairments of aphasics in picture sorting and matching tasks.

    Science.gov (United States)

    Cohen, R; Glöckner-Rist, A; Lutz, M; Maier, T; Meier, E

    1982-01-01

    On the basis of earlier experiments showing a differential deficit of aphasics in picture sorting and matching tasks, two experiments were conducted to test the conjecture of a specific deficit of aphasics in the analytical appraisal of individual features. Broca's and Wernicke's aphasics--according to clinical diagnoses and the Aachener Aphasie Test--were compared with patients having right-hemisphere lesions or left-hemisphere lesions without aphasia. Both groups of aphasics differed from the control groups in the sorting task, irrespective of the sorting criterion, but the differences were small. The picture matching task did not discriminate between groups. Obviously, the basic assumption has to be modified with respect to specific conditions of task requirements. The experimental literature is reviewed.

  4. "Clothed in triple blues": sorting out the Italian blues.

    Science.gov (United States)

    Bimler, David; Uusküla, Mari

    2014-04-01

    Cross-cultural comparisons of color perception and cognition often feature versions of the "similarity sorting" procedure. By interpreting the assignment of two color samples to different groups as an indication that the dissimilarity between them exceeds some threshold, sorting data can be regarded as low-resolution similarity judgments. Here we analyze sorting data from speakers of Italian, Russian, and English, applying multidimensional scaling to delineate the boundaries between perceptual categories while highlighting differences between the three populations. Stimuli were 55 color swatches, predominantly from the blue region. Results suggest that at least two Italian words for "blue" are basic, a similar situation to Russian, in contrast to English where a single "blue" term is basic.

  5. Particle migration and sorting in microbubble streaming flows.

    Science.gov (United States)

    Thameem, Raqeeb; Rallabandi, Bhargav; Hilgenfeldt, Sascha

    2016-01-01

    Ultrasonic driving of semicylindrical microbubbles generates strong streaming flows that are robust over a wide range of driving frequencies. We show that in microchannels, these streaming flow patterns can be combined with Poiseuille flows to achieve two distinctive, highly tunable methods for size-sensitive sorting and trapping of particles much smaller than the bubble itself. This method allows higher throughput than typical passive sorting techniques, since it does not require the inclusion of device features on the order of the particle size. We propose a simple mechanism, based on channel and flow geometry, which reliably describes and predicts the sorting behavior observed in experiment. It is also shown that an asymptotic theory that incorporates the device geometry and superimposed channel flow accurately models key flow features such as peak speeds and particle trajectories, provided it is appropriately modified to account for 3D effects caused by the axial confinement of the bubble.

  6. Basic Study on Color Sorting of Fresh Market Tomatoes

    OpenAIRE

    Mohri, Kentaro; UMEDA, Shigeo; TSURUMI, Gaku

    1987-01-01

    The surface colors of tomatoes are one of decision factor for the ripeness rate of fruits, and that is mainly graded by the human sense in fact. The color sorting based on the surface colors of tomatoes was attemped by using an opto-electronic system consisting of the color sensors. The sample tomatoes of each ripeness rate were prepared and tested by the color sorting system. As the ripeness rate of tomatoes develops from unripe to ripe, the surface colors change from green to pink or red. W...

  7. Decision trees with minimum average depth for sorting eight elements

    KAUST Repository

    AbouEisha, Hassan

    2015-11-19

    We prove that the minimum average depth of a decision tree for sorting 8 pairwise different elements is equal to 620160/8!. We show also that each decision tree for sorting 8 elements, which has minimum average depth (the number of such trees is approximately equal to 8.548×10^326365), has also minimum depth. Both problems were considered by Knuth (1998). To obtain these results, we use tools based on extensions of dynamic programming which allow us to make sequential optimization of decision trees relative to depth and average depth, and to count the number of decision trees with minimum average depth.

  8. A full parallel radix sorting algorithm for multicore processors

    OpenAIRE

    Maus, Arne

    2011-01-01

    The problem addressed in this paper is that we want to sort an integer array a [] of length n on a multi core machine with k cores. Amdahl’s law tells us that the inherent sequential part of any algorithm will in the end dominate and limit the speedup we get from parallelisation of that algorithm. This paper introduces PARL, a parallel left radix sorting algorithm for use on ordinary shared memory multi core machines, that has just one simple statement in its sequential part. It can be seen a...

  9. A Novel Auto-Sorting System for Chinese Cabbage Seeds

    OpenAIRE

    Kuo-Yi Huang; Jian-Feng Cheng

    2017-01-01

    This paper presents a novel machine vision-based auto-sorting system for Chinese cabbage seeds. The system comprises an inlet-outlet mechanism, machine vision hardware and software, and control system for sorting seed quality. The proposed method can estimate the shape, color, and textural features of seeds that are provided as input neurons of neural networks in order to classify seeds as “good” and “not good” (NG). The results show the accuracies of classification to be 91.53% and 88.95% fo...

  10. Excitation-energy sorting in superfluid fission dynamics

    Directory of Open Access Journals (Sweden)

    Schmidt K.-H.

