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Sample records for raft-dependent endocytic pathway

  1. Recombinant VSV G proteins reveal a novel raft-dependent endocytic pathway in resorbing osteoclasts

    SciTech Connect

    Mulari, Mika T.K. Nars, Martin; Laitala-Leinonen, Tiina; Kaisto, Tuula; Metsikkoe, Kalervo; Sun Yi; Vaeaenaenen, H. Kalervo

    2008-05-01

    Transcytotic membrane flow delivers degraded bone fragments from the ruffled border to the functional secretory domain, FSD, in bone resorbing osteoclasts. Here we show that there is also a FSD-to-ruffled border trafficking pathway that compensates for the membrane loss during the matrix uptake process and that rafts are essential for this ruffled border-targeted endosomal pathway. Replacing the cytoplasmic tail of the vesicular stomatitis virus G protein with that of CD4 resulted in partial insolubility in Triton X-100 and retargeting from the peripheral non-bone facing plasma membrane to the FSD. Recombinant G proteins were subsequently endosytosed and delivered from the FSD to the peripheral fusion zone of the ruffled border, which were both rich in lipid rafts as suggested by viral protein transport analysis and visualizing the rafts with fluorescent recombinant cholera toxin. Cholesterol depletion by methyl-{beta}-cyclodextrin impaired the ruffled border-targeted vesicle trafficking pathway and inhibited bone resorption dose-dependently as quantified by measuring the CTX and TRACP 5b secreted to the culture medium and by measuring the resorbed area visualized with a bi-phasic labeling method using sulpho-NHS-biotin and WGA-lectin. Thus, rafts are vital for membrane recycling from the FSD to the late endosomal/lysosomal ruffled border and bone resorption.

  2. Signaling on the endocytic pathway.

    PubMed

    McPherson, P S; Kay, B K; Hussain, N K

    2001-06-01

    Ligand binding to receptor tyrosine kinases and G-protein-coupled receptors initiates signal transduction events and induces receptor endocytosis via clathrin-coated pits and vesicles. While receptor-mediated endocytosis has been traditionally considered an effective mechanism to attenuate ligand-activated responses, more recent studies demonstrate that signaling continues on the endocytic pathway. In fact, certain signaling events, such as the activation of the extracellular signal-regulated kinases, appear to require endocytosis. Protein components of signal transduction cascades can assemble at clathrin coated pits and remain associated with endocytic vesicles following their dynamin-dependent release from the plasma membrane. Thus, endocytic vesicles can function as a signaling compartment distinct from the plasma membrane. These observations demonstrate that endocytosis plays an important role in the activation and propagation of signaling pathways. PMID:11389765

  3. Effects of PDT on the endocytic pathway

    NASA Astrophysics Data System (ADS)

    Kessel, David

    2010-02-01

    Two lines of evidence point to an early effect of photodamage on membrane trafficking. [1] Internalization of a fluorescent probe for hydrophobic membrane loci was impaired by prior photodamage. [2] Interference with the endocytic pathway by the PI-3 kinase antagonist wortmannin led to accumulation of cytoplasmic vacuoles suggesting a block in the recycling of plasma membrane components. Prior photodamage blocked this pathway so that no vacuoles were formed upon exposure of cells to wortmannin. In a murine hepatoma line, the endocytic pathway was preferentially sensitive to lysosomal photodamage. The role of photodamage to the endocytic pathway as a factor in PDT efficacy remains to be assessed.

  4. Molecular aspects of the endocytic pathway.

    PubMed Central

    Clague, M J

    1998-01-01

    Observation of the flow of material along the endocytic pathway has lead to the description of the basic architecture of the pathway and provided insight into the relationship between compartments. Significant advances have been made in the study of endocytic transport steps at the molecular level, of which studies of cargo selection, vesicle budding and membrane fusion events comprise the major part. Progress in this area has been driven by two approaches, yeast genetics and in vitro or cell-free assays, which reconstitute particular transport steps and allow biochemical manipulation. The complex protein machineries that control vesicle budding and fusion are significantly conserved between the secretory and endocytic pathways such that proteins that regulate particular steps are often part of a larger family of proteins which exercise a conserved function at other locations within the cell. Well characterized examples include vesicle coat proteins, rabs (small GTPases) and soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (SNAREs). Intracompartmental pH, lipid composition and cytoskeletal organization have also been identified as important determinants of the orderly flow of material within the endocytic pathway. PMID:9820800

  5. GPCR signaling along the endocytic pathway

    PubMed Central

    Irannejad, Roshanak; von Zastrow, Mark

    2016-01-01

    Many G protein-coupled receptors (GPCRs) internalize after agonist-induced activation. While endocytosis has long been associated with homeostatic attenuation of cellular responsiveness, accumulating evidence from study of a wide range of eukaryotes reveals that the endocytic pathway also contributes to generating receptor-initiated signals themselves. Here we review recent progress in this area, discussing primarily but not exclusively GPCR signaling in mammalian cells. PMID:24680436

  6. Lymphocytic choriomeningitis virus uses a novel endocytic pathway for infectious entry via late endosomes

    SciTech Connect

    Quirin, Katharina; Eschli, Bruno; Scheu, Isabella; Poort, Linda; Kartenbeck, Juergen; Helenius, Ari

    2008-08-15

    The endocytic entry of lymphocytic choriomeningitis virus (LCMV) into host cells was compared to the entry of viruses known to exploit clathrin or caveolae/raft-dependent pathways. Pharmacological inhibitors, expression of pathway-specific dominant-negative constructs, and siRNA silencing of clathrin together with electron and light microscopy provided evidence that although a minority population followed a classical clathrin-mediated mechanism of entry, the majority of these enveloped RNA viruses used a novel endocytic route to late endosomes. The pathway was clathrin, dynamin-2, actin, Arf6, flotillin-1, caveolae, and lipid raft independent but required membrane cholesterol. Unaffected by perturbation of Rab5 or Rab7 and apparently without passing through Rab5/EEA1-positive early endosomes, the viruses reached late endosomes and underwent acid-induced penetration. This membrane trafficking route between the plasma membrane and late endosomes may function in the turnover of a select group of surface glycoproteins such as the dystroglycan complex, which serves as the receptor of LCMV.

  7. Pathways and mechanisms of endocytic recycling

    PubMed Central

    Grant, Barth D.; Donaldson, Julie G.

    2011-01-01

    Endocytic recycling is coordinated with endocytic uptake to control the composition of the plasma membrane. Although much of our understanding of endocytic recycling has come from studies on the transferrin receptor, a protein internalized through clathrin-dependent endocytosis, increased interest in clathrin-independent endocytosis has led to the discovery of new endocytic recycling systems. Recent insights into the regulatory mechanisms that control endocytic recycling have focused on recycling through tubular carriers and the return to the cell surface of cargo that enters cells through clathrin-independent mechanisms. Recent work emphasizes the importance of regulated recycling in such diverse processes as cytokinesis, cell adhesion and morphogenesis, cell fusion, and learning and memory. PMID:19696797

  8. Decreased function of survival motor neuron protein impairs endocytic pathways.

    PubMed

    Dimitriadi, Maria; Derdowski, Aaron; Kalloo, Geetika; Maginnis, Melissa S; O'Hern, Patrick; Bliska, Bryn; Sorkaç, Altar; Nguyen, Ken C Q; Cook, Steven J; Poulogiannis, George; Atwood, Walter J; Hall, David H; Hart, Anne C

    2016-07-26

    Spinal muscular atrophy (SMA) is caused by depletion of the ubiquitously expressed survival motor neuron (SMN) protein, with 1 in 40 Caucasians being heterozygous for a disease allele. SMN is critical for the assembly of numerous ribonucleoprotein complexes, yet it is still unclear how reduced SMN levels affect motor neuron function. Here, we examined the impact of SMN depletion in Caenorhabditis elegans and found that decreased function of the SMN ortholog SMN-1 perturbed endocytic pathways at motor neuron synapses and in other tissues. Diminished SMN-1 levels caused defects in C. elegans neuromuscular function, and smn-1 genetic interactions were consistent with an endocytic defect. Changes were observed in synaptic endocytic proteins when SMN-1 levels decreased. At the ultrastructural level, defects were observed in endosomal compartments, including significantly fewer docked synaptic vesicles. Finally, endocytosis-dependent infection by JC polyomavirus (JCPyV) was reduced in human cells with decreased SMN levels. Collectively, these results demonstrate for the first time, to our knowledge, that SMN depletion causes defects in endosomal trafficking that impair synaptic function, even in the absence of motor neuron cell death. PMID:27402754

  9. Raft-Dependent Endocytosis of Autocrine Motility Factor/Phosphoglucose Isomerase: A Potential Drug Delivery Route for Tumor Cells

    PubMed Central

    Kojic, Liliana D.; Wiseman, Sam M.; Ghaidi, Fariba; Joshi, Bharat; Nedev, Hinyu; Saragovi, H. Uri; Nabi, Ivan R.

    2008-01-01

    Background Autocrine motility factor/phosphoglucose isomerase (AMF/PGI) is the extracellular ligand for the gp78/AMFR receptor overexpressed in a variety of human cancers. We showed previously that raft-dependent internalization of AMF/PGI is elevated in metastatic MDA-435 cells, but not metastatic, caveolin-1-expressing MDA-231 cells, relative to non-metastatic MCF7 and dysplastic MCF10A cells suggesting that it might represent a tumor cell-specific endocytic pathway. Methodology/Principal Findings Similarly, using flow cytometry, we demonstrate that raft-dependent endocytosis of AMF/PGI is increased in metastatic HT29 cancer cells expressing low levels of caveolin-1 relative to metastatic, caveolin-1-expressing, HCT116 colon cells and non-metastatic Caco-2 cells. Therefore, we exploited the raft-dependent internalization of AMF/PGI as a potential tumor-cell specific targeting mechanism. We synthesized an AMF/PGI-paclitaxel conjugate and found it to be as efficient as free paclitaxel in inducing cytotoxicity and apoptosis in tumor cells that readily internalize AMF/PGI compared to tumor cells that poorly internalize AMF/PGI. Murine K1735-M1 and B16-F1 melanoma cells internalize FITC-conjugated AMF/PGI and are acutely sensitive to AMF/PGI-paclitaxel mediated cytotoxicity in vitro. Moreover, following in vivo intratumoral injection, FITC-conjugated AMF/PGI is internalized in K1735-M1 tumors. Intratumoral injection of AMF/PGI-paclitaxel induced significantly higher tumor regression compared to free paclitaxel, even in B16-F1 cells, known to be resistant to taxol treatment. Treatment with AMF/PGI-paclitaxel significantly prolonged the median survival time of tumor bearing mice. Free AMF/PGI exhibited a pro-survival role, reducing the cytotoxic effect of both AMF/PGI-paclitaxel and free paclitaxel suggesting that AMF/PGI-paclitaxel targets a pathway associated with resistance to chemotherapeutic agents. AMF/PGI-FITC uptake by normal murine spleen and thymus cells was

  10. Bifurcation of the endocytic pathway into Rab5-dependent and -independent transport to the vacuole

    NASA Astrophysics Data System (ADS)

    Toshima, Junko Y.; Nishinoaki, Show; Sato, Yoshifumi; Yamamoto, Wataru; Furukawa, Daiki; Siekhaus, Daria Elisabeth; Sawaguchi, Akira; Toshima, Jiro

    2014-03-01

    The yeast Rab5 homologue, Vps21p, is known to be involved both in the vacuolar protein sorting (VPS) pathway from the trans-Golgi network to the vacuole, and in the endocytic pathway from the plasma membrane to the vacuole. However, the intracellular location at which these two pathways converge remains unclear. In addition, the endocytic pathway is not completely blocked in yeast cells lacking all Rab5 genes, suggesting the existence of an unidentified route that bypasses the Rab5-dependent endocytic pathway. Here we show that convergence of the endocytic and VPS pathways occurs upstream of the requirement for Vps21p in these pathways. We also identify a previously unidentified endocytic pathway mediated by the AP-3 complex. Importantly, the AP-3-mediated pathway appears mostly intact in Rab5-disrupted cells, and thus works as an alternative route to the vacuole/lysosome. We propose that the endocytic traffic branches into two routes to reach the vacuole: a Rab5-dependent VPS pathway and a Rab5-independent AP-3-mediated pathway.

  11. Role of specific endocytic pathways in electrotransfection of cells.

    PubMed

    Chang, Chun-Chi; Wu, Mina; Yuan, Fan

    2014-01-01

    Electrotransfection is a technique utilized for gene delivery in both preclinical and clinical studies. However, its mechanisms are not fully understood. The goal of this study was to investigate specific pathways of endocytosis involved in electrotransfection. In the study, three different human cell lines (HEK293, HCT116, and HT29) were either treated with ice cold medium postelectrotransfection or endocytic inhibitors prior to electrotransfection. The inhibitors were pharmacological agents (chlorpromazine, genistein, and amiloride) or different small interfering RNA (siRNA) molecules that could knockdown expression of clathrin heavy chain (CLTC), caveolin-1, and Rab34, respectively. The reduction in gene expressions was confirmed with western blot analysis at 48-72h post-siRNA treatment. It was observed that treatments with either ice cold medium, chlorpromazine, or genistein resulted in significant reductions in electrotransfection efficiency (eTE) in all three cell lines, compared to the matched controls, but amiloride treatment had insignificant effects on eTE. For cells treated with siRNA, only CLTC knockdown resulted in eTE reduction for all three cell lines. Together, these data demonstrated that the clathrin-mediated endocytosis played an important role in electrotransfection. PMID:26052524

  12. Brucella alters endocytic pathway in J774 macrophages.

    PubMed

    Arenas, Graciela N; Grilli, Diego J; Samartino, Luis E; Magadán, Javier; Mayorga, Luis S

    2010-01-01

    Brucella is a facultative intracellular bacterium which causes chronic infections in mammals by surviving and replicating within host cells. The putative role of the endoplasmic reticulum (ER) in the formation of the phagosome in non-professional phagocytes is supported by several research groups, but still leaves open the question of the fate of Brucella inside professional phagocytes and its resistance mechanisms therein. Macrophages are particularly important for the survival and spreading of Brucella during infection. The intracellular transport of Brucella in these cells has not been thoroughly characterized. To study the maturation process of Brucella-containing phagosomes in phagocytes, we comparatively monitored the intracellular transport of a virulent strain (2308) with two vaccine strains (S19 and RB51) in J 774 macrophages. Then, we compared the behavior of all three strains studied through transmission electron microscopy. The results indicate that the virulent strain not only occupies two different kinds of compartments but also alters the endocytic pathway of the cell it parasitizes, unlike what has been reported for non-professional phagocytes, like HeLa cell. Besides, differences are observed in the behavior of both Brucella abortus vaccine strains. PMID:21178473

  13. Effects of photodynamic therapy on the endocytic pathway

    PubMed Central

    Kessel, David; Price, Michael; Caruso, Joseph; Reiners, John

    2011-01-01

    In this report, we describe an effect of photodynamic therapy (PDT) on membrane trafficking in murine 1c1c7 hepatoma cells. A brief exposure of 1c1c7 cells to a 20 nM concentration of the phosphatidylinositol kinase class-3 antagonist wortmannin led to the rapid appearance of cytoplasmic vacuoles. Fluorescence monitoring of plasma membrane-associated 1-[4-(trimethylamino)-phenyl]-6-phenylhexa-1,3,5-triene (TDPH) over time demonstrated that the wortmannin-induced vacuoles were derived from endocytosed plasma membrane. Low-dose photodamage catalyzed by the lysosomal photosensitizer NPe6, prior to the addition of wortmannin, prevented formation of these vacuoles. NPe6 was found to suppress for several hours the normal trafficking of TDPH-labeled plasma membrane to the cytosol, and the formation of punctate TDPH-labeled cytoplasmic vesicles. The ability of NPe6-induced photodamage to suppress wortmannin-induced vacuolization occurred under conditions that did not disrupt lysosomes and were at or below the threshold of cytostatic/cytotoxic effects. Furthermore, the suppressive effects of NPe6-PDT were not prevented by inclusion of an agent that stabilized lysosomal membranes, or by E64d, an inhibitor of lysosomal cathepsin proteases. Mitochondrial photodamage was less effective at preventing wortmannin-induced vacuole formation and PDT directed against the ER had no effect. The role of photodamage to the endocytic pathway may be a hitherto unexplored effect on cells that selectively accumulate photosensitizing agents. These results indicate that photodamage directed against endosomes/lysosomes has effects independent of the release of lysosomal proteases. PMID:21125114

  14. Vibrio vulnificus VvhA induces autophagy-related cell death through the lipid raft-dependent c-Src/NOX signaling pathway.

    PubMed

    Song, Eun Ju; Lee, Sei-Jung; Lim, Hyeon Su; Kim, Jun Sung; Jang, Kyung Ku; Choi, Sang Ho; Han, Ho Jae

    2016-01-01

    VvhA, a virulent factor of Vibrio (V.) vulnificus, induces acute cell death in a destructive manner. Autophagy plays an important role in cell death, but the functional role of VvhA in autophagy-related cell death has not been elucidated yet. We found that rVvhA significantly increased LC3 puncta formation and autophagic flux in promoting the cell death of human intestinal epithelial Caco-2 cells. The cell death induced by rVvhA was independent of lysosomal permeabilizaton and caspase activation. rVvhA induced rapid phosphorylation of c-Src in the membrane lipid raft, which resulted in an increased interaction between lipid raft molecule caveolin-1 and NADPH oxidase (NOX) complex Rac1 for ROS production. NOX-mediated ROS signaling induced by rVvhA increased the phosphorylation of extracellular signal-regulated kinase (ERK) and eukaryotic translation initiation factor 2α (eIF2α) which are required for mRNA expression of Atg5 and Atg16L1 involved in autophagosome formation. In an in vivo model, VvhA increased autophagy activation and paracellular permeabilization in intestinal epithelium. Collectively, the results here show that VvhA plays a pivotal role in the pathogenesis and dissemination of V. vulnificus by autophagy upregulation, through the lipid raft-mediated c-Src/NOX signaling pathway and ERK/eIF2α activation. PMID:27250250

  15. Vibrio vulnificus VvhA induces autophagy-related cell death through the lipid raft-dependent c-Src/NOX signaling pathway

    PubMed Central

    Song, Eun Ju; Lee, Sei-Jung; Lim, Hyeon Su; Kim, Jun Sung; Jang, Kyung Ku; Choi, Sang Ho; Han, Ho Jae

    2016-01-01

    VvhA, a virulent factor of Vibrio (V.) vulnificus, induces acute cell death in a destructive manner. Autophagy plays an important role in cell death, but the functional role of VvhA in autophagy-related cell death has not been elucidated yet. We found that rVvhA significantly increased LC3 puncta formation and autophagic flux in promoting the cell death of human intestinal epithelial Caco-2 cells. The cell death induced by rVvhA was independent of lysosomal permeabilizaton and caspase activation. rVvhA induced rapid phosphorylation of c-Src in the membrane lipid raft, which resulted in an increased interaction between lipid raft molecule caveolin-1 and NADPH oxidase (NOX) complex Rac1 for ROS production. NOX-mediated ROS signaling induced by rVvhA increased the phosphorylation of extracellular signal-regulated kinase (ERK) and eukaryotic translation initiation factor 2α (eIF2α) which are required for mRNA expression of Atg5 and Atg16L1 involved in autophagosome formation. In an in vivo model, VvhA increased autophagy activation and paracellular permeabilization in intestinal epithelium. Collectively, the results here show that VvhA plays a pivotal role in the pathogenesis and dissemination of V. vulnificus by autophagy upregulation, through the lipid raft-mediated c-Src/NOX signaling pathway and ERK/eIF2α activation. PMID:27250250

  16. Salt-Induced Remodeling of Spatially Restricted Clathrin-Independent Endocytic Pathways in Arabidopsis Root

    PubMed Central

    Baral, Anirban; Irani, Niloufer G.; Fujimoto, Masaru; Nakano, Akihiko; Mayor, Satyajit; Mathew, M.K.

    2015-01-01

    Endocytosis is a ubiquitous cellular process that is characterized well in animal cells in culture but poorly across intact, functioning tissue. Here, we analyze endocytosis throughout the Arabidopsis thaliana root using three classes of probes: a lipophilic dye, tagged transmembrane proteins, and a lipid-anchored protein. We observe a stratified distribution of endocytic processes. A clathrin-dependent endocytic pathway that internalizes transmembrane proteins functions in all cell layers, while a sterol-dependent, clathrin-independent pathway that takes up lipid and lipid-anchored proteins but not transmembrane proteins is restricted to the epidermal layer. Saline stress induces a third pathway that is clathrin-independent, nondiscriminatory in its choice of cargo, and operates across all layers of the root. Concomitantly, small acidic compartments in inner cell layers expand to form larger vacuole-like structures. Plants lacking function of the Rab-GEF (guanine nucleotide exchange factor) VPS9a (vacuolar protein sorting 9A) neither induce the third endocytic pathway nor expand the vacuolar system in response to salt stress. The plants are also hypersensitive to salt. Thus, saline stress reconfigures clathrin-independent endocytosis and remodels endomembrane systems, forming large vacuoles in the inner cell layers, both processes correlated by the requirement of VPS9a activity. PMID:25901088

  17. Population Distribution Analyses Reveal a Hierarchy of Molecular Players Underlying Parallel Endocytic Pathways

    PubMed Central

    Gupta, Gagan D.; Howes, Mark T.; Chandran, Ruma; Das, Anupam; Menon, Sindhu; Parton, Robert G.; Sowdhamini, R.; Thattai, Mukund; Mayor, Satyajit

    2014-01-01

    Single-cell-resolved measurements reveal heterogeneous distributions of clathrin-dependent (CD) and -independent (CLIC/GEEC: CG) endocytic activity in Drosophila cell populations. dsRNA-mediated knockdown of core versus peripheral endocytic machinery induces strong changes in the mean, or subtle changes in the shapes of these distributions, respectively. By quantifying these subtle shape changes for 27 single-cell features which report on endocytic activity and cell morphology, we organize 1072 Drosophila genes into a tree-like hierarchy. We find that tree nodes contain gene sets enriched in functional classes and protein complexes, providing a portrait of core and peripheral control of CD and CG endocytosis. For 470 genes we obtain additional features from separate assays and classify them into early- or late-acting genes of the endocytic pathways. Detailed analyses of specific genes at intermediate levels of the tree suggest that Vacuolar ATPase and lysosomal genes involved in vacuolar biogenesis play an evolutionarily conserved role in CG endocytosis. PMID:24971745

  18. Infectious Entry by Amphotropic as well as Ecotropic Murine Leukemia Viruses Occurs through an Endocytic Pathway

    PubMed Central

    Katen, Louis J.; Januszeski, Michael M.; Anderson, W. French; Hasenkrug, Kim J.; Evans, Leonard H.

    2001-01-01

    Infectious entry of enveloped viruses is thought to proceed by one of two mechanisms. pH-dependent viruses enter the cells by receptor-mediated endocytosis and are inhibited by transient treatment with agents that prevent acidification of vesicles in the endocytic pathway, while pH-independent viruses are not inhibited by such agents and are thought to enter the cell by direct fusion with the plasma membrane. Nearly all retroviruses, including amphotropic murine leukemia virus (MuLV) and human immunodeficiency virus type 1, are classified as pH independent. However, ecotropic MuLV is considered to be a pH-dependent virus. We have examined the infectious entry of ecotropic and amphotropic MuLVs and found that they were equally inhibited by NH4Cl and bafilomycin A. These agents inhibited both viruses only partially over the course of the experiments. Agents that block the acidification of endocytic vesicles also arrest vesicular trafficking. Thus, partial inhibition of the MuLVs could be the result of virus inactivation during arrest in this pathway. In support of this contention, we found that that the loss of infectivity of the MuLVs during treatment of target cells with the drugs closely corresponded to the loss of activity due to spontaneous inactivation at 37°C in the same period of time. Furthermore, the drugs had no effect on the efficiency of infection under conditions in which the duration of infection was held to a very short period to minimize the effects of spontaneous inactivation. These results indicate that the infectious processes of both ecotropic and amphotropic MuLVs were arrested rather than aborted by transient treatment of the cells with the drugs. We also found that infectious viruses were efficiently internalized during treatment. This indicated that the arrest occurred in an intracellular compartment and that the infectious process of both the amphotropic and ecotropic MuLVs very likely involved endocytosis. An important aspect of this study

  19. The minute virus of mice exploits different endocytic pathways for cellular uptake

    SciTech Connect

    Garcin, Pierre O.; Panté, Nelly

    2015-08-15

    The minute virus of mice, prototype strain (MVMp), is a non-enveloped, single-stranded DNA virus of the family Parvoviridae. Unlike other parvoviruses, the mechanism of cellular uptake of MVMp has not been studied in detail. We analyzed MVMp endocytosis in mouse LA9 fibroblasts and a tumor cell line derived from epithelial–mesenchymal transition through polyomavirus middle T antigen transformation in transgenic mice. By a combination of immunofluorescence and electron microscopy, we found that MVMp endocytosis occurs at the leading edge of migrating cells in proximity to focal adhesion sites. By using drug inhibitors of various endocytic pathways together with immunofluorescence microscopy and flow cytometry analysis, we discovered that MVMp can use a number of endocytic pathways, depending on the host cell type. At least three different mechanisms were identified: clathrin-, caveolin-, and clathrin-independent carrier-mediated endocytosis, with the latter occurring in transformed cells but not in LA9 fibroblasts. - Highlights: • MVMp uptake takes place at the leading edge of migrating cells. • MVMp exploits a variety of endocytic pathways. • MVMp could use clathrin- and caveolin-mediated endocytosis. • MVMp could also use clathrin-independent carriers for cellular uptake.

  20. Two Plant–Viral Movement Proteins Traffic in the Endocytic Recycling PathwayW⃞

    PubMed Central

    Haupt, Sophie; Cowan, Graham H.; Ziegler, Angelika; Roberts, Alison G.; Oparka, Karl J.; Torrance, Lesley

    2005-01-01

    Many plant viruses exploit a conserved group of proteins known as the triple gene block (TGB) for cell-to-cell movement. Here, we investigated the interaction of two TGB proteins (TGB2 and TGB3) of Potato mop-top virus (PMTV), with components of the secretory and endocytic pathways when expressed as N-terminal fusions to green fluorescent protein or monomeric red fluorescent protein (mRFP). Our studies revealed that fluorophore-labeled TGB2 and TGB3 showed an early association with the endoplasmic reticulum (ER) and colocalized in motile granules that used the ER-actin network for intracellular movement. Both proteins increased the size exclusion limit of plasmodesmata, and TGB3 accumulated at plasmodesmata in the absence of TGB2. TGB3 contains a putative Tyr-based sorting motif, mutations in which abolished ER localization and plasmodesmatal targeting. Later in the expression cycle, both fusion proteins were incorporated into vesicular structures. TGB2 associated with these structures on its own, but TGB3 could not be incorporated into the vesicles in the absence of TGB2. Moreover, in addition to localization to the ER and motile granules, mRFP-TGB3 was incorporated into vesicles when expressed in PMTV-infected epidermal cells, indicating recruitment by virus-expressed TGB2. The TGB fusion protein-containing vesicles were labeled with FM4-64, a marker for plasma membrane internalization and components of the endocytic pathway. TGB2 also colocalized in vesicles with Ara7, a Rab5 ortholog that marks the early endosome. Protein interaction analysis revealed that recombinant TGB2 interacted with a tobacco protein belonging to the highly conserved RME-8 family of J-domain chaperones, shown to be essential for endocytic trafficking in Caenorhabditis elegans and Drosophila melanogaster. Collectively, the data indicate the involvement of the endocytic pathway in viral intracellular movement, the implications of which are discussed. PMID:15608333

  1. Filamin A and Big2: a shared endocytic pathway.

    PubMed

    Sheen, Volney L

    2014-01-01

    Neural proliferation, migration and differentiation require reorganization of the actin cytoskeleton and regulation of vesicle trafficking to provide stability in maintaining cell adhesions, allow for changes in cell shape, and establishing cell polarity. Human disorders involving the actin-binding Filamin A (FLNA) and vesicle trafficking Brefeldin-associated guanine exchange factor 2 (BIG2 is encoded by the ARFGEF2 gene) proteins are implicated in these various developmental processes, resulting in a malformation of cortical development called periventricular heterotopia (nodules along the ventricular lining) and microcephaly (small brain). Here we discuss several recent reports from our laboratory that demonstrate a shared role for both proteins in actin-associated vesicle trafficking, which is required to maintain the expression and stability of cell adhesion and cell cycle associated molecules during cortical development. While changes in FLNA and BIG2 have first been linked to disorders involving the central nervous system, increasing reports suggest they are associated with aberrant development of various other organ systems in the body. These studies suggest that vesicle trafficking defects in FLN-GEF dependent pathways may contribute to a much broader phenotype than previously realized. PMID:24709996

  2. An Abp1-dependent route of endocytosis functions when the classical endocytic pathway in yeast is inhibited.

    PubMed

    Aghamohammadzadeh, Soheil; Smaczynska-de Rooij, Iwona I; Ayscough, Kathryn R

    2014-01-01

    Clathrin-mediated endocytosis (CME) is a well characterized pathway in both yeast and mammalian cells. An increasing number of alternative endocytic pathways have now been described in mammalian cells that can be both clathrin, actin, and Arf6- dependent or independent. In yeast, a single clathrin-mediated pathway has been characterized in detail. However, disruption of this pathway in many mutant strains indicates that other uptake pathways might exist, at least for bulk lipid and fluid internalization. Using a combination of genetics and live cell imaging, here we show evidence for a novel endocytic pathway in S. cerevisiae that does not involve several of the proteins previously shown to be associated with the 'classic' pathway of endocytosis. This alternative pathway functions in the presence of low levels of the actin-disrupting drug latrunculin-A which inhibits movement of the proteins Sla1, Sla2, and Sac6, and is independent of dynamin function. We reveal that in the absence of the 'classic' pathway, the actin binding protein Abp1 is now essential for bulk endocytosis. This novel pathway appears to be distinct from another described alternative endocytic route in S. cerevisiae as it involves at least some proteins known to be associated with cortical actin patches rather than being mediated at formin-dependent endocytic sites. These data indicate that cells have the capacity to use overlapping sets of components to facilitate endocytosis under a range of conditions. PMID:25072293

  3. An Abp1-Dependent Route of Endocytosis Functions when the Classical Endocytic Pathway in Yeast Is Inhibited

    PubMed Central

    Aghamohammadzadeh, Soheil; Smaczynska-de Rooij, Iwona I.; Ayscough, Kathryn R.

    2014-01-01

    Clathrin-mediated endocytosis (CME) is a well characterized pathway in both yeast and mammalian cells. An increasing number of alternative endocytic pathways have now been described in mammalian cells that can be both clathrin, actin, and Arf6- dependent or independent. In yeast, a single clathrin-mediated pathway has been characterized in detail. However, disruption of this pathway in many mutant strains indicates that other uptake pathways might exist, at least for bulk lipid and fluid internalization. Using a combination of genetics and live cell imaging, here we show evidence for a novel endocytic pathway in S. cerevisiae that does not involve several of the proteins previously shown to be associated with the ‘classic’ pathway of endocytosis. This alternative pathway functions in the presence of low levels of the actin-disrupting drug latrunculin-A which inhibits movement of the proteins Sla1, Sla2, and Sac6, and is independent of dynamin function. We reveal that in the absence of the ‘classic’ pathway, the actin binding protein Abp1 is now essential for bulk endocytosis. This novel pathway appears to be distinct from another described alternative endocytic route in S. cerevisiae as it involves at least some proteins known to be associated with cortical actin patches rather than being mediated at formin-dependent endocytic sites. These data indicate that cells have the capacity to use overlapping sets of components to facilitate endocytosis under a range of conditions. PMID:25072293

  4. The endocytic uptake pathways of targeted toxins are influenced by synergistically acting Gypsophila saponins.

    PubMed

    Bachran, Diana; Schneider, Stefanie; Bachran, Christopher; Weng, Alexander; Melzig, Matthias F; Fuchs, Hendrik

    2011-12-01

    The expression of the epidermal growth factor (EGF) receptor is upregulated in many human tumors. We developed the targeted toxin SE, consisting of the plant toxin saporin-3 and human EGF. The cytotoxic effect of SE drastically increases in a synergistic manner by a combined treatment with Saponinum album (Spn), a saponin composite from Gypsophila paniculata L. Here we analyzed which endocytic pathways are involved in the uptake of SE and which are mandatory for the Spn-mediated enhancement. We treated HER14 cells (NIH-3T3 cells transfected with human EGF receptor) with either chlorpromazine, dynasore, latrunculin A, chloroquine, bafilomycin A1 or filipin and analyzed the effect on the cytotoxicity of SE alone or in combination with Spn. We demonstrated that SE in combination with Spn enters cells via clathrin- and actin-dependent pathways and the acidification of the endosomes after endocytosis is relevant for the cytotoxicity of SE. Notably, our data suggest that SE without Spn follows a different endocytic uptake pathway. SE cytotoxicity is independent of blocking of clathrin or actin, and the decrease in endosomal pH is irrelevant for SE cytotoxicity. Furthermore, Spn has no influence on the retrograde transport. This work is important for the better understanding of the underlying mechanism of Spn-enhanced cytotoxicity and helps to describe the role of Spn better. PMID:21981719

  5. A CCR2 macrophage endocytic pathway mediates extravascular fibrin clearance in vivo

    PubMed Central

    Motley, Michael P.; Madsen, Daniel H.; Jürgensen, Henrik J.; Spencer, David E.; Szabo, Roman; Holmbeck, Kenn; Flick, Matthew J.; Lawrence, Daniel A.; Castellino, Francis J.; Weigert, Roberto

    2016-01-01

    Extravascular fibrin deposition accompanies many human diseases and causes chronic inflammation and organ damage, unless removed in a timely manner. Here, we used intravital microscopy to investigate how fibrin is removed from extravascular space. Fibrin placed into the dermis of mice underwent cellular endocytosis and lysosomal targeting, revealing a novel intracellular pathway for extravascular fibrin degradation. A C-C chemokine receptor type 2 (CCR2)-positive macrophage subpopulation constituted the majority of fibrin-uptaking cells. Consequently, cellular fibrin uptake was diminished by elimination of CCR2-expressing cells. The CCR2-positive macrophage subtype was different from collagen-internalizing M2-like macrophages. Cellular fibrin uptake was strictly dependent on plasminogen and plasminogen activator. Surprisingly, however, fibrin endocytosis was unimpeded by the absence of the fibrin(ogen) receptors, αMβ2 and ICAM-1, the myeloid cell integrin-binding site on fibrin or the endocytic collagen receptor, the mannose receptor. The study identifies a novel fibrin endocytic pathway engaged in extravascular fibrin clearance and shows that interstitial fibrin and collagen are cleared by different subsets of macrophages employing distinct molecular pathways. PMID:26647393

  6. A CCR2 macrophage endocytic pathway mediates extravascular fibrin clearance in vivo.

    PubMed

    Motley, Michael P; Madsen, Daniel H; Jürgensen, Henrik J; Spencer, David E; Szabo, Roman; Holmbeck, Kenn; Flick, Matthew J; Lawrence, Daniel A; Castellino, Francis J; Weigert, Roberto; Bugge, Thomas H

    2016-03-01

    Extravascular fibrin deposition accompanies many human diseases and causes chronic inflammation and organ damage, unless removed in a timely manner. Here, we used intravital microscopy to investigate how fibrin is removed from extravascular space. Fibrin placed into the dermis of mice underwent cellular endocytosis and lysosomal targeting, revealing a novel intracellular pathway for extravascular fibrin degradation. A C-C chemokine receptor type 2 (CCR2)-positive macrophage subpopulation constituted the majority of fibrin-uptaking cells. Consequently, cellular fibrin uptake was diminished by elimination of CCR2-expressing cells. The CCR2-positive macrophage subtype was different from collagen-internalizing M2-like macrophages. Cellular fibrin uptake was strictly dependent on plasminogen and plasminogen activator. Surprisingly, however, fibrin endocytosis was unimpeded by the absence of the fibrin(ogen) receptors, αMβ2 and ICAM-1, the myeloid cell integrin-binding site on fibrin or the endocytic collagen receptor, the mannose receptor. The study identifies a novel fibrin endocytic pathway engaged in extravascular fibrin clearance and shows that interstitial fibrin and collagen are cleared by different subsets of macrophages employing distinct molecular pathways. PMID:26647393

  7. The endocytic pathway and therapeutic efficiency of doxorubicin conjugated cholesterol-derived polymers.

    PubMed

    Sevimli, Sema; Sagnella, Sharon; Macmillan, Alexander; Whan, Renee; Kavallaris, Maria; Bulmus, Volga; Davis, Thomas P

    2015-02-01

    Previously synthesized poly(methacrylic acid-co-cholesteryl methacrylate) P(MAA-co-CMA) copolymers were examined as potential drug delivery vehicles. P(MAA-co-CMA) copolymers were fluorescently labelled and imaged in SHEP and HepG2 cells. To understand their cell internalization pathway endocytic inhibition studies were conducted. It was concluded that P(MAA-co-CMA) are taken up by the cells via clathrin-independent endocytosis (CIE) (both caveolae mediated and cholesterol dependent endocytosis) mechanisms. The formation and characterization of P(MAA-co-CMA)-doxorubicin (DOX) nanocomplexes was investigated by fluorescence lifetime imaging microscopy (FLIM), UV-Visible spectroscopy (UV-Vis) and dynamic light scattering (DLS) studies. The toxicity screening between P(MAA-co-CMA)-DOX nanocomplexes (at varying w/w ratios) and free DOX, revealed nanocomplexes to exhibit higher cytotoxicity towards cancer cells in comparison to normal cells. FLIM and confocal microscopy were employed for investigating the time-dependent release of DOX in SHEP cells and the cellular uptake profile of P(MAA-co-CMA)-DOX nanocomplexes in cancer and normal cell lines, respectively. The endocytic pathway of P(MAA-co-CMA)-DOX nanocomplexes were examined in SHEP and HepG2 cells via flow cytometry revealing the complexes to be internalized through both clathrin-dependent (CDE) and CIE mechanisms. The drug delivery profile, reported herein, illuminates the specific endocytic route and therapeutic efficiency of P(MAA-co-CMA)-DOX nanocomplexes strongly suggesting these particles to be promising candidates for in vivo applications. PMID:26218123

  8. The Toll-like receptor 2 (TLR2) ligand FSL-1 is internalized via the clathrin-dependent endocytic pathway triggered by CD14 and CD36 but not by TLR2

    PubMed Central

    Shamsul, Haque M; Hasebe, Akira; Iyori, Mitsuhiro; Ohtani, Makoto; Kiura, Kazuto; Zhang, Diya; Totsuka, Yasunori; Shibata, Ken- ichiro

    2010-01-01

    Little is known of how Toll-like receptor (TLR) ligands are processed after recognition by TLRs. This study was therefore designed to investigate how the TLR2 ligand FSL-1 is processed in macrophages after recognition by TLR2. FSL-1 was internalized into the murine macrophage cell line, RAW264.7. Both chlorpromazine and methyl-β-cyclodextrin, which inhibit clathrin-dependent endocytosis, reduced FSL-1 uptake by RAW264.7 cells in a dose-dependent manner but nystatin, which inhibits caveolae- and lipid raft-dependent endocytosis, did not. FSL-1 was co-localized with clathrin but not with TLR2 in the cytosol of RAW264.7 cells. These results suggest that internalization of FSL-1 is clathrin dependent. In addition, FSL-1 was internalized by peritoneal macrophages from TLR2-deficient mice. FSL-1 was internalized by human embryonic kidney 293 cells transfected with CD14 or CD36 but not by the non-transfected cells. Also, knockdown of CD14 or CD36 in the transfectants reduced FSL-1 uptake. In this study, we suggest that (i) FSL-1 is internalized into macrophages via a clathrin-dependent endocytic pathway, (ii) the FSL-1 uptake by macrophages occurs irrespective of the presence of TLR2, and (iii) CD14 and CD36 are responsible for the internalization of FSL-1. PMID:20113368

  9. Endocytic pathway mediates refractoriness of insect Bactrocera dorsalis to RNA interference

    PubMed Central

    Li, Xiaoxue; Dong, Xiaolong; Zou, Cong; Zhang, Hongyu

    2015-01-01

    RNA interference (RNAi) is a powerful and convenient tool for sequence-specific gene silencing, and it is triggered by double-stranded RNA (dsRNA). RNAi can be easily achieved in many eukaryotes by either injecting or feeding dsRNAs. This mechanism has demonstrated its potential in fundamental research on genetics, medicine and agriculture. However, the possibility that insects might develop refractoriness to RNAi remains unexplored. In this study, we report that the oriental fruit fly, Bactrocera dorsalis, became refractory to RNAi using orally administered dsRNA targeting endogenous genes. Furthermore, refractoriness to RNAi is not gene-specific, and its duration depends on the dsRNA concentration. RNAi blockage requires the endocytic pathway. Fluorescence microscopy indicated that in RNAi refractory flies, dsRNA uptake is blocked. Genes involved in the entry of dsRNAs into cells, including chc, cog3, light and others, are down-regulated in RNAi refractory flies. Increasing the endocytic capacity by improving F-actin polymerization disrupts RNAi refractoriness after both primary and secondary dsRNA exposures. Our results demonstrate that an insect can become refractory to RNAi by preventing the entry of dsRNA into its cells. PMID:25731667

  10. Endocytic pathway mediates refractoriness of insect Bactrocera dorsalis to RNA interference.

    PubMed

    Li, Xiaoxue; Dong, Xiaolong; Zou, Cong; Zhang, Hongyu

    2015-01-01

    RNA interference (RNAi) is a powerful and convenient tool for sequence-specific gene silencing, and it is triggered by double-stranded RNA (dsRNA). RNAi can be easily achieved in many eukaryotes by either injecting or feeding dsRNAs. This mechanism has demonstrated its potential in fundamental research on genetics, medicine and agriculture. However, the possibility that insects might develop refractoriness to RNAi remains unexplored. In this study, we report that the oriental fruit fly, Bactrocera dorsalis, became refractory to RNAi using orally administered dsRNA targeting endogenous genes. Furthermore, refractoriness to RNAi is not gene-specific, and its duration depends on the dsRNA concentration. RNAi blockage requires the endocytic pathway. Fluorescence microscopy indicated that in RNAi refractory flies, dsRNA uptake is blocked. Genes involved in the entry of dsRNAs into cells, including chc, cog3, light and others, are down-regulated in RNAi refractory flies. Increasing the endocytic capacity by improving F-actin polymerization disrupts RNAi refractoriness after both primary and secondary dsRNA exposures. Our results demonstrate that an insect can become refractory to RNAi by preventing the entry of dsRNA into its cells. PMID:25731667

  11. The Spike Protein VP4 Defines the Endocytic Pathway Used by Rotavirus To Enter MA104 Cells

    PubMed Central

    Díaz-Salinas, Marco A.; Romero, Pedro; Espinosa, Rafaela; Hoshino, Yasutaka; López, Susana

    2013-01-01

    Rotaviruses are internalized into MA104 cells by endocytosis, with different endocytic pathways used depending on the virus strain. The bovine rotavirus UK strain enters cells through a clathrin-mediated endocytic process, while the simian rhesus rotavirus (RRV) strain uses a poorly defined endocytic pathway that is clathrin and caveolin independent. The viral surface protein VP7 and the spike protein VP4 interact with cellular receptors during cell binding and penetration. To determine the viral protein that defines the mechanism of internalization, we used a panel of UK × RRV reassortant viruses having different combinations of the viral structural proteins. Characterization of the infectivities of these reassortants in MA104 cells either transfected with a small interfering RNA (siRNA) against the heavy chain of clathrin or incubated with hypertonic medium that destabilizes the clathrin coat clearly showed that VP4 determines the pathway of virus entry. Of interest, the characterization of Nar3, a sialic acid-independent variant of RRV, showed that a single amino acid change in VP4 shifts the route of entry from being clathrin dependent to clathrin independent. Furthermore, characterizations of several additional rotavirus strains that differ in their use of cellular receptors showed that all entered cells by clathrin-mediated endocytosis, suggesting that diverse VP4-cell surface interactions can lead to rotavirus cell entry through this endocytic pathway. PMID:23175367

  12. Recycling compartments and the internal vesicles of multivesicular bodies harbor most of the cholesterol found in the endocytic pathway.

    PubMed

    Möbius, W; van Donselaar, E; Ohno-Iwashita, Y; Shimada, Y; Heijnen, H F G; Slot, J W; Geuze, H J

    2003-04-01

    We employed our recently developed immuno-electron microscopic method (W. Möbius, Y. Ohno-Iwashita, E. G. van Donselaar, V. M. Oorschot, Y. Shimada, T. Fujimoto, H. F. Heijnen, H. J. Geuze and J. W. Slot, J Histochem Cytochem 2002; 50: 43-55) to analyze the distribution of cholesterol in the endocytic pathway of human B lymphocytes. We could distinguish 6 categories of endocytic compartments on the basis of morphology, BSA gold uptake kinetics and organelle marker analysis. Of all cholesterol detected in the endocytic pathway, we found 20% in the recycling tubulo-vesicles and 63% present in two types of multivesicular bodies. In the multivesicular bodies, most of the cholesterol was contained in the internal membrane vesicles, the precursors of exosomes secreted by B cells. Cholesterol was almost absent from lysosomes, that contained the bulk of the lipid bis(monoacylglycero)phosphate, also termed lysobisphosphatidic acid. Thus, cholesterol displays a highly differential distribution in the various membrane domains of the endocytic pathway. PMID:12694561

  13. Amyloid precursor protein-mediated endocytic pathway disruption induces axonal dysfunction and neurodegeneration.

    PubMed

    Xu, Wei; Weissmiller, April M; White, Joseph A; Fang, Fang; Wang, Xinyi; Wu, Yiwen; Pearn, Matthew L; Zhao, Xiaobei; Sawa, Mariko; Chen, Shengdi; Gunawardena, Shermali; Ding, Jianqing; Mobley, William C; Wu, Chengbiao

    2016-05-01

    The endosome/lysosome pathway is disrupted early in the course of both Alzheimer's disease (AD) and Down syndrome (DS); however, it is not clear how dysfunction in this pathway influences the development of these diseases. Herein, we explored the cellular and molecular mechanisms by which endosomal dysfunction contributes to the pathogenesis of AD and DS. We determined that full-length amyloid precursor protein (APP) and its β-C-terminal fragment (β-CTF) act though increased activation of Rab5 to cause enlargement of early endosomes and to disrupt retrograde axonal trafficking of nerve growth factor (NGF) signals. The functional impacts of APP and its various products were investigated in PC12 cells, cultured rat basal forebrain cholinergic neurons (BFCNs), and BFCNs from a mouse model of DS. We found that the full-length wild-type APP (APPWT) and β-CTF both induced endosomal enlargement and disrupted NGF signaling and axonal trafficking. β-CTF alone induced atrophy of BFCNs that was rescued by the dominant-negative Rab5 mutant, Rab5S34N. Moreover, expression of a dominant-negative Rab5 construct markedly reduced APP-induced axonal blockage in Drosophila. Therefore, increased APP and/or β-CTF impact the endocytic pathway to disrupt NGF trafficking and signaling, resulting in trophic deficits in BFCNs. Our data strongly support the emerging concept that dysregulation of Rab5 activity contributes importantly to early pathogenesis of AD and DS. PMID:27064279

  14. Amyloid precursor protein–mediated endocytic pathway disruption induces axonal dysfunction and neurodegeneration

    PubMed Central

    Xu, Wei; Weissmiller, April M.; White, Joseph A.; Fang, Fang; Wang, Xinyi; Wu, Yiwen; Pearn, Matthew L.; Zhao, Xiaobei; Chen, Shengdi; Gunawardena, Shermali; Ding, Jianqing; Mobley, William C.

    2016-01-01

    The endosome/lysosome pathway is disrupted early in the course of both Alzheimer’s disease (AD) and Down syndrome (DS); however, it is not clear how dysfunction in this pathway influences the development of these diseases. Herein, we explored the cellular and molecular mechanisms by which endosomal dysfunction contributes to the pathogenesis of AD and DS. We determined that full-length amyloid precursor protein (APP) and its β-C-terminal fragment (β-CTF) act though increased activation of Rab5 to cause enlargement of early endosomes and to disrupt retrograde axonal trafficking of nerve growth factor (NGF) signals. The functional impacts of APP and its various products were investigated in PC12 cells, cultured rat basal forebrain cholinergic neurons (BFCNs), and BFCNs from a mouse model of DS. We found that the full-length wild-type APP (APPWT) and β-CTF both induced endosomal enlargement and disrupted NGF signaling and axonal trafficking. β-CTF alone induced atrophy of BFCNs that was rescued by the dominant-negative Rab5 mutant, Rab5S34N. Moreover, expression of a dominant-negative Rab5 construct markedly reduced APP-induced axonal blockage in Drosophila. Therefore, increased APP and/or β-CTF impact the endocytic pathway to disrupt NGF trafficking and signaling, resulting in trophic deficits in BFCNs. Our data strongly support the emerging concept that dysregulation of Rab5 activity contributes importantly to early pathogenesis of AD and DS. PMID:27064279

  15. Shibire mutations reveal distinct dynamin-independent and -dependent endocytic pathways in primary cultures of Drosophila hemocytes.

    PubMed

    Guha, A; Sriram, V; Krishnan, K S; Mayor, S

    2003-08-15

    We have developed a primary cell culture system derived from embryonic and larval stages of Drosophila. This allows for high-resolution imaging and genetic analyses of endocytic processes. Here, we have investigated endocytic pathways of three types of molecules: an endogenous receptor that binds anionic ligands (ALs), glycosylphosphatidylinositol (GPI)-anchored protein (GPI-AP), and markers of the fluid phase in primary hemocytes. We find that the endogenous AL-binding receptor (ALBR) is internalized into Rab5-positive endosomes, whereas the major portion of the fluid phase is taken up into Rab5-negative endosomes; GPI-APs are endocytosed into both classes of endosomes. ALBR and fluid-phase-containing early endosomes subsequently fuse to yield a population of Rab7-positive late endosomes. In primary culture, the endocytic phenotype of ALBR internalization in cells carrying mutations in Drosophila Dynamin (dDyn) at the shibire locus (shits) parallels the temperature-sensitive behavior of shits animals. At the restrictive temperature in shits cells, receptor-bound ALs remain completely surface accessible, localized to clathrin and alpha-adaptin-positive structures. On lowering the temperature, ALs are rapidly sequestered, suggesting a reversible block at a late step in dDyn-dependent endocytosis. By contrast, GPI-AP and fluid-phase endocytosis are quantitatively unaffected at the restrictive temperature in shits hemocytes, demonstrating a constitutive dDyn and Rab5-independent endocytic pathway in Drosophila. PMID:12857788

  16. Cellular uptake of cyclotide MCoTI-I follows multiple endocytic pathways.

    PubMed

    Contreras, Janette; Elnagar, Ahmed Y O; Hamm-Alvarez, Sarah F; Camarero, Julio A

    2011-10-30

    Cyclotides are plant-derived proteins that naturally exhibit various biological activities and whose unique cyclic structure makes them remarkably stable and resistant to denaturation or degradation. These attributes, among others, make them ideally suited for use as drug development tools. This study investigated the cellular uptake of cyclotide, MCoTI-I in live HeLa cells. Using real time confocal fluorescence microscopy imaging, we show that MCoTI-I is readily internalized in live HeLa cells and that its endocytosis is temperature-dependent. Endocytosis of MCoTI-I in HeLa cells is achieved primarily through fluid-phase endocytosis, as evidenced by its significant colocalization with 10K-dextran, but also through other pathways as well, as evidenced by its colocalization with markers for cholesterol-dependent and clathrin-mediated endocytosis, cholera toxin B and EGF respectively. Uptake does not appear to occur only via macropinocytosis as inhibition of this pathway by Latrunculin B-induced disassembly of actin filaments did not affect MCoTI-I uptake and treatment with EIPA which also seemed to inhibit other pathways collectively inhibited approximately 80% of cellular uptake. As well, a significant amount of MCoTI-I accumulates in late endosomal and lysosomal compartments and MCoTI-I-containing vesicles continue to exhibit directed movements. These findings demonstrate internalization of MCoTI-I through multiple endocytic pathways that are dominant in the cell type investigated, suggesting that this cyclotide has ready access to general endosomal/lysosomal pathways but could readily be re-targeted to specific receptors through addition of targeting ligands. PMID:21906641

  17. Live cell imaging of FM4-64, a tool for tracing the endocytic pathways in Arabidopsis root cells.

    PubMed

    Rigal, Adeline; Doyle, Siamsa M; Robert, Stéphanie

    2015-01-01

    Confocal live imaging of the amphiphilic styryl dye FM4-64 is a valuable technique to monitor organelle dynamics and in particular endocytic pathways. After application in plants, FM4-64 immediately stains the plasma membrane and is then integrated on vesicles following endomembrane system-dependent internalization processes. Over time, FM4-64 becomes distributed throughout the full vesicular network from the plasma membrane to the vacuole, including the components of the secretory pathways. Here we provide succinct examples of the many important developmental processes in plants that rely on endocytosis and describe two suitable methods to trace the endocytic pathways in Arabidopsis thaliana root cells based on the uptake of FM4-64. PMID:25408447

  18. Morphology and Dynamics of the Endocytic Pathway in Dictyostelium discoideumV⃞

    PubMed Central

    Neuhaus, Eva M.; Almers, Wolfhard; Soldati, Thierry

    2002-01-01

    Dictyostelium discoideum is a genetically and biochemically tractable social amoeba belonging to the crown group of eukaryotes. It performs some of the tasks characteristic of a leukocyte such as chemotactic motility, macropinocytosis, and phagocytosis that are not performed by other model organisms or are difficult to study. D. discoideum is becoming a popular system to study molecular mechanisms of endocytosis, but the morphological characterization of the organelles along this pathway and the comparison with equivalent and/or different organelles in animal cells and yeasts were lagging. Herein, we used a combination of evanescent wave microscopy and electron microscopy of rapidly frozen samples to visualize primary endocytic vesicles, vesicular-tubular structures of the early and late endo-lysosomal system, such as multivesicular bodies, and the specialized secretory lysosomes. In addition, we present biochemical and morphological evidence for the existence of a micropinocytic pathway, which contributes to the uptake of membrane along side macropinocytosis, which is the major fluid phase uptake process. This complex endosomal compartment underwent continuous cycles of tubulation/vesiculation as well as homo- and heterotypic fusions, in a way reminiscent of mechanisms and structures documented in leukocytes. Finally, egestion of fluid phase from the secretory lysosomes was directly observed. PMID:11950947

  19. Probiotic-derived polyphosphate improves the intestinal barrier function through the caveolin-dependent endocytic pathway.

    PubMed

    Tanaka, Kazuyuki; Fujiya, Mikihiro; Konishi, Hiroaki; Ueno, Nobuhiro; Kashima, Shin; Sasajima, Junpei; Moriichi, Kentaro; Ikuta, Katsuya; Tanabe, Hiroki; Kohgo, Yutaka

    2015-11-20

    Probiotics exhibit beneficial functions for host homeostasis maintenance. We herein investigated the mechanism by which Lactobacillus brevis-derived poly P exhibited a beneficial function. Immunostaining indicated that poly P was captured in the plasma membrane via integrin β1 in Caco2/bbe cells. The uptake of poly P was reduced by the inhibition of integrin β1 as well as caveolin-1, a major component of lipid rafts. The function of poly P, including the induction of HSP27 and enhancement of the intestinal barrier function, was suppressed by the inhibition of caveolin-1, illustrating that the function of poly P was mediated by the endocytic pathway. High-throughput sequencing revealed that poly P induced tumor necrosis factor alpha-induced protein 3, which contributes to cytoprotection, including upregulation of the intestinal barrier function. The present study demonstrates a novel host-probiotic interaction through the uptake of bacterial substance into host cells, which is distinct from pattern recognition receptor pathways. PMID:26459590

  20. γ-SNAP stimulates disassembly of endosomal SNARE complexes and regulates endocytic trafficking pathways.

    PubMed

    Inoue, Hiroki; Matsuzaki, Yuka; Tanaka, Ayaka; Hosoi, Kaori; Ichimura, Kaoru; Arasaki, Kohei; Wakana, Yuichi; Asano, Kenichi; Tanaka, Masato; Okuzaki, Daisuke; Yamamoto, Akitsugu; Tani, Katsuko; Tagaya, Mitsuo

    2015-08-01

    Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) that reside in the target membranes and transport vesicles assemble into specific SNARE complexes to drive membrane fusion. N-ethylmaleimide-sensitive factor (NSF) and its attachment protein, α-SNAP (encoded by NAPA), catalyze disassembly of the SNARE complexes in the secretory and endocytic pathways to recycle them for the next round of fusion events. γ-SNAP (encoded by NAPG) is a SNAP isoform, but its function in SNARE-mediated membrane trafficking remains unknown. Here, we show that γ-SNAP regulates the endosomal trafficking of epidermal growth factor (EGF) receptor (EGFR) and transferrin. Immunoprecipitation and mass spectrometry analyses revealed that γ-SNAP interacts with a limited range of SNAREs, including endosomal ones. γ-SNAP, as well as α-SNAP, mediated the disassembly of endosomal syntaxin-7-containing SNARE complexes. Overexpression and small interfering (si)RNA-mediated depletion of γ-SNAP changed the morphologies and intracellular distributions of endosomes. Moreover, the depletion partially suppressed the exit of EGFR and transferrin from EEA1-positive early endosomes to delay their degradation and uptake. Taken together, our findings suggest that γ-SNAP is a unique SNAP that functions in a limited range of organelles - including endosomes - and their trafficking pathways. PMID:26101353

  1. Recombinant antibody mediated delivery of organelle-specific DNA pH sensors along endocytic pathways

    NASA Astrophysics Data System (ADS)

    Modi, Souvik; Halder, Saheli; Nizak, Clément; Krishnan, Yamuna

    2013-12-01

    DNA has been used to build nanomachines with potential in cellulo and in vivo applications. However their different in cellulo applications are limited by the lack of generalizable strategies to deliver them to precise intracellular locations. Here we describe a new molecular design of DNA pH sensors with response times that are nearly 20 fold faster. Further, by changing the sequence of the pH sensitive domain of the DNA sensor, we have been able to tune their pH sensitive regimes and create a family of DNA sensors spanning ranges from pH 4 to 7.6. To enable a generalizable targeting methodology, this new sensor design also incorporates a `handle' domain. We have identified, using a phage display screen, a set of three recombinant antibodies (scFv) that bind sequence specifically to the handle domain. Sequence analysis of these antibodies revealed several conserved residues that mediate specific interactions with the cognate DNA duplex. We also found that all three scFvs clustered into different branches indicating that their specificity arises from mutations in key residues. When one of these scFvs is fused to a membrane protein (furin) that traffics via the cell surface, the scFv-furin chimera binds the `handle' and ferries a family of DNA pH sensors along the furin endocytic pathway. Post endocytosis, all DNA nanodevices retain their functionality in cellulo and provide spatiotemporal pH maps of retrogradely trafficking furin inside living cells. This new molecular technology of DNA-scFv-protein chimeras can be used to site-specifically complex DNA nanostructures for bioanalytical applications.DNA has been used to build nanomachines with potential in cellulo and in vivo applications. However their different in cellulo applications are limited by the lack of generalizable strategies to deliver them to precise intracellular locations. Here we describe a new molecular design of DNA pH sensors with response times that are nearly 20 fold faster. Further, by changing

  2. Endospanins Regulate a Postinternalization Step of the Leptin Receptor Endocytic Pathway*

    PubMed Central

    Séron, Karin; Couturier, Cyril; Belouzard, Sandrine; Bacart, Johan; Monté, Didier; Corset, Laetitia; Bocquet, Olivier; Dam, Julie; Vauthier, Virginie; Lecœur, Cécile; Bailleul, Bernard; Hoflack, Bernard; Froguel, Philippe; Jockers, Ralf; Rouillé, Yves

    2011-01-01

    Endospanin-1 is a negative regulator of the cell surface expression of leptin receptor (OB-R), and endospanin-2 is a homologue of unknown function. We investigated the mechanism for endospanin-1 action in regulating OB-R cell surface expression. Here we show that endospanin-1 and -2 are small integral membrane proteins that localize in endosomes and the trans-Golgi network. Antibody uptake experiments showed that both endospanins are transported to the plasma membrane and then internalized into early endosomes but do not recycle back to the trans-Golgi network. Overexpression of endospanin-1 or endospanin-2 led to a decrease of OB-R cell surface expression, whereas shRNA-mediated depletion of each protein increased OB-R cell surface expression. This increased cell surface expression was not observed with OB-Ra mutants defective in endocytosis or with transferrin and EGF receptors. Endospanin-1 or endospanin-2 depletion did not change the internalization rate of OB-Ra but slowed down its lysosomal degradation. Thus, both endospanins are regulators of postinternalization membrane traffic of the endocytic pathway of OB-R. PMID:21454707

  3. Endospanins regulate a postinternalization step of the leptin receptor endocytic pathway.

    PubMed

    Séron, Karin; Couturier, Cyril; Belouzard, Sandrine; Bacart, Johan; Monté, Didier; Corset, Laetitia; Bocquet, Olivier; Dam, Julie; Vauthier, Virginie; Lecœur, Cécile; Bailleul, Bernard; Hoflack, Bernard; Froguel, Philippe; Jockers, Ralf; Rouillé, Yves

    2011-05-20

    Endospanin-1 is a negative regulator of the cell surface expression of leptin receptor (OB-R), and endospanin-2 is a homologue of unknown function. We investigated the mechanism for endospanin-1 action in regulating OB-R cell surface expression. Here we show that endospanin-1 and -2 are small integral membrane proteins that localize in endosomes and the trans-Golgi network. Antibody uptake experiments showed that both endospanins are transported to the plasma membrane and then internalized into early endosomes but do not recycle back to the trans-Golgi network. Overexpression of endospanin-1 or endospanin-2 led to a decrease of OB-R cell surface expression, whereas shRNA-mediated depletion of each protein increased OB-R cell surface expression. This increased cell surface expression was not observed with OB-Ra mutants defective in endocytosis or with transferrin and EGF receptors. Endospanin-1 or endospanin-2 depletion did not change the internalization rate of OB-Ra but slowed down its lysosomal degradation. Thus, both endospanins are regulators of postinternalization membrane traffic of the endocytic pathway of OB-R. PMID:21454707

  4. Transport through the yeast endocytic pathway occurs through morphologically distinct compartments and requires an active secretory pathway and Sec18p/N-ethylmaleimide-sensitive fusion protein.

    PubMed Central

    Hicke, L; Zanolari, B; Pypaert, M; Rohrer, J; Riezman, H

    1997-01-01

    Molecules travel through the yeast endocytic pathway from the cell surface to the lysosome-like vacuole by passing through two sequential intermediates. Immunofluorescent detection of an endocytosed pheromone receptor was used to morphologically identify these intermediates, the early and late endosomes. The early endosome is a peripheral organelle that is heterogeneous in appearance, whereas the late endosome is a large perivacuolar compartment that corresponds to the prevacuolar compartment previously shown to be an endocytic intermediate. We demonstrate that inhibiting transport through the early secretory pathway in sec mutants quickly impedes transport from the early endosome. Treatment of sensitive cells with brefeldin A also blocks transport from this compartment. We provide evidence that Sec18p/N-ethylmaleimide-sensitive fusion protein, a protein required for membrane fusion, is directly required in vivo for forward transport early in the endocytic pathway. Inhibiting protein synthesis does not affect transport from the early endosome but causes endocytosed proteins to accumulate in the late endosome. As newly synthesized proteins and the late steps of secretion are not required for early to late endosome transport, but endoplasmic reticulum through Golgi traffic is, we propose that efficient forward transport in the early endocytic pathway requires delivery of lipid from secretory organelles to endosomes. Images PMID:9017592

  5. Differential interaction with endocytic and exocytic pathways distinguish parasitophorous vacuoles of Coxiella burnetii and Chlamydia trachomatis.

    PubMed Central

    Heinzen, R A; Scidmore, M A; Rockey, D D; Hackstadt, T

    1996-01-01

    Coxiella burnetii and Chlamydia trachomatis are bacterial obligate intracellular parasites that occupy distinct vacuolar niches within eucaryotic host cells. We have employed immunofluorescence, cytochemistry, fluorescent vital stains, and fluid-phase markers in conjunction with electron, confocal, and conventional microscopy to characterize the vacuolar environments of these pathogens. The acidic nature of the C. burnetii-containing vacuole was confirmed by its acquisition of the acidotropic base acridine orange (AO). The presence of the vacuolar-type (H+) ATPase (V-ATPase) within the Coxiella vacuolar membrane was demonstrated by indirect immunofluorescence, and growth of C. burnetii was inhibited by bafilomycin A1 (Baf A), a specific inhibitor of the V-ATPase. In contrast, AO did not accumulate in C. trachomatis inclusions nor was the V-ATPase found in the inclusion membrane. Moreover, chlamydial growth was not inhibited by Baf A or the lysosomotropic amines methylamine, ammonium chloride, and chloroquine. Vacuoles harboring C. burnetii incorporated the fluorescent fluid- phase markers, fluorescein isothiocyanate-dextran (FITC-dex) and Lucifer yellow (LY), indicating trafficking between that vacuole and the endocytic pathway. Neither FITC-dex nor LY was sequestered by chlamydial inclusions. The late endosomal-prelysosomal marker cation-independent mannose 6-phosphate receptor was not detectable in the vacuolar membranes encompassing either parasite. However, the lysosomal enzymes acid phosphatase and cathepsin D and the lysosomal glycoproteins LAMP-1 and LAMP-2 localized to the C. burnetii vacuole but not the chlamydial vacuole. Interaction of C. trachomatis inclusions with the Golgi-derived vesicles was demonstrated by the transport of sphingomyelin, endogenously synthesized from C6-NBD-ceramide, to the chlamydial inclusion and incorporation into the bacterial cell wall. Similar trafficking of C-NBD-ceramide was not evident in C. burnetii-infected cells

  6. Sac2/INPP5F is an inositol 4-phosphatase that functions in the endocytic pathway

    PubMed Central

    Nakatsu, Fubito; Messa, Mirko; Nández, Ramiro; Czapla, Heather; Zou, Yixiao; Strittmatter, Stephen M.

    2015-01-01

    The recruitment of inositol phosphatases to endocytic membranes mediates dephosphorylation of PI(4,5)P2, a phosphoinositide concentrated in the plasma membrane, and prevents its accumulation on endosomes. The importance of the conversion of PI(4,5)P2 to PtdIns during endocytosis is demonstrated by the presence of both a 5-phosphatase and a 4-phosphatase (Sac domain) module in the synaptojanins, endocytic PI(4,5)P2 phosphatases conserved from yeast to humans and the only PI(4,5)P2 phosphatases in yeast. OCRL, another 5-phosphatase that couples endocytosis to PI(4,5)P2 dephosphorylation, lacks a Sac domain. Here we show that Sac2/INPP5F is a PI4P phosphatase that colocalizes with OCRL on endocytic membranes, including vesicles formed by clathrin-mediated endocytosis, macropinosomes, and Rab5 endosomes. An OCRL–Sac2/INPP5F interaction could be demonstrated by coimmunoprecipitation and was potentiated by Rab5, whose activity is required to recruit Sac2/INPP5F to endosomes. Sac2/INPP5F and OCRL may cooperate in the sequential dephosphorylation of PI(4,5)P2 at the 5 and 4 position of inositol in a partnership that mimics that of the two phosphatase modules of synaptojanin. PMID:25869668

  7. Regulation of the V-ATPase along the Endocytic Pathway Occurs through Reversible Subunit Association and Membrane Localization

    PubMed Central

    Lafourcade, Céline; Sobo, Komla; Kieffer-Jaquinod, Sylvie; Garin, Jérome; van der Goot, F. Gisou

    2008-01-01

    The lumen of endosomal organelles becomes increasingly acidic when going from the cell surface to lysosomes. Luminal pH thereby regulates important processes such as the release of internalized ligands from their receptor or the activation of lysosomal enzymes. The main player in endosomal acidification is the vacuolar ATPase (V-ATPase), a multi-subunit transmembrane complex that pumps protons from the cytoplasm to the lumen of organelles, or to the outside of the cell. The active V-ATPase is composed of two multi-subunit domains, the transmembrane V0 and the cytoplasmic V1. Here we found that the ratio of membrane associated V1/Vo varies along the endocytic pathway, the relative abundance of V1 being higher on late endosomes than on early endosomes, providing an explanation for the higher acidity of late endosomes. We also found that all membrane-bound V-ATPase subunits were associated with detergent resistant membranes (DRM) isolated from late endosomes, raising the possibility that association with lipid-raft like domains also plays a role in regulating the activity of the proton pump. In support of this, we found that treatment of cells with U18666A, a drug that leads to the accumulation of cholesterol in late endosomes, affected acidification of late endosome. Altogether our findings indicate that the activity of the vATPase in the endocytic pathway is regulated both by reversible association/dissociation and the interaction with specific lipid environments. PMID:18648502

  8. AtVPS41-mediated endocytic pathway is essential for pollen tube–stigma interaction in Arabidopsis

    PubMed Central

    Hao, Lihong; Liu, Jingjing; Zhong, Sheng; Gu, Hongya; Qu, Li-Jia

    2016-01-01

    In flowering plants, extensive male–female interactions are required for successful fertilization in which various signaling cascades are involved. Prevacuolar compartments (PVC) and vacuoles are two types of subcellular compartments that terminate signal transduction by sequestrating signaling molecules in yeast and mammalian cells; however, the manner in which they might be involved in male–female interactions in plants is unknown. In this study, we identified Arabidopsis thaliana vacuolar protein sorting 41 (AtVPS41), encoded by a single-copy gene with sequence similarity to yeast Vps41p, as a new factor controlling pollen tube–stigma interaction. Loss of AtVPS41 function disrupted penetration of pollen tubes into the transmitting tissue and thus led to failed male transmission. In the pollen tubes, AtVPS41 protein is associated with PVCs and the tonoplast. We demonstrate that AtVPS41 is required for the late stage of the endocytic pathway (i.e., endomembrane trafficking from PVCs to vacuoles) because internalization of cell-surface molecules was normal in the vps41-deficient pollen tubes, whereas PVC-to-vacuole trafficking was impaired. We further show that the CHCR domain is required for subcellular localization and biological functioning of AtVPS41. These results indicate that the AtVPS41-mediated late stage of the endocytic pathway is essential for pollen tube–stigma interaction in Arabidopsis. PMID:27185920

  9. AtVPS41-mediated endocytic pathway is essential for pollen tube-stigma interaction in Arabidopsis.

    PubMed

    Hao, Lihong; Liu, Jingjing; Zhong, Sheng; Gu, Hongya; Qu, Li-Jia

    2016-05-31

    In flowering plants, extensive male-female interactions are required for successful fertilization in which various signaling cascades are involved. Prevacuolar compartments (PVC) and vacuoles are two types of subcellular compartments that terminate signal transduction by sequestrating signaling molecules in yeast and mammalian cells; however, the manner in which they might be involved in male-female interactions in plants is unknown. In this study, we identified Arabidopsis thaliana vacuolar protein sorting 41 (AtVPS41), encoded by a single-copy gene with sequence similarity to yeast Vps41p, as a new factor controlling pollen tube-stigma interaction. Loss of AtVPS41 function disrupted penetration of pollen tubes into the transmitting tissue and thus led to failed male transmission. In the pollen tubes, AtVPS41 protein is associated with PVCs and the tonoplast. We demonstrate that AtVPS41 is required for the late stage of the endocytic pathway (i.e., endomembrane trafficking from PVCs to vacuoles) because internalization of cell-surface molecules was normal in the vps41-deficient pollen tubes, whereas PVC-to-vacuole trafficking was impaired. We further show that the CHCR domain is required for subcellular localization and biological functioning of AtVPS41. These results indicate that the AtVPS41-mediated late stage of the endocytic pathway is essential for pollen tube-stigma interaction in Arabidopsis. PMID:27185920

  10. Functionalized quantum dots induce proinflammatory responses in vitro: the role of terminal functional group-associated endocytic pathways

    NASA Astrophysics Data System (ADS)

    Zhang, Yijuan; Pan, Hong; Zhang, Pengfei; Gao, Ningning; Lin, Yi; Luo, Zichao; Li, Ping; Wang, Ce; Liu, Lanlan; Pang, Daiwen; Cai, Lintao; Ma, Yifan

    2013-06-01

    PEGylation has been applied as an effective strategy of surface functionalization to improve the stability and reduce non-specific binding of quantum dots (QDs). However, its effects on the proinflammatory properties of QDs and the underlying mechanism have not been well elucidated yet. Herein, the proinflammatory effects of PEGylated CdSe/ZnS QDs with an amphiphilic polymer coating (PEG-pQDs) were investigated in human pulmonary epithelial cells and macrophages by evaluating the cytokine/chemokine production. The results showed that the proinflammatory effects of PEG-pQDs were strongly associated with the functional groups (-COOH, -NH2, -OH, and -OCH3) at the end of PEG chain. COOH-PEG-pQDs demonstrated the most proinflammatory effects followed by NH2-PEG-pQDs and HO-PEG-pQDs with CH3O-PEG-pQDs exhibiting the least proinflammatory effects. The proinflammatory effects of PEG-pQDs relied on lipid raft- and class A scavenger receptor (SRA)-dependent endocytic pathways as well as the downstream NF-κB and MAPK signaling cascades. COOH-PEG-pQDs were selectively internalized by lipid raft- and SRA-mediated endocytosis, which consequently activated NF-κB signaling pathway. On the other hand, NH2-PEG-pQDs and HO-PEG-pQDs were mostly internalized via lipid raft-mediated endocytosis, thereby activating p38 MAPK/AP-1 signaling cascades. These data revealed a critical role of terminal functional group-associated endocytic pathways in the proinflammatory responses induced by PEGylated QDs in human pulmonary epithelial cells and macrophages.PEGylation has been applied as an effective strategy of surface functionalization to improve the stability and reduce non-specific binding of quantum dots (QDs). However, its effects on the proinflammatory properties of QDs and the underlying mechanism have not been well elucidated yet. Herein, the proinflammatory effects of PEGylated CdSe/ZnS QDs with an amphiphilic polymer coating (PEG-pQDs) were investigated in human pulmonary epithelial

  11. Intravital Microscopy Reveals Differences in the Kinetics of Endocytic Pathways between Cell Cultures and Live Animals

    PubMed Central

    Masedunskas, Andrius; Porat-Shliom, Natalie; Rechache, Kamil; Aye, Myo-Pale’; Weigert, Roberto

    2012-01-01

    Intravital microscopy has enabled imaging of the dynamics of subcellular structures in live animals, thus opening the door to investigating membrane trafficking under physiological conditions. Here, we sought to determine whether the architecture and the environment of a fully developed tissue influences the dynamics of endocytic processes. To this aim, we imaged endocytosis in the stromal cells of rat salivary glands both in situ and after they were isolated and cultured on a solid surface. We found that the internalization of transferrin and dextran, two molecules that traffic via distinct mechanisms, is substantially altered in cultured cells, supporting the idea that the three dimensional organization of the tissue and the cues generated by the surrounding environment strongly affect membrane trafficking events. PMID:24710546

  12. GLUT4 and transferrin receptor are differentially sorted along the endocytic pathway in CHO cells.

    PubMed

    Wei, M L; Bonzelius, F; Scully, R M; Kelly, R B; Herman, G A

    1998-02-01

    The trafficking of GLUT4, a facilitative glucose transporter, is examined in transfected CHO cells. In previous work, we expressed GLUT4 in neuroendocrine cells and fibroblasts and found that it was targeted to a population of small vesicles slightly larger than synaptic vesicles (Herman, G.A, F. Bonzelius, A.M. Cieutat, and R.B. Kelly. 1994. Proc. Natl. Acad. Sci. USA. 91: 12750-12754.). In this study, we demonstrate that at 37 degrees C, GLUT4-containing small vesicles (GSVs) are detected after cell surface radiolabeling of GLUT4 whereas uptake of radioiodinated human transferrin does not show appreciable accumulation within these small vesicles. Immunofluorescence microscopy experiments show that at 37 degrees C, cell surface-labeled GLUT4 as well as transferrin is internalized into peripheral and perinuclear structures. At 15 degrees C, endocytosis of GLUT4 continues to occur at a slowed rate, but whereas fluorescently labeled GLUT4 is seen to accumulate within large peripheral endosomes, no perinuclear structures are labeled, and no radiolabeled GSVs are detectable. Shifting cells to 37 degrees C after accumulating labeled GLUT4 at 15 degrees C results in the reappearance of GLUT4 in perinuclear structures and GSV reformation. Cytosol acidification or treatment with hypertonic media containing sucrose prevents the exit of GLUT4 from peripheral endosomes as well as GSV formation, suggesting that coat proteins may be involved in the endocytic trafficking of GLUT4. In contrast, at 15 degrees C, transferrin continues to traffic to perinuclear structures and overall labels structures similar in distribution to those observed at 37 degrees C. Furthermore, treatment with hypertonic media has no apparent effect on transferrin trafficking from peripheral endosomes. Double-labeling experiments after the internalization of both transferrin and surface-labeled GLUT4 show that GLUT4 accumulates within peripheral compartments that exclude the transferrin receptor (TfR) at

  13. Desmosome Assembly and Disassembly Are Membrane Raft-Dependent

    PubMed Central

    Faundez, Victor; Koval, Michael; Mattheyses, Alexa L.; Kowalczyk, Andrew P.