    2010-10-01

    Full Text Available It is now well established that at moderate excitation energies the nucleus temperature does not vary with increasing excitation energy. We show that, as a consequence, two nuclei with different temperatures brought into contact show a rather surprising energy-sorting mechanism where the hotter nucleus transfers all its excitation energy to the colder one. The scission configuration of the fission process offers a unique possibility to observe this phenomenon. The energy-sorting mechanism is clearly reflected by the mean number of prompt neutrons as a function of the fragment mass and by the dependence of the local even-odd effect with mass asymmetry.

  11. FPGA-based implementation of sorting networks in MMC applications

    DEFF Research Database (Denmark)

    Ricco, Mattia; Máthé, Lászlo; Teodorescu, Remus

    2016-01-01

    , and they are usually implemented in microcontrollers or DSPs. However, they are not convenient for hardware implementation due to their inherent sequential operation. Instead, the proposed SNs, are suitable for FPGA devices thanks to their fixed parallel structure that allows improving the timing performance......In this paper an implementation technique for Field Programmable Gate Array (FPGA) devices of two Sorting Networks (SNs) used for control of Modular Multilevel Converter (MMC) is presented. In such applications, the classical sorting algorithms are based on repetitive/recursive loops...

  12. Faster magnet sorting with a threshold acceptance algorithm

    Science.gov (United States)

    Lidia, Steve; Carr, Roger

    1995-02-01

    We introduce here a new technique for sorting magnets to minimize the field errors in permanent magnet insertion devices. Simulated annealing has been used in this role, but we find the technique of threshold acceptance produces results of equal quality in less computer time. Threshold accepting would be of special value in designing very long insertion devices, such as long free electron lasers (FELs). Our application of threshold acceptance to magnet sorting showed that it converged to equivalently low values of the cost function, but that it converged significantly faster. We present typical cases showing time to convergence for various error tolerances, magnet numbers, and temperature schedules.

  13. Characterization of color texture: color texture based sorting of tiles

    Science.gov (United States)

    Bourada, Y.; Lafon, Dominique; Eterradossi, O.

    1998-09-01

    Many materials used by the building industry show a color texture which affects the product commercial value. This texture can be seen as the spatial arrangement of regions of acceptable color differences. This work describes an appearance based automated sorting via color texture analysis, using ceramic tiles as example. Textural analysis of the tiles digital images expressed in CIEL*a*b* color system is performed through the analysis of intrinsic features of each region and relationships between regions. Results obtained through the automated process are compared to a visual sorting which leads to calculation of application dependant color and texture tolerances.

  14. Finding all sorting tandem duplication random loss operations

    DEFF Research Database (Denmark)

    Bernt, Matthias; Chen, Kuan Yu; Chen, Ming Chiang

    2011-01-01

    A tandem duplication random loss (TDRL) operation duplicates a contiguous segment of genes, followed by the random loss of one copy of each of the duplicated genes. Although the importance of this operation is founded by several recent biological studies, it has been investigated only rarely from...... a theoretical point of view. Of particular interest are sorting TDRLs which are TDRLs that, when applied to a permutation representing a genome, reduce the distance towards another given permutation. The identification of sorting genome rearrangement operations in general is a key ingredient of many algorithms...

  15. Exocytosis of Varicella-Zoster Virus Virions Involves a Convergence of Endosomal and Autophagy Pathways

    Science.gov (United States)

    Buckingham, Erin M.; Jarosinski, Keith W.; Jackson, Wallen; Carpenter, John E.

    2016-01-01

    ABSTRACT Varicella-zoster virus (VZV) is an extremely cell-associated herpesvirus with limited egress of viral particles. The induction of autophagy in VZV-infected monolayers is easily detectable; inhibition of autophagy leads to decreased VZV glycoprotein biosynthesis and diminished viral titers. To explain how autophagic flux could exert a proviral effect on the VZV infectious cycle, we postulated that the VZV exocytosis pathway following secondary envelopment may converge with the autophagy pathway. This hypothesis depended on known similarities between VZV gE and autophagy-related (Atg) Atg9/Atg16L1 trafficking pathways. Investigations were carried out with highly purified fractions of VZV virions. When the virion fraction was tested for the presence of autophagy and endosomal proteins, microtubule-associated protein 1 light chain (MAP1LC3B) and Ras-like GTPase 11 (Rab11) were detected. By two-dimensional (2D) and 3D imaging after immunolabeling, both proteins also colocalized with VZV gE in a proportion of cytoplasmic vesicles. When purified VZV virions were enumerated after immunoelectron microscopy, gold beads were detected on viruses following incubation with antibodies to VZV gE (∼100%), Rab11 (50%), and LC3B (30%). Examination of numerous electron micrographs demonstrated that enveloped virions were housed in single-membraned vesicles; viral particles were not observed in autophagosomes. Taken together, our data suggested that some viral particles after secondary envelopment accumulated in a heterogeneous population of single-membraned vesicular compartments, which were decorated with components from both the endocytic pathway (Rab11) and the autophagy pathway (LC3B). The latter cytoplasmic viral vesicles resembled an amphisome. IMPORTANCE VZV infection leads to increased autophagic flux, while inhibition of autophagy leads to a marked reduction in virus spread. In this investigation of the proviral role of autophagy, we found evidence for an

  16. Characterizing traits of coordination

    NARCIS (Netherlands)

    Poss, R.

    2013-01-01

    How can one recognize coordination languages and technologies? As this report shows, the common approach that contrasts coordination with computation is intellectually unsound: depending on the selected understanding of the word "computation", it either captures too many or too few programming

  17. Coordination models and languages

    NARCIS (Netherlands)

    Papadopoulos, G.A.; Arbab, F.

    1998-01-01

    A new class of models, formalisms and mechanisms has recently evolved for describing concurrent and distributed computations based on the concept of ``coordination''. The purpose of a coordination model and associated language is to provide a means of integrating a number of possibly heterogeneous c

  18. Team coordination dynamics.

    Science.gov (United States)

    Gorman, Jamie C; Amazeen, Polemnia G; Cooke, Nancy J

    2010-07-01

    Team coordination consists of both the dynamics of team member interaction and the environmental dynamics to which a team is subjected. Focusing on dynamics, an approach is developed that contrasts with traditional aggregate-static concepts of team coordination as characterized by the shared mental model approach. A team coordination order parameter was developed to capture momentary fluctuations in coordination. Team coordination was observed in three-person uninhabited air vehicle teams across two experimental sessions. The dynamics of the order parameter were observed under changes of a team familiarity control parameter. Team members returned for the second session to either the same (Intact) or different (Mixed) team. 'Roadblock' perturbations, or novel changes in the task environment, were introduced in order to probe the stability of team coordination. Nonlinear dynamic methods revealed differences that a traditional approach did not: Intact and Mixed team coordination dynamics looked very different; Mixed teams were more stable than Intact teams and explored the space of solutions without the need for correction. Stability was positively correlated with the number of roadblock perturbations that were overcome successfully. The novel and non-intuitive contribution of a dynamical analysis was that Mixed teams, who did not have a long history working together, were more adaptive. Team coordination dynamics carries new implications for traditional problems such as training adaptive teams.

  19. Coordinate measuring machines

    DEFF Research Database (Denmark)

    De Chiffre, Leonardo

    This document is used in connection with three exercises of 2 hours duration as a part of the course GEOMETRICAL METROLOGY AND MACHINE TESTING. The exercises concern three aspects of coordinate measuring: 1) Measuring and verification of tolerances on coordinate measuring machines, 2) Traceability...

  20. Social Postural Coordination

    Science.gov (United States)

    Varlet, Manuel; Marin, Ludovic; Lagarde, Julien; Bardy, Benoit G.

    2011-01-01

    The goal of the current study was to investigate whether a visual coupling between two people can produce spontaneous interpersonal postural coordination and change their intrapersonal postural coordination involved in the control of stance. We examined the front-to-back head displacements of participants and the angular motion of their hip and…

  1. Social Postural Coordination

    Science.gov (United States)

    Varlet, Manuel; Marin, Ludovic; Lagarde, Julien; Bardy, Benoit G.

    2011-01-01

    The goal of the current study was to investigate whether a visual coupling between two people can produce spontaneous interpersonal postural coordination and change their intrapersonal postural coordination involved in the control of stance. We examined the front-to-back head displacements of participants and the angular motion of their hip and…

  2. Wisconsin card sorting test: a new global score, with Italian norms, and its relationship with the Weigl sorting test.

    Science.gov (United States)

    Laiacona, M; Inzaghi, M G; De Tanti, A; Capitani, E

    2000-10-01

    The Wisconsin card sorting test and the Weigl test are two neuropsychological tools widely used in clinical practice to assess frontal lobe functions. In this study we present norms useful for Italian subjects aged from 15 to 85 years, with 5-17 years of education. Concerning the Wisconsin card sorting test, a new measure of global efficiency (global score) is proposed as well as norms for some well known qualitative aspects of the performance, i.e. perseverative responses, failure to maintain the set and non-perseverative errors. In setting normative values, we followed a statistical methodology (equivalent scores) employed in Italy for other neuropsychological tests, in order to favour the possibility of comparison among these tests. A correlation study between the global score of the Wisconsin card sorting test and the score on the Weigl test was carried out and it emerges that some cognitive aspects are not overlapping in these two measures.