    2014-01-01

    Strong intercellular adhesion is critical for tissues that experience mechanical stress, such as the skin and heart. Desmosomes provide adhesive strength to tissues by anchoring desmosomal cadherins of neighboring cells to the intermediate filament cytoskeleton. Alterations in assembly and disassembly compromise desmosome function and may contribute to human diseases, such as the autoimmune skin blistering disease pemphigus vulgaris (PV). We previously demonstrated that PV auto-antibodies directed against the desmosomal cadherin desmoglein 3 (Dsg3) cause loss of adhesion by triggering membrane raft-mediated Dsg3 endocytosis. We hypothesized that raft membrane microdomains play a broader role in desmosome homeostasis by regulating the dynamics of desmosome assembly and disassembly. In human keratinocytes, Dsg3 is raft associated as determined by biochemical and super resolution immunofluorescence microscopy methods. Cholesterol depletion, which disrupts rafts, prevented desmosome assembly and adhesion, thus functionally linking rafts to desmosome formation. Interestingly, Dsg3 did not associate with rafts in cells lacking desmosomal proteins. Additionally, PV IgG-induced desmosome disassembly occurred by redistribution of Dsg3 into raft-containing endocytic membrane domains, resulting in cholesterol-dependent loss of adhesion. These findings demonstrate that membrane rafts are required for desmosome assembly and disassembly dynamics, suggesting therapeutic potential for raft targeting agents in desmosomal diseases such as PV. PMID:24498201

  14. Two DNA nanomachines map pH changes along intersecting endocytic pathways inside the same cell

    NASA Astrophysics Data System (ADS)

    Modi, Souvik; Nizak, Clément; Surana, Sunaina; Halder, Saheli; Krishnan, Yamuna

    2013-06-01

    DNA is a versatile scaffold for molecular sensing in living cells, and various cellular applications of DNA nanodevices have been demonstrated. However, the simultaneous use of different DNA nanodevices within the same living cell remains a challenge. Here, we show that two distinct DNA nanomachines can be used simultaneously to map pH gradients along two different but intersecting cellular entry pathways. The two nanomachines, which are molecularly programmed to enter cells via different pathways, can map pH changes within well-defined subcellular environments along both pathways inside the same cell. We applied these nanomachines to probe the pH of early endosomes and the trans-Golgi network, in real time. When delivered either sequentially or simultaneously, both nanomachines localized into and independently captured the pH of the organelles for which they were designed. The successful functioning of DNA nanodevices within living systems has important implications for sensing and therapies in a diverse range of contexts.

  15. Cationic polystyrene nanosphere toxicity depends on cell-specific endocytic and mitochondrial injury pathways.

    PubMed

    Xia, Tian; Kovochich, Michael; Liong, Monty; Zink, Jeffrey I; Nel, Andre E

    2008-01-01

    The exponential increase in the number of new nanomaterials that are being produced increases the likelihood of adverse biological effects in humans and the environment. In this study we compared the effects of cationic nanoparticles in five different cell lines that represent portal-of-entry or systemic cellular targets for engineered nanoparticles. Although 60 nm NH(2)-labeled polystyrene (PS) nanospheres were highly toxic in macrophage (RAW 264.7) and epithelial (BEAS-2B) cells, human microvascular endothelial (HMEC), hepatoma (HEPA-1), and pheochromocytoma (PC-12) cells were relatively resistant to particle injury. While the death pathway in RAW 264.7 cells involves caspase activation, the cytotoxic response in BEAS-2B cells is more necrotic in nature. Using fluorescent-labeled NH(2)-PS, we followed the routes of particle uptake. Confocal microscopy showed that the cationic particles entered a LAMP-1 positive lysosomal compartment in RAW 264.7 cells from where the particles could escape by lysosomal rupture. A proton pump inhibitor interfered in this pathway. Subsequent deposition of the particles in the cytosol induced an increase in mitochondrial Ca(2+) uptake and cell death that could be suppressed by cyclosporin A (CsA). In contrast, NH(2)-PS toxicity in BEAS-2B cells did not involve the LAMP-1 endosomal compartment, stimulation of proton pump activity, or an increase in mitochondrial Ca(2+). Particles were taken up by caveolae, and their toxicity could be disrupted by cholesterol extraction from the surface membrane. Although the particles induced mitochondrial damage and ATP depletion, CsA did not affect cytotoxicity. Cationic particles were taken up into HEPA-1, HMEC, and PC-12 cells, but this did not lead to lysosomal permeabilization, increased Ca(2+) flux, or mitochondrial damage. Taken together, the results of this study demonstrate the importance of cell-specific uptake mechanisms and pathways that could lead to sensitivity or resistance to cationic

  16. Characterization of a Nonclathrin Endocytic Pathway: Membrane Cargo and Lipid RequirementsD⃞

    PubMed Central

    Naslavsky, Naava; Weigert, Roberto; Donaldson, Julie G.

    2004-01-01

    Clathrin-independent endocytosis internalizes plasma membrane proteins that lack cytoplasmic sequences recognized by clathrin adaptor proteins. There is evidence for different clathrin-independent pathways but whether they share common features has not been systematically tested. Here, we examined whether CD59, an endogenous glycosylphosphatidyl inositol-anchored protein (GPI-AP), and major histocompatibility protein class I (MHCI), an endogenous, integral membrane protein, entered cells through a common mechanism and followed a similar itinerary. At early times of internalization, CD59 and MHCI were found in the same Arf6-associated endosomes before joining clathrin cargo proteins such as transferrin in common sorting endosomes. CD59 and MHCI, but not transferrin, also were observed in the Arf6-associated tubular recycling membranes. Endocytosis of CD59 and MHCI required free membrane cholesterol because it was inhibited by filipin binding to the cell surface. Expression of active Arf6 stimulated endocytosis of GPI-APs and MHCI to the same extent and led to their accumulation in Arf6 endosomes that labeled intensely with filipin. This blocked delivery of GPI-APs and MHCI to early sorting endosomes and to lysosomes for degradation. Endocytosis of transferrin was not affected by any of these treatments. These observations suggest common mechanisms for endocytosis without clathrin. PMID:15146059

  17. Exit of intracellular Porphyromonas gingivalis from gingival epithelial cells is mediated by endocytic recycling pathway.

    PubMed

    Takeuchi, Hiroki; Furuta, Nobumichi; Morisaki, Ichijiro; Amano, Atsuo

    2011-05-01

    Gingival epithelial cells function as an innate host defence system to prevent intrusion by periodontal bacteria. Nevertheless, Porphyromonas gingivalis, the most well-known periodontal pathogen, can enter gingival epithelial cells and pass through the epithelial barrier into deeper tissues. However, it is poorly understood how this pathogen exits from infected cells for further transcellular spreading. The present study was performed to elucidate the cellular machinery exploited by P. gingivalis to exit from immortalized human gingival epithelial cells. P. gingivalis was shown to be internalized with early endosomes positive for the FYVE domain of EEA1 and transferrin receptor, and about half of the intracellular bacteria were then sorted to lytic compartments, including autolysosomes and late endosomes/lysosomes, while a considerable number of the remaining organisms were sorted to Rab11- and RalA-positive recycling endosomes. Inhibition experiments revealed that bacterial exit was dependent on actin polymerization, lipid rafts and microtubule assembly. Dominant negative forms and RNAi knockdown of Rab11, RalA and exocyst complex subunits (Sec5, Sec6 and Exo84) significantly disturbed the exit of P. gingivalis. These results strongly suggest that the recycling pathway is exploited by intracellular P. gingivalis to exit from infected cells to neighbouring cells as a mechanism of cell-to-cell spreading. PMID:21155963

  18. Direct Visualization of Ebola Virus Fusion Triggering in the Endocytic Pathway

    PubMed Central

    Spence, Jennifer S.; Krause, Tyler B.; Mittler, Eva; Jangra, Rohit K.

    2016-01-01

    ABSTRACT Ebola virus (EBOV) makes extensive and intricate use of host factors in the cellular endosomal/lysosomal pathway to release its genome into the cytoplasm and initiate infection. Following viral internalization into endosomes, host cysteine proteases cleave the EBOV fusion glycoprotein (GP) to unmask the binding site for its intracellular receptor, the cholesterol transporter Niemann-Pick C1 (NPC1). GP-NPC1 interaction is required for viral entry. Despite these and other recent discoveries, late events in EBOV entry following GP-NPC1 binding and culminating in GP-catalyzed fusion between viral and cellular lipid bilayers remain enigmatic. A mechanistic understanding of EBOV membrane fusion has been hampered by the failure of previous efforts to reconstitute fusion in vitro or at the cell surface. This report describes an assay to monitor initial steps directly in EBOV membrane fusion—triggering of GP and virus-cell lipid mixing—by single virions in live cells. Fusogenic triggering of GP occurs predominantly in Rab7-positive (Rab7+) endosomes, absolutely requires interaction between proteolytically primed GP and NPC1, and is blocked by key GP-specific neutralizing antibodies with therapeutic potential. Unexpectedly, cysteine protease inhibitors do not inhibit lipid mixing by virions bearing precleaved GP, even though they completely block cytoplasmic entry by these viruses, as shown previously. These results point to distinct cellular requirements for different steps in EBOV membrane fusion and suggest a model in which host cysteine proteases are dispensable for GP fusion triggering after NPC1 binding but are required for the formation of fusion pores that permit genome delivery. PMID:26861015

  19. Novel role of the small GTPase Rheb: its implication in endocytic pathway independent of the activation of mammalian target of rapamycin.

    PubMed

    Saito, Kota; Araki, Yasuhiro; Kontani, Kenji; Nishina, Hiroshi; Katada, Toshiaki

    2005-03-01

    The Ras-homologous GTPase Rheb that is conserved from yeast to human appears to be involved not only in cell growth but also in nutrient uptake. Recent biochemical analysis revealed that tuberous sclerosis complex (TSC), a GTPase-activating protein (GAP), deactivates Rheb and that phosphatidylinositol 3'-kinase (PI3k)-Akt/PKB kinase pathway activates Rheb through inhibition of the GAP-mediated deactivation. Although mammalian target of rapamycin (mTOR) kinase is implicated in the downstream target of Rheb, the direct effector(s) and exact functions of Rheb have not been fully elucidated. Here we identified that Rheb expression in cultured cells induces the formation of large cytoplasmic vacuoles, which are characterized as late endocytic (late endosome- and lysosome-like) components. The vacuole formation required the GTP form of Rheb, but not the activation of the downstream mTOR kinase. These results suggest that Rheb regulates endocytic trafficking pathway independent of the previously identified mTOR pathway. The physiological roles of the two Rheb-dependent signaling pathways are discussed in terms of nutrient uptake and cell growth or cell cycle progression. PMID:15809346

  20. HLB1 Is a Tetratricopeptide Repeat Domain-Containing Protein That Operates at the Intersection of the Exocytic and Endocytic Pathways at the TGN/EE in Arabidopsis.

    PubMed

    Sparks, J Alan; Kwon, Taegun; Renna, Luciana; Liao, Fuqi; Brandizzi, Federica; Blancaflor, Elison B

    2016-03-01

    The endomembrane system plays essential roles in plant development, but the proteome responsible for its function and organization remains largely uncharacterized in plants. Here, we identified and characterized the HYPERSENSITIVE TO LATRUNCULIN B1 (HLB1) protein isolated through a forward-genetic screen in Arabidopsis thaliana for mutants with heightened sensitivity to actin-disrupting drugs. HLB1 is a plant-specific tetratricopeptide repeat domain-containing protein of unknown function encoded by a single Arabidopsis gene. HLB1 associated with the trans-Golgi network (TGN)/early endosome (EE) and tracked along filamentous actin, indicating that it could link post-Golgi traffic with the actin cytoskeleton in plants. HLB1 was found to interact with the ADP-ribosylation-factor guanine nucleotide exchange factor, MIN7/BEN1 (HOPM INTERACTOR7/BREFELDIN A-VISUALIZED ENDOCYTIC TRAFFICKING DEFECTIVE1) by coimmunoprecipitation. The min7/ben1 mutant phenocopied the mild root developmental defects and latrunculin B hypersensitivity of hlb1, and analyses of ahlb1/ min7/ben1 double mutant showed that hlb1 and min7/ben1 operate in common genetic pathways. Based on these data, we propose that HLB1 together with MIN7/BEN1 form a complex with actin to modulate the function of the TGN/EE at the intersection of the exocytic and endocytic pathways in plants. PMID:26941089

  1. Impaired caveolae function and upregulation of alternative endocytic pathways induced by experimental modulation of intersectin-1s expression in mouse lung endothelium.

    PubMed

    Predescu, Dan N; Neamu, Radu; Bardita, Cristina; Wang, Minhua; Predescu, Sanda A

    2012-01-01

    Intersectin-1s (ITSN-1s), a protein containing five SH3 (A-E) domains, regulates via the SH3A the function of dynamin-2 (dyn2) at the endocytic site. ITSN-1s expression was modulated in mouse lung endothelium by liposome delivery of either a plasmid cDNA encoding myc-SH3A or a specific siRNA targeting ITSN-1 gene. The lung vasculature of SH3A-transduced and ITSN-1s- deficient mice was perfused with gold albumin (Au-BSA) to analyze by electron microscopy the morphological intermediates and pathways involved in transendothelial transport or with dinitrophenylated (DNP)-BSA to quantify by ELISA its transport. Acute modulation of ITSN-1s expression decreased the number of caveolae, impaired their transport, and opened the interendothelial junctions, while upregulating compensatory nonconventional endocytic/transcytotic structures. Chronic inhibition of ITSN-1s further increased the occurrence of nonconventional intermediates and partially restored the junctional integrity. These findings indicate that ITSN-1s expression is required for caveolae function and efficient transendothelial transport. Moreover, our results demonstrate that ECs are highly adapted to perform their transport function while maintaining lung homeostasis. PMID:22506115

  2. Impaired Caveolae Function and Upregulation of Alternative Endocytic Pathways Induced by Experimental Modulation of Intersectin-1s Expression in Mouse Lung Endothelium

    PubMed Central

    Predescu, Dan N.; Neamu, Radu; Bardita, Cristina; Wang, Minhua; Predescu, Sanda A.

    2012-01-01

    Intersectin-1s (ITSN-1s), a protein containing five SH3 (A-E) domains, regulates via the SH3A the function of dynamin-2 (dyn2) at the endocytic site. ITSN-1s expression was modulated in mouse lung endothelium by liposome delivery of either a plasmid cDNA encoding myc-SH3A or a specific siRNA targeting ITSN-1 gene. The lung vasculature of SH3A-transduced and ITSN-1s- deficient mice was perfused with gold albumin (Au-BSA) to analyze by electron microscopy the morphological intermediates and pathways involved in transendothelial transport or with dinitrophenylated (DNP)-BSA to quantify by ELISA its transport. Acute modulation of ITSN-1s expression decreased the number of caveolae, impaired their transport, and opened the interendothelial junctions, while upregulating compensatory nonconventional endocytic/transcytotic structures. Chronic inhibition of ITSN-1s further increased the occurrence of nonconventional intermediates and partially restored the junctional integrity. These findings indicate that ITSN-1s expression is required for caveolae function and efficient transendothelial transport. Moreover, our results demonstrate that ECs are highly adapted to perform their transport function while maintaining lung homeostasis. PMID:22506115

  3. Ephedrae herba stimulates hepatocyte growth factor-induced MET endocytosis and downregulation via early/late endocytic pathways in gefitinib-resistant human lung cancer cells.

    PubMed

    Nishimura, Yukio; Hyuga, Sumiko; Takiguchi, Soichi; Hyuga, Masashi; Itoh, Kazuyuki; Hanawa, Toshihiko

    2016-05-01

    The MET tyrosine kinase receptor and its ligand, hepatocyte growth factor (HGF), are known to be overexpressed in a variety of malignant tumor cells, and are implicated in the development of gefitinib-resistance in human non-small cell lung cancer (NSCLC) cells. Ephedrae herba was previously reported to prevent HGF-induced cancer cell motility by directly suppressing HGF/MET signaling through the inhibition of MET tyrosine kinase, and treatment with its extract also considerably reduced MET protein levels. To further investigate the mechanism underlying the Ephedrae herba-induced inhibition of MET phosphorylation as well as its degradation and subsequent disappearance, we examined the effect of Ephedrae herba on HGF-stimulated MET endocytosis and downregulation via early/late endocytic pathways in an NSCLC cell line. Using immunofluorescence microscopy, we found that pretreatment of cells with Ephedrae herba extract dramatically changed the intracellular distribution of plasma membrane-associated MET, and that the resultant MET staining was distributed throughout the cytoplasm. Pretreatment of the cells with Ephedrae herba extract also led to the rapid loss of MET and phosphorylated (p)-MET in HGF-stimulated cells. In contrast, inefficient endocytic delivery of MET and p-MET from early to late endosomes was observed in the absence of Ephedrae herba extract, since considerable amounts of the internalized MET accumulated in the early endosomes and were not delivered to lysosomes up to 1 h after HGF-stimulation. Furthermore, large amounts of MET and p-MET that had accumulated in late endosomes of Ephedrae herba-pretreated cells after HGF stimulation were observed along with bafilomycin A1. Therefore, we inferred that degradation of MET occurred in the late endosome/lysosome pathway. Moreover, western blot analysis revealed the accelerated degradation of MET and p-MET proceeds in cells pretreated with Ephedrae herba extract. Collectively, our results suggest that

  4. Visualization of the endocytic pathway in the filamentous fungus Aspergillus oryzae using an EGFP-fused plasma membrane protein

    SciTech Connect

    Higuchi, Yujiro; Nakahama, Tomoyuki; Shoji, Jun-ya; Arioka, Manabu; Kitamoto, Katsuhiko . E-mail: akitamo@mail.ecc.u-tokyo.ac.jp

    2006-02-17

    Endocytosis is an important process for cellular activities. However, in filamentous fungi, the existence of endocytosis has been so far elusive. In this study, we used AoUapC-EGFP, the fusion protein of a putative uric acid-xanthine permease with enhanced green fluorescent protein (EGFP) in Aspergillus oryzae, to examine whether the endocytic process occurs or not. Upon the addition of ammonium into the medium the fusion protein was internalized from the plasma membrane. The internalization of AoUapC-EGFP was completely blocked by sodium azide, cold, and cytochalasin A treatments, suggesting that the internalization possesses the general features of endocytosis. These results demonstrate the occurrence of endocytosis in filamentous fungi. Moreover, we discovered that the endosomal compartments appeared upon the induction of endocytosis and moved in a microtubule-dependent manner.

  5. Sterol dynamics during endocytic trafficking in Arabidopsis.

    PubMed

    Stanislas, Thomas; Grebe, Markus; Boutté, Yohann

    2014-01-01

    Sterols are lipids found in membranes of eukaryotic cells. Functions of sterols have been demonstrated for various cellular processes including endocytic trafficking in animal, fungal, and plant cells. The ability to visualize sterols at the subcellular level is crucial to understand sterol distribution and function during endocytic trafficking. In plant cells, the polyene antibiotic filipin is the most extensively used tool for the specific detection of fluorescently labeled 3-β-hydroxysterols in situ. Filipin can to some extent be used to track sterol internalization in live cells, but this application is limited, due to the inhibitory effects filipin exerts on sterol-dependent endocytosis. Nevertheless, filipin-sterol labeling can be performed on aldehyde-fixed cells which allows for sterol detection in endocytic compartments. This approach can combine studies correlating sterol distribution with experimental manipulations of endocytic trafficking pathways. Here, we describe step-by-step protocols and troubleshooting for procedures on live and fixed cells to visualize sterols during endocytic trafficking. We also provide a detailed discussion of advantages and limitations of both methods. Moreover, we illustrate the use of the endocytic recycling inhibitor brefeldin A and a genetically modified version of one of its target molecules for studying endocytic sterol trafficking. PMID:25117272

  6. The paramyxovirus simian virus 5 hemagglutinin-neuraminidase glycoprotein, but not the fusion glycoprotein, is internalized via coated pits and enters the endocytic pathway.

    PubMed Central

    Leser, G P; Ector, K J; Lamb, R A

    1996-01-01

    The hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins of the paramyxovirus simian virus 5 (SV5) are expressed on the surface of virus-infected cells. Although the F protein was found to be expressed stably, the HN protein was internalized from the plasma membrane. HN protein lacks known internalization signals in its cytoplasmic domain that are common to many integral membrane proteins that are internalized via clathrin-coated pits. Thus, the cellular pathway of HN protein internalization was examined. Biochemical analysis indicated that HN was lost from the cell surface with a t1/2 of approximately 45-50 min and turned over with a t1/2 of approximately 2 h. Immunofluorescent analysis showed internalized SV5 HN in vesicle-like structures in a juxtanuclear pattern coincident with the localization of ovalbumin. In contrast the SV5 F glycoprotein and the HN glycoprotein of the highly related parainfluenza virus 3 (hPIV-3) were found only on the cell surface. Immunogold staining of HN on the surface of SV5-infected CV-1 cells and examination using electron microscopy, showed heavy surface labeling that gradually decreased with time. Concomitantly, gold particles were detected in the endosomal system and with increasing time, gold-labeled structures having the morphology of lysosomes were observed. On the plasma membrane approximately 5% of the gold-labeled HN was found in coated pits. The inhibition of the pinching-off of coated pits from the plasma membrane by cytosol acidification significantly reduced HN internalization. Internalized HN was co-localized with gold-conjugated transferrin, a marker for the early endosomal compartments, and with gold-conjugated bovine serum albumin, a marker for late endosomal compartments. Taken together, these data strongly suggest that the HN glycoprotein is internalized via clathrin-coated pits and delivered to the endocytic pathway. Images PMID:8741847

  7. Inhibition of Endocytic Processes by Photodynamic Therapy

    PubMed Central

    Kessel, David

    2011-01-01

    Background and Objective Recent studies have demonstrated an effect of photodamage on the endocytic pathway involved in recycling of membrane components. Using a series of agents with known sub-cellular targets, we explored the determinants of photodynamic inhibition of endocytic processes in three cell lines: a murine leukemia, a murine hepatoma and a non-malignant epithelial cell line of human origin. Study Design/Materials and Methods The PI-3 kinase antagonist wortmannin blocks endosomal processing pathway dependent on this enzyme, providing an indication of the ‘flux’ of endocytosis. Microscopic observations were used to assess the effect of photodamage on this pathway. Photosensitizing agents specific for mitochondrial, endoplasmic reticulum (ER), lysosomal and endosomal photodamage were employed. Conclusions Sub-lethal photodamage directed against endosomes or lysosomes interrupted early steps in this endocytic process in the hepatoma cell line. A mechanism for these effects is proposed. Mitochondrial photodamage could interrupt endocytosis, but at levels that also induced apoptosis. ER photodamage did not affect endocytosis even at lethal levels. Somewhat similar results were obtained with other cell lines, but there were sufficient differences to indicate that the cell phenotype is, in part, a determinant of the endocytic response to PDT. Further work will be needed to delineate the role of these endocytic effects in the array of responses to photodynamic therapy. PMID:22057481

  8. Enhanced intracellular uptake and endocytic pathway selection mediated by hemocompatible ornithine grafted chitosan polycation for gene delivery.

    PubMed

    Alex, Susan M; Sharma, Chandra P

    2014-10-01

    Nanotechnology is adopted in gene therapy research to create gene vectors that will facilitate gene transfer to cells with utmost efficacy and safety. For vector design, polymers are the preferred nonviral colloidal systems as they are feasible for any chemical modifications. In this study, chitosan, a versatile biopolymer has been subjected to chemical conjugation with the amino acid ornithine to generate chitosan-ornithine conjugate (CON) for gene delivery. With the help of FTIR and (1)H NMR spectra the chemical composition of the chitosan derivative was confirmed. Buffering capacity was found enhanced with the synthesised chitosan derivative when compared to the parent unmodified chitosan. The cationic derivative formed nanoparticles when mixed with negatively charged DNA. The nanoparticles showed good DNA retardation ability in agarose gel electrophoresis and sizes were ascertained by DLS and TEM observations. The derivative on interaction with blood plasma showed negligible protein adsorption and did not cause either hemolysis or RBC aggregation in blood. In vitro cell culture also revealed the CON derivative to be nontoxic to cells and capable of transfection with an explicit increase in cellular uptake of nanoparticles. An uptake study in the presence of endocytosis inhibitors indicated the specific pathway used for cell entry. The results revealed that the clathrin mediated pathway and dynamin played a role in the internalisation of these specific nanoparticles. PMID:25193152

  9. Probing the endocytic pathways of the filamentous bacteriophage in live cells using ratiometric pH fluorescent indicator.

    PubMed

    Tian, Ye; Wu, Man; Liu, Xiangxiang; Liu, Zhi; Zhou, Quan; Niu, Zhongwei; Huang, Yong

    2015-02-18

    Viral nanoparticles have attracted extensive research interests in diverse applications of diagnosis and therapy. In particular, filamentous M13 bacteriophages have shown great potential in biomedical applications. However, its pathways entering into cells still remain unclear, and this greatly hinders its further use as a drug or gene carrier. Here, a ratiometric M13 pH probe is designed by conjugating two fluorescent dyes onto the surface of M13. Since the intensity ratio is not influenced by probe concentration, ion strength, temperature, photobleaching, and optical path length, this ratiometric probe can be used to investigate the intracellular pH map of M13. More importantly, the internalization mechanism of M13 can be elucidated. It is found that this filamentous phage shows great cell-type dependence in interaction with cells and internalization mechanism. The phage tends to be bounded on the cell membrane of only epithelial cells, not endothelial cells. Furthermore, the M13 phage enters into cells through endocytosis with specific mechanism: clathrin-mediated endocytosis and macropinocytosis for HeLa; vesicular transport, clathrin-mediated endocytosis, and macropinocytosis for MCF-7; caveolae-mediated endocytosis for human dermal microvascular endothelial cell (HDMEC). This work provides key notes for cancer diagnosis and therapy based on filamentous bacteriophage, especially for design of pH-sensitive drug delivery systems. PMID:25308797

  10. Cholesterol and ORP1L-mediated ER contact sites control autophagosome transport and fusion with the endocytic pathway

    PubMed Central

    Wijdeven, Ruud H.; Janssen, Hans; Nahidiazar, Leila; Janssen, Lennert; Jalink, Kees; Berlin, Ilana; Neefjes, Jacques

    2016-01-01

    Autophagy is the main homeostatic pathway guiding cytosolic materials for degradation by the lysosome. Maturation of autophagosomes requires their transport towards the perinuclear region of the cell, with key factors underlying both processes still poorly understood. Here we show that transport and positioning of late autophagosomes depends on cholesterol by way of the cholesterol-sensing Rab7 effector ORP1L. ORP1L localizes to late autophagosomes and—under low-cholesterol conditions—contacts the ER protein VAP-A, forming ER-autophagosome contact sites, which prevent minus-end transport by the Rab7–RILP–dynein complex. ORP1L-mediated contact sites also inhibit localization of PLEKHM1 to Rab7. PLEKHM1, together with RILP, then recruits the homotypic fusion and vacuole protein-sorting (HOPS) complex for fusion of autophagosomes with late endosomes and lysosomes. Thus, ORP1L, via its liganding by lipids and the formation of contacts between autophagic vacuoles and the ER, governs the last steps in autophagy that lead to the lysosomal degradation of cytosolic material. PMID:27283760

  11. Cholesterol and ORP1L-mediated ER contact sites control autophagosome transport and fusion with the endocytic pathway.

    PubMed

    Wijdeven, Ruud H; Janssen, Hans; Nahidiazar, Leila; Janssen, Lennert; Jalink, Kees; Berlin, Ilana; Neefjes, Jacques

    2016-01-01

    Autophagy is the main homeostatic pathway guiding cytosolic materials for degradation by the lysosome. Maturation of autophagosomes requires their transport towards the perinuclear region of the cell, with key factors underlying both processes still poorly understood. Here we show that transport and positioning of late autophagosomes depends on cholesterol by way of the cholesterol-sensing Rab7 effector ORP1L. ORP1L localizes to late autophagosomes and-under low-cholesterol conditions-contacts the ER protein VAP-A, forming ER-autophagosome contact sites, which prevent minus-end transport by the Rab7-RILP-dynein complex. ORP1L-mediated contact sites also inhibit localization of PLEKHM1 to Rab7. PLEKHM1, together with RILP, then recruits the homotypic fusion and vacuole protein-sorting (HOPS) complex for fusion of autophagosomes with late endosomes and lysosomes. Thus, ORP1L, via its liganding by lipids and the formation of contacts between autophagic vacuoles and the ER, governs the last steps in autophagy that lead to the lysosomal degradation of cytosolic material. PMID:27283760

  12. Benzyl-N-acetyl-alpha-D-galactosaminide induces a storage disease-like phenotype by perturbing the endocytic pathway.

    PubMed

    Ulloa, Fausto; Real, Francisco X

    2003-04-01

    The sugar analog O-benzyl-N-acetyl-alpha-d-galactosaminide (BG) is an inhibitor of glycan chain elongation and inhibits alpha2,3-sialylation in mucus-secreting HT-29 cells. Long-term exposure of these cells to BG is associated with the accumulation of apical glycoproteins in cytoplasmic vesicles. The mechanisms involved therein and the nature of the vesicles have not been elucidated. In these cells, a massive amount of BG metabolites is synthesized. Because sialic acid is mainly distributed apically in epithelial cells, it has been proposed that the BG-induced undersialylation of apical membrane glycoproteins is responsible for their intracellular accumulation due to a defect in anterograde traffic and that sialic acid may constitute an apical targeting signal. In this work, we demonstrate that the intracellular accumulation of membrane glycoproteins does not result mainly from defects in anterograde traffic. By contrast, in BG-treated cells, endocytosed membrane proteins were retained intracellularly for longer periods of time than in control cells and colocalized with accumulated MUC1 and beta(1) integrin in Rab7/lysobisphosphatidic acid(+) vesicles displaying features of late endosomes. The phenotype of BG-treated cells is reminiscent of that observed in lysosomal storage disorders. Sucrose induced a BG-like, lysosomal storage disease-like phenotype without affecting sialylation, indicating that undersialylation is not a requisite for the intracellular accumulation of membrane glycoproteins. Our findings strongly support the notion that the effects observed in BG-treated cells result from the accumulation of BG-derived metabolites and from defects in the endosomal pathway. We propose that abnormal subcellular distribution of membrane glycoproteins involved in cellular communication and/or signaling may also take place in lysosomal storage disorders and may contribute to their pathogenesis. PMID:12538583

  13. Megapinocytosis: a novel endocytic pathway.

    PubMed

    Bauer, Andrea; Subramanian, Narmadha; Villinger, Clarissa; Frascaroli, Giada; Mertens, Thomas; Walther, Paul

    2016-06-01

    M2 macrophages showed large endocytotic structures, very different from classical macropinosomes that we named megapinosomes. As observed in the scanning electron microscope, megapinosome formation started with a large (diameter of several micrometers) invagination of the plasma membrane. When the invagination was almost completed, the remaining opening was closed by an actinomorphous centripetal arrangement of many (about 50-100) microvilli-like structures. In transmission electron microscopy using high-pressure freezing, we observed that the megapinosome was filled with a trabecular meshwork that originated from the highly structured plasma membrane. The trabecular meshwork was topologically part of the cytosol and separated from the extracellular fluid by a lipid bilayer. According to ultrastructural features, we could define different phases of megapinosome formation and decay. Megapinosomes became more frequent when M2 macrophages were inoculated with human cytomegalovirus. We did not find megapinosome formation in M1 macrophages. PMID:26733077

  14. Pseudotyping human immunodeficiency virus type 1 (HIV-1) by the glycoprotein of vesicular stomatitis virus targets HIV-1 entry to an endocytic pathway and suppresses both the requirement for Nef and the sensitivity to cyclosporin A.

    PubMed Central

    Aiken, C

    1997-01-01

    Human immunodeficiency virus type 1 (HIV-1) normally enters cells by direct fusion with the plasma membrane. In this report, HIV-1 particles capable of infecting cells through an endocytic pathway are described. Chimeric viruses composed of the HIV-1 core and the envelope glycoprotein of vesicular stomatitis virus (VSV-G) were constructed and are herein termed HIV-1(VSV) pseudotypes. HIV-1(VSV) pseudotypes were 20- to 130-fold more infectious than nonpseudotyped HIV-1. Infection by HIV-1(VSV) pseudotypes was markedly diminished by ammonium chloride and concanamycin A, a selective inhibitor of vacuolar H+ ATPases, demonstrating that these viruses require endosomal acidification to achieve productive infection. HIV-1 is thus capable of performing all of the viral functions necessary for infection when entry is targeted to an endocytic route. Maximal HIV-1 infectivity requires the presence of the viral Nef protein and the cellular protein cyclophilin A (CyPA) during virus assembly. Pseudotyping by VSV-G markedly suppressed the requirement for Nef. HIV-1(VSV) particles were also resistant to inhibition by cyclosporin A; however, the deleterious effect of a gag mutation inhibiting CyPA incorporation was not relieved by VSV-G. These results suggest that Nef acts at a step of the HIV-1 life cycle that is either circumvented or facilitated by targeting virus entry to an endocytic pathway. The findings also support the hypothesis that Nef and CyPA enhance HIV-1 infectivity through independent processes and demonstrate a mechanistic difference between reduction of HIV-1 infectivity by cyclosporin A and gag mutations that decrease HIV-1 incorporation of CyPA. PMID:9223476

  15. HLB1 Is a Tetratricopeptide Repeat Domain-Containing Protein That Operates at the Intersection of the Exocytic and Endocytic Pathways at the TGN/EE in Arabidopsis[OPEN

    PubMed Central

    Sparks, J. Alan; Renna, Luciana; Liao, Fuqi; Brandizzi, Federica

    2016-01-01

    The endomembrane system plays essential roles in plant development, but the proteome responsible for its function and organization remains largely uncharacterized in plants. Here, we identified and characterized the HYPERSENSITIVE TO LATRUNCULIN B1 (HLB1) protein isolated through a forward-genetic screen in Arabidopsis thaliana for mutants with heightened sensitivity to actin-disrupting drugs. HLB1 is a plant-specific tetratricopeptide repeat domain-containing protein of unknown function encoded by a single Arabidopsis gene. HLB1 associated with the trans-Golgi network (TGN)/early endosome (EE) and tracked along filamentous actin, indicating that it could link post-Golgi traffic with the actin cytoskeleton in plants. HLB1 was found to interact with the ADP-ribosylation-factor guanine nucleotide exchange factor, MIN7/BEN1 (HOPM INTERACTOR7/BREFELDIN A-VISUALIZED ENDOCYTIC TRAFFICKING DEFECTIVE1) by coimmunoprecipitation. The min7/ben1 mutant phenocopied the mild root developmental defects and latrunculin B hypersensitivity of hlb1, and analyses of a hlb1/ min7/ben1 double mutant showed that hlb1 and min7/ben1 operate in common genetic pathways. Based on these data, we propose that HLB1 together with MIN7/BEN1 form a complex with actin to modulate the function of the TGN/EE at the intersection of the exocytic and endocytic pathways in plants. PMID:26941089

  16. Identification of nucleolin as a lipid-raft-dependent β1-integrin-interacting protein in A375 cell migration.

    PubMed

    Bi, Jiajia; Wang, Ruifei; Zhang, Yue; Han, Xiaoqing; Ampah, Khamal Kwesi; Liu, Wenguang; Zeng, Xianlu

    2013-12-01

    Lipid rafts are related to cell surface receptor function. Integrin is a major surface receptor protein in cell adhesion and migration on the extracellular matrix (ECM). Here, we showed that lipid rafts played a critical role in human melanoma A375 cell spreading and migration on fibronectin; an important component of the ECM that interacts with β1 integrin. We found that the disruption of lipid rafts did not markedly inhibit the expression and activation of β1 integrin. By coimmunoprecipitation and mass spectrometry, we investigated the influence of lipid rafts on the β1 integrin complex and identified nucleolin as a potential lipid-raft-dependent β1-integrin-interacting protein. Upon confirmation of the interaction between β1 integrin and nucleolin, further studies revealed that nucleolin colocalized with β1 integrin in lipid rafts and raft disruption interrupted their association. In addition, knockdown of nucleolin markedly attenuated A375 cell spreading and migration on fibronectin. Taken together, we demonstrated that nucleolin is a critical lipid-raft-dependent β1-integrin-interacting protein in A375 cell spreading and migration on fibronectin. PMID:24292944

  17. Qualitative and quantitative analysis of endocytic recycling.

    PubMed

    Reineke, James B; Xie, Shuwei; Naslavsky, Naava; Caplan, Steve

    2015-01-01

    Endocytosis, which encompasses the internalization and sorting of plasma membrane (PM) lipids and proteins to distinct membrane-bound intracellular compartments, is a highly regulated and fundamental cellular process by which eukaryotic cells dynamically regulate their PM composition. Indeed, endocytosis is implicated in crucial cellular processes that include proliferation, migration, and cell division as well as maintenance of tissue homeostasis such as apical-basal polarity. Once PM constituents have been taken up into the cell, either via clathrin-dependent endocytosis (CDE) or clathrin-independent endocytosis (CIE), they typically have two fates: degradation through the late-endosomal/lysosomal pathway or returning to the PM via endocytic recycling pathways. In this review, we will detail experimental procedures that allow for both qualitative and quantitative assessment of endocytic recycling of transmembrane proteins internalized by CDE and CIE, using the HeLa cervical cancer cell line as a model system. PMID:26360033

  18. Deletions of Endocytic Components VPS28 and VPS32 Affect Growth at Alkaline pH and Virulence through both RIM101-Dependent and RIM101-Independent Pathways in Candida albicans

    PubMed Central

    Cornet, Muriel; Bidard, Frédérique; Schwarz, Patrick; Da Costa, Grégory; Blanchin-Roland, Sylvie; Dromer, Françoise; Gaillardin, Claude

    2005-01-01

    Ambient pH signaling involves a cascade of conserved Rim or Pal products in ascomycetous yeasts or filamentous fungi, respectively. Recent evidences in the fungi Aspergillus nidulans, Saccharomyces cerevisiae, Yarrowia lipolytica, and Candida albicans suggested that components of endosomal sorting complexes required for transport (ESCRT) involved in endocytic trafficking were needed for signal transduction along the Rim pathway. In this study, we confirm these findings with C. albicans and show that Vps28p (ESCRT-I) and Vps32p/Snf7p (ESCRT-III) are required for the transcriptional regulation of known targets of the Rim pathway, such as the PHR1 and PHR2 genes encoding cell surface proteins, which are expressed at alkaline and acidic pH, respectively. We additionally show that deletion of these two VPS genes, particularly VPS32, has a more drastic effect than a RIM101 deletion on growth at alkaline pH and that this effect is only partially suppressed by expression of a constitutively active form of Rim101p. Finally, in an in vivo mouse model, both vps null mutants were significantly less virulent than a rim101 mutant, suggesting that VPS28 and VPS32 gene products affect virulence both through Rim-dependent and Rim-independent pathways. PMID:16299290

  19. shRNA-Based Screen Identifies Endocytic Recycling Pathway Components That Act as Genetic Modifiers of Alpha-Synuclein Aggregation, Secretion and Toxicity

    PubMed Central

    Macedo, Diana; Raquel, Helena; Simões, Pedro D.; Giorgini, Flaviano; Ramalho, José S.; Barral, Duarte C.; Ferreira Moita, Luís; Outeiro, Tiago Fleming

    2016-01-01

    Alpha-Synuclein (aSyn) misfolding and aggregation is common in several neurodegenerative diseases, including Parkinson’s disease and dementia with Lewy bodies, which are known as synucleinopathies. Accumulating evidence suggests that secretion and cell-to-cell trafficking of pathological forms of aSyn may explain the typical patterns of disease progression. However, the molecular mechanisms controlling aSyn aggregation and spreading of pathology are still elusive. In order to obtain unbiased information about the molecular regulators of aSyn oligomerization, we performed a microscopy-based large-scale RNAi screen in living cells. Interestingly, we identified nine Rab GTPase and kinase genes that modulated aSyn aggregation, toxicity and levels. From those, Rab8b, Rab11a, Rab13 and Slp5 were able to promote the clearance of aSyn inclusions and rescue aSyn induced toxicity. Furthermore, we found that endocytic recycling and secretion of aSyn was enhanced upon Rab11a and Rab13 expression in cells accumulating aSyn inclusions. Overall, our study resulted in the identification of new molecular players involved in the aggregation, toxicity, and secretion of aSyn, opening novel avenues for our understanding of the molecular basis of synucleinopathies. PMID:27123591

  20. Nucleocytoplasmic Shuttling of Endocytic Proteins

    PubMed Central

    Vecchi, Manuela; Polo, Simona; Poupon, Viviane; van de Loo, Jan-Willem; Benmerah, Alexandre; Di Fiore, Pier Paolo

    2001-01-01

    Many cellular processes rely on the ordered assembly of macromolecular structures. Here, we uncover an unexpected link between two such processes, endocytosis and transcription. Many endocytic proteins, including eps15, epsin1, the clathrin assembly lymphoid myeloid leukemia (CALM), and α-adaptin, accumulate in the nucleus when nuclear export is inhibited. Endocytosis and nucleocytoplasmic shuttling of endocytic proteins are apparently independent processes, since inhibition of endocytosis did not appreciably alter nuclear translocation of endocytic proteins, and blockade of nuclear export did not change the initial rate of endocytosis. In the nucleus, eps15 and CALM acted as positive modulators of transcription in a GAL4-based transactivation assay, thus raising the intriguing possibility that some endocytic proteins play a direct or indirect role in transcriptional regulation. PMID:11425879

  1. The novel chlamydial adhesin CPn0473 mediates the lipid raft-dependent uptake of Chlamydia pneumoniae.

    PubMed

    Fechtner, Tim; Galle, Jan N; Hegemann, Johannes H

    2016-08-01

    Chlamydiae are Gram-negative, obligate intracellular pathogens that pose a serious threat to public health worldwide. Chlamydial surface molecules are essential for host cell invasion. The first interaction with the host cell is thereby accomplished by the Outer membrane complex protein B (OmcB) binding to heparan sulfate moieties on the host cell surface, followed by the interaction of the chlamydial polymorphic membrane proteins (Pmps) with host cell receptors. Specifically, the interaction of the Pmp21 adhesin and invasin with its human interaction partner, the epidermal growth factor receptor, results in receptor activation, down-stream signalling and finally internalization of the bacteria. Blocking both, the OmcB and Pmp21 adhesion pathways, did not completely abolish infection, suggesting the presence of additional factors relevant for host cell invasion. Here, we show that the novel surface protein CPn0473 of Chlamydia pneumoniae contributes to the binding and invasion of infectious chlamydial particles. CPn0473 is expressed late in the infection cycle and located on the infectious chlamydial cell surface. Soluble recombinant CPn0473 as well as rCPn0473-coupled fluorescent latex beads adhere to human epithelial HEp-2 cells. Interestingly, in classical infection blocking experiments pretreatment of HEp-2 cells with rCPn0473 does not attenuate adhesion but promotes dose-dependently internalization by C. pneumoniae suggesting an unusual mode of action for this adhesin. This CPn0473-dependent promotion of infection by C. pneumoniae depends on two different domains within the protein and requires intact lipid rafts. Thus, inhibition of the interaction of CPn0473 with the host cell could provide a way to reduce the virulence of C. pneumoniae. PMID:26780295

  2. Spatio-temporal Model of Endogenous ROS and Raft-Dependent WNT/Beta-Catenin Signaling Driving Cell Fate Commitment in Human Neural Progenitor Cells

    PubMed Central

    Haack, Fiete; Lemcke, Heiko; Ewald, Roland; Rharass, Tareck; Uhrmacher, Adelinde M.

    2015-01-01

    Canonical WNT/β-catenin signaling is a central pathway in embryonic development, but it is also connected to a number of cancers and developmental disorders. Here we apply a combined in-vitro and in-silico approach to investigate the spatio-temporal regulation of WNT/β-catenin signaling during the early neural differentiation process of human neural progenitors cells (hNPCs), which form a new prospect for replacement therapies in the context of neurodegenerative diseases. Experimental measurements indicate a second signal mechanism, in addition to canonical WNT signaling, being involved in the regulation of nuclear β-catenin levels during the cell fate commitment phase of neural differentiation. We find that the biphasic activation of β-catenin signaling observed experimentally can only be explained through a model that combines Reactive Oxygen Species (ROS) and raft dependent WNT/β-catenin signaling. Accordingly after initiation of differentiation endogenous ROS activates DVL in a redox-dependent manner leading to a transient activation of down-stream β-catenin signaling, followed by continuous auto/paracrine WNT signaling, which crucially depends on lipid rafts. Our simulation studies further illustrate the elaborate spatio-temporal regulation of DVL, which, depending on its concentration and localization, may either act as direct inducer of the transient ROS/β-catenin signal or as amplifier during continuous auto-/parcrine WNT/β-catenin signaling. In addition we provide the first stochastic computational model of WNT/β-catenin signaling that combines membrane-related and intracellular processes, including lipid rafts/receptor dynamics as well as WNT- and ROS-dependent β-catenin activation. The model’s predictive ability is demonstrated under a wide range of varying conditions for in-vitro and in-silico reference data sets. Our in-silico approach is realized in a multi-level rule-based language, that facilitates the extension and modification of the

  3. Spatio-temporal model of endogenous ROS and raft-dependent WNT/beta-catenin signaling driving cell fate commitment in human neural progenitor cells.

    PubMed

    Haack, Fiete; Lemcke, Heiko; Ewald, Roland; Rharass, Tareck; Uhrmacher, Adelinde M

    2015-03-01

    Canonical WNT/β-catenin signaling is a central pathway in embryonic development, but it is also connected to a number of cancers and developmental disorders. Here we apply a combined in-vitro and in-silico approach to investigate the spatio-temporal regulation of WNT/β-catenin signaling during the early neural differentiation process of human neural progenitors cells (hNPCs), which form a new prospect for replacement therapies in the context of neurodegenerative diseases. Experimental measurements indicate a second signal mechanism, in addition to canonical WNT signaling, being involved in the regulation of nuclear β-catenin levels during the cell fate commitment phase of neural differentiation. We find that the biphasic activation of β-catenin signaling observed experimentally can only be explained through a model that combines Reactive Oxygen Species (ROS) and raft dependent WNT/β-catenin signaling. Accordingly after initiation of differentiation endogenous ROS activates DVL in a redox-dependent manner leading to a transient activation of down-stream β-catenin signaling, followed by continuous auto/paracrine WNT signaling, which crucially depends on lipid rafts. Our simulation studies further illustrate the elaborate spatio-temporal regulation of DVL, which, depending on its concentration and localization, may either act as direct inducer of the transient ROS/β-catenin signal or as amplifier during continuous auto-/parcrine WNT/β-catenin signaling. In addition we provide the first stochastic computational model of WNT/β-catenin signaling that combines membrane-related and intracellular processes, including lipid rafts/receptor dynamics as well as WNT- and ROS-dependent β-catenin activation. The model's predictive ability is demonstrated under a wide range of varying conditions for in-vitro and in-silico reference data sets. Our in-silico approach is realized in a multi-level rule-based language, that facilitates the extension and modification of the

  4. DNA internalized via caveolae requires microtubule-dependent, Rab7-independent transport to the late endocytic pathway for delivery to the nucleus.

    PubMed

    Wong, Athena W; Scales, Suzie J; Reilly, Dorothea E

    2007-08-01

    Using cationic liposomes to mediate gene delivery by transfection has the advantages of improved safety and simplicity of use over viral gene therapy. Understanding the mechanism by which cationic liposome:DNA complexes are internalized and delivered to the nucleus should help identify which transport steps might be manipulated in order to improve transfection efficiencies. We therefore examined the endocytosis and trafficking of two cationic liposomes, DMRIE-C and Lipofectamine LTX, in CHO cells. We found that DMRIE-C-transfected DNA is internalized via caveolae, while LTX-transfected DNA is internalized by clathrin-mediated endocytosis, with both pathways converging at the late endosome or lysosome. Inhibition of microtubule-dependent transport with nocodazole revealed that DMRIE-C:DNA complexes cannot enter the cytosol directly from caveosomes. Lysosomal degradation of transfected DNA has been proposed to be a major reason for poor transfection efficiency. However, in our system dominant negatives of both Rab7 and its effector RILP inhibited late endosome to lysosome transport of DNA complexes and LDL, but did not affect DNA delivery to the nucleus. This suggests that DNA is able to escape from late endosomes without traversing lysosomes and that caveosome to late endosome transport does not require Rab7 function. Lysosomal inhibition with chloroquine likewise had no effect on transfection product titers. These data suggest that DMRIE-C and LTX transfection complexes are endocytosed by separate pathways that converge at the late endosome or lysosome, but that blocking lysosomal traffic does not improve transfection product yields, identifying late endosome/lysosome to nuclear delivery as a step for future study. PMID:17562704

  5. IMAGING ENDOCYTIC CLATHRIN STRUCTURES IN LIVING CELLS

    PubMed Central

    Kirchhausen, Tom

    2009-01-01

    Our understanding of the clathrin-dependent endocytic pathway owes much to new visualization techniques. Budding coated pits and clathrin-coated structures are transient molecular machines with distinctive morphological characteristics, and fluorescently labeled versions of a variety of marker proteins have given us a tantalizing glimpse of the dynamics of the system in living cells. Recent live-cell imaging studies reveal unexpected modes of coat assembly, with distinct kinetics, recruitment of associated proteins, requirements for the participation of actin and its accessory proteins, and apparently, distinct mechanisms of membrane deformation. A crucial issue is to connect the events detected by light microscopy with the structures and properties of the molecular constituents. Here, I outline descriptions of coat assembly in different circumstances that are consistent with what is known from x-ray crystallography and electron microscopy. PMID:19836955

  6. Cargo- and compartment-selective endocytic scaffold proteins

    PubMed Central

    2004-01-01

    The endocytosis of membrane receptors is a complex and tightly controlled process that is essential for maintaining cellular homoeostasis. The removal of receptors from the cell surface can be constitutive or ligand-induced, and occurs in a clathrin-dependent or -independent manner. The recruitment of receptors into specialized membrane domains, the formation of vesicles and the trafficking of receptors together with their ligands within endocytic compartments are regulated by reversible protein modifications, and multiple protein–protein and protein–lipid interactions. Recent reports describe a variety of multidomain molecules that facilitate receptor endocytosis and function as platforms for the assembly of protein complexes. These scaffold proteins typically act in a cargo-specific manner, recognizing one or more receptor types, or function at the level of endocytic cellular microcompartments by controlling the movement of cargo molecules and linking endocytic machineries to signalling pathways. In the present review we summarize present knowledge on endocytic scaffold molecules and discuss their functions. PMID:15219178

  7. Efficient Endocytic Uptake and Maturation in Drosophila Oocytes Requires Dynamitin/p50.

    PubMed

    Liu, Guojun; Sanghavi, Paulomi; Bollinger, Kathryn E; Perry, Libby; Marshall, Brendan; Roon, Penny; Tanaka, Tsubasa; Nakamura, Akira; Gonsalvez, Graydon B

    2015-10-01

    Dynactin is a multi-subunit complex that functions as a regulator of the Dynein motor. A central component of this complex is Dynamitin/p50 (Dmn). Dmn is required for endosome motility in mammalian cell lines. However, the extent to which Dmn participates in the sorting of cargo via the endosomal system is unknown. In this study, we examined the endocytic role of Dmn using the Drosophila melanogaster oocyte as a model. Yolk proteins are internalized into the oocyte via clathrin-mediated endocytosis, trafficked through the endocytic pathway, and stored in condensed yolk granules. Oocytes that were depleted of Dmn contained fewer yolk granules than controls. In addition, these oocytes accumulated numerous endocytic intermediate structures. Particularly prominent were enlarged endosomes that were relatively devoid of Yolk proteins. Ultrastructural and genetic analyses indicate that the endocytic intermediates are produced downstream of Rab5. Similar phenotypes were observed upon depleting Dynein heavy chain (Dhc) or Lis1. Dhc is the motor subunit of the Dynein complex and Lis1 is a regulator of Dynein activity. We therefore propose that Dmn performs its function in endocytosis via the Dynein motor. Consistent with a role for Dynein in endocytosis, the motor colocalized with the endocytic machinery at the oocyte cortex in an endocytosis-dependent manner. Our results suggest a model whereby endocytic activity recruits Dynein to the oocyte cortex. The motor along with its regulators, Dynactin and Lis1, functions to ensure efficient endocytic uptake and maturation. PMID:26265702

  8. An ARF6/Rab35 GTPase cascade for endocytic recycling and successful cytokinesis.

    PubMed

    Chesneau, Laurent; Dambournet, Daphné; Machicoane, Mickaël; Kouranti, Ilektra; Fukuda, Mitsunori; Goud, Bruno; Echard, Arnaud

    2012-01-24

    Cytokinesis bridge instability leads to binucleated cells that can promote tumorigenesis in vivo. Membrane trafficking is crucial for animal cell cytokinesis, and several endocytic pathways regulated by distinct GTPases (Rab11, Rab21, Rab35, ARF6, RalA/B) contribute to the postfurrowing steps of cytokinesis. However, little is known about how these pathways are coordinated for successful cytokinesis. The Rab35 GTPase controls a fast endocytic recycling pathway and must be activated for SEPTIN cytoskeleton localization at the intercellular bridge, and thus for completion of cytokinesis. Here, we report that the ARF6 GTPase negatively regulates Rab35 activation and hence the Rab35 pathway. Human cells expressing a constitutively activated, GTP-bound ARF6 mutant display identical endocytic recycling and cytokinesis defects as those observed upon overexpression of the inactivated, GDP-bound Rab35 mutant. As a molecular mechanism, we identified the Rab35 GAP EPI64B as an effector of ARF6 in negatively regulating Rab35 activation. Unexpectedly, this regulation takes place at clathrin-coated pits, and activated ARF6 reduces Rab35 loading into the endocytic pathway. Thus, an effector of an ARF protein is a GAP for a downstream Rab protein, and we propose that this hierarchical ARF/Rab GTPase cascade controls the proper activation of a common endocytic pathway essential for cytokinesis. PMID:22226746

  9. Important relationships between Rab and MICAL proteins in endocytic trafficking

    PubMed Central

    Rahajeng, Juliati; Giridharan, Sai Srinivas Panapakkam; Cai, Bishuang; Naslavsky, Naava; Caplan, Steve

    2010-01-01

    The internalization of essential nutrients, lipids and receptors is a crucial process for all eukaryotic cells. Accordingly, endocytosis is highly conserved across cell types and species. Once internalized, small cargo-containing vesicles fuse with early endosomes (also known as sorting endosomes), where they undergo segregation to distinct membrane regions and are sorted and transported on through the endocytic pathway. Although the mechanisms that regulate this sorting are still poorly understood, some receptors are directed to late endosomes and lysosomes for degradation, whereas other receptors are recycled back to the plasma membrane; either directly or through recycling endosomes. The Rab family of small GTP-binding proteins plays crucial roles in regulating these trafficking pathways. Rabs cycle from inactive GDP-bound cytoplasmic proteins to active GTP-bound membrane-associated proteins, as a consequence of the activity of multiple specific GTPase-activating proteins (GAPs) and GTP exchange factors (GEFs). Once bound to GTP, Rabs interact with a multitude of effector proteins that carry out Rab-specific functions. Recent studies have shown that some of these effectors are also interaction partners for the C-terminal Eps15 homology (EHD) proteins, which are also intimately involved in endocytic regulation. A particularly interesting example of common Rab-EHD interaction partners is the MICAL-like protein, MICAL-L1. MICAL-L1 and its homolog, MICAL-L2, belong to the larger MICAL family of proteins, and both have been directly implicated in regulating endocytic recycling of cell surface receptors and junctional proteins, as well as controlling cytoskeletal rearrangement and neurite outgrowth. In this review, we summarize the functional roles of MICAL and Rab proteins, and focus on the significance of their interactions and the implications for endocytic transport. PMID:21537482

  10. EARP, a multisubunit tethering complex involved in endocytic recycling

    PubMed Central

    Schindler, Christina; Chen, Yu; Pu, Jing; Guo, Xiaoli; Bonifacino, Juan S.

    2015-01-01

    Recycling of endocytic receptors to the cell surface involves passage through a series of membrane-bound compartments by mechanisms that are poorly understood. In particular, it is unknown if endocytic recycling requires the function of multisubunit tethering complexes, as is the case for other intracellular trafficking pathways. Herein we describe a tethering complex named Endosome-Associated Recycling Protein (EARP) that is structurally related to the previously described Golgi-Associated Retrograde Protein (GARP) complex. Both complexes share the Ang2, Vps52 and Vps53 subunits, but EARP comprises an uncharacterized protein, Syndetin, in place of the Vps54 subunit of GARP. This change determines differential localization of EARP to recycling endosomes and GARP to the Golgi complex. EARP interacts with the target-SNARE Syntaxin 6 and various cognate SNAREs. Depletion of Syndetin or Syntaxin 6 delays recycling of internalized transferrin to the cell surface. These findings implicate EARP in canonical membrane-fusion events in the process of endocytic recycling. PMID:25799061

  11. NECAP2 controls clathrin coat recruitment to early endosomes for fast endocytic recycling.

    PubMed

    Chamberland, John P; Antonow, Lauren T; Dias Santos, Michel; Ritter, Brigitte

    2016-07-01

    Endocytic recycling returns receptors to the plasma membrane following internalization and is essential to maintain receptor levels on the cell surface, re-sensitize cells to extracellular ligands and for continued nutrient uptake. Yet, the protein machineries and mechanisms that drive endocytic recycling remain ill-defined. Here, we establish that NECAP2 regulates the endocytic recycling of EGFR and transferrin receptor. Our analysis of the recycling dynamics revealed that NECAP2 functions in the fast recycling pathway that directly returns cargo from early endosomes to the cell surface. In contrast, NECAP2 does not regulate the clathrin-mediated endocytosis of these cargos, the degradation of EGFR or the recycling of transferrin along the slow, Rab11-dependent recycling pathway. We show that protein knockdown of NECAP2 leads to enlarged early endosomes and causes the loss of the clathrin adapter AP-1 from the organelle. Through structure-function analysis, we define the protein-binding interfaces in NECAP2 that are crucial for AP-1 recruitment to early endosomes. Together, our data identify NECAP2 as a pathway-specific regulator of clathrin coat formation on early endosomes for fast endocytic recycling. PMID:27206861

  12. Trypanosoma brucei brucei: endocytic recycling is important for mouse infectivity.

    PubMed

    Natesan, Senthil Kumar A; Black, Alana; Matthews, Keith R; Mottram, Jeremy C; Field, Mark C

    2011-04-01

    Endocytosis in the African trypanosome, Trypanosoma brucei, is intimately involved in maintaining homeostasis of the cell surface proteome, morphology of the flagellar pocket and has recently been demonstrated as a bona fide drug target. RNAi-mediated knockdown of many factors required for endocytic transport, including several small GTPases, the major coat protein clathrin and a clathrin-associated receptor, epsinR, results in rapid cell death in vitro. Rapid loss of viability in vitro precludes meaningful investigation by RNAi of the roles of trypanosome endocytosis in vivo. Here we have sought to address this issue using strategies designed to produce milder effects on the endocytic system than complete functional ablation. We created a trypanosome clathrin heavy chain hemizygote and several lines expressing mutant forms of Rab5 and Rab11, described previously. All are viable in in vitro culture, with negligible impact to proliferative rates or cell cycle. Clathrin hemizygotes express clathrin heavy chain at ∼50% of wild type levels, but despite this demonstrate no defect to growth in mice, while none of the Rab5 mutants affected proliferation in vivo, despite clear evidence for effects on endocytosis. By contrast we find that expressing a dominantly active Rab11 mutant led to compromised growth in mice. These data indicate that trypanosomes likely tolerate the effects of partly decreased clathrin expression and alterations in early endocytosis, but are more sensitive to alterations in the recycling arm of the pathway. PMID:21256128

  13. Salicylic acid interferes with clathrin-mediated endocytic protein trafficking.

    PubMed

    Du, Yunlong; Tejos, Ricardo; Beck, Martina; Himschoot, Ellie; Li, Hongjiang; Robatzek, Silke; Vanneste, Steffen; Friml, Jirí

    2013-05-01

    Removal of cargos from the cell surface via endocytosis is an efficient mechanism to regulate activities of plasma membrane (PM)-resident proteins, such as receptors or transporters. Salicylic acid (SA) is an important plant hormone that is traditionally associated with pathogen defense. Here, we describe an unanticipated effect of SA on subcellular endocytic cycling of proteins. Both exogenous treatments and endogenously enhanced SA levels repressed endocytosis of different PM proteins. The SA effect on endocytosis did not involve transcription or known components of the SA signaling pathway for transcriptional regulation. SA likely targets an endocytic mechanism that involves the coat protein clathrin, because SA interfered with the clathrin incidence at the PM and clathrin-deficient mutants were less sensitive to the impact of SA on the auxin distribution and root bending during the gravitropic response. By contrast, SA did not affect the ligand-induced endocytosis of the flagellin sensing2 (FLS2) receptor during pathogen responses. Our data suggest that the established SA impact on transcription in plant immunity and the nontranscriptional effect of SA on clathrin-mediated endocytosis are independent mechanisms by which SA regulates distinct aspects of plant physiology. PMID:23613581

  14. Identification of an endocytic signal essential for the antiviral action of IFITM3

    PubMed Central

    Jia, Rui; Xu, Fengwen; Qian, Jin; Yao, Yunfang; Miao, Chunhui; Zheng, Yi-Min; Liu, Shan-Lu; Guo, Fei; Geng, Yunqi; Qiao, Wentao; Liang, Chen

    2014-01-01

    Members of the interferon-induced transmembrane (IFITM) protein family inhibit the entry of a wide range of viruses. Viruses often exploit the endocytosis pathways to invade host cells and escape from the endocytic vesicles often in response to low pH. Localization to these endocytic vesicles is essential for IFITM3 to interfere with the cytosolic entry of pH-dependent viruses. However, the nature of the sorting signal that targets IFITM3 to these vesicles is poorly defined. In this study, we report that IFITM3 possesses a YxxΦ sorting motif, i.e., 20-YEML-23, that enables IFITM3 to undergo endocytosis through binding to the μ2 subunit of the AP-2 complex. IFITM3 accumulates at the plasma membrane as a result of either mutating 20-YEML-23, depleting the μ2 subunit, or overexpressing μ2 mutants. Importantly, blocking endocytosis of IFITM3 abrogates its ability to inhibit pH-dependent viruses. We have therefore identified a critical sorting signal, namely 20-YEML-23, that controls both the endocytic trafficking and the antiviral action of IFITM3. This finding also reveals that as an endocytic protein, IFITM3 first arrives at the plasma membrane before it is endocytosed and further traffics to the late endosomes where it acts to impede virus entry. PMID:24521078

  15. A Systematic Analysis Reveals Heterogeneous Changes in the Endocytic Activities of Cancer Cells

    PubMed Central

    Elkin, Sarah R.; Bendris, Nawal; Reis, Carlos R.; Zhou, Yunyun; Xie, Yang; Huffman, Kenneth E.; Minna, John D.; Schmid, Sandra L.

    2016-01-01

    Metastasis is a multistep process requiring cancer cell signaling, invasion, migration, survival, and proliferation. These processes require dynamic modulation of cell surface proteins by endocytosis. Given this functional connection, it has been suggested that endocytosis is dysregulated in cancer. To test this, we developed In-Cell ELISA assays to measure three different endocytic pathways: clathrin-mediated endocytosis, caveolae-mediated endocytosis, and clathrin-independent endocytosis and compared these activities using two different syngeneic models for normal and oncogene-transformed human lung epithelial cells. We found that all endocytic activities were reduced in the transformed versus normal counterparts. However, when we screened 29 independently isolated non–small cell lung cancer (NSCLC) cell lines to determine whether these changes were systematic, we observed significant heterogeneity. Nonetheless, using hierarchical clustering based on their combined endocytic properties, we identified two phenotypically distinct clusters of NSCLCs. One co-clustered with mutations in KRAS, a mesenchymal phenotype, increased invasion through collagen and decreased growth in soft agar, whereas the second was enriched in cells with an epithelial phenotype. Interestingly, the two clusters also differed significantly in clathrin-independent internalization and surface expression of CD44 and CD59. Taken together, our results suggest that endocytotic alterations in cancer cells that affect cell surface expression of critical molecules have a significant influence on cancer-relevant phenotypes, with potential implications for interventions to control cancer by modulating endocytic dynamics. PMID:26359453

  16. INTERSECTINg Pathways in Cell Biology

    PubMed Central

    O'Bryan, John P.