  3. Topographic control of sorted circle morphology on Svalbard

    Science.gov (United States)

    Voigt, Joana; Hauber, Ernst; Reiss, Dennis; Hiesinger, Harald; Johnsson, Andreas; van Gasselt, Stephan; Balme, Matt; Head, Jim; de Verra, Jean-Pierre; Steinbrügge, Gregor; Jaumann, Ralf

    2015-04-01

    Patterned ground is a typical phenomenon in polar, subpolar and alpine regions [1]. As it is commonly (but not necessarily!) related to freeze-thaw cycles, its presence on Mars could possibly point to locations and periods where and when liquid water existed in the recent past [2]. Sorted circles are a class of patterned ground that was tentatively identified in Elysium Planitia (Mars) [3], but this interpretation has been challenged on the basis of physical considerations [4]. Without direct access to potential patterned ground on Mars, the analysis of terrestrial analogues can inform the interpretation of Martian landforms. Svalbard (Norway) offers a wide variety of permafrost features that are morphologically analogous to Martian cold-climate landforms [5]. It hosts some of the best examples of sorted circles on Earth, which are located on the westernmost tip of Brøgger peninsula, on a broad strand flat that is characterized by a series of postglacial beach ridges [6]. Here we report on our analysis of sorted circle morphology (especially their plan-view shape, i.e. their "roundness" or ellipticity) and its correlation with local topography (slopes, curvature). Sorted circle morphology was determined from HRSC-AX images (for details on the flight campaign and image properties see ref [5]) and through field work. Topographic information comes from a 50 cm gridded DEM derived from HRSC-AX stereo images. We measured sorted circle morphology (ellipticity, azimuth of major axis) along a WNW-ESE traverse that runs from the inland towards the sea and is oriented perpendicular to the local beach ridge trend. Selected areas with homogeneous sorted circle appearance were visually mapped, and compared to the average slope, aspect, and the calculated topographic wetness index (TWI). Furthermore the whole traverse was classified into four different morphologies of the sorted patterned ground (sorted circles, sorted "ellipses", sorted nets and areas without patterned ground

  4. Ligand Conformation Dictates Membrane and Endosomal Trafficking of Arginine-Glycine-Aspartate (RGD)-Functionalized Mesoporous Silica Nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Fang, I-Ju; Slowing, Igor I; Wu, Kevin C.W.; Lin, Victor S.Y.; Trewyn, Brian

    2012-05-15

    Recent breakthrough research on mesoporous silica nanoparticle (MSN) materials has illustrated their significant potential in biological applications due to their excellent drug delivery and endocytotic behavior. We set out to determine if MSN, covalently functionalized with conformation specific bioactive molecules (either linear or cyclic RGD ligands), behave towards mammalian cells in a similar manner as the free ligands. We discovered that RGD immobilized on the MSN surface did not influence the integrity of the porous matrix and improved the endocytosis efficiency of the MSN materials. Through competition experiments with free RGD ligands, we also discovered a conformation specific receptor–integrin association. The interaction between RGD immobilized on the MSN surface and integrins plays an important role in endosome trafficking, specifically dictating the kinetics of endosomal escape. Thus, covalent functionalization of biomolecules on MSN assists in the design of a system for controlling the interface with cancer cells.

  5. Development of a novel cell sorting method that samples population diversity in flow cytometry.

    Science.gov (United States)

    Osborne, Geoffrey W; Andersen, Stacey B; Battye, Francis L

    2015-11-01

    Flow cytometry based electrostatic cell sorting is an important tool in the separation of cell populations. Existing instruments can sort single cells into multi-well collection plates, and keep track of cell of origin and sorted well location. However currently single sorted cell results reflect the population distribution and fail to capture the population diversity. Software was designed that implements a novel sorting approach, "Slice and Dice Sorting," that links a graphical representation of a multi-well plate to logic that ensures that single cells are sampled and sorted from all areas defined by the sort region/s. Therefore the diversity of the total population is captured, and the more frequently occurring or rarer cell types are all sampled. The sorting approach was tested computationally, and using functional cell based assays. Computationally we demonstrate that conventional single cell sorting can sample as little as 50% of the population diversity dependant on the population distribution, and that Slice and Dice sorting samples much more of the variety present within a cell population. We then show by sorting single cells into wells using the Slice and Dice sorting method that there are cells sorted using this method that would be either rarely sorted, or not sorted at all using conventional single cell sorting approaches. The present study demonstrates a novel single cell sorting method that samples much more of the population diversity than current methods. It has implications in clonal selection, stem cell sorting, single cell sequencing and any areas where population heterogeneity is of importance.

  6. Amyloid precursor protein and endosomal-lysosomal dysfunction in Alzheimer's disease: inseparable partners in a multifactorial disease.

    Science.gov (United States)

    Nixon, Ralph A

    2017-07-01

    Abnormalities of the endosomal-lysosomal network (ELN) are a signature feature of Alzheimer's disease (AD). These include the earliest known cytopathology that is specific to AD and that affects endosomes and induces the progressive failure of lysosomes, each of which are directly linked by distinct mechanisms to neurodegeneration. The origins of ELN dysfunction and β-amyloidogenesis closely overlap, which reflects their common genetic basis, the established early involvement of endosomes and lysosomes in amyloid precursor protein (APP) processing and clearance, and the pathologic effect of certain APP metabolites on ELN functions. Genes that promote β-amyloidogenesis in AD (APP, PSEN1/2, and APOE4) have primary effects on ELN function. The importance of primary ELN dysfunction to pathogenesis is underscored by the mutations in more than 35 ELN-related genes that, thus far, are known to cause familial neurodegenerative diseases even though different pathogenic proteins may be involved. In this article, I discuss growing evidence that implicates AD gene-driven ELN disruptions as not only the antecedent pathobiology that underlies β-amyloidogenesis but also as the essential partner with APP and its metabolites that drive the development of AD, including tauopathy, synaptic dysfunction, and neurodegeneration. The striking amelioration of diverse deficits in animal AD models by remediating ELN dysfunction further supports a need to integrate APP and ELN relationships, including the role of amyloid-β, into a broader conceptual framework of how AD arises, progresses, and may be effectively therapeutically targeted.-Nixon, R. A. Amyloid precursor protein and endosomal-lysosomal dysfunction in Alzheimer's disease: inseparable partners in a multifactorial disease. © FASEB.

  7. Cholinergic Abnormalities, Endosomal Alterations and Up-Regulation of Nerve Growth Factor Signaling in Niemann-Pick Type C Disease

    Directory of Open Access Journals (Sweden)

    Cabeza Carolina

    2012-03-01

    Full Text Available Abstract Background Neurotrophins and their receptors regulate several aspects of the developing and mature nervous system, including neuronal morphology and survival. Neurotrophin receptors are active in signaling endosomes, which are organelles that propagate neurotrophin signaling along neuronal processes. Defects in the Npc1 gene are associated with the accumulation of cholesterol and lipids in late endosomes and lysosomes, leading to neurodegeneration and Niemann-Pick type C (NPC disease. The aim of this work was to assess whether the endosomal and lysosomal alterations observed in NPC disease disrupt neurotrophin signaling. As models, we used i NPC1-deficient mice to evaluate the central cholinergic septo-hippocampal pathway and its response to nerve growth factor (NGF after axotomy and ii PC12 cells treated with U18666A, a pharmacological cellular model of NPC, stimulated with NGF. Results NPC1-deficient cholinergic cells respond to NGF after axotomy and exhibit increased levels of choline acetyl transferase (ChAT, whose gene is under the control of NGF signaling, compared to wild type cholinergic neurons. This finding was correlated with increased ChAT and phosphorylated Akt in basal forebrain homogenates. In addition, we found that cholinergic neurons from NPC1-deficient mice had disrupted neuronal morphology, suggesting early signs of neurodegeneration. Consistently, PC12 cells treated with U18666A presented a clear NPC cellular phenotype with a prominent endocytic dysfunction that includes an increased size of TrkA-containing endosomes and reduced recycling of the receptor. This result correlates with increased sensitivity to NGF, and, in particular, with up-regulation of the Akt and PLC-γ signaling pathways, increased neurite extension, increased phosphorylation of tau protein and cell death when PC12 cells are differentiated and treated with U18666A. Conclusions Our results suggest that the NPC cellular phenotype causes neuronal

  8. Modeling the endosomal escape of cell-penetrating peptides: transmembrane pH gradient driven translocation across phospholipid bilayers.

    Science.gov (United States)

    Magzoub, Mazin; Pramanik, Aladdin; Gräslund, Astrid

    2005-11-15

    Cell-penetrating peptides (CPPs) are able to mediate the efficient cellular uptake of a wide range of cargoes. Internalization of a number of CPPs requires uptake by endocytosis, initiated by binding to anionic cell surface heparan sulfate (HS), followed by escape from endosomes. To elucidate the endosomal escape mechanism, we have modeled the process for two CPPs: penetratin (pAntp) and the N-terminal signal peptide of the unprocessed bovine prion protein (bPrPp). Large unilamellar phospholipid vesicles (LUVs) were produced encapsulating either peptide, and an ionophore, nigericin, was used to create a transmembrane pH gradient (DeltapH(mem), inside acidic) similar to the one arising in endosomes in vivo. In the absence of DeltapH(mem), no pAntp escape from the LUVs is observed, while a fraction of bPrPp escapes. In the presence of DeltapH(mem), a significant amount of pAntp escapes and an even higher degree of bPrPp escape takes place. These results, together with the differences in kinetics of escape, indicate different escape mechanisms for the two peptides. A minimum threshold peptide concentration exists for the escape of both peptides. Coupling of the peptides to a cargo reduces the fraction escaping, while complexation with HS significantly hinders the escape. Fluorescence correlation spectroscopy results show that during the escape process the LUVs are intact. Taken together, these results suggest a model for endosomal escape of CPPs: DeltapH(mem)-mediated mechanism, following dissociation from HS of the peptides, above a minimum threshold peptide concentration, in a process that does not involve lysis of the vesicles.

  9. Magnetic cell sorting and flow cytometry sorting methods for the isolation and function analysis of mouse CD4+ CD25+ Treg cells*

    OpenAIRE

    2009-01-01

    Objective: In this paper we compared the two methods of cell sorting (magnetic cell sorting and flow cytometry sorting) for the isolation and function analysis of mouse CD4+ CD25+ regulatory T (Treg) cells, in order to inform further studies in Treg cell function. Methods: We separately used magnetic cell sorting and flow cytometry sorting to identify CD4+ CD25+ Treg cells. After magnetic cell separation, we further used flow cytometry to analyze the purity of CD4+ CD25+ Treg cells, trypan bl...