    2010-01-01

    The endocytic pathway is involved in activation and inhibition of cellular signaling. Thus, defining the regulatory mechanisms that link endocytosis and cellular signaling is of interest. An emerging link between these processes is a family of proteins called intersectins (ITSNs). These multi-domain proteins serve as scaffolds in the assembly of endocytic vesicles, and also regulate components of various signaling pathways, including kinases, GTPases, and ubiquitin ligases. This review will summarize research on the role of ITSNs in regulating both endocytic and signal transduction pathways, discuss the link of ITSNs with human disease, and highlight future directions in the study of ITSNs. PMID:21156937

  17. The CD63-Syntenin-1 Complex Controls Post-Endocytic Trafficking of Oncogenic Human Papillomaviruses.

    PubMed

    Gräßel, Linda; Fast, Laura Aline; Scheffer, Konstanze D; Boukhallouk, Fatima; Spoden, Gilles A; Tenzer, Stefan; Boller, Klaus; Bago, Ruzica; Rajesh, Sundaresan; Overduin, Michael; Berditchevski, Fedor; Florin, Luise

    2016-01-01

    Human papillomaviruses enter host cells via a clathrin-independent endocytic pathway involving tetraspanin proteins. However, post-endocytic trafficking required for virus capsid disassembly remains unclear. Here we demonstrate that the early trafficking pathway of internalised HPV particles involves tetraspanin CD63, syntenin-1 and ESCRT-associated adaptor protein ALIX. Following internalisation, viral particles are found in CD63-positive endosomes recruiting syntenin-1, a CD63-interacting adaptor protein. Electron microscopy and immunofluorescence experiments indicate that the CD63-syntenin-1 complex controls delivery of internalised viral particles to multivesicular endosomes. Accordingly, infectivity of high-risk HPV types 16, 18 and 31 as well as disassembly and post-uncoating processing of viral particles was markedly suppressed in CD63 or syntenin-1 depleted cells. Our analyses also present the syntenin-1 interacting protein ALIX as critical for HPV infection and CD63-syntenin-1-ALIX complex formation as a prerequisite for intracellular transport enabling viral capsid disassembly. Thus, our results identify the CD63-syntenin-1-ALIX complex as a key regulatory component in post-endocytic HPV trafficking. PMID:27578500

  18. The CD63-Syntenin-1 Complex Controls Post-Endocytic Trafficking of Oncogenic Human Papillomaviruses

    PubMed Central

    Gräßel, Linda; Fast, Laura Aline; Scheffer, Konstanze D.; Boukhallouk, Fatima; Spoden, Gilles A.; Tenzer, Stefan; Boller, Klaus; Bago, Ruzica; Rajesh, Sundaresan; Overduin, Michael; Berditchevski, Fedor; Florin, Luise

    2016-01-01

    Human papillomaviruses enter host cells via a clathrin-independent endocytic pathway involving tetraspanin proteins. However, post-endocytic trafficking required for virus capsid disassembly remains unclear. Here we demonstrate that the early trafficking pathway of internalised HPV particles involves tetraspanin CD63, syntenin-1 and ESCRT-associated adaptor protein ALIX. Following internalisation, viral particles are found in CD63-positive endosomes recruiting syntenin-1, a CD63-interacting adaptor protein. Electron microscopy and immunofluorescence experiments indicate that the CD63-syntenin-1 complex controls delivery of internalised viral particles to multivesicular endosomes. Accordingly, infectivity of high-risk HPV types 16, 18 and 31 as well as disassembly and post-uncoating processing of viral particles was markedly suppressed in CD63 or syntenin-1 depleted cells. Our analyses also present the syntenin-1 interacting protein ALIX as critical for HPV infection and CD63-syntenin-1-ALIX complex formation as a prerequisite for intracellular transport enabling viral capsid disassembly. Thus, our results identify the CD63-syntenin-1-ALIX complex as a key regulatory component in post-endocytic HPV trafficking. PMID:27578500

  19. Spatiotemporal control of phosphatidylinositol 4-phosphate by Sac2 regulates endocytic recycling

    PubMed Central

    Hsu, FoSheng; Hu, Fenghua

    2015-01-01

    It is well established that the spatial- and temporal-restricted generation and turnover of phosphoinositides (PIs) by a cascade of PI-metabolizing enzymes is a key regulatory mechanism in the endocytic pathway. Here, we demonstrate that the Sac1 domain–containing protein Sac2 is a PI 4-phosphatase that specifically hydrolyzes phosphatidylinositol 4-phosphate in vitro. We further show that Sac2 colocalizes with early endosomal markers and is recruited to transferrin (Tfn)-containing vesicles during endocytic recycling. Exogenous expression of the catalytically inactive mutant Sac2C458S resulted in altered cellular distribution of Tfn receptors and delayed Tfn recycling. Furthermore, genomic ablation of Sac2 caused a similar perturbation on Tfn and integrin recycling as well as defects in cell migration. Structural characterization of Sac2 revealed a unique pleckstrin-like homology Sac2 domain conserved in all Sac2 orthologues. Collectively, our findings provide evidence for the tight regulation of PIs by Sac2 in the endocytic recycling pathway. PMID:25869669

  20. Myosin VI and its interacting protein LMTK2 regulate tubule formation and transport to the endocytic recycling compartment

    PubMed Central

    Chibalina, Margarita V.; Seaman, Matthew N.J.; Miller, Christopher C.; Kendrick-Jones, John; Buss, Folma

    2009-01-01

    Summary Myosin VI is an actin-based retrograde motor protein, which plays a crucial role in both endocytic and secretory membrane trafficking pathways. Myosin VI’s targeting to and function in these intracellular pathways is mediated by a number of specific binding partners. In this paper we have identified a new myosin VI binding partner, Lemur tyrosine kinase 2 (LMTK2), which is the first transmembrane protein and kinase that directly binds to myosin VI. LMTK2 binds to the WWY site in the C-terminal myosin VI tail, the same site as the endocytic adaptor protein Dab2. When either myosin VI or LMTK2 is depleted by siRNA, the transferrin receptor (TfR) is trapped in swollen endosomes and tubule formation in the endocytic recycling pathway is dramatically reduced, showing that both proteins are required for the transport of cargo such as the TfR from early endosomes to the endocytic recycling compartment. PMID:18029400

  1. Yeast Eps15-like endocytic protein Pan1p regulates the interaction between endocytic vesicles, endosomes and the actin cytoskeleton

    PubMed Central

    Toshima, Junko Y; Furuya, Eri; Nagano, Makoto; Kanno, Chisa; Sakamoto, Yuta; Ebihara, Masashi; Siekhaus, Daria Elisabeth; Toshima, Jiro

    2016-01-01

    The actin cytoskeleton plays important roles in the formation and internalization of endocytic vesicles. In yeast, endocytic vesicles move towards early endosomes along actin cables, however, the molecular machinery regulating interaction between endocytic vesicles and actin cables is poorly understood. The Eps15-like protein Pan1p plays a key role in actin-mediated endocytosis and is negatively regulated by Ark1 and Prk1 kinases. Here we show that pan1 mutated to prevent phosphorylation at all 18 threonines, pan1-18TA, displayed almost the same endocytic defect as ark1Δ prk1Δ cells, and contained abnormal actin concentrations including several endocytic compartments. Early endosomes were highly localized in the actin concentrations and displayed movement along actin cables. The dephosphorylated form of Pan1p also caused stable associations between endocytic vesicles and actin cables, and between endocytic vesicles and endosomes. Thus Pan1 phosphorylation is part of a novel mechanism that regulates endocytic compartment interactions with each other and with actin cables. DOI: http://dx.doi.org/10.7554/eLife.10276.001 PMID:26914139

  2. The Mammalian Orthologs of Drosophila Lgd, CC2D1A and CC2D1B, Function in the Endocytic Pathway, but Their Individual Loss of Function Does Not Affect Notch Signalling

    PubMed Central

    Drusenheimer, Nadja; Migdal, Bernhard; Jäckel, Sandra; Tveriakhina, Lena; Scheider, Kristina; Schulz, Katharina; Gröper, Jieny; Köhrer, Karl; Klein, Thomas

    2015-01-01

    CC2D1A and CC2D1B belong to the evolutionary conserved Lgd protein family with members in all multi-cellular animals. Several functions such as centrosomal cleavage, involvement in signalling pathways, immune response and synapse maturation have been described for CC2D1A. Moreover, the Drosophila melanogaster ortholog Lgd was shown to be involved in the endosomal trafficking of the Notch receptor and other transmembrane receptors and physically interacts with the ESCRT-III component Shrub/CHMP4. To determine if this function is conserved in mammals we generated and characterized Cc2d1a and Cc2d1b conditional knockout mice. While Cc2d1b deficient mice displayed no obvious phenotype, we found that Cc2d1a deficient mice as well as conditional mutants that lack CC2D1A only in the nervous system die shortly after birth due to respiratory distress. This finding confirms the suspicion that the breathing defect is caused by the central nervous system. However, an involvement in centrosomal function could not be confirmed in Cc2d1a deficient MEF cells. To analyse an influence on Notch signalling, we generated intestine specific Cc2d1a mutant mice. These mice did not display any alterations in goblet cell number, proliferating cell number or expression of the Notch reporter Hes1-emGFP, suggesting that CC2D1A is not required for Notch signalling. However, our EM analysis revealed that the average size of endosomes of Cc2d1a mutant cells, but not Cc2d1b mutant cells, is increased, indicating a defect in endosomal morphogenesis. We could show that CC2D1A and its interaction partner CHMP4B are localised on endosomes in MEF cells, when the activity of the endosomal protein VPS4 is reduced. This indicates that CC2D1A cycles between the cytosol and the endosomal membrane. Additionally, in rescue experiments in D. melanogaster, CC2D1A and CC2D1B were able to functionally replace Lgd. Altogether our data suggest a functional conservation of the Lgd protein family in the ESCRT

  3. Endocytic trafficking of laminin is controlled by dystroglycan and is disrupted in cancers.

    PubMed

    Leonoudakis, Dmitri; Huang, Ge; Akhavan, Armin; Fata, Jimmie E; Singh, Manisha; Gray, Joe W; Muschler, John L

    2014-11-15

    The dynamic interactions between cells and basement membranes serve as essential regulators of tissue architecture and function in metazoans, and perturbation of these interactions contributes to the progression of a wide range of human diseases, including cancers. Here, we reveal the pathway and mechanism for the endocytic trafficking of a prominent basement membrane protein, laminin-111 (referred to here as laminin), and their disruption in disease. Live-cell imaging of epithelial cells revealed pronounced internalization of laminin into endocytic vesicles. Laminin internalization was receptor mediated and dynamin dependent, and laminin proceeded to the lysosome through the late endosome. Manipulation of laminin receptor expression revealed that the dominant regulator of laminin internalization is dystroglycan, a laminin receptor that is functionally perturbed in muscular dystrophies and in many cancers. Correspondingly, laminin internalization was found to be deficient in aggressive cancer cells displaying non-functional dystroglycan, and restoration of dystroglycan function strongly enhanced the endocytosis of laminin in both breast cancer and glioblastoma cells. These results establish previously unrecognized mechanisms for the modulation of cell-basement-membrane communication in normal cells and identify a profound disruption of endocytic laminin trafficking in aggressive cancer subtypes. PMID:25217627

  4. Small rho GTPases mediate tumor-induced inhibition of endocytic activity of dendritic cells.

    PubMed

    Tourkova, Irina L; Shurin, Galina V; Wei, Sheng; Shurin, Michael R

    2007-06-15

    The generation, maturation, and function of dendritic cells (DC) have been shown to be markedly compromised in the tumor microenvironment in animals and humans. However, the molecular mechanisms and intracellular pathways involved in the regulation of the DC system in cancer are not yet fully understood. Recently, we have reported on the role of the small Rho GTPase family members Cdc42, Rac1, and RhoA in regulating DC adherence, motility, and Ag presentation. To investigate involvement of small Rho GTPases in dysregulation of DC function by tumors, we next evaluated how Cdc42, Rac1, and RhoA regulated endocytic activity of DC in the tumor microenvironment. We revealed a decreased uptake of dextran 40 and polystyrene beads by DC generated in the presence of different tumor cell lines, including RM1 prostate, MC38 colon, 3LL lung, and B7E3 oral squamous cell carcinomas in vitro and by DC prepared from tumor-bearing mice ex vivo. Impaired endocytic activity of DC cocultured with tumor cells was associated with decreased levels of active Cdc42 and Rac1. Transduction of DC with the dominant negative Cdc42 and Rac1 genes also led to reduced phagocytosis and receptor-mediated endocytosis. Furthermore, transduction of DC with the constitutively active Cdc42 and Rac1 genes restored endocytic activity of DC that was inhibited by the tumors. Thus, our results suggest that tumor-induced dysregulation of endocytic activity of DC is mediated by reduced activity of several members of the small Rho GTPase family, which might serve as new targets for improving the efficacy of DC vaccines. PMID:17548616

  5. Visualizing the functional architecture of the endocytic machinery

    PubMed Central

    Picco, Andrea; Mund, Markus; Ries, Jonas; Nédélec, François; Kaksonen, Marko

    2015-01-01

    Clathrin-mediated endocytosis is an essential process that forms vesicles from the plasma membrane. Although most of the protein components of the endocytic protein machinery have been thoroughly characterized, their organization at the endocytic site is poorly understood. We developed a fluorescence microscopy method to track the average positions of yeast endocytic proteins in relation to each other with a time precision below 1 s and with a spatial precision of ∼10 nm. With these data, integrated with shapes of endocytic membrane intermediates and with superresolution imaging, we could visualize the dynamic architecture of the endocytic machinery. We showed how different coat proteins are distributed within the coat structure and how the assembly dynamics of N-BAR proteins relate to membrane shape changes. Moreover, we found that the region of actin polymerization is located at the base of the endocytic invagination, with the growing ends of filaments pointing toward the plasma membrane. DOI: http://dx.doi.org/10.7554/eLife.04535.001 PMID:25675087

  6. Unconventional Functions for Clathrin, ESCRTs, and Other Endocytic Regulators in the Cytoskeleton, Cell Cycle, Nucleus, and Beyond: Links to Human Disease

    PubMed Central

    Brodsky, Frances M.; Sosa, R. Thomas; Ybe, Joel A.; O’Halloran, Theresa J.

    2014-01-01

    The roles of clathrin, its regulators, and the ESCRT (endosomal sorting complex required for transport) proteins are well defined in endocytosis. These proteins can also participate in intracellular pathways that are independent of endocytosis and even independent of the membrane trafficking function of these proteins. These nonendocytic functions involve unconventional biochemical interactions for some endocytic regulators, but can also exploit known interactions for nonendocytic functions. The molecular basis for the involvement of endocytic regulators in unconventional functions that influence the cytoskeleton, cell cycle, signaling, and gene regulation are described here. Through these additional functions, endocytic regulators participate in pathways that affect infection, glucose metabolism, development, and cellular transformation, expanding their significance in human health and disease. PMID:25183831

  7. Pulse Dynamics in Endocytic Protein Patches

    NASA Astrophysics Data System (ADS)

    Carlsson, Anders; Wang, Xinxin

    2015-03-01

    During the process of endocytosis in yeast, submicron-sized protein patches assemble, exert forces on the membrane to bend it, and finally disassemble. The patches contain an initial coat that establishes the endocytic site and binds cargo, polymers of the protein actin, ``nucleation-promoting factors'' (NPFs) that catalyze actin polymerization, and curvature-generating proteins. We model the dynamics of protein patches in yeast using a variant of the activator-inhibitor ``Fitzhugh-Nagumo'' model. We treat NPFs as the activator, and polymerized actin as the inhibitor, on the basis of findings that the lifetime of NPF patches is extended when actin polymerization is inhibited. Using this model, we find that as the polymerization rate is reduced, there is a discontinuous transition from protein pulses to persistent patches. We also find, surprisingly, that in some parameter regimes reducing the polymerization rate can increase the polymerized-actin content of the patch. We present data for NPF dynamics budding yeast, which confirm some of the predictions of the model. Supported by NIH under Grant R01-GM107667.

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

    PubMed Central

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

    2011-01-01

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

  9. Endocytic response of type I alveolar epithelial cells to hypertonic stress

    PubMed Central

    Wang, Shaohua; Singh, Raman Deep; Godin, Lindsay; Pagano, Richard E.

    2011-01-01

    We present plasma membrane (PM) internalization responses of type I alveolar epithelial cells to a 50 mosmol/l increase in tonicity. Our research is motivated by interest in ATI repair, for which endocytic retrieval of PM appears to be critical. We validated pharmacological and molecular tools to dissect the endocytic machinery of these cells and used these tools to test the hypothesis that osmotic stress triggers a pathway-specific internalization of PM domains. Validation experiments confirmed the fluorescent analogs of lactosyl-ceramide, transferrin, and dextran as pathway-specific cargo of caveolar, clathrin, and fluid-phase uptake, respectively. Pulse-chase experiments indicate that hypertonic exposure causes a downregulation of clathrin and fluid-phase endocytosis while stimulating caveolar endocytosis. The tonicity-mediated increase in caveolar endocytosis was associated with the translocation of caveolin-1 from the PM and was absent in cells that had been transfected with dominant-negative dynamin constructs. In separate experiments we show that hypertonic exposure increases the probability of PM wound repair following micropuncture from 82 ± 4 to 94 ± 2% (P < 0.01) and that this effect depends on Src pathway activation-mediated caveolar endocytosis. The therapeutic and biological implications of our findings are discussed. PMID:21257731

  10. Autophagy-mediated reentry of Francisella tularensis into the endocytic compartment after cytoplasmic replication

    PubMed Central

    Checroun, Claire; Wehrly, Tara D.; Fischer, Elizabeth R.; Hayes, Stanley F.; Celli, Jean

    2006-01-01

    Intracellular bacterial pathogens evade the bactericidal functions of mammalian cells by physical escape from their phagosome and replication into the cytoplasm or through the modulation of phagosome maturation and biogenesis of a membrane-bound replicative organelle. Here, we detail in murine primary macrophages the intracellular life cycle of Francisella tularensis, a highly infectious bacterium that survives and replicates within mammalian cells. After transient interactions with the endocytic pathway, bacteria escaped from their phagosome by 1 h after infection and underwent replication in the cytoplasm from 4 to 20 h after infection. Unexpectedly, the majority of bacteria were subsequently found to be enclosed within large, juxtanuclear, LAMP-1-positive vacuoles called Francisella-containing vacuoles (FCVs). FCV formation required intracytoplasmic replication of bacteria. Using electron and fluorescence microscopy, we observed that the FCVs contained morphologically intact bacteria, despite fusing with lysosomes. FCVs are multimembranous structures that accumulate monodansylcadaverine and display the autophagy-specific protein LC3 on their membrane. Formation of FCVs was significantly inhibited by 3-methyladenine, confirming a role for the autophagic pathway in the biogenesis of these organelles. Taken together, our results demonstrate that, via autophagy, F. tularensis reenters the endocytic pathway after cytoplasmic replication, a process thus far undescribed for intracellular pathogens. PMID:16983090

  11. Human B lymphoblastoid cells contain distinct patterns of cathepsin activity in endocytic compartments and regulate MHC class II transport in a cathepsin S-independent manner.

    PubMed

    Lautwein, Alfred; Kraus, Marianne; Reich, Michael; Burster, Timo; Brandenburg, J; Overkleeft, Herman S; Schwarz, Gerold; Kammer, Winfried; Weber, Ekkehard; Kalbacher, Hubert; Nordheim, Alfred; Driessen, Christoph

    2004-05-01

    Endocytic proteolysis represents a major functional component of the major histocompatibility complex class II antigen-presentation machinery. Although transport and assembly of class II molecules in the endocytic compartment are well characterized, we lack information about the pattern of endocytic protease activity along this pathway. Here, we used chemical tools that visualize endocytic proteases in an activity-dependent manner in combination with subcellular fractionation to dissect the subcellular distribution of the major cathepsins (Cat) CatS, CatB, CatH, CatD, CatC, and CatZ as well as the asparagine-specific endoprotease (AEP) in human B-lymphoblastoid cells (BLC). Endocytic proteases were distributed in two distinct patterns: CatB and CatZ were most prominent in early and late endosomes but absent from lysosomes, and CatH, CatS, CatD, CatC, and AEP distributed between late endosomes and lysosomes, suggesting that CatB and CatZ might be involved in the initial proteolytic attack on a given antigen. The entire spectrum of protease activity colocalized with human leukocyte antigen-DM and the C-terminal and N-terminal processing of invariant chain (Ii) in late endosomes. CatS was active in all endocytic compartments. Surprisingly and in contrast with results from dendritic cells, inhibition of CatS activity by leucine-homophenylalanine-vinylsulfone-phenol prevented N-terminal processing of Ii but did not alter the subcellular trafficking or surface delivery of class II complexes, as deferred from pulse-chase analysis in combination with subcellular fractionation and biotinylation of cell-surface protein. Thus, BLC contain distinct activity patterns of proteases in endocytic compartments and regulate the intracellular transport and surface-delivery of class II in a CatS-independent manner. PMID:14966190

  12. The F-box protein Rcy1p is involved in endocytic membrane traffic and recycling out of an early endosome in Saccharomyces cerevisiae.

    PubMed

    Wiederkehr, A; Avaro, S; Prescianotto-Baschong, C; Haguenauer-Tsapis, R; Riezman, H

    2000-04-17

    In Saccharomyces cerevisiae, endocytic material is transported through different membrane-bound compartments before it reaches the vacuole. In a screen for mutants that affect membrane trafficking along the endocytic pathway, we have identified a novel mutant disrupted for the gene YJL204c that we have renamed RCY1 (recycling 1). Deletion of RCY1 leads to an early block in the endocytic pathway before the intersection with the vacuolar protein sorting pathway. Mutation of RCY1 leads to the accumulation of an enlarged compartment that contains the t-SNARE Tlg1p and lies close to areas of cell expansion. In addition, endocytic markers such as Ste2p and the fluorescent dyes, Lucifer yellow and FM4-64, were found in a similar enlarged compartment after their internalization. To determine whether rcy1Delta is defective for recycling, we have developed an assay that measures the recycling of previously internalized FM4-64. This method enables us to follow the recycling pathway in yeast in real time. Using this assay, it could be demonstrated that recycling of membranes is rapid in S. cerevisiae and that a major fraction of internalized FM4-64 is secreted back into the medium within a few minutes. The rcy1Delta mutant is strongly defective in recycling. PMID:10769031

  13. The F-Box Protein Rcy1p Is Involved in Endocytic Membrane Traffic and Recycling Out of an Early Endosome in Saccharomyces cerevisiae

    PubMed Central

    Wiederkehr, Andreas; Avaro, Sandrine; Prescianotto-Baschong, Cristina; Haguenauer-Tsapis, Rosine; Riezman, Howard

    2000-01-01

    In Saccharomyces cerevisiae, endocytic material is transported through different membrane-bound compartments before it reaches the vacuole. In a screen for mutants that affect membrane trafficking along the endocytic pathway, we have identified a novel mutant disrupted for the gene YJL204c that we have renamed RCY1 (recycling 1). Deletion of RCY1 leads to an early block in the endocytic pathway before the intersection with the vacuolar protein sorting pathway. Mutation of RCY1 leads to the accumulation of an enlarged compartment that contains the t-SNARE Tlg1p and lies close to areas of cell expansion. In addition, endocytic markers such as Ste2p and the fluorescent dyes, Lucifer yellow and FM4-64, were found in a similar enlarged compartment after their internalization. To determine whether rcy1Δ is defective for recycling, we have developed an assay that measures the recycling of previously internalized FM4-64. This method enables us to follow the recycling pathway in yeast in real time. Using this assay, it could be demonstrated that recycling of membranes is rapid in S. cerevisiae and that a major fraction of internalized FM4-64 is secreted back into the medium within a few minutes. The rcy1Δ mutant is strongly defective in recycling. PMID:10769031

  14. Endocytic trafficking routes of wild type and DeltaF508 cystic fibrosis transmembrane conductance regulator.

    PubMed

    Gentzsch, Martina; Chang, Xiu-Bao; Cui, Liying; Wu, Yufeng; Ozols, Victor V; Choudhury, Amit; Pagano, Richard E; Riordan, John R

    2004-06-01

    Intracellular trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) is a focus of attention because it is defective in most patients with cystic fibrosis. DeltaF508 CFTR, which does not mature conformationally, normally does not exit the endoplasmic reticulum, but if induced to do so at reduced temperature is short-lived at the surface. We used external epitope-tagged constructs to elucidate the itinerary and kinetics of wild type and DeltaF508 CFTR in the endocytic pathway and visualized movement of CFTR from the surface to intracellular compartments. Modulation of different endocytic steps with low temperature (16 degrees C) block, protease inhibitors, and overexpression of wild type and mutant Rab GTPases revealed that surface CFTR enters several different routes, including a Rab5-dependent initial step to early endosomes, then either Rab11-dependent recycling back to the surface or Rab7-regulated movement to late endosomes or alternatively Rab9-mediated transit to the trans-Golgi network. Without any of these modulations DeltaF508 CFTR rapidly disappears from and does not return to the cell surface, confirming that its altered structure is detected in the distal as well as proximal secretory pathway. Importantly, however, the mutant protein can be rescued at the plasma membrane by Rab11 overexpression, proteasome inhibitors, or inhibition of Rab5-dependent endocytosis. PMID:15075371

  15. Ultrastructural Imaging of Endocytic Sites in Saccharomyces cerevisiae by Transmission Electron Microscopy and Immunolabeling

    PubMed Central

    Buser, Christopher; Drubin, David G.

    2014-01-01

    Defining the ultrastructure of endocytic sites and localization of endocytic proteins in Saccharomyces cerevisiae by immunoelectron microscopy is central in understanding the mechanisms of membrane deformation and scission during endocytosis. We show that an improved sample preparation protocol based on high-pressure freezing, freeze substitution, and low-temperature embedding allows us to maintain the cellular fine structure and to immunolabel green fluorescent protein–tagged endocytic proteins or actin in the same sections. Using this technique we analyzed the stepwise deformation of endocytic membranes and immuno-localized the endocytic proteins Abp1p, Sla1p, Rvs167p, and actin, and were able to draw a clear ultrastructural distinction between endocytic sites and eisosomes by immunolocalizing Pil1p. In addition to defining the geometry and the fine structure of budding yeast endocytic sites, we observed associated actin filaments forming a cage-like meshwork around the endocytic membrane. PMID:23458500

  16. Drosophila Rolling Blackout Displays Lipase Domain-Dependent and Independent Endocytic Functions Downstream of Dynamin

    PubMed Central

    Vijayakrishnan, Niranjana; Phillips, Scott E.; Broadie, Kendal

    2010-01-01

    Drosophila temperature-sensitive rolling blackout (rbots) mutants display a total block of endocytosis in non-neuronal cells and a weaker, partial defect at neuronal synapses. RBO is an integral plasma membrane protein and is predicted to be a serine esterase. To determine if lipase activity is required for RBO function, we mutated the catalytic serine 358 to alanine in the G-X-S-X-G active site, and assayed genomic rescue of rbo mutant non-neuronal and neuronal phenotypes. The rboS358A mutant is unable to rescue rbo null 100% embryonic lethality, indicating that the lipase-domain is critical for RBO essential function. Likewise, the rboS358A mutant cannot provide any rescue of endocytic blockade in rbots Garland cells, demonstrating that the lipase-domain is indispensable for non-neuronal endocytosis. In contrast, rbots conditional paralysis, synaptic transmission block and synapse endocytic defects are all fully rescued by the rboS358A mutant, showing that the RBO lipase-domain is dispensable in neuronal contexts. We identified a synthetic lethal interaction between rbots and the well-characterized dynamin GTPase conditional shibire (shits1) mutant. In both non-neuronal cells and neuronal synapses, shits1;rbots phenocopies shits1 endocytic defects, indicating that dynamin and RBO act in the same pathway, with dynamin functioning upstream of RBO. We conclude that RBO possesses both lipase-domain dependent and scaffolding functions with differential requirements in non-neuronal versus neuronal endocytosis mechanisms downstream of dynamin GTPase activity. PMID:21029287

  17. The epsin family of endocytic adaptors promotes fibrosarcoma migration and invasion.

    PubMed

    Coon, Brian G; Burgner, John; Camonis, Jacques H; Aguilar, R Claudio

    2010-10-22

    Abnormalities in the process of endocytosis are classically linked to malignant transformation through the deficient down-regulation of signaling receptors. The present study describes a non-classical mechanism that does not require internalization by which endocytic proteins affect cell migration and basement membrane invasion. Specifically, we found that the endocytic adaptor epsin binds and regulates the biological properties of the signaling molecule RalBP1 (Ral-binding protein 1). Epsin interacted with the N terminus of RalBP1 via its characteristic epsin N-terminal homology (ENTH) domain. A combination of siRNA-mediated knock-down and transfection of siRNA-resistant constructs in fibrosarcoma cells demonstrated that impairment of the epsin-RalBP1 interaction led to cell migration and basement membrane invasion defects. We found the ENTH domain was necessary and sufficient to sustain normal cell migration and invasion. Because all the epsin endocytic motifs reside in the C-terminal part of the molecule, these results suggest that this novel regulatory circuit does not require endocytosis. In addition, cells depleted of epsin-RalBP1 complex displayed deficient activation of Rac1 and Arf6 suggesting a signaling function for this novel interaction. Further, overexpression of either epsin or RalBP1 enhanced migration and invasion of fibrosarcoma cells. Collectively, our results indicate that epsin regulates RalBP1 function in Rac1- and Arf6-dependent pathways to ultimately affect cell migration and invasion. We propose that the observed up-regulation of both epsin and RalBP1 in certain cancers contributes to their invasive characteristics. PMID:20709745

  18. The Epsin Family of Endocytic Adaptors Promotes Fibrosarcoma Migration and Invasion*

    PubMed Central

    Coon, Brian G.; Burgner, John; Camonis, Jacques H.; Aguilar, R. Claudio

    2010-01-01

    Abnormalities in the process of endocytosis are classically linked to malignant transformation through the deficient down-regulation of signaling receptors. The present study describes a non-classical mechanism that does not require internalization by which endocytic proteins affect cell migration and basement membrane invasion. Specifically, we found that the endocytic adaptor epsin binds and regulates the biological properties of the signaling molecule RalBP1 (Ral-binding protein 1). Epsin interacted with the N terminus of RalBP1 via its characteristic epsin N-terminal homology (ENTH) domain. A combination of siRNA-mediated knock-down and transfection of siRNA-resistant constructs in fibrosarcoma cells demonstrated that impairment of the epsin-RalBP1 interaction led to cell migration and basement membrane invasion defects. We found the ENTH domain was necessary and sufficient to sustain normal cell migration and invasion. Because all the epsin endocytic motifs reside in the C-terminal part of the molecule, these results suggest that this novel regulatory circuit does not require endocytosis. In addition, cells depleted of epsin-RalBP1 complex displayed deficient activation of Rac1 and Arf6 suggesting a signaling function for this novel interaction. Further, overexpression of either epsin or RalBP1 enhanced migration and invasion of fibrosarcoma cells. Collectively, our results indicate that epsin regulates RalBP1 function in Rac1- and Arf6-dependent pathways to ultimately affect cell migration and invasion. We propose that the observed up-regulation of both epsin and RalBP1 in certain cancers contributes to their invasive characteristics. PMID:20709745

  19. Fast neurotransmitter release regulated by the endocytic scaffold intersectin.

    PubMed

    Sakaba, Takeshi; Kononenko, Natalia L; Bacetic, Jelena; Pechstein, Arndt; Schmoranzer, Jan; Yao, Lijun; Barth, Holger; Shupliakov, Oleg; Kobler, Oliver; Aktories, Klaus; Haucke, Volker

    2013-05-14

    Sustained fast neurotransmission requires the rapid replenishment of release-ready synaptic vesicles (SVs) at presynaptic active zones. Although the machineries for exocytic fusion and for subsequent endocytic membrane retrieval have been well characterized, little is known about the mechanisms underlying the rapid recruitment of SVs to release sites. Here we show that the Down syndrome-associated endocytic scaffold protein intersectin 1 is a crucial factor for the recruitment of release-ready SVs. Genetic deletion of intersectin 1 expression or acute interference with intersectin function inhibited the replenishment of release-ready vesicles, resulting in short-term depression, without significantly affecting the rate of endocytic membrane retrieval. Acute perturbation experiments suggest that intersectin-mediated vesicle replenishment involves the association of intersectin with the fissioning enzyme dynamin and with the actin regulatory GTPase CDC42. Our data indicate a role for the endocytic scaffold intersectin in fast neurotransmitter release, which may be of prime importance for information processing in the brain. PMID:23633571

  20. Systematic spatial mapping of proteins at exocytic and endocytic structures

    PubMed Central

    Larson, Ben T.; Sochacki, Kem A.; Kindem, Jonathan M.; Taraska, Justin W.

    2014-01-01

    Vesicular secretion (exocytosis) involves the release and then compensatory recycling of vesicle components through endocytosis. This fundamental cellular process is controlled by the coordinated assembly and interactions of dozens of proteins at the plasma membrane. Understanding the molecular composition of individual exocytic and endocytic structures and their organization across the plasma membrane is critical to understanding the behavior and regulation of these two cellular processes. Here we develop a high-resolution and high-throughput fluorescence imaging–based approach for the unbiased mapping of 78 proteins at single exocytic vesicles and endocytic structures in neuroendocrine PC12 cells. This analysis uses two-color single-frame images to provide a systems-level map of the steady-state distributions of proteins at individual exocytic and endocytic structures in the cell. Along with this quantitative map, we find that both calcium-regulated exocytic vesicles (dense core vesicles) and endocytic structures (clathrin-coated structures) and the proteins associated with these structures exhibit a random spatial distribution in unstimulated neuroendocrine PC12 cells. This approach is broadly applicable for quantitatively mapping the molecular composition and spatial organization of discrete cellular processes with central molecular hubs. PMID:24807904

  1. APPL endosomes are not obligatory endocytic intermediates but act as stable cargo-sorting compartments

    PubMed Central

    Kalaidzidis, Inna; Miaczynska, Marta; Brewińska-Olchowik, Marta; Hupalowska, Anna; Ferguson, Charles; Parton, Robert G.; Kalaidzidis, Yannis

    2015-01-01

    Endocytosis allows cargo to enter a series of specialized endosomal compartments, beginning with early endosomes harboring Rab5 and its effector EEA1. There are, however, additional structures labeled by the Rab5 effector APPL1 whose role in endocytic transport remains unclear. It has been proposed that APPL1 vesicles are transport intermediates that convert into EEA1 endosomes. Here, we tested this model by analyzing the ultrastructural morphology, kinetics of cargo transport, and stability of the APPL1 compartment over time. We found that APPL1 resides on a tubulo-vesicular compartment that is capable of sorting cargo for recycling or degradation and that displays long lifetimes, all features typical of early endosomes. Fitting mathematical models to experimental data rules out maturation of APPL1 vesicles into EEA1 endosomes as a primary mechanism for cargo transport. Our data suggest instead that APPL1 endosomes represent a distinct population of Rab5-positive sorting endosomes, thus providing important insights into the compartmental organization of the early endocytic pathway. PMID:26459602

  2. Regulation of ubiquitin-dependent cargo sorting by multiple endocytic adaptors at the plasma membrane

    PubMed Central

    Mayers, Jonathan R.; Wang, Lei; Pramanik, Jhuma; Johnson, Adam; Sarkeshik, Ali; Wang, Yueju; Saengsawang, Witchuda; Yates, John R.; Audhya, Anjon

    2013-01-01

    Endocytic protein trafficking is directed by sorting signals on cargo molecules that are recognized by cytosolic adaptor proteins. However, the steps necessary to segregate the variety of cargoes during endocytosis remain poorly defined. Using Caenorhabditis elegans, we demonstrate that multiple plasma membrane endocytic adaptors function redundantly to regulate clathrin-mediated endocytosis and to recruit components of the endosomal sorting complex required for transport (ESCRT) machinery to the cell surface to direct the sorting of ubiquitin-modified substrates. Moreover, our data suggest that preassembly of cargoes with the ESCRT-0 complex at the plasma membrane enhances the efficiency of downstream sorting events in the endolysosomal system. In the absence of a heterooligomeric adaptor complex composed of FCHO, Eps15, and intersectin, ESCRT-0 accumulation at the cell surface is diminished, and the degradation of a ubiquitin-modified cargo slows significantly without affecting the rate of its clathrin-mediated internalization. Consistent with a role for the ESCRT machinery during cargo endocytosis, we further show that the ESCRT-0 complex accumulates at a subset of clathrin-coated pits on the surface of human cells. Our findings suggest a unique mechanism by which ubiquitin-modified cargoes are sequestered into the endolysosomal pathway. PMID:23818590

  3. Exocyst requirement for endocytic traffic directed toward the apical and basolateral poles of polarized MDCK cells.

    PubMed

    Oztan, Asli; Silvis, Mark; Weisz, Ora A; Bradbury, Neil A; Hsu, Shu-Chan; Goldenring, James R; Yeaman, Charles; Apodaca, Gerard

    2007-10-01

    The octameric exocyst complex is associated with the junctional complex and recycling endosomes and is proposed to selectively tether cargo vesicles directed toward the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. We observed that the exocyst subunits Sec6, Sec8, and Exo70 were localized to early endosomes, transferrin-positive common recycling endosomes, and Rab11a-positive apical recycling endosomes of polarized MDCK cells. Consistent with its localization to multiple populations of endosomes, addition of function-blocking Sec8 antibodies to streptolysin-O-permeabilized cells revealed exocyst requirements for several endocytic pathways including basolateral recycling, apical recycling, and basolateral-to-apical transcytosis. The latter was selectively dependent on interactions between the small GTPase Rab11a and Sec15A and was inhibited by expression of the C-terminus of Sec15A or down-regulation of Sec15A expression using shRNA. These results indicate that the exocyst complex may be a multipurpose regulator of endocytic traffic directed toward both poles of polarized epithelial cells and that transcytotic traffic is likely to require Rab11a-dependent recruitment and modulation of exocyst function, likely through interactions with Sec15A. PMID:17686995

  4. USP9X Controls EGFR Fate by Deubiquitinating the Endocytic Adaptor Eps15.

    PubMed

    Savio, Michol Giovanna; Wollscheid, Nadine; Cavallaro, Elena; Algisi, Veronica; Di Fiore, Pier Paolo; Sigismund, Sara; Maspero, Elena; Polo, Simona

    2016-01-25

    Following activation by its cognate ligand(s), the epidermal growth factor receptor (EGFR) is rapidly routed to the lysosome for degradation in a ubiquitination-dependent fashion. This pathway represents the major mechanism of long-term attenuation of EGFR signaling, and its deregulation is a significant feature in different types of cancers. Here we demonstrate, through a systematic RNAi-based approach, that several deubiquitinating (DUB) enzymes extend or decrease EGFR half-life upon EGF stimulation. We focus on USP9X, whose depletion severely affects EGFR turnover, interfering with its internalization and trafficking. We identify the endocytic protein Eps15 as one of the critical substrates of USP9X, and we map the Eps15 ubiquitination sites. We found that Eps15 monoubiquitination occurs already at minimal dose of EGF stimulation and is essential for EGFR internalization. Overall, our findings identify USP9X as a novel regulator of EGFR endocytosis and suggest a model whereby cycles of ubiquitination and deubiquitination events on endocytic accessory proteins may regulate the internalization and trafficking of the EGFR toward the lysosomes. PMID:26748853

  5. Endocytic membrane trafficking and neurodegenerative disease.

    PubMed

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

    2016-04-01

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

  6. Polar localization and endocytic degradation of a boron transporter, BOR1, is dependent on specific tyrosine residues.

    PubMed

    Yoshinari, Akira; Kasai, Koji; Fujiwara, Toru; Naito, Satoshi; Takano, Junpei

    2012-01-01

    Boron (B) is essential for plants, but is toxic in excess. Plants have to strictly regulate the uptake and translocation of B. In Arabidopsis thaliana root cells, a boric acid channel, NIP5;1, and a boric acid/borate exporter, BOR1, localize to the outer (facing soil) and inner plasma membrane domains, respectively, under B limitation. The opposite polar localizations of the importer and exporter would enable plant roots to transport B efficiently towards the xylem. In addition, accumulation of the B transporters is controlled by B conditions. When plants are shifted from low to high B conditions, NIP5;1 transcript accumulation is down-regulated through mRNA degradation. The BOR1 protein is transported to the trans-Golgi network/early endosome and multivesicular body and finally degraded in the vacuole. We have recently shown that both the polar localization and the endocytic degradation of BOR1 are controlled by at least two tyrosine residues in a large loop located in the cytosol. We also showed that ubiquitination is required for the endocytic degradation of BOR1. Here, we analyzed possible involvement of an additional tyrosine residue (Y414) in the loop region and discuss the pathway of the BOR1 trafficking for polar localization and endocytic degradation of BOR1. PMID:22301967

  7. Macropinocytosis: searching for an endocytic identity and role in the uptake of cell penetrating peptides

    PubMed Central

    Jones, Arwyn Tomos

    2007-01-01

    Abstract Macropinocytosis defines a series of events initiated by extensive plasma membrane reorganization or ruffling to form an external macropinocytic structure that is then enclosed and internalized. The process is constitutive in some organisms and cell types but in others it is only pronounced after growth factor stimulation. Internalized macropinosomes share many features with phagosomes and both are distinguished from other forms of pinocytic vesicles by their large size, morphological heterogeneity and lack of coat structures. A paucity of information is available on other distinguishing features for macropinocytosis such as specific marker proteins and drugs that interfere with its mechanism over other endocytic processes. This has hampered efforts to characterize the dynamics of this pathway and to identify regulatory proteins that are expressed in order to allow it to proceed. Upon internalization, macropinosomes acquire regulatory proteins common to other endocytic pathways, suggesting that their identities as unique structures are short-lived. There is however less consensus regarding the overall fate of the macropinosome cargo or its limiting membrane and processes such as fusion, tubulation, recycling and regulated exocytosis have all been implicated in shaping the macropinosome and directing cargo traffic. Macropinocytosis has also been implicated in the internalization of cell penetrating peptides that are of significant interest to researchers aiming to utilize their translocation abilities to deliver therapeutic entities such as genes and proteins into cells. This review focuses on recent findings on the regulation of macropinocytosis, the intracellular fate of the macropinosome and discusses evidence for the role of this pathway as a mechanism of entry for cell penetrating peptides. PMID:17760832

  8. Stem-cell-specific endocytic degradation defects lead to intestinal dysplasia in Drosophila

    PubMed Central

    Nagy, Péter; Kovács, Laura; Sándor, Gyöngyvér O.

    2016-01-01

    ABSTRACT UV radiation resistance-associated gene (UVRAG) is a tumor suppressor involved in autophagy, endocytosis and DNA damage repair, but how its loss contributes to colorectal cancer is poorly understood. Here, we show that UVRAG deficiency in Drosophila intestinal stem cells leads to uncontrolled proliferation and impaired differentiation without preventing autophagy. As a result, affected animals suffer from gut dysfunction and short lifespan. Dysplasia upon loss of UVRAG is characterized by the accumulation of endocytosed ligands and sustained activation of STAT and JNK signaling, and attenuation of these pathways suppresses stem cell hyperproliferation. Importantly, the inhibition of early (dynamin-dependent) or late (Rab7-dependent) steps of endocytosis in intestinal stem cells also induces hyperproliferation and dysplasia. Our data raise the possibility that endocytic, but not autophagic, defects contribute to UVRAG-deficient colorectal cancer development in humans. PMID:26921396

  9. Possible roles of the endocytic cycle in cell motility.

    PubMed

    Traynor, David; Kay, Robert R

    2007-07-15

    Starving, highly motile Dictyostelium cells maintain an active endocytic cycle, taking up their surface about every 11 minutes. Cell motility depends on a functional NSF (N-ethylmaleimide sensitive factor) protein--also essential for endocytosis and membrane trafficking generally--and we, therefore, investigated possible ways in which the endocytic cycle might be required for cell movement. First, NSF, and presumably membrane trafficking, are not required for the initial polarization of the leading edge in a cyclic-AMP gradient. Second, we can detect no evidence for membrane flow from the leading edge, as photobleached or photoactivated marks in the plasma membrane move forward roughly in step with the leading edge, rather than backwards from it. Third, we find that the surface area of a cell--measured from confocal reconstructions--constantly fluctuates during movement as it projects pseudopodia and otherwise changes shape; increases of 20-30% can often occur over a few minutes. These fluctuations cannot be explained by reciprocal changes in filopodial surface area and they substantially exceed the 2-3% by which membranes can stretch. We propose that the endocytic cycle has a key function in motility by allowing adjustment of cell surface area to match changes in shape and that, without this function, movement is severely impaired. PMID:17606987

  10. Rab1a regulates sorting of early endocytic vesicles

    PubMed Central

    Mukhopadhyay, Aparna; Quiroz, Jose A.

    2014-01-01

    We previously reported that Rab1a is associated with asialoorosomucoid (ASOR)-containing early endocytic vesicles, where it is required for their microtubule-based motility. In Rab1a knockdown (KD) cell lines, ASOR failed to segregate from its receptor and, consequently, did not reach lysosomes for degradation, indicating a defect in early endosome sorting. Although Rab1 is required for Golgi/endoplasmic reticulum trafficking, this process was unaffected, likely due to retained expression of Rab1b in these cells. The present study shows that Rab1a has a more general role in endocytic vesicle processing that extends to EGF and transferrin (Tfn) trafficking. Compared with results in control Huh7 cells, EGF accumulated in aggregates within Rab1a KD cells, failing to reach lysosomal compartments. Tfn, a prototypical example of recycling cargo, accumulated in a Rab11-mediated slow-recycling compartment in Rab1a KD cells, in contrast to control cells, which sort Tfn into a fast-recycling Rab4 compartment. These data indicate that Rab1a is an important regulator of early endosome sorting for multiple cargo species. The effectors and accessory proteins recruited by Rab1a to early endocytic vesicles include the minus-end-directed kinesin motor KifC1, while others remain to be discovered. PMID:24407591

  11. Rab14 and Its Exchange Factor FAM116 Link Endocytic Recycling and Adherens Junction Stability in Migrating Cells

    PubMed Central

    Linford, Andrea; Yoshimura, Shin-ichiro; Bastos, Ricardo Nunes; Langemeyer, Lars; Gerondopoulos, Andreas; Rigden, Daniel J.; Barr, Francis A.

    2012-01-01

    Summary Rab GTPases define the vesicle trafficking pathways underpinning cell polarization and migration. Here, we find that Rab4, Rab11, and Rab14 and the candidate Rab GDP-GTP exchange factors (GEFs) FAM116A and AVL9 are required for cell migration. Rab14 and its GEF FAM116A localize to and act on an intermediate compartment of the transferrin-recycling pathway prior to Rab11 and after Rab5 and Rab4. This Rab14 intermediate recycling compartment has specific functions in migrating cells discrete from early and recycling endosomes. Rab14-depleted cells show increased N-cadherin levels at junctional complexes and cannot resolve cell-cell junctions. This is due to decreased shedding of cell-surface N-cadherin by the ADAM family protease ADAM10/Kuzbanian. In FAM116A- and Rab14-depleted cells, ADAM10 accumulates in a transferrin-positive endocytic compartment, and the cell-surface level of ADAM10 is correspondingly reduced. FAM116 and Rab14 therefore define an endocytic recycling pathway needed for ADAM protease trafficking and regulation of cell-cell junctions. PMID:22595670

  12. Endocytic uptake of a large array of HPMA copolymers: Elucidation into the dependence on the physicochemical characteristics

    PubMed Central

    Liu, Jihua; Bauer, Hillevi; Callahan, Jon; Kopečková, Pavla; Pan, Huaizhong; Kopeček, Jindřich

    2010-01-01

    Endocytic uptake and subcellular trafficking of a large array of HPMA (N-(2-hydroxypropyl)methacrylamide) based copolymers possessing positively or negatively charged residues, or hydrophobic groups were evaluated by flow cytometry and living cell confocal microscopy in cultured prostate cancer cells. The degrees of cellular uptake of various copolymer fractions with narrow polydispersities were quantified. The copolymer charge was the predominant physicochemical feature in terms of cellular uptake. Fast and efficient uptake occurred in positively charged copolymers due to non-specific adsorptive endocytosis, whereas slow uptake of negatively charged copolymers was observed. The uptake of copolymers was also molecular weight dependent. The copolymers were internalized into the cells through multiple endocytic pathways: positively charged copolymers robustly engaged clathrin-mediated endocytosis, macropinocytosis and dynamin-dependent endocytosis, while weakly negatively charged copolymers weakly employed these pathways; strongly negatively charged copolymers only mobilized macropinocytosis. HPMA copolymer possessing 4 mol% of moderately hydrophobic functional groups did not show preferential uptake. All copolymers ultimately localized in late endosomes/lysosomes via early endosomes; with varying kinetics among the copolymers. This study indicates that cell entry and subsequent intracellular trafficking of polymeric drug carriers are strongly dependent on the physicochemical characteristics of the nanocarrier, such as charge and molecular weight. PMID:20043962

  13. The role of endocytic Rab GTPases in regulation of growth factor signaling and the migration and invasion of tumor cells

    PubMed Central

    Porther, N; Barbieri, MA

    2015-01-01

    Metastasis is characterized pathologically by uncontrolled cell invasion, proliferation, migration and angiogenesis. It is a multistep process that encompasses the modulation of membrane permeability and invasion, cell spreading, cell migration and proliferation of the extracellular matrix, increase in cell adhesion molecules and interaction, decrease in cell attachment and induced survival signals and propagation of nutrient supplies (blood vessels). In cancer, a solid tumor cannot expand and spread without a series of synchronized events. Changes in cell adhesion receptor molecules (e.g., integrins, cadherin-catenins) and protease expressions have been linked to tumor invasion and metastasis. It has also been determined that ligand-growth factor receptor interactions have been associated with cancer development and metastasis via the endocytic pathway. Specifically, growth factors, which include IGF-1 and IGF-2 therapy, have been associated with most if not all of the features of metastasis. In this review, we will revisit some of the key findings on perhaps one of the most important hallmarks of cancer metastasis: cell migration and cell invasion and the role of the endocytic pathway in mediating this phenomenon PMID:26317377

  14. Endocytic Trafficking Routes of Wild Type and ΔF508 Cystic Fibrosis Transmembrane Conductance RegulatorD⃞

    PubMed Central

    Gentzsch, Martina; Chang, Xiu-Bao; Cui, Liying; Wu, Yufeng; Ozols, Victor V.; Choudhury, Amit; Pagano, Richard E.; Riordan, John R.

    2004-01-01

    Intracellular trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) is a focus of attention because it is defective in most patients with cystic fibrosis. ΔF508 CFTR, which does not mature conformationally, normally does not exit the endoplasmic reticulum, but if induced to do so at reduced temperature is short-lived at the surface. We used external epitope-tagged constructs to elucidate the itinerary and kinetics of wild type and ΔF508 CFTR in the endocytic pathway and visualized movement of CFTR from the surface to intracellular compartments. Modulation of different endocytic steps with low temperature (16°C) block, protease inhibitors, and overexpression of wild type and mutant Rab GTPases revealed that surface CFTR enters several different routes, including a Rab5-dependent initial step to early endosomes, then either Rab11-dependent recycling back to the surface or Rab7-regulated movement to late endosomes or alternatively Rab9-mediated transit to the trans-Golgi network. Without any of these modulations ΔF508 CFTR rapidly disappears from and does not return to the cell surface, confirming that its altered structure is detected in the distal as well as proximal secretory pathway. Importantly, however, the mutant protein can be rescued at the plasma membrane by Rab11 overexpression, proteasome inhibitors, or inhibition of Rab5-dependent endocytosis. PMID:15075371

  15. Loss of SNAP29 Impairs Endocytic Recycling and Cell Motility

    PubMed Central

    Rapaport, Debora; Lugassy, Yevgenia; Sprecher, Eli; Horowitz, Mia

    2010-01-01

    Intracellular membrane trafficking depends on the ordered formation and consumption of transport intermediates and requires that membranes fuse with each other in a tightly regulated and highly specific manner. Membrane anchored SNAREs assemble into SNARE complexes that bring membranes together to promote fusion. SNAP29 is a ubiquitous synaptosomal-associated SNARE protein. It interacts with several syntaxins and with the EH domain containing protein EHD1. Loss of functional SNAP29 results in CEDNIK syndrome (Cerebral Dysgenesis, Neuropathy, Ichthyosis and Keratoderma). Using fibroblast cell lines derived from CEDNIK patients, we show that SNAP29 mediates endocytic recycling of transferrin and β1-integrin. Impaired β1-integrin recycling affected cell motility, as reflected by changes in cell spreading and wound healing. No major changes were detected in exocytosis of VSVG protein from the Golgi apparatus, although the Golgi system acquired a dispersed morphology in SNAP29 deficient cells. Our results emphasize the importance of SNAP29 mediated membrane fusion in endocytic recycling and consequently, in cell motility. PMID:20305790

  16. Down syndrome fibroblast model of Alzheimer-related endosome pathology: accelerated endocytosis promotes late endocytic defects.

    PubMed

    Cataldo, Anne M; Mathews, Paul M; Boiteau, Anne Boyer; Hassinger, Linda C; Peterhoff, Corrinne M; Jiang, Ying; Mullaney, Kerry; Neve, Rachael L; Gruenberg, Jean; Nixon, Ralph A

    2008-08-01

    Endocytic dysfunction is an early pathological change in Alzheimer's disease (AD) and Down's syndrome (DS). Using primary fibroblasts from DS individuals, we explored the interactions among endocytic compartments that are altered in AD and assessed their functional consequences in AD pathogenesis. We found that, like neurons in both AD and DS brains, DS fibroblasts exhibit increased endocytic uptake, fusion, and recycling, and trafficking of lysosomal hydrolases to rab5-positive early endosomes. Moreover, late endosomes identified using antibodies to rab7 and lysobisphosphatidic acid increased in number and appeared as enlarged, perinuclear vacuoles, resembling those in neurons of both AD and DS brains. In control fibroblasts, similar enlargement of rab5-, rab7-, and lysobisphosphatidic acid-positive endosomes was induced when endocytosis and endosomal fusion were increased by expression of either a rab5 or an active rab5 mutant, suggesting that persistent endocytic activation results in late endocytic dysfunction. Conversely, expression of a rab5 mutant that inhibits endocytic uptake reversed early and late endosomal abnormalities in DS fibroblasts. Our results indicate that DS fibroblasts recapitulate the neuronal endocytic dysfunction of AD and DS, suggesting that increased trafficking from early endosomes can account, in part, for downstream endocytic perturbations that occur in neurons in both AD and DS brains. PMID:18535180

  17. Collapsin Response Mediator Protein-2 (Crmp2) Regulates Trafficking by Linking Endocytic Regulatory Proteins to Dynein Motors*

    PubMed Central

    Rahajeng, Juliati; Giridharan, Sai S. P.; Naslavsky, Naava; Caplan, Steve

    2010-01-01

    Endocytosis is a conserved cellular process in which nutrients, lipids, and receptors are internalized and transported to early endosomes, where they are sorted and either channeled to degradative pathways or recycled to the plasma membrane. MICAL-L1 and EHD1 are important regulatory proteins that control key endocytic transport steps. However, the precise mechanisms by which they mediate transport, and particularly the mode by which they connect to motor proteins, have remained enigmatic. Here we have identified the collapsin response mediator protein-2 (Crmp2) as an interaction partner of MICAL-L1 in non-neuronal cells. Crmp2 interacts with tubulin dimers and kinesin and negatively regulates dynein-based transport in neuronal cells, but its expression and function in non-neuronal cells have remained poorly characterized. Upon Crmp2 depletion, we observed dramatic relocalization of internalized transferrin (Tf) from peripheral vesicles to the endocytic recycling compartment (ERC), similar to the effect of depleting either MICAL-L1 or EHD1. Moreover, Tf relocalization to the ERC could be inhibited by interfering with microtubule polymerization, consistent with a role for uncoupled motor protein-based transport upon depletion of Crmp2, MICAL-L1, or EHD1. Finally, transfection of dynamitin, a component of the dynactin complex whose overexpression inhibits dynein activity, prevented the relocalization of internalized Tf to the ERC upon depletion of Crmp2, MICAL-L1, or EHD1. These data provide the first trafficking regulatory role for Crmp2 in non-neuronal cells and support a model in which Crmp2 is an important endocytic regulatory protein that links MICAL-L1·EHD1-based vesicular transport to dynein motors. PMID:20801876

  18. Constitutive Endocytic Recycling and Protein Kinase C-mediated Lysosomal Degradation Control KATP Channel Surface Density*

    PubMed Central

    Manna, Paul T.; Smith, Andrew J.; Taneja, Tarvinder K.; Howell, Gareth J.; Lippiat, Jonathan D.; Sivaprasadarao, Asipu

    2010-01-01

    Pancreatic ATP-sensitive potassium (KATP) channels control insulin secretion by coupling the excitability of the pancreatic β-cell to glucose metabolism. Little is currently known about how the plasma membrane density of these channels is regulated. We therefore set out to examine in detail the endocytosis and recycling of these channels and how these processes are regulated. To achieve this goal, we expressed KATP channels bearing an extracellular hemagglutinin epitope in human embryonic kidney cells and followed their fate along the endocytic pathway. Our results show that KATP channels undergo multiple rounds of endocytosis and recycling. Further, activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate significantly decreases KATP channel surface density by reducing channel recycling and diverting the channel to lysosomal degradation. These findings were recapitulated in the model pancreatic β-cell line INS1e, where activation of PKC leads to a decrease in the surface density of native KATP channels. Because sorting of internalized channels between lysosomal and recycling pathways could have opposite effects on the excitability of pancreatic β-cells, we propose that PKC-regulated KATP channel trafficking may play a role in the regulation of insulin secretion. PMID:20026601

  19. Endocytic Trafficking of Membrane-Bound Cargo: A Flotillin Point of View

    PubMed Central

    Meister, Melanie; Tikkanen, Ritva

    2014-01-01

    The ubiquitous and highly conserved flotillin proteins, flotillin-1 and flotillin-2, have been shown to be involved in various cellular processes such as cell adhesion, signal transduction through receptor tyrosine kinases as well as in cellular trafficking pathways. Due to the fact that flotillins are acylated and form hetero-oligomers, they constitutively associate with cholesterol-enriched lipid microdomains. In recent years, such microdomains have been appreciated as platforms that participate in endocytosis and other cellular trafficking steps. This review summarizes the current findings on the role of flotillins in membrane-bound cargo endocytosis and endosomal trafficking events. We will discuss the proposed function of flotillins in endocytosis in the light of recent findings that point towards a role for flotillins in a step that precedes the actual endocytic uptake of cargo molecules. Recent findings have also revealed that flotillins may be important for endosomal sorting and recycling of specific cargo molecules. In addition to these aspects, the cellular trafficking pathway of flotillins themselves as potential cargo in the context of growth factor signaling will be discussed. PMID:25019426

  20. Methods to study endocytic trafficking of the EGF receptor

    PubMed Central

    Pinilla-Macua, Itziar; Sorkin, Alexander

    2016-01-01

    Endocytosis and postendocytic sorting of epidermal growth factor (EGF) receptor (EGFR) are the major regulators of EGFR signaling. EGFR endocytosis and ubiquitin-dependent lysosomal targeting are also considered to be the prototypic experimental system for studying the molecular mechanisms of stimulus-induced and constitutive endocytic trafficking. Therefore, elucidation of the mechanisms of EGFR endocytosis and its regulation of the signaling network is essential not only for better understanding of the EGFR biology but also for defining general regulatory principles in the endocytosis system. Comprehensive analysis of these mechanisms requires quantitative and physiologically relevant methodological approaches for measuring the rates of EGFR internalization, degradation, and recycling. Basic experimental protocols described in this chapter cover a combination of single-cell microscopy and biochemical methods that are used to follow EGF-induced endocytosis of EGFR in real time, measure the kinetic rate parameters of EGFR internalization and recycling, and analyze EGF-dependent ubiquitination and degradation of EGFR. PMID:26360045

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

    PubMed

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

    2014-01-01

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

  2. Proteomics of Secretory and Endocytic Organelles in Giardia lamblia

    PubMed Central

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

    2014-01-01

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

  3. Selective high-level expression of epsin 3 in gastric parietal cells, where it is localized at endocytic sites of apical canaliculi

    PubMed Central

    Ko, Genevieve; Paradise, Summer; Chen, Hong; Graham, Morven; Vecchi, Manuela; Bianchi, Fabrizio; Cremona, Ottavio; Di Fiore, Pier Paolo; De Camilli, Pietro

    2010-01-01

    Epsin is a ubiquitin-binding endocytic adaptor, which is highly concentrated at clathrin-coated pits and coordinates acquisition of bilayer curvature with coat recruitment and cargo selection. Epsin is encoded by three distinct genes in mammals. Epsin 1 and 2 have broad tissue distribution with high-level expression in the brain. In contrast, epsin 3 was reported to be expressed primarily in immature keratinocytes. Here, we show that epsin 3 is selectively expressed at high levels in the stomach (including the majority of gastric cancers), where it is concentrated in parietal cells. In these cells, epsin 3 is enriched and colocalized with clathrin around apical canaliculi, the sites that control acidification of the stomach lumen via the exo-endocytosis of vesicles containing the H/K ATPase. Deletion of the epsin 3 gene in mice did not result in obvious pathological phenotypes in either the stomach or other organs, possibly because of overlapping functions of the other two epsins. However, levels of EHD1 and EHD2, two membrane tubulating proteins with a role in endocytic recycling, were elevated in epsin 3 knock-out stomachs, pointing to a functional interplay of epsin 3 with EHD proteins in the endocytic pathway of parietal cells. We suggest that epsin 3 cooperates with other bilayer binding proteins with curvature sensing/generating properties in the specialized traffic and membrane remodeling processes typical of gastric parietal cells. PMID:21115825

  4. An RNAi screen identifies KIF15 as a novel regulator of the endocytic trafficking of integrin.

    PubMed

    Eskova, Anastasia; Knapp, Bettina; Matelska, Dorota; Reusing, Susanne; Arjonen, Antti; Lisauskas, Tautvydas; Pepperkok, Rainer; Russell, Robert; Eils, Roland; Ivaska, Johanna; Kaderali, Lars; Erfle, Holger; Starkuviene, Vytaute

    2014-06-01

    α2β1 integrin is one of the most important collagen-binding receptors, and it has been implicated in numerous thrombotic and immune diseases. α2β1 integrin is a potent tumour suppressor, and its downregulation is associated with increased metastasis and poor prognosis in breast cancer. Currently, very little is known about the mechanism that regulates the cell-surface expression and trafficking of α2β1 integrin. Here, using a quantitative fluorescence-microscopy-based RNAi assay, we investigated the impact of 386 cytoskeleton-associated or -regulatory genes on α2 integrin endocytosis and found that 122 of these affected the intracellular accumulation of α2 integrin. Of these, 83 were found to be putative regulators of α2 integrin trafficking and/or expression, with no observed effect on the internalization of epidermal growth factor (EGF) or transferrin. Further interrogation and validation of the siRNA screen revealed a role for KIF15, a microtubule-based molecular motor, as a significant inhibitor of the endocytic trafficking of α2 integrin. Our data suggest a novel role for KIF15 in mediating plasma membrane localization of the alternative clathrin adaptor Dab2, thus impinging on pathways that regulate α2 integrin internalization. PMID:24659801

  5. The polarity protein Par3 regulates APP trafficking and processing through the endocytic adaptor protein Numb.

    PubMed

    Sun, Miao; Asghar, Suwaiba Z; Zhang, Huaye

    2016-09-01

    The processing of amyloid precursor protein (APP) into β-amyloid peptide (Aβ) is a key step in the pathogenesis of Alzheimer's disease (AD), and trafficking dysregulations of APP and its secretases contribute significantly to altered APP processing. Here we show that the cell polarity protein Par3 plays an important role in APP processing and trafficking. We found that the expression of full length Par3 is significantly decreased in AD patients. Overexpression of Par3 promotes non-amyloidogenic APP processing, while depletion of Par3 induces intracellular accumulation of Aβ. We further show that Par3 functions by regulating APP trafficking. Loss of Par3 decreases surface expression of APP by targeting APP to the late endosome/lysosome pathway. Finally, we show that the effects of Par3 are mediated through the endocytic adaptor protein Numb, and Par3 functions by interfering with the interaction between Numb and APP. Together, our studies show a novel role for Par3 in regulating APP processing and trafficking. PMID:27072891

  6. Identification of a Novel Lysosomal Trafficking Peptide using Phage Display Biopanning Coupled with Endocytic Selection Pressure

    PubMed Central

    2015-01-01

    Methods to select ligands that accumulate specifically in cancer cells and traffic through a defined endocytic pathway may facilitate rapid pairing of ligands with linkers suitable for drug conjugate therapies. We performed phage display biopanning on cancer cells that are treated with selective inhibitors of a given mechanism of endocytosis. Using chlorpromazine to inhibit clathrin-mediated endocytosis in H1299 nonsmall cell lung cancer cells, we identified two clones, ATEPRKQYATPRVFWTDAPG (15.1) and a novel peptide LQWRRDDNVHNFGVWARYRL (H1299.3). The peptides segregate by mechanism of endocytosis and subsequent location of subcellular accumulation. The H1299.3 peptide primarily utilizes clathrin-mediated endocytosis and colocalizes with Lamp1, a lysosomal marker. Conversely, the 15.1 peptide is clathrin-independent and localizes to a perinuclear region. Thus, this novel phage display scheme allows for selection of peptides that selectively internalize into cells via a known mechanism of endocytosis. These types of selections may allow for better matching of linker with targeting ligand by selecting ligands that internalize and traffic to known subcellular locations. PMID:25188559

  7. Cell-free fusion of bacteria-containing phagosomes with endocytic compartments

    PubMed Central

    Becken, Ulrike; Jeschke, Andreas; Veltman, Katharina; Haas, Albert

    2010-01-01

    Uptake of microorganisms by professional phagocytic cells leads to formation of a new subcellular compartment, the phagosome, which matures by sequential fusion with early and late endocytic compartments, resulting in oxidative and nonoxidative killing of the enclosed microbe. Few tools are available to study membrane fusion between phagocytic and late endocytic compartments in general and with pathogen-containing phagosomes in particular. We have developed and applied a fluorescence microscopy assay to study fusion of microbe-containing phagosomes with different-aged endocytic compartments in vitro. This revealed that fusion of phagosomes containing nonpathogenic Escherichia coli with lysosomes requires Rab7 and SNARE proteins but not organelle acidification. In vitro fusion experiments with phagosomes containing pathogenic Salmonella enterica serovar Typhimurium indicated that reduced fusion of these phagosomes with early and late endocytic compartments was independent of endosome and cytosol sources and, hence, a consequence of altered phagosome quality. PMID:21071675

  8. Cell-free fusion of bacteria-containing phagosomes with endocytic compartments.

    PubMed

    Becken, Ulrike; Jeschke, Andreas; Veltman, Katharina; Haas, Albert

    2010-11-30

    Uptake of microorganisms by professional phagocytic cells leads to formation of a new subcellular compartment, the phagosome, which matures by sequential fusion with early and late endocytic compartments, resulting in oxidative and nonoxidative killing of the enclosed microbe. Few tools are available to study membrane fusion between phagocytic and late endocytic compartments in general and with pathogen-containing phagosomes in particular. We have developed and applied a fluorescence microscopy assay to study fusion of microbe-containing phagosomes with different-aged endocytic compartments in vitro. This revealed that fusion of phagosomes containing nonpathogenic Escherichia coli with lysosomes requires Rab7 and SNARE proteins but not organelle acidification. In vitro fusion experiments with phagosomes containing pathogenic Salmonella enterica serovar Typhimurium indicated that reduced fusion of these phagosomes with early and late endocytic compartments was independent of endosome and cytosol sources and, hence, a consequence of altered phagosome quality. PMID:21071675

  9. Evaluation of nanoparticles as endocytic tracers in cellular microbiology

    NASA Astrophysics Data System (ADS)

    Zhang, Yuying; Hensel, Michael

    2013-09-01

    The study of pathogen interactions with eukaryotic host cells requires the introduction of fluorescent probes to visualize processes such as endocytosis, intracellular transport or host cell manipulation by the pathogen. Here, three types of fluorescent nanoparticles (NPs), i.e. Rhodamine-labeled polymethacrylate (PMA) NPs, silica NPs and gold NPs, were employed to label the host cellular endolysosomal system and monitor manipulations by the pathogen Salmonella enterica. Using live cell imaging, we investigated the performance of NPs in cellular uptake, labeling of endocytic vesicles and lysosomes, as well as interaction with the pathogen. We show that fluorescent gold and silica, but not PMA NPs appropriately label host cell structures and efficiently track rearrangements of the host endosomal system by the activities of intracellular Salmonella. Silica NPs slightly aggregated and located in Salmonella-induced compartments as isolated dots, while gold NPs distributed uniformly inside such structures. Both silica and gold NPs exhibited no adverse impact on either host cells or pathogens, and are versatile tools for infection biology.The study of pathogen interactions with eukaryotic host cells requires the introduction of fluorescent probes to visualize processes such as endocytosis, intracellular transport or host cell manipulation by the pathogen. Here, three types of fluorescent nanoparticles (NPs), i.e. Rhodamine-labeled polymethacrylate (PMA) NPs, silica NPs and gold NPs, were employed to label the host cellular endolysosomal system and monitor manipulations by the pathogen Salmonella enterica. Using live cell imaging, we investigated the performance of NPs in cellular uptake, labeling of endocytic vesicles and lysosomes, as well as interaction with the pathogen. We show that fluorescent gold and silica, but not PMA NPs appropriately label host cell structures and efficiently track rearrangements of the host endosomal system by the activities of intracellular

  10. CULD is required for rhodopsin and TRPL channel endocytic trafficking and survival of photoreceptor cells

    PubMed Central

    Xu, Ying; Wang, Tao

    2016-01-01

    ABSTRACT Endocytosis of G-protein-coupled receptors (GPCRs) and associated channels contributes to desensitization and adaptation of a variety of signaling cascades. In Drosophila melanogaster, the main light-sensing rhodopsin (Rh1; encoded by ninaE) and the downstream ion channel, transient receptor potential like (TRPL), are endocytosed in response to light, but the mechanism is unclear. By using an RNA-Sequencing (RNA-Seq) approach, we discovered a protein we named CULD, a photoreceptor-cell enriched CUB- and LDLa-domain transmembrane protein, that is required for endocytic trafficking of Rh1 and TRPL. CULD localized to endocytic Rh1-positive or TRPL-positive vesicles. Mutations in culd resulted in the accumulation of Rh1 and TRPL within endocytic vesicles, and disrupted the regular turnover of endocytic Rh1 and TRPL. In addition, loss of CULD induced light- and age-dependent retinal degeneration, and reduced levels of Rh1, but not of TRPL, suppressed retinal degeneration in culd-null mutant flies. Our data demonstrate that CULD plays an important role in the endocytic turnover of Rh1 and TRPL, and suggest that CULD-dependent rhodopsin endocytic trafficking is required for maintaining photoreceptor integrity. PMID:26598556

  11. GGA1-mediated endocytic traffic of LR11/SorLA alters APP intracellular distribution and amyloid-β production.

    PubMed

    Herskowitz, Jeremy H; Offe, Katrin; Deshpande, Aniruddha; Kahn, Richard A; Levey, Allan I; Lah, James J

    2012-07-01

    Proteolytic processing of the amyloid-β precursor protein (APP) and generation of amyloid-β peptide (Aβ) are key events in Alzheimer's disease (AD) pathogenesis. Cell biological and genetic evidence has implicated the low-density lipoprotein and sorting receptor LR11/SorLA in AD through mechanisms related to APP and Aβ production. Defining the cellular pathway(s) by which LR11 modulates Aβ production is critical to understanding how changes in LR11 expression affect the development of Aβ pathology in AD progression. We report that the LR11 ectodomain is required for LR11-mediated reduction of Aβ and that mutagenesis of the LR11 Golgi-localizing, γ-adaptin ear homology domain, ADP-ribosylation factor (GGA)-binding motif affects the endosomal distribution of LR11, as well as LR11's effects on APP traffic and Aβ production. Targeted small interfering RNA (siRNA) knockdown studies of GGA1, GGA2, and GGA3 indicate a surprising degree of specificity toward GGA1, suggesting that GGA1 is a candidate regulator of LR11 traffic. Additional siRNA knockdown experiments reveal that GGA1 is necessary for both LR11 and β-site APP-cleaving enzyme-1 (BACE1) modulation of APP processing to Aβ. Mutagenesis of BACE1 serine 498 to alanine enhances BACE1 targeting to LR11-positive compartments and nullifies LR11-mediated reduction of Aβ. On basis of these results, we propose that GGA1 facilitates LR11 endocytic traffic and that LR11 modulates Aβ levels by promoting APP traffic to the endocytic recycling compartment. PMID:22621900

  12. Probiotics promote endocytic allergen degradation in gut epithelial cells

    SciTech Connect

    Song, Chun-Hua; Liu, Zhi-Qiang; Huang, Shelly; Zheng, Peng-Yuan; Yang, Ping-Chang

    2012-09-14

    Highlights: Black-Right-Pointing-Pointer Knockdown of A20 compromised the epithelial barrier function. Black-Right-Pointing-Pointer The fusion of endosome/lysosome was disturbed in the A20-deficient HT-29 cells. Black-Right-Pointing-Pointer Antigens transported across A20-deficient HT-29 monolayers conserved antigenicity. Black-Right-Pointing-Pointer Probiotic proteins increased the expression of A20 in HT-29 cells. -- Abstract: Background and aims: Epithelial barrier dysfunction plays a critical role in the pathogenesis of allergic diseases; the mechanism is to be further understood. The ubiquitin E3 ligase A20 (A20) plays a role in the endocytic protein degradation in the cells. This study aims to elucidate the role of A20 in the maintenance of gut epithelial barrier function. Methods: Gut epithelial cell line, HT-29 cell, was cultured into monolayers to evaluate the barrier function in transwells. RNA interference was employed to knock down the A20 gene in HT-29 cells to test the role of A20 in the maintenance of epithelial barrier function. Probiotic derived proteins were extracted from the culture supernatants using to enhance the expression of A20 in HT-29 cells. Results: The results showed that the knockdown of A20 compromised the epithelial barrier function in HT-29 monolayers, mainly increased the intracellular permeability. The fusion of endosome/lysosome was disturbed in the A20-deficient HT-29 cells. Allergens collected from the transwell basal chambers of A20-deficient HT-29 monolayers still conserved functional antigenicity. Treating with probiotic derived proteins increased the expression of A20 in HT-29 cells and promote the barrier function. Conclusion: A20 plays an important role in the maintenance of epithelial barrier function as shown by HT-29 monolayer. Probiotic derived protein increases the expression of A20 and promote the HT-29 monolayer barrier function.

  13. Endocytic vesicle scission by lipid phase boundary forces

    PubMed Central

    Liu, Jian; Kaksonen, Marko; Drubin, David G.; Oster, George

    2006-01-01

    Endocytosis in budding yeast is thought to occur in several phases. First, the membrane invaginates and then elongates into a tube. A vesicle forms at the end of the tube, eventually pinching off to form a “free” vesicle. Experiments show that actin polymerization is an active participant in the endocytic process, along with a number of membrane-associated proteins. Here we investigate the possible roles of these components in driving vesiculation by constructing a quantitative model of the process beginning at the stage where the membrane invagination has elongated into a tube encased in a sheath of membrane-associated protein. This protein sheath brings about the scission step where the vesicle separates from the tube. When the protein sheath is dynamin, it is commonly assumed that scission is brought about by the constriction of the sheath. Here, we show that an alternative scenario can work as well: The protein sheath acts as a “filter” to effect a phase separation of lipid species. The resulting line tension tends to minimize the interface between the tube region and the vesicle region. Interestingly, large vesicle size can further facilitate the reduction of the interfacial diameter down to a few nanometers, small enough so that thermal fluctuations can fuse the membrane and pinch off the vesicle. To deform the membrane into the tubular vesicle shape, the membrane elastic resistance forces must be balanced by some additional forces that we show can be generated by actin polymerization and/or myosin I. These active forces are shown to be important in successful scission processes as well. PMID:16801551

  14. Cholesterol oxidase and the hydroxymethylglutaryl coenzyme A reductase inhibitor mevinolin perturb endocytic trafficking in cultured vascular smooth muscle cells.

    PubMed

    Thyberg, J

    2003-10-01

    Cholesterol is a component of cellular membranes and especially abundant in caveolae (50-80 nm flask-shaped invaginations of the plasma membrane). Caveolae are highly numerous in vascular endothelial and smooth muscle cells and have been implicated in a variety of functions, including signal transduction, lipid transport and uptake of macromolecules. Here, the effects of cholesterol oxidase (an enzyme that oxidizes cholesterol in caveolae of living cells) and mevinolin (an inhibitor of cholesterol synthesis) on fine structure and internalization of exogenous markers were studied in rat aortic smooth muscle cells grown on a substrate of fibronectin in serum-free primary cultures. Cholesterol oxidase caused a growth in size of the endocytic compartment with accumulation of enlarged endosomes and lysosomes containing tracer molecules. In parallel, the number of caveolae was reduced by about one fifth. Moreover, the morphology of the Golgi complex was altered with swollen cisternae surrounded by empty-looking vacuoles. Mevinolin suppressed transition of the cells from a differentiated or contractile to a dedifferentiated or synthetic phenotype. In addition, contractile cells were found to ingest horseradish peroxidase (HRP) not only into endosomes and lysosomes but also into Golgi cisternae, especially on the convex/cis side of the stacks, and the endoplasmic reticulum. A similar pathway was noted in contractile cells exposed to cholera toxin B subunit (CTB)-HRP conjugates, a ligand that binds to ganglioside GM1 and at least in part is ingested via caveolae. Mevinolin did not prevent the transport of CTB-HRP to the Golgi complex, but the conjugates were in this case concentrated to the concave/trans side of the cisternal stacks. However, no clear effect on the number of caveolae was noted. The observations indicate an important role of cholesterol and caveolae in the control of endocytic traffic in smooth muscle cells. This function appears most significant when the

  15. Ubiquitin-Related Roles of β-Arrestins in Endocytic Trafficking and Signal Transduction.

    PubMed

    Jean-Charles, Pierre-Yves; Rajiv, Vishwaesh; Shenoy, Sudha K

    2016-10-01

    The non-visual arrestins, β-arrestin1, and β-arrestin2 were originally identified as proteins that bind to seven-transmembrane receptors (7TMRs, also called G protein-coupled receptors, GPCRs) and block heterotrimeric G protein activation, thus leading to desensitization of transmembrane signaling. However, as subsequent discoveries have continually demonstrated, their functionality is not constrained to desensitization. They are now recognized for their critical roles in mediating intracellular trafficking of 7TMRs, growth factor receptors, ion transporters, ion channels, nuclear receptors, and non-receptor proteins. Additionally, they function as crucial mediators of ubiquitination of 7TMRs as well as other receptors and non-receptor proteins. Recently, emerging studies suggest that a class of proteins with predicted structural features of β-arrestins regulate substrate ubiquitination in yeast and higher mammals, lending support to the idea that the adaptor role of β-arrestins in protein ubiquitination is evolutionarily conserved. β-arrestins also function as scaffolds for kinases and transduce signals from 7TMRs through pathways that do not require G protein activation. Remarkably, the endocytic and scaffolding functions of β-arrestin are intertwined with its ubiquitination status; the dynamic and site specific ubiquitination on β-arrestin plays a critical role in stabilizing β-arrestin-7TMR association and the formation of signalosomes. This review summarizes the current findings on ubiquitin-dependent regulation of 7TMRs as well as β-arrestins and the potential role of reversible ubiquitination as a "biological switch" in signal transduction. J. Cell. Physiol. 231: 2071-2080, 2016. © 2016 Wiley Periodicals, Inc. PMID:26790995

  16. Epithelial-to-Mesenchymal Plasticity Harnesses Endocytic Circuitries

    PubMed Central

    Corallino, Salvatore; Malabarba, Maria Grazia; Zobel, Martina; Di Fiore, Pier Paolo; Scita, Giorgio

    2015-01-01

    The ability of cells to alter their phenotypic and morphological characteristics, known as cellular plasticity, is critical in normal embryonic development and adult tissue repair and contributes to the pathogenesis of diseases, such as organ fibrosis and cancer. The epithelial-to-mesenchymal transition (EMT) is a type of cellular plasticity. This transition involves genetic and epigenetic changes as well as alterations in protein expression and post-translational modifications. These changes result in reduced cell-cell adhesion, enhanced cell adhesion to the extracellular matrix, and altered organization of the cytoskeleton and of cell polarity. Among these modifications, loss of cell polarity represents the nearly invariable, distinguishing feature of EMT that frequently precedes the other traits or might even occur in their absence. EMT transforms cell morphology and physiology, and hence cell identity, from one typical of cells that form a tight barrier, like epithelial and endothelial cells, to one characterized by a highly motile mesenchymal phenotype. Time-resolved proteomic and phosphoproteomic analyses of cells undergoing EMT recently identified thousands of changes in proteins involved in many cellular processes, including cell proliferation and motility, DNA repair, and – unexpectedly – membrane trafficking (1). These results have highlighted a picture of great complexity. First, the EMT transition is not an all-or-none response but rather a gradual process that develops over time. Second, EMT events are highly dynamic and frequently reversible, involving both cell-autonomous and non-autonomous mechanisms. The net results is that EMT generates populations of mixed cells, with partial or full phenotypes, possibly accounting (at least in part) for the physiological as well as pathological cellular heterogeneity of some tissues. Endocytic circuitries have emerged as complex connectivity infrastructures for numerous cellular networks required for the

  17. Eng2 is a component of a dynamic protein complex required for endocytic uptake in fission yeast.

    PubMed

    Encinar del Dedo, Javier; Idrissi, Fatima-Zahra; Arnáiz-Pita, Yolanda; James, Michael; Dueñas-Santero, Encarnación; Orellana-Muñoz, Sara; del Rey, Francisco; Sirotkin, Vladimir; Geli, M Isabel; Vázquez de Aldana, Carlos R

    2014-10-01

    Eng2 is a glucanase required for spore release, although it is also expressed during vegetative growth, suggesting that it might play other cellular functions. Its homology to the Saccharomyces cerevisiae Acf2 protein, previously shown to promote actin polymerization at endocytic sites in vitro, prompted us to investigate its role in endocytosis. Interestingly, depletion of Eng2 caused profound defects in endocytic uptake, which were not due to the absence of its glucanase activity. Analysis of the dynamics of endocytic proteins by fluorescence microscopy in the eng2Δ strain unveiled a previously undescribed phenotype, in which assembly of the Arp2/3 complex appeared uncoupled from the internalization of the endocytic coat and resulted in a fission defect. Strikingly also, we found that Eng2-GFP dynamics did not match the pattern of other endocytic proteins. Eng2-GFP localized to bright cytosolic spots that moved around the cellular poles and occasionally contacted assembling endocytic patches just before recruitment of Wsp1, the Schizosaccharomyces pombe WASP. Interestingly, Csh3-YFP, a WASP-interacting protein, interacted with Eng2 by co-immunoprecipitation and was recruited to Eng2 in bright cytosolic spots. Altogether, our work defines a novel endocytic functional module, which probably couples the endocytic coat to the actin module. PMID:25040903

  18. The Endocytic Fate of the Transferrin Receptor Is Regulated by c-Abl Kinase.

    PubMed

    Cao, Hong; Schroeder, Barbara; Chen, Jing; Schott, Micah B; McNiven, Mark A

    2016-08-01

    Clathrin-mediated endocytosis of transferrin (Tf) and its cognate receptor (TfR1) is a central pathway supporting the uptake of trophic iron. It has generally been assumed that this is a constitutive process. However, we have reported that the non-receptor tyrosine kinase, Src, is activated by Tf to facilitate the internalization of the Tf-TfR1 ligand-receptor complex. As an extension of these findings, we have tested whether subsequent trafficking steps might be regulated by additional kinase-dependent cascades, and we observed a significant endocytic block by inhibiting c-Abl kinase by a variety of methods. Importantly, Tf internalization was reduced significantly in all of these cell models and could be restored by re-expression of WT c-Abl. Surprisingly, this attenuated Tf-TfR1 endocytosis was due to a substantial drop in both the surface and total cellular receptor levels. Additional studies with the LDL receptor showed a similar effect. Surprisingly, immunofluorescence microscopy of imatinib-treated cells revealed a marked colocalization of internalized TfR1 with late endosomes/lysosomes, whereas attenuating the lysosome function with several inhibitors reduced this receptor loss. Importantly, inhibition of c-Abl resulted in a striking redistribution of the chaperone Hsc70 from a diffuse cytosolic localization to an association with the TfR1 at the late endosome-lysosome. Pharmacological inhibition of Hsc70 ATPase activity in cultured cells by the drug VER155008 prevents this chaperone-receptor interaction, resulting in an accumulation of the TfR1 in the early endosome. Thus, inhibition of c-Abl minimizes receptor recycling pathways and results in chaperone-dependent trafficking of the TfR1 to the lysosome for degradation. These findings implicate a novel role for c-Abl and Hsc70 as an unexpected regulator of Hsc70-mediated transport of trophic receptor cargo between the early and late endosomal compartments. PMID:27226592

  19. SNX18 shares a redundant role with SNX9 and modulates endocytic trafficking at the plasma membrane.

    PubMed

    Park, Joohyun; Kim, Yoonju; Lee, Suho; Park, Jae Jun; Park, Zee Yong; Sun, Woong; Kim, Hyun; Chang, Sunghoe

    2010-05-15

    SNX18 and SNX9 are members of a subfamily of SNX (sorting nexin) proteins with the same domain structure. Although a recent report showed that SNX18 and SNX9 localize differently in cells and appear to function in different trafficking pathways, concrete evidence regarding whether they act together or separately in intracellular trafficking is still lacking. Here, we show that SNX18 has a similar role to SNX9 in endocytic trafficking at the plasma membrane, rather than having a distinct role. SNX18 and SNX9 are expressed together in most cell lines, but to a different extent. Like SNX9, SNX18 interacts with dynamin and stimulates the basal GTPase activity of dynamin. It also interacts with neuronal Wiskott-Aldrich syndrome protein (N-WASP) and synaptojanin, as does SNX9. SNX18 and SNX9 can form a heterodimer and colocalize in tubular membrane structures. Depletion of SNX18 by small hairpin RNA inhibited transferrin uptake. SNX18 successfully compensates for SNX9 deficiency during clathrin-mediated endocytosis and vice versa. Total internal reflection fluorescence microscopy in living cells shows that a transient burst of SNX18 recruitment to clathrin-coated pits coincides spatiotemporally with a burst of dynamin and SNX9. Taken together, our results suggest that SNX18 functions with SNX9 in multiple pathways of endocytosis at the plasma membrane and that they are functionally redundant. PMID:20427313

  20. Phosphorylation Regulates the Endocytic Function of the Yeast Dynamin-Related Protein Vps1.

    PubMed

    Smaczynska-de Rooij, Iwona I; Marklew, Christopher J; Allwood, Ellen G; Palmer, Sarah E; Booth, Wesley I; Mishra, Ritu; Goldberg, Martin W; Ayscough, Kathryn R

    2016-03-01

    The family of dynamin proteins is known to function in many eukaryotic membrane fusion and fission events. The yeast dynamin-related protein Vps1 functions at several stages of membrane trafficking, including Golgi apparatus to endosome and vacuole, peroxisomal fission, and endocytic scission. We have previously shown that in its endocytic role, Vps1 functions with the amphiphysin heterodimer Rvs161/Rvs167 to facilitate scission and release of vesicles. Phosphoproteome studies of Saccharomyces cerevisiae have identified a phosphorylation site in Vps1 at serine 599. In this study, we confirmed this phosphorylation event, and we reveal that, like Rvs167, Vps1 can be phosphorylated by the yeast cyclin-associated kinase Pho85 in vivo and in vitro. The importance of this posttranslational modification was revealed when mutagenesis of S599 to a phosphomimetic or nonphosphorylatable form caused defects in endocytosis but not in other functions associated with Vps1. Mutation to nonphosphorylatable valine inhibited the Rvs167 interaction, while both S599V and S599D caused defects in vesicle scission, as shown by both live-cell imaging and electron microscopy of endocytic invaginations. Our data support a model in which phosphorylation and dephosphorylation of Vps1 promote distinct interactions and highlight the importance of such regulatory events in facilitating sequential progression of the endocytic process. PMID:26711254

  1. Endocytic downregulation of ErbB receptors: mechanisms and relevance in cancer

    PubMed Central

    Roepstorff, Kirstine; Grøvdal, Lene; Grandal, Michael; Lerdrup, Mads

    2008-01-01

    ErbB receptors (EGFR (ErbB1), ErbB2, ErbB3, and ErbB4) are important regulators of normal growth and differentiation, and they are involved in the pathogenesis of cancer. Following ligand binding and receptor activation, EGFR is endocytosed and transported to lysosomes where the receptor is degraded. This downregulation of EGFR is a complex and tightly regulated process. The functions of ErbB2, ErbB3, and ErbB4 are also regulated by endocytosis to some extent, although the current knowledge of these processes is sparse. Impaired endocytic downregulation of signaling receptors is frequently associated with cancer, since it can lead to increased and uncontrolled receptor signaling. In this review we describe the current knowledge of ErbB receptor endocytic downregulation. In addition, we outline how ErbB receptors can escape endocytic downregulation in cancer, and we discuss how targeted anti-cancer therapy may induce endocytic downregulation of ErbB receptors. PMID:18288481

  2. Phosphorylation Regulates the Endocytic Function of the Yeast Dynamin-Related Protein Vps1

    PubMed Central

    Smaczynska-de Rooij, Iwona I.; Marklew, Christopher J.; Allwood, Ellen G.; Palmer, Sarah E.; Booth, Wesley I.; Mishra, Ritu; Goldberg, Martin W.

    2015-01-01

    The family of dynamin proteins is known to function in many eukaryotic membrane fusion and fission events. The yeast dynamin-related protein Vps1 functions at several stages of membrane trafficking, including Golgi apparatus to endosome and vacuole, peroxisomal fission, and endocytic scission. We have previously shown that in its endocytic role, Vps1 functions with the amphiphysin heterodimer Rvs161/Rvs167 to facilitate scission and release of vesicles. Phosphoproteome studies of Saccharomyces cerevisiae have identified a phosphorylation site in Vps1 at serine 599. In this study, we confirmed this phosphorylation event, and we reveal that, like Rvs167, Vps1 can be phosphorylated by the yeast cyclin-associated kinase Pho85 in vivo and in vitro. The importance of this posttranslational modification was revealed when mutagenesis of S599 to a phosphomimetic or nonphosphorylatable form caused defects in endocytosis but not in other functions associated with Vps1. Mutation to nonphosphorylatable valine inhibited the Rvs167 interaction, while both S599V and S599D caused defects in vesicle scission, as shown by both live-cell imaging and electron microscopy of endocytic invaginations. Our data support a model in which phosphorylation and dephosphorylation of Vps1 promote distinct interactions and highlight the importance of such regulatory events in facilitating sequential progression of the endocytic process. PMID:26711254

  3. Preparation of a low-density species of endocytic vesicle containing immunoglobulin A.

    PubMed Central

    Mullock, B M; Luzio, J P; Hinton, R H

    1983-01-01

    Immunoglobulin A is transported across hepatocytes in specialized vesicles. A population of endocytic vesicles of approx. 140 nm diameter, containing immunoglobulin A, has now been separated from all other major cytoplasmic organelles, including plasma membrane and lysosomes, by sequential centrifugation on Ficoll/sucrose and Metrizamide gradients. Images Fig. 2. PMID:6626159

  4. Fluorescence-Lifetime Imaging Microscopy for Visualization of Quantum Dots’ Endocytic Pathway

    PubMed Central

    Damalakiene, Leona; Karabanovas, Vitalijus; Bagdonas, Saulius; Rotomskis, Ricardas

    2016-01-01

    Accumulation of carboxylated polyethylene glycol (PEG) CdSe/ZnSquantum dots (QDs) has been monitored in living fibroblasts using confocal microscopy for fluorescence intensity and fluorescence-lifetime imaging (FLIM). The wide range of mean photoluminescence (PL) lifetime values was observed for the intracellular QDs in different intracellular microenvironment, which revealed structural heterogeneity of endosomes and enabled the distinguishing among endosomes of different maturity.

  5. Activity-dependent BDNF release via endocytic pathways is regulated by synaptotagmin-6 and complexin

    PubMed Central

    Wong, Yu-Hui; Lee, Chia-Ming; Xie, Wenjun; Cui, Bianxiao; Poo, Mu-ming

    2015-01-01

    Brain-derived neurotrophic factor (BDNF) is known to modulate synapse development and plasticity, but the source of synaptic BDNF and molecular mechanisms regulating BDNF release remain unclear. Using exogenous BDNF tagged with quantum dots (BDNF-QDs), we found that endocytosed BDNF-QDs were preferentially localized to postsynaptic sites in the dendrite of cultured hippocampal neurons. Repetitive neuronal spiking induced the release of BDNF-QDs at these sites, and this process required activation of glutamate receptors. Down-regulating complexin 1/2 (Cpx1/2) expression eliminated activity-induced BDNF-QD secretion, although the overall activity-independent secretion was elevated. Among eight synaptotagmin (Syt) isoforms examined, down-regulation of only Syt6 impaired activity-induced BDNF-QD secretion. In contrast, activity-induced release of endogenously synthesized BDNF did not depend on Syt6. Thus, neuronal activity could trigger the release of endosomal BDNF from postsynaptic dendrites in a Cpx- and Syt6-dependent manner, and endosomes containing BDNF may serve as a source of BDNF for activity-dependent synaptic modulation. PMID:26216953

  6. Activity-dependent BDNF release via endocytic pathways is regulated by synaptotagmin-6 and complexin.

    PubMed

    Wong, Yu-Hui; Lee, Chia-Ming; Xie, Wenjun; Cui, Bianxiao; Poo, Mu-ming

    2015-08-11

    Brain-derived neurotrophic factor (BDNF) is known to modulate synapse development and plasticity, but the source of synaptic BDNF and molecular mechanisms regulating BDNF release remain unclear. Using exogenous BDNF tagged with quantum dots (BDNF-QDs), we found that endocytosed BDNF-QDs were preferentially localized to postsynaptic sites in the dendrite of cultured hippocampal neurons. Repetitive neuronal spiking induced the release of BDNF-QDs at these sites, and this process required activation of glutamate receptors. Down-regulating complexin 1/2 (Cpx1/2) expression eliminated activity-induced BDNF-QD secretion, although the overall activity-independent secretion was elevated. Among eight synaptotagmin (Syt) isoforms examined, down-regulation of only Syt6 impaired activity-induced BDNF-QD secretion. In contrast, activity-induced release of endogenously synthesized BDNF did not depend on Syt6. Thus, neuronal activity could trigger the release of endosomal BDNF from postsynaptic dendrites in a Cpx- and Syt6-dependent manner, and endosomes containing BDNF may serve as a source of BDNF for activity-dependent synaptic modulation. PMID:26216953

  7. Simultaneous pH measurement in endocytic and cytosolic compartments in living cells using confocal microscopy.

    PubMed

    Lucien, Fabrice; Harper, Kelly; Pelletier, Pierre-Paul; Volkov, Leonid; Dubois, Claire M

    2014-01-01

    Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported(1). Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases(2). Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells(3). This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis(3,4). Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can

  8. Vibrio vulnificus VvpE Stimulates IL-1β Production by the Hypomethylation of the IL-1β Promoter and NF-κB Activation via Lipid Raft-Dependent ANXA2 Recruitment and Reactive Oxygen Species Signaling in Intestinal Epithelial Cells.

    PubMed

    Lee, Sei-Jung; Jung, Young Hyun; Song, Eun Ju; Jang, Kyung Ku; Choi, Sang Ho; Han, Ho Jae

    2015-09-01

    An inflammatory response is a hallmark of necrosis evoked by bacterial pathogens. Vibrio vulnificus, VvpE, is an elastase that is responsible for tissue necrosis and inflammation; however, the molecular mechanism by which it regulates host cell death has not been characterized. In the present study, we investigate the cellular mechanism of VvpE with regard to host cell death and the inflammatory response of human intestinal epithelial (INT-407) cells. The recombinant protein (r)VvpE (50 pg/ml) caused cytotoxicity mainly via necrosis coupled with IL-1β production. The necrotic cell death induced by rVvpE is highly susceptible to the knockdown of annexin A (ANXA)2 and the sequestration of membrane cholesterol. We found that rVvpE induces the recruitment of NADPH oxidase 2 and neutrophil cytosolic factor 1 into membrane lipid rafts coupled with ANXA2 to facilitate the production of reactive oxygen species (ROS). The bacterial signaling of rVvpE through ROS production is uniquely mediated by the phosphorylation of redox-sensitive transcription factor NF-κB. The silencing of NF-κB inhibited IL-1β production during necrosis. rVvpE induced hypomethylation and region-specific transcriptional occupancy by NF-κB in the IL-1β promoter and has the ability to induce pyroptosis via NOD-, LRR-, and pyrin domain-containing 3 inflammasome. In a mouse model of V. vulnificus infection, the mutation of the vvpE gene from V. vulnificus negated the proinflammatory responses and maintained the physiological levels of the proliferation and migration of enterocytes. These results demonstrate that VvpE induces the hypomethylation of the IL-1β promoter and the transcriptional regulation of NF-κB through lipid raft-dependent ANXA2 recruitment and ROS signaling to promote IL-1β production in intestinal epithelial cells. PMID:26223656

  9. The Cell Fate Determinant Numb Interacts with EHD/Rme-1 Family Proteins and Has a Role in Endocytic Recycling

    PubMed Central

    Smith, Christian A.; Dho, Sascha E.; Donaldson, Julie; Tepass, Ulrich; McGlade, C. Jane

    2004-01-01

    The adaptor protein Numb is necessary for the cell fate specification of progenitor cells in the Drosophila nervous system. Numb is evolutionarily conserved and previous studies have provided evidence for a similar functional role during mammalian development. The Numb protein has multiple protein-protein interaction regions including a phosphotyrosine binding (PTB) domain and a carboxy-terminal domain that contains conserved interaction motifs including an EH (Eps15 Homology) domain binding motif and α-adaptin binding site. In this study we identify the EHD/Rme-1/Pincher family of endocytic proteins as Numb interacting partners in mammals and Drosophila. The EHD/Rme-1 proteins function in recycling of plasma membrane receptors internalized by both clathrin-mediated endocytosis and a clathrin-independent pathway regulated by ADP ribosylation factor 6 (Arf6). Here we report that Numb colocalizes with endogenous EHD4/Pincher and Arf6 and that Arf6 mutants alter Numb subcellular localization. In addition, we present evidence that Numb has a novel function in endosomal recycling and intracellular trafficking of receptors. PMID:15155807

  10. Identification of a New Exo-Endocytic Mechanism Triggered by Corticotropin-Releasing Hormone in Mast Cells.

    PubMed

    Balseiro-Gomez, Santiago; Flores, Juan A; Acosta, Jorge; Ramirez-Ponce, M Pilar; Ales, Eva

    2015-09-01

    The key role of mast cells (MC), either in development of inflammatory pathologies or in response to environmental stress, has been widely reported in recent years. Previous studies have described the effects of corticotropin-releasing hormone (CRH), which is released from inflamed tissues by cellular stress signals, on MC degranulation, a process possibly driven by selective secretion of mediators (piecemeal degranulation). In this study, we introduce a novel granular exo-endocytic pathway induced by CRH on peritoneal MC. We found that CRH triggers substantial exocytosis, which is even stronger than that induced by Ag stimulation and is characterized by large quantal size release events. Membrane fluorescence increases during stimulation in the presence of FM1-43 dye, corroborating the strength of this exocytosis, given that discrete upward fluorescence steps are often observed and suggesting that secretory granules are preferentially released by compound exocytosis. Additionally, the presence of a depot of large tubular organelles in the cytoplasm suggests that the exocytotic process is tightly coupled to a fast compound endocytosis. This CRH-stimulated mechanism is mediated through activation of adenylate cyclase and an increase of cAMP and intracellular Ca(2+), as evidenced by the fact that the effect of CRH is mimicked by forskolin and 8-bromo-cAMP. Thus, these outcomes constitute new evidence for the critical role of MC in pathophysiological conditions within a cellular stress environment and an alternative membrane trafficking route mediated by CRH. PMID:26202981

  11. The single dynamin family protein in the primitive protozoan Giardia lamblia is essential for stage conversion and endocytic transport.

    PubMed

    Gaechter, Verena; Schraner, Elisabeth; Wild, Peter; Hehl, Adrian B

    2008-01-01

    Dynamins are universally conserved large guanosine triphosphatases, which function as mechanoenzymes in membrane scission. The primitive protozoan Giardia lamblia has a single dynamin-related protein (GlDRP) with an unusual domain structure. Giardia lacks a Golgi apparatus but generates transient Golgi-like delay compartments dubbed encystation-specific vesicles (ESVs), which serve to accumulate and mature cyst wall proteins during differentiation to infectious cyst forms. Here, we analyze the function of GlDRP during growth and encystation and demonstrate that it relocalizes from peripheral endosomal-lysosomal compartments to nascent ESVs. We show that GlDRP is necessary for secretion of the cyst wall material and ESV homeostasis. Expression of a dominant-negative GlDRP variant does not interfere with ESV formation but blocks cyst formation completely prior to regulated exocytosis. GlDRP colocalizes with clathrin at the cell periphery and is necessary for endocytosis of surface proteins to endosomal-lysosomal organelles in trophozoites. Electron microscopy and live cell imaging reveal gross morphological changes as well as functional impairment of the endocytic system in cells expressing the dominant-negative GlDRP. Thus, giardial DRP plays a key role in two distinct trafficking pathways and in organelle homeostasis, both essential functions for the proliferation of the parasite in the gut and its transmission to a new host. PMID:17892527

  12. Adapting for endocytosis: roles for endocytic sorting adaptors in directing neural development

    PubMed Central

    Yap, Chan Choo; Winckler, Bettina

    2015-01-01

    Proper cortical development depends on the orchestrated actions of a multitude of guidance receptors and adhesion molecules and their downstream signaling. The levels of these receptors on the surface and their precise locations can greatly affect guidance outcomes. Trafficking of receptors to a particular surface locale and removal by endocytosis thus feed crucially into the final guidance outcomes. In addition, endocytosis of receptors can affect downstream signaling (both quantitatively and qualitatively) and regulated endocytosis of guidance receptors is thus an important component of ensuring proper neural development. We will discuss the cell biology of regulated endocytosis and the impact on neural development. We focus our discussion on endocytic accessory proteins (EAPs) (such as numb and disabled) and how they regulate endocytosis and subsequent post-endocytic trafficking of their cognate receptors (such as Notch, TrkB, β-APP, VLDLR, and ApoER2). PMID:25904845

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

    PubMed

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

    2015-09-01

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

  14. Inactivation of Caenorhabditis elegans aminopeptidase DNPP-1 restores endocytic sorting and recycling in tat-1 mutants

    PubMed Central

    Li, Xin; Chen, Baohui; Yoshina, Sawako; Cai, Tanxi; Yang, Fuquan; Mitani, Shohei; Wang, Xiaochen

    2013-01-01

    In Caenorhabditis elegans, the P4-ATPase TAT-1 and its chaperone, the Cdc50 family protein CHAT-1, maintain membrane phosphatidylserine (PS) asymmetry, which is required for membrane tubulation during endocytic sorting and recycling. Loss of tat-1 and chat-1 disrupts endocytic sorting, leading to defects in both cargo recycling and degradation. In this study, we identified the C. elegans aspartyl aminopeptidase DNPP-1, loss of which suppresses the sorting and recycling defects in tat-1 mutants without reversing the PS asymmetry defect. We found that tubular membrane structures containing recycling cargoes were restored in dnpp-1 tat-1 double mutants and that these tubules overlap with RME-1–positive recycling endosomes. The restoration of the tubular structures in dnpp-1 tat-1 mutants requires normal functions of RAB-5, RAB-10, and RME-1. In tat-1 mutants, we observed alterations in membrane surface charge and targeting of positively charged proteins that were reversed by loss of dnpp-1. DNPP-1 displays a specific aspartyl aminopeptidase activity in vitro, and its enzymatic activity is required for its function in vivo. Our data reveal the involvement of an aminopeptidase in regulating endocytic sorting and recycling and suggest possible roles of peptide signaling and/or protein metabolism in these processes. PMID:23427264

  15. A role of the sulfonylurea receptor 1 in endocytic trafficking of ATP-sensitive potassium channels

    PubMed Central

    Bruederle, Cathrin E.; Gay, Joel; Shyng, Show-Ling

    2011-01-01

    The ATP-sensitive potassium (KATP) channel consisting of sulfonylurea receptor 1 (SUR1) and inward rectifier potassium channel 6.2 (Kir6.2) has a well-established role in insulin secretion. Mutations in either subunit can lead to disease due to aberrant channel gating, altered channel density at the cell surface or a combination of both. Endocytic trafficking of channels at the plasma membrane is one way to influence surface channel numbers. It has been previously reported that channel endocytosis is dependent on a tyrosine-based motif in Kir6.2 while SUR1 alone is unable to internalize. In this study, we followed endocytic trafficking of surface channels in real time by live cell imaging of channel subunits tagged with an extracellular minimal α-bungarotoxin binding peptide labeled with a fluorescent dye. We demonstrate that SUR1 undergoes endocytosis independent of Kir6.2. Moreover, mutations in the putative endocytosis motif of Kir6.2, Y330C, Y330A and F333I are unable to prevent channel endocytosis. These findings challenge the notion that Kir6.2 bears the sole endocytic signal for KATP channels and support a role of SUR1 in this trafficking process. PMID:21649805

  16. Annexin A2 facilitates endocytic trafficking of antisense oligonucleotides

    PubMed Central

    Wang, Shiyu; Sun, Hong; Tanowitz, Michael; Liang, Xue-hai; Crooke, Stanley T.

    2016-01-01

    Chemically modified antisense oligonucleotides (ASOs) designed to mediate site-specific cleavage of RNA by RNase H1 are used as research tools and as therapeutics. ASOs modified with phosphorothioate (PS) linkages enter cells via endocytotic pathways. The mechanisms by which PS-ASOs are released from membrane-enclosed endocytotic organelles to reach target RNAs remain largely unknown. We recently found that annexin A2 (ANXA2) co-localizes with PS-ASOs in late endosomes (LEs) and enhances ASO activity. Here, we show that co-localization of ANXA2 with PS-ASO is not dependent on their direct interactions or mediated by ANXA2 partner protein S100A10. Instead, ANXA2 accompanies the transport of PS-ASOs to LEs, as ANXA2/PS-ASO co-localization was observed inside LEs. Although ANXA2 appears not to affect levels of PS-ASO internalization, ANXA2 reduction caused significant accumulation of ASOs in early endosomes (EEs) and reduced localization in LEs and decreased PS-ASO activity. Importantly, the kinetics of PS-ASO activity upon free uptake show that target mRNA reduction occurs at least 4 hrs after PS-ASOs exit from EEs and is coincident with release from LEs. Taken together, our results indicate that ANXA2 facilitates PS-ASO trafficking from early to late endosomes where it may also contribute to PS-ASO release. PMID:27378781

  17. Endosome-mediated endocytic mechanism replenishes the majority of synaptic vesicles at mature CNS synapses in an activity-dependent manner

    PubMed Central

    Park, Joohyun; Cho, Oh Yeon; Kim, Jung Ah; Chang, Sunghoe

    2016-01-01

    Whether synaptic vesicles (SVs) are recovered via endosome-mediated pathways is a matter of debate; however, recent evidence suggests that clathrin-independent bulk endocytosis (CIE) via endosomes is functional and preferentially replenishes SV pools during strong stimulation. Here, using brefeldin-A (BFA) to block CIE, we found that CIE retrieved a minority of SVs at developing CNS synapses during strong stimulation, but its contribution increased up to 61% at mature CNS synapses. Contrary to previous views, BFA not only blocked SV formation from the endosome but also blocked the endosome formation at the plasma membrane. Adaptor protein 1 and 3 (AP-1/3) have key roles in SV reformation from endosomes during CIE, and AP-1 also affects bulk endosome formation from the plasma membrane. Finally, temporary blocking of chronic or acute neuronal activity with tetrodotoxin in mature neurons redirected most SV retrieval to endosome-independent pathways. These results show that during high neuronal activity, CIE becomes the major endocytic pathway at mature CNS synapses. Moreover, mature neurons use clathrin-mediated endocytosis and the CIE pathway to different extents depending on their previous activity; this may result in activity-dependent alterations of the SV composition which ultimately influence transmitter release and contribute to synaptic plasticity. PMID:27534442

  18. Endosome-mediated endocytic mechanism replenishes the majority of synaptic vesicles at mature CNS synapses in an activity-dependent manner.

    PubMed

    Park, Joohyun; Cho, Oh Yeon; Kim, Jung Ah; Chang, Sunghoe

    2016-01-01

    Whether synaptic vesicles (SVs) are recovered via endosome-mediated pathways is a matter of debate; however, recent evidence suggests that clathrin-independent bulk endocytosis (CIE) via endosomes is functional and preferentially replenishes SV pools during strong stimulation. Here, using brefeldin-A (BFA) to block CIE, we found that CIE retrieved a minority of SVs at developing CNS synapses during strong stimulation, but its contribution increased up to 61% at mature CNS synapses. Contrary to previous views, BFA not only blocked SV formation from the endosome but also blocked the endosome formation at the plasma membrane. Adaptor protein 1 and 3 (AP-1/3) have key roles in SV reformation from endosomes during CIE, and AP-1 also affects bulk endosome formation from the plasma membrane. Finally, temporary blocking of chronic or acute neuronal activity with tetrodotoxin in mature neurons redirected most SV retrieval to endosome-independent pathways. These results show that during high neuronal activity, CIE becomes the major endocytic pathway at mature CNS synapses. Moreover, mature neurons use clathrin-mediated endocytosis and the CIE pathway to different extents depending on their previous activity; this may result in activity-dependent alterations of the SV composition which ultimately influence transmitter release and contribute to synaptic plasticity. PMID:27534442

  19. Ack1 is a dopamine transporter endocytic brake that rescues a trafficking-dysregulated ADHD coding variant

    PubMed Central

    Wu, Sijia; Bellve, Karl D.; Fogarty, Kevin E.; Melikian, Haley E.

    2015-01-01

    The dopamine (DA) transporter (DAT) facilitates high-affinity presynaptic DA reuptake that temporally and spatially constrains DA neurotransmission. Aberrant DAT function is implicated in attention-deficit/hyperactivity disorder and autism spectrum disorder. DAT is a major psychostimulant target, and psychostimulant reward strictly requires binding to DAT. DAT function is acutely modulated by dynamic membrane trafficking at the presynaptic terminal and a PKC-sensitive negative endocytic mechanism, or “endocytic brake,” controls DAT plasma membrane stability. However, the molecular basis for the DAT endocytic brake is unknown, and it is unknown whether this braking mechanism is unique to DAT or common to monoamine transporters. Here, we report that the cdc42-activated, nonreceptor tyrosine kinase, Ack1, is a DAT endocytic brake that stabilizes DAT at the plasma membrane and is released in response to PKC activation. Pharmacologic and shRNA-mediated Ack1 silencing enhanced basal DAT internalization and blocked PKC-stimulated DAT internalization, but had no effects on SERT endocytosis. Both cdc42 activation and PKC stimulation converge on Ack1 to control Ack1 activity and DAT endocytic capacity, and Ack1 inactivation is required for stimulated DAT internalization downstream of PKC activation. Moreover, constitutive Ack1 activation is sufficient to rescue the gain-of-function endocytic phenotype exhibited by the ADHD DAT coding variant, R615C. These findings reveal a unique endocytic control switch that is highly specific for DAT. Moreover, the ability to rescue the DAT(R615C) coding variant suggests that manipulating DAT trafficking mechanisms may be a potential therapeutic approach to correct DAT coding variants that exhibit trafficking dysregulation. PMID:26621748

  20. ΔF508 CFTR Surface Stability Is Regulated by DAB2 and CHIP-Mediated Ubiquitination in Post-Endocytic Compartments

    PubMed Central

    Fu, Lianwu; Rab, Andras; Tang, Li ping; Bebok, Zsuzsa; Rowe, Steven M.; Bartoszewski, Rafal; Collawn, James F.

    2015-01-01

    The ΔF508 mutant form of the cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR) that is normally degraded by the ER-associated degradative pathway can be rescued to the cell surface through low-temperature (27°C) culture or small molecular corrector treatment. However, it is unstable on the cell surface, and rapidly internalized and targeted to the lysosomal compartment for degradation. To understand the mechanism of this rapid turnover, we examined the role of two adaptor complexes (AP-2 and Dab2) and three E3 ubiquitin ligases (c-Cbl, CHIP, and Nedd4-2) on low-temperature rescued ΔF508 CFTR endocytosis and degradation in human airway epithelial cells. Our results demonstrate that siRNA depletion of either AP-2 or Dab2 inhibits ΔF508 CFTR endocytosis by 69% and 83%, respectively. AP-2 or Dab2 depletion also increases the rescued protein half-life of ΔF508 CFTR by ~18% and ~91%, respectively. In contrast, the depletion of each of the E3 ligases had no effect on ΔF508 CFTR endocytosis, whereas CHIP depletion significantly increased the surface half-life of ΔF508 CFTR. To determine where and when the ubiquitination occurs during ΔF508 CFTR turnover, we monitored the ubiquitination of rescued ΔF508 CFTR during the time course of CFTR endocytosis. Our results indicate that ubiquitination of the surface pool of ΔF508 CFTR begins to increase 15 min after internalization, suggesting that CFTR is ubiquitinated in a post-endocytic compartment. This post-endocytic ubiquination of ΔF508 CFTR could be blocked by either inhibiting endocytosis, by siRNA knockdown of CHIP, or by treating cells with the CFTR corrector, VX-809. Our results indicate that the post-endocytic ubiquitination of CFTR by CHIP is a critical step in the peripheral quality control of cell surface ΔF508 CFTR. PMID:25879443

  1. ΔF508 CFTR surface stability is regulated by DAB2 and CHIP-mediated ubiquitination in post-endocytic compartments.

    PubMed

    Fu, Lianwu; Rab, Andras; Tang, Li ping; Bebok, Zsuzsa; Rowe, Steven M; Bartoszewski, Rafal; Collawn, James F

    2015-01-01

    The ΔF508 mutant form of the cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR) that is normally degraded by the ER-associated degradative pathway can be rescued to the cell surface through low-temperature (27°C) culture or small molecular corrector treatment. However, it is unstable on the cell surface, and rapidly internalized and targeted to the lysosomal compartment for degradation. To understand the mechanism of this rapid turnover, we examined the role of two adaptor complexes (AP-2 and Dab2) and three E3 ubiquitin ligases (c-Cbl, CHIP, and Nedd4-2) on low-temperature rescued ΔF508 CFTR endocytosis and degradation in human airway epithelial cells. Our results demonstrate that siRNA depletion of either AP-2 or Dab2 inhibits ΔF508 CFTR endocytosis by 69% and 83%, respectively. AP-2 or Dab2 depletion also increases the rescued protein half-life of ΔF508 CFTR by ~18% and ~91%, respectively. In contrast, the depletion of each of the E3 ligases had no effect on ΔF508 CFTR endocytosis, whereas CHIP depletion significantly increased the surface half-life of ΔF508 CFTR. To determine where and when the ubiquitination occurs during ΔF508 CFTR turnover, we monitored the ubiquitination of rescued ΔF508 CFTR during the time course of CFTR endocytosis. Our results indicate that ubiquitination of the surface pool of ΔF508 CFTR begins to increase 15 min after internalization, suggesting that CFTR is ubiquitinated in a post-endocytic compartment. This post-endocytic ubiquination of ΔF508 CFTR could be blocked by either inhibiting endocytosis, by siRNA knockdown of CHIP, or by treating cells with the CFTR corrector, VX-809. Our results indicate that the post-endocytic ubiquitination of CFTR by CHIP is a critical step in the peripheral quality control of cell surface ΔF508 CFTR. PMID:25879443

  2. Endocytic deficiency induced by ITSN-1s knockdown alters the Smad2/3-Erk1/2 signaling balance downstream of Alk5

    PubMed Central

    Bardita, Cristina; Predescu, Dan N.; Sha, Fei; Patel, Monal; Balaji, Ganesh; Predescu, Sanda A.

    2015-01-01

    Recently, we demonstrated in cultured endothelial cells and in vivo that deficiency of an isoform of intersectin-1, ITSN-1s, impairs caveolae and clathrin-mediated endocytosis and functionally upregulates compensatory pathways and their morphological carriers (i.e. enlarged endocytic structures, membranous rings or tubules) that are normally underrepresented. We now show that these endocytic structures internalize the broadly expressed transforming growth factor β receptor I (TGFβ-RI or TGFBR1), also known as Alk5, leading to its ubiquitylation and degradation. Moreover, the apoptotic or activated vascular cells of the ITSN-1s-knockdown mice release Alk5-bearing microparticles to the systemic circulation. These interact with and transfer Alk5 to endocytosis-deficient endothelial cells, resulting in lung endothelial cell survival and phenotypic alteration towards proliferation through activation of Erk1 and Erk2 (also known as MAPK3 and MAPK1, respectively). We also show that non-productive assembly of the Alk5–Smad–SARA (Smad anchor for receptor activation, also known as ZFYVE9) signaling complex and preferential formation of the Alk5–mSos–Grb2 complex account for Erk1/2 activation downstream of Alk5 and proliferation of pulmonary endothelial cells. Taken together, our studies demonstrate a functional relationship between the intercellular transfer of Alk5 by microparticles and endothelial cell survival and proliferation, and define a novel molecular mechanism for TGFβ and Alk5-dependent Erk1/2MAPK signaling that is significant for proliferative signaling and abnormal growth. PMID:25720380

  3. Endocytic deficiency induced by ITSN-1s knockdown alters the Smad2/3-Erk1/2 signaling balance downstream of Alk5.

    PubMed

    Bardita, Cristina; Predescu, Dan N; Sha, Fei; Patel, Monal; Balaji, Ganesh; Predescu, Sanda A

    2015-04-15

    Recently, we demonstrated in cultured endothelial cells and in vivo that deficiency of an isoform of intersectin-1, ITSN-1s, impairs caveolae and clathrin-mediated endocytosis and functionally upregulates compensatory pathways and their morphological carriers (i.e. enlarged endocytic structures, membranous rings or tubules) that are normally underrepresented. We now show that these endocytic structures internalize the broadly expressed transforming growth factor β receptor I (TGFβ-RI or TGFBR1), also known as Alk5, leading to its ubiquitylation and degradation. Moreover, the apoptotic or activated vascular cells of the ITSN-1s-knockdown mice release Alk5-bearing microparticles to the systemic circulation. These interact with and transfer Alk5 to endocytosis-deficient endothelial cells, resulting in lung endothelial cell survival and phenotypic alteration towards proliferation through activation of Erk1 and Erk2 (also known as MAPK3 and MAPK1, respectively). We also show that non-productive assembly of the Alk5-Smad-SARA (Smad anchor for receptor activation, also known as ZFYVE9) signaling complex and preferential formation of the Alk5-mSos-Grb2 complex account for Erk1/2 activation downstream of Alk5 and proliferation of pulmonary endothelial cells. Taken together, our studies demonstrate a functional relationship between the intercellular transfer of Alk5 by microparticles and endothelial cell survival and proliferation, and define a novel molecular mechanism for TGFβ and Alk5-dependent Erk1/2(MAPK) signaling that is significant for proliferative signaling and abnormal growth. PMID:25720380

  4. Localization and role of MYO-1, an endocytic protein in hyphae of Neurospora crassa.

    PubMed

    Lara-Rojas, Fernando; Bartnicki-García, Salomón; Mouriño-Pérez, Rosa R

    2016-03-01

    The subapical endocytic collar is a prominent feature of hyphae of Neurospora crassa. It comprises a dynamic collection of actin patches associated with a number of proteins required for endocytosis, namely, ARP-2/3 complex, fimbrin, coronin, etc. We presently show that MYO-1 is another key component of this endocytic collar. A myo-1 sequence was identified in the genome of N. crassa and used it to generate a strain with a myo-1-sgfp allele under the ccg1 promoter. Examination of living hyphae by confocal microscopy, revealed MYO-1-GFP located mainly as a dynamic collection of small patches arranged in collar-like fashion in the hyphal subapex. Dual tagging showed MYO-1-GFP partially colocalized with two other endocytic proteins, fimbrin and coronin. MYO-1 was also present during septum formation. By recovering a viable strain, albeit severely inhibited, after deletion of myo-1, it was possible to investigate the phenotypic consequences of the elimination of MYO-1. Deletion of myo-1 caused a severe reduction in growth rate (95%), near absence of aerial mycelium and no conidiation. A reduced uptake of the lipophilic dye FM4-64 indicated a deficiency in endocytosis in the Δmyo-1 mutant. Hyphae were produced by the Δmyo-1 mutant but their morphogenesis was severely affected; hyphal morphology was distorted displaying irregular periods of isotropic and polarized growth. The morphological alterations were accompanied, and presumably caused, by a disruption in the organization and dynamics of a myosin-deprived actin cytoskeleton that, ultimately, compromised the stability and function of the Spitzenkörper as a vesicle supply center. PMID:26805950

  5. Modes and Regulation of Endocytic Membrane Retrieval in Mouse Auditory Hair Cells

    PubMed Central

    Jung, SangYong; Wong, Aaron B.; Reuter, Kirsten; Pangršič, Tina; Chakrabarti, Rituparna; Kügler, Sebastian; Lenz, Christine; Nouvian, Régis; Boumil, Rebecca M.; Frankel, Wayne N.; Wichmann, Carolin

    2014-01-01

    Synaptic vesicle recycling sustains high rates of neurotransmission at the ribbon-type active zones (AZs) of mouse auditory inner hair cells (IHCs), but its modes and molecular regulation are poorly understood. Electron microscopy indicated the presence of clathrin-mediated endocytosis (CME) and bulk endocytosis. The endocytic proteins dynamin, clathrin, and amphiphysin are expressed and broadly distributed in IHCs. We used confocal vglut1–pHluorin imaging and membrane capacitance (Cm) measurements to study the spatial organization and dynamics of IHC exocytosis and endocytosis. Viral gene transfer expressed vglut1–pHluorin in IHCs and targeted it to synaptic vesicles. The intravesicular pH was ∼6.5, supporting only a modest increase of vglut1–pHluorin fluorescence during exocytosis and pH neutralization. Ca2+ influx triggered an exocytic increase of vglut1–pHluorin fluorescence at the AZs, around which it remained for several seconds. The endocytic Cm decline proceeded with constant rate (linear component) after exocytosis of the readily releasable pool (RRP). When exocytosis exceeded three to four RRP equivalents, IHCs additionally recruited a faster Cm decline (exponential component) that increased with the amount of preceding exocytosis and likely reflects bulk endocytosis. The dynamin inhibitor Dyngo-4a and the clathrin blocker pitstop 2 selectively impaired the linear component of endocytic Cm decline. A missense mutation of dynamin 1 (fitful) inhibited endocytosis to a similar extent as Dyngo-4a. We propose that IHCs use dynamin-dependent endocytosis via CME to support vesicle cycling during mild stimulation but recruit bulk endocytosis to balance massive exocytosis. PMID:24431429

  6. CD163-L1 is an endocytic macrophage protein strongly regulated by mediators in the inflammatory response.

    PubMed

    Moeller, Jesper B; Nielsen, Marianne J; Reichhardt, Martin P; Schlosser, Anders; Sorensen, Grith L; Nielsen, Ole; Tornøe, Ida; Grønlund, Jørn; Nielsen, Maria E; Jørgensen, Jan S; Jensen, Ole N; Mollenhauer, Jan; Moestrup, Søren K; Holmskov, Uffe

    2012-03-01

    CD163-L1 belongs to the group B scavenger receptor cysteine-rich family of proteins, where the CD163-L1 gene arose by duplication of the gene encoding the hemoglobin scavenger receptor CD163 in late evolution. The current data demonstrate that CD163-L1 is highly expressed and colocalizes with CD163 on large subsets of macrophages, but in contrast to CD163 the expression is low or absent in monocytes and in alveolar macrophages, glia, and Kupffer cells. The expression of CD163-L1 increases when cultured monocytes are M-CSF stimulated to macrophages, and the expression is further increased by the acute-phase mediator IL-6 and the anti-inflammatory mediator IL-10 but is suppressed by the proinflammatory mediators IL-4, IL-13, TNF-α, and LPS/IFN-γ. Furthermore, we show that CD163-L1 is an endocytic receptor, which internalizes independently of cross-linking through a clathrin-mediated pathway. Two cytoplasmic splice variants of CD163-L1 are differentially expressed and have different subcellular distribution patterns. Despite its many similarities to CD163, CD163-L1 does not possess measurable affinity for CD163 ligands such as the haptoglobin-hemoglobin complex or various bacteria. In conclusion, CD163-L1 exhibits similarity to CD163 in terms of structure and regulated expression in cultured monocytes but shows clear differences compared with the known CD163 ligand preferences and expression pattern in the pool of tissue macrophages. We postulate that CD163-L1 functions as a scavenger receptor for one or several ligands that might have a role in resolution of inflammation. PMID:22279103

  7. AMPH-1/Amphiphysin/Bin1 functions with RME-1/Ehd in endocytic recycling

    PubMed Central

    Pant, Saumya; Sharma, Mahak; Patel, Kruti; Caplan, Steve; Carr, Chavela M.; Grant, Barth D.

    2009-01-01

    RME-1/EHD1 family proteins are key residents of the recycling endosome required for endosome to plasma membrane transport in C. elegans and mammals. Recent studies suggest parallels of the RME-1/EHD proteins to the Dynamin GTPase superfamily of mechanochemical pinchases that promote membrane fission. Here we show that that endogenous C. elegans AMPH-1, the only C. elegans member of Amphiphysin/BIN1 family of BAR-domain proteins, colocalizes with RME-1 on recycling endosomes in vivo, that amph-1 deletion mutants are defective in recycling endosome morphology and function, and that binding of AMPH-1 NPF (D/E) sequences to the RME-1 EH-domain promotes the recycling of transmembrane cargo. We also show a requirement for human BIN1/Amphyphysin 2 in EHD1-regulated endocytic recycling. In vitro we find that the purified recombinant AMPH-1/RME-1 complexes produce short, coated, membrane tubules that are qualitatively distinct from those produced by either protein alone. Our results indicate that AMPH-1 and RME-1 cooperatively regulate endocytic recycling, likely through functions required for the production of cargo carriers exiting the recycling endosome for the cell surface. PMID:19915558

  8. Endophilin B1 regulates EGFR endocytic degradation in prostate cancer cell.

    PubMed

    Zhu, J-Y; Xiong, Y; Zhang, W; Wan, J; Wan, J

    2016-01-01

    Prostate cancer (Pca) is one of the most common types of cancer for elder men. Aberrant expression of epidermal growth factor receptor (EGFR) and EGFR downstream signaling have been known to contribute to disease progression in prostate cancer. EGF-stimulated EGFR is internalized and the process of endocytic degradation of EGFR mediates its signaling which is frequently dysregulated in many kinds of cancer. In the present study, we demonstrated that endophilin B1 expression was inhibited and EGFR expression was significantly increased in prostate cancer cell lines. We demonstrated that suppression of endophilin B1 increased EGFR levels via delaying EGFR internalization triggered by EGF and its intracellular degradation. Endophilin B1 decreased also sustained EGFR downstream signaling such as Erk1/2 phosphorylation in response to EGF stimulation and promoted prostate cancer cell proliferation which is EGF independent. Our data indicated that endophilin B1 mediated the biological function of EGFR in cancer cell proliferation through regulating the EGFR endocytic trafficking and downstream signaling. PMID:27609472

  9. The endocytic recycling compartment maintains cargo segregation acquired upon exit from the sorting endosome

    PubMed Central

    Xie, Shuwei; Bahl, Kriti; Reinecke, James B.; Hammond, Gerald R. V.; Naslavsky, Naava; Caplan, Steve

    2016-01-01

    The endocytic recycling compartment (ERC) is a series of perinuclear tubular and vesicular membranes that regulates recycling to the plasma membrane. Despite evidence that cargo is sorted at the early/sorting endosome (SE), whether cargo mixes downstream at the ERC or remains segregated is an unanswered question. Here we use three-dimensional (3D) structured illumination microscopy and dual-channel and 3D direct stochastic optical reconstruction microscopy (dSTORM) to obtain new information about ERC morphology and cargo segregation. We show that cargo internalized either via clathrin-mediated endocytosis (CME) or independently of clathrin (CIE) remains segregated in the ERC, likely on distinct carriers. This suggests that no further sorting occurs upon cargo exit from SE. Moreover, 3D dSTORM data support a model in which some but not all ERC vesicles are tethered by contiguous “membrane bridges.” Furthermore, tubular recycling endosomes preferentially traffic CIE cargo and may originate from SE membranes. These findings support a significantly altered model for endocytic recycling in mammalian cells in which sorting occurs in peripheral endosomes and segregation is maintained at the ERC. PMID:26510502

  10. Myristoyl-CoA:protein N-myristoyltransferase depletion in trypanosomes causes avirulence and endocytic defects

    PubMed Central

    Price, Helen P.; Güther, M. Lucia S.; Ferguson, Michael A.J.; Smith, Deborah F.

    2010-01-01

    The enzyme myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyses the co-translational covalent attachment of the fatty acid myristate to the N-terminus of target proteins. NMT is known to be essential for viability in Trypanosoma brucei and Leishmania major. Here we describe phenotypic analysis of T. brucei bloodstream form cells following knockdown of NMT expression by tetracycline-inducible RNA interference. Cell death occurs from 72 h post-induction, with approximately 50% of cells displaying a defect in endocytic uptake by this time. The majority of these induced cells do not have an enlarged flagellar pocket typical of a block in endocytosis but vesicle accumulation around the flagellar pocket indicates a defect in vesicular progression following endocytic fusion. Induced parasites have a wild-type or slightly enlarged Golgi apparatus, unlike the phenotype of cells with reduced expression of a major N-myristoylated protein, ARL1. Critically we show that following NMT knockdown, T. brucei bloodstream form cells are unable to establish an infection in a mouse model, therefore providing further validation of this enzyme as a target for drug development. PMID:19782106

  11. A-RAF Kinase Functions in ARF6 Regulated Endocytic Membrane Traffic

    PubMed Central

    Nekhoroshkova, Elena; Albert, Stefan; Becker, Matthias; Rapp, Ulf R.

    2009-01-01

    Background RAF kinases direct ERK MAPK signaling to distinct subcellular compartments in response to growth factor stimulation. Methodology/Principal Findings Of the three mammalian isoforms A-RAF is special in that one of its two lipid binding domains mediates a unique pattern of membrane localization. Specific membrane binding is retained by an N-terminal fragment (AR149) that corresponds to a naturally occurring splice variant termed DA-RAF2. AR149 colocalizes with ARF6 on tubular endosomes and has a dominant negative effect on endocytic trafficking. Moreover actin polymerization of yeast and mammalian cells is abolished. AR149/DA-RAF2 does not affect the internalization step of endocytosis, but trafficking to the recycling compartment. Conclusions/Significance A-RAF induced ERK activation is required for this step by activating ARF6, as A-RAF depletion or inhibition of the A-RAF controlled MEK-ERK cascade blocks recycling. These data led to a new model for A-RAF function in endocytic trafficking. PMID:19247477

  12. The Endocytic Receptor Megalin and its Associated Proteins in Proximal Tubule Epithelial Cells

    PubMed Central

    De, Shankhajit; Kuwahara, Shoji; Saito, Akihiko

    2014-01-01

    Receptor-mediated endocytosis in renal proximal tubule epithelial cells (PTECs) is important for the reabsorption and metabolization of proteins and other substances, including carrier-bound vitamins and trace elements, in glomerular filtrates. Impairment of this endocytic process results in the loss of such substances and development of proteinuria, which is an important clinical indicator of kidney diseases and is also a risk marker for cardiovascular disease. Megalin, a member of the low-density lipoprotein receptor gene family, is a multiligand receptor expressed in the apical membrane of PTECs and plays a central role in the endocytic process. Megalin interacts with various intracellular adaptor proteins for intracellular trafficking and cooperatively functions with other membrane molecules, including the cubilin-amnionless complex. Evidence suggests that megalin and the cubilin-amnionless complex are involved in the uptake of toxic substances into PTECs, which leads to the development of kidney disease. Studies of megalin and its associated molecules will be useful for future development of novel strategies for the diagnosis and treatment of kidney diseases. PMID:25019425

  13. Endocytic recycling protein EHD1 regulates primary cilia morphogenesis and SHH signaling during neural tube development

    PubMed Central

    Bhattacharyya, Sohinee; Rainey, Mark A; Arya, Priyanka; Dutta, Samikshan; George, Manju; Storck, Matthew D.; McComb, Rodney D.; Muirhead, David; Todd, Gordon L.; Gould, Karen; Datta, Kaustubh; Waes, Janee Gelineau-van; Band, Vimla; Band, Hamid

    2016-01-01

    Members of the four-member C-terminal EPS15-Homology Domain-containing (EHD) protein family play crucial roles in endocytic recycling of cell surface receptors from endosomes to the plasma membrane. In this study, we show that Ehd1 gene knockout in mice on a predominantly B6 background is embryonic lethal. Ehd1-null embryos die at mid-gestation with a failure to complete key developmental processes including neural tube closure, axial turning and patterning of the neural tube. We found that Ehd1-null embryos display short and stubby cilia on the developing neuroepithelium at embryonic day 9.5 (E9.5). Loss of EHD1 also deregulates the ciliary SHH signaling with Ehd1-null embryos displaying features indicative of increased SHH signaling, including a significant downregulation in the formation of the GLI3 repressor and increase in the ventral neuronal markers specified by SHH. Using Ehd1-null MEFS we found that EHD1 protein co-localizes with the SHH receptor Smoothened in the primary cilia upon ligand stimulation. Under the same conditions, EHD1 was shown to co-traffic with Smoothened into the developing primary cilia and we identify EHD1 as a direct binding partner of Smoothened. Overall, our studies identify the endocytic recycling regulator EHD1 as a novel regulator of the primary cilium-associated trafficking of Smoothened and Hedgehog signaling. PMID:26884322

  14. Residues in the hendra virus fusion protein transmembrane domain are critical for endocytic recycling.

    PubMed

    Popa, Andreea; Carter, James R; Smith, Stacy E; Hellman, Lance; Fried, Michael G; Dutch, Rebecca Ellis

    2012-03-01

    Hendra virus is a highly pathogenic paramyxovirus classified as a biosafety level four agent. The fusion (F) protein of Hendra virus is critical for promoting viral entry and cell-to-cell fusion. To be fusogenically active, Hendra virus F must undergo endocytic recycling and cleavage by the endosomal/lysosomal protease cathepsin L, but the route of Hendra virus F following internalization and the recycling signals involved are poorly understood. We examined the intracellular distribution of Hendra virus F following endocytosis and showed that it is primarily present in Rab5- and Rab4-positive endosomal compartments, suggesting that cathepsin L cleavage occurs in early endosomes. Hendra virus F transmembrane domain (TMD) residues S490 and Y498 were found to be important for correct Hendra virus F recycling, with the hydroxyl group of S490 and the aromatic ring of Y498 important for this process. In addition, changes in association of isolated Hendra virus F TMDs correlated with alterations to Hendra virus F recycling, suggesting that appropriate TMD interactions play an important role in endocytic trafficking. PMID:22238299

  15. Constitutive activated Cdc42-associated kinase (Ack) phosphorylation at arrested endocytic clathrin-coated pits of cells that lack dynamin

    PubMed Central

    Shen, Hongying; Ferguson, Shawn M.; Dephoure, Noah; Park, Ryan; Yang, Yan; Volpicelli-Daley, Laura; Gygi, Steven; Schlessinger, Joseph; De Camilli, Pietro

    2011-01-01

    Clathrin-mediated endocytosis is a fundamental cellular process conserved from yeast to mammals and is an important endocytic route for the internalization of many specific cargos, including activated growth factor receptors. Here we examined changes in tyrosine phosphorylation, a representative output of growth factor receptor signaling, in cells in which endocytic clathrin-coated pits are frozen at a deeply invaginated state, that is, cells that lack dynamin (fibroblasts from dynamin 1, dynamin 2 double conditional knockout mice). The major change observed in these cells relative to wild-type cells was an increase in the phosphorylation state, and thus activation, of activated Cdc42-associated kinase (Ack), a nonreceptor tyrosine kinase. Ack is concentrated at clathrin-coated pits, and binds clathrin heavy chain via two clathrin boxes. RNA interference–based approaches and pharmacological manipulations further demonstrated that the phosphorylation of Ack requires both clathrin assembly into endocytic clathrin-coated pits and active Cdc42. These findings reveal a link between progression of clathrin-coated pits to endocytic vesicles and an activation–deactivation cycle of Ack. PMID:21169560

  16. A SPOPL/Cullin-3 ubiquitin ligase complex regulates endocytic trafficking by targeting EPS15 at endosomes

    PubMed Central

    Gschweitl, Michaela; Ulbricht, Anna; Barnes, Christopher A; Enchev, Radoslav I; Stoffel-Studer, Ingrid; Meyer-Schaller, Nathalie; Huotari, Jatta; Yamauchi, Yohei; Greber, Urs F; Helenius, Ari; Peter, Matthias

    2016-01-01

    Cullin-3 (CUL3)-based ubiquitin ligases regulate endosome maturation and trafficking of endocytic cargo to lysosomes in mammalian cells. Here, we report that these functions depend on SPOPL, a substrate-specific CUL3 adaptor. We find that SPOPL associates with endosomes and is required for both the formation of multivesicular bodies (MVBs) and the endocytic host cell entry of influenza A virus. In SPOPL-depleted cells, endosomes are enlarged and fail to acquire intraluminal vesicles (ILVs). We identify a critical substrate ubiquitinated by CUL3-SPOPL as EPS15, an endocytic adaptor that also associates with the ESCRT-0 complex members HRS and STAM on endosomes. Indeed, EPS15 is ubiquitinated in a SPOPL-dependent manner, and accumulates with HRS in cells lacking SPOPL. Together, our data indicates that a CUL3-SPOPL E3 ubiquitin ligase complex regulates endocytic trafficking and MVB formation by ubiquitinating and degrading EPS15 at endosomes, thereby influencing influenza A virus infection as well as degradation of EGFR and other EPS15 targets. DOI: http://dx.doi.org/10.7554/eLife.13841.001 PMID:27008177

  17. A SPOPL/Cullin-3 ubiquitin ligase complex regulates endocytic trafficking by targeting EPS15 at endosomes.

    PubMed

    Gschweitl, Michaela; Ulbricht, Anna; Barnes, Christopher A; Enchev, Radoslav I; Stoffel-Studer, Ingrid; Meyer-Schaller, Nathalie; Huotari, Jatta; Yamauchi, Yohei; Greber, Urs F; Helenius, Ari; Peter, Matthias

    2016-01-01

    Cullin-3 (CUL3)-based ubiquitin ligases regulate endosome maturation and trafficking of endocytic cargo to lysosomes in mammalian cells. Here, we report that these functions depend on SPOPL, a substrate-specific CUL3 adaptor. We find that SPOPL associates with endosomes and is required for both the formation of multivesicular bodies (MVBs) and the endocytic host cell entry of influenza A virus. In SPOPL-depleted cells, endosomes are enlarged and fail to acquire intraluminal vesicles (ILVs). We identify a critical substrate ubiquitinated by CUL3-SPOPL as EPS15, an endocytic adaptor that also associates with the ESCRT-0 complex members HRS and STAM on endosomes. Indeed, EPS15 is ubiquitinated in a SPOPL-dependent manner, and accumulates with HRS in cells lacking SPOPL. Together, our data indicates that a CUL3-SPOPL E3 ubiquitin ligase complex regulates endocytic trafficking and MVB formation by ubiquitinating and degrading EPS15 at endosomes, thereby influencing influenza A virus infection as well as degradation of EGFR and other EPS15 targets. PMID:27008177

  18. The relationship between lumenal and limiting membranes in swollen late endocytic compartments formed after wortmannin treatment or sucrose accumulation.

    PubMed

    Bright, N A; Lindsay, M R; Stewart, A; Luzio, J P

    2001-09-01

    Immunofluorescence and electron microscopy were used to evaluate the formation of swollen endosomes in NRK cells after treatment with wortmannin or sucrose and to study the relationship between lumenal and limiting membrane. Both treatments resulted in the formation of two populations of swollen late endocytic vacuoles, positive for lysosomal glycoproteins or cation-independent mannose 6-phosphate receptors, but those induced by wortmannin were characterised by time-dependent accumulation of lumenal vesicles, whereas those induced by sucrose uptake did not accumulate lumenal vesicles. In both cases, the distribution of the late endosomal marker, lysobisphosphatidic acid, remained unchanged and was present within the lumen of the swollen vacuoles. Consumption of plasma membrane and peripheral early endosomes, and the appearance of transferrin receptors in swollen late endosomes, indicated that continued membrane influx from early endocytic compartments, together with inhibition of membrane traffic out of the swollen compartments, is sufficient to account for the observed phenotype of cells treated with wortmannin. The accumulation of organelles with the characteristic morphology of endocytic carrier vesicles in cells that have taken up sucrose offers an explanation for the paucity of lumenal vesicles in swollen sucrosomes. Our data suggest that in fibroblast cells the swollen endosome phenotype induced by wortmannin is a consequence of endocytic membrane influx, coupled with the failure to recycle membrane to other cellular destinations, and not the inhibition of multivesicular body biogenesis. PMID:11555417

  19. Compensatory Flux Changes within an Endocytic Trafficking Network Maintain Thermal Robustness of Notch Signaling

    PubMed Central

    Shimizu, Hideyuki; Woodcock, Simon A.; Wilkin, Marian B.; Trubenová, Barbora; Monk, Nicholas A.M.; Baron, Martin

    2014-01-01

    Summary Developmental signaling is remarkably robust to environmental variation, including temperature. For example, in ectothermic animals such as Drosophila, Notch signaling is maintained within functional limits across a wide temperature range. We combine experimental and computational approaches to show that temperature compensation of Notch signaling is achieved by an unexpected variety of endocytic-dependent routes to Notch activation which, when superimposed on ligand-induced activation, act as a robustness module. Thermal compensation arises through an altered balance of fluxes within competing trafficking routes, coupled with temperature-dependent ubiquitination of Notch. This flexible ensemble of trafficking routes supports Notch signaling at low temperature but can be switched to restrain Notch signaling at high temperature and thus compensates for the inherent temperature sensitivity of ligand-induced activation. The outcome is to extend the physiological range over which normal development can occur. Similar mechanisms may provide thermal robustness for other developmental signals. PMID:24855951

  20. Modulation of endocytic trafficking and apical stability of CFTR in primary human airway epithelial cultures.

    PubMed

    Cholon, Deborah M; O'Neal, Wanda K; Randell, Scott H; Riordan, John R; Gentzsch, Martina

    2010-03-01

    CFTR is a highly regulated apical chloride channel of epithelial cells that is mutated in cystic fibrosis (CF). In this study, we characterized the apical stability and intracellular trafficking of wild-type and mutant CFTR in its native environment, i.e., highly differentiated primary human airway epithelial (HAE) cultures. We labeled the apical pool of CFTR and subsequently visualized the protein in intracellular compartments. CFTR moved from the apical surface to endosomes and then efficiently recycled back to the surface. CFTR endocytosis occurred more slowly in polarized than in nonpolarized HAE cells or in a polarized epithelial cell line. The most common mutation in CF, DeltaF508 CFTR, was rescued from endoplasmic reticulum retention by low-temperature incubation but transited from the apical membrane to endocytic compartments more rapidly and recycled less efficiently than wild-type CFTR. Incubation with small-molecule correctors resulted in DeltaF508 CFTR at the apical membrane but did not restore apical stability. To stabilize the mutant protein at the apical membrane, we found that the dynamin inhibitor Dynasore and the cholesterol-extracting agent cyclodextrin dramatically reduced internalization of DeltaF508, whereas the proteasomal inhibitor MG-132 completely blocked endocytosis of DeltaF508. On examination of intrinsic properties of CFTR that may affect its apical stability, we found that N-linked oligosaccharides were not necessary for transport to the apical membrane but were required for efficient apical recycling and, therefore, influenced the turnover of surface CFTR. Thus apical stability of CFTR in its native environment is affected by properties of the protein and modulation of endocytic trafficking. PMID:20008117

  1. Modulation of endocytic trafficking and apical stability of CFTR in primary human airway epithelial cultures

    PubMed Central

    Cholon, Deborah M.; O'Neal, Wanda K.; Randell, Scott H.; Riordan, John R.

    2010-01-01

    CFTR is a highly regulated apical chloride channel of epithelial cells that is mutated in cystic fibrosis (CF). In this study, we characterized the apical stability and intracellular trafficking of wild-type and mutant CFTR in its native environment, i.e., highly differentiated primary human airway epithelial (HAE) cultures. We labeled the apical pool of CFTR and subsequently visualized the protein in intracellular compartments. CFTR moved from the apical surface to endosomes and then efficiently recycled back to the surface. CFTR endocytosis occurred more slowly in polarized than in nonpolarized HAE cells or in a polarized epithelial cell line. The most common mutation in CF, ΔF508 CFTR, was rescued from endoplasmic reticulum retention by low-temperature incubation but transited from the apical membrane to endocytic compartments more rapidly and recycled less efficiently than wild-type CFTR. Incubation with small-molecule correctors resulted in ΔF508 CFTR at the apical membrane but did not restore apical stability. To stabilize the mutant protein at the apical membrane, we found that the dynamin inhibitor Dynasore and the cholesterol-extracting agent cyclodextrin dramatically reduced internalization of ΔF508, whereas the proteasomal inhibitor MG-132 completely blocked endocytosis of ΔF508. On examination of intrinsic properties of CFTR that may affect its apical stability, we found that N-linked oligosaccharides were not necessary for transport to the apical membrane but were required for efficient apical recycling and, therefore, influenced the turnover of surface CFTR. Thus apical stability of CFTR in its native environment is affected by properties of the protein and modulation of endocytic trafficking. PMID:20008117

  2. Macropinocytosis: a pathway to protozoan infection.

    PubMed

    de Carvalho, Tecia M U; Barrias, Emile S; de Souza, Wanderley

    2015-01-01

    Among the various endocytic mechanisms in mammalian cells, macropinocytosis involves internalization of large amounts of plasma membrane together with extracellular medium, leading to macropinosome formation. These structures are formed when plasma membrane ruffles are assembled after actin filament rearrangement. In dendritic cells, macropinocytosis has been reported to play a role in antigen presentation. Several intracellular pathogens are internalized by host cells via multiple endocytic pathways and macropinocytosis has been described as an important entry site for various organisms. Some bacteria, such as Legionella pneumophila, as well as various viruses, use this pathway to penetrate and subvert host cells. Some protozoa, which are larger than bacteria and virus, can also use this pathway to invade host cells. As macropinocytosis is characterized by the formation of large uncoated vacuoles and is triggered by various signaling pathways, which is similar to what occurs during the formation of the majority of parasitophorous vacuoles, it is believed that this phenomenon may be more widely used by parasites than is currently appreciated. Here we review protozoa host cell invasion via macropinocytosis. PMID:25914647

  3. Macropinocytosis: a pathway to protozoan infection

    PubMed Central

    de Carvalho, Tecia M. U.; Barrias, Emile S.; de Souza, Wanderley

    2015-01-01

    Among the various endocytic mechanisms in mammalian cells, macropinocytosis involves internalization of large amounts of plasma membrane together with extracellular medium, leading to macropinosome formation. These structures are formed when plasma membrane ruffles are assembled after actin filament rearrangement. In dendritic cells, macropinocytosis has been reported to play a role in antigen presentation. Several intracellular pathogens are internalized by host cells via multiple endocytic pathways and macropinocytosis has been described as an important entry site for various organisms. Some bacteria, such as Legionella pneumophila, as well as various viruses, use this pathway to penetrate and subvert host cells. Some protozoa, which are larger than bacteria and virus, can also use this pathway to invade host cells. As macropinocytosis is characterized by the formation of large uncoated vacuoles and is triggered by various signaling pathways, which is similar to what occurs during the formation of the majority of parasitophorous vacuoles, it is believed that this phenomenon may be more widely used by parasites than is currently appreciated. Here we review protozoa host cell invasion via macropinocytosis. PMID:25914647

  4. Endocytic trafficking towards the vacuole plays a key role in the auxin receptor SCF(TIR)-independent mechanism of lateral root formation in A. thaliana.

    PubMed

    Pérez-Henríquez, Patricio; Raikhel, Natasha V; Norambuena, Lorena

    2012-11-01

    Plants' developmental plasticity plays a pivotal role in responding to environmental conditions. One of the most plastic plant organs is the root system. Different environmental stimuli such as nutrients and water deficiency may induce lateral root formation to compensate for a low level of water and/or nutrients. It has been shown that the hormone auxin tunes lateral root development and components for its signaling pathway have been identified. Using chemical biology, we discovered an Arabidopsis thaliana lateral root formation mechanism that is independent of the auxin receptor SCF(TIR). The bioactive compound Sortin2 increased lateral root occurrence by acting upstream from the morphological marker of lateral root primordium formation, the mitotic activity. The compound did not display auxin activity. At the cellular level, Sortin2 accelerated endosomal trafficking, resulting in increased trafficking of plasma membrane recycling proteins to the vacuole. Sortin2 affected Late endosome/PVC/MVB trafficking and morphology. Combining Sortin2 with well-known drugs showed that endocytic trafficking of Late E/PVC/MVB towards the vacuole is pivotal for Sortin2-induced SCF(TIR)-independent lateral root initiation. Our results revealed a distinctive role for endosomal trafficking in the promotion of lateral root formation via a process that does not rely on the auxin receptor complex SCF(TIR). PMID:22848095

  5. Distinct and separable activities of the endocytic clathrin coat components Fcho1/2 and AP-2 in developmental patterning

    PubMed Central

    Umasankar, P. K.; Sanker, Subramaniam; Thieman, James R.; Chakraborty, Souvik; Wendland, Beverly; Tsang, Michael; Traub, Linton M.

    2012-01-01

    Clathrin-mediated endocytosis occurs at multiple independent import sites on the plasma membrane, but how these positions are selected and how different cargo is simultaneously recognized is obscure. FCHO1 and FCHO2 are early-arriving proteins at surface clathrin assemblies and are speculated to act as compulsory coat nucleators, preceding the core clathrin adaptor AP-2. Here, we show the μ-homology domain (μHD) of FCHO1/2 represents a novel endocytic interaction hub. Translational silencing of fcho1 in zebrafish embryos causes strong dorsoventral patterning defects analogous to Bmp signal failure. The Fcho1 μHD interacts with the Bmp receptor Alk8, uncovering a new endocytic component that positively modulates Bmp signal transmission. Still, the fcho1 morphant phenotype is distinct from severe embryonic defects apparent when AP-2 is depleted. Our data thus contradict the primacy of FCHO1/2 in coat initiation. PMID:22484487

  6. Endocytic Sorting of CFTR variants Monitored by Single Cell Fluorescence Ratio Image Analysis (FRIA) in Living Cells

    PubMed Central

    Barriere, H.; Apaja, P.; Okiyoneda, T.; Lukacs, G. L.

    2016-01-01

    Summary The wild-type CFTR channel undergoes constitutive internalization and recycling at the plasma membrane. This process is initiated by the recognition of the Tyr- and di-Leu-based endocytic motifs of CFTR by the AP-2 adaptor complex, leading to the formation of clathrin-coated vesicles and the channel delivery to sorting/recycling endosomes. Accumulating evidence suggests that conformationally defective mutant CFTRs (e.g. rescued ΔF508 and glycosylation-deficient channel) are unstable at the plasma membrane and undergo augmented ubiquitination in post-Golgi compartments. Ubiquitination conceivably accounts for the metabolic instability at cell surface by provoking accelerated internalization, as well as rerouting the channel from recycling towards lysosomal degradation. We developed an in vivo fluorescence ratio imaging assay (FRIA) that in concert with genetic manipulation can be utilized to establish the post-endocytic fate and sorting determinants of mutant CFTRs. PMID:21594793

  7. The late chlamydial inclusion membrane is not derived from the endocytic pathway and is relatively deficient in host proteins.