  10. Endobrevin, a Novel Synaptobrevin/VAMP-Like Protein Preferentially Associated with the Early Endosome

    Science.gov (United States)

    Wong, Siew Heng; Zhang, Tao; Xu, Yue; Subramaniam, V. Nathan; Griffiths, Gareth; Hong, Wanjin

    1998-01-01

    Synaptobrevins/vesicle-associated membrane proteins (VAMPs) together with syntaxins and a synaptosome-associated protein of 25 kDa (SNAP-25) are the main components of a protein complex involved in the docking and/or fusion of synaptic vesicles with the presynaptic membrane. We report here the molecular, biochemical, and cell biological characterization of a novel member of the synaptobrevin/VAMP family. The amino acid sequence of endobrevin has 32, 33, and 31% identity to those of synaptobrevin/VAMP-1, synaptobrevin/VAMP-2, and cellubrevin, respectively. Membrane fractionation studies demonstrate that endobrevin is enriched in membrane fractions that are also enriched in the asialoglycoprotein receptor. Indirect immunofluorescence microscopy establishes that endobrevin is primarily associated with the perinuclear vesicular structures of the early endocytic compartment. The preferential association of endobrevin with the early endosome was further established by electron microscopy (EM) immunogold labeling. In vitro binding assays show that endobrevin interacts with immobilized recombinant α-SNAP fused to glutathione S-transferase (GST). Our results highlight the general importance of members of the synaptobrevin/VAMP protein family in membrane traffic and provide new avenues for future functional and mechanistic studies of this protein as well as the endocytotic pathway. PMID:9614193

  11. Neuroblastoma tyrosine kinase signaling networks involve FYN and LYN in endosomes and lipid rafts.

    Directory of Open Access Journals (Sweden)

    Juan Palacios-Moreno

    2015-04-01

    Full Text Available Protein phosphorylation plays a central role in creating a highly dynamic network of interacting proteins that reads and responds to signals from growth factors in the cellular microenvironment. Cells of the neural crest employ multiple signaling mechanisms to control migration and differentiation during development. It is known that defects in these mechanisms cause neuroblastoma, but how multiple signaling pathways interact to govern cell behavior is unknown. In a phosphoproteomic study of neuroblastoma cell lines and cell fractions, including endosomes and detergent-resistant membranes, 1622 phosphorylated proteins were detected, including more than half of the receptor tyrosine kinases in the human genome. Data were analyzed using a combination of graph theory and pattern recognition techniques that resolve data structure into networks that incorporate statistical relationships and protein-protein interaction data. Clusters of proteins in these networks are indicative of functional signaling pathways. The analysis indicates that receptor tyrosine kinases are functionally compartmentalized into distinct collaborative groups distinguished by activation and intracellular localization of SRC-family kinases, especially FYN and LYN. Changes in intracellular localization of activated FYN and LYN were observed in response to stimulation of the receptor tyrosine kinases, ALK and KIT. The results suggest a mechanism to distinguish signaling responses to activation of different receptors, or combinations of receptors, that govern the behavior of the neural crest, which gives rise to neuroblastoma.

  12. Apical transport of influenza A virus ribonucleoprotein requires Rab11-positive recycling endosome.

    Directory of Open Access Journals (Sweden)

    Fumitaka Momose

    Full Text Available Influenza A virus RNA genome exists as eight-segmented ribonucleoprotein complexes containing viral RNA polymerase and nucleoprotein (vRNPs. Packaging of vRNPs and virus budding take place at the apical plasma membrane (APM. However, little is known about the molecular mechanisms of apical transport of newly synthesized vRNP. Transfection of fluorescent-labeled antibody and subsequent live cell imaging revealed that punctate vRNP signals moved along microtubules rapidly but intermittently in both directions, suggestive of vesicle trafficking. Using a series of Rab family protein, we demonstrated that progeny vRNP localized to recycling endosome (RE in an active/GTP-bound Rab11-dependent manner. The vRNP interacted with Rab11 through viral RNA polymerase. The localization of vRNP to RE and subsequent accumulation to the APM were impaired by overexpression of Rab binding domains (RBD of Rab11 family interacting proteins (Rab11-FIPs. Similarly, no APM accumulation was observed by overexpression of class II Rab11-FIP mutants lacking RBD. These results suggest that the progeny vRNP makes use of Rab11-dependent RE machinery for APM trafficking.

  13. Niclosamide is a proton carrier and targets acidic endosomes with broad antiviral effects.

    Directory of Open Access Journals (Sweden)

    Andreas Jurgeit

    Full Text Available Viruses use a limited set of host pathways for infection. These pathways represent bona fide antiviral targets with low likelihood of viral resistance. We identified the salicylanilide niclosamide as a broad range antiviral agent targeting acidified endosomes. Niclosamide is approved for human use against helminthic infections, and has anti-neoplastic and antiviral effects. Its mode of action is unknown. Here, we show that niclosamide, which is a weak lipophilic acid inhibited infection with pH-dependent human rhinoviruses (HRV and influenza virus. Structure-activity studies showed that antiviral efficacy and endolysosomal pH neutralization co-tracked, and acidification of the extracellular medium bypassed the virus entry block. Niclosamide did not affect the vacuolar H(+-ATPase, but neutralized coated vesicles or synthetic liposomes, indicating a proton carrier mode-of-action independent of any protein target. This report demonstrates that physico-chemical interference with host pathways has broad range antiviral effects, and provides a proof of concept for the development of host-directed antivirals.