    PubMed Central

    Taraska, T; Ward, D M; Ajioka, R S; Wyrick, P B; Davis-Kaplan, S R; Davis, C H; Kaplan, J

    1996-01-01

    Chlamydiae are obligate intracellular parasites which multiply within infected cells in a membrane-bound structure termed an inclusion. Newly internalized bacteria are surrounded by host plasma membrane; however, the source of membrane for the expansion of the inclusion is unknown. To determine if the membrane for the mature inclusion was derived by fusion with cellular organelles, we stained infected cells with fluorescent or electron-dense markers specific for organelles and examined inclusions for those markers. We observed no evidence for the presence of endoplasmic reticulum, Golgi, late endosomal, or lysosomal proteins in the inclusion. These data suggest that the expansion of the inclusion membrane, beginning 24 h postinoculation, does not occur by the addition of host proteins resulting from either de novo host synthesis or by fusion with preexisting membranes. To determine the source of the expanding inclusion membrane, antibodies were produced against isolated membranes from Chlamydia-infected mouse cells. The antibodies were demonstrated to be solely against Chlamydia-specified proteins by both immunoprecipitation of [35S]methionine-labeled extracts and Western blotting (immunoblotting). Techniques were used to semipermeabilize Chlamydia-infected cells without disrupting the permeability of the inclusion, allowing antibodies access to the outer surface of the inclusion membrane. Immunofluorescent staining demonstrated a ring-like fluorescence around inclusions in semipermeabilized cells, whereas Triton X-100-permeabilized cells showed staining throughout the inclusion. These studies demonstrate that the inclusion membrane is made up, in part, of Chlamydia-specified proteins and not of existing host membrane proteins. PMID:8751921

  8. The AP-2 complex is required for proper temporal and spatial dynamics of endocytic patches in fission yeast.

    PubMed

    de León, Nagore; Hoya, Marta; Curto, M-Angeles; Moro, Sandra; Yanguas, Francisco; Doncel, Cristina; Valdivieso, M-Henar

    2016-05-01

    In metazoans the AP-2 complex has a well-defined role in clathrin-mediated endocytosis. By contrast, its direct role in endocytosis in unicellular eukaryotes has been questioned. Here, we report co- immunoprecipitation between the fission yeast AP-2 component Apl3p and clathrin, as well as the genetic interactions between apl3Δ and clc1 and sla2Δ/end4Δ mutants. Furthermore, a double clc1 apl3Δ mutant was found to be defective in FM4-64 uptake. In an otherwise wild-type strain, apl3Δ cells exhibit altered dynamics of the endocytic sites, with a heterogeneous and extended lifetime of early and late markers at the patches. Additionally, around 50% of the endocytic patches exhibit abnormal spatial dynamics, with immobile patches and patches that bounce backwards to the cell surface, showing a pervasive effect of the absence of AP-2. These alterations in the endocytic machinery result in abnormal cell wall synthesis and morphogenesis. Our results complement those found in budding yeast and confirm that a direct role of AP-2 in endocytosis has been conserved throughout evolution. PMID:26749213

  9. Endocytic Trafficking of Nanoparticles Delivered by Cell-penetrating Peptides Comprised of Nona-arginine and a Penetration Accelerating Sequence

    PubMed Central

    Liu, Betty R.; Lo, Shih-Yen; Liu, Chia-Chin; Chyan, Chia-Lin; Huang, Yue-Wern; Aronstam, Robert S.; Lee, Han-Jung

    2013-01-01

    Cell-penetrating peptides (CPPs) can traverse cellular membranes and deliver biologically active molecules into cells. In this study, we demonstrate that CPPs comprised of nona-arginine (R9) and a penetration accelerating peptide sequence (Pas) that facilitates escape from endocytic lysosomes, denoted as PR9, greatly enhance the delivery of noncovalently associated quantum dots (QDs) into human A549 cells. Mechanistic studies, intracellular trafficking analysis and a functional gene assay reveal that endocytosis is the main route for intracellular delivery of PR9/QD complexes. Endocytic trafficking of PR9/QD complexes was monitored using both confocal and transmission electron microscopy (TEM). Zeta-potential and size analyses indicate the importance of electrostatic forces in the interaction of PR9/QD complexes with plasma membranes. Circular dichroism (CD) spectroscopy reveals that the secondary structural elements of PR9 have similar conformations in aqueous buffer at pH 7 and 5. This study of nontoxic PR9 provides a basis for the design of optimized cargo delivery that allows escape from endocytic vesicles. PMID:23840594

  10. Rabankyrin-5 interacts with EHD1 and Vps26 to regulate endocytic trafficking and retromer function

    PubMed Central

    Zhang, Jing; Reiling, Calliste; Reinecke, James B.; Prislan, Iztok; Marky, Luis A.; Sorgen, Paul L.; Naslavsky, Naava; Caplan, Steve

    2012-01-01

    Rabankyrin-5 (Rank-5) has been implicated as an effector of the small GTPase Rab5 and plays an important role in macropinocytosis. We have now identified Rank-5 as an interaction partner for the recycling regulatory protein EHD1. We have demonstrated this interaction by GST-pulldown, yeast two-hybrid assay, isothermal calorimetry, and co-immunoprecipitation and found that the binding occurs between the EH-domain of EHD1 and the NPFED motif of Rank-5. Similar to EHD1, we found that Rank-5 co-localizes and interacts with components of the retromer complex such as Vps26, suggesting a role for Rank-5 in retromer-based transport. Indeed, depletion of Rank-5 causes mislocalization of Vps26 and affects both the retrieval of mannose 6-phosphate receptor (M6PR) transport to the Golgi from endosomes and biosynthetic transport. Moreover, Rank-5 is required for normal retromer distribution, as over-expression of a wild-type Rank-5-siRNA-resistant construct rescues retromer mislocalization. Finally, we show that depletion of either Rank-5 or EHD1 impairs secretion of VSV-G. Overall, our data identify a new interaction between Rank-5 and EHD1, and novel endocytic regulatory roles that include retromer-based transport and secretion. PMID:22284051

  11. SERS nanosensors that report pH of endocytic compartments during FcεRI transit

    PubMed Central

    Nowak-Lovato, K.L.; Wilson, Bridget S.; Rector, K.D.

    2011-01-01

    Recently, the development of an IgE receptor (FcεRI)-targeted, pH sensitive, surface-enhanced Raman spectroscopy (SERS) nanosensor has been demonstrated [1]. The targeted nanosensor enables spatial and temporal pH measurements as internalized receptors progress through endosomal compartments in live cells. Trafficking of receptor-bound nanosensors was compared at physiological temperature (37°C) versus room temperature (25°C). As expected, we observed markedly slower progression of receptors through low pH endocytic compartments at the lower temperature. We also demonstrate the utility of the nanosensors to measure directly changes in the pH of intracellular compartments after treatment with bafilomycin or amiloride. We report an increase in endosome compartment pH after treatment with bafilomycin, an H+ ATPase pump inhibitor. Decreased endosomal luminal pH was measured in cells treated with amiloride, an inhibitor of Na+/H+ exchange. The decrease in amiloride-treated cells was transient, followed by a recovery period of approximately 15–20 minutes to restore endosomal pH. These experiments demonstrate the novel application of Raman spectroscopy to monitor local pH environment in live cells with the use of targeted SERS nanosensors. PMID:20842349

  12. Exocytosis of post-Golgi vesicles is regulated by components of the endocytic machinery

    PubMed Central

    Jaiswal, Jyoti K.; Rivera, Victor M.; Simon, Sanford M.

    2009-01-01

    Post-Golgi vesicles target and deliver most biosynthetic cargoes to the cell surface. However, the molecules and mechanism involved in fusion of these vesicles is not well understood in mammalian cells. We have employed a system to simultaneously monitor release of luminal and membrane biosynthetic cargoes from individual post-Golgi vesicles. Exocytosis of these vesicles is not calcium-triggered and can result in complete, partial or no release of membrane cargo. Partial or no release of membrane cargo of a fusing vesicle are fates associated with kiss-and-run exocytosis and are the predominant mode of post-Golgi vesicle exocytosis. Partial cargo release by post-Golgi vesicles occurs due to premature closure of the fusion pore and is modulated by the activity of clathrin, actin and dynamin. Our results demonstrate that the components of the endocytic machinery modulate the nature and extent of secretion of biosynthetic cargo by affecting fusion of post-Golgi vesicles to the cell membrane. PMID:19563761

  13. The Rab5 Effector Rabankyrin-5 Regulates and Coordinates Different Endocytic Mechanisms

    PubMed Central

    2004-01-01

    The small GTPase Rab5 is a key regulator of clathrin-mediated endocytosis. On early endosomes, within a spatially restricted domain enriched in phosphatydilinositol-3-phosphate [PI(3)P], Rab5 coordinates a complex network of effectors that functionally cooperate in membrane tethering, fusion, and organelle motility. Here we discovered a novel PI(3)P-binding Rab5 effector, Rabankyrin-5, which localises to early endosomes and stimulates their fusion activity. In addition to early endosomes, however, Rabankyrin-5 localises to large vacuolar structures that correspond to macropinosomes in epithelial cells and fibroblasts. Overexpression of Rabankyrin-5 increases the number of macropinosomes and stimulates fluid-phase uptake, whereas its downregulation inhibits these processes. In polarised epithelial cells, this function is primarily restricted to the apical membrane. Rabankyrin-5 localises to large pinocytic structures underneath the apical surface of kidney proximal tubule cells, and its overexpression in polarised Madin-Darby canine kidney cells stimulates apical but not basolateral, non-clathrin-mediated pinocytosis. In demonstrating a regulatory role in endosome fusion and (macro)pinocytosis, our studies suggest that Rab5 regulates and coordinates different endocytic mechanisms through its effector Rabankyrin-5. Furthermore, its active role in apical pinocytosis in epithelial cells suggests an important function of Rabankyrin-5 in the physiology of polarised cells. PMID:15328530

  14. The tetraspanin CD63/lamp3 cycles between endocytic and secretory compartments in human endothelial cells.

    PubMed

    Kobayashi, T; Vischer, U M; Rosnoblet, C; Lebrand, C; Lindsay, M; Parton, R G; Kruithof, E K; Gruenberg, J

    2000-05-01

    In the present study, we show that in human endothelial cells the tetraspanin CD63/lamp3 distributes predominantly to the internal membranes of multivesicular-multilamellar late endosomes, which contain the unique lipid lysobisphosphatidic acid. Some CD63/lamp3 is also present in Weibel-Palade bodies, the characteristic secretory organelle of these cells. We find that CD63/lamp3 molecules can be transported from late endosomes to Weibel-Palade bodies and thus that CD63/lamp3 cycles between endocytic and biosynthetic compartments; however, movement of CD63/lamp3 is much slower than that of P-selectin, which is known to cycle between plasma membrane and Weibel-Palade bodies. When cells are treated with U18666A, a drug that mimics the Niemann-Pick type C syndrome, both proteins accumulate in late endosomes and fail to reach Weibel-Palade bodies efficiently, suggesting that P-selectin, like CD63/lamp3, cycles via late endosomes. Our data suggest that CD63/lamp3 partitions preferentially within late endosome internal membranes, thus causing its accumulation, and that this mechanism contributes to CD63/lamp3 retention in late endosomes; however, our data also indicate that the protein can eventually escape from these internal membranes and recycle toward Weibel-Palade bodies to be reused. Our observations thus uncover the existence of a selective trafficking route from late endosomes to Weibel-Palade bodies. PMID:10793155

  15. RILP interacts with HOPS complex via VPS41 subunit to regulate endocytic trafficking

    PubMed Central

    Lin, Xiaosi; Yang, Ting; Wang, Shicong; Wang, Zhen; Yun, Ye; Sun, Lixiang; Zhou, Yunhe; Xu, Xiaohui; Akazawa, Chihiro; Hong, Wanjin; Wang, Tuanlao

    2014-01-01

    The HOPS complex serves as a tethering complex with GEF activity for Ypt7p in yeast to regulate late endosomal membrane maturation. While the role of HOPS complex is well established in yeast cells, its functional and mechanistic aspects in mammalian cells are less well defined. In this study, we report that RILP, a downstream effector of Rab7, interacts with HOPS complex and recruits HOPS subunits to the late endosomal compartment. Structurally, the amino-terminal portion of RILP interacts with HOPS complex. Unexpectedly, this interaction is independent of Rab7. VPS41 subunit of HOPS complex was defined to be the major partner for interacting with RILP. The carboxyl-terminal region of VPS41 was mapped to be responsible for the interaction. Functionally, either depletion of VPS41 by shRNA or overexpression of VPS41 C-terminal half retarded EGF-induced degradation of EGFR. These results suggest that interaction of RILP with HOPS complex via VPS41 plays a role in endocytic trafficking of EGFR. PMID:25445562

  16. Molecular basis of synaptic vesicle cargo recognition by the endocytic sorting adaptor stonin 2.

    PubMed

    Jung, Nadja; Wienisch, Martin; Gu, Mingyu; Rand, James B; Müller, Sebastian L; Krause, Gerd; Jorgensen, Erik M; Klingauf, Jürgen; Haucke, Volker

    2007-12-31

    Synaptic transmission depends on clathrin-mediated recycling of synaptic vesicles (SVs). How select SV proteins are targeted for internalization has remained elusive. Stonins are evolutionarily conserved adaptors dedicated to endocytic sorting of the SV protein synaptotagmin. Our data identify the molecular determinants for recognition of synaptotagmin by stonin 2 or its Caenorhabditis elegans orthologue UNC-41B. The interaction involves the direct association of clusters of basic residues on the surface of the cytoplasmic domain of synaptotagmin 1 and a beta strand within the mu-homology domain of stonin 2. Mutation of K783, Y784, and E785 to alanine within this stonin 2 beta strand results in failure of the mutant stonin protein to associate with synaptotagmin, to accumulate at synapses, and to facilitate synaptotagmin internalization. Synaptotagmin-binding-defective UNC-41B is unable to rescue paralysis in C. elegans stonin mutant animals, suggesting that the mechanism of stonin-mediated SV cargo recognition is conserved from worms to mammals. PMID:18166656

  17. Common and distinct roles for the binding partners Rabenosyn-5 and Vps45 in the regulation of endocytic trafficking in mammalian cells

    PubMed Central

    Rahajeng, Juliati; Caplan, Steve; Naslavsky, Naava

    2010-01-01

    In several invertebrate organisms, the Sec1p/Munc18-like protein Vps45 interacts with the divalent Rab4/Rab5 effector, Rabenosyn-5 and carries out multiple functions in the endocytic/secretory pathways. In mammalian cells, Vps45 and Rabenosyn-5 also interact, but the molecular characterization of this binding, and the functional relationship between these two proteins has not been well defined. Here we identify a novel sequence within Rabenosyn-5 required for its interaction with Vps45. We demonstrate that hVps45-depletion decreases expression of Rabenosyn-5, likely resulting from Rabenosyn-5 degradation through the proteasomal pathway. Furthermore, we demonstrate that similar to Rabenosyn-5-depletion, hVps45-depletion causes impaired recycling of β1 integrins, and a subsequent delay in human fibroblast cell migration on fibronectin-coated plates. Moreover, β1 integrin recycling could be rescued by reintroduction of siRNA-resistant wild-type Rabenosyn-5, but not a mutant deficient in Vps45 binding. However, unlike Rabenosyn-5-depletion, which induces Golgi fragmentation and decreased recruitment of sorting nexin retromer subunits to the Golgi, hVps45-depletion induces Golgi condensation and accumulation of retromer subunits in the vicinity of the Golgi. In part, these phenomena could be attributed to reduced Syntaxin16 expression and altered localization of both Syntaxin16 and Syntaxin6 upon Vps45-depletion. Overall, these findings implicate hVps45 and Rabenosyn-5 in post early endosome transport, and we propose that their interaction serves as a nexus to promote bidirectional transport along the endosome-to-recycling compartment and endosome-to-Golgi axes. PMID:19931244

  18. Loss of the BBSome perturbs endocytic trafficking and disrupts virulence of Trypanosoma brucei

    PubMed Central

    Langousis, Gerasimos; Shimogawa, Michelle M.; Saada, Edwin A.; Vashisht, Ajay A.; Spreafico, Roberto; Nager, Andrew R.; Barshop, William D.; Nachury, Maxence V.; Wohlschlegel, James A.; Hill, Kent L.

    2016-01-01

    Cilia (eukaryotic flagella) are present in diverse eukaryotic lineages and have essential motility and sensory functions. The cilium’s capacity to sense and transduce extracellular signals depends on dynamic trafficking of ciliary membrane proteins. This trafficking is often mediated by the Bardet–Biedl Syndrome complex (BBSome), a protein complex for which the precise subcellular distribution and mechanisms of action are unclear. In humans, BBSome defects perturb ciliary membrane protein distribution and manifest clinically as Bardet–Biedl Syndrome. Cilia are also important in several parasites that cause tremendous human suffering worldwide, yet biology of the parasite BBSome remains largely unexplored. We examined BBSome functions in Trypanosoma brucei, a flagellated protozoan parasite that causes African sleeping sickness in humans. We report that T. brucei BBS proteins assemble into a BBSome that interacts with clathrin and is localized to membranes of the flagellar pocket and adjacent cytoplasmic vesicles. Using BBS gene knockouts and a mouse infection model, we show the T. brucei BBSome is dispensable for flagellar assembly, motility, bulk endocytosis, and cell viability but required for parasite virulence. Quantitative proteomics reveal alterations in the parasite surface proteome of BBSome mutants, suggesting that virulence defects are caused by failure to maintain fidelity of the host–parasite interface. Interestingly, among proteins altered are those with ubiquitination-dependent localization, and we find that the BBSome interacts with ubiquitin. Collectively, our data indicate that the BBSome facilitates endocytic sorting of select membrane proteins at the base of the cilium, illuminating BBSome roles at a critical host–pathogen interface and offering insights into BBSome molecular mechanisms. PMID:26721397

  19. Loss of the BBSome perturbs endocytic trafficking and disrupts virulence of Trypanosoma brucei.

    PubMed

    Langousis, Gerasimos; Shimogawa, Michelle M; Saada, Edwin A; Vashisht, Ajay A; Spreafico, Roberto; Nager, Andrew R; Barshop, William D; Nachury, Maxence V; Wohlschlegel, James A; Hill, Kent L

    2016-01-19

    Cilia (eukaryotic flagella) are present in diverse eukaryotic lineages and have essential motility and sensory functions. The cilium's capacity to sense and transduce extracellular signals depends on dynamic trafficking of ciliary membrane proteins. This trafficking is often mediated by the Bardet-Biedl Syndrome complex (BBSome), a protein complex for which the precise subcellular distribution and mechanisms of action are unclear. In humans, BBSome defects perturb ciliary membrane protein distribution and manifest clinically as Bardet-Biedl Syndrome. Cilia are also important in several parasites that cause tremendous human suffering worldwide, yet biology of the parasite BBSome remains largely unexplored. We examined BBSome functions in Trypanosoma brucei, a flagellated protozoan parasite that causes African sleeping sickness in humans. We report that T. brucei BBS proteins assemble into a BBSome that interacts with clathrin and is localized to membranes of the flagellar pocket and adjacent cytoplasmic vesicles. Using BBS gene knockouts and a mouse infection model, we show the T. brucei BBSome is dispensable for flagellar assembly, motility, bulk endocytosis, and cell viability but required for parasite virulence. Quantitative proteomics reveal alterations in the parasite surface proteome of BBSome mutants, suggesting that virulence defects are caused by failure to maintain fidelity of the host-parasite interface. Interestingly, among proteins altered are those with ubiquitination-dependent localization, and we find that the BBSome interacts with ubiquitin. Collectively, our data indicate that the BBSome facilitates endocytic sorting of select membrane proteins at the base of the cilium, illuminating BBSome roles at a critical host-pathogen interface and offering insights into BBSome molecular mechanisms. PMID:26721397

  20. Glycoprotein H and α4β1 Integrins Determine the Entry Pathway of Alphaherpesviruses

    PubMed Central

    Azab, Walid; Lehmann, Maik J.

    2013-01-01

    Herpesviruses enter cells either by direct fusion at the plasma membrane or from within endosomes, depending on the cell type and receptor(s). We investigated two closely related herpesviruses of horses, equine herpesvirus type 1 (EHV-1) and EHV-4, for which the cellular and viral determinants routing virus entry are unknown. We show that EHV-1 enters equine epithelial cells via direct fusion at the plasma membrane, while EHV-4 does so via an endocytic pathway, which is dependent on dynamin II, cholesterol, caveolin 1, and tyrosine kinase activity. Exchange of glycoprotein H (gH) between EHV-1 and EHV-4 resulted in rerouting of EHV-1 to the endocytic pathway, as did blocking of α4β1 integrins on the cell surface. Furthermore, a point mutation in the SDI integrin-binding motif of EHV-1 gH also directed EHV-1 to the endocytic pathway. Cumulatively, we show that viral gH and cellular α4β1 integrins are important determinants in the choice of alphaherpesvirus cellular entry pathways. PMID:23514881

  1. Isolation of endocytic and exocytic populations of coated vesicles from perfused rat liver using an acetylcholinesterase mediated density shift technique

    SciTech Connect

    Helmy, S.I.

    1985-01-01

    Rat liver contains endogenous acetylcholinesterase (AChE). The authors have isolated rat liver coated vesicles (CVs) and incubated them with the Karnovsky-Roots reagents and the AChE substrate, acetylthiocholine. A dense iron-copper precipitate is deposited at hydrolysis sites. When the CVs are subjected to sucrose-Ficoll-D20 density gradient ultracentrifugation, the AChE containing CVs are shifted to a denser region of the gradient. The molecular forms of AChE present in the CVs resemble secretory AChE not serum AChE. CVs isolated from perfused liver, treated with diisopropylfluorophosphate (DFP), to inactivate endogenous AChE, and allowed to resynthesize AChE for 30 minutes, contain AChE which is shown to be exocytic. When /sup 125/I-insulin and gal-AChE are co-perfused into DFP treated liver for 3 minutes, the CVs isolated and density shifted, approximately 80% of both Gal-AChE and /sup 125/I-insulin are in the shifted fraction which also contains approximately 50% of the CVs. Similar experiments have been carried out using /sup 35/S-methionine to label newly synthesized secretory proteins. They have measured the diameter of the endocytic and exocytic CVs and have found that the endocytic CVs are significantly larger than the exocytic CVs and more heterogeneous in size. When the cholesterol/phospholipid ratio was compared in both populations, the endocytic CVs had a significantly higher ratio than the exocytic CVs. Finally both populations were subjected to one dimensional and two dimensional SDS gel electrophoresis and were found to be remarkably similar in protein composition.

  2. V-ATPase-dependent luminal acidification is required for endocytic recycling of a yeast cell wall stress sensor, Wsc1p

    SciTech Connect

    Ueno, Kazuma; Saito, Mayu; Nagashima, Makiko; Kojima, Ai; Nishinoaki, Show; Toshima, Junko Y.; Toshima, Jiro

    2014-01-10

    Highlights: •A targeted genome screen identified 5 gene groups affecting Wsc1p recycling. •V-ATPase-dependent luminal acidification is required for Wsc1p recycling. •Activity of V-ATPase might be required for cargo recognition by the retromer complex. -- Abstract: Wsc1p is a major cell wall sensor protein localized at the polarized cell surface. The localization of Wsc1p is maintained by endocytosis and recycling from endosomes back to the cell surface, but changes to the vacuole when cells are subjected to heat stress. Exploiting this unique property of Wsc1p, we screened for yeast single-gene deletion mutants exhibiting defects in Wsc1p trafficking. By expressing 3GFP-tagged Wsc1p in mutants with deleted genes whose function is related to intracellular trafficking, we identified 5 gene groups affecting Wsc1p trafficking, impaired respectively in endocytic internalization, multivesicular body sorting, the GARP complex, endosomal maturation/vacuolar fusion, and V-ATPase. Interestingly, deletion of the VPH1 gene, encoding the V{sub o} subunit of vacuolar-type H{sup +}-ATPase (V-ATPase), led to mis-localization of Wsc1p from the plasma membrane to the vacuole. In addition, disruption of other V-ATPase subunits (vma mutants) also caused defects of Wsc1p trafficking and vacuolar acidification similar to those seen in the vph1Δ mutant. Moreover, we found that deletion of the VPS26 gene, encoding a subunit of the retromer complex, also caused a defect in Wsc1p recycling and mis-localization of Wsc1p to the vacuole. These findings clarified the previously unidentified Wsc1p recycling pathway and requirement of V-ATPase-dependent luminal acidification for Wsc1p recycling.

  3. Probing heterobivalent binding to the endocytic AP-2 adaptor complex by DNA-based spatial screening.

    PubMed

    Diezmann, F; von Kleist, L; Haucke, V; Seitz, O

    2015-08-01

    The double helical DNA scaffold offers a unique set of properties, which are particularly useful for studies of multivalency in biomolecular interactions: (i) multivalent ligand displays can be formed upon nucleic acid hybridization in a self-assembly process, which facilitates spatial screening (ii) valency and spatial arrangement of the ligand display can be precisely controlled and (iii) the flexibility of the ligand display can be adjusted by integrating nick sites and unpaired template regions. Herein we describe the use of DNA-based spatial screening for the characterization of the adaptor complex 2 (AP-2), a central interaction hub within the endocytic protein network in clathrin-mediated endocytosis. AP-2 is comprised of a core domain and two, so-called appendage domains, the α- and the β2-ear, which associate with cytoplasmatic proteins required for the formation or maturation of clathrin/AP-2 coated pits. Each appendage domain has two binding grooves which recognize distinct peptide motives with micromolar affinity. This provides opportunities for enhanced interactions with protein molecules that contain two (or more) different peptide motives. To determine whether a particular, spatial arrangement of binding motifs is required for high affinity binding we probed the distance-affinity relationships by means of DNA-programmed spatial screening with self-assembled peptide-DNA complexes. By using trimolecular and tetramolecular assemblies two different peptides were positioned in 2-22 nucleotide distance. The binding data obtained with both recombinant protein in well-defined buffer systems and native AP-2 in brain extract suggests that the two binding sites of the AP-2 α-appendage can cooperate to provide up to 40-fold enhancement of affinity compared to the monovalent interaction. The distance between the two recognized peptide motives was less important provided that the DNA duplex segments were connected by flexible, single strand segments. By

  4. Clathrin-dependent pathways and the cytoskeleton network are involved in ceramide endocytosis by a parasitic protozoan, Giardia lamblia.

    PubMed

    Hernandez, Yunuen; Castillo, Cynthia; Roychowdhury, Sukla; Hehl, Adrian; Aley, Stephen B; Das, Siddhartha

    2007-01-01

    Although identified as an early-diverged protozoan, Giardia lamblia shares many similarities with higher eukaryotic cells, including an internal membrane system and cytoskeleton, as well as secretory pathways. However, unlike many other eukaryotes, Giardia does not synthesize lipids de novo, but rather depends on exogenous sources for both energy production and organelle or membrane biogenesis. It is not known how lipid molecules are taken up by this parasite and if endocytic pathways are involved in this process. In this investigation, we tested the hypothesis that highly regulated and selective lipid transport machinery is present in Giardia and necessary for the efficient internalization and intracellular targeting of ceramide molecules, the major sphingolipid precursor. Using metabolic and pathway inhibitors, we demonstrate that ceramide is internalized through endocytic pathways and is primarily targeted into perinuclear/endoplasmic reticulum membranes. Further investigations suggested that Giardia uses both clathrin-dependent pathways and the actin cytoskeleton for ceramide uptake, as well as microtubule filaments for intracellular localization and targeting. We speculate that this parasitic protozoan has evolved cytoskeletal and clathrin-dependent endocytic mechanisms for importing ceramide molecules from the cell exterior for the synthesis of membranes and vesicles during growth and differentiation. PMID:17087963

  5. Syp1 is a conserved endocytic adaptor that contains domains involved in cargo selection and membrane tubulation

    SciTech Connect

    Reider, Amanda; Barker, Sarah L.; Mishra, Sanjay K.; Im, Young Jun; Maldonado-Báez, Lymarie; Hurley, James H.; Traub, Linton M.; Wendland, Beverly

    2010-10-28

    Internalization of diverse transmembrane cargos from the plasma membrane requires a similarly diverse array of specialized adaptors, yet only a few adaptors have been characterized. We report the identification of the muniscin family of endocytic adaptors that is conserved from yeast to human beings. Solving the structures of yeast muniscin domains confirmed the unique combination of an N-terminal domain homologous to the crescent-shaped membrane-tubulating EFC/F-BAR domains and a C-terminal domain homologous to cargo-binding {mu} homology domains ({mu}HDs). In vitro and in vivo assays confirmed membrane-tubulation activity for muniscin EFC/F-BAR domains. The {mu}HD domain has conserved interactions with the endocytic adaptor/scaffold Ede1/eps15, which influences muniscin localization. The transmembrane protein Mid2, earlier implicated in polarized Rho1 signalling, was identified as a cargo of the yeast adaptor protein. These and other data suggest a model in which the muniscins provide a combined adaptor/membrane-tubulation activity that is important for regulating endocytosis.

  6. Slit-Dependent Endocytic Trafficking of the Robo Receptor Is Required for Son of Sevenless Recruitment and Midline Axon Repulsion

    PubMed Central

    Chance, Rebecca K.; Bashaw, Greg J.

    2015-01-01

    Understanding how axon guidance receptors are activated by their extracellular ligands to regulate growth cone motility is critical to learning how proper wiring is established during development. Roundabout (Robo) is one such guidance receptor that mediates repulsion from its ligand Slit in both invertebrates and vertebrates. Here we show that endocytic trafficking of the Robo receptor in response to Slit-binding is necessary for its repulsive signaling output. Dose-dependent genetic interactions and in vitro Robo activation assays support a role for Clathrin-dependent endocytosis, and entry into both the early and late endosomes as positive regulators of Slit-Robo signaling. We identify two conserved motifs in Robo’s cytoplasmic domain that are required for its Clathrin-dependent endocytosis and activation in vitro; gain of function and genetic rescue experiments provide strong evidence that these trafficking events are required for Robo repulsive guidance activity in vivo. Our data support a model in which Robo’s ligand-dependent internalization from the cell surface to the late endosome is essential for receptor activation and proper repulsive guidance at the midline by allowing recruitment of the downstream effector Son of Sevenless in a spatially constrained endocytic trafficking compartment. PMID:26335920

  7. Structure of Sla1p homology domain 1 and interaction with the NPFxD endocytic internalization motif

    PubMed Central

    Mahadev, Ravi K; Di Pietro, Santiago M; Olson, John M; Piao, Hai Lan; Payne, Gregory S; Overduin, Michael

    2007-01-01

    Adaptor proteins play important endocytic roles including recognition of internalization signals in transmembrane cargo. Sla1p serves as the adaptor for uptake of transmembrane proteins containing the NPFxD internalization signal, and is essential for normal functioning of the actin cytoskeleton during endocytosis. The Sla1p homology domain 1 (SHD1) within Sla1p is responsible for recognition of the NPFxD signal. This study presents the NMR structure of the NPFxD-bound state of SHD1 and a model for the protein–ligand complex. The α+β structure of the protein reveals an SH3-like topology with a solvent-exposed hydrophobic ligand binding site. NMR chemical shift perturbations and effects of structure-based mutations on ligand binding in vitro define residues that are key for NPFxD binding. Mutations that abolish ligand recognition in vitro also abolish NPFxD-mediated receptor internalization in vivo. Thus, SHD1 is a novel functional domain based on SH3-like topology, which employs a unique binding site to recognize the NPFxD endocytic internalization signal. Its distant relationship with the SH3 fold endows this superfamily with a new role in endocytosis. PMID:17363896

  8. Exocyst Subunits Exo70 and Exo84 Cooperate with Small GTPases to Regulate Behavior and Endocytic Trafficking in C. elegans

    PubMed Central

    Jiu, Yaming; Jin, Congyu; Liu, Yanbo; Holmberg, Carina I.; Jäntti, Jussi

    2012-01-01

    The exocyst complex is required for cell polarity regulation and the targeting and tethering of transport vesicles to the plasma membrane. The complex is structurally well conserved, however, the functions of individual subunits and their regulation is poorly understood. Here we characterize the mutant phenotypes for the exocyst complex genes exoc-7 (exo70) and exoc-8 (exo84) in Caenorhabditis elegans. The mutants display pleiotropic behavior defects that resemble those observed in cilia mutants (slow growth, uncoordinated movement, defects in chemo-, mechano- and thermosensation). However, no obvious morphological defects in cilia were observed. A targeted RNAi screen for small GTPases identified eleven genes with enhanced phenotypes when combined with exoc-7, exoc-8 single and exoc-7;exoc-8 double mutants. The screen verified previously identified functional links between the exocyst complex and small GTPases and, in addition, identified several novel potential regulators of exocyst function. The exoc-8 and exoc-7;exoc-8 mutations caused a significant size increase in the rab-10 RNAi-induced endocytic vacuoles in the intestinal epithelial cells. In addition, exoc-8 and exoc-7;exoc-8 mutations resulted in up-regulation of RAB-10 expression and affected the accumulation of endocytic marker proteins in these cells in response to rab-10 RNAi. The findings identify novel, potential regulators for exocyst function and show that exoc-7 and exoc-8 are functionally linked to rab-10 in endosomal trafficking in intestinal epithelial cells in C. elegans. PMID:22389680

  9. Exocyst subunits Exo70 and Exo84 cooperate with small GTPases to regulate behavior and endocytic trafficking in C. elegans.

    PubMed

    Jiu, Yaming; Jin, Congyu; Liu, Yanbo; Holmberg, Carina I; Jäntti, Jussi

    2012-01-01

    The exocyst complex is required for cell polarity regulation and the targeting and tethering of transport vesicles to the plasma membrane. The complex is structurally well conserved, however, the functions of individual subunits and their regulation is poorly understood. Here we characterize the mutant phenotypes for the exocyst complex genes exoc-7 (exo70) and exoc-8 (exo84) in Caenorhabditis elegans. The mutants display pleiotropic behavior defects that resemble those observed in cilia mutants (slow growth, uncoordinated movement, defects in chemo-, mechano- and thermosensation). However, no obvious morphological defects in cilia were observed. A targeted RNAi screen for small GTPases identified eleven genes with enhanced phenotypes when combined with exoc-7, exoc-8 single and exoc-7;exoc-8 double mutants. The screen verified previously identified functional links between the exocyst complex and small GTPases and, in addition, identified several novel potential regulators of exocyst function. The exoc-8 and exoc-7;exoc-8 mutations caused a significant size increase in the rab-10 RNAi-induced endocytic vacuoles in the intestinal epithelial cells. In addition, exoc-8 and exoc-7;exoc-8 mutations resulted in up-regulation of RAB-10 expression and affected the accumulation of endocytic marker proteins in these cells in response to rab-10 RNAi. The findings identify novel, potential regulators for exocyst function and show that exoc-7 and exoc-8 are functionally linked to rab-10 in endosomal trafficking in intestinal epithelial cells in C. elegans. PMID:22389680

  10. Endocytic membrane turnover at the leading edge is driven by a transient interaction between Cdc42 and GRAF1

    PubMed Central

    Francis, Monika K.; Holst, Mikkel R.; Vidal-Quadras, Maite; Henriksson, Sara; Santarella-Mellwig, Rachel; Sandblad, Linda; Lundmark, Richard

    2015-01-01

    ABSTRACT Changes in cell morphology require coordination of plasma membrane turnover and cytoskeleton dynamics, processes that are regulated by Rho GTPases. Here, we describe how a direct interaction between the Rho GTPase Cdc42 and the GTPase-activating protein (GAP) GRAF1 (also known as ARHGAP26), facilitates rapid cell surface turnover at the leading edge. Both Cdc42 and GRAF1 were required for fluid-phase uptake and regulated the generation of transient GRAF1-coated endocytic carriers, which were distinct from clathrin-coated vesicles. GRAF1 was found to transiently assemble at discrete Cdc42-enriched punctae at the plasma membrane, resulting in a corresponding decrease in the microdomain association of Cdc42. However, Cdc42 captured in its active state was, through a GAP-domain-mediated interaction, localised together with GRAF1 on accumulated internal structures derived from the cell surface. Correlative fluorescence and electron tomography microscopy revealed that these structures were clusters of small membrane carriers with defective endosomal processing. We conclude that a transient interaction between Cdc42 and GRAF1 drives endocytic turnover and controls the transition essential for endosomal maturation of plasma membrane internalised by this mechanism. PMID:26446261

  11. Microarray analysis of E9.5 reduced folate carrier (RFC1; Slc19a1) knockout embryos reveals altered expression of genes in the cubilin-megalin multiligand endocytic receptor complex

    PubMed Central

    Gelineau-van Waes, Janee; Maddox, Joyce R; Smith, Lynette M; van Waes, Michael; Wilberding, Justin; Eudy, James D; Bauer, Linda K; Finnell, Richard H

    2008-01-01

    Background The reduced folate carrier (RFC1) is an integral membrane protein and facilitative anion exchanger that mediates delivery of 5-methyltetrahydrofolate into mammalian cells. Adequate maternal-fetal transport of folate is necessary for normal embryogenesis. Targeted inactivation of the murine RFC1 gene results in post-implantation embryolethality, but daily folic acid supplementation of pregnant dams prolongs survival of homozygous embryos until mid-gestation. At E10.5 RFC1-/- embryos are developmentally delayed relative to wildtype littermates, have multiple malformations, including neural tube defects, and die due to failure of chorioallantoic fusion. The mesoderm is sparse and disorganized, and there is a marked absence of erythrocytes in yolk sac blood islands. The identification of alterations in gene expression and signaling pathways involved in the observed dysmorphology following inactivation of RFC1-mediated folate transport are the focus of this investigation. Results Affymetrix microarray analysis of the relative gene expression profiles in whole E9.5 RFC1-/- vs. RFC1+/+ embryos identified 200 known genes that were differentially expressed. Major ontology groups included transcription factors (13.04%), and genes involved in transport functions (ion, lipid, carbohydrate) (11.37%). Genes that code for receptors, ligands and interacting proteins in the cubilin-megalin multiligand endocytic receptor complex accounted for 9.36% of the total, followed closely by several genes involved in hematopoiesis (8.03%). The most highly significant gene network identified by Ingenuity™ Pathway analysis included 12 genes in the cubilin-megalin multiligand endocytic receptor complex. Altered expression of these genes was validated by quantitative RT-PCR, and immunohistochemical analysis demonstrated that megalin protein expression disappeared from the visceral yolk sac of RFC1-/- embryos, while cubilin protein was widely misexpressed. Conclusion Inactivation of

  12. ARF6 and GASP-1 are post-endocytic sorting proteins selectively involved in the intracellular trafficking of dopamine D2 receptors mediated by GRK and PKC in transfected cells

    PubMed Central

    Cho, DI; Zheng, M; Min, C; Kwon, KJ; Shin, CY; Choi, HK; Kim, KM

    2013-01-01

    Background and Purpose GPCRs undergo both homologous and heterologous regulatory processes in which receptor phosphorylation plays a critical role. The protein kinases responsible for each pathway are well established; however, other molecular details that characterize each pathway remain unclear. In this study, the molecular mechanisms that determine the differences in the functional roles and intracellular trafficking between homologous and PKC-mediated heterologous internalization pathways for the dopamine D2 receptor were investigated. Experimental Approach All of the S/T residues located within the intracellular loops of D2 receptor were mutated, and the residues responsible for GRK- and PKC-mediated internalization were determined in HEK-293 cells and SH-SY5Y cells. The functional role of receptor internalization and the cellular components that determine the post-endocytic fate of internalized D2 receptors were investigated in the transfected cells. Key Results T134, T225/S228/S229 and S325 were involved in PKC-mediated D2 receptor desensitization. S229 and adjacent S/T residues mediated the PKC-dependent internalization of D2 receptors, which induced down-regulation and desensitization. S/T residues within the second intracellular loop and T225 were the major residues involved in GRK-mediated internalization of D2 receptors, which induced receptor resensitization. ARF6 mediated the recycling of D2 receptors internalized in response to agonist stimulation. In contrast, GASP-1 mediated the down-regulation of D2 receptors internalized in a PKC-dependent manner. Conclusions and Implications GRK- and PKC-mediated internalizations of D2 receptors occur through different intracellular trafficking pathways and mediate distinct functional roles. Distinct S/T residues within D2 receptors and different sorting proteins are involved in the dissimilar regulation of D2 receptors by GRK2 and PKC. PMID:23082996

  13. Enteroviruses harness the cellular endocytic machinery to remodel the host cell cholesterol landscape for effective viral replication

    PubMed Central

    Ilnytska, Olha; Santiana, Marianita; Hsu, Nai-Yun; Du, Wen-Li; Chen, Ying-Han; Viktorova, Ekaterina G.; Belov, Georgy; Brinker, Anita; Storch, Judith; Moore, Christopher; Dixon, Joseph L.; Altan-Bonnet, Nihal

    2013-01-01

    Cholesterol is a critical component of cellular membranes, regulating assembly and function of membrane-based protein/lipid complexes. Many RNA viruses, including enteroviruses, remodel host membranes to generate organelles with unique lipid blueprints on which they assemble replication complexes and synthesize viral RNA. Here we find that clathrin-mediated endocytosis (CME) is harnessed by enteroviruses to traffic cholesterol from the plasma membrane (PM) and extracellular medium to replication organelles where cholesterol then regulates viral polyprotein processing and facilitates genome synthesis. When CME is disrupted, cellular cholesterol pools are instead stored in lipid droplets; cholesterol cannot be trafficked to replication organelles; and replication is inhibited. In contrast, replication is stimulated in cholesterol-elevated cells like those lacking caveolins or those from Niemann-Pick disease patients. Our findings indicate cholesterol as a critical determinant for enteroviral replication and outline roles for the endocytic machinery in both the enteroviral lifecycle and host cell cholesterol homeostasis. PMID:24034614

  14. Rationally Engineering Phototherapy Modules of Eosin-Conjugated Responsive Polymeric Nanocarriers via Intracellular Endocytic pH Gradients.

    PubMed

    Liu, Guhuan; Hu, Jinming; Zhang, Guoying; Liu, Shiyong

    2015-07-15

    Spatiotemporal switching of respective phototherapy modes at the cellular level with minimum side effects and high therapeutic efficacy is a major challenge for cancer phototherapy. Herein we demonstrate how to address this issue by employing photosensitizer-conjugated pH-responsive block copolymers in combination with intracellular endocytic pH gradients. At neutral pH corresponding to extracellular and cytosol milieu, the copolymers self-assemble into micelles with prominently quenched fluorescence emission and low (1)O2 generation capability, favoring a highly efficient photothermal module. Under mildly acidic pH associated with endolysosomes, protonation-triggered micelle-to-unimer transition results in recovered emission and enhanced photodynamic (1)O2 efficiency, which synergistically actuates release of encapsulated drugs, endosomal escape, and photochemical internalization processes. PMID:25514473

  15. The Microtubule-associated Histone Deacetylase 6 (HDAC6) Regulates Epidermal Growth Factor Receptor (EGFR) Endocytic Trafficking and Degradation*

    PubMed Central

    Gao, Ya-sheng; Hubbert, Charlotte C.; Yao, Tso-Pang

    2010-01-01

    Histone deacetylase 6 (HDAC6) is a microtubule-associated deacetylase with tubulin deacetylase activity, and it binds dynein motors. Recent studies revealed that microtubule acetylation affects the affinity and processivity of microtubule motors. These unique properties implicate a role for HDAC6 in intracellular organelle transport. Here, we show that HDAC6 associates with the endosomal compartments and controls epidermal growth factor receptor (EGFR) trafficking and degradation. We found that loss of HDAC6 promoted EGFR degradation. Mechanistically, HDAC6 deficiency did not cause aberrant EGFR internalization and recycling. Rather, it resulted in accelerated segregation of EGFR from early endosomes and premature delivery of EGFR to the late endosomal and lysosomal compartments. The deregulated EGFR endocytic trafficking was accompanied by an increase in microtubule-dependent movement of EGFR-bearing vesicles, revealing a novel regulation of EGFR vesicular trafficking and degradation by the microtubule deacetylase HDAC6. PMID:20133936

  16. Chemical Modulation of Endocytic Sorting Augments Adeno-associated Viral Transduction.

    PubMed

    Berry, Garrett E; Asokan, Aravind

    2016-01-01

    Intracellular trafficking of viruses can be influenced by a variety of inter-connected cellular sorting and degradation pathways involving endo-lysosomal vesicles, the ubiquitin-proteasome system, and autophagy-based or endoplasmic reticulum-associated machinery. In the case of recombinant adeno-associated viruses (AAV), proteasome inhibitors are known to prevent degradation of ubiquitinated AAV capsids, thereby leading to increased nuclear accumulation and transduction. However, the impact of other cellular degradation pathways on AAV trafficking is not well understood. In the current study, we screened a panel of small molecules focused on modulating different cellular degradation pathways and identified eeyarestatin I (EerI) as a novel reagent that enhances AAV transduction. EerI improved AAV transduction by an order of magnitude regardless of vector dose, genome architecture, cell type, or serotype. This effect was preceded by sequestration of AAV within enlarged vesicles that were dispersed throughout the cytoplasm. Specifically, EerI treatment redirected AAV particles toward large vesicles positive for late endosomal (Rab7) and lysosomal (LAMP1) markers. Notably, MG132 and EerI (proteasomal and endoplasmic reticulum-associated degradation inhibitors, respectively) appear to enhance AAV transduction by increasing the intracellular accumulation of viral particles in a mutually exclusive fashion. Taken together, our results expand on potential strategies to redirect recombinant AAV vectors toward more productive trafficking pathways by deregulating cellular degradation mechanisms. PMID:26527686

  17. Dysregulated post-synaptic density and endocytic zone in the amygdala of human heroin and cocaine abusers

    PubMed Central

    Ökvist, Anna; Fagergren, Pernilla; Whittard, John; Garcia-Osta, Ana; Drakenberg, Katarina; Horvath, Monika Cs.; Schmidt, Carl J.; Keller, Eva; Bannon, Michael J.; Hurd, Yasmin L.

    2010-01-01

    Background Glutamatergic transmission in the amygdala is hypothesized as an important mediator of stimulus-reward associations contributing to drug-seeking behavior and relapse. Insight is, however, lacking regarding the amygdala glutamatergic system in human drug abusers. Methods We examined glutamate receptors and scaffolding proteins associated with the post-synaptic density (PSD) of excitatory synapses in the human post-mortem amygdala. mRNA or protein levels were studied in a multi-drug (7 heroin, 8 cocaine, 7 heroin/cocaine and 7 control) or predominant heroin (29 heroin and 15 control) population of subjects. Results The amygdala of drug abusers was characterized by a striking positive correlation (r > 0.8) between AMPA GluA1 and post-synaptic protein-95 (PSD-95) mRNA levels, which was not evident in controls. Structural equation multi-group analysis of protein correlations also identified the relationship between GluA1 and PSD-95 protein levels as the distinguishing feature of abusers. In line with the GluA1—PSD-95 implications of enhanced synaptic plasticity, Homer 1b/c protein expression was significantly increased in both heroin and cocaine users as was its binding partner dynamin-3, localized to the endocytic zone. Furthermore, there was a positive relationship between Homer 1b/c and dynamin-3 in drug abusers that reflected an increase in the direct physical coupling between the proteins. A noted age-related decline of Homer 1b/c—dynamin-3 interactions, as well as GluA1 levels, was blunted in abusers. Conclusions Impairment of key components of the amygdala PSD and coupling to the endocytic zone, critical for the regulation of glutamate receptor cycling, may underlie heightened synaptic plasticity in human drug abusers. PMID:21126734

  18. Porcine Sialoadhesin (CD169/Siglec-1) Is an Endocytic Receptor that Allows Targeted Delivery of Toxins and Antigens to Macrophages

    PubMed Central

    Favoreel, Herman W.; Hoebeke, Inge; Delrue, Iris; Dewerchin, Hannah; Verdonck, Frank; Verhasselt, Bruno; Cox, Eric; Nauwynck, Hans J.

    2011-01-01

    Sialoadhesin is exclusively expressed on specific subpopulations of macrophages. Since sialoadhesin-positive macrophages are involved in inflammatory autoimmune diseases, such as multiple sclerosis, and potentially in the generation of immune responses, targeted delivery of drugs, toxins or antigens via sialoadhesin-specific immunoconjugates may prove a useful therapeutic strategy. Originally, sialoadhesin was characterized as a lymphocyte adhesion molecule, though recently its involvement in internalization of sialic acid carrying pathogens was shown, suggesting that sialoadhesin is an endocytic receptor. In this report, we show that porcine sialoadhesin-specific antibodies and F(ab')2 fragments trigger sialoadhesin internalization, both in primary porcine macrophages and in cells expressing recombinant porcine sialoadhesin. Using chemical inhibitors, double immunofluorescence stainings and dominant-negative constructs, porcine sialoadhesin internalization was shown to be clathrin- and Eps15-dependent and to result in targeting to early endosomes but not lysosomes. Besides characterizing the sialoadhesin endocytosis mechanism, two sialoadhesin-specific immunoconjugates were evaluated. We observed that porcine sialoadhesin-specific immunotoxins efficiently kill sialoadhesin-expressing macrophages. Furthermore, porcine sialoadhesin-specific albumin immunoconjugates were shown to be internalized in macrophages and immunization with these immunoconjugates resulted in a rapid and robust induction of albumin-specific antibodies, this compared to immunization with albumin alone. Together, these data expand sialoadhesin functionality and show that it can function as an endocytic receptor, a feature that cannot only be misused by sialic acid carrying pathogens, but that may also be used for specific targeting of toxins or antigens to sialoadhesin-expressing macrophages. PMID:21359217

  19. Cell entry and exit by periodontal pathogen via recycling pathway.

    PubMed

    Takeuchi, Hiroki; Furuta, Nobumichi; Amano, Atsuo

    2011-09-01

    In the oral cavity, gingival epithelial cell (GEC) layers function as an innate host defense system to prevent intrusion by periodontal bacteria. Nevertheless, Porphyromonas gingivalis, the most well-known periodontal pathogen, can enter GECs and pass through the epithelial barrier into deeper tissues. An intracellular location is considered advantageous for bacteria to escape from immune surveillance by the host as well as antibiotic pressure, leading to intracellular persistence, multiplication and dissemination to adjacent tissues. P. gingivalis are invaginated by gingival epithelial cells via the endocytic pathway, and some intracellular bacteria are sorted to lytic compartments, including autolysosomes and late endosomes/lysosomes, while a considerable number of the remaining organisms are sorted to Rab11- and RalA-positive recycling endosomes, followed by bacterial exit from the cells. Exited bacteria can re-enter fresh cells. However, dominant negative forms and RNAi-knockdown of Rab11, RalA and exocyst complex subunits (Sec5, Sec6 and Exo84) significantly disturb the exit of P. gingivalis. These are the first known results to show that the endocytic recycling pathway mediates bacterial exit from infected cells to neighboring cells and may provide important information regarding the exit mechanisms of various invasive pathogens. PMID:22046471

  20. Parkin Modulates Endosomal Organization and Function of the Endo-Lysosomal Pathway.

    PubMed

    Song, Pingping; Trajkovic, Katarina; Tsunemi, Taiji; Krainc, Dimitri

    2016-02-24

    Mutations in PARK2 (parkin), which encodes Parkin protein, an E3 ubiquitin ligase, are associated with autosomal recessive early-onset Parkinson's disease (PD). While several studies implicated Parkin in the regulation of mitophagy and proteasomal degradation, the precise mechanism leading to neurodegeneration upon Parkin loss of function remains incompletely understood. In this study, we found that Parkin modulates the endocytic pathway through the regulation of endosomal structure and function. We showed that loss of Parkin function led to decreased endosomal tubulation and membrane association of vesicle protein sorting 35 (VPS35) and sorting nexin 1 (SNX1), as well as decreased mannose 6 phosphate receptor (M6PR), suggesting the impairment of retromer pathway in Parkin-deficient cells. We also found increased formation of intraluminal vesicles coupled with enhanced release of exosomes in the presence of mutant Parkin. To elucidate the molecular mechanism of these alterations in the endocytic pathway in Parkin-deficient cells, we found that Parkin regulates the levels and activity of Rab7 by promoting its ubiquitination on lysine 38 residue. Both endogenous Rab7 in Parkin-deficient cells and overexpressed K38 R-Rab7 mutant displayed decreased effector binding and membrane association. Furthermore, overexpression of K38R-Rab7 in HEK293 cells phenocopied the increased secretion of exosomes observed in Parkin-deficient cells, suggesting that Rab7 deregulation may be at least partially responsible for the endocytic phenotype observed in Parkin-deficient cells. These findings establish a role for Parkin in regulating the endo-lysosomal pathway and retromer function and raise the possibility that alterations in these pathways contribute to the development of pathology in Parkin-linked Parkinson's disease. PMID:26911690

  1. Direct Observation of α-Synuclein Amyloid Aggregates in Endocytic Vesicles of Neuroblastoma Cells.

    PubMed

    Apetri, Mihaela M; Harkes, Rolf; Subramaniam, Vinod; Canters, Gerard W; Schmidt, Thomas; Aartsma, Thijs J

    2016-01-01

    Aggregation of α-synuclein has been linked to both familial and sporadic Parkinson's disease. Recent studies suggest that α-synuclein aggregates may spread from cell to cell and raise questions about the propagation of neurodegeneration. While continuous progress has been made characterizing α-synuclein aggregates in vitro, there is a lack of information regarding the structure of these species inside the cells. Here, we use confocal fluorescence microscopy in combination with direct stochastic optical reconstruction microscopy, dSTORM, to investigate α-synuclein uptake when added exogenously to SH-SY5Y neuroblastoma cells, and to probe in situ morphological features of α-synuclein aggregates with near nanometer resolution. We demonstrate that using dSTORM, it is possible to follow noninvasively the uptake of extracellularly added α-synuclein aggregates by the cells. Once the aggregates are internalized, they move through the endosomal pathway and accumulate in lysosomes to be degraded. Our dSTORM data show that α-synuclein aggregates remain assembled after internalization and they are shortened as they move through the endosomal pathway. No further aggregation was observed inside the lysosomes as speculated in the literature, nor in the cytoplasm of the cells. Our study thus highlights the super-resolution capability of dSTORM to follow directly the endocytotic uptake of extracellularly added amyloid aggregates and to probe the morphology of in situ protein aggregates even when they accumulate in small vesicular compartments. PMID:27105068

  2. Direct Observation of α-Synuclein Amyloid Aggregates in Endocytic Vesicles of Neuroblastoma Cells

    PubMed Central

    Subramaniam, Vinod; Canters, Gerard W.; Schmidt, Thomas; Aartsma, Thijs J.

    2016-01-01

    Aggregation of α-synuclein has been linked to both familial and sporadic Parkinson’s disease. Recent studies suggest that α-synuclein aggregates may spread from cell to cell and raise questions about the propagation of neurodegeneration. While continuous progress has been made characterizing α-synuclein aggregates in vitro, there is a lack of information regarding the structure of these species inside the cells. Here, we use confocal fluorescence microscopy in combination with direct stochastic optical reconstruction microscopy, dSTORM, to investigate α-synuclein uptake when added exogenously to SH-SY5Y neuroblastoma cells, and to probe in situ morphological features of α-synuclein aggregates with near nanometer resolution. We demonstrate that using dSTORM, it is possible to follow noninvasively the uptake of extracellularly added α-synuclein aggregates by the cells. Once the aggregates are internalized, they move through the endosomal pathway and accumulate in lysosomes to be degraded. Our dSTORM data show that α-synuclein aggregates remain assembled after internalization and they are shortened as they move through the endosomal pathway. No further aggregation was observed inside the lysosomes as speculated in the literature, nor in the cytoplasm of the cells. Our study thus highlights the super-resolution capability of dSTORM to follow directly the endocytotic uptake of extracellularly added amyloid aggregates and to probe the morphology of in situ protein aggregates even when they accumulate in small vesicular compartments. PMID:27105068

  3. Secretory Pathway of Trypanosomatid Parasites

    PubMed Central

    McConville, Malcolm J.; Mullin, Kylie A.; Ilgoutz, Steven C.; Teasdale, Rohan D.

    2002-01-01

    The Trypanosomatidae comprise a large group of parasitic protozoa, some of which cause important diseases in humans. These include Trypanosoma brucei (the causative agent of African sleeping sickness and nagana in cattle), Trypanosoma cruzi (the causative agent of Chagas' disease in Central and South America), and Leishmania spp. (the causative agent of visceral and [muco]cutaneous leishmaniasis throughout the tropics and subtropics). The cell surfaces of these parasites are covered in complex protein- or carbohydrate-rich coats that are required for parasite survival and infectivity in their respective insect vectors and mammalian hosts. These molecules are assembled in the secretory pathway. Recent advances in the genetic manipulation of these parasites as well as progress with the parasite genome projects has greatly advanced our understanding of processes that underlie secretory transport in trypanosomatids. This article provides an overview of the organization of the trypanosomatid secretory pathway and connections that exist with endocytic organelles and multiple lytic and storage vacuoles. A number of the molecular components that are required for vesicular transport have been identified, as have some of the sorting signals that direct proteins to the cell surface or organelles in the endosome-vacuole system. Finally, the subcellular organization of the major glycosylation pathways in these parasites is reviewed. Studies on these highly divergent eukaryotes provide important insights into the molecular processes underlying secretory transport that arose very early in eukaryotic evolution. They also reveal unusual or novel aspects of secretory transport and protein glycosylation that may be exploited in developing new antiparasite drugs. PMID:11875130

  4. A Kinase Inhibitor Screen Reveals Protein Kinase C-dependent Endocytic Recycling of ErbB2 in Breast Cancer Cells*

    PubMed Central

    Bailey, Tameka A.; Luan, Haitao; Tom, Eric; Bielecki, Timothy Alan; Mohapatra, Bhopal; Ahmad, Gulzar; George, Manju; Kelly, David L.; Natarajan, Amarnath; Raja, Srikumar M.; Band, Vimla; Band, Hamid

    2014-01-01

    ErbB2 overexpression drives oncogenesis in 20–30% cases of breast cancer. Oncogenic potential of ErbB2 is linked to inefficient endocytic traffic into lysosomes and preferential recycling. However, regulation of ErbB2 recycling is incompletely understood. We used a high-content immunofluorescence imaging-based kinase inhibitor screen on SKBR-3 breast cancer cells to identify kinases whose inhibition alters the clearance of cell surface ErbB2 induced by Hsp90 inhibitor 17-AAG. Less ErbB2 clearance was observed with broad-spectrum PKC inhibitor Ro 31-8220. A similar effect was observed with Go 6976, a selective inhibitor of classical Ca2+-dependent PKCs (α, β1, βII, and γ). PKC activation by PMA promoted surface ErbB2 clearance but without degradation, and ErbB2 was observed to move into a juxtanuclear compartment where it colocalized with PKC-α and PKC-δ together with the endocytic recycling regulator Arf6. PKC-α knockdown impaired the juxtanuclear localization of ErbB2. ErbB2 transit to the recycling compartment was also impaired upon PKC-δ knockdown. PMA-induced Erk phosphorylation was reduced by ErbB2 inhibitor lapatinib, as well as by knockdown of PKC-δ but not that of PKC-α. Our results suggest that activation of PKC-α and -δ mediates a novel positive feedback loop by promoting ErbB2 entry into the endocytic recycling compartment, consistent with reported positive roles for these PKCs in ErbB2-mediated tumorigenesis. As the endocytic recycling compartment/pericentrion has emerged as a PKC-dependent signaling hub for G-protein-coupled receptors, our findings raise the possibility that oncogenesis by ErbB2 involves previously unexplored PKC-dependent endosomal signaling. PMID:25225290

  5. HARE-Mediated Endocytosis of Hyaluronan and Heparin Is Targeted by Different Subsets of Three Endocytic Motifs

    PubMed Central

    Pandey, Madhu S.; Harris, Edward N.; Weigel, Paul H.

    2015-01-01

    The hyaluronan (HA) receptor for endocytosis (HARE) is a multifunctional recycling clearance receptor for 14 different ligands, including HA and heparin (Hep), which bind to discrete nonoverlapping sites. Four different functional endocytic motifs (M) in the cytoplasmic domain (CD) target coated pit mediated uptake: (YSYFRI2485 (M1), FQHF2495 (M2), NPLY2519 (M3), and DPF2534 (M4)). We previously found (Pandey et al. J. Biol. Chem. 283, 21453, 2008) that M1, M2, and M3 mediate endocytosis of HA. Here we assessed the ability of HARE variants with a single-motif deletion or containing only a single motif to endocytose HA or Hep. Single-motif deletion variants lacking M1, M3, or M4 (a different subset than involved in HA uptake) showed decreased Hep endocytosis, although M3 was the most active; the remaining redundant motifs did not compensate for loss of other motifs. Surprisingly, a HARE CD variant with only M3 internalized both HA and Hep, whereas variants with either M2 or M4 alone did not endocytose either ligand. Internalization of HA and Hep by HARE CD mutants was dynamin-dependent and was inhibited by hyperosmolarity, confirming clathrin-mediated endocytosis. The results indicate a complicated relationship among multiple CD motifs that target coated pit uptake and a more fundamental role for motif M3. PMID:25883656

  6. Direct Delivery of Antigens to Dendritic Cells via Antibodies Specific for Endocytic Receptors as a Promising Strategy for Future Therapies

    PubMed Central

    Lehmann, Christian H. K.; Heger, Lukas; Heidkamp, Gordon F.; Baranska, Anna; Lühr, Jennifer J.; Hoffmann, Alana; Dudziak, Diana

    2016-01-01

    Dendritic cells (DCs) are the most potent professional antigen presenting cells and are therefore indispensable for the control of immunity. The technique of antibody mediated antigen targeting to DC subsets has been the basis of intense research for more than a decade. Many murine studies have utilized this approach of antigen delivery to various kinds of endocytic receptors of DCs both in vitro and in vivo. Today, it is widely accepted that different DC subsets are important for the induction of select immune responses. Nevertheless, many questions still remain to be answered, such as the actual influence of the targeted receptor on the initiation of the immune response to the delivered antigen. Further efforts to better understand the induction of antigen-specific immune responses will support the transfer of this knowledge into novel treatment strategies for human diseases. In this review, we will discuss the state-of-the-art aspects of the basic principles of antibody mediated antigen targeting approaches. A table will also provide a broad overview of the latest studies using antigen targeting including addressed DC subset, targeted receptors, outcome, and applied coupling techniques. PMID:27043640

  7. Characterization of Salmonella-induced filaments (Sifs) reveals a delayed interaction between Salmonella-containing vacuoles and late endocytic compartments.

    PubMed

    Brumell, J H; Tang, P; Mills, S D; Finlay, B B

    2001-09-01

    Salmonella typhimurium is a facultative intracellular pathogen that colonizes host cells throughout the course of infection. A unique feature of this pathogen is its ability to enter into (invade) epithelial cells and elongate the vacuole within which it resides into tubular structures called Salmonella-induced filaments (Sifs). In this study we sought to characterize the mechanism of Sif formation by immunofluorescence analysis using subcellular markers. The late endosomal lipid lysobisphosphatidic acid associated in a punctate pattern with the Salmonella-containing vacuole, starting 90 min after infection and increasing thereafter. Lysobisphosphatidic acid-rich vesicles were also found to interact with Sifs, at numerous sites along the tubules. Similarly, cholesterol-rich vesicles were also found in association with intracellular bacteria and Sifs. The lysosomal hydrolase cathepsin D was present in Sifs, both in a punctate pattern and, at later times, predominantly in an uninterrupted linear pattern. Rab7 associated with Sifs and expression of the N125I dominant negative mutant of this GTPase inhibited Sif formation. Transfection of HeLa cells with a vector encoding SifA fused to the green fluorescent protein caused swelling and aggregation of lysobisphosphatidic acid-containing compartments, suggesting that this virulence factor directs membrane fusion events involving late endosomes. Our findings demonstrate that Sif formation involves fusion of late endocytic compartments with the Salmonella-containing vacuole, and suggest that SifA modulates this event. PMID:11555418

  8. Endocytic function is critical for influenza A virus infection via DC-SIGN and L-SIGN

    PubMed Central

    Gillespie, Leah; Roosendahl, Paula; Ng, Wy Ching; Brooks, Andrew G.; Reading, Patrick C.; Londrigan, Sarah L.

    2016-01-01

    The ubiquitous presence of cell-surface sialic acid (SIA) has complicated efforts to identify specific transmembrane glycoproteins that function as bone fide entry receptors for influenza A virus (IAV) infection. The C-type lectin receptors (CLRs) DC-SIGN (CD209) and L-SIGN (CD209L) enhance IAV infection however it is not known if they act as attachment factors, passing virions to other unknown receptors for virus entry, or as authentic entry receptors for CLR-mediated virus uptake and infection. Sialic acid-deficient Lec2 Chinese Hamster Ovary (CHO) cell lines were resistant to IAV infection whereas expression of DC-SIGN/L-SIGN restored susceptibility of Lec2 cells to pH- and dynamin-dependent infection. Moreover, Lec2 cells expressing endocytosis-defective DC-SIGN/L-SIGN retained capacity to bind IAV but showed reduced susceptibility to infection. These studies confirm that DC-SIGN and L-SIGN are authentic endocytic receptors for IAV entry and infection. PMID:26763587

  9. Influenza Virus Induces Cholesterol-Enriched Endocytic Recycling Compartments for Budozone Formation via Cell Cycle-Independent Centrosome Maturation

    PubMed Central

    Harada, Yoshimi; Osari, Suguru; Nagata, Kyosuke

    2015-01-01

    Influenza virus particles are assembled at the plasma membrane in concert with incorporation of the virus genome, but the details of its spatio-temporal regulation are not understood. Here we showed that influenza virus infection induces the assembly of pericentrosomal endocytic recycling compartment (ERC) through the activation of Rab11a GTPase and cell cycle-independent maturation of centrosome by YB-1, a multifunctional protein that is involved in mitotic division, RNA metabolism and tumorigenesis. YB-1 is recruited to the centrosome in infected cells and is required for anchoring microtubules to the centrosome. We also found that viral infection accumulates cholesterol in ERC and is dependent on YB-1. Depletion of YB-1 shows reduced cholesterol-enriched ERC and prevented budozone formation at the plasma membrane. These results suggest that cholesterol in recycling endosomes, which are emanated from ERC, may trigger the virus assembly concomitantly with the packaging of the virus genome. We propose that the virus genome is transported to the plasma membrane by cholesterol-enriched recycling endosomes through cell cycle-independent activation of the centrosome by YB-1. PMID:26575487

  10. Endocytic function is critical for influenza A virus infection via DC-SIGN and L-SIGN.

    PubMed

    Gillespie, Leah; Roosendahl, Paula; Ng, Wy Ching; Brooks, Andrew G; Reading, Patrick C; Londrigan, Sarah L

    2016-01-01

    The ubiquitous presence of cell-surface sialic acid (SIA) has complicated efforts to identify specific transmembrane glycoproteins that function as bone fide entry receptors for influenza A virus (IAV) infection. The C-type lectin receptors (CLRs) DC-SIGN (CD209) and L-SIGN (CD209L) enhance IAV infection however it is not known if they act as attachment factors, passing virions to other unknown receptors for virus entry, or as authentic entry receptors for CLR-mediated virus uptake and infection. Sialic acid-deficient Lec2 Chinese Hamster Ovary (CHO) cell lines were resistant to IAV infection whereas expression of DC-SIGN/L-SIGN restored susceptibility of Lec2 cells to pH- and dynamin-dependent infection. Moreover, Lec2 cells expressing endocytosis-defective DC-SIGN/L-SIGN retained capacity to bind IAV but showed reduced susceptibility to infection. These studies confirm that DC-SIGN and L-SIGN are authentic endocytic receptors for IAV entry and infection. PMID:26763587

  11. Integrin-beta3 clusters recruit clathrin-mediated endocytic machinery in the absence of traction force

    PubMed Central

    Yu, Cheng-han; Rafiq, Nisha Bte Mohd; Cao, Fakun; Zhou, Yuhuan; Krishnasamy, Anitha; Biswas, Kabir Hassan; Ravasio, Andrea; Chen, Zhongwen; Wang, Yu-Hsiu; Kawauchi, Keiko; Jones, Gareth E.; Sheetz, Michael P.

    2015-01-01

    The turnover of integrin receptors is critical for cell migration and adhesion dynamics. Here we find that force development at integrins regulates adaptor protein recruitment and endocytosis. Using mobile RGD (Arg-Gly-Asp) ligands on supported lipid membranes (RGD membranes) and rigid RGD ligands on glass (RGD-glass), we find that matrix force-dependent integrin signals block endocytosis. Dab2, an adaptor protein of clathrin-mediated endocytosis, is not recruited to activated integrin-beta3 clusters on RGD-glass; however, it is recruited to integrin-mediated adhesions on RGD membranes. Further, when force generation is inhibited on RGD-glass, Dab2 binds to integrin-beta3 clusters. Dab2 binding to integrin-beta3 excludes other adhesion-related adaptor proteins, such as talin. The clathrin-mediated endocytic machinery combines with Dab2 to facilitate the endocytosis of RGD-integrin-beta3 clusters. From these observations, we propose that loss of traction force on ligand-bound integrin-beta3 causes recruitment of Dab2/clathrin, resulting in endocytosis of integrins. PMID:26507506

  12. GGA3 mediates TrkA endocytic recycling to promote sustained Akt phosphorylation and cell survival

    PubMed Central

    Li, Xuezhi; Lavigne, Pierre; Lavoie, Christine

    2015-01-01

    Although TrkA postendocytic sorting significantly influences neuronal cell survival and differentiation, the molecular mechanism underlying TrkA receptor sorting in the recycling or degradation pathways remains poorly understood. Here we demonstrate that Golgi-localized, γ adaptin-ear–containing ADP ribosylation factor-binding protein 3 (GGA3) interacts directly with the TrkA cytoplasmic tail through an internal DXXLL motif and mediates the functional recycling of TrkA to the plasma membrane. We find that GGA3 depletion by siRNA delays TrkA recycling, accelerates TrkA degradation, attenuates sustained NGF-induced Akt activation, and reduces cell survival. We also show that GGA3’s effect on TrkA recycling is dependent on the activation of Arf6. This work identifies GGA3 as a key player in a novel DXXLL-mediated endosomal sorting machinery that targets TrkA to the plasma membrane, where it prolongs the activation of Akt signaling and survival responses. PMID:26446845

  13. Phorbol 12-myristate 13-acetate-induced endocytosis of the Na-K-2Cl cotransporter in MDCK cells is associated with a clathrin-dependent pathway

    PubMed Central

    Mykoniatis, Andreas; Shen, Le; Fedor-Chaiken, Mary; Tang, Jun; Tang, Xu; Worrell, Roger T.; Delpire, Eric; Turner, Jerrold R.; Matlin, Karl S.

    2010-01-01

    In secretory epithelial cells, the basolateral Na+-K+-2Cl− cotransporter (NKCC1) plays a major role in salt and fluid secretion. Our laboratory has identified NKCC1 surface expression as an important regulatory mechanism for Cl− secretion in the colonic crypt cell line T84, a process also present in native human colonic crypts. We previously showed that activation of protein kinase C (PKC) by carbachol and phorbol 12-myristate 13-acetate (PMA) decreases NKCC1 surface expression in T84 cells. However, the specific endocytic entry pathway has not been defined. We used a Madin-Darby canine kidney (MDCK) cell line stably transfected with enhanced green fluorescent protein (EGFP)-NKCC1 to map NKCC1 entry during PMA exposure. At given times, we fixed and stained the cells with specific markers (e.g., dynamin II, clathrin heavy chain, and caveolin-1). We also used chlorpromazine, methyl-β-cyclodextrin, amiloride, and dynasore, blockers of the clathrin, caveolin, and macropinocytosis pathways and the vesicle “pinchase” dynamin, respectively. We found that PMA caused dose- and time-dependent NKCC1 endocytosis. After 2.5 min of PMA exposure, ∼80% of EGFP-NKCC1 endocytic vesicles colocalized with clathrin and ∼40% colocalized with dynamin II and with the transferrin receptor, the uptake of which is also mediated by clathrin-coated vesicles. We did not observe significant colocalization of EGFP-NKCC1 endocytic vesicles with caveolin-1, a marker of the caveolae-mediated endocytic pathway. We quantified the effect of each inhibitor on PMA-induced EGFP-NKCC1 endocytosis and found that only chlorpromazine and dynasore caused significant inhibition compared with the untreated control (61% and 25%, respectively, at 2.5 min). Together, these results strongly support the conclusion that PMA-stimulated NKCC1 endocytosis is associated with a clathrin pathway. PMID:19864322

  14. A TOCA/CDC-42/PAR/WAVE functional module required for retrograde endocytic recycling

    PubMed Central

    Bai, Zhiyong; Grant, Barth D.

    2015-01-01

    Endosome-to-Golgi transport is required for the function of many key membrane proteins and lipids, including signaling receptors, small-molecule transporters, and adhesion proteins. The retromer complex is well-known for its role in cargo sorting and vesicle budding from early endosomes, in most cases leading to cargo fusion with the trans-Golgi network (TGN). Transport from recycling endosomes to the TGN has also been reported, but much less is understood about the molecules that mediate this transport step. Here we provide evidence that the F-BAR domain proteins TOCA-1 and TOCA-2 (Transducer of Cdc42 dependent actin assembly), the small GTPase CDC-42 (Cell division control protein 42), associated polarity proteins PAR-6 (Partitioning defective 6) and PKC-3/atypical protein kinase C, and the WAVE actin nucleation complex mediate the transport of MIG-14/Wls and TGN-38/TGN38 cargo proteins from the recycling endosome to the TGN in Caenorhabditis elegans. Our results indicate that CDC-42, the TOCA proteins, and the WAVE component WVE-1 are enriched on RME-1–positive recycling endosomes in the intestine, unlike retromer components that act on early endosomes. Furthermore, we find that retrograde cargo TGN-38 is trapped in early endosomes after depletion of SNX-3 (a retromer component) but is mainly trapped in recycling endosomes after depletion of CDC-42, indicating that the CDC-42–associated complex functions after retromer in a distinct organelle. Thus, we identify a group of interacting proteins that mediate retrograde recycling, and link these proteins to a poorly understood trafficking step, recycling endosome-to-Golgi transport. We also provide evidence for the physiological importance of this pathway in WNT signaling. PMID:25775511

  15. Uptake and intracytoplasmic storage of pigmented particles by human CD34+ stromal cells/telocytes: endocytic property of telocytes

    PubMed Central

    Díaz-Flores, Lucio; Gutiérrez, Ricardo; García, Mª Pino; Sáez, Francisco J; Aparicio, Fernando; Díaz-Flores, Lucio; Madrid, Juan F

    2014-01-01

    We studied the phagocytic-like capacity of human CD34+ stromal cells/telocytes (TCs). For this, we examined segments of the colon after injection of India ink to help surgeons localize lesions identified at endoscopy. Our results demonstrate that CD34+ TCs have endocytic properties (phagocytic-like TCs: phTCs), with the capacity to uptake and store India ink particles. phTCs conserve the characteristics of TCs (long, thin, bipolar or multipolar, moniliform cytoplasmic processes/telopodes, with linear distribution of the pigment) and maintain their typical distribution. Likewise, they are easily distinguished from pigment-loaded macrophages (CD68+ macrophages, with oval morphology and coarse granules of pigment clustered in their cytoplasm). A few c-kit/CD117+ interstitial cells of Cajal also incorporate pigment and may conserve the phagocytic-like property of their probable TC precursors. CD34+ stromal cells in other locations (skin and periodontal tissues) also have the phagocytic-like capacity to uptake and store pigments (hemosiderin, some components of dental amalgam and melanin). This suggests a function of TCs in general, which may be related to the transfer of macromolecules in these cells. Our ultrastructural observation of melanin-storing stromal cells with characteristics of TCs (telopodes with dichotomous branching pattern) favours this possibility. In conclusion, intestinal TCs have a phagocytic-like property, a function that may be generalized to TCs in other locations. This function (the ability to internalize small particles), together with the capacity of these cells to release extracellular vesicles with macromolecules, could close the cellular bidirectional cooperative circle of informative exchange and intercellular interactions. PMID:25266164

  16. Oxidation by Neutrophils-Derived HOCl Increases Immunogenicity of Proteins by Converting Them into Ligands of Several Endocytic Receptors Involved in Antigen Uptake by Dendritic Cells and Macrophages

    PubMed Central

    Biedroń, Rafał; Konopiński, Maciej K.; Marcinkiewicz, Janusz; Józefowski, Szczepan

    2015-01-01

    The initiation of adaptive immune responses to protein antigens has to be preceded by their uptake by antigen presenting cells and intracellular proteolytic processing. Paradoxically, endocytic receptors involved in antigen uptake do not bind the majority of proteins, which may be the main reason why purified proteins stimulate at most weak immune responses. A shared feature of different types of adjuvants, capable of boosting immunogenicity of protein vaccines, is their ability to induce acute inflammation, characterized by early influx of activated neutrophils. Neutrophils are also rapidly recruited to sites of tissue injury or infection. These cells are the source of potent oxidants, including hypochlorous acid (HOCl), causing oxidation of proteins present in inflammatory foci. We demonstrate that oxidation of proteins by endogenous, neutrophils-derived HOCl increases their immunogenicity. Upon oxidation, different, randomly chosen simple proteins (yeast alcohol dehydrogenase, human and bovine serum albumin) and glycoproteins (human apo-transferrin, ovalbumin) gain the ability to bind with high affinity to several endocytic receptors on antigen presenting cells, which seems to be the major mechanism of their increased immunogenicity. The mannose receptor (CD206), scavenger receptors A (CD204) and CD36 were responsible for the uptake and presentation of HOCl-modified proteins by murine dendritic cells and macrophages. Other scavenger receptors, SREC-I and LOX-1, as well as RAGE were also able to bind HOCl-modified proteins, but they did not contribute significantly to these ligands uptake by dendritic cells because they were either not expressed or exhibited preference for more heavily oxidised proteins. Our results indicate that oxidation by neutrophils-derived HOCl may be a physiological mechanism of conferring immunogenicity on proteins which in their native forms do not bind to endocytic receptors. This mechanism might enable the immune system to detect

  17. Oxidation by neutrophils-derived HOCl increases immunogenicity of proteins by converting them into ligands of several endocytic receptors involved in antigen uptake by dendritic cells and macrophages.

    PubMed

    Biedroń, Rafał; Konopiński, Maciej K; Marcinkiewicz, Janusz; Józefowski, Szczepan

    2015-01-01

    The initiation of adaptive immune responses to protein antigens has to be preceded by their uptake by antigen presenting cells and intracellular proteolytic processing. Paradoxically, endocytic receptors involved in antigen uptake do not bind the majority of proteins, which may be the main reason why purified proteins stimulate at most weak immune responses. A shared feature of different types of adjuvants, capable of boosting immunogenicity of protein vaccines, is their ability to induce acute inflammation, characterized by early influx of activated neutrophils. Neutrophils are also rapidly recruited to sites of tissue injury or infection. These cells are the source of potent oxidants, including hypochlorous acid (HOCl), causing oxidation of proteins present in inflammatory foci. We demonstrate that oxidation of proteins by endogenous, neutrophils-derived HOCl increases their immunogenicity. Upon oxidation, different, randomly chosen simple proteins (yeast alcohol dehydrogenase, human and bovine serum albumin) and glycoproteins (human apo-transferrin, ovalbumin) gain the ability to bind with high affinity to several endocytic receptors on antigen presenting cells, which seems to be the major mechanism of their increased immunogenicity. The mannose receptor (CD206), scavenger receptors A (CD204) and CD36 were responsible for the uptake and presentation of HOCl-modified proteins by murine dendritic cells and macrophages. Other scavenger receptors, SREC-I and LOX-1, as well as RAGE were also able to bind HOCl-modified proteins, but they did not contribute significantly to these ligands uptake by dendritic cells because they were either not expressed or exhibited preference for more heavily oxidised proteins. Our results indicate that oxidation by neutrophils-derived HOCl may be a physiological mechanism of conferring immunogenicity on proteins which in their native forms do not bind to endocytic receptors. This mechanism might enable the immune system to detect

  18. Raft endocytosis of AMF regulates mitochondrial dynamics through Rac1 signaling and the Gp78 ubiquitin ligase.

    PubMed

    Shankar, Jay; Kojic, Liliana D; St-Pierre, Pascal; Wang, Peter T C; Fu, Min; Joshi, Bharat; Nabi, Ivan R

    2013-08-01

    Gp78 is a cell surface receptor that also functions as an E3 ubiquitin ligase in the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway. The Gp78 ligand, the glycolytic enzyme phosphoglucose isomerase (PGI; also called autocrine motility factor, AMF), functions as a cytokine upon secretion by tumor cells. AMF is internalized through a PI3K- and dynamin-dependent raft endocytic pathway to the smooth ER; however, the relationship between AMF and Gp78 ubiquitin ligase activity remains unclear. AMF uptake to the smooth ER is inhibited by the dynamin inhibitor, dynasore, is reduced in Gp78 knockdown cells and induces the dynamin-dependent downregulation of its cell surface receptor. AMF uptake is Rac1-dependent and is inhibited by expression of dominant-negative Rac1 and the Rac1 inhibitor NSC23766, and is therefore distinct from Cdc42- and RhoA-dependent raft endocytic pathways. AMF stimulates Rac1 activation, but this is reduced by dynasore treatment and is absent in Gp78-knockdown cells; therefore, AMF activities require Gp78-mediated endocytosis. AMF also prevents Gp78-induced degradation of the mitochondrial fusion proteins, mitofusin 1 and 2 in a dynamin-, Rac1- and phosphoinositide 3-kinase (PI3K)-dependent manner. Gp78 induces mitochondrial clustering and fission in a manner dependent on GP78 ubiquitin ligase activity, and this is also reversed by uptake of AMF. The raft-dependent endocytosis of AMF, therefore, promotes Rac1-PI3K signaling that feeds back to promote AMF endocytosis and also inhibits the ability of Gp78 to target the mitofusins for degradation, thereby preventing Gp78-dependent mitochondrial fission. Through regulation of an ER-localized ubiquitin ligase, the raft-dependent endocytosis of AMF represents an extracellular regulator of mitochondrial fusion and dynamics. PMID:23690547

  19. The Viral G Protein-Coupled Receptor ORF74 Hijacks β-Arrestins for Endocytic Trafficking in Response to Human Chemokines

    PubMed Central

    de Munnik, Sabrina M.; Kooistra, Albert J.; van Offenbeek, Jody; Nijmeijer, Saskia; de Graaf, Chris; Smit, Martine J.; Leurs, Rob; Vischer, Henry F.

    2015-01-01

    Kaposi’s sarcoma-associated herpesvirus-infected cells express the virally encoded G protein-coupled receptor ORF74. Although ORF74 is constitutively active, it binds human CXC chemokines that modulate this basal activity. ORF74-induced signaling has been demonstrated to underlie the development of the angioproliferative tumor Kaposi’s sarcoma. Whereas G protein-dependent signaling of ORF74 has been the subject of several studies, the interaction of this viral GPCR with β-arrestins has hitherto not been investigated. Bioluminescence resonance energy transfer experiments demonstrate that ORF74 recruits β-arrestins and subsequently internalizes in response to human CXCL1 and CXCL8, but not CXCL10. Internalized ORF74 traffics via early endosomes to recycling and late endosomes. Site-directed mutagenesis and homology modeling identified four serine and threonine residues at the distal end of the intracellular carboxyl-terminal of ORF74 that are required for β-arrestin recruitment and subsequent endocytic trafficking. Hijacking of the human endocytic trafficking machinery is a previously unrecognized action of ORF74. PMID:25894435

  20. Entry Pathways of Herpes Simplex Virus Type 1 into Human Keratinocytes Are Dynamin- and Cholesterol-Dependent

    PubMed Central

    Hsu, Mei-Ju; Rixon, Frazer J.; Knebel-Mörsdorf, Dagmar

    2011-01-01

    Herpes simplex virus type 1 (HSV-1) can enter cells via endocytic pathways or direct fusion at the plasma membrane depending on the cell line and receptor(s). Most studies into virus entry have used cultured fibroblasts but since keratinocytes represent the primary entry site for HSV-1 infection in its human host, we initiated studies to characterize the entry pathway of HSV-1 into human keratinocytes. Electron microscopy studies visualized free capsids in the cytoplasm and enveloped virus particles in vesicles suggesting viral uptake both by direct fusion at the plasma membrane and by endocytic vesicles. The ratio of the two entry modes differed in primary human keratinocytes and in the keratinocyte cell line HaCaT. Inhibitor studies further support a role for endocytosis during HSV-1 entry. Infection was inhibited by the cholesterol-sequestering drug methyl-β-cyclodextrin, which demonstrates the requirement for host cholesterol during virus entry. Since the dynamin-specific inhibitor dynasore and overexpression of a dominant-negative dynamin mutant blocked infection, we conclude that the entry pathways into keratinocytes are dynamin-mediated. Electron microscopy studies confirmed that virus uptake is completely blocked when the GTPase activity of dynamin is inhibited. Ex vivo infection of murine epidermis that was treated with dynasore further supports the essential role of dynamin during entry into the epithelium. Thus, we conclude that HSV-1 can enter human keratinocytes by alternative entry pathways that require dynamin and host cholesterol. PMID:22022400

  1. Advances in analysis of low signal-to-noise images link dynamin and AP2 to the functions of an endocytic checkpoint.

    PubMed

    Aguet, François; Antonescu, Costin N; Mettlen, Marcel; Schmid, Sandra L; Danuser, Gaudenz

    2013-08-12

    Numerous endocytic accessory proteins (EAPs) mediate assembly and maturation of clathrin-coated pits (CCPs) into cargo-containing vesicles. Analysis of EAP function through bulk measurement of cargo uptake has been hampered due to potential redundancy among EAPs and, as we show here, the plasticity and resilience of clathrin-mediated endocytosis (CME). Instead, EAP function is best studied by uncovering the correlation between variations in EAP association to individual CCPs and the resulting variations in maturation. However, most EAPs bind to CCPs in low numbers, making the measurement of EAP association via fused fluorescent reporters highly susceptible to detection errors. Here, we present a framework for unbiased measurement of EAP recruitment to CCPs and their direct effects on CCP dynamics. We identify dynamin and the EAP-binding α-adaptin appendage domain of the AP2 adaptor as switches in a regulated, multistep maturation process and provide direct evidence for a molecular checkpoint in CME. PMID:23891661

  2. Endocytosis and toxicity of clostridial binary toxins depend on a clathrin-independent pathway regulated by Rho-GDI.

    PubMed

    Gibert, Maryse; Monier, Marie-Noëlle; Ruez, Richard; Hale, Martha L; Stiles, Bradley G; Benmerah, Alexandre; Johannes, Ludger; Lamaze, Christophe; Popoff, Michel R

    2011-01-01

    Clostridial binary toxins, such as Clostridium perfringens Iota and Clostridium botulinum C2, are composed of a binding protein (Ib and C2II respectively) that recognizes distinct membrane receptors and mediates internalization of a catalytic protein (Ia and C2-I respectively) with ADP-ribosyltransferase activity that disrupts the actin cytoskeleton. We show here that the endocytic pathway followed by these toxins is independent of clathrin but requires the activity of dynamin and is regulated by Rho-GDI. This endocytic pathway is similar to a recently characterized clathrin-independent pathway followed by the interleukin-2 (IL2) receptor. We found indeed that Ib and C2II colocalized intracellularly with the IL2 receptor but not the transferrin receptor after different times of endocytosis. Accordingly, the intracellular effects of Iota and C2 on the cytoskeleton were inhibited by inactivation of dynamin or by Rho-GDI whereas inhibitors of clathrin-dependent endocytosis had no protective effect. PMID:20846184

  3. Emerging Roles for Intersectin (ITSN) in Regulating Signaling and Disease Pathways

    PubMed Central

    Hunter, Michael P.; Russo, Angela; O’Bryan, John P.

    2013-01-01

    Intersectins (ITSNs) represent a family of multi-domain adaptor proteins that regulate endocytosis and cell signaling. ITSN genes are highly conserved and present in all metazoan genomes examined thus far. Lower eukaryotes have only one ITSN gene, whereas higher eukaryotes have two ITSN genes. ITSN was first identified as an endocytic scaffold protein, and numerous studies reveal a conserved role for ITSN in endocytosis. Subsequently, ITSNs were found to regulate multiple signaling pathways including receptor tyrosine kinases (RTKs), GTPases, and phosphatidylinositol 3-kinase Class 2beta (PI3KC2β). ITSN has also been implicated in diseases such as Down Syndrome (DS), Alzheimer Disease (AD), and other neurodegenerative disorders. This review summarizes the evolutionary conservation of ITSN, the latest research on the role of ITSN in endocytosis, the emerging roles of ITSN in regulating cell signaling pathways, and the involvement of ITSN in human diseases such as DS, AD, and cancer. PMID:23574942

  4. HIV-1 Tat Protein Induces Production of Proinflammatory Cytokines by Human Dendritic Cells and Monocytes/Macrophages through Engagement of TLR4-MD2-CD14 Complex and Activation of NF-κB Pathway

    PubMed Central

    Leghmari, Kaoutar; Serrero, Manutea; Delobel, Pierre; Izopet, Jacques; BenMohamed, Lbachir; Bahraoui, Elmostafa

    2015-01-01

    We recently reported that the human immunodeficiency virus type-1 (HIV-1) Tat protein induced the expression of programmed death ligand-1 (PD-L1) on dendritic cells (DCs) through a TLR4 pathway. However, the underlying mechanisms by which HIV-1 Tat protein induces the abnormal hyper-activation of the immune system seen in HIV-1 infected patients remain to be fully elucidated. In the present study, we report that HIV-1 Tat protein induced the production of significant amounts of the pro-inflammatory IL-6 and IL-8 cytokines by DCs and monocytes from both healthy and HIV-1 infected patients. Such production was abrogated in the presence of anti-TLR4 blocking antibodies or soluble recombinant TLR4-MD2 as a decoy receptor, suggesting TLR4 was recruited by Tat protein. Tat-induced murine IL-6 and CXCL1/KC a functional homologue of human IL-8 was abolished in peritoneal macrophages derived from TLR4 KO but not from Wt mice, confirming the involvement of the TLR4 pathway. Furthermore, the recruitment of TLR4-MD2-CD14 complex by Tat protein was demonstrated by the activation of TLR4 downstream pathways including NF-κB and SOCS-1 and by down-modulation of cell surface TLR4 by endocytosis in dynamin and lipid-raft-dependent manners. Collectively, these findings demonstrate, for the first time, that HIV-1 Tat interacts with TLR4-MD2-CD14 complex and activates the NF-κB pathway, leading to overproduction of IL-6 and IL-8 pro-inflammatory cytokines by myeloid cells from both healthy and HIV-1 infected patients. This study reveals a novel mechanism by which HIV-1, via its early expressed Tat protein, hijacks the TLR4 pathway, hence establishing abnormal hyper-activation of the immune system. PMID:26090662

  5. RAB2A controls MT1-MMP endocytic and E-cadherin polarized Golgi trafficking to promote invasive breast cancer programs.

    PubMed

    Kajiho, Hiroaki; Kajiho, Yuko; Frittoli, Emanuela; Confalonieri, Stefano; Bertalot, Giovanni; Viale, Giuseppe; Di Fiore, Pier Paolo; Oldani, Amanda; Garre, Massimiliano; Beznoussenko, Galina V; Palamidessi, Andrea; Vecchi, Manuela; Chavrier, Philippe; Perez, Frank; Scita, Giorgio

    2016-07-01

    The mechanisms of tumor cell dissemination and the contribution of membrane trafficking in this process are poorly understood. Through a functional siRNA screening of human RAB GTPases, we found that RAB2A, a protein essential for ER-to-Golgi transport, is critical in promoting proteolytic activity and 3D invasiveness of breast cancer (BC) cell lines. Remarkably, RAB2A is amplified and elevated in human BC and is a powerful and independent predictor of disease recurrence in BC patients. Mechanistically, RAB2A acts at two independent trafficking steps. Firstly, by interacting with VPS39, a key component of the late endosomal HOPS complex, it controls post-endocytic trafficking of membrane-bound MT1-MMP, an essential metalloprotease for matrix remodeling and invasion. Secondly, it further regulates Golgi transport of E-cadherin, ultimately controlling junctional stability, cell compaction, and tumor invasiveness. Thus, RAB2A is a novel trafficking determinant essential for regulation of a mesenchymal invasive program of BC dissemination. PMID:27255086

  6. Revisiting the Role of Cystic Fibrosis Transmembrane Conductance Regulator and Counterion Permeability in the pH Regulation of Endocytic Organelles

    PubMed Central

    Barriere, Herve; Bagdany, Miklos; Bossard, Florian; Okiyoneda, Tsukasa; Wojewodka, Gabriella; Gruenert, Dieter; Radzioch, Danuta

    2009-01-01

    Organellar acidification by the electrogenic vacuolar proton-ATPase is coupled to anion uptake and cation efflux to preserve electroneutrality. The defective organellar pH regulation, caused by impaired counterion conductance of the mutant cystic fibrosis transmembrane conductance regulator (CFTR), remains highly controversial in epithelia and macrophages. Restricting the pH-sensitive probe to CFTR-containing vesicles, the counterion and proton permeability, and the luminal pH of endosomes were measured in various cells, including genetically matched CF and non-CF human respiratory epithelia, as well as cftr+/+ and cftr−/− mouse alveolar macrophages. Passive proton and relative counterion permeabilities, determinants of endosomal, lysosomal, and phagosomal pH-regulation, were probed with FITC-conjugated transferrin, dextran, and Pseudomonas aeruginosa, respectively. Although CFTR function could be documented in recycling endosomes and immature phagosomes, neither channel activation nor inhibition influenced the pH in any of these organelles. CFTR heterologous overexpression also failed to alter endocytic organellar pH. We propose that the relatively large CFTR-independent counterion and small passive proton permeability ensure efficient shunting of the proton-ATPase–generated membrane potential. These results have implications in the regulation of organelle acidification in general and demonstrate that perturbations of the endolysosomal organelles pH homeostasis cannot be linked to the etiology of the CF lung disease. PMID:19420138

  7. Real-Time Sensing of Enteropathogenic E. coli-Induced Effects on Epithelial Host Cell Height, Cell-Substrate Interactions, and Endocytic Processes by Infrared Surface Plasmon Spectroscopy

    PubMed Central

    Zlotkin-Rivkin, Efrat; Rund, David; Melamed-Book, Naomi; Zahavi, Eitan Erez; Perlson, Eran; Mercone, Silvana; Golosovsky, Michael; Davidov, Dan; Aroeti, Benjamin

    2013-01-01

    Enteropathogenic Escherichia coli (EPEC) is an important, generally non-invasive, bacterial pathogen that causes diarrhea in humans. The microbe infects mainly the enterocytes of the small intestine. Here we have applied our newly developed infrared surface plasmon resonance (IR-SPR) spectroscopy approach to study how EPEC infection affects epithelial host cells. The IR-SPR experiments showed that EPEC infection results in a robust reduction in the refractive index of the infected cells. Assisted by confocal and total internal reflection microscopy, we discovered that the microbe dilates the intercellular gaps and induces the appearance of fluid-phase-filled pinocytic vesicles in the lower basolateral regions of the host epithelial cells. Partial cell detachment from the underlying substratum was also observed. Finally, the waveguide mode observed by our IR-SPR analyses showed that EPEC infection decreases the host cell's height to some extent. Together, these observations reveal novel impacts of the pathogen on the host cell architecture and endocytic functions. We suggest that these changes may induce the infiltration of a watery environment into the host cell, and potentially lead to failure of the epithelium barrier functions. Our findings also indicate the great potential of the label-free IR-SPR approach to study the dynamics of host-pathogen interactions with high spatiotemporal sensitivity. PMID:24194932

  8. White spot syndrome virus entry is dependent on multiple endocytic routes and strongly facilitated by Cq-GABARAP in a CME-dependent manner.

    PubMed

    Chen, Rong-Yuan; Shen, Kai-Li; Chen, Zhen; Fan, Wei-Wei; Xie, Xiao-Lu; Meng, Chuang; Chang, Xue-Jiao; Zheng, Li-Bing; Jeswin, Joseph; Li, Cheng-Hua; Wang, Ke-Jian; Liu, Hai-Peng

    2016-01-01

    White spot syndrome virus (WSSV) is a lethal pathogen of shrimp and many other crustaceans, including crayfish. However, the molecular mechanism underlying its cellular entry remains elusive due to the lack of shrimp cell lines for viral propagation. Crayfish hematopoietic tissue (Hpt) cell culture was recently established as a good model for WSSV infection study. Here, we showed that multiple endocytic routes, including clathrin-mediated endocytosis (CME), macropinocytosis and caveolae-mediated endocytosis, were indispensably employed for the viral entry into Hpt cell of the crayfish Cherax quadricarinatus. Intriguingly, cellular autophagic activity was positively correlated with efficient viral entry, in which a key autophagy-related protein, γ-aminobutyric acid receptor-associated protein (Cq-GABARAP), that not only localized but also co-localized with WSSV on the Hpt cell membrane, strongly facilitated WSSV entry by binding to the viral envelope VP28 in a CME-dependent manner that was negatively regulated by Cq-Rac1. Furthermore, cytoskeletal components, including Cq-β-tubulin and Cq-β-actin, bound to both recombinant rCq-GABARAP and WSSV envelope proteins, which likely led to viral entry promotion via cooperation with rCq-GABARAP. Even under conditions that promoted viral entry, rCq-GABARAP significantly reduced viral replication at an early stage of infection, which was probably caused by the formation of WSSV aggregates in the cytoplasm. PMID:27385304

  9. Endocytic Adaptor Epidermal Growth Factor Receptor Substrate 15 (Eps15) Is Involved in the Trafficking of Ubiquitinated α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptors*

    PubMed Central

    Lin, Amy; Man, Heng-Ye

    2014-01-01

    AMPA-type glutamate receptors (AMPARs) play a critical role in mediating fast excitatory synaptic transmission in the brain. Alterations in receptor expression, distribution, and trafficking have been shown to underlie synaptic plasticity and higher brain functions, including learning and memory, as well as brain dysfunctions such as drug addiction and psychological disorders. Therefore, it is essential to elucidate the molecular mechanisms that regulate AMPAR dynamics. We have shown previously that mammalian AMPARs are subject to posttranslational modification by ubiquitin, with AMPAR ubiquitination enhancing receptor internalization and reducing AMPAR cell surface expression. Here we report a crucial role for epidermal growth factor receptor substrate 15 (Eps15), an endocytic adaptor, in ubiquitination-dependent AMPAR internalization. We find that suppression or overexpression of Eps15 results in changes in AMPAR surface expression. Eps15 interacts with AMPARs, which requires Nedd4-mediated GluA1 ubiquitination and the ubiquitin-interacting motif of Eps15. Importantly, we find that Eps15 plays an important role in AMPAR internalization. Knockdown of Eps15 suppresses the internalization of GluA1 but not the mutant GluA1 that lacks ubiquitination sites, indicating a role of Eps15 for the internalization of ubiquitinated AMPARs. These results reveal a novel molecular mechanism employed specifically for the trafficking of the ubiquitin-modified AMPARs. PMID:25023288

  10. White spot syndrome virus entry is dependent on multiple endocytic routes and strongly facilitated by Cq-GABARAP in a CME-dependent manner

    PubMed Central

    Chen, Rong-yuan; Shen, Kai-li; Chen, Zhen; Fan, Wei-wei; Xie, Xiao-lu; Meng, Chuang; Chang, Xue-jiao; Zheng, Li-bing; Jeswin, Joseph; Li, Cheng-hua; Wang, Ke-jian; Liu, Hai-peng

    2016-01-01

    White spot syndrome virus (WSSV) is a lethal pathogen of shrimp and many other crustaceans, including crayfish. However, the molecular mechanism underlying its cellular entry remains elusive due to the lack of shrimp cell lines for viral propagation. Crayfish hematopoietic tissue (Hpt) cell culture was recently established as a good model for WSSV infection study. Here, we showed that multiple endocytic routes, including clathrin-mediated endocytosis (CME), macropinocytosis and caveolae-mediated endocytosis, were indispensably employed for the viral entry into Hpt cell of the crayfish Cherax quadricarinatus. Intriguingly, cellular autophagic activity was positively correlated with efficient viral entry, in which a key autophagy-related protein, γ-aminobutyric acid receptor-associated protein (Cq-GABARAP), that not only localized but also co-localized with WSSV on the Hpt cell membrane, strongly facilitated WSSV entry by binding to the viral envelope VP28 in a CME-dependent manner that was negatively regulated by Cq-Rac1. Furthermore, cytoskeletal components, including Cq-β-tubulin and Cq-β-actin, bound to both recombinant rCq-GABARAP and WSSV envelope proteins, which likely led to viral entry promotion via cooperation with rCq-GABARAP. Even under conditions that promoted viral entry, rCq-GABARAP significantly reduced viral replication at an early stage of infection, which was probably caused by the formation of WSSV aggregates in the cytoplasm. PMID:27385304

  11. Interleukin-1-induced gene expression requires the membrane-raft-dependent internalization of the interleukin-1 receptor.

    PubMed

    Windheim, Mark

    2016-10-01

    Interleukin-1 (IL-1) binding to its receptor triggers signaling events at the plasma membrane that are essential but not sufficient for the induction of the IL-1-dependent gene expression. In addition, the ligand-induced endocytosis of the IL-1 receptor and signaling events that are initiated after the internalization of the IL-1 receptor presumably involving signaling endosomes are critical for the IL-1-induced gene expression. In this study, we investigate the role of membrane domains, commonly denoted as lipid rafts, in the IL-1-induced signal transduction. We demonstrate that the internalization of the IL-1 receptor depends on the integrity of lipid rafts and that the disruption of lipid rafts strongly reduces the IL-1-induced gene expression. Interestingly, the IL-1-dependent signaling events activated at the plasma membrane are not influenced by the disruption of lipid rafts suggesting that IL-1 signaling is initiated in a non-raft domain of the plasma membrane. Subsequently, the IL-1 receptor is translocated to lipid rafts where receptor endocytosis occurs to enable the internalization-dependent IL-1 signaling to activate the IL-1-induced gene expression. PMID:27327966

  12. CHOLINE PARTIALLY PREVENTS THE IMPACT OF ETHANOL ON THE LIPID RAFT DEPENDENT FUNCTIONS OF L1 CELL ADHESION MOLECULE

    PubMed Central

    Tang, Ningfeng; Bamford, Penny; Jones, Jace; He, Min; Kane, Maureen A.; Mooney, Sandra M.; Bearer, Cynthia F.

    2014-01-01

    Background Fetal Alcohol Spectrum Disorder, the leading known cause of mental retardation, is caused by alcohol exposure during pregnancy. One mechanism of ethanol teratogenicity is the disruption of the function of L1 cell adhesion molecule (L1). These functions include enhancement of neurite outgrowth, trafficking through lipid rafts, and signal transduction. Recent data have shown that choline supplementation of rat pups reduces the effects of ethanol on neurobehavior. We sought to determine if choline could prevent the effect of ethanol on L1 function using a simple experimental system. Methods Cerebellar granule neurons (CGN) from postnatal day 6 rat pups were cultured with and without supplemental choline, and the effects on L1 signaling, lipid raft distribution and neurite outgrowth were measured in the presence or absence of ethanol. Results Choline significantly reduced the effect of ethanol on L1 signaling, the distribution of L1 in lipid rafts and L1 mediated neurite outgrowth. However, choline supplemented ethanol exposed cultures remained significantly different than controls. Conclusions Choline pretreatment of CGN significantly reduces the disruption of L1 function by ethanol, but does not completely return L1 function to baseline. This experimental system will enable discovery of the mechanism of the neuroprotective effect of choline. PMID:25421509

  13. Alemtuzumab induces caspase-independent cell death in human chronic lymphocytic leukemia cells through a lipid raft-dependent mechanism.

    PubMed

    Mone, A P; Cheney, C; Banks, A L; Tridandapani, S; Mehter, N; Guster, S; Lin, T; Eisenbeis, C F; Young, D C; Byrd, J C

    2006-02-01

    Alemtuzumab is a humanized IgG1 kappa antibody directed against CD52, a glycosyl-phosphatidylinositol linked cell-membrane protein of unknown function. Herein, we demonstrate that alemtuzumab promotes rapid death of chronic lymphocytic leukemia (CLL) cells in vitro, in a complement and accessory cell free system. Using minimal detergent solubilization of CLL membranes, we found that CD52 colocalizes with ganglioside GM-1, a marker of membrane rafts. Fluorescence microscopy revealed that upon crosslinking CD52 with alemtuzumab+anti-Fc IgG, large patches, and in many cases caps, enriched in CD52 and GM-1 formed upon the CLL cell plasma membrane. Depletion of membrane cholesterol or inhibition of actin polymerization significantly diminished the formation of alemtuzumab-induced caps and reduced alemtuzumab-mediated CLL cell death. We compared alemtuzumab-induced direct cytotoxicity, effector cell-mediated toxicity and complement-mediated cytotoxicity of CLL cells to normal T cells. The direct cytotoxicity and observed capping was significantly greater for CLL cells as compared to normal T cells. Cell-mediated and complement-mediated cytotoxicity did not significantly differ between the two cell types. In summary, our data support the hypothesis that alemtuzumab can initiate CLL cell death by crosslinking CD52-enriched lipid rafts. Furthermore, the differential direct cytotoxic effect suggests that CD52 directed antibodies could possibly be engineered to more specifically target CLL cells. PMID:16341049

  14. The Endosomal Pathway and the Golgi Complex Are Involved in the Infectious Bursal Disease Virus Life Cycle

    PubMed Central

    Delgui, Laura R.; Rodríguez, José F.

    2013-01-01

    Infectious bursal disease virus (IBDV), a double-stranded RNA virus belonging to the Birnaviridae family, causes immunosuppression in chickens. In this study, we defined the localization of IBDV replication complexes based on colocalization analysis of VP3, the major protein component of IBDV ribonucleoproteins (RNPs). Our results indicate that VP3 localizes to vesicular structures bearing features of early and late endocytic compartments located in the juxtanuclear region. Interfering with the endocytic pathway with a dominant negative version of Rab5 after the internalization step leads to a reduction in virus titer. Triple-immunostaining studies between VP3, the viral RNA-dependent RNA polymerase VP1, and viral double-stranded RNA (dsRNA) showed a well-defined colocalization, indicating that the three critical components of the RNPs colocalize in the same structure, likely representing replication complexes. Interestingly, recombinant expressed VP3 also localizes to endosomes. Employing Golgi markers, we found that VP3-containing vesicles were closely associated with this organelle. Depolymerization of microtubules with nocodazole caused a profound change in VP3 localization, showing a punctate distribution scattered throughout the cytoplasm. However, these VP3-positive structures remained associated with Golgi ministacks. Similarly, brefeldin A (BFA) treatment led to a punctate distribution of VP3, scattered throughout the cytoplasm of infected cells. In addition, analysis of intra- and extracellular viral infective particles after BFA treatment of avian cells suggested a role for the Golgi complex in viral assembly. These results constitute the first study elucidating the localization of IBDV replication complexes (i.e., in endocytic compartments) and establishing a role for the Golgi apparatus in the assembly step of a birnavirus. PMID:23741000

  15. Controlling the intracellular fate of nano-bioconjugates: pathways for realizing nanoparticle-mediated theranostics

    NASA Astrophysics Data System (ADS)

    Delehanty, James B.; Blanco-Canosa, Juan B.; Bradburne, Christopher E.; Susumu, Kimihiro; Stewart, Michael H.; Prasuhn, Duane E.; Dawson, Philip E.; Medintz, Igor L.

    2014-08-01

    For nanomaterials to realize their full potential in disease diagnosis and drug delivery applications, one must be able to exert fine control over their cellular delivery, localization and long-term fate in biological systems. Our laboratory has been active in developing methodologies for the controlled and site-specific delivery of a range of nanomaterials (e.g., quantum dots, colloidal gold, nematic liquid crystals) for cellular labeling, imaging and sensing. This talk will highlight several examples from these efforts and will demonstrate the use of peptide- and protein-mediated facilitated delivery of nanomaterials to discrete cellular locations including the endocytic pathway, the plasma membrane and the cellular cytosol. The implications of the ability to exert fine control over nanomaterial constructs in biological settings will be discussed with a particular focus on their use in nanoparticle-based theranostics.

  16. Vitellogenesis in the fruit fly, Drosophila melanogaster: antagonists demonstrate that the PLC, IP3/DAG, PK-C pathway is triggered by calmodulin.

    PubMed

    Brubaker-Purkey, Bethany J; Woodruff, Richard I

    2013-01-01

    In Drosophila melanogaster M. (Diptera: Drosophilidae), a phospholipase-C to proteininase-C signal cascade leads to the endocytic uptake of yolk precursor molecules. The data suggest that D. melanogaster has a phospholipase-C/proteinkinase-C signaling pathway similar to that previously shown to be required for vitellogenesis in the milkweed bug, Oncopeltus fasciatus Dallas (Hemiptera: Lygaeidae). Calmodulin, derived from epithelial cells and transported to the oocytes via gap junctions, may trigger this pathway. To investigate this, a series of known antagonists to various elements of the pathway were used. W-7 (which prevents calmodulin binding to phospholipase-C), U-73122 (which prevents activation of phospholipase-C), verapamil (which blocks Ca(2+) release by IP3), HAG (which blocks diacylglycerol), and staurosporine (which inactivates proteinkinase-C) were each shown to inhibit endocytosis, thereby blocking formation of nascent yolk spheres. PMID:24228869

  17. Vitellogenesis in the Fruit Fly, Drosophila melanogaster: Antagonists Demonstrate that the PLC, IP3/DAG, PK-C Pathway is Triggered by Calmodulin

    PubMed Central

    Brubaker-Purkey, Bethany J.; Woodruff, Richard I.

    2013-01-01

    In Drosophila melanogaster M. (Diptera: Drosophilidae), a phospholipase-C to proteininase-C signal cascade leads to the endocytic uptake of yolk precursor molecules. The data suggest that D. melanogaster has a phospholipase-C/proteinkinase-C signaling pathway similar to that previously shown to be required for vitellogenesis in the milkweed bug, Oncopeltus fasciatus Dallas (Hemiptera: Lygaeidae). Calmodulin, derived from epithelial cells and transported to the oocytes via gap junctions, may trigger this pathway. To investigate this, a series of known antagonists to various elements of the pathway were used. W-7 (which prevents calmodulin binding to phospholipase-C), U-73122 (which prevents activation of phospholipase-C), verapamil (which blocks Ca2+ release by IP3), HAG (which blocks diacylglycerol), and staurosporine (which inactivates proteinkinase-C) were each shown to inhibit endocytosis, thereby blocking formation of nascent yolk spheres. PMID:24228869

  18. G Protein-coupled Receptor Kinase-mediated Phosphorylation Regulates Post-endocytic Trafficking of the D2 Dopamine Receptor*S⃞

    PubMed Central

    Namkung, Yoon; Dipace, Concetta; Javitch, Jonathan A.; Sibley, David R.

    2009-01-01

    We investigated the role of G protein-coupled receptor kinase (GRK)-mediated phosphorylation in agonist-induced desensitization, arrestin association, endocytosis, and intracellular trafficking of the D2 dopamine receptor (DAR). Agonist activation of D2 DARs results in rapid and sustained receptor phosphorylation that is solely mediated by GRKs. A survey of GRKs revealed that only GRK2 or GRK3 promotes D2 DAR phosphorylation. Mutational analyses resulted in the identification of eight serine/threonine residues within the third cytoplasmic loop of the receptor that are phosphorylated by GRK2/3. Simultaneous mutation of these eight residues results in a receptor construct, GRK(-), that is completely devoid of agonist-promoted GRK-mediated receptor phosphorylation. We found that both wild-type (WT) and GRK(-) receptors underwent a similar degree of agonist-induced desensitization as assessed using [35S]GTPγS binding assays. Similarly, both receptor constructs internalized to the same extent in response to agonist treatment. Furthermore, using bioluminescence resonance energy transfer assays to directly assess receptor association with arrestin3, we found no differences between the WT and GRK(-) receptors. Thus, phosphorylation is not required for arrestin-receptor association or agonist-induced desensitization or internalization. In contrast, when we examined recycling of the D2 DARs to the cell surface, subsequent to agonist-induced endocytosis, the GRK(-) construct exhibited less recycling in comparison with the WT receptor. This impairment appears to be due to a greater propensity of the GRK(-) receptors to down-regulate once internalized. In contrast, if the receptor is highly phosphorylated, then receptor recycling is promoted. These results reveal a novel role for GRK-mediated phosphorylation in regulating the post-endocytic trafficking of a G protein-coupled receptor. PMID:19332542

  19. The CD20 homologue MS4A4 directs trafficking of KIT toward clathrin-independent endocytosis pathways and thus regulates receptor signaling and recycling

    PubMed Central

    Cruse, Glenn; Beaven, Michael A.; Music, Stephen C.; Bradding, Peter; Gilfillan, Alasdair M.; Metcalfe, Dean D.

    2015-01-01

    MS4A family members differentially regulate the cell cycle, and aberrant, or loss of, expression of MS4A family proteins has been observed in colon and lung cancer. However, the precise functions of MS4A family proteins and their mechanistic interactions remain unsolved. Here we report that MS4A4 facilitates trafficking of the receptor tyrosine kinase KIT through endocytic recycling rather than degradation pathways by a mechanism that involves recruitment of KIT to caveolin-1–enriched microdomains. Silencing of MS4A4 in human mast cells altered ligand-induced KIT endocytosis pathways and reduced receptor recycling to the cell surface, thus promoting KIT signaling in the endosomes while reducing that in the plasma membrane, as exemplified by Akt and PLCγ1 phosphorylation, respectively. The altered endocytic trafficking of KIT also resulted in an increase in SCF-induced mast cell proliferation and migration, which may reflect altered signaling in these cells. Our data reveal a novel function for MS4A family proteins in regulating trafficking and signaling, which could have implications in both proliferative and immunological diseases. PMID:25717186

  20. The CD20 homologue MS4A4 directs trafficking of KIT toward clathrin-independent endocytosis pathways and thus regulates receptor signaling and recycling.

    PubMed

    Cruse, Glenn; Beaven, Michael A; Music, Stephen C; Bradding, Peter; Gilfillan, Alasdair M; Metcalfe, Dean D

    2015-05-01

    MS4A family members differentially regulate the cell cycle, and aberrant, or loss of, expression of MS4A family proteins has been observed in colon and lung cancer. However, the precise functions of MS4A family proteins and their mechanistic interactions remain unsolved. Here we report that MS4A4 facilitates trafficking of the receptor tyrosine kinase KIT through endocytic recycling rather than degradation pathways by a mechanism that involves recruitment of KIT to caveolin-1-enriched microdomains. Silencing of MS4A4 in human mast cells altered ligand-induced KIT endocytosis pathways and reduced receptor recycling to the cell surface, thus promoting KIT signaling in the endosomes while reducing that in the plasma membrane, as exemplified by Akt and PLCγ1 phosphorylation, respectively. The altered endocytic trafficking of KIT also resulted in an increase in SCF-induced mast cell proliferation and migration, which may reflect altered signaling in these cells. Our data reveal a novel function for MS4A family proteins in regulating trafficking and signaling, which could have implications in both proliferative and immunological diseases. PMID:25717186

  1. Cold Temperature Induces the Reprogramming of Proteolytic Pathways in Yeast.

    PubMed

    Isasa, Marta; Suñer, Clara; Díaz, Miguel; Puig-Sàrries, Pilar; Zuin, Alice; Bichman, Anne; Gygi, Steven P; Rebollo, Elena; Crosas, Bernat

    2016-01-22

    Despite much evidence of the involvement of the proteasome-ubiquitin signaling system in temperature stress response, the dynamics of the ubiquitylome during cold response has not yet been studied. Here, we have compared quantitative ubiquitylomes from a strain deficient in proteasome substrate recruitment and a reference strain during cold response. We have observed that a large group of proteins showing increased ubiquitylation in the proteasome mutant at low temperature is comprised by reverses suppressor of Ty-phenotype 5 (Rsp5)-regulated plasma membrane proteins. Analysis of internalization and degradation of plasma membrane proteins at low temperature showed that the proteasome becomes determinant for this process, whereas, at 30 °C, the proteasome is dispensable. Moreover, our observations indicate that proteasomes have increased capacity to interact with lysine 63-polyubiquitylated proteins during low temperature in vivo. These unanticipated observations indicate that, during cold response, there is a proteolytic cellular reprogramming in which the proteasome acquires a role in the endocytic-vacuolar pathway. PMID:26601941

  2. Visualization of Intracellular Pathways of Engineered Baculovirus in Mammalian Cells

    PubMed Central

    Liu, Yarong; Joo, Kye-Il; Lei, Yuning; Wang, Pin

    2014-01-01

    Baculoviruses are a promising gene delivery vector. They have the ability to express large transgenes in mammalian cells without displaying pathogenicity in humans; however, little is known about their transduction mechanisms in target cells. In this study, we use colocalization and live-cell imaging studies to elucidate the internalization and intracellular trafficking pathways of baculoviruses through direct visualization of VP39-GFP-labeled viral particles and various endocytic structures within target cells. Drug inhibition and confocal microscopy results suggested that baculoviruses enter the cells via clathrin-mediated endocytosis in a dynamin-dependent manner. Viral particles were shown to traffic through early endosomes, triggering a low-pH-dependent endosomal fusion process of viruses. Suppressed autophagy activity enhanced viral transduction and overexpression of autophagosomes reduced viral transduction, suggesting that autophagy is involved in degradation process of viral particles. Actin filaments were involved in the viral transduction, while microtubules negatively regulated viral transduction by facilitating the fusion of autophagosomes with lysosomes to form autolysosomes, where degradation of viral particles occurs. These results shed some light on the essential cellular factors limiting viral transduction, which can be used to improve the use of baculoviral vectors in cell and gene therapy. PMID:24457070

  3. Infectious Entry Pathway Mediated by the Human Endogenous Retrovirus K Envelope Protein

    PubMed Central

    Robinson, Lindsey R.

    2016-01-01

    ABSTRACT Endogenous retroviruses (ERVs), the majority of which exist as degraded remnants of ancient viruses, comprise approximately 8% of the human genome. The youngest human ERVs (HERVs) belong to the HERV-K(HML-2) subgroup and were endogenized within the past 1 million years. The viral envelope protein (ENV) facilitates the earliest events of endogenization (cellular attachment and entry), and here, we characterize the requirements for HERV-K ENV to mediate infectious cell entry. Cell-cell fusion assays indicate that a minimum of two events are required for fusion, proteolytic processing by furin-like proteases and exposure to acidic pH. We generated an infectious autonomously replicating recombinant vesicular stomatitis virus (VSV) in which the glycoprotein was replaced by HERV-K ENV. HERV-K ENV imparts an endocytic entry pathway that requires dynamin-mediated membrane scission and endosomal acidification but is distinct from clathrin-dependent or macropinocytic uptake pathways. The lack of impediments to the replication of the VSV core in eukaryotic cells allowed us to broadly survey the HERV-K ENV-dictated tropism. Unlike extant betaretroviral envelopes, which impart a narrow species tropism, we found that HERV-K ENV mediates broad tropism encompassing cells from multiple mammalian and nonmammalian species. We conclude that HERV-K ENV dictates an evolutionarily conserved entry pathway and that the restriction of HERV-K to primate genomes reflects downstream stages of the viral replication cycle. IMPORTANCE Approximately 8% of the human genome is of retroviral origin. While many of those viral genomes have become inactivated, some copies of the most recently endogenized human retrovirus, HERV-K, can encode individual functional proteins. Here, we characterize the envelope protein (ENV) of the virus to define how it mediates infection of cells. We demonstrate that HERV-K ENV undergoes a proteolytic processing step and triggers membrane fusion in response to

  4. Activation of ERK and NF-κB during HARE-Mediated Heparin Uptake Require Only One of the Four Endocytic Motifs

    PubMed Central

    Pandey, Madhu S.; Miller, Colton M.; Harris, Edward N.; Weigel, Paul H.

    2016-01-01

    Fifteen different ligands, including heparin (Hep), are cleared from lymph and blood by the Hyaluronan (HA) Receptor for Endocytosis (HARE; derived from Stabilin-2 by proteolysis), which contains four endocytic motifs (M1-M4). Endocytosis of HARE•Hep complexes is targeted to coated pits by M1, M2, and M3 (Pandey et al, Int. J. Cell Biol. 2015, article ID 524707), which activates ERK1/2 and NF-κB (Pandey et al J. Biol. Chem. 288, 14068–79, 2013). Here, we used a NF-κB promoter-driven luciferase gene assay and cell lines expressing different HARE cytoplasmic domain mutants to identify motifs needed for Hep-mediated signaling. Deletion of M1, M2 or M4 singly had no effect on Hep-mediated ERK1/2 activation, whereas signaling (but not uptake) was eliminated in HARE(ΔM3) cells lacking NPLY2519. ERK1/2 signaling in cells expressing WT HARE(Y2519A) or HARE(Y2519A) lacking M1, M2 and M4 (containing M3-only) was decreased by 75% or eliminated, respectively. Deletion of M3 (but not M1, M2 or M4) also inhibited the formation of HARE•Hep•ERK1/2 complexes by 67%. NF-κB activation by HARE-mediated uptake of Hep, HA, dermatan sulfate or acetylated LDL was unaffected in single-motif deletion mutants lacking M1, M2 or M4. In contrast, cells expressing HARE(ΔM3) showed loss of HARE-mediated NF-κB activation during uptake of each of these four ligands. NF-κB activation by the four signaling ligands was also eliminated in HARE(Y2519A) or HARE(M3-only;Y2519A) cells. We conclude that the HARE NPLY2519 motif is necessary for both ERK1/2 and NF-κB signaling and that Tyr2519 is critical for these functions. PMID:27100626

  5. Insights into endosomal maturation of human holo-transferrin in the enteric parasite Entamoeba histolytica: essential roles of Rab7A and Rab5 in biogenesis of giant early endocytic vacuoles.

    PubMed

    Verma, Kuldeep; Saito-Nakano, Yumiko; Nozaki, Tomoyoshi; Datta, Sunando

    2015-12-01

    The pathogenic amoeba Entamoeba histolytica is one of the causative agents of health hazards in tropical countries. It causes amoebic dysentery, colitis and liver abscesses in human. Iron is one of the essential nutritional resources for survival and chronic infection caused by the amoeba. The parasite has developed multiple ways to import, sequester and utilize iron from various iron-binding proteins from its host. In spite of its central role in pathogenesis, the mechanism of iron uptake by the parasite is largely unknown. Here, we carried out a systematic study to understand the role of some of the amoebic homologues of mammalian endocytic Rab GTPases (Rab5 and Rab21, Rab7A and Rab7B) in intracellular transport of human holo-transferrin by the parasite. Flow cytometry and quantitative microscopic image analysis revealed that Rab5 and Rab7A are required for the biogenesis of amoebic giant endocytic vacuoles (GEVs) and regulate the early phase of intracellular trafficking of transferrin. Rab7B is involved in the late phase, leading to the degradation of transferrin in the amoebic lysosome-like compartments. Using time-lapse fluorescence imaging in fixed trophozoites, we determined the kinetics of the vesicular transport of transferrin through Rab5-, Rab7A- and Rab7B-positive compartments. The involvement of Rab7A in the early phase of endocytosis by the parasite marks a significant divergence from its host in terms of spatiotemporal regulation by the Rab GTPases. PMID:26096601

  6. Mechanisms of JAK/STAT pathway negative regulation by the short coreceptor Eye Transformer/Latran

    PubMed Central

    Fisher, Katherine H.; Stec, Wojciech; Brown, Stephen; Zeidler, Martin P.

    2016-01-01

    Transmembrane receptors interact with extracellular ligands to transduce intracellular signaling cascades, modulate target gene expression, and regulate processes such as proliferation, apoptosis, differentiation, and homeostasis. As a consequence, aberrant signaling events often underlie human disease. Whereas the vertebrate JAK/STAT signaling cascade is transduced via multiple receptor combinations, the Drosophila pathway has only one full-length signaling receptor, Domeless (Dome), and a single negatively acting receptor, Eye Transformer/Latran (Et/Lat). Here we investigate the molecular mechanisms underlying Et/Lat activity. We demonstrate that Et/Lat negatively regulates the JAK/STAT pathway activity and can bind to Dome, thus reducing Dome:Dome homodimerization by creating signaling-incompetent Dome:Et/Lat heterodimers. Surprisingly, we find that Et/Lat is able to bind to both JAK and STAT92E but, despite the presence of putative cytokine-binding motifs, does not detectably interact with pathway ligands. We find that Et/Lat is trafficked through the endocytic machinery for lysosomal degradation but at a much slower rate than Dome, a difference that may enhance its ability to sequester Dome into signaling-incompetent complexes. Our data offer new insights into the molecular mechanism and regulation of Et/Lat in Drosophila that may inform our understanding of how short receptors function in other organisms. PMID:26658615

  7. Mechanisms of JAK/STAT pathway negative regulation by the short coreceptor Eye Transformer/Latran.

    PubMed

    Fisher, Katherine H; Stec, Wojciech; Brown, Stephen; Zeidler, Martin P

    2016-02-01

    Transmembrane receptors interact with extracellular ligands to transduce intracellular signaling cascades, modulate target gene expression, and regulate processes such as proliferation, apoptosis, differentiation, and homeostasis. As a consequence, aberrant signaling events often underlie human disease. Whereas the vertebrate JAK/STAT signaling cascade is transduced via multiple receptor combinations, the Drosophila pathway has only one full-length signaling receptor, Domeless (Dome), and a single negatively acting receptor, Eye Transformer/Latran (Et/Lat). Here we investigate the molecular mechanisms underlying Et/Lat activity. We demonstrate that Et/Lat negatively regulates the JAK/STAT pathway activity and can bind to Dome, thus reducing Dome:Dome homodimerization by creating signaling-incompetent Dome:Et/Lat heterodimers. Surprisingly, we find that Et/Lat is able to bind to both JAK and STAT92E but, despite the presence of putative cytokine-binding motifs, does not detectably interact with pathway ligands. We find that Et/Lat is trafficked through the endocytic machinery for lysosomal degradation but at a much slower rate than Dome, a difference that may enhance its ability to sequester Dome into signaling-incompetent complexes. Our data offer new insights into the molecular mechanism and regulation of Et/Lat in Drosophila that may inform our understanding of how short receptors function in other organisms. PMID:26658615

  8. An endocytosis pathway initiated through neuropilin-1 and regulated by nutrient availability

    PubMed Central

    Pang, Hong-Bo; Braun, Gary B.; Friman, Tomas; Aza-Blanc, Pedro; Ruidiaz, Manuel E.; Sugahara, Kazuki N.; Teesalu, Tambet; Ruoslahti, Erkki

    2014-01-01

    Neuropilins (NRPs) are trans-membrane receptors involved in axon guidance and vascular development. Many growth factors and other signaling molecules bind to NRPs through a C-terminal, basic sequence motif (C-end Rule or CendR motif). Peptides with this motif (CendR peptides) are taken up into cells by endocytosis. Tumor-homing CendR peptides penetrate through tumor tissue and have shown utility in enhancing drug delivery into tumors. Here we show, using RNAi screening and subsequent validation studies, that NRP1-mediated endocytosis of CendR peptides is distinct from known endocytic pathways. Ultrastructurally, CendR endocytosis resembles macropinocytosis, but is mechanistically different. We also show that nutrient-sensing networks such as mTOR signaling regulate CendR endocytosis and subsequent intercellular transport of CendR cargo, both of which are stimulated by nutrient depletion. As CendR is a bulk transport pathway, our results suggest a role for it in nutrient transport; CendR-enhanced drug delivery then makes use of this natural pathway. PMID:25277522

  9. Neurophysiology and itch pathways.

    PubMed

    Schmelz, Martin

    2015-01-01

    As we all can easily differentiate the sensations of itch and pain, the most straightforward neurophysiologic concept would consist of two specific pathways that independently encode itch and pain. Indeed, a neuronal pathway for histamine-induced itch in the peripheral and central nervous system has been described in animals and humans, and recently several non-histaminergic pathways for itch have been discovered in rodents that support a dichotomous concept differentiated into a pain and an itch pathway, with both pathways being composed of different "flavors." Numerous markers and mediators have been found that are linked to itch processing pathways. Thus, the delineation of neuronal pathways for itch from pain pathways seemingly proves that all sensory aspects of itch are based on an itch-specific neuronal pathway. However, such a concept is incomplete as itch can also be induced by the activation of the pain pathway in particular when the stimulus is applied in a highly localized spatial pattern. These opposite views reflect the old dispute between specificity and pattern theories of itch. Rather than only being of theoretic interest, this conceptual problem has key implication for the strategy to treat chronic itch as key therapeutic targets would be either itch-specific pathways or unspecific nociceptive pathways. PMID:25861773

  10. PATHWAYS - ELECTRON TUNNELING PATHWAYS IN PROTEINS

    NASA Technical Reports Server (NTRS)

    Beratan, D. N.

    1994-01-01

    The key to understanding the mechanisms of many important biological processes such as photosynthesis and respiration is a better understanding of the electron transfer processes which take place between metal atoms (and other groups) fixed within large protein molecules. Research is currently focused on the rate of electron transfer and the factors that influence it, such as protein composition and the distance between metal atoms. Current models explain the swift transfer of electrons over considerable distances by postulating bridge-mediated tunneling, or physical tunneling pathways, made up of interacting bonds in the medium around and between donor and acceptor sites. The program PATHWAYS is designed to predict the route along which electrons travel in the transfer processes. The basic strategy of PATHWAYS is to begin by recording each possible path element on a connectivity list, including in each entry which two atoms are connected and what contribution the connection would make to the overall rate if it were included in a pathway. The list begins with the bonded molecular structure (including the backbone sequence and side chain connectivity), and then adds probable hydrogen bond links and through-space contacts. Once this list is completed, the program runs a tree search from the donor to the acceptor site to find the dominant pathways. The speed and efficiency of the computer search offers an improvement over manual techniques. PATHWAYS is written in FORTRAN 77 for execution on DEC VAX series computers running VMS. The program inputs data from four data sets and one structure file. The software was written to input BIOGRAF (old format) structure files based on x-ray crystal structures and outputs ASCII files listing the best pathways and BIOGRAF vector files containing the paths. Relatively minor changes could be made in the input format statements for compatibility with other graphics software. The executable and source code are included with the

  11. The Legionella Kinase LegK2 Targets the ARP2/3 Complex To Inhibit Actin Nucleation on Phagosomes and Allow Bacterial Evasion of the Late Endocytic Pathway

    PubMed Central

    Michard, Céline; Sperandio, Daniel; Baïlo, Nathalie; Pizarro-Cerdá, Javier; LeClaire, Lawrence; Chadeau-Argaud, Elise; Pombo-Grégoire, Isabel; Hervet, Eva; Vianney, Anne; Gilbert, Christophe; Faure, Mathias; Cossart, Pascale

    2015-01-01

    ABSTRACT Legionella pneumophila, the etiological agent of legionellosis, replicates within phagocytic cells. Crucial to biogenesis of the replicative vacuole is the Dot/Icm type 4 secretion system, which translocates a large number of effectors into the host cell cytosol. Among them is LegK2, a protein kinase that plays a key role in Legionella infection. Here, we identified the actin nucleator ARP2/3 complex as a target of LegK2. LegK2 phosphorylates the ARPC1B and ARP3 subunits of the ARP2/3 complex. LegK2-dependent ARP2/3 phosphorylation triggers global actin cytoskeleton remodeling in cells, and it impairs actin tail formation by Listeria monocytogenes, a well-known ARP2/3-dependent process. During infection, LegK2 is addressed to the Legionella-containing vacuole surface and inhibits actin polymerization on the phagosome, as revealed by legK2 gene inactivation. Consequently, LegK2 prevents late endosome/lysosome association with the phagosome and finally contributes to remodeling of the bacterium-containing phagosome into a replicative niche. The inhibition of actin polymerization by LegK2 and its effect on endosome trafficking are ARP2/3 dependent since it can be phenocopied by a specific chemical inhibitor of the ARP2/3 complex. Thus, LegK2-ARP2/3 interplay highlights an original mechanism of bacterial virulence with an unexpected role in local actin remodeling that allows bacteria to control vesicle trafficking in order to escape host defenses. PMID:25944859

  12. Epidermal growth factor receptors destined for the nucleus are internalized via a clathrin-dependent pathway

    SciTech Connect

    De Angelis Campos, Ana Carolina; Rodrigues, Michele Angela; Andrade, Carolina de; Miranda de Goes, Alfredo; Nathanson, Michael H.; Gomes, Dawidson A.

    2011-08-26

    Highlights: {yields} EGF and its receptor translocates to the nucleus in liver cells. {yields} Real time imaging shows that EGF moves to the nucleus. {yields} EGF moves with its receptor to the nucleus. {yields} Dynamin and clathrin are necessary for EGFR nuclear translocation. -- Abstract: The epidermal growth factor (EGF) transduces its actions via the EGF receptor (EGFR), which can traffic from the plasma membrane to either the cytoplasm or the nucleus. However, the mechanism by which EGFR reaches the nucleus is unclear. To investigate these questions, liver cells were analyzed by immunoblot of cell fractions, confocal immunofluorescence and real time confocal imaging. Cell fractionation studies showed that EGFR was detectable in the nucleus after EGF stimulation with a peak in nuclear receptor after 10 min. Movement of EGFR to the nucleus was confirmed by confocal immunofluorescence and labeled EGF moved with the receptor to the nucleus. Small interference RNA (siRNA) was used to knockdown clathrin in order to assess the first endocytic steps of EGFR nuclear translocation in liver cells. A mutant dynamin (dynamin K44A) was also used to determine the pathways for this traffic. Movement of labeled EGF or EGFR to the nucleus depended upon dynamin and clathrin. This identifies the pathway that mediates the first steps for EGFR nuclear translocation in liver cells.

  13. Misassembled mutant DeltaF508 CFTR in the distal secretory pathway alters cellular lipid trafficking.

    PubMed

    Gentzsch, Martina; Choudhury, Amit; Chang, Xiu-bao; Pagano, Richard E; Riordan, John R

    2007-02-01

    Most patients with cystic fibrosis (CF) have a single codon deletion (DeltaF508) in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) that impairs assembly of the multidomain glycoprotein. The mutant protein escapes endoplasmic reticulum (ER) quality control at low temperature, but is rapidly cleared from the distal secretory pathway and degraded in lysosomes. CF cells accumulate free cholesterol similar to Niemann-Pick disease type C cells. We show that this lipid alteration is caused by the presence of misassembled mutant CFTR proteins, including DeltaF508, in the distal secretory pathway rather than the absence of functional CFTR. By contrast, cholesterol distribution is not changed by either D572N CFTR, which does not mature even at low temperature, or G551D, which is processed normally but is inactive. On expression of the DeltaF508 mutant, cholesterol and glycosphingolipids accumulate in punctate endosomal structures and cholesterol esters are reduced, indicating a block in the translocation of cholesterol to the ER for esterification. This is overcome by Rab9 overexpression, resulting in clearance of accumulating intracellular cholesterol. Similar but less pronounced alterations in intracellular cholesterol distribution are observed on expression of a temperature-rescued mutant variant of the related ATP-binding cassette (ABC) protein multidrug resistance-associated protein 1 (MRP1). Thus, on escape from ER quality control, misassembled mutants of CFTR and MRP1 impair lipid homeostasis in endocytic compartments. PMID:17213331

  14. Pathways from Poverty.

    ERIC Educational Resources Information Center

    Baldwin, Barbara, Ed.

    1995-01-01

    Articles in this theme issue are based on presentations at the Pathways from Poverty Workshop held in Albuquerque, New Mexico, on May 18-25, 1995. The event aimed to foster development of a network to address rural poverty issues in the Western Rural Development Center (WRDC) region. Articles report on outcomes from the Pathways from Poverty…

  15. Pituitary Adenylate Cyclase-activating Polypeptide (PACAP)/PAC1HOP1 Receptor Activation Coordinates Multiple Neurotrophic Signaling Pathways

    PubMed Central

    May, Victor; Lutz, Eve; MacKenzie, Christopher; Schutz, Kristin C.; Dozark, Kate; Braas, Karen M.

    2010-01-01

    MAPK and Akt pathways are predominant mediators of trophic signaling for many neuronal systems. Among the vasoactive intestinal peptide/secretin/glucagon family of related peptides, pituitary adenylate cyclase-activating polypeptide (PACAP) binding to specific PAC1 receptor isoforms can engage multiple signaling pathways and promote neuroprotection through mechanisms that are not well understood. Using a primary sympathetic neuronal system, the current studies demonstrate that PACAP activation of PAC1HOP1 receptors engages both MAPK and Akt neurotrophic pathways in an integrated program to facilitate neuronal survival after growth factor withdrawal. PACAP not only stimulated prosurvival ERK1/2 and ERK5 activation but also abrogated SAPK/JNK and p38 MAPK signaling in parallel. In contrast to the potent and rapid effects of PACAP in ERK1/2 phosphorylation, PACAP stimulated Akt phosphorylation in a late phase of PAC1HOP1 receptor signaling. From inhibitor and immunoprecipitation analyses, the PACAP/PAC1HOP1 receptor-mediated Akt responses did not represent transactivation mechanisms but appeared to depend on Gαq/phosphatidylinositol 3-kinase γ activity and vesicular internalization pathways. Phosphatidylinositol 3-kinase γ-selective inhibitors blocked PACAP-stimulated Akt phosphorylation in primary neuronal cultures and in PAC1HOP1-overexpressing cell lines; RNA interference-mediated knockdown of the receptor effectors attenuated PACAP-mediated Akt activation. Similarly, perturbation of endocytic pathways also blocked Akt phosphorylation. Between ERK and Akt pathways, PACAP-stimulated Akt signaling was the primary cascade that attenuated cultured neuron apoptosis after growth factor withdrawal. The partitioning of PACAP-mediated Akt signaling in endosomes may be a key mechanism contributing to the high spatial and temporal specificity in signal transduction necessary for survival pathways. PMID:20093365

  16. Identification of multiple cellular uptake pathways of polystyrene nanoparticles and factors affecting the uptake: relevance for drug delivery systems.

    PubMed

    Firdessa, Rebuma; Oelschlaeger, Tobias A; Moll, Heidrun

    2014-01-01

    Nanoparticles may address challenges by human diseases through improving diagnosis, vaccination and treatment. The uptake mechanism regulates the type of threat a particle poses on the host cells and how a cell responds to it. Hence, understanding the uptake mechanisms and cellular interactions of nanoparticles at the cellular and subcellular level is a prerequisite for their effective biomedical applications. The present study shows the uptake mechanisms of polystyrene nanoparticles and factors affecting their uptake in bone marrow-derived macrophages, 293T kidney epithelial cells and L929 fibroblasts. Labeling with the endocytic marker FM4-64 and transmission electron microscopy studies show that the nanoparticles were internalized rapidly via endocytosis and accumulated in intracellular vesicles. Soon after their internalizations, nanoparticles trafficked to organelles with acidic pH. Analysis of the ultrastructural morphology of the plasma membrane invaginations or extravasations provides clear evidence for the involvement of several uptake routes in parallel to internalize a given type of nanoparticles by mammalian cells, highlighting the complexity of the nanoparticle-cell interactions. Blocking the specific endocytic pathways by different pharmacological inhibitors shows similar outcomes. The potential to take up nanoparticles varies highly among different cell types in a particle sizes-, time- and energy-dependent manner. Furthermore, infection and the activation status of bone marrow-derived macrophages significantly affect the uptake potential of the cells, indicating the need to understand the diseases' pathogenesis to establish effective and rational drug-delivery systems. This study enhances our understanding of the application of nanotechnology in biomedical sciences. PMID:25224362

  17. Lysoplex: An efficient toolkit to detect DNA sequence variations in the autophagy-lysosomal pathway

    PubMed Central

    Di Fruscio, Giuseppina; Schulz, Angela; De Cegli, Rossella; Savarese, Marco; Mutarelli, Margherita; Parenti, Giancarlo; Banfi, Sandro; Braulke, Thomas; Nigro, Vincenzo; Ballabio, Andrea

    2015-01-01

    The autophagy-lysosomal pathway (ALP) regulates cell homeostasis and plays a crucial role in human diseases, such as lysosomal storage disorders (LSDs) and common neurodegenerative diseases. Therefore, the identification of DNA sequence variations in genes involved in this pathway and their association with human diseases would have a significant impact on health. To this aim, we developed Lysoplex, a targeted next-generation sequencing (NGS) approach, which allowed us to obtain a uniform and accurate coding sequence coverage of a comprehensive set of 891 genes involved in lysosomal, endocytic, and autophagic pathways. Lysoplex was successfully validated on 14 different types of LSDs and then used to analyze 48 mutation-unknown patients with a clinical phenotype of neuronal ceroid lipofuscinosis (NCL), a genetically heterogeneous subtype of LSD. Lysoplex allowed us to identify pathogenic mutations in 67% of patients, most of whom had been unsuccessfully analyzed by several sequencing approaches. In addition, in 3 patients, we found potential disease-causing variants in novel NCL candidate genes. We then compared the variant detection power of Lysoplex with data derived from public whole exome sequencing (WES) efforts. On average, a 50% higher number of validated amino acid changes and truncating variations per gene were identified. Overall, we identified 61 truncating sequence variations and 488 missense variations with a high probability to cause loss of function in a total of 316 genes. Interestingly, some loss-of-function variations of genes involved in the ALP pathway were found in homozygosity in the normal population, suggesting that their role is not essential. Thus, Lysoplex provided a comprehensive catalog of sequence variants in ALP genes and allows the assessment of their relevance in cell biology as well as their contribution to human disease. PMID:26075876

  18. Transformation pathways of liposomes.

    PubMed

    Hotani, H

    1984-09-01

    Liposomes undergoing transformation were observed by dark-field light microscopy in order to study the role of lipid in morphogenesis of biological vesicular structures. Liposomes were found to transform sequentially in a well-defined manner through one of several transformation pathways. A circular biconcave form was an initial shape in all the pathways and it transformed into a stable thin flexible filament or small spheres via a variety of regularly shaped vesicles which possessed geometrical symmetry. The transformation was reversible up to a certain point in each pathway. Osmotic pressure was found to be the driving force for the transformations. Biological membrane vesicles such as trypsinized red cell ghosts also transformed by similar pathways. PMID:6548263

  19. Updating the Wnt pathways

    PubMed Central

    Yu, Jia; Virshup, David M.

    2014-01-01

    In the three decades since the discovery of the Wnt1 proto-oncogene in virus-induced mouse mammary tumours, our understanding of the signalling pathways that are regulated by the Wnt proteins has progressively expanded. Wnts are involved in an complex signalling network that governs multiple biological processes and cross-talk with multiple additional signalling cascades, including the Notch, FGF (fibroblast growth factor), SHH (Sonic hedgehog), EGF (epidermal growth factor) and Hippo pathways. The Wnt signalling pathway also illustrates the link between abnormal regulation of the developmental processes and disease manifestation. Here we provide an overview of Wnt-regulated signalling cascades and highlight recent advances. We focus on new findings regarding the dedicated Wnt production and secretion pathway with potential therapeutic targets that might be beneficial for patients with Wnt-related diseases. PMID:25208913

  20. Identifying Branched Metabolic Pathways by Merging Linear Metabolic Pathways

    NASA Astrophysics Data System (ADS)

    Heath, Allison P.; Bennett, George N.; Kavraki, Lydia E.

    This paper presents a graph-based algorithm for identifying complex metabolic pathways in multi-genome scale metabolic data. These complex pathways are called branched pathways because they can arrive at a target compound through combinations of pathways that split compounds into smaller ones, work in parallel with many compounds, and join compounds into larger ones. While most previous work has focused on identifying linear metabolic pathways, branched metabolic pathways predominate in metabolic networks. Automatic identification of branched pathways has a number of important applications in areas that require deeper understanding of metabolism, such as metabolic engineering and drug target identification. Our algorithm utilizes explicit atom tracking to identify linear metabolic pathways and then merges them together into branched metabolic pathways. We provide results on two well-characterized metabolic pathways that demonstrate that this new merging approach can efficiently find biologically relevant branched metabolic pathways with complex structures.

  1. Inflammatory pathways in spondyloarthritis.

    PubMed

    Hreggvidsdottir, Hulda S; Noordenbos, Troy; Baeten, Dominique L

    2014-01-01

    Spondyloarthritis is the second most common form of chronic inflammatory arthritis and a unique hallmark of the disease is pathologic new bone formation. Several cytokine pathways have been genetically associated with ankylosing spondylitis (AS), the prototypic subtype of SpA, and additional evidence from human and animal studies support a role of these pathways in the disease. TNF has a key role in SpA as blockade significantly reduces inflammation and destruction, however the treatment does not halt new bone formation. New insights into the TNF pathway were recently obtained from an animal model specifically overexpressing the transmembrane form of TNF. This model leads to axial and peripheral new bone formation which is not seen in soluble TNF overexpression models, indicating different pathogenic roles of soluble and transmembrane TNF in arthritis development. Besides TNF, the IL-23/IL-17 axis is emerging as an important inflammatory pathway in SpA, as a SNP in the IL-23R locus has been associated with developing AS, mice overexpressing IL-23 develop SpA-like features and IL-17 blockade has been shown to be efficacious for AS patients in a phase II trial. In this review, we focus on the cytokine pathways that have recently been genetically associated with SpA, i.e. TNF, IL-1, IL-6 and IL-23/IL-17. We review the current genetic, experimental and human in vivo data available and discuss how these different pathways are involved in the pathophysiology of SpA. Additionally, we discuss how these pathways relate to the pathogenic new bone formation in SpA. PMID:23969080

  2. Clinical Pathway for Thyroidectomy.

    PubMed

    Villar del Moral, Jesús María; Soria Aledo, Víctor; Colina Alonso, Alberto; Flores Pastor, Benito; Gutiérrez Rodríguez, María Teresa; Ortega Serrano, Joaquín; Parra Hidalgo, Pedro; Ros López, Susana

    2015-05-01

    Clinical pathways are care plans applicable to patient care procedures that present variations in practice and a predictable clinical course. They are designed not as a substitute for clinical judgment, but rather as a means to improve the effectiveness and efficiency of the procedures. This clinical pathway is the result of a collaborative work of the Sections of Endocrine Surgery and Quality Management of the Spanish Association of Surgeons. It attempts to provide a framework for standardizing the performance of thyroidectomy, the most frequently performed operation in endocrine surgery. Along with the usual documents of clinical pathways (temporary matrix, variance tracking and information sheets, assessment indicators and a satisfaction questionnaire) it includes a review of the scientific evidence around different aspects of pre, intra and postoperative management. Among others, antibiotic and antithrombotic prophylaxis, preoperative preparation in hyperthyroidism, intraoperative neuromonitoring and systems for obtaining hemostasis are included, along with management of postoperative hypocalcemia. PMID:25732107

  3. Pathways to chromothripsis

    PubMed Central

    Ivkov, Robert; Bunz, Fred

    2015-01-01

    Chromothripsis is a recently recognized mode of genetic instability that generates chromosomes with strikingly large numbers of segmental re-arrangements. While the characterization of these derivative chromosomes has provided new insights into the processes by which cancer genomes can evolve, the underlying signaling events and molecular mechanisms remain unknown. In medulloblastomas, chromothripsis has been observed to occur in the context of mutational inactivation of p53 and activation of the canonical Hedgehog (Hh) pathway. Recent studies have illuminated mechanistic links between these 2 signaling pathways, including a novel PTCH1 homolog that is regulated by p53. Here, we integrate this new pathway into a hypothetical model for the catastrophic DNA breakage that appears to trigger profound chromosomal rearrangements. PMID:26178348

  4. Characterization of a transport and detoxification pathway for the antitumour drug bleomycin in Saccharomyces cerevisiae

    PubMed Central

    2004-01-01

    BLM (bleomycin) is effective in combination therapy against various cancers including testicular cancer. However, several other cancers such as colon cancer are refractory to BLM treatment. The exact mechanism for this differential response of cancer cells to the drug is not known. In the present study, we created fluorescently labelled BLM-A5, which retained nearly full genotoxic potential, and used this molecule to conduct the first study to understand the transport pathway of the drug in Saccharomyces cerevisiae. Uptake studies revealed that fluoro-BLM-A5 is transported into the cell in a concentration-dependent manner. Transport of a non-saturating concentration of fluoro-BLM-A5 was modest for the first 90 min, but thereafter it was sharply induced until 300 min. The inducible transport was completely abolished by the addition of cycloheximide, suggesting that BLM-A5 uptake into the cell is dependent on new protein synthesis. Interestingly, transport of fluoro-BLM-A5 was blocked if the cells were preincubated with increasing concentrations of spermine. Moreover, a mutant lacking the Ptk2 kinase, necessary for positively regulating polyamine transport, was defective in fluoro-BLM-A5 uptake and exhibited extreme resistance to the drug. A simple interpretation of these results is that BLM-A5 may enter the cell through the polyamine transport system. We showed further that after the uptake, fluoro-BLM-A5 accumulated into the vacuole of the parent, but localized to the cytoplasm of mutants disrupted for the END3 gene required for an early step of the endocytotic pathway. In general, mutants with a defect in the endocytic pathway to the vacuole were hypersensitive to BLM-A5. We suggest that BLM-A5 is transported across the yeast plasma membrane and sequestered into the vacuole for detoxification. PMID:15248838

  5. Endocytosis of Ligand-Activated Sphingosine 1-Phosphate Receptor 1 Mediated by the Clathrin-Pathway.

    PubMed

    Reeves, Patrick M; Kang, Yuan-Lin; Kirchhausen, Tom

    2016-01-01

    The sphingosine 1-phosphate receptor 1 (S1PR1) is one of five G protein-coupled receptors activated by the lipid sphingosine 1-phosphate (S1P). Stimulation of S1PR1 by binding S1P or the synthetic agonist FTY720P results in rapid desensitization, associated in part with depletion of receptor from the cell surface. We report here combining spinning disc confocal fluorescence microscopy and flow cytometry to show that rapid internalization of activated S1PR1 relies on a functional clathrin-mediated endocytic pathway. Uptake of activated S1PR1 was strongly inhibited in cells disrupted in their clathrin-mediated endocytosis by depleting clathrin or AP-2 or by treating cells with dynasore-OH. The uptake of activated S1P1R was strongly inhibited in cells lacking both β-arrestin 1 and β-arrestin 2, indicating that activated S1PR1 follows the canonical route of endocytosis for G-protein coupled receptor's (GPCR)'s. PMID:26481905

  6. Macrophage Receptor with Collagenous Structure (MARCO) Is Processed by either Macropinocytosis or Endocytosis-Autophagy Pathway

    PubMed Central

    Hirano, Seishiro; Kanno, Sanae

    2015-01-01

    The Macrophage Receptor with COllagenous structure (MARCO) protein is a plasma membrane receptor for un-opsonized or environmental particles on phagocytic cells. Here, we show that MARCO was internalized either by ruffling of plasma membrane followed by macropinocytosis or by endocytosis followed by fusion with autophagosome in CHO-K1 cells stably transfected with GFP-MARCO. The macropinocytic process generated large vesicles when the plasma membrane subsided. The endocytosis/autophagosome (amphisome) generated small fluorescent puncta which were visible in the presence of glutamine, chloroquine, bafilomycin, ammonia, and other amines. The small puncta, but not the large vesicles, co-localized with LC3B and lysosomes. The LC3-II/LC3-I ratio increased in the presence of glutamine, ammonia, and chloroquine in various cells. The small puncta trafficked between the peri-nuclear region and the distal ends of cells back and forth at rates of up to 2–3 μm/sec; tubulin, but not actin, regulated the trafficking of the small puncta. Besides phagocytosis MARCO, an adhesive plasma membrane receptor, may play a role in incorporation of various extracellular materials into the cell via both macropinocytic and endocytic pathways. PMID:26545255

  7. Pathways to School Success

    ERIC Educational Resources Information Center

    University of Pittsburgh Office of Child Development, 2012

    2012-01-01

    In 2006, the University of Pittsburgh Office of Child Development began implementing a multi-year school readiness project in several area schools. Evidence from both research and the field point to several key elements that foster school readiness and create pathways to school success for all children. This paper presents components of a…

  8. Pathways to Prosperity

    ERIC Educational Resources Information Center

    Symonds, William C.