  14. Inositol phosphates and phosphoinositides activate insulin-degrading enzyme, while phosphoinositides also mediate binding to endosomes.

    Science.gov (United States)

    Song, Eun Suk; Jang, HyeIn; Guo, Hou-Fu; Juliano, Maria A; Juliano, Luiz; Morris, Andrew J; Galperin, Emilia; Rodgers, David W; Hersh, Louis B

    2017-04-04

    Insulin-degrading enzyme (IDE) hydrolyzes bioactive peptides, including insulin, amylin, and the amyloid β peptides. Polyanions activate IDE toward some substrates, yet an endogenous polyanion activator has not yet been identified. Here we report that inositol phosphates (InsPs) and phosphatdidylinositol phosphates (PtdInsPs) serve as activators of IDE. InsPs and PtdInsPs interact with the polyanion-binding site located on an inner chamber wall of the enzyme. InsPs activate IDE by up to ∼95-fold, affecting primarily Vmax The extent of activation and binding affinity correlate with the number of phosphate groups on the inositol ring, with phosphate positional effects observed. IDE binds PtdInsPs from solution, immobilized on membranes, or presented in liposomes. Interaction with PtdInsPs, likely PtdIns(3)P, plays a role in localizing IDE to endosomes, where the enzyme reportedly encounters physiological substrates. Thus, InsPs and PtdInsPs can serve as endogenous modulators of IDE activity, as well as regulators of its intracellular spatial distribution.

  15. The Parkinson's Disease-Associated Protein Kinase LRRK2 Modulates Notch Signaling through the Endosomal Pathway.

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    Yuzuru Imai

    2015-09-01

    Full Text Available Leucine-rich repeat kinase 2 (LRRK2 is a key molecule in the pathogenesis of familial and idiopathic Parkinson's disease (PD. We have identified two novel LRRK2-associated proteins, a HECT-type ubiquitin ligase, HERC2, and an adaptor-like protein with six repeated Neuralized domains, NEURL4. LRRK2 binds to NEURL4 and HERC2 via the LRRK2 Ras of complex proteins (ROC domain and NEURL4, respectively. HERC2 and NEURL4 link LRRK2 to the cellular vesicle transport pathway and Notch signaling, through which the LRRK2 complex promotes the recycling of the Notch ligand Delta-like 1 (Dll1/Delta (Dl through the modulation of endosomal trafficking. This process negatively regulates Notch signaling through cis-inhibition by stabilizing Dll1/Dl, which accelerates neural stem cell differentiation and modulates the function and survival of differentiated dopaminergic neurons. These effects are strengthened by the R1441G ROC domain-mutant of LRRK2. These findings suggest that the alteration of Notch signaling in mature neurons is a component of PD etiology linked to LRRK2.

  16. Activation of Notch in lgd mutant cells requires the fusion of late endosomes with the lysosome.

    Science.gov (United States)

    Schneider, Markus; Troost, Tobias; Grawe, Ferdi; Martinez-Arias, Alfonso; Klein, Thomas

    2013-01-15

    The tumour suppressor Lethal (2) giant discs (Lgd) is a regulator of endosomal trafficking of the Notch signalling receptor as well as other transmembrane proteins in Drosophila. The loss of its function results in an uncontrolled ligand-independent activation of the Notch signalling receptor. Here, we investigated the consequences of loss of lgd function and the requirements for the activation of Notch. We show that the activation of Notch in lgd cells is independent of Kuz and dependent on γ-secretase. We found that the lgd cells have a defect that delays degradation of transmembrane proteins, which are residents of the plasma membrane. Furthermore, our results show that the activation of Notch in lgd cells occurs in the lysosome. By contrast, the pathway is activated at an earlier phase in mutants of the gene that encodes the ESCRT-III component Shrub, which is an interaction partner of Lgd. We further show that activation of Notch appears to be a general consequence of loss of lgd function. In addition, electron microscopy of lgd cells revealed that they contain enlarged multi-vesicular bodies. The presented results further elucidate the mechanism of uncontrolled Notch activation upon derailed endocytosis.

  17. African Swine Fever Virus Undergoes Outer Envelope Disruption, Capsid Disassembly and Inner Envelope Fusion before Core Release from Multivesicular Endosomes.