    2012-01-01

    Symonds, director of the Pathways to Prosperity Project, asserts that the current U.S. system of preparing students for successful adult lives fails too many youth. Citing data showing that only 40 percent of young Americans earn an associate's or bachelor's degree by age 27--and that many employers say they see candidates who don't have…

  9. Dexter energy transfer pathways.

    PubMed

    Skourtis, Spiros S; Liu, Chaoren; Antoniou, Panayiotis; Virshup, Aaron M; Beratan, David N

    2016-07-19

    Energy transfer with an associated spin change of the donor and acceptor, Dexter energy transfer, is critically important in solar energy harvesting assemblies, damage protection schemes of photobiology, and organometallic opto-electronic materials. Dexter transfer between chemically linked donors and acceptors is bridge mediated, presenting an enticing analogy with bridge-mediated electron and hole transfer. However, Dexter coupling pathways must convey both an electron and a hole from donor to acceptor, and this adds considerable richness to the mediation process. We dissect the bridge-mediated Dexter coupling mechanisms and formulate a theory for triplet energy transfer coupling pathways. Virtual donor-acceptor charge-transfer exciton intermediates dominate at shorter distances or higher tunneling energy gaps, whereas virtual intermediates with an electron and a hole both on the bridge (virtual bridge excitons) dominate for longer distances or lower energy gaps. The effects of virtual bridge excitons were neglected in earlier treatments. The two-particle pathway framework developed here shows how Dexter energy-transfer rates depend on donor, bridge, and acceptor energetics, as well as on orbital symmetry and quantum interference among pathways. PMID:27382185

  10. Hedgehog Pathway Inhibition.

    PubMed

    Sekulic, Aleksandar; Von Hoff, Daniel

    2016-02-25

    The hedgehog (Hh) signaling pathway is aberrantly activated in a majority of basal cell carcinomas (BCC). Vismodegib and sonidegib are targeted inhibitors of Smoothened (SMO). Both drugs are approved for use in locally advanced BCC (laBCC), with vismodegib also approved for metastatic BCC (mBCC). PMID:26919418

  11. Phosphorylation of the Deubiquitinase USP20 by Protein Kinase A Regulates Post-endocytic Trafficking of β2 Adrenergic Receptors to Autophagosomes during Physiological Stress*

    PubMed Central

    Kommaddi, Reddy Peera; Jean-Charles, Pierre-Yves; Shenoy, Sudha K.

    2015-01-01

    Ubiquitination by the E3 ligase Nedd4 and deubiquitination by the deubiquitinases USP20 and USP33 have been shown to regulate the lysosomal trafficking and recycling of agonist-activated β2 adrenergic receptors (β2ARs). In this work, we demonstrate that, in cells subjected to physiological stress by nutrient starvation, agonist-activated ubiquitinated β2ARs traffic to autophagosomes to colocalize with the autophagy marker protein LC3-II. Furthermore, this trafficking is synchronized by dynamic posttranslational modifications of USP20 that, in turn, are induced in a β2AR-dependent manner. Upon β2AR activation, a specific isoform of the second messenger cAMP-dependent protein kinase A (PKAα) rapidly phosphorylates USP20 on serine 333 located in its unique insertion domain. This phosphorylation of USP20 correlates with a characteristic SDS-PAGE mobility shift of the protein, blocks its deubiquitinase activity, promotes its dissociation from the activated β2AR complex, and facilitates trafficking of the ubiquitinated β2AR to autophagosomes, which fuse with lysosomes to form autolysosomes where receptors are degraded. Dephosphorylation of USP20 has reciprocal effects and blocks trafficking of the β2AR to autophagosomes while promoting plasma membrane recycling of internalized β2ARs. Our findings reveal a dynamic regulation of USP20 by site-specific phosphorylation as well as the interdependence of signal transduction and trafficking pathways in balancing adrenergic stimulation and maintaining cellular homeostasis. PMID:25666616

  12. Transmembrane Protein (Perfringolysin O) Association with Ordered Membrane Domains (Rafts) Depends Upon the Raft-Associating Properties of Protein-Bound Sterol

    PubMed Central

    Lin, Qingqing; London, Erwin

    2013-01-01

    Because transmembrane (TM) protein localization, or nonlocalization, in ordered membrane domains (rafts) is a key to understanding membrane domain function, it is important to define the origin of protein-raft interaction. One hypothesis is that a tight noncovalent attachment of TM proteins to lipids that have a strong affinity for ordered domains can be sufficient to induce raft-protein interaction. The sterol-binding protein perfringolysin O (PFO) was used to test this hypothesis. PFO binds both to sterols that tend to localize in ordered domains (e.g., cholesterol), and to those that do not (e.g., coprostanol), but it does not bind to epicholesterol, a raft-promoting 3α-OH sterol. Using a fluorescence resonance energy transfer assay in model membrane vesicles containing coexisting ordered and disordered lipid domains, both TM and non-TM forms of PFO were found to concentrate in ordered domains in vesicles containing high and low-Tm lipids plus cholesterol or 1:1 (mol/mol) cholesterol/epicholesterol, whereas they concentrate in disordered domains in vesicles containing high-Tm and low-Tm lipids plus 1:1 (mol/mol) coprostanol/epicholesterol. Combined with previous studies this behavior indicates that TM protein association with ordered domains is dependent upon both the association of the protein-bound sterol with ordered domains and hydrophobic match between TM segments and rafts. PMID:24359745

  13. Glypican-1 regulates myoblast response to HGF via Met in a lipid raft-dependent mechanism: effect on migration of skeletal muscle precursor cells

    PubMed Central

    2014-01-01

    Background Via the hepatocyte growth factor receptor (Met), hepatocyte growth factor (HGF) exerts key roles involving skeletal muscle development and regeneration. Heparan sulfate proteoglycans (HSPGs) are critical modulators of HGF activity, but the role of specific HSPGs in HGF regulation is poorly understood. Glypican-1 is the only HSPG expressed in myoblasts that localize in lipid raft membrane domains, controlling cell responses to extracellular stimuli. We determined if glypican-1 in these domains is necessary to stabilize the HGF-Met signaling complex and myoblast response to HGF. Methods C2C12 myoblasts and a derived clone (C6) with low glypican-1 expression were used as an experimental model. The activation of Met, ERK1/2 and AKT in response to HGF was evaluated. The distribution of Met and its activated form in lipid raft domains, as well as its dependence on glypican-1, were characterized by sucrose density gradient fractionation in both cell types. Rescue experiments reexpressing glypican-1 or a chimeric glypican-1 fused to the transmembrane and cytoplasmic domains of mouse syndecan-1 or myoblast pretreatment with MβCD were conducted. In vitro and in vivo myoblast migration assays in response to HGF were also performed. Results Glypican-1 localization in membrane raft domains was required for a maximum cell response to HGF. It stabilized Met and HGF in lipid raft domains, forming a signaling complex where the active phospho-Met receptor was concentrated. Glypican-1 also stabilized CD44 in a HGF-dependent manner. In addition, glypican-1 was required for in vitro and in vivo HGF-dependent myoblast migration. Conclusions Glypican-1 is a regulator of HGF-dependent signaling via Met in lipid raft domains. PMID:24517345

  14. Carbon nanoparticles induce ceramide- and lipid raft-dependent signalling in lung epithelial cells: a target for a preventive strategy against environmentally-induced lung inflammation

    PubMed Central

    2012-01-01

    Background Particulate air pollution in lung epithelial cells induces pathogenic endpoints like proliferation, apoptosis, and pro-inflammatory reactions. The activation of the epidermal growth factor receptor (EGFR) is a key event responsible for signalling events involving mitogen activated protein kinases specific for these endpoints. The molecular events leading to receptor activation however are not well understood. These events are relevant for the toxicological evaluation of inhalable particles as well as for potential preventive strategies in situations when particulate air pollution cannot be avoided. The current study therefore had the objective to elucidate membrane-coupled events leading to EGFR activation and the subsequent signalling cascade in lung epithelial cells. Furthermore, we aimed to identify the molecular target of ectoine, a biophysical active substance which we described to prevent carbon nanoparticle-induced lung inflammation. Methods Membrane signalling events were investigated in isolated lipid rafts from lung epithelial cells with regard to lipid and protein content of the signalling platforms. Using positive and negative intervention approaches, lipid raft changes, subsequent signalling events, and lung inflammation were investigated in vitro in lung epithelial cells (RLE-6TN) and in vivo in exposed animals. Results Carbon nanoparticle treatment specifically led to an accumulation of ceramides in lipid rafts. Detailed analyses demonstrated a causal link of ceramides and subsequent EGFR activation coupled with a loss of the receptor in the lipid raft fractions. In vitro and in vivo investigations demonstrate the relevance of these events for carbon nanoparticle-induced lung inflammation. Moreover, the compatible solute ectoine was able to prevent ceramide-mediated EGFR phosphorylation and subsequent signalling as well as lung inflammation in vivo. Conclusion The data identify a so far unknown event in pro-inflammatory signalling and contribute to the understanding of particle cell interaction and therefore to risk identification and risk assessment of inhalable xenobiotics. Moreover, as this cellular reaction can be prevented by the well tolerated substance ectoine, a molecular preventive strategy for susceptible persons against airway inflammation is proposed. PMID:23228165

  15. AIP Career Pathways

    NASA Astrophysics Data System (ADS)

    Palchak, Amanda

    2012-02-01

    American Institute of Physics (AIP) Career Pathways is a new project funded by the National Science Foundation. One of the goals of AIP Career Pathways is to prepare students to compete for Science, Technology, Engineering, and Mathematics (STEM) careers with a bachelor's degree in physics. In order to do so, I reviewed and compiled useful resources on finding a STEM career with a bachelor's degree in physics. These resources not only supply the job seeker with job postings in STEM careers but also provide them with information on resumes, interviewing skills, and networking. Recently at the 2011 Industrial Physics Forum, I interviewed companies in the private sector to obtain a unique perspective on what types of skills potential employers expect an applicant to posses with a bachelor's degree in physics. Ultimately, these components will be used as supplements at student career workshops held at annual Society of Physics Students Zone Meetings.

  16. Molecular pathways in dystonia

    PubMed Central

    Bragg, D. Cristopher; Armata, Ioanna A.; Nery, Flavia C.; Breakefield, Xandra O.; Sharma, Nutan

    2011-01-01

    The hereditary dystonias comprise a set of diseases defined by a common constellation of motor deficits. These disorders are most likely associated with different molecular etiologies, many of which have yet to be elucidated. Here we discuss recent advances in three forms of hereditary dystonia, DYT1, DYT6 and DYT16, which share a similar clinical picture: onset in childhood or adolescence, progressive spread of symptoms with generalized involvement of body regions and a steady state affliction without treatment. Unlike DYT1, the genes responsible for DYT6 and DYT16 have only recently been identified, with relatively little information about the function of the encoded proteins. Nevertheless, recent data suggest that these proteins may fit together within interacting pathways involved in dopaminergic signaling, transcriptional regulation, and cellular stress responses. This review focuses on these molecular pathways, highlighting potential common themes among these dystonias which may serve as areas for future research. PMID:21134457

  17. Pathways of tau fibrillization.

    PubMed

    Kuret, Jeff; Chirita, Carmen N; Congdon, Erin E; Kannanayakal, Theresa; Li, Guibin; Necula, Mihaela; Yin, Haishan; Zhong, Qi

    2005-01-01

    New methods for analyzing tau fibrillization have yielded insights into the biochemical transitions involved in the process. Here we review the parallels between the sequential progression of tau fibrillization observed macroscopically in Alzheimer's disease (AD) lesions and the pathway of tau aggregation observed in vitro with purified tau preparations. In addition, pharmacological agents for further dissection of fibrillization mechanism and lesion formation are discussed. PMID:15615636

  18. Alternative Respiratory Pathway

    PubMed Central

    Siedow, James N.; Girvin, Mark E.

    1980-01-01

    Oxygen uptake during the first hours of imbibition in intact soybean and mung bean seeds showed a marked sensitivity to potassium cyanide but was unaffected by addition of either salicylhydroxamic acid or propyl gallate. However O2 uptake by finely ground seed particles was very sensitive to the addition of either compound. The results indicated that O2 uptake in intact, imbibing seeds was associated with a cyanide-sensitive process, most probably mitochondrial mediated respiration, and not the result of the cyanide-insensitive lipoxygenase activity which was readily detectable in ground seed particles. The antioxidant propyl gallate was found to inhibit specifically alternative pathway electron transfer in isolated mung bean mitochondria. Half-maximal inhibition occurred with 2 to 5 micromolar propyl gallate. Kinetic analysis indicated that propyl gallate inhibition of the alternative pathway occurred at, or very near, the site of inhibition of the alternative pathway by salicylhydroxamic acid. A high level of lipoxygenase activity was found to be associated with washed mitochondria isolated from a variety of etiolated plant tissues. Most of this lipoxygenase activity could be eliminated from mung bean mitochondria if the mitochondria were purified on a discontinuous sucrose gradient. This indicated that the mitochondrial-associated activity was probably the result of nonspecific adsorption of lipoxygenase onto the mitochondrial membranes during isolation. PMID:16661259

  19. Pathway and Resource Overview (Presentation)

    SciTech Connect

    Ruth, M. F.

    2009-11-16

    This presentation provides information about hydrogen pathway analysis, which is analysis of the total levelized cost (including return on investment). Well-to-wheels (WTW) energy use, and WTW emissions for hydrogen production, delivery, and distribution pathways.

  20. An early secretory pathway mediated by GNOM-LIKE 1 and GNOM is essential for basal polarity establishment in Arabidopsis thaliana

    PubMed Central

    Doyle, Siamsa M.; Haeger, Ash; Vain, Thomas; Rigal, Adeline; Viotti, Corrado; Łangowska, Małgorzata; Ma, Qian; Friml, Jiří; Raikhel, Natasha V.; Hicks, Glenn R.; Robert, Stéphanie

    2015-01-01

    Spatial regulation of the plant hormone indole-3-acetic acid (IAA, or auxin) is essential for plant development. Auxin gradient establishment is mediated by polarly localized auxin transporters, including PIN-FORMED (PIN) proteins. Their localization and abundance at the plasma membrane are tightly regulated by endomembrane machinery, especially the endocytic and recycling pathways mediated by the ADP ribosylation factor guanine nucleotide exchange factor (ARF-GEF) GNOM. We assessed the role of the early secretory pathway in establishing PIN1 polarity in Arabidopsis thaliana by pharmacological and genetic approaches. We identified the compound endosidin 8 (ES8), which selectively interferes with PIN1 basal polarity without altering the polarity of apical proteins. ES8 alters the auxin distribution pattern in the root and induces a strong developmental phenotype, including reduced root length. The ARF-GEF–defective mutants gnom-like 1 (gnl1-1) and gnom (van7) are significantly resistant to ES8. The compound does not affect recycling or vacuolar trafficking of PIN1 but leads to its intracellular accumulation, resulting in loss of PIN1 basal polarity at the plasma membrane. Our data confirm a role for GNOM in endoplasmic reticulum (ER)–Golgi trafficking and reveal that a GNL1/GNOM-mediated early secretory pathway selectively regulates PIN1 basal polarity establishment in a manner essential for normal plant development. PMID:25646449

  1. Pathways with PathWhiz.

    PubMed

    Pon, Allison; Jewison, Timothy; Su, Yilu; Liang, Yongjie; Knox, Craig; Maciejewski, Adam; Wilson, Michael; Wishart, David S

    2015-07-01

    PathWhiz (http://smpdb.ca/pathwhiz) is a web server designed to create colourful, visually pleasing and biologically accurate pathway diagrams that are both machine-readable and interactive. As a web server, PathWhiz is accessible from almost any place and compatible with essentially any operating system. It also houses a public library of pathways and pathway components that can be easily viewed and expanded upon by its users. PathWhiz allows users to readily generate biologically complex pathways by using a specially designed drawing palette to quickly render metabolites (including automated structure generation), proteins (including quaternary structures, covalent modifications and cofactors), nucleic acids, membranes, subcellular structures, cells, tissues and organs. Both small-molecule and protein/gene pathways can be constructed by combining multiple pathway processes such as reactions, interactions, binding events and transport activities. PathWhiz's pathway replication and propagation functions allow for existing pathways to be used to create new pathways or for existing pathways to be automatically propagated across species. PathWhiz pathways can be saved in BioPAX, SBGN-ML and SBML data exchange formats, as well as PNG, PWML, HTML image map or SVG images that can be viewed offline or explored using PathWhiz's interactive viewer. PathWhiz has been used to generate over 700 pathway diagrams for a number of popular databases including HMDB, DrugBank and SMPDB. PMID:25934797

  2. Pathways with PathWhiz

    PubMed Central

    Pon, Allison; Jewison, Timothy; Su, Yilu; Liang, Yongjie; Knox, Craig; Maciejewski, Adam; Wilson, Michael; Wishart, David S.

    2015-01-01

    PathWhiz (http://smpdb.ca/pathwhiz) is a web server designed to create colourful, visually pleasing and biologically accurate pathway diagrams that are both machine-readable and interactive. As a web server, PathWhiz is accessible from almost any place and compatible with essentially any operating system. It also houses a public library of pathways and pathway components that can be easily viewed and expanded upon by its users. PathWhiz allows users to readily generate biologically complex pathways by using a specially designed drawing palette to quickly render metabolites (including automated structure generation), proteins (including quaternary structures, covalent modifications and cofactors), nucleic acids, membranes, subcellular structures, cells, tissues and organs. Both small-molecule and protein/gene pathways can be constructed by combining multiple pathway processes such as reactions, interactions, binding events and transport activities. PathWhiz's pathway replication and propagation functions allow for existing pathways to be used to create new pathways or for existing pathways to be automatically propagated across species. PathWhiz pathways can be saved in BioPAX, SBGN-ML and SBML data exchange formats, as well as PNG, PWML, HTML image map or SVG images that can be viewed offline or explored using PathWhiz's interactive viewer. PathWhiz has been used to generate over 700 pathway diagrams for a number of popular databases including HMDB, DrugBank and SMPDB. PMID:25934797

  3. Modulation of the Surface Proteome through Multiple Ubiquitylation Pathways in African Trypanosomes

    PubMed Central

    Alsford, Sam; Horn, David; Field, Mark C.

    2015-01-01

    Recently we identified multiple suramin-sensitivity genes with a genome wide screen in Trypanosoma brucei that includes the invariant surface glycoprotein ISG75, the adaptin-1 (AP-1) complex and two deubiquitylating enzymes (DUBs) orthologous to ScUbp15/HsHAUSP1 and pVHL-interacting DUB1 (type I), designated TbUsp7 and TbVdu1, respectively. Here we have examined the roles of these genes in trafficking of ISG75, which appears key to suramin uptake. We found that, while AP-1 does not influence ISG75 abundance, knockdown of TbUsp7 or TbVdu1 leads to reduced ISG75 abundance. Silencing TbVdu1 also reduced ISG65 abundance. TbVdu1 is a component of an evolutionarily conserved ubiquitylation switch and responsible for rapid receptor modulation, suggesting similar regulation of ISGs in T. brucei. Unexpectedly, TbUsp7 knockdown also blocked endocytosis. To integrate these observations we analysed the impact of TbUsp7 and TbVdu1 knockdown on the global proteome using SILAC. For TbVdu1, ISG65 and ISG75 are the only significantly modulated proteins, but for TbUsp7 a cohort of integral membrane proteins, including the acid phosphatase MBAP1, that is required for endocytosis, and additional ISG-related proteins are down-regulated. Furthermore, we find increased expression of the ESAG6/7 transferrin receptor and ESAG5, likely resulting from decreased endocytic activity. Therefore, multiple ubiquitylation pathways, with a complex interplay with trafficking pathways, control surface proteome expression in trypanosomes. PMID:26492041

  4. Developmental pathways in colon cancer

    PubMed Central

    Bertrand, Fred E.; Angus, C. William; Partis, William J.; Sigounas, George

    2012-01-01

    A hallmark of cancer is reactivation/alteration of pathways that control cellular differentiation during developmental processes. Evidence indicates that WNT, Notch, BMP and Hedgehog pathways have a role in normal epithelial cell differentiation, and that alterations in these pathways accompany establishment of the tumorigenic state. Interestingly, there is recent evidence that these pathways are intertwined at the molecular level, and these nodes of intersection may provide opportunities for effective targeted therapies. This review will highlight the role of the WNT, Notch, BMP and Hedgehog pathways in colon cancer. PMID:23032367

  5. Improving Carbon Fixation Pathways

    PubMed Central

    Ducat, Daniel C.

    2012-01-01

    A recent resurgence in basic and applied research on photosynthesis has been driven in part by recognition that fulfilling future food and energy requirements will necessitate improvements in crop carbon-fixation efficiencies. Photosynthesis in traditional terrestrial crops is being reexamined in light of molecular strategies employed by photosynthetic microbes to enhance the activity of the Calvin cycle. Synthetic biology is well-situated to provide original approaches for compartmentalizing and enhancing photosynthetic reactions in a species independent manner. Furthermore, the elucidation of alternative carbon-fixation routes distinct from the Calvin cycle raises possibilities that alternative pathways and organisms can be utilized to fix atmospheric carbon dioxide into useful materials. PMID:22647231

  6. Growth hormone signaling pathways.

    PubMed

    Carter-Su, Christin; Schwartz, Jessica; Argetsinger, Lawrence S

    2016-06-01

    Over 20years ago, our laboratory showed that growth hormone (GH) signals through the GH receptor-associated tyrosine kinase JAK2. We showed that GH binding to its membrane-bound receptor enhances binding of JAK2 to the GHR, activates JAK2, and stimulates tyrosyl phosphorylation of both JAK2 and GHR. The activated JAK2/GHR complex recruits a variety of signaling proteins, thereby initiating multiple signaling pathways and cellular responses. These proteins and pathways include: 1) Stat transcription factors implicated in the expression of multiple genes, including the gene encoding insulin-like growth factor 1; 2) Shc adapter proteins that lead to activation of the grb2-SOS-Ras-Raf-MEK-ERK1,2 pathway; 3) insulin receptor substrate proteins implicated in the phosphatidylinositol-3-kinase and Akt pathway; 4) signal regulatory protein α, a transmembrane scaffold protein that recruits proteins including the tyrosine phosphatase SHP2; and 5) SH2B1, a scaffold protein that can activate JAK2 and enhance GH regulation of the actin cytoskeleton. Our recent work has focused on the function of SH2B1. We have shown that SH2B1β is recruited to and phosphorylated by JAK2 in response to GH. SH2B1 localizes to the plasma membrane, cytoplasm and focal adhesions; it also cycles through the nucleus. SH2B1 regulates the actin cytoskeleton and promotes GH-dependent motility of RAW264.7 macrophages. Mutations in SH2B1 have been found in humans exhibiting severe early-onset childhood obesity and insulin resistance. These mutations impair SH2B1 enhancement of GH-induced macrophage motility. As SH2B1 is expressed ubiquitously and is also recruited to a variety of receptor tyrosine kinases, our results raise the possibility that effects of SH2B1 on the actin cytoskeleton in various cell types, including neurons, may play a role in regulating body weight. PMID:26421979

  7. Improving carbon fixation pathways

    SciTech Connect

    Ducat, DC; Silver, PA

    2012-08-01

    A recent resurgence in basic and applied research on photosynthesis has been driven in part by recognition that fulfilling future food and energy requirements will necessitate improvements in crop carbon-fixation efficiencies. Photosynthesis in traditional terrestrial crops is being reexamined in light of molecular strategies employed by photosynthetic microbes to enhance the activity of the Calvin cycle. Synthetic biology is well-situated to provide original approaches for compartmentalizing and enhancing photosynthetic reactions in a species independent manner. Furthermore, the elucidation of alternative carbon-fixation routes distinct from the Calvin cycle raises possibilities that novel pathways and organisms can be utilized to fix atmospheric carbon dioxide into useful materials.

  8. The Reactome pathway knowledgebase.

    PubMed

    Croft, David; Mundo, Antonio Fabregat; Haw, Robin; Milacic, Marija; Weiser, Joel; Wu, Guanming; Caudy, Michael; Garapati, Phani; Gillespie, Marc; Kamdar, Maulik R; Jassal, Bijay; Jupe, Steven; Matthews, Lisa; May, Bruce; Palatnik, Stanislav; Rothfels, Karen; Shamovsky, Veronica; Song, Heeyeon; Williams, Mark; Birney, Ewan; Hermjakob, Henning; Stein, Lincoln; D'Eustachio, Peter

    2014-01-01

    Reactome (http://www.reactome.org) is a manually curated open-source open-data resource of human pathways and reactions. The current version 46 describes 7088 human proteins (34% of the predicted human proteome), participating in 6744 reactions based on data extracted from 15 107 research publications with PubMed links. The Reactome Web site and analysis tool set have been completely redesigned to increase speed, flexibility and user friendliness. The data model has been extended to support annotation of disease processes due to infectious agents and to mutation. PMID:24243840

  9. WikiPathways: capturing the full diversity of pathway knowledge

    PubMed Central

    Kutmon, Martina; Riutta, Anders; Nunes, Nuno; Hanspers, Kristina; Willighagen, Egon L.; Bohler, Anwesha; Mélius, Jonathan; Waagmeester, Andra; Sinha, Sravanthi R.; Miller, Ryan; Coort, Susan L.; Cirillo, Elisa; Smeets, Bart; Evelo, Chris T.; Pico, Alexander R.

    2016-01-01

    WikiPathways (http://www.wikipathways.org) is an open, collaborative platform for capturing and disseminating models of biological pathways for data visualization and analysis. Since our last NAR update, 4 years ago, WikiPathways has experienced massive growth in content, which continues to be contributed by hundreds of individuals each year. New aspects of the diversity and depth of the collected pathways are described from the perspective of researchers interested in using pathway information in their studies. We provide updates on extensions and services to support pathway analysis and visualization via popular standalone tools, i.e. PathVisio and Cytoscape, web applications and common programming environments. We introduce the Quick Edit feature for pathway authors and curators, in addition to new means of publishing pathways and maintaining custom pathway collections to serve specific research topics and communities. In addition to the latest milestones in our pathway collection and curation effort, we also highlight the latest means to access the content as publishable figures, as standard data files, and as linked data, including bulk and programmatic access. PMID:26481357

  10. WikiPathways: capturing the full diversity of pathway knowledge.

    PubMed

    Kutmon, Martina; Riutta, Anders; Nunes, Nuno; Hanspers, Kristina; Willighagen, Egon L; Bohler, Anwesha; Mélius, Jonathan; Waagmeester, Andra; Sinha, Sravanthi R; Miller, Ryan; Coort, Susan L; Cirillo, Elisa; Smeets, Bart; Evelo, Chris T; Pico, Alexander R

    2016-01-01

    WikiPathways (http://www.wikipathways.org) is an open, collaborative platform for capturing and disseminating models of biological pathways for data visualization and analysis. Since our last NAR update, 4 years ago, WikiPathways has experienced massive growth in content, which continues to be contributed by hundreds of individuals each year. New aspects of the diversity and depth of the collected pathways are described from the perspective of researchers interested in using pathway information in their studies. We provide updates on extensions and services to support pathway analysis and visualization via popular standalone tools, i.e. PathVisio and Cytoscape, web applications and common programming environments. We introduce the Quick Edit feature for pathway authors and curators, in addition to new means of publishing pathways and maintaining custom pathway collections to serve specific research topics and communities. In addition to the latest milestones in our pathway collection and curation effort, we also highlight the latest means to access the content as publishable figures, as standard data files, and as linked data, including bulk and programmatic access. PMID:26481357

  11. The Chordin Morphogenetic Pathway.

    PubMed

    De Robertis, Edward M; Moriyama, Yuki

    2016-01-01

    The ancestral Chordin/bone morphogenetic protein (BMP) signaling pathway that establishes dorsal-ventral (D-V) patterning in animal development is one of the best understood morphogenetic gradients, and is established by multiple proteins that interact with each other in the extracellular space-including several BMPs, Chordin, Tolloid, Ont-1, Crossveinless-2, and Sizzled. The D-V gradient is adjusted redundantly by regulating the synthesis of its components, by direct protein-protein interactions between morphogens, and by long-range diffusion. The entire embryo participates in maintaining the D-V BMP gradient, so that for each action in the dorsal side there is a reaction in the ventral side. A gradient of Chordin is formed in the extracellular matrix that separates ectoderm from endomesoderm, called Brachet's cleft in Xenopus. The Chordin/BMP pathway is self-organizing and able to scale pattern in the dorsal half of bisected embryos or in Spemann dorsal lip transplantation experiments. PMID:26970622

  12. Pathways of lateral spreading.

    PubMed

    Jacobi, U; Schanzer, S; Weigmann, H-J; Patzelt, A; Vergou, T; Sterry, W; Lademann, J

    2011-01-01

    In the case of topically applied substances, usually both lateral spreading and competitive penetration into the skin occur in parallel. In the present study, the pathways of lateral spreading were studied quantitatively and visually. The local distribution and lateral spreading of the UV filter substance butyl methoxydibenzoylmethane applied in an o/w emulsion was studied on the forearm and the back. The tape stripping procedure was used to determine the recovery rates inside and outside the area of application. The skin characteristics of transepidermal water loss, pH value, hydration of the stratum corneum and sebum rate were determined at both anatomic sites. Photography and laser scanning microscopy were used to visually investigate the lateral spreading of topically applied dyes. On the back, a preferred direction of lateral spreading parallel to the body axis was observed. This result was caused by differences in the network of furrows. The furrows functioned as a pathway for lateral spreading, whereas the follicles formed a reservoir for the topically applied substance. PMID:21455016

  13. The Reactome pathway Knowledgebase.

    PubMed

    Fabregat, Antonio; Sidiropoulos, Konstantinos; Garapati, Phani; Gillespie, Marc; Hausmann, Kerstin; Haw, Robin; Jassal, Bijay; Jupe, Steven; Korninger, Florian; McKay, Sheldon; Matthews, Lisa; May, Bruce; Milacic, Marija; Rothfels, Karen; Shamovsky, Veronica; Webber, Marissa; Weiser, Joel; Williams, Mark; Wu, Guanming; Stein, Lincoln; Hermjakob, Henning; D'Eustachio, Peter

    2016-01-01

    The Reactome Knowledgebase (www.reactome.org) provides molecular details of signal transduction, transport, DNA replication, metabolism and other cellular processes as an ordered network of molecular transformations-an extended version of a classic metabolic map, in a single consistent data model. Reactome functions both as an archive of biological processes and as a tool for discovering unexpected functional relationships in data such as gene expression pattern surveys or somatic mutation catalogues from tumour cells. Over the last two years we redeveloped major components of the Reactome web interface to improve usability, responsiveness and data visualization. A new pathway diagram viewer provides a faster, clearer interface and smooth zooming from the entire reaction network to the details of individual reactions. Tool performance for analysis of user datasets has been substantially improved, now generating detailed results for genome-wide expression datasets within seconds. The analysis module can now be accessed through a RESTFul interface, facilitating its inclusion in third party applications. A new overview module allows the visualization of analysis results on a genome-wide Reactome pathway hierarchy using a single screen page. The search interface now provides auto-completion as well as a faceted search to narrow result lists efficiently. PMID:26656494

  14. Signaling Pathways in Melanogenesis

    PubMed Central

    D’Mello, Stacey A. N.; Finlay, Graeme J.; Baguley, Bruce C.; Askarian-Amiri, Marjan E.

    2016-01-01

    Melanocytes are melanin-producing cells found in skin, hair follicles, eyes, inner ear, bones, heart and brain of humans. They arise from pluripotent neural crest cells and differentiate in response to a complex network of interacting regulatory pathways. Melanins are pigment molecules that are endogenously synthesized by melanocytes. The light absorption of melanin in skin and hair leads to photoreceptor shielding, thermoregulation, photoprotection, camouflage and display coloring. Melanins are also powerful cation chelators and may act as free radical sinks. Melanin formation is a product of complex biochemical events that starts from amino acid tyrosine and its metabolite, dopa. The types and amounts of melanin produced by melanocytes are determined genetically and are influenced by a variety of extrinsic and intrinsic factors such as hormonal changes, inflammation, age and exposure to UV light. These stimuli affect the different pathways in melanogenesis. In this review we will discuss the regulatory mechanisms involved in melanogenesis and explain how intrinsic and extrinsic factors regulate melanin production. We will also explain the regulatory roles of different proteins involved in melanogenesis. PMID:27428965

  15. Signaling Pathways in Melanogenesis.

    PubMed

    D'Mello, Stacey A N; Finlay, Graeme J; Baguley, Bruce C; Askarian-Amiri, Marjan E

    2016-01-01

    Melanocytes are melanin-producing cells found in skin, hair follicles, eyes, inner ear, bones, heart and brain of humans. They arise from pluripotent neural crest cells and differentiate in response to a complex network of interacting regulatory pathways. Melanins are pigment molecules that are endogenously synthesized by melanocytes. The light absorption of melanin in skin and hair leads to photoreceptor shielding, thermoregulation, photoprotection, camouflage and display coloring. Melanins are also powerful cation chelators and may act as free radical sinks. Melanin formation is a product of complex biochemical events that starts from amino acid tyrosine and its metabolite, dopa. The types and amounts of melanin produced by melanocytes are determined genetically and are influenced by a variety of extrinsic and intrinsic factors such as hormonal changes, inflammation, age and exposure to UV light. These stimuli affect the different pathways in melanogenesis. In this review we will discuss the regulatory mechanisms involved in melanogenesis and explain how intrinsic and extrinsic factors regulate melanin production. We will also explain the regulatory roles of different proteins involved in melanogenesis. PMID:27428965

  16. AIP's Career Pathways Project

    NASA Astrophysics Data System (ADS)

    Avila, Jose

    2014-03-01

    The American Institute of Physics (AIP) Career Pathways Project, funded by the National Science Foundation, aims to increase the number of undergraduates going into STEM careers. The main purposes of this project are to show students the professional opportunities for a STEM career, understand what departments can do to better prepare physics bachelor's degree recipients to enter the workforce, understand what students can do to better prepare themselves, and develop resources based on these findings. I was chosen by the Society of Physics Students (SPS) to be the 2013 summer intern of the AIP's Career Pathways Project. In this talk I will discuss several resources I worked on with the Statistical Research Center of the American Institute of Physics and SPS. These resources include how to write a resume and cover letter, how to perform an informational interview, common job titles for physics bachelors, how to find career information in physics and STEM, how to search and use job postings, and how to network.

  17. Summer 2014 Pathways Report

    NASA Technical Reports Server (NTRS)

    Hand, Zachary

    2014-01-01

    Over the summer I had the exciting opportunity to work for NASA at the Kennedy Space Center as a Mission Assurance Engineering intern. When I was offered a position in mission assurance for the Safety and Mission Assurance directorate's Launch Services Division, I didn't really know what I would be doing, but I knew it would be an excellent opportunity to learn and grow professionally. In this report I will provide some background information on the Launch Services Division, as well as detail my duties and accomplishments during my time as an intern. Additionally, I will relate the significance of my work experience to my current academic work and future career goals. This report contains background information on Mission Assurance Engineering, a description of my duties and accomplishments over the summer of 2014, and relates the significance of my work experience to my school work and future career goals. It is a required document for the Pathways program.

  18. Pathways of Antigen Processing

    PubMed Central

    Blum, Janice S.; Wearsch, Pamela A.; Cresswell, Peter

    2014-01-01

    T cell recognition of antigen presenting cells depends on their expression of a spectrum of peptides bound to Major Histocompatibility Complex class I (MHC-I) and class II (MHC-II) molecules. Conversion of antigens from pathogens or transformed cells into MHC-I and MHC-II-bound peptides is critical for mounting protective T cell responses, and similar processing of self proteins is necessary to establish and maintain tolerance. Cells use a variety of mechanisms to acquire protein antigens, from translation in the cytosol to variations on the theme of endocytosis, and to degrade them once acquired. In this review we highlight the aspects of MHC-I and MHC-II biosynthesis and assembly that have evolved to intersect these pathways and sample the peptides that are produced. PMID:23298205

  19. The Peroxide Pathway

    NASA Technical Reports Server (NTRS)

    McNeal, Curtis I., Jr.; Anderson, William

    1999-01-01

    NASA's current focus on technology roadmaps as a tool for guiding investment decisions leads naturally to a discussion of NASA's roadmap for peroxide propulsion system development. NASA's new Second Generation Space Transportation System roadmap calls for an integrated Reusable Upper-Stage (RUS) engine technology demonstration in the FY03/FY04 time period. Preceding this integrated demonstration are several years of component developments and subsystem technology demonstrations. NASA and the Air Force took the first steps at developing focused upper stage technologies with the initiation of the Upper Stage Flight Experiment with Orbital Sciences in December 1997. A review of this program's peroxide propulsion development is a useful first step in establishing the peroxide propulsion pathway that could lead to a RUS demonstration in 2004.

  20. Pathways Intern Work Report

    NASA Technical Reports Server (NTRS)

    Midulla, Laura P.

    2014-01-01

    For the spring of 2014, I continued my position at NASA as a Pathways Engineering Student Trainee in the Engineering and Technology Directorate (NE), in the Test, Operations & Support Software Engineering Branch of the Control and Data Systems Division. The Control and Data Systems Division provides control, monitor, checkout, processing, display, and simulation capabilities to support vehicle element testing, checkout and launch. In addition the division provides significant leadership associated with engineering processes and Information Technology Security management. My assignment during this semester was to continue to support the Engineering Information Technology (IT) Security team, providing IT Security support to the Ground Systems Development and Operations Spaceport Command & Control System and Ground Systems, NE Labs and other systems.

  1. Pathways of iron absorption.

    PubMed

    Conrad, Marcel E; Umbreit, Jay N

    2002-01-01

    Iron is vital for all living organisms but excess iron can be lethal because it facilitates free radical formation. Thus iron absorption is carefully regulated to maintain an equilibrium between absorption and body loss of iron. In countries where meat is a significant part of the diet, most body iron is derived from dietary heme because heme binds few of the dietary chelators that bind inorganic iron. Uptake of heme into enterocytes occurs as a metalloporphyrin in an endosomal process. Intracellular iron is released from heme by heme oxygenase to enter plasma as inorganic iron. Ferric iron is absorbed via a beta(3) integrin and mobilferrin pathway (IMP) which is unshared with other nutritional metals. Ferrous iron uptake is facilitated by a DMT-1 pathway which is shared with manganese. In the iron deficient gut, large quantities of both mobilferrin and DMT-1 are found in goblet cells and intraluminal mucins suggesting that they are secreted with mucin into the intestinal lumen to bind iron to facilitate uptake by the cells. In the cytoplasm, IMP and DMT associate in a large protein complex called paraferritin which serves as a ferrireductase. Paraferritin solublizes iron binding proteins and reduces iron to make iron available for production of iron containing proteins such as heme. Iron uptake by intestinal absorptive cells is regulated by the iron concentration within the cell. Except in hemochromatosis it remains in equilibrium with total body stores via transferrin receptors on the basolateral membrane of absorptive cells. Increased intracellular iron either up-regulates or satiates iron binding proteins on regulatory proteins to alter their location in the intestinal mucosa. PMID:12547224

  2. Insulin-induced redistribution of GLUT4 glucose carriers in the muscle fiber. In search of GLUT4 trafficking pathways.

    PubMed

    Zorzano, A; Muñoz, P; Camps, M; Mora, C; Testar, X; Palacín, M

    1996-01-01

    Insulin rapidly stimulates glucose transport in muscle fiber. This process controls the utilization of glucose in skeletal muscle, and it is deficient in various insulin-resistant states, such as non-insulin-dependent diabetes mellitus. The effect of insulin on muscle glucose transport is mainly due to the recruitment of GLUT4 glucose carriers to the cell surface of the muscle fiber. There is increasing evidence that the recruitment of GLUT4 carriers triggered by insulin affects selective domains of sarcolemma and transverse tubules. In contrast, GLUT1 is located mainly in sarcolemma and is absent in transverse tubules, and insulin does not alter its cellular distribution in muscle fiber. The differential distribution of GLUT1 and GLUT4 in the cell surface raises new questions regarding the precise endocytic and exocytic pathways that are functional in the muscle fiber. The current view of insulin-induced GLUT4 translocation is based mainly on studies performed in adipocytes. These studies have proposed the existence of intracellular compartments of GLUT4 that respond to insulin in a highly homogeneous manner. However, studies performed in skeletal muscle have identified insulin-sensitive as well as insulin-insensitive intracellular GLUT4-containing membranes. These data open a new perspective on the dynamics of intracellular GLUT4 compartments in insulin-sensitive cells. PMID:8529804

  3. A dynamin 1-, dynamin 3- and clathrin-independent pathway of synaptic vesicle recycling mediated by bulk endocytosis

    PubMed Central

    Wu, Yumei; O'Toole, Eileen T; Girard, Martine; Ritter, Brigitte; Messa, Mirko; Liu, Xinran; McPherson, Peter S; Ferguson, Shawn M; De Camilli, Pietro

    2014-01-01

    The exocytosis of synaptic vesicles (SVs) elicited by potent stimulation is rapidly compensated by bulk endocytosis of SV membranes leading to large endocytic vacuoles (‘bulk’ endosomes). Subsequently, these vacuoles disappear in parallel with the reappearance of new SVs. We have used synapses of dynamin 1 and 3 double knock-out neurons, where clathrin-mediated endocytosis (CME) is dramatically impaired, to gain insight into the poorly understood mechanisms underlying this process. Massive formation of bulk endosomes was not defective, but rather enhanced, in the absence of dynamin 1 and 3. The subsequent conversion of bulk endosomes into SVs was not accompanied by the accumulation of clathrin coated buds on their surface and this process proceeded even after further clathrin knock-down, suggesting its independence of clathrin. These findings support the existence of a pathway for SV reformation that bypasses the requirement for clathrin and dynamin 1/3 and that operates during intense synaptic activity. DOI: http://dx.doi.org/10.7554/eLife.01621.001 PMID:24963135

  4. A pathway to spirituality.

    PubMed

    Shaw, Jon A

    2005-01-01

    The phenomenology of mystical experiences has been described throughout all the ages and in all religions. All mystical traditions identify some sense of union with the absolute as the ultimate spiritual goal. I assume that the pathway to both theistic and secular spirituality and our readiness to seek a solution in a psychological merger with something beyond the self evolves out of our human experience. Spirituality is one of man's strategies for dealing with the limitations of the life cycle, separation and loss, biological fragility, transience, and non-existence. Spirituality may serve as the affective component to a belief system or myth that is not rooted in scientific evidence but is lived as if it is true. Spirituality may take many forms, but I will suggest that in some instances it may serve as a reparative process in which one creates in the external world, through symbolic form, a nuance or facet of an internalized mental representation which has become lost or is no longer available to the self; or it may represent the continuity of the self-representation after death through a self-object merger. Lastly I will illustrate from the writings of two of our greatest poets, Dante Alighieri and William Wordsworth, how their poetry became interwoven with a profound spirituality. In Dante we will see the elaboration of a religious spirituality, while in the writings of Wordsworth a secular spirituality emerges interwoven with nature and belatedly his identification with "tragic man" as his mythos. PMID:16599401

  5. Pathways to legal immigration

    PubMed Central

    MASSEY, DOUGLAS S.; MALONE, NOLAN

    2010-01-01

    In this paper we use the New Immigrant Survey Pilot Study (NISP) to describe the amount and kind of experience that immigrants accumulate in the United States before they become permanent resident aliens. The NISP surveyed a representative sample of legal immigrants who acquired residence papers during July and August of 1996, yielding a completed sample of 1,135 adults. Our analysis revealed that roughly two-thirds of these newly arrived immigrants had prior experience in the United States within one of six basic categories: illegal border-crossers, visa abusers, non-resident visitors, non-resident workers, students or exchange visitors, and refugees/asylees. Each of these pathways to legal immigration was associated with a different profile with respect to nationality, social background, and economic status. Using simple earnings regressions we demonstrate how these differences can yield misleading conclusions about the process of immigrant adaptation and assimilation, even if measured effects are reasonably accurate. We suggest that social scientists should change the way they think and ask about immigrants’ arrival in the United States. PMID:20830313

  6. Binding of Alphaherpesvirus Glycoprotein H to Surface α4β1-Integrins Activates Calcium-Signaling Pathways and Induces Phosphatidylserine Exposure on the Plasma Membrane

    PubMed Central

    Gramatica, Andrea; Herrmann, Andreas; Osterrieder, Nikolaus

    2015-01-01

    ABSTRACT Intracellular signaling connected to integrin activation is known to induce cytoplasmic Ca2+ release, which in turn mediates a number of downstream signals. The cellular entry pathways of two closely related alphaherpesviruses, equine herpesviruses 1 and 4 (EHV-1 and EHV-4), are differentially regulated with respect to the requirement of interaction of glycoprotein H (gH) with α4β1-integrins. We show here that binding of EHV-1, but not EHV-4, to target cells resulted in a rapid and significant increase in cytosolic Ca2+ levels. EHV-1 expressing EHV-4 gH (gH4) in lieu of authentic gH1 failed to induce Ca2+ release, while EHV-4 with gH1 triggered significant Ca2+ release. Blocking the interaction between gH1 and α4β1-integrins, inhibiting phospholipase C (PLC) activation, or blocking binding of inositol 1,4,5-triphosphate (IP3) to its receptor on the endoplasmic reticulum (ER) abrogated Ca2+ release. Interestingly, phosphatidylserine (PS) was exposed on the plasma membrane in response to cytosolic calcium increase after EHV-1 binding through a scramblase-dependent mechanism. Inhibition of both Ca2+ release from the ER and scramblase activation blocked PS scrambling and redirected virus entry to the endocytic pathway, indicating that PS may play a role in facilitating virus entry directly at the plasma membrane. PMID:26489864

  7. Representations of metabolic knowledge: pathways.

    PubMed

    Karp, P D; Paley, S M

    1994-01-01

    The automatic generation of drawings of metabolic pathways is a challenging problem that depends intimately on exactly what information has been recorded for each pathway, and on how that information is encoded. The chief contributions of the paper are a minimized representation for biochemical pathways called the predecessor list, and inference procedures for converting the predecessor list into a pathway-graph representation that can serve as input to a pathway-drawing algorithm. The predecessor list has several advantages over the pathway graph, including its compactness and its lack of redundancy. The conversion between the two representations can be formulated as both a constraint-satisfaction problem and a logical inference problem, whose goal is to assign directions to reactions, and to determine which are the main chemical compounds in the reaction. We describe a set of production rules that solves this inference problem. We also present heuristics for inferring whether the exterior compounds that are substrates of reactions at the periphery of a pathway are side or main compounds. These techniques were evaluated on 18 metabolic pathways from the EcoCyc knowledge base. PMID:7584392

  8. Vestibular pathways involved in cognition

    PubMed Central

    Hitier, Martin; Besnard, Stephane; Smith, Paul F.

    2014-01-01

    Recent discoveries have emphasized the role of the vestibular system in cognitive processes such as memory, spatial navigation and bodily self-consciousness. A precise understanding of the vestibular pathways involved is essential to understand the consequences of vestibular diseases for cognition, as well as develop therapeutic strategies to facilitate recovery. The knowledge of the “vestibular cortical projection areas”, defined as the cortical areas activated by vestibular stimulation, has dramatically increased over the last several years from both anatomical and functional points of view. Four major pathways have been hypothesized to transmit vestibular information to the vestibular cortex: (1) the vestibulo-thalamo-cortical pathway, which probably transmits spatial information about the environment via the parietal, entorhinal and perirhinal cortices to the hippocampus and is associated with spatial representation and self-versus object motion distinctions; (2) the pathway from the dorsal tegmental nucleus via the lateral mammillary nucleus, the anterodorsal nucleus of the thalamus to the entorhinal cortex, which transmits information for estimations of head direction; (3) the pathway via the nucleus reticularis pontis oralis, the supramammillary nucleus and the medial septum to the hippocampus, which transmits information supporting hippocampal theta rhythm and memory; and (4) a possible pathway via the cerebellum, and the ventral lateral nucleus of the thalamus (perhaps to the parietal cortex), which transmits information for spatial learning. Finally a new pathway is hypothesized via the basal ganglia, potentially involved in spatial learning and spatial memory. From these pathways, progressively emerges the anatomical network of vestibular cognition. PMID:25100954

  9. Pathways from Poverty Educational Network.

    ERIC Educational Resources Information Center

    Northeast Regional Center for Rural Development, University Park, PA.

    Pathways from Poverty is a public policy education and research initiative organized by the Rural Sociological Society's Task Force on Persistent Rural Poverty and the four regional rural development centers. This publication focuses on project efforts in the Northeast and includes three sections. The first section describes the Pathways from…

  10. MPW : the metabolic pathways database.

    SciTech Connect

    Selkov, E., Jr.; Grechkin, Y.; Mikhailova, N.; Selkov, E.; Mathematics and Computer Science; Russian Academy of Sciences

    1998-01-01

    The Metabolic Pathways Database (MPW) (www.biobase.com/emphome.html/homepage. html.pags/pathways.html) a derivative of EMP (www.biobase.com/EMP) plays a fundamental role in the technology of metabolic reconstructions from sequenced genomes under the PUMA (www.mcs.anl.gov/home/compbio/PUMA/Production/ ReconstructedMetabolism/reconstruction.html), WIT (www.mcs.anl.gov/home/compbio/WIT/wit.html ) and WIT2 (beauty.isdn.msc.anl.gov/WIT2.pub/CGI/user.cgi) systems. In October 1997, it included some 2800 pathway diagrams covering primary and secondary metabolism, membrane transport, signal transduction pathways, intracellular traffic, translation and transcription. In the current public release of MPW (beauty.isdn.mcs.anl.gov/MPW), the encoding is based on the logical structure of the pathways and is represented by the objects commonly used in electronic circuit design. This facilitates drawing and editing the diagrams and makes possible automation of the basic simulation operations such as deriving stoichiometric matrices, rate laws, and, ultimately, dynamic models of metabolic pathways. Individual pathway diagrams, automatically derived from the original ASCII records, are stored as SGML instances supplemented by relational indices. An auxiliary database of compound names and structures, encoded in the SMILES format, is maintained to unambiguously connect the pathways to the chemical structures of their intermediates.

  11. Pathway Interaction Database (PID) —

    Cancer.gov

    The National Cancer Institute (NCI) in collaboration with Nature Publishing Group has established the Pathway Interaction Database (PID) in order to provide a highly structured, curated collection of information about known biomolecular interactions and key cellular processes assembled into signaling pathways.

  12. Nematode endogenous small RNA pathways

    PubMed Central

    Hoogstrate, Suzanne W; Volkers, Rita JM; Sterken, Mark G; Kammenga, Jan E; Snoek, L Basten

    2014-01-01

    The discovery of small RNA silencing pathways has greatly extended our knowledge of gene regulation. Small RNAs have been presumed to play a role in every field of biology because they affect many biological processes via regulation of gene expression and chromatin remodeling. Most well-known examples of affected processes are development, fertility, and maintenance of genome stability. Here we review the role of the three main endogenous small RNA silencing pathways in Caenorhabditis elegans: microRNAs, endogenous small interfering RNAs, and PIWI-interacting RNAs. After providing an entry-level overview on how these pathways function, we discuss research on other nematode species providing insight into the evolution of these small RNA pathways. In understanding the differences between the endogenous small RNA pathways and their evolution, a more comprehensive picture is formed of the functions and effects of small RNAs. PMID:25340013

  13. On generalized distributions and pathways

    NASA Astrophysics Data System (ADS)

    Mathai, A. M.; Haubold, H. J.

    2008-03-01

    The scalar version of the pathway model of Mathai [A.M. Mathai, Linear Alg. Appl. 396 (2005) 317] is shown to be associated with a large number of probability models used in physics. Different families of densities are listed here, which are all connected through the pathway parameter α, generating a distributional pathway. The idea is to switch from one functional form to another through this parameter and it is shown that one can proceed from the generalized type-1 beta family to generalized type-2 beta family to generalized gamma family. It is also shown that the pathway model is available by maximizing a generalized measure of entropy, leading to an entropic pathway, covering the particularly interesting cases of Tsallis statistics [C. Tsallis, J. Stat. Phys. 52 (1988) 479] and superstatistics [C. Beck, E.G.D. Cohen, Physica A 322 (2003) 267].

  14. A local coastal adaptation pathway

    NASA Astrophysics Data System (ADS)

    Barnett, J.; Graham, S.; Mortreux, C.; Fincher, R.; Waters, E.; Hurlimann, A.

    2014-12-01

    Local governments are not adapting to sea-level rise because it is difficult to build consensus on the need for change and the best way to implement it. In theory, adaptation pathways can resolve this impasse. Adaptation pathways are a sequence of linked strategies that are triggered by a change in environmental conditions, and in which initial decisions can have low regrets and preserve options for future generations. We report on a project that sought to empirically test the relevance and feasibility of a local pathway for adapting to sea-level rise. We find that triggers of change that have social impacts are salient to local people, and developing a local adaptation pathway helps build consensus among diverse constituencies. Our results show that adaptation pathways are feasible at the local scale, offering a low-risk, low-cost way to begin the long process of adaptation to sea-level rise.

  15. Refining the quantitative pathway of the Pathways to Mathematics model.

    PubMed

    Sowinski, Carla; LeFevre, Jo-Anne; Skwarchuk, Sheri-Lynn; Kamawar, Deepthi; Bisanz, Jeffrey; Smith-Chant, Brenda

    2015-03-01

    In the current study, we adopted the Pathways to Mathematics model of LeFevre et al. (2010). In this model, there are three cognitive domains--labeled as the quantitative, linguistic, and working memory pathways--that make unique contributions to children's mathematical development. We attempted to refine the quantitative pathway by combining children's (N=141 in Grades 2 and 3) subitizing, counting, and symbolic magnitude comparison skills using principal components analysis. The quantitative pathway was examined in relation to dependent numerical measures (backward counting, arithmetic fluency, calculation, and number system knowledge) and a dependent reading measure, while simultaneously accounting for linguistic and working memory skills. Analyses controlled for processing speed, parental education, and gender. We hypothesized that the quantitative, linguistic, and working memory pathways would account for unique variance in the numerical outcomes; this was the case for backward counting and arithmetic fluency. However, only the quantitative and linguistic pathways (not working memory) accounted for unique variance in calculation and number system knowledge. Not surprisingly, only the linguistic pathway accounted for unique variance in the reading measure. These findings suggest that the relative contributions of quantitative, linguistic, and working memory skills vary depending on the specific cognitive task. PMID:25521665

  16. Pathways Intern Report

    NASA Technical Reports Server (NTRS)

    Bell, Evan A.

    2015-01-01

    During my time at NASA, I worked with the Granular Mechanics and Regolith Organization (GMRO), better known as Swamp Works. The goal of the lab is to find ways to utilize resources found after the astronaut or robot has landed on another planet or asteroid. This concept is known as in-situ resource utilization and it is critical to long term missions such as those to Mars. During my time here I worked on the Asteroid and Lava Tube Free Flyer project (ALTFF). A lava tube, such as the one shown in figure 1, is a long tear drop shaped cavern that is produced when molten lava tunnels through the surrounding rock creating large unground pathways. Before mining for resources on Mars or on asteroids, a sampling mission must be done to scout out useful resource deposits. ALTFF's goal is to provide a low cost, autonomous scout robot that can sample the surface and return to the mother ship or lander for further processing of the samples. The vehicle will be looking for water ice in the regolith that can be processed into either potable water, hydrogen and oxygen fuel, or a binder material for 3D printing. By using a low cost craft to sample, there is much less risk to the more expensive mother ship or lander. While my main task was the construction of a simulation environment to test control code in and the construction of the asteroid free flyer prototype, there were other tasks that I performed relating to the ALTFF project.

  17. Ascorbate Synthesis Pathway

    PubMed Central

    Gabbay, Kenneth H.; Bohren, Kurt M.; Morello, Roy; Bertin, Terry; Liu, Jeff; Vogel, Peter

    2010-01-01

    Using mouse gene knock-out models, we identify aldehyde reductase (EC 1.1.1.2, Akr1a4 (GR)) and aldose reductase (EC 1.1.1.21, Akr1b3 (AR)) as the enzymes responsible for conversion of d-glucuronate to l-gulonate, a key step in the ascorbate (ASC) synthesis pathway in mice. The gene knock-out (KO) mice show that the two enzymes, GR and AR, provide ∼85 and ∼15% of l-gulonate, respectively. GRKO/ARKO double knock-out mice are unable to synthesize ASC (>95% ASC deficit) and develop scurvy. The GRKO mice (∼85% ASC deficit) develop and grow normally when fed regular mouse chow (ASC content = 0) but suffer severe osteopenia and spontaneous fractures with stresses that increase ASC requirements, such as pregnancy or castration. Castration greatly increases osteoclast numbers and activity in GRKO mice and promotes increased bone loss as compared with wild-type controls and additionally induces proliferation of immature dysplastic osteoblasts likely because of an ASC-sensitive block(s) in early differentiation. ASC and the antioxidants pycnogenol and resveratrol block osteoclast proliferation and bone loss, but only ASC feeding restores osteoblast differentiation and prevents their dysplastic proliferation. This is the first in vivo demonstration of two independent roles for ASC as an antioxidant suppressing osteoclast activity and number as well as a cofactor promoting osteoblast differentiation. Although humans have lost the ability to synthesize ASC, our mouse models suggest the mechanisms by which suboptimal ASC availability facilitates the development of osteoporosis, which has important implications for human osteoporosis. PMID:20410296

  18. Mechanisms of Hippo pathway regulation

    PubMed Central

    Meng, Zhipeng; Moroishi, Toshiro; Guan, Kun-Liang

    2016-01-01

    The Hippo pathway was initially identified in Drosophila melanogaster screens for tissue growth two decades ago and has been a subject extensively studied in both Drosophila and mammals in the last several years. The core of the Hippo pathway consists of a kinase cascade, transcription coactivators, and DNA-binding partners. Recent studies have expanded the Hippo pathway as a complex signaling network with >30 components. This pathway is regulated by intrinsic cell machineries, such as cell–cell contact, cell polarity, and actin cytoskeleton, as well as a wide range of signals, including cellular energy status, mechanical cues, and hormonal signals that act through G-protein-coupled receptors. The major functions of the Hippo pathway have been defined to restrict tissue growth in adults and modulate cell proliferation, differentiation, and migration in developing organs. Furthermore, dysregulation of the Hippo pathway leads to aberrant cell growth and neoplasia. In this review, we focus on recent developments in our understanding of the molecular actions of the core Hippo kinase cascade and discuss key open questions in the regulation and function of the Hippo pathway. PMID:26728553

  19. Mechanisms of Hippo pathway regulation.

    PubMed

    Meng, Zhipeng; Moroishi, Toshiro; Guan, Kun-Liang

    2016-01-01

    The Hippo pathway was initially identified in Drosophila melanogaster screens for tissue growth two decades ago and has been a subject extensively studied in both Drosophila and mammals in the last several years. The core of the Hippo pathway consists of a kinase cascade, transcription coactivators, and DNA-binding partners. Recent studies have expanded the Hippo pathway as a complex signaling network with >30 components. This pathway is regulated by intrinsic cell machineries, such as cell-cell contact, cell polarity, and actin cytoskeleton, as well as a wide range of signals, including cellular energy status, mechanical cues, and hormonal signals that act through G-protein-coupled receptors. The major functions of the Hippo pathway have been defined to restrict tissue growth in adults and modulate cell proliferation, differentiation, and migration in developing organs. Furthermore, dysregulation of the Hippo pathway leads to aberrant cell growth and neoplasia. In this review, we focus on recent developments in our understanding of the molecular actions of the core Hippo kinase cascade and discuss key open questions in the regulation and function of the Hippo pathway. PMID:26728553

  20. Signaling pathways in diabetic nephropathy.

    PubMed

    Kawanami, Daiji; Matoba, Keiichiro; Utsunomiya, Kazunori

    2016-10-01

    Diabetic nephropathy (DN) is a major cause of end-stage renal disease (ESRD), however, specific treatment for DN has not yet been elucidated. Therefore, it is critically important to understand the molecular mechanism underlying DN to develop cause-related therapeutic strategy. To date, various factors such as hemodynamic changes and metabolic pathways have been shown to be involved in the pathogenesis of DN. Excessive glucose influx activates cellular signaling pathways, including the diacylglycerol (DAG)-protein kinase C (PKC) pathway, advanced glycation end-products (AGE), polyol pathway, hexosamine pathway and oxidative stress. These factors interact with one another, thereby facilitating inflammatory processes, leading to the development of glomerulosclerosis under diabetic conditions. In addition to metabolic pathways, Rho-kinase, an effector of small-GTPase binding protein Rho, has been implicated as an important factor in the pathogenesis of DN. A number of studies have demonstrated that Rho-kinase plays key roles in the development of DN by inducing endothelial dysfunction, mesangial excessive extracellular matrix (ECM) production, podocyte abnormality, and tubulointerstitial fibrosis. In this review article, we describe our current understanding of the signaling pathways in DN. PMID:27094540

  1. Protein design for pathway engineering

    SciTech Connect

    Eriksen, DT; Lian, JZ; Zhao, HM

    2014-02-01

    Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds. (C) 2013 Elsevier Inc. All rights reserved.

  2. Protein Design for Pathway Engineering

    PubMed Central

    Eriksen, Dawn T.; Lian, Jiazhang; Zhao, Huimin

    2013-01-01

    Design and construction of biochemical pathways has increased the complexity of biosynthetically-produced compounds when compared to single enzyme biocatalysis. However, the coordination of multiple enzymes can introduce a complicated set of obstacles to overcome in order to achieve a high titer and yield of the desired compound. Metabolic engineering has made great strides in developing tools to optimize the flux through a target pathway, but the inherent characteristics of a particular enzyme within the pathway can still limit the productivity. Thus, judicious protein design is critical for metabolic and pathway engineering. This review will describe various strategies and examples of applying protein design to pathway engineering to optimize the flux through the pathway. The proteins can be engineered for altered substrate specificity/selectivity, increased catalytic activity, reduced mass transfer limitations through specific protein localization, and reduced substrate/product inhibition. Protein engineering can also be expanded to design biosensors to enable high through-put screening and to customize cell signaling networks. These strategies have successfully engineered pathways for significantly increased productivity of the desired product or in the production of novel compounds. PMID:23558037

  3. Examination of the endosomal and lysosomal pathways in Dictyostelium discoideum myosin I mutants.

    PubMed

    Temesvari, L A; Bush, J M; Peterson, M D; Novak, K D; Titus, M A; Cardelli, J A

    1996-03-01

    The role of myosin Is in endosomal trafficking and the lysosomal system was investigated in a Dictyostelium discoideum myosin I double mutant myoB-/C-, that has been previously shown to exhibit defects in fluid-phase endocytosis during growth in suspension culture (Novak et al., 1995). Various properties of the endosomal pathway in the myoB-/C- double mutant as well as in the myoB- and myoC- single mutants, including intravesicular pH, and intracellular retention time and exocytosis of a fluid phase marker, were found to be indistinguishable from wild-type parental cells. The intimate connection between the contractile vacuole complex and the endocytic pathway in Dictyostelium, and the localization of a myosin I to the contractile vacuole in Acanthamoeba, led us to also examine the structure and function of this organelle in the three myosin I mutants. No alteration in contractile vacuole structure or function was observed in the myoB-, myoC- or myoB-/C- cell lines. The transport, processing, and localization of a lysosomal enzyme, alpha-mannosidase, were also unaltered in all three mutants. However, the myoB- and myoB-/C- cell lines, but not the myoC- cell line, were found to oversecrete the lysosomal enzymes alpha-mannosidase and acid phosphatase, during growth and starvation. None of the mutants oversecreted proteins following the constitutive secretory pathway. Two additional myosin I mutants, myoA- and myoA-/B-, were also found to oversecrete the lysosomally localized enzymes alpha-mannosidase and acid phosphatase. Taken together, these results suggest that these myosins do not play a role in the intracellular movement of vesicles, but that they may participate in controlling events that occur at the actin-rich cortical region of the cell. While no direct evidence has been found for the association of myosin Is with lysosomes, we predict that the integrity of the lysosomal system is tied to the fidelity of the actin cortex, and changes in cortical organization

  4. Nutrient Sensing Mechanisms and Pathways

    PubMed Central

    Efeyan, Alejo; Comb, William C.; Sabatini, David M.

    2015-01-01

    PREFACE The ability to sense and respond to fluctuations in environmental nutrient levels is a requisite for life. Nutrient scarcity is a selective pressure that has shaped the evolution of most cellular processes. Different pathways that detect intracellular and extracellular levels of sugars, amino acids and lipids, and surrogate metabolites, are then integrated and coordinated at the organismal level via hormonal signals. During food abundance, nutrient sensing pathways engage anabolism and storage, and scarcity triggers homeostatic mechanisms, like the mobilization of internal stores through mechanisms such as autophagy. Nutrient sensing pathways are commonly deregulated in human metabolic diseases. PMID:25592535

  5. Targeting the p53 pathway.

    PubMed

    Golubovskaya, Vita M; Cance, William G

    2013-10-01

    This article summarizes data on translational studies to target the p53 pathway in cancer. It describes the functions of the p53 and Mdm-2 signaling pathways, and discusses current therapeutic approaches to target p53 pathways, including reactivation of p53. In addition, direct interaction and colocalization of the p53 and focal adhesion kinase proteins in cancer cells have been demonstrated, and different approaches to target this interaction are reviewed. This is a broad review of p53 function as it relates to the diagnosis and treatment of a wide range of cancers. PMID:24012397

  6. THE PATHWAY OF ARSENIC METABLISM

    EPA Science Inventory

    The Pathway of Arsenic Methylation

    David J. Thomas, Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC

    Understanding ...

  7. An Endosome-to-Plasma Membrane Pathway Involved in Trafficking of a Mutant Plasma Membrane ATPase in Yeast

    PubMed Central

    Luo, Wen-jie; Chang, Amy

    2000-01-01

    The plasma membrane ATPase, encoded by PMA1, is delivered to the cell surface via the secretory pathway. Previously, we characterized a temperature-sensitive pma1 mutant in which newly synthesized Pma1-7 is not delivered to the plasma membrane but is mislocalized instead to the vacuole at 37°C. Several vps mutants, which are defective in vacuolar protein sorting, suppress targeting-defective pma1 by allowing mutant Pma1 to move once again to the plasma membrane. In this study, we have analyzed trafficking in the endosomal system by monitoring the movement of Pma1-7 in vps36, vps1, and vps8 mutants. Upon induction of expression, mutant Pma1 accumulates in the prevacuolar compartment in vps36 cells. After chase, a fraction of newly synthesized Pma1-7 is delivered to the plasma membrane. In both vps1 and vps8 cells, newly synthesized mutant Pma1 appears in small punctate structures before arrival at the cell surface. Nevertheless, biosynthetic membrane traffic appears to follow different routes in vps8 and vps1: the vacuolar protein-sorting receptor Vps10p is stable in vps8 but not in vps1. Furthermore, a defect in endocytic delivery to the vacuole was revealed in vps8 (and vps36) but not vps1 by endocytosis of the bulk membrane marker FM 4-64. Moreover, in vps8 cells, there is defective down-regulation from the cell surface of the mating receptor Ste3, consistent with persistent receptor recycling from an endosomal compartment to the plasma membrane. These data support a model in which mutant Pma1 is diverted from the Golgi to the surface in vps1 cells. We hypothesize that in vps8 and vps36, in contrast to vps1, mutant Pma1 moves to the surface via endosomal intermediates, implicating an endosome-to-surface traffic pathway. PMID:10679016

  8. Lectin-dependent enhancement of Ebola virus infection via soluble and transmembrane C-type lectin receptors.

    PubMed

    Brudner, Matthew; Karpel, Marshall; Lear, Calli; Chen, Li; Yantosca, L Michael; Scully, Corinne; Sarraju, Ashish; Sokolovska, Anna; Zariffard, M Reza; Eisen, Damon P; Mungall, Bruce A; Kotton, Darrell N; Omari, Amel; Huang, I-Chueh; Farzan, Michael; Takahashi, Kazue; Stuart, Lynda; Stahl, Gregory L; Ezekowitz, Alan B; Spear, Gregory T; Olinger, Gene G; Schmidt, Emmett V; Michelow, Ian C

    2013-01-01

    Mannose-binding lectin (MBL) is a key soluble effector of the innate immune system that recognizes pathogen-specific surface glycans. Surprisingly, low-producing MBL genetic variants that may predispose children and immunocompromised individuals to infectious diseases are more common than would be expected in human populations. Since certain immune defense molecules, such as immunoglobulins, can be exploited by invasive pathogens, we hypothesized that MBL might also enhance infections in some circumstances. Consequently, the low and intermediate MBL levels commonly found in human populations might be the result of balancing selection. Using model infection systems with pseudotyped and authentic glycosylated viruses, we demonstrated that MBL indeed enhances infection of Ebola, Hendra, Nipah and West Nile viruses in low complement conditions. Mechanistic studies with Ebola virus (EBOV) glycoprotein pseudotyped lentiviruses confirmed that MBL binds to N-linked glycan epitopes on viral surfaces in a specific manner via the MBL carbohydrate recognition domain, which is necessary for enhanced infection. MBL mediates lipid-raft-dependent macropinocytosis of EBOV via a pathway that appears to require less actin or early endosomal processing compared with the filovirus canonical endocytic pathway. Using a validated RNA interference screen, we identified C1QBP (gC1qR) as a candidate surface receptor that mediates MBL-dependent enhancement of EBOV infection. We also identified dectin-2 (CLEC6A) as a potentially novel candidate attachment factor for EBOV. Our findings support the concept of an innate immune haplotype that represents critical interactions between MBL and complement component C4 genes and that may modify susceptibility or resistance to certain glycosylated pathogens. Therefore, higher levels of native or exogenous MBL could be deleterious in the setting of relative hypocomplementemia which can occur genetically or because of immunodepletion during active

  9. Lectin-Dependent Enhancement of Ebola Virus Infection via Soluble and Transmembrane C-type Lectin Receptors

    PubMed Central

    Lear, Calli; Chen, Li; Yantosca, L. Michael; Scully, Corinne; Sarraju, Ashish; Sokolovska, Anna; Zariffard, M. Reza; Eisen, Damon P.; Mungall, Bruce A.; Kotton, Darrell N.; Omari, Amel; Huang, I-Chueh; Farzan, Michael; Takahashi, Kazue; Stuart, Lynda; Stahl, Gregory L.; Ezekowitz, Alan B.; Spear, Gregory T.; Olinger, Gene G.; Schmidt, Emmett V.; Michelow, Ian C.