    Directory of Open Access Journals (Sweden)

    Bruno Hernáez

    2016-04-01

    Full Text Available African swine fever virus (ASFV is a nucleocytoplasmic large DNA virus (NCLDV that causes a highly lethal disease in domestic pigs. As other NCLDVs, the extracellular form of ASFV possesses a multilayered structure consisting of a genome-containing nucleoid successively wrapped by a thick protein core shell, an inner lipid membrane, an icosahedral protein capsid and an outer lipid envelope. This structural complexity suggests an intricate mechanism of internalization in order to deliver the virus genome into the cytoplasm. By using flow cytometry in combination with pharmacological entry inhibitors, as well as fluorescence and electron microscopy approaches, we have dissected the entry and uncoating pathway used by ASFV to infect the macrophage, its natural host cell. We found that purified extracellular ASFV is internalized by both constitutive macropinocytosis and clathrin-mediated endocytosis. Once inside the cell, ASFV particles move from early endosomes or macropinosomes to late, multivesicular endosomes where they become uncoated. Virus uncoating requires acidic pH and involves the disruption of the outer membrane as well as of the protein capsid. As a consequence, the inner viral membrane becomes exposed and fuses with the limiting endosomal membrane to release the viral core into the cytosol. Interestingly, virus fusion is dependent on virus protein pE248R, a transmembrane polypeptide of the inner envelope that shares sequence similarity with some members of the poxviral entry/fusion complex. Collective evidence supports an entry model for ASFV that might also explain the uncoating of other multienveloped icosahedral NCLDVs.

  18. African Swine Fever Virus Undergoes Outer Envelope Disruption, Capsid Disassembly and Inner Envelope Fusion before Core Release from Multivesicular Endosomes

    Science.gov (United States)

    Hernáez, Bruno; Guerra, Milagros; Salas, María L.

    2016-01-01

    African swine fever virus (ASFV) is a nucleocytoplasmic large DNA virus (NCLDV) that causes a highly lethal disease in domestic pigs. As other NCLDVs, the extracellular form of ASFV possesses a multilayered structure consisting of a genome-containing nucleoid successively wrapped by a thick protein core shell, an inner lipid membrane, an icosahedral protein capsid and an outer lipid envelope. This structural complexity suggests an intricate mechanism of internalization in order to deliver the virus genome into the cytoplasm. By using flow cytometry in combination with pharmacological entry inhibitors, as well as fluorescence and electron microscopy approaches, we have dissected the entry and uncoating pathway used by ASFV to infect the macrophage, its natural host cell. We found that purified extracellular ASFV is internalized by both constitutive macropinocytosis and clathrin-mediated endocytosis. Once inside the cell, ASFV particles move from early endosomes or macropinosomes to late, multivesicular endosomes where they become uncoated. Virus uncoating requires acidic pH and involves the disruption of the outer membrane as well as of the protein capsid. As a consequence, the inner viral membrane becomes exposed and fuses with the limiting endosomal membrane to release the viral core into the cytosol. Interestingly, virus fusion is dependent on virus protein pE248R, a transmembrane polypeptide of the inner envelope that shares sequence similarity with some members of the poxviral entry/fusion complex. Collective evidence supports an entry model for ASFV that might also explain the uncoating of other multienveloped icosahedral NCLDVs. PMID:27110717

  19. A fusogenic peptide from a sea urchin fertilization protein promotes intracellular delivery of biomacromolecules by facilitating endosomal escape.

    Science.gov (United States)

    Niikura, Keisuke; Horisawa, Kenichi; Doi, Nobuhide

    2015-08-28

    The low efficiency of endosomal escape has been considered a bottleneck for the cytosolic delivery of biomacromolecules such as proteins and DNA. Although fusogenic peptides (FPs) such as HA2 have been employed to improve the intracellular delivery of biomacromolecules, the FPs studied thus far are not adequately efficient in enabling endosomal escape; therefore, novel FPs with higher activity are required. In this context, we focused on FPs derived from a sea urchin fertilization protein, bindin, which is involved in gamete recognition (B18, residues 103-120 and B55, residues 83-137 of mature bindin). We show that enhanced green fluorescent protein (EGFP)-fused B55 peptide binds to plasma membranes more strongly than EGFP-B18 and promotes the intracellular delivery of dextrans, which were co-administered using the trans method in a pH-dependent manner without affecting cell viability and proliferation, whereas conventional EGFP-HA2 did not affect dextran internalization. Furthermore, EGFP-B55 promoted the intracellular delivery of biomacromolecules such as antibodies, ribonuclease and plasmidic DNA using the trans method. Because the promotion of intracellular delivery by EGFP-B55 was suppressed by endocytosis inhibitors, EGFP-B55 is considered to have facilitated the endosomal escape of co-administered cargos. These results suggested that an FP that promotes the intracellular delivery of a variety of biomacromolecules with no detectable cytotoxicity should be useful for the cytosolic delivery of membrane-impermeable molecules for biomedical and biotechnological applications.

  20. Incentives versus sorting in tournaments: evidence from a field experiment

    NARCIS (Netherlands)

    Leuven, E.; Oosterbeek, H.; Sonnemans, J.; van der Klaauw, B.

    2011-01-01

    Existing field evidence on rank-order tournaments typically does not allow disentangling incentive and sorting effects. We conduct a field experiment illustrating the confounding effect. Students in an introductory microeconomics course selected themselves into tournaments with low, medium, or high