    2013-01-01

    Mannose-binding lectin (MBL) is a key soluble effector of the innate immune system that recognizes pathogen-specific surface glycans. Surprisingly, low-producing MBL genetic variants that may predispose children and immunocompromised individuals to infectious diseases are more common than would be expected in human populations. Since certain immune defense molecules, such as immunoglobulins, can be exploited by invasive pathogens, we hypothesized that MBL might also enhance infections in some circumstances. Consequently, the low and intermediate MBL levels commonly found in human populations might be the result of balancing selection. Using model infection systems with pseudotyped and authentic glycosylated viruses, we demonstrated that MBL indeed enhances infection of Ebola, Hendra, Nipah and West Nile viruses in low complement conditions. Mechanistic studies with Ebola virus (EBOV) glycoprotein pseudotyped lentiviruses confirmed that MBL binds to N-linked glycan epitopes on viral surfaces in a specific manner via the MBL carbohydrate recognition domain, which is necessary for enhanced infection. MBL mediates lipid-raft-dependent macropinocytosis of EBOV via a pathway that appears to require less actin or early endosomal processing compared with the filovirus canonical endocytic pathway. Using a validated RNA interference screen, we identified C1QBP (gC1qR) as a candidate surface receptor that mediates MBL-dependent enhancement of EBOV infection. We also identified dectin-2 (CLEC6A) as a potentially novel candidate attachment factor for EBOV. Our findings support the concept of an innate immune haplotype that represents critical interactions between MBL and complement component C4 genes and that may modify susceptibility or resistance to certain glycosylated pathogens. Therefore, higher levels of native or exogenous MBL could be deleterious in the setting of relative hypocomplementemia which can occur genetically or because of immunodepletion during active

  10. SRNL ALL-PATHWAYS APPLICATION

    SciTech Connect

    Koffman, L; Elmer Wilhite, E; Leonard Collard, L

    2007-05-29

    The Environmental Analysis and Performance Modeling group of Savannah River National Laboratory (SRNL) performs performance assessments of the Savannah River Site (SRS) low-level waste facilities to meet the requirements of DOE Order 435.1. One of the performance objectives in the DOE Order is that the radiological dose to representative members of the public shall not exceed 25 mrem in a year total effective dose equivalent from all exposure pathways, excluding radon. Analysis to meet this performance objective is generally referred to as all-pathways analysis. SRNL performs detailed transient groundwater transport analysis for the waste disposal units, which has been used as input for the groundwater part of all-pathways analysis. The desire to better integrate all-pathways analysis with the groundwater transport analysis lead to the development of a software application named the SRNL All-Pathways Application. Another requirement of DOE Order 435.1 is to assess the impact of nuclear waste disposal on water resources, which SRS has interpreted for groundwater protection as meeting the EPA regulations for radionuclides in drinking water. EPA specifies four separate criteria as part of their implementation guidance for radionuclides, which are specified as maximum contaminant levels (MCL). (1) Beta/gamma emitters have a combined dose limit of 4 mrem/year. (2) Alpha emitters have a combined concentration limit of 15 pCi/L (called gross alpha), excluding uranium and radon, but including radium-226. (3) Combined radium-226 and radium-228 have a concentration limit of 5 pCi/L. (4) Isotopes of uranium have a combined concentration limit of 30 {micro}g/L. The All-Pathways Application was designed to be an easy-to-use software application that utilizes transient concentration results from groundwater transport analysis to (1) calculate the groundwater part of all-pathways dose and to (2) evaluate the four EPA criteria for groundwater protection.

  11. HealthPathways: creating a pathway for health systems reform.

    PubMed

    Robinson, Suzanne; Varhol, Richard; Bell, Colin; Quirk, Frances; Durrington, Learne

    2015-02-01

    Inefficiencies in the co-ordination and integration of primary and secondary care services in Australia, have led to increases in waiting times, unnecessary presentations to emergency departments and issues around poor discharge of patients. HealthPathways is a program developed in Canterbury, New Zealand, that builds relationships between General Practitioners and Specialists and uses information technology so that efficiency is maximised and the right patient is given the right care at the right time. Healthpathways is being implemented by a number of Medicare Locals across Australia however, little is known about the impact HealthPathways may have in Australia. This article provides a short description of HealthPathways and considers what it may offer in the Australian context and some of the barriers and facilitators to implementation. PMID:25433515

  12. SRNL All-Pathways Application

    SciTech Connect

    Koffman, Larry D.; Wilhite, Elmer L.; Collard, Leonard B.

    2008-01-15

    The Environmental Analysis and Performance Modeling group of Savannah River National Laboratory (SRNL) performs performance assessments of the Savannah River Site (SRS) low-level waste facilities to meet the requirements of DOE Order 435.1. One of the performance objectives in the DOE Order is that the radiological dose to representative members of the public shall not exceed 25 mrem in a year total effective dose equivalent from all exposure pathways, excluding radon. Analysis to meet this performance objective is generally referred to as all-pathways analysis. SRNL performs detailed transient groundwater transport analysis for the waste disposal units, which has been used as input for the groundwater part of all-pathways analysis. The desire to better integrate all-pathways analysis with the groundwater transport analysis lead to the development of a software application named the SRNL All-Pathways Application. Another requirement of DOE Order 435.1 is to assess the impact of nuclear waste disposal on water resources, which SRS has interpreted for groundwater protection as meeting the EPA regulations for radionuclides in drinking water. EPA specifies four separate criteria as part of their implementation guidance for radionuclides, which are specified as maximum contaminant levels (MCL). 1. Beta/gamma emitters have a combined dose limit of 4 mrem/year. 2. Alpha emitters have a combined concentration limit of 15 pCi/L (called gross alpha), excluding uranium and radon, but including radium-226. 3. Combined radium-226 and radium-228 have a concentration limit of 5 pCi/L. 4. Isotopes of uranium have a combined concentration limit of 30 {mu}g/L. The All-Pathways Application was designed to be an easy-to-use software application that utilizes transient concentration results from groundwater transport analysis to (1) calculate the groundwater part of all-pathways dose and to (2) evaluate the four EPA criteria for groundwater protection. An application has been developed

  13. Coherence in electron transfer pathways.

    PubMed

    Skourtis, Spiros S; Beratan, David N; Waldeck, David H

    2011-01-01

    Central to the view of electron-transfer reactions is the idea that nuclear motion generates a transition state geometry at which the electron/hole amplitude propagates coherently from the electron donor to the electron acceptor. In the weakly coupled or nonadiabatic regime, the electron amplitude tunnels through an electronic barrier between the donor and acceptor. The structure of the barrier is determined by the covalent and noncovalent interactions of the bridge. Because the tunneling barrier depends on the nuclear coordinates of the reactants (and on the surrounding medium), the tunneling barrier is highly anisotropic, and it is useful to identify particular routes, or pathways, along which the transmission amplitude propagates. Moreover, when more than one such pathway exists, and the paths give rise to comparable transmission amplitude magnitudes, one may expect to observe quantum interferences among pathways if the propagation remains coherent. Given that the effective tunneling barrier height and width are affected by the nuclear positions, the modulation of the nuclear coordinates will lead to a modulation of the tunneling barrier and hence of the electron flow. For long distance electron transfer in biological and biomimetic systems, nuclear fluctuations, arising from flexible protein moieties and mobile water bridges, can become quite significant. We discuss experimental and theoretical results that explore the quantum interferences among coupling pathways in electron-transfer kinetics; we emphasize recent data and theories associated with the signatures of chirality and inelastic processes, which are manifested in the tunneling pathway coherence (or absence of coherence). PMID:23833692

  14. A biosynthetic pathway for anandamide

    PubMed Central

    Liu, Jie; Wang, Lei; Harvey-White, Judith; Osei-Hyiaman, Douglas; Razdan, Raj; Gong, Qian; Chan, Andrew C.; Zhou, Zhifeng; Huang, Bill X.; Kim, Hee-Yong; Kunos, George

    2006-01-01

    The endocannabinoid arachidonoyl ethanolamine (anandamide) is a lipid transmitter synthesized and released “on demand” by neurons in the brain. Anandamide is also generated by macrophages where its endotoxin (LPS)-induced synthesis has been implicated in the hypotension of septic shock and advanced liver cirrhosis. Anandamide can be generated from its membrane precursor, N-arachidonoyl phosphatidylethanolamine (NAPE) through cleavage by a phospholipase D (NAPE–PLD). Here we document a biosynthetic pathway for anandamide in mouse brain and RAW264.7 macrophages that involves the phospholipase C (PLC)-catalyzed cleavage of NAPE to generate a lipid, phosphoanandamide, which is subsequently dephosphorylated by phosphatases, including PTPN22, previously described as a protein tyrosine phosphatase. Bacterial endotoxin (LPS)-induced synthesis of anandamide in macrophages is mediated exclusively by the PLC/phosphatase pathway, which is up-regulated by LPS, whereas NAPE–PLD is down-regulated by LPS and functions as a salvage pathway of anandamide synthesis when the PLC/phosphatase pathway is compromised. Both PTPN22 and endocannabinoids have been implicated in autoimmune diseases, suggesting that the PLC/phosphatase pathway of anandamide synthesis may be a pharmacotherapeutic target. PMID:16938887

  15. The HEART Pathway Randomized Trial

    PubMed Central

    Mahler, Simon A.; Riley, Robert F.; Hiestand, Brian C.; Russell, Gregory B.; Hoekstra, James W.; Lefebvre, Cedric W.; Nicks, Bret A.; Cline, David M.; Askew, Kim L.; Elliott, Stephanie B.; Herrington, David M.; Burke, Gregory L.; Miller, Chadwick D.

    2015-01-01

    Background The HEART Pathway is a decision aid designed to identify emergency department patients with acute chest pain for early discharge. No randomized trials have compared the HEART Pathway with usual care. Methods and Results Adult emergency department patients with symptoms related to acute coronary syndrome without ST-elevation on ECG (n=282) were randomized to the HEART Pathway or usual care. In the HEART Pathway arm, emergency department providers used the HEART score, a validated decision aid, and troponin measures at 0 and 3 hours to identify patients for early discharge. Usual care was based on American College of Cardiology/American Heart Association guidelines. The primary outcome, objective cardiac testing (stress testing or angiography), and secondary outcomes, index length of stay, early discharge, and major adverse cardiac events (death, myocardial infarction, or coronary revascularization), were assessed at 30 days by phone interview and record review. Participants had a mean age of 53 years, 16% had previous myocardial infarction, and 6% (95% confidence interval, 3.6%–9.5%) had major adverse cardiac events within 30 days of randomization. Compared with usual care, use of the HEART Pathway decreased objective cardiac testing at 30 days by 12.1% (68.8% versus 56.7%; P=0.048) and length of stay by 12 hours (9.9 versus 21.9 hours; P=0.013) and increased early discharges by 21.3% (39.7% versus 18.4%; P<0.001). No patients identified for early discharge had major adverse cardiac events within 30 days. Conclusions The HEART Pathway reduces objective cardiac testing during 30 days, shortens length of stay, and increases early discharges. These important efficiency gains occurred without any patients identified for early discharge suffering MACE at 30 days. PMID:25737484

  16. The Alström Syndrome Protein, ALMS1, Interacts with α-Actinin and Components of the Endosome Recycling Pathway

    PubMed Central

    Collin, Gayle B.; Marshall, Jan D.; King, Benjamin L.; Milan, Gabriella; Maffei, Pietro; Jagger, Daniel J.; Naggert, Jürgen K.

    2012-01-01

    Alström syndrome (ALMS) is a progressive multi-systemic disorder characterized by cone-rod dystrophy, sensorineural hearing loss, childhood obesity, insulin resistance and cardiac, renal, and hepatic dysfunction. The gene responsible for Alström syndrome, ALMS1, is ubiquitously expressed and has multiple splice variants. The protein encoded by this gene has been implicated in ciliary function, cell cycle control, and intracellular transport. To gain better insight into the pathways through which ALMS1 functions, we carried out a yeast two hybrid (Y2H) screen in several mouse tissue libraries to identify ALMS1 interacting partners. The majority of proteins found to interact with the murine carboxy-terminal end (19/32) of ALMS1 were α-actinin isoforms. Interestingly, several of the identified ALMS1 interacting partners (α-actinin 1, α-actinin 4, myosin Vb, rad50 interacting 1 and huntingtin associated protein1A) have been previously associated with endosome recycling and/or centrosome function. We examined dermal fibroblasts from human subjects bearing a disruption in ALMS1 for defects in the endocytic pathway. Fibroblasts from these patients had a lower uptake of transferrin and reduced clearance of transferrin compared to controls. Antibodies directed against ALMS1 N- and C-terminal epitopes label centrosomes and endosomal structures at the cleavage furrow of dividing MDCK cells, respectively, suggesting isoform-specific cellular functions. Our results suggest a role for ALMS1 variants in the recycling endosome pathway and give us new insights into the pathogenesis of a subset of clinical phenotypes associated with ALMS. PMID:22693585

  17. Collateral Pathways in Portal Hypertension

    PubMed Central

    Sharma, Malay; Rameshbabu, Chittapuram S.

    2012-01-01

    Presence of portosystemic collateral veins (PSCV) is common in portal hypertension due to cirrhosis. Physiologically, normal portosystemic anastomoses exist which exhibit hepatofugal flow. With the development of portal hypertension, transmission of backpressure leads to increased flow in these patent normal portosystemic anastomoses. In extrahepatic portal vein obstruction collateral circulation develops in a hepatopetal direction and portoportal pathways are frequently found. The objective of this review is to illustrate the various PSCV and portoportal collateral vein pathways pertinent to portal hypertension in liver cirrhosis and EHPVO. PMID:25755456

  18. [Pathways of flowering regulation in plants].

    PubMed

    Liu, Yongping; Yang, Jing; Yang, Mingfeng

    2015-11-01

    Flowering, the floral transition from vegetative growth to reproductive growth, is induced by diverse endogenous and exogenous cues, such as photoperiod, temperature, hormones and age. Precise flowering time is critical to plant growth and evolution of species. The numerous renewal molecular and genetic results have revealed five flowering time pathways, including classical photoperiod pathway, vernalization pathway, autonomous pathway, gibberellins (GA) pathway and newly identified age pathway. These pathways take on relatively independent role, and involve extensive crosstalks and feedback loops. This review describes the complicated regulatory network of this floral transition to understand the molecular mechanism of flowering and provide references for further research in more plants. PMID:26939439

  19. SMPDB: The Small Molecule Pathway Database.

    PubMed

    Frolkis, Alex; Knox, Craig; Lim, Emilia; Jewison, Timothy; Law, Vivian; Hau, David D; Liu, Phillip; Gautam, Bijaya; Ly, Son; Guo, An Chi; Xia, Jianguo; Liang, Yongjie; Shrivastava, Savita; Wishart, David S

    2010-01-01

    The Small Molecule Pathway Database (SMPDB) is an interactive, visual database containing more than 350 small-molecule pathways found in humans. More than 2/3 of these pathways (>280) are not found in any other pathway database. SMPDB is designed specifically to support pathway elucidation and pathway discovery in clinical metabolomics, transcriptomics, proteomics and systems biology. SMPDB provides exquisitely detailed, hyperlinked diagrams of human metabolic pathways, metabolic disease pathways, metabolite signaling pathways and drug-action pathways. All SMPDB pathways include information on the relevant organs, organelles, subcellular compartments, protein cofactors, protein locations, metabolite locations, chemical structures and protein quaternary structures. Each small molecule is hyperlinked to detailed descriptions contained in the Human Metabolome Database (HMDB) or DrugBank and each protein or enzyme complex is hyperlinked to UniProt. All SMPDB pathways are accompanied with detailed descriptions, providing an overview of the pathway, condition or processes depicted in each diagram. The database is easily browsed and supports full text searching. Users may query SMPDB with lists of metabolite names, drug names, genes/protein names, SwissProt IDs, GenBank IDs, Affymetrix IDs or Agilent microarray IDs. These queries will produce lists of matching pathways and highlight the matching molecules on each of the pathway diagrams. Gene, metabolite and protein concentration data can also be visualized through SMPDB's mapping interface. All of SMPDB's images, image maps, descriptions and tables are downloadable. SMPDB is available at: http://www.smpdb.ca. PMID:19948758

  20. Interaction of Notch Signaling Modulator Numb with α-Adaptin Regulates Endocytosis of Notch Pathway Components and Cell Fate Determination of Neural Stem Cells*

    PubMed Central

    Song, Yan; Lu, Bingwei

    2012-01-01

    The ability to balance self-renewal and differentiation is a hallmark of stem cells. In Drosophila neural stem cells (NSCs), Numb/Notch (N) signaling plays a key role in this process. However, the molecular and cellular mechanisms underlying Numb function in a stem cell setting remain poorly defined. Here we show that α-Adaptin (α-Ada), a subunit of the endocytic AP-2 complex, interacts with Numb through a new mode of interaction to regulate NSC homeostasis. In α-ada mutants, N pathway component Sanpodo and the N receptor itself exhibited altered trafficking, and N signaling was up-regulated in the intermediate progenitors of type II NSC lineages, leading to their transformation into ectopic NSCs. Surprisingly, although the Ear domain of α-Ada interacts with the C terminus of Numb and is important for α-Ada function in the sensory organ precursor lineage, it was dispensable in the NSCs. Instead, α-Ada could regulate Sanpodo, N trafficking, and NSC homeostasis by interacting with Numb through new domains in both proteins previously not known to mediate their interaction. This interaction could be bypassed when α-Ada was directly fused to the phospho-tyrosine binding domain of Numb. Our results identify a critical role for the AP-2-mediated endocytosis in regulating NSC behavior and reveal a new mechanism by which Numb regulates NSC behavior through N. These findings are likely to have important implications for cancer biology. PMID:22474327

  1. The human prothrombin kringle-2 derived peptide, NSA9, is internalized into bovine capillary endothelial cells through endocytosis and energy-dependent pathways

    SciTech Connect

    Hwang, Hyun Sook; Kim, Soung Soo . E-mail: kimss518@yonsei.ac.kr

    2005-09-23

    Human prothrombin kringle-2 and its partial peptide, NSA9 (NSAVQLVEN), have been reported to have potent anti-angiogenic activities. Here, the internalization mechanism of NSA9 into bovine capillary endothelial (BCE) cells was examined using lactate dehydrogenase (LDH) release assay, fluorescence microscopy, and flow cytometry. LDH release assay results suggested that the integrity of the BCE cell membrane was unaffected by NSA9. Fluorescence microscopy indicated that internalized NSA9 was localized in the cytoplasm around the nucleus, and showed a punctuated fluorescence pattern, which is indicative of endocytic vesicles. Also, the cellular internalization of NSA9 is significantly inhibited by depletion of the cellular ATP pool, endocytosis inhibitors such as chloroquine and nocodazole, and incubation at low temperature (4 deg C). In addition, the anti-proliferative activity of NSA9 against BCE cells was diminished in the presence of endocytosis or metabolic inhibitors. In conclusion, these results strongly suggest that NSA9 might exert its anti-proliferative activity through internalization into BCE cells by endocytosis and energy-dependent pathways.

  2. BsdA(Bsd2) -dependent vacuolar turnover of a misfolded version of the UapA transporter along the secretory pathway: prominent role of selective autophagy.

    PubMed

    Evangelinos, Minoas; Martzoukou, Olga; Chorozian, Koar; Amillis, Sotiris; Diallinas, George

    2016-06-01

    Transmembrane proteins translocate cotranslationally in the endoplasmic reticulum (ER) membrane and traffic as vesicular cargoes, via the Golgi, in their final membrane destination. Misfolding in the ER leads to protein degradation basically through the ERAD/proteasome system. Here, we use a mutant version of the purine transporter UapA (ΔR481) to show that specific misfolded versions of plasma membrane cargoes undergo vacuolar turnover prior to localization in the plasma membrane. We show that non-endocytic vacuolar turnover of ΔR481 is dependent on BsdA(Bsd2) , an ER transmembrane adaptor of HulA(Rsp5) ubiquitin ligase. We obtain in vivo evidence that BsdA(Bsd2) interacts with HulA(Rsp5) and ΔR481, primarily in the ER. Importantly, accumulation of ΔR481 in the ER triggers delivery of the selective autophagy marker Atg8 in vacuoles along with ΔR481. Genetic block of autophagy (atg9Δ, rabO(ts) ) reduces, but does not abolish, sorting of ΔR481 in the vacuoles, suggesting that a fraction of the misfolded transporter might be redirected for vacuolar degradation via the Golgi. Our results support that multiple routes along the secretory pathway operate for the detoxification of Aspergillus nidulans cells from misfolded membrane proteins and that BsdA is a key factor for marking specific misfolded cargoes. PMID:26917498

  3. UniPathway: a resource for the exploration and annotation of metabolic pathways.

    PubMed

    Morgat, Anne; Coissac, Eric; Coudert, Elisabeth; Axelsen, Kristian B; Keller, Guillaume; Bairoch, Amos; Bridge, Alan; Bougueleret, Lydie; Xenarios, Ioannis; Viari, Alain

    2012-01-01

    UniPathway (http://www.unipathway.org) is a fully manually curated resource for the representation and annotation of metabolic pathways. UniPathway provides explicit representations of enzyme-catalyzed and spontaneous chemical reactions, as well as a hierarchical representation of metabolic pathways. This hierarchy uses linear subpathways as the basic building block for the assembly of larger and more complex pathways, including species-specific pathway variants. All of the pathway data in UniPathway has been extensively cross-linked to existing pathway resources such as KEGG and MetaCyc, as well as sequence resources such as the UniProt KnowledgeBase (UniProtKB), for which UniPathway provides a controlled vocabulary for pathway annotation. We introduce here the basic concepts underlying the UniPathway resource, with the aim of allowing users to fully exploit the information provided by UniPathway. PMID:22102589

  4. UniPathway: a resource for the exploration and annotation of metabolic pathways

    PubMed Central

    Morgat, Anne; Coissac, Eric; Coudert, Elisabeth; Axelsen, Kristian B.; Keller, Guillaume; Bairoch, Amos; Bridge, Alan; Bougueleret, Lydie; Xenarios, Ioannis; Viari, Alain

    2012-01-01

    UniPathway (http://www.unipathway.org) is a fully manually curated resource for the representation and annotation of metabolic pathways. UniPathway provides explicit representations of enzyme-catalyzed and spontaneous chemical reactions, as well as a hierarchical representation of metabolic pathways. This hierarchy uses linear subpathways as the basic building block for the assembly of larger and more complex pathways, including species-specific pathway variants. All of the pathway data in UniPathway has been extensively cross-linked to existing pathway resources such as KEGG and MetaCyc, as well as sequence resources such as the UniProt KnowledgeBase (UniProtKB), for which UniPathway provides a controlled vocabulary for pathway annotation. We introduce here the basic concepts underlying the UniPathway resource, with the aim of allowing users to fully exploit the information provided by UniPathway. PMID:22102589

  5. Reverse Engineering Adverse Outcome Pathways

    SciTech Connect

    Perkins, Edward; Chipman, J.K.; Edwards, Stephen; Habib, Tanwir; Falciani, Francesco; Taylor, Ronald C.; Van Aggelen, Graham; Vulpe, Chris; Antczak, Philipp; Loguinov, Alexandre

    2011-01-30

    The toxicological effects of many stressors are mediated through unknown, or poorly characterized, mechanisms of action. We describe the application of reverse engineering complex interaction networks from high dimensional omics data (gene, protein, metabolic, signaling) to characterize adverse outcome pathways (AOPs) for chemicals that disrupt the hypothalamus-pituitary-gonadal endocrine axis in fathead minnows. Gene expression changes in fathead minnow ovaries in response to 7 different chemicals, over different times, doses, and in vivo versus in vitro conditions were captured in a large data set of 868 arrays. We examined potential AOPs of the antiandrogen flutamide using two mutual information theory methods, ARACNE and CLR to infer gene regulatory networks and potential adverse outcome pathways. Representative networks from these studies were used to predict a network path from stressor to adverse outcome as a candidate AOP. The relationship of individual chemicals to an adverse outcome can be determined by following perturbations through the network in response to chemical treatment leading to the nodes associated with the adverse outcome. Identification of candidate pathways allows for formation of testable hypotheses about key biologic processes, biomarkers or alternative endpoints, which could be used to monitor an adverse outcome pathway. Finally, we identify the unique challenges facing the application of this approach in ecotoxicology, and attempt to provide a road map for the utilization of these tools. Key Words: mechanism of action, toxicology, microarray, network inference

  6. Capstone Design Project Course Pathways

    ERIC Educational Resources Information Center

    Eppes, Tom A.; Milanovic, Ivana

    2011-01-01

    Capstones are open-ended undertakings where students are expected to creatively analyze, synthesize, and apply a wide-variety of learning outcomes from prior coursework. This paper discusses the structure, approach and evolution of the capstone project pathways within our College. Specifically two programs, MET and EET, have adopted different…

  7. Pathways to poly-victimization.

    PubMed

    Finkelhor, David; Ormrod, Richard; Turner, Heather; Holt, Melissa

    2009-11-01

    Some children, whom we have labeled poly-victims, experience very high levels of victimizations of different types. This article finds support for a conceptual model suggesting that there may be four distinct pathways to becoming such a poly-victim: (a) residing in a dangerous community, (b) living in a dangerous family, (c) having a chaotic, multiproblem family environment, or (d) having emotional problems that increase risk behavior, engender antagonism, and compromise the capacity to protect oneself. It uses three waves of the Developmental Victimization Survey, a nationally representative sample of children aged 2-17 years. All four hypothesized pathways showed significant independent association with poly-victim onset. For the younger children, the symptom score representing emotional problems was the only significant predictor. For the older children, the other three pathway variables were significant predictors--dangerous communities, dangerous families, and problem families--but not symptom score. Poly-victimization onset was also disproportionately likely to occur in the year prior to children's 7th and 15th birthday, corresponding roughly to the entry into elementary school and high school. The identification of such pathways and the ages of high onset should help practitioners design programs for preventing vulnerable children from becoming poly-victims. PMID:19837972

  8. Cancer stem cell signaling pathways.

    PubMed

    Matsui, William H

    2016-09-01

    Tissue development and homeostasis are governed by the actions of stem cells. Multipotent cells are capable of self-renewal during the course of one's lifetime. The accurate and appropriate regulation of stem cell functions is absolutely critical for normal biological activity. Several key developmental or signaling pathways have been shown to play essential roles in this regulatory capacity. Specifically, the Janus-activated kinase/signal transducer and activator of transcription, Hedgehog, Wnt, Notch, phosphatidylinositol 3-kinase/phosphatase and tensin homolog, and nuclear factor-κB signaling pathways have all been shown experimentally to mediate various stem cell properties, such as self-renewal, cell fate decisions, survival, proliferation, and differentiation. Unsurprisingly, many of these crucial signaling pathways are dysregulated in cancer. Growing evidence suggests that overactive or abnormal signaling within and among these pathways may contribute to the survival of cancer stem cells (CSCs). CSCs are a relatively rare population of cancer cells capable of self-renewal, differentiation, and generation of serially transplantable heterogeneous tumors of several types of cancer. PMID:27611937

  9. Career Technical Education Pathways Initiative

    ERIC Educational Resources Information Center

    California Community Colleges, Chancellor's Office, 2013

    2013-01-01

    California's education system--the largest in the United States--is an essential resource for ensuring strong economic growth in the state. The Career Technical Education Pathways Initiative (referred to as the Initiative in this report), which became law in 2005, brings together community colleges, K-12 school districts, employers, organized…

  10. Overskilling Dynamics and Education Pathways

    ERIC Educational Resources Information Center

    Mavromaras, Kostas; McGuinness, Seamus

    2012-01-01

    This paper uses panel data and econometric methods to estimate the incidence and the dynamic properties of overskilling among employed individuals. The paper begins by asking whether there is extensive overskilling in the labour market, and whether overskilling differs by education pathway. The answer to both questions is yes. The paper continues…

  11. Signalling pathways in endometrial cancer.

    PubMed

    Markowska, Anna; Pawałowska, Monika; Lubin, Jolanta; Markowska, Janina

    2014-01-01

    Carcinogenesis is a multistage process, during which the activity of signalling pathways responsible for cell cycle regulation and division is disrupted which leads to inhibition of apoptosis and enhanced proliferation. Improper activation of Wnt/β-catenin and PI3K. Akt pathways play essential role in endometrial cancers (EC), mainly type I. Mutations in APC, axin or CTNBB1 may lead to β-catenin overactivation leading to excessive gene expression. PTEN inactivation, mutations in the PIK3CA or Akt result in increased transmission in the PI3K/Akt pathway, apoptosis inhibition, intensive cell division, mTOR excitation. In non-endometrioid cancers, key mutations include suppressor gene TP53 responsible for repairing damaged DNA or apoptosis initiation. Irregularities in gene P16, encoding a protein forming the p16-cyclinD/CDK-pRb have also been described. Understanding the complex relations between specific proteins taking part in signal transduction of the abovementioned pathways is key to research on drugs used in targeted therapy. PMID:25520571

  12. The Phenylpropanoid Pathway in Arabidopsis

    PubMed Central

    Fraser, Christopher M.; Chapple, Clint

    2011-01-01

    The phenylpropanoid pathway serves as a rich source of metabolites in plants, being required for the biosynthesis of lignin, and serving as a starting point for the production of many other important compounds, such as the flavonoids, coumarins, and lignans. In spite of the fact that the phenylpropanoids and their derivatives are sometimes classified as secondary metabolites, their relevance to plant survival has been made clear via the study of Arabidopsis and other plant species. As a model system, Arabidopsis has helped to elucidate many details of the phenylpropanoid pathway, its enzymes and intermediates, and the interconnectedness of the pathway with plant metabolism as a whole. These advances in our understanding have been made possible in large part by the relative ease with which mutations can be generated, identified, and studied in Arabidopsis. Herein, we provide an overview of the research progress that has been made in recent years, emphasizing both the genes (and gene families) associated with the phenylpropanoid pathway in Arabidopsis, and the end products that have contributed to the identification of many mutants deficient in the phenylpropanoid metabolism: the sinapate esters. PMID:22303276

  13. Multiple pathways regulate shoot branching

    PubMed Central

    Rameau, Catherine; Bertheloot, Jessica; Leduc, Nathalie; Andrieu, Bruno; Foucher, Fabrice; Sakr, Soulaiman

    2015-01-01

    Shoot branching patterns result from the spatio-temporal regulation of axillary bud outgrowth. Numerous endogenous, developmental and environmental factors are integrated at the bud and plant levels to determine numbers of growing shoots. Multiple pathways that converge to common integrators are most probably involved. We propose several pathways involving not only the classical hormones auxin, cytokinins and strigolactones, but also other signals with a strong influence on shoot branching such as gibberellins, sugars or molecular actors of plant phase transition. We also deal with recent findings about the molecular mechanisms and the pathway involved in the response to shade as an example of an environmental signal controlling branching. We propose the TEOSINTE BRANCHED1, CYCLOIDEA, PCF transcription factor TB1/BRC1 and the polar auxin transport stream in the stem as possible integrators of these pathways. We finally discuss how modeling can help to represent this highly dynamic system by articulating knowledges and hypothesis and calculating the phenotype properties they imply. PMID:25628627

  14. Rapid prototype extruded conductive pathways

    DOEpatents

    Bobbitt, III, John T.

    2016-06-21

    A process of producing electrically conductive pathways within additively manufactured parts and similar parts made by plastic extrusion nozzles. The process allows for a three-dimensional part having both conductive and non-conductive portions and allows for such parts to be manufactured in a single production step.

  15. Multiple Pathways for All Students

    ERIC Educational Resources Information Center

    Stirling, Lee Anna

    2012-01-01

    Maine has been focusing on the importance of postsecondary training. Maine's Skowhegan Area High School (SAHS) and Somerset Career and Technical Center (SCTC) have partnered in a Multiple Pathways initiative (funded by the Nellie Mae Education Foundation) to increase students' high school completion rate and to increase enrollment in postsecondary…

  16. Two-Electron Transfer Pathways.

    PubMed

    Lin, Jiaxing; Balamurugan, D; Zhang, Peng; Skourtis, Spiros S; Beratan, David N

    2015-06-18

    The frontiers of electron-transfer chemistry demand that we develop theoretical frameworks to describe the delivery of multiple electrons, atoms, and ions in molecular systems. When electrons move over long distances through high barriers, where the probability for thermal population of oxidized or reduced bridge-localized states is very small, the electrons will tunnel from the donor (D) to acceptor (A), facilitated by bridge-mediated superexchange interactions. If the stable donor and acceptor redox states on D and A differ by two electrons, it is possible that the electrons will propagate coherently from D to A. While structure-function relations for single-electron superexchange in molecules are well established, strategies to manipulate the coherent flow of multiple electrons are largely unknown. In contrast to one-electron superexchange, two-electron superexchange involves both one- and two-electron virtual intermediate states, the number of virtual intermediates increases very rapidly with system size, and multiple classes of pathways interfere with one another. In the study described here, we developed simple superexchange models for two-electron transfer. We explored how the bridge structure and energetics influence multielectron superexchange, and we compared two-electron superexchange interactions to single-electron superexchange. Multielectron superexchange introduces interference between singly and doubly oxidized (or reduced) bridge virtual states, so that even simple linear donor-bridge-acceptor systems have pathway topologies that resemble those seen for one-electron superexchange through bridges with multiple parallel pathways. The simple model systems studied here exhibit a richness that is amenable to experimental exploration by manipulating the multiple pathways, pathway crosstalk, and changes in the number of donor and acceptor species. The features that emerge from these studies may assist in developing new strategies to deliver multiple

  17. Molecular Pathways: Targeting DNA Repair Pathway Defects Enriched in Metastasis.

    PubMed

    Corcoran, Niall M; Clarkson, Michael J; Stuchbery, Ryan; Hovens, Christopher M

    2016-07-01

    The maintenance of a pristine genome, free from errors, is necessary to prevent cellular transformation and degeneration. When errors in DNA are detected, DNA damage repair (DDR) genes and their regulators are activated to effect repair. When these DDR pathways are themselves mutated or aberrantly downregulated, cancer and neurodegenerative disorders can ensue. Multiple lines of evidence now indicate, however, that defects in key regulators of DNA repair pathways are highly enriched in human metastasis specimens and hence may be a key step in the acquisition of metastasis and the ability of localized disease to disseminate. Some of the key regulators of checkpoints in the DNA damage response are the TP53 protein and the PARP enzyme family. Targeting of these pathways, especially through PARP inhibition, is now being exploited therapeutically to effect significant clinical responses in subsets of individuals, particularly in patients with ovarian cancer or prostate cancer, including cancers with a marked metastatic burden. Targeting DNA repair-deficient tumors with drugs that take advantage of the fundamental differences between normal repair-proficient cells and repair-deficient tumors offers new avenues for treating advanced disease in the future. Clin Cancer Res; 22(13); 3132-7. ©2016 AACR. PMID:27169997

  18. Reactome from a WikiPathways Perspective.

    PubMed

    Bohler, Anwesha; Wu, Guanming; Kutmon, Martina; Pradhana, Leontius Adhika; Coort, Susan L; Hanspers, Kristina; Haw, Robin; Pico, Alexander R; Evelo, Chris T

    2016-05-01

    Reactome and WikiPathways are two of the most popular freely available databases for biological pathways. Reactome pathways are centrally curated with periodic input from selected domain experts. WikiPathways is a community-based platform where pathways are created and continually curated by any interested party. The nascent collaboration between WikiPathways and Reactome illustrates the mutual benefits of combining these two approaches. We created a format converter that converts Reactome pathways to the GPML format used in WikiPathways. In addition, we developed the ComplexViz plugin for PathVisio which simplifies looking up complex components. The plugin can also score the complexes on a pathway based on a user defined criterion. This score can then be visualized on the complex nodes using the visualization options provided by the plugin. Using the merged collection of curated and converted Reactome pathways, we demonstrate improved pathway coverage of relevant biological processes for the analysis of a previously described polycystic ovary syndrome gene expression dataset. Additionally, this conversion allows researchers to visualize their data on Reactome pathways using PathVisio's advanced data visualization functionalities. WikiPathways benefits from the dedicated focus and attention provided to the content converted from Reactome and the wealth of semantic information about interactions. Reactome in turn benefits from the continuous community curation available on WikiPathways. The research community at large benefits from the availability of a larger set of pathways for analysis in PathVisio and Cytoscape. The pathway statistics results obtained from PathVisio are significantly better when using a larger set of candidate pathways for analysis. The conversion serves as a general model for integration of multiple pathway resources developed using different approaches. PMID:27203685

  19. Reactome from a WikiPathways Perspective

    PubMed Central

    Bohler, Anwesha; Wu, Guanming; Pradhana, Leontius Adhika; Hanspers, Kristina; Haw, Robin; Pico, Alexander R.

    2016-01-01

    Reactome and WikiPathways are two of the most popular freely available databases for biological pathways. Reactome pathways are centrally curated with periodic input from selected domain experts. WikiPathways is a community-based platform where pathways are created and continually curated by any interested party. The nascent collaboration between WikiPathways and Reactome illustrates the mutual benefits of combining these two approaches. We created a format converter that converts Reactome pathways to the GPML format used in WikiPathways. In addition, we developed the ComplexViz plugin for PathVisio which simplifies looking up complex components. The plugin can also score the complexes on a pathway based on a user defined criterion. This score can then be visualized on the complex nodes using the visualization options provided by the plugin. Using the merged collection of curated and converted Reactome pathways, we demonstrate improved pathway coverage of relevant biological processes for the analysis of a previously described polycystic ovary syndrome gene expression dataset. Additionally, this conversion allows researchers to visualize their data on Reactome pathways using PathVisio’s advanced data visualization functionalities. WikiPathways benefits from the dedicated focus and attention provided to the content converted from Reactome and the wealth of semantic information about interactions. Reactome in turn benefits from the continuous community curation available on WikiPathways. The research community at large benefits from the availability of a larger set of pathways for analysis in PathVisio and Cytoscape. The pathway statistics results obtained from PathVisio are significantly better when using a larger set of candidate pathways for analysis. The conversion serves as a general model for integration of multiple pathway resources developed using different approaches. PMID:27203685

  20. Characterization and Visualization of Vesicles in the Endo-Lysosomal Pathway with Surface-Enhanced Raman Spectroscopy and Chemometrics.

    PubMed

    Huefner, Anna; Kuan, Wei-Li; Müller, Karin H; Skepper, Jeremy N; Barker, Roger A; Mahajan, Sumeet

    2016-01-26

    Surface-enhanced Raman spectroscopy (SERS) is an ultrasensitive vibrational fingerprinting technique widely used in analytical and biosensing applications. For intracellular sensing, typically gold nanoparticles (AuNPs) are employed as transducers to enhance the otherwise weak Raman spectroscopy signals. Thus, the signature patterns of the molecular nanoenvironment around intracellular unlabeled AuNPs can be monitored in a reporter-free manner by SERS. The challenge of selectively identifying molecular changes resulting from cellular processes in large and multidimensional data sets and the lack of simple tools for extracting this information has resulted in limited characterization of fundamental cellular processes by SERS. Here, this shortcoming in analysis of SERS data sets is tackled by developing a suitable methodology of reference-based PCA-LDA (principal component analysis-linear discriminant analysis). This method is validated and exemplarily used to extract spectral features characteristic of the endocytic compartment inside cells. The voluntary uptake through vesicular endocytosis is widely used for the internalization of AuNPs into cells, but the characterization of the individual stages of this pathway has not been carried out. Herein, we use reporter-free SERS to identify and visualize the stages of endocytosis of AuNPs in cells and map the molecular changes via the adaptation and advantageous use of chemometric methods in combination with tailored sample preparation. Thus, our study demonstrates the capabilities of reporter-free SERS for intracellular analysis and its ability to provide a way of characterizing intracellular composition. The developed analytical approach is generic and enables the application of reporter-free SERS to identify unknown components in different biological matrices and materials. PMID:26649752

  1. Signaling pathways mediating alcohol effects.

    PubMed

    Ron, Dorit; Messing, Robert O

    2013-01-01

    Ethanol's effects on intracellular signaling pathways contribute to acute effects of ethanol as well as to neuroadaptive responses to repeated ethanol exposure. In this chapter we review recent discoveries that demonstrate how ethanol alters signaling pathways involving several receptor tyrosine kinases and intracellular tyrosine and serine-threonine kinases, with consequences for regulation of cell surface receptor function, gene expression, protein translation, neuronal excitability and animal behavior. We also describe recent work that demonstrates a key role for ethanol in regulating the function of scaffolding proteins that organize signaling complexes into functional units. Finally, we review recent exciting studies demonstrating ethanol modulation of DNA and histone modification and the expression of microRNAs, indicating epigenetic mechanisms by which ethanol regulates neuronal gene expression and addictive behaviors. PMID:21877259

  2. Fundamental reaction pathways during coprocessing

    SciTech Connect

    Stock, L.M.; Gatsis, J.G.

    1992-12-01

    The objective of this research was to investigate the fundamental reaction pathways in coal petroleum residuum coprocessing. Once the reaction pathways are defined, further efforts can be directed at improving those aspects of the chemistry of coprocessing that are responsible for the desired results such as high oil yields, low dihydrogen consumption, and mild reaction conditions. We decided to carry out this investigation by looking at four basic aspects of coprocessing: (1) the effect of fossil fuel materials on promoting reactions essential to coprocessing such as hydrogen atom transfer, carbon-carbon bond scission, and hydrodemethylation; (2) the effect of varied mild conditions on the coprocessing reactions; (3) determination of dihydrogen uptake and utilization under severe conditions as a function of the coal or petroleum residuum employed; and (4) the effect of varied dihydrogen pressure, temperature, and residence time on the uptake and utilization of dihydrogen and on the distribution of the coprocessed products. Accomplishments are described.

  3. Signaling Pathways in Cartilage Repair

    PubMed Central

    Mariani, Erminia; Pulsatelli, Lia; Facchini, Andrea

    2014-01-01

    In adult healthy cartilage, chondrocytes are in a quiescent phase characterized by a fine balance between anabolic and catabolic activities. In ageing, degenerative joint diseases and traumatic injuries of cartilage, a loss of homeostatic conditions and an up-regulation of catabolic pathways occur. Since cartilage differentiation and maintenance of homeostasis are finely tuned by a complex network of signaling molecules and biophysical factors, shedding light on these mechanisms appears to be extremely relevant for both the identification of pathogenic key factors, as specific therapeutic targets, and the development of biological approaches for cartilage regeneration. This review will focus on the main signaling pathways that can activate cellular and molecular processes, regulating the functional behavior of cartilage in both physiological and pathological conditions. These networks may be relevant in the crosstalk among joint compartments and increased knowledge in this field may lead to the development of more effective strategies for inducing cartilage repair. PMID:24837833

  4. Developmental pathways of motor dysfunction.

    PubMed

    Kleven, Gale A; Bellinger, Seanceray A

    2015-05-01

    Recent evidence has revealed unique patterns of behavioral development after prenatal insult similar to those outlined in studies of adult metabolic dysfunction after prenatal malnutrition. The hallmark features of this Developmental Pathway include a prenatal insult to the nervous system (environmental or genetic) followed by a period of Silent Vulnerability, where no or few functional deficits are observed, and finally emergence of later dysfunction. Possible mechanisms leading to later dysfunction from prenatal insult may include secondary or cascade effects due to the timing of prenatal insults relative to later developing structures in the brain. Methods best employed to study the mechanisms of these pathways are microgenetic and longitudinal designs that include behavioral assessment during the prenatal period of development, and animal models such as the guinea pig. PMID:25864561

  5. Fragmentation pathways of polymer ions.

    PubMed

    Wesdemiotis, Chrys; Solak, Nilüfer; Polce, Michael J; Dabney, David E; Chaicharoen, Kittisak; Katzenmeyer, Bryan C

    2011-01-01

    Tandem mass spectrometry (MS/MS) is increasingly applied to synthetic polymers to characterize chain-end or in-chain substituents, distinguish isobaric and isomeric species, and determine macromolecular connectivities and architectures. For confident structural assignments, the fragmentation mechanisms of polymer ions must be understood, as they provide guidelines on how to deduce the desired information from the fragments observed in MS/MS spectra. This article reviews the fragmentation pathways of synthetic polymer ions that have been energized to decompose via collisionally activated dissociation (CAD), the most widely used activation method in polymer analysis. The compounds discussed encompass polystyrenes, poly(2-vinyl pyridine), polyacrylates, poly(vinyl acetate), aliphatic polyester copolymers, polyethers, and poly(dimethylsiloxane). For a number of these polymers, several substitution patterns and architectures are considered, and questions regarding the ionization agent and internal energy of the dissociating precursor ions are also addressed. Competing and consecutive dissociations are evaluated in terms of the structural insight they provide about the macromolecular structure. The fragmentation pathways of the diverse array of polymer ions examined fall into three categories, viz. (1) charge-directed fragmentations, (2) charge-remote rearrangements, and (3) charge-remote fragmentations via radical intermediates. Charge-remote processes predominate. Depending on the ionizing agent and the functional groups in the polymer, the incipient fragments arising by pathways (1)-(3) may form ion-molecule complexes that survive long enough to permit inter-fragment hydrogen atom, proton, or hydride transfers. PMID:20623599

  6. Quantifying macromolecular conformational transition pathways

    NASA Astrophysics Data System (ADS)

    Seyler, Sean; Kumar, Avishek; Thorpe, Michael; Beckstein, Oliver

    2015-03-01

    Diverse classes of proteins function through large-scale conformational changes that are challenging for computer simulations. A range of fast path-sampling techniques have been used to generate transitions, but it has been difficult to compare paths from (and assess the relative strengths of) different methods. We introduce a comprehensive method (pathway similarity analysis, PSA) for quantitatively characterizing and comparing macromolecular pathways. The Hausdorff and Fréchet metrics (known from computational geometry) are used to quantify the degree of similarity between polygonal curves in configuration space. A strength of PSA is its use of the full information available from the 3 N-dimensional configuration space trajectory without requiring additional specific knowledge about the system. We compare a sample of eleven different methods for the closed-to-open transitions of the apo enzyme adenylate kinase (AdK) and also apply PSA to an ensemble of 400 AdK trajectories produced by dynamic importance sampling MD and the Geometrical Pathways algorithm. We discuss the method's potential to enhance our understanding of transition path sampling methods, validate them, and help guide future research toward deeper physical insights into conformational transitions.

  7. Identification of Metabolic Pathway Systems

    PubMed Central

    Dolatshahi, Sepideh; Voit, Eberhard O.

    2016-01-01

    The estimation of parameters in even moderately large biological systems is a significant challenge. This challenge is greatly exacerbated if the mathematical formats of appropriate process descriptions are unknown. To address this challenge, the method of dynamic flux estimation (DFE) was proposed for the analysis of metabolic time series data. Under ideal conditions, the first phase of DFE yields numerical representations of all fluxes within a metabolic pathway system, either as values at each time point or as plots against their substrates and modulators. However, this numerical result does not reveal the mathematical format of each flux. Thus, the second phase of DFE selects functional formats that are consistent with the numerical trends obtained from the first phase. While greatly facilitating metabolic data analysis, DFE is only directly applicable if the pathway system contains as many dependent variables as fluxes. Because most actual systems contain more fluxes than metabolite pools, this requirement is seldom satisfied. Auxiliary methods have been proposed to alleviate this issue, but they are not general. Here we propose strategies that extend DFE toward general, slightly underdetermined pathway systems. PMID:26904095

  8. Alcohol Effects on Stress Pathways

    PubMed Central

    Blaine, Sara K.; Milivojevic, Verica; Fox, Helen

    2016-01-01

    A significant amount of neurobiological research regarding the development of alcohol use disorders (AUDs) has focused on alcohol-related activation and long-term alterations in the mesocortical dopaminergic reward pathways. However, alcohol does not only interact with brain reward systems. Many of its acute and chronic effects may be related to allostatic adaptations in hypothalamic and extrahypothalamic stress regulation pathways. For example, acute binge intoxication is associated with hypothalamically driven increases in blood cortisol, norepinephrine, and sex steroid metabolite levels. This may contribute to the development of mesocortical sensitization to alcohol. Furthermore, chronic alcohol exposure is associated with systemic dysregulation of the hypothalamic pituitary adrenal axis, sympathetic adrenal medullary system, and sex steroid systems. This dysregulation appears to manifest as neuroendocrine tolerance. In this review, we first summarize the literature suggesting that alcohol-induced alterations in these hypothalamic systems influence craving and contribute to the development of AUDs. We note that for women, the effects of alcohol on these neuroendocrine stress regulation systems may be influenced by the rhythmic variations of hormones and steroids across the menstrual cycle. Second, we discuss how changes in these systems may indicate progression of AUDs and increased risk of relapse in both sexes. Specifically, neuroendocrine tolerance may contribute to mesocortical sensitization, which in turn may lead to decreased prefrontal inhibitory control of the dopaminergic reward and hypothalamic stress systems. Thus, pharmacological strategies that counteract alcohol-associated changes in hypothalamic and extrahypothalamic stress regulation pathways may slow the development and progression of AUDs. PMID:27254089

  9. Imbalanced kynurenine pathway in schizophrenia.

    PubMed

    Kegel, Magdalena E; Bhat, Maria; Skogh, Elisabeth; Samuelsson, Martin; Lundberg, Kristina; Dahl, Marja-Liisa; Sellgren, Carl; Schwieler, Lilly; Engberg, Göran; Schuppe-Koistinen, Ina; Erhardt, Sophie

    2014-01-01

    Several studies suggest a role for kynurenic acid (KYNA) in the pathophysiology of schizophrenia. It has been proposed that increased brain KYNA levels in schizophrenia result from a pathological shift in the kynurenine pathway toward enhanced KYNA formation, away from the other branch of the pathway leading to quinolinic acid (QUIN). Here we investigate the levels of QUIN in cerebrospinal fluid (CSF) of patients with schizophrenia and healthy controls, and relate those to CSF levels of KYNA and other kynurenine metabolites from the same individuals. CSF QUIN levels from stable outpatients treated with olanzapine (n = 22) and those of controls (n = 26) were analyzed using liquid chromatography-mass spectrometry. No difference in CSF QUIN levels between patients and controls was observed (20.6 ± 1.5 nM vs. 18.2 ± 1.1 nM, P = 0.36). CSF QUIN was positively correlated to CSF kynurenine and CSF KYNA in patients but not in controls. The CSF QUIN/KYNA ratio was lower in patients than in controls (P = 0.027). In summary, the present study offers support for an over-activated and imbalanced kynurenine pathway, favoring the production of KYNA over QUIN in patients with schizophrenia. PMID:25288889

  10. Imbalanced Kynurenine Pathway in Schizophrenia

    PubMed Central

    Kegel, Magdalena E; Bhat, Maria; Skogh, Elisabeth; Samuelsson, Martin; Lundberg, Kristina; Dahl, Marja-Liisa; Sellgren, Carl; Schwieler, Lilly; Engberg, Göran; Schuppe-Koistinen, Ina; Erhardt, Sophie

    2014-01-01

    Several studies suggest a role for kynurenic acid (KYNA) in the pathophysiology of schizophrenia. It has been proposed that increased brain KYNA levels in schizophrenia result from a pathological shift in the kynurenine pathway toward enhanced KYNA formation, away from the other branch of the pathway leading to quinolinic acid (QUIN). Here we investigate the levels of QUIN in cerebrospinal fluid (CSF) of patients with schizophrenia and healthy controls, and relate those to CSF levels of KYNA and other kynurenine metabolites from the same individuals. CSF QUIN levels from stable outpatients treated with olanzapine (n = 22) and those of controls (n = 26) were analyzed using liquid chromatography-mass spectrometry. No difference in CSF QUIN levels between patients and controls was observed (20.6 ± 1.5 nM vs. 18.2 ± 1.1 nM, P = 0.36). CSF QUIN was positively correlated to CSF kynurenine and CSF KYNA in patients but not in controls. The CSF QUIN/KYNA ratio was lower in patients than in controls (P = 0.027). In summary, the present study offers support for an over-activated and imbalanced kynurenine pathway, favoring the production of KYNA over QUIN in patients with schizophrenia. PMID:25288889

  11. Synergy between methylerythritol phosphate pathway and mevalonate pathway for isoprene production in Escherichia coli.

    PubMed

    Yang, Chen; Gao, Xiang; Jiang, Yu; Sun, Bingbing; Gao, Fang; Yang, Sheng

    2016-09-01

    Isoprene, a key building block of synthetic rubber, is currently produced entirely from petrochemical sources. In this work, we engineered both the methylerythritol phosphate (MEP) pathway and the mevalonate (MVA) pathway for isoprene production in E. coli. The synergy between the MEP pathway and the MVA pathway was demonstrated by the production experiment, in which overexpression of both pathways improved the isoprene yield about 20-fold and 3-fold, respectively, compared to overexpression of the MEP pathway or the MVA pathway alone. The (13)C metabolic flux analysis revealed that simultaneous utilization of the two pathways resulted in a 4.8-fold increase in the MEP pathway flux and a 1.5-fold increase in the MVA pathway flux. The synergy of the dual pathway was further verified by quantifying intracellular flux responses of the MEP pathway and the MVA pathway to fosmidomycin treatment and mevalonate supplementation. Our results strongly suggest that coupling of the complementary reducing equivalent demand and ATP requirement plays an important role in the synergy of the dual pathway. Fed-batch cultivation of the engineered strain overexpressing the dual pathway resulted in production of 24.0g/L isoprene with a yield of 0.267g/g of glucose. The synergy of the MEP pathway and the MVA pathway also successfully increased the lycopene productivity in E. coli, which demonstrates that it can be used to improve the production of a broad range of terpenoids in microorganisms. PMID:27174717

  12. MOLECULAR PATHWAYS: JAK/STAT PATHWAY: MUTATIONS, INHIBITORS, AND RESISTANCE

    PubMed Central

    Quintás-Cardama, Alfonso; Verstovsek, Srdan

    2016-01-01

    Aberrant activation of the JAK/STAT pathway has been reported in a variety of disease states, including inflammatory conditions, hematologic malignancies, and solid tumors. For instance, a large proportion of patients with myeloproliferative neoplasms (MPNs) carry the acquired gain-of-function JAK2 V617F somatic mutation. This knowledge has dramatically improved our understanding of the pathogenesis of MPNs and it has facilitated the development of therapeutics capable of suppressing the constitutive activation of the JAK/STAT pathway, now recognized as a common underlying biological abnormality in MPNs. Ruxolitinib is an oral JAK1 and JAK2 inhibitor that has recently been approved for the treatment of myelofibrosis and has been tested against other hematologic malignancies. A series of agents with different specificities against different members of the JAK family of proteins is currently undergoing evaluation in clinical trials for patients with MPNs, lymphoma, and solid tumors such as breast or pancreatic cancer. Despite their significant clinical activity exhibited in myelofibrosis, some patients fail to respond or progress during JAK kinase inhibitor therapy. Recent reports have shed light into the mechanisms of resistance to JAK kinase inhibitor therapy. Several approaches hold promise to overcome such resistance. PMID:23406773

  13. Pharmacology of intracellular signalling pathways

    PubMed Central

    Nahorski, Stefan R

    2006-01-01

    This article provides a brief and somewhat personalized review of the dramatic developments that have occurred over the last 45 years in our understanding of intracellular signalling pathways associated with G-protein-coupled receptor activation. Signalling via cyclic AMP, the phosphoinositides and Ca2+ is emphasized and these systems have already been revealed as new pharmacological targets. The therapeutic benefits of most of such targets are, however, yet to be realized, but it is certain that the discipline of pharmacology needs to widen its boundaries to meet these challenges in the future. PMID:16402119

  14. Signaling Pathways in Osteoclast Differentiation.

    PubMed

    Kim, Jung Ha; Kim, Nacksung

    2016-01-01

    Osteoclasts are multinucleated cells of hematopoietic origin that are responsible for the degradation of old bone matrix. Osteoclast differentiation and activity are controlled by two essential cytokines, macrophage colony-stimulating factor (M-CSF) and the receptor activator of nuclear factor-κB ligand (RANKL). M-CSF and RANKL bind to their respective receptors c-Fms and RANK to stimulate osteoclast differentiation through regulation of delicate signaling systems. Here, we summarize the critical or essential signaling pathways for osteoclast differentiation including M-CSF-c-Fms signaling, RANKL-RANK signaling, and costimulatory signaling for RANK. PMID:26865996

  15. Exploring Biological Electron Transfer Pathway Dynamics with the Pathways Plugin for VMD

    PubMed Central

    Balabin, Ilya A.; Hu, Xiangqian; Beratan, David N.

    2012-01-01

    We describe the new Pathways plugin for the molecular visualization program VMD. The plugin identifies and visualizes tunneling pathways and pathway families in biomolecules and calculates relative electronic couplings. The plugin includes unique features to estimate the importance of individual atoms for mediating the coupling, to analyze the coupling sensitivity to thermal motion, and to visualize pathway fluctuations. The Pathways plugin is open source software distributed under the terms of the GNU public license. PMID:22298319

  16. Proteolysis in the secretory pathway

    SciTech Connect

    Guzowski, D.E.; Bienkowski, R.S.

    1987-05-01

    Many secretory proteins are degraded intracellularly rather than secreted, however the location of this catabolic process is not known. The authors have tested the hypothesis that the degradation occurs in the organelles of the secretory pathway. Slices of rat liver were incubated with (/sup 14/C)leucine for 3 h and then incubated under chase conditions for 30 min. The tissue was homogenized and the Golgi apparatus, smooth endoplasmic reticulum (sER) and rough endoplasmic reticulum (rER) were isolated by ultracentrifugation on a discontinuous sucrose gradient. The organelles were incubated in 0.3M sucrose-50 mM citrate (pH 4) for 8-12 h at 37 C; control samples were incubated at 4 C. Percent degradation was calculated as the amount of acid soluble radioactivity released relative to total radioactivity in the sample. Proteolysis in the organelles incubated at 37 C was as follows: Golgi: 15-25%; sER: 10-20%; rER: 10-20%. Proteolysis at 4 C was negligible in all cases. These results support the hypothesis that the compartments of the secretory pathway are capable of degrading newly synthesized secretory proteins.

  17. Model pathways in lignin thermolysis

    SciTech Connect

    Klein, M.T.; Virk, P.S.

    1981-02-01

    A fundamental description of lignin thermolysis was attempted. Analysis of the chemical topology of lignin suggested likely reaction pathways of import to lignin pyrolysis. In turn, 20 model compound pyrolysis substrates were selected to mimic the important reactive functional groups present in whole-lignin thermolysis. The more salient models were: phenethyl phenyl ether (PPE), which depicts the most prevalent lignin interunit linkage, guaiacol, model of the predominant aromatic methoxyl, and saligenol and cinnamyl alcohol, models of important propanoid side chains. Detailed pathway and kinetic analyses and determination of reaction Arrhenius parameters provided mechanistic insights into the model compound pyrolyses. Several pericyclic reaction mechanisms, hitherto not mentioned in the lignin pyrolysis literature, were suggested. In particular, PPE likely pyrolyses via a concerted retro-ene mechanism, whereas guaiacol and saligenol may respectively eliminate methane and water by concerted group transfers. A statistical interpretation of the lignin substrate coupled with the experimental model compound pyrolyses allowed simulation of whole-lignin thermolysis. The simulations were in substantial agreement with experimental pyrolyses reported in the literature in regard to overall gas, methane, carbon monoxide, individual phenols, and carbonaceous residue yields. Weight loss kinetics deduced from the time dependency of the latter yield also accorded well with the experimental literature.

  18. Combustion kinetics and reaction pathways

    SciTech Connect

    Klemm, R.B.; Sutherland, J.W.

    1993-12-01

    This project is focused on the fundamental chemistry of combustion. The overall objectives are to determine rate constants for elementary reactions and to elucidate the pathways of multichannel reactions. A multitechnique approach that features three independent experiments provides unique capabilities in performing reliable kinetic measurements over an exceptionally wide range in temperature, 300 to 2500 K. Recent kinetic work has focused on experimental studies and theoretical calculations of the methane dissociation system (CH{sub 4} + Ar {yields} CH{sub 3} + H + Ar and H + CH{sub 4} {yields} CH{sub 3} + H{sub 2}). Additionally, a discharge flow-photoionization mass spectrometer (DF-PIMS) experiment is used to determine branching fractions for multichannel reactions and to measure ionization thresholds of free radicals. Thus, these photoionization experiments generate data that are relevant to both reaction pathways studies (reaction dynamics) and fundamental thermochemical research. Two distinct advantages of performing PIMS with high intensity, tunable vacuum ultraviolet light at the National Synchrotron Light Source are high detection sensitivity and exceptional selectivity in monitoring radical species.

  19. Pathway analysis of coronary atherosclerosis.

    PubMed

    King, Jennifer Y; Ferrara, Rossella; Tabibiazar, Raymond; Spin, Joshua M; Chen, Mary M; Kuchinsky, Allan; Vailaya, Aditya; Kincaid, Robert; Tsalenko, Anya; Deng, David Xing-Fei; Connolly, Andrew; Zhang, Peng; Yang, Eugene; Watt, Clifton; Yakhini, Zohar; Ben-Dor, Amir; Adler, Annette; Bruhn, Laurakay; Tsao, Philip; Quertermous, Thomas; Ashley, Euan A

    2005-09-21

    Large-scale gene expression studies provide significant insight into genes differentially regulated in disease processes such as cancer. However, these investigations offer limited understanding of multisystem, multicellular diseases such as atherosclerosis. A systems biology approach that accounts for gene interactions, incorporates nontranscriptionally regulated genes, and integrates prior knowledge offers many advantages. We performed a comprehensive gene level assessment of coronary atherosclerosis using 51 coronary artery segments isolated from the explanted hearts of 22 cardiac transplant patients. After histological grading of vascular segments according to American Heart Association guidelines, isolated RNA was hybridized onto a customized 22-K oligonucleotide microarray, and significance analysis of microarrays and gene ontology analyses were performed to identify significant gene expression profiles. Our studies revealed that loss of differentiated smooth muscle cell gene expression is the primary expression signature of disease progression in atherosclerosis. Furthermore, we provide insight into the severe form of coronary artery disease associated with diabetes, reporting an overabundance of immune and inflammatory signals in diabetics. We present a novel approach to pathway development based on connectivity, determined by language parsing of the published literature, and ranking, determined by the significance of differentially regulated genes in the network. In doing this, we identify highly connected "nexus" genes that are attractive candidates for therapeutic targeting and followup studies. Our use of pathway techniques to study atherosclerosis as an integrated network of gene interactions expands on traditional microarray analysis methods and emphasizes the significant advantages of a systems-based approach to analyzing complex disease. PMID:15942018

  20. Transneuronal pathways to the vestibulocerebellum

    NASA Technical Reports Server (NTRS)

    Kaufman, G. D.; Mustari, M. J.; Miselis, R. R.; Perachio, A. A.

    1996-01-01

    The alpha-herpes virus (pseudorabies, PRV) was used to observe central nervous system (CNS) pathways associated with the vestibulocerebellar system. Retrograde transneuronal migration of alpha-herpes virions from specific lobules of the gerbil and rat vestibulo-cerebellar cortex was detected immunohistochemically. Using a time series analysis, progression of infection along polyneuronal cerebellar afferent pathways was examined. Pressure injections of > 20 nanoliters of a 10(8) plaque forming units (pfu) per ml solution of virus were sufficient to initiate an infectious locus which resulted in labeled neurons in the inferior olivary subnuclei, vestibular nuclei, and their afferent cell groups in a progressive temporal fashion and in growing complexity with increasing incubation time. We show that climbing fibers and some other cerebellar afferent fibers transported the virus retrogradely from the cerebellum within 24 hours. One to three days after cerebellar infection discrete cell groups were labeled and appropriate laterality within crossed projections was preserved. Subsequent nuclei labeled with PRV after infection of the flocculus/paraflocculus, or nodulus/uvula, included the following: vestibular (e.g., z) and inferior olivary nuclei (e.g., dorsal cap), accessory oculomotor (e.g., Darkschewitsch n.) and accessory optic related nuclei, (e.g., the nucleus of the optic tract, and the medial terminal nucleus); noradrenergic, raphe, and reticular cell groups (e.g., locus coeruleus, dorsal raphe, raphe pontis, and the lateral reticular tract); other vestibulocerebellum sites, the periaqueductal gray, substantia nigra, hippocampus, thalamus and hypothalamus, amygdala, septal nuclei, and the frontal, cingulate, entorhinal, perirhinal, and insular cortices. However, there were differences in the resulting labeling between infection in either region. Double-labeling experiments revealed that vestibular efferent neurons are located adjacent to, but are not included

  1. PathwayMatrix: visualizing binary relationships between proteins in biological pathways

    PubMed Central

    2015-01-01

    Background Molecular activation pathways are inherently complex, and understanding relations across many biochemical reactions and reaction types is difficult. Visualizing and analyzing a pathway is a challenge due to the network size and the diversity of relations between proteins and molecules. Results In this paper, we introduce PathwayMatrix, a visualization tool that presents the binary relations between proteins in the pathway via the use of an interactive adjacency matrix. We provide filtering, lensing, clustering, and brushing and linking capabilities in order to present relevant details about proteins within a pathway. Conclusions We evaluated PathwayMatrix by conducting a series of in-depth interviews with domain experts who provided positive feedback, leading us to believe that our visualization technique could be helpful for the larger community of researchers utilizing pathway visualizations. PathwayMatrix is freely available at https://github.com/CreativeCodingLab/PathwayMatrix. PMID:26361499

  2. Oxidative biodegradation pathways of PAHs

    SciTech Connect

    Cerniglia, C.E.

    1993-12-31

    Polycyclic aromatic hydrocarbons (PAHs) constitute a class of hazardous organic chemical consisting of three of more fused benzene rings in linear, angular and cluster arrangements. PAHs mostly occur as a result of fossil fuel combustion, as by-product of industrial processing and during the cooking of foods. A catabolically diverse microbial community, consisting of bacteria, fungi and algae, metabolizes aromatic compounds. Molecular oxygen is essential for the initial hydroxylation of polycyclic aromatic hydrocarbons by microorganisms. In contrast to bacteria, filamentous fungi use hydroxylation as a prelude to detoxification rather than to catabolism and assimilation. The biochemical principles underlying the degradation of polycyclic aromatic hydrocarbons will be discussed. The oxidative pathways of polycyclic aromatic hydrocarbon catabolism will be discussed. Studies will be presented on the relationship between the chemical structure of the polycyclic aromatic hydrocarbon and the rate of polycyclic aromatic hydrocarbon biodegradation in aquatic and terrestrial ecosystems.

  3. Nonicosahedral pathways for capsid expansion

    NASA Astrophysics Data System (ADS)

    Cermelli, Paolo; Indelicato, Giuliana; Twarock, Reidun

    2013-09-01

    For a significant number of viruses a structural transition of the protein container that encapsulates the viral genome forms an important part of the life cycle and is a prerequisite for the particle becoming infectious. Despite many recent efforts the mechanism of this process is still not fully understood, and a complete characterization of the expansion pathways is still lacking. We present here a coarse-grained model that captures the essential features of the expansion process and allows us to investigate the conditions under which a viral capsid becomes unstable. Based on this model we demonstrate that the structural transitions in icosahedral viral capsids are likely to occur through a low-symmetry cascade of local expansion events spreading in a wavelike manner over the capsid surface.

  4. Asparagine Metabolic Pathways in Arabidopsis.

    PubMed

    Gaufichon, Laure; Rothstein, Steven J; Suzuki, Akira

    2016-04-01

    Inorganic nitrogen in the form of ammonium is assimilated into asparagine via multiple steps involving glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AspAT) and asparagine synthetase (AS) in Arabidopsis. The asparagine amide group is liberated by the reaction catalyzed by asparaginase (ASPG) and also the amino group of asparagine is released by asparagine aminotransferase (AsnAT) for use in the biosynthesis of amino acids. Asparagine plays a primary role in nitrogen recycling, storage and transport in developing and germinating seeds, as well as in vegetative and senescence organs. A small multigene family encodes isoenzymes of each step of asparagine metabolism in Arabidopsis, except for asparagine aminotransferase encoded by a single gene. The aim of this study is to highlight the structure of the genes and encoded enzyme proteins involved in asparagine metabolic pathways; the regulation and role of different isogenes; and kinetic and physiological properties of encoded enzymes in different tissues and developmental stages. PMID:26628609

  5. The mitochondrial p53 pathway

    PubMed Central

    Vaseva, Angelina V.; Moll, Ute M.

    2010-01-01

    p53 is one of the most mutated tumor suppressors in human cancers and as such has been intensively studied for a long time. p53 is a major orchestrator of the cellular response to a broad array of stress types by regulating apoptosis, cell cycle arrest, senescence, DNA repair and genetic stability. For a long time it was thought that these functions of p53 solely rely on its function as a transcription factor, and numerous p53 target genes have been identified [1]. In the last 8 years however, a novel transcription-independent proapoptotic function mediated by the cytoplasmic pool of p53 has been revealed. p53 participates directly in the intrinsic apoptosis pathway by interacting with the multidomain members of the Bcl-2 family to induce mitochondrial outer membrane permeabilization. Our review will discuss these studies, focusing on recent advances in the field. PMID:19007744

  6. Biosynthetic pathways of ergot alkaloids.

    PubMed

    Gerhards, Nina; Neubauer, Lisa; Tudzynski, Paul; Li, Shu-Ming

    2014-01-01

    Ergot alkaloids are nitrogen-containing natural products belonging to indole alkaloids. The best known producers are fungi of the phylum Ascomycota, e.g., Claviceps, Epichloë, Penicillium and Aspergillus species. According to their structures, ergot alkaloids can be divided into three groups: clavines, lysergic acid amides and peptides (ergopeptines). All of them share the first biosynthetic steps, which lead to the formation of the tetracyclic ergoline ring system (except the simplest, tricyclic compound: chanoclavine). Different modifications on the ergoline ring by specific enzymes result in an abundance of bioactive natural products, which are used as pharmaceutical drugs or precursors thereof. From the 1950s through to recent years, most of the biosynthetic pathways have been elucidated. Gene clusters from several ergot alkaloid producers have been identified by genome mining and the functions of many of those genes have been demonstrated by knock-out experiments or biochemical investigations of the overproduced enzymes. PMID:25513893

  7. [Stress and the kynurenine pathway].

    PubMed

    Majláth, Zsófia; Vécsei, László

    2015-08-30

    The kynurenine pathway is the main route of tryptophan degradation which gives rise to several neuroactive metabolites. Kynurenic acid is an endogenous antagonist of excitatory receptors, which proved to be neuroprotective in the preclinical settings. Kynurenines have been implicated in the neuroendocrine regulatory processes. Stress induces several alterations in the kynurenine metabolism and this process may contribute to the development of stress-related pathological processes. Irritable bowel disease and gastric ulcer are well-known disorders which are related to psychiatric comorbidity and stress. In experimental conditions kynurenic acid proved to be beneficial by reducing inflammatory processes and normalizing microcirculation in the bowel. Further investigations are needed to better understand the relations of stress and the kynurenines, with the aim of developing novel therapeutic tools for stress-related pathologies. PMID:26299831

  8. Mars - Pathway to the stars

    NASA Astrophysics Data System (ADS)

    Angelo, J. A., Jr.; Buden, D.

    Mars has and will continue to play a key role in our exploration and conquest of the Solar System. Within the context of the creation of humanity's extraterrestrial civilization, the major technical features of the following Mars programs are reviewed: the Mars Geoscience/Climatology Orbiter; the Mars Aeronomy Orbiter; the Mars airplane; the Mars Penetrator Network; Mars surface rovers and mobility systems; human exploration of Mars; and permanent Martian bases and settlements. Mars properly explored and utilized opens the way to the resources of the asteroid belt and the outer planets; supports the creation of smart machines for space exploration and exploitation; and encourages the creation of autonomous niches of intelligent life within heliocentric space. All of these developments, in turn, establish the technological pathway for the first interstellar missions.

  9. Biosynthetic Pathways of Ergot Alkaloids

    PubMed Central

    Gerhards, Nina; Neubauer, Lisa; Tudzynski, Paul; Li, Shu-Ming

    2014-01-01

    Ergot alkaloids are nitrogen-containing natural products belonging to indole alkaloids. The best known producers are fungi of the phylum Ascomycota, e.g., Claviceps, Epichloë, Penicillium and Aspergillus species. According to their structures, ergot alkaloids can be divided into three groups: clavines, lysergic acid amides and peptides (ergopeptines). All of them share the first biosynthetic steps, which lead to the formation of the tetracyclic ergoline ring system (except the simplest, tricyclic compound: chanoclavine). Different modifications on the ergoline ring by specific enzymes result in an abundance of bioactive natural products, which are used as pharmaceutical drugs or precursors thereof. From the 1950s through to recent years, most of the biosynthetic pathways have been elucidated. Gene clusters from several ergot alkaloid producers have been identified by genome mining and the functions of many of those genes have been demonstrated by knock-out experiments or biochemical investigations of the overproduced enzymes. PMID:25513893

  10. Steering electrons on moving pathways.

    PubMed

    Beratan, David N; Skourtis, Spiros S; Balabin, Ilya A; Balaeff, Alexander; Keinan, Shahar; Venkatramani, Ravindra; Xiao, Dequan

    2009-10-20

    Electron transfer (ET) reactions provide a nexus among chemistry, biochemistry, and physics. These reactions underpin the "power plants" and "power grids" of bioenergetics, and they challenge us to understand how evolution manipulates structure to control ET kinetics. Ball-and-stick models for the machinery of electron transfer, however, fail to capture the rich electronic and nuclear dynamics of ET molecules: these static representations disguise, for example, the range of thermally accessible molecular conformations. The influence of structural fluctuations on electron-transfer kinetics is amplified by the exponential decay of electron tunneling probabilities with distance, as well as the delicate interference among coupling pathways. Fluctuations in the surrounding medium can also switch transport between coherent and incoherent ET mechanisms--and may gate ET so that its kinetics is limited by conformational interconversion times, rather than by the intrinsic ET time scale. Moreover, preparation of a charge-polarized donor state or of a donor state with linear or angular momentum can have profound dynamical and kinetic consequences. In this Account, we establish a vocabulary to describe how the conformational ensemble and the prepared donor state influence ET kinetics in macromolecules. This framework is helping to unravel the richness of functional biological ET pathways, which have evolved within fluctuating macromolecular structures. The conceptual framework for describing nonadiabatic ET seems disarmingly simple: compute the ensemble-averaged (mean-squared) donor-acceptor (DA) tunneling interaction, , and the Franck-Condon weighted density of states, rho(FC), to describe the rate, (2pi/variant Planck's over 2pi)rho(FC). Modern descriptions of the thermally averaged electronic coupling and of the Franck-Condon factor establish a useful predictive framework in biology, chemistry, and nanoscience. Describing the influence of geometric and

  11. A pathway to academic accreditation

    SciTech Connect

    Seitz, M.R.

    1994-09-01

    The pathways to successfully accrediting programs through a partnership with a local college can be convoluted and offer many dead ends. Those pathways can be made straighter and have fewer false starts by following a plan that has worked. Accreditation of courses and programs can add credibility and prestige to a program. The process can be facilitated by following a basic plan such as the one outlined. The discussion will track the preliminary activities that form the ground work for the beginning of the accreditation process through final approval by a college`s State Board of trustees or regents. On the road to approval, the packaging of courses for presentation, the formulation and composition of an advisory committee, the subsequent use of the advisors, presentation to the faculty committees, the presentation to the college`s governing board of trustees or regents, and final approval by the State Board are covered. An important benefit of accreditation is the formation of a partnership with the local college. Teaming with a local college to provide an accredited certificate in a field of employee training is an excellent opportunity to establish an educational partnership within the local community that will be of benefit to the participating entities. It also represents a training/retraining opportunity in direct support of the US Department of Energy`s current missions of partnership and localization. The accredited modules can be taught where appropriate by college personnel or loaned instructors from the work site. By using the company employees who are working with the topics covered in the modules, the courses are kept up-to-date.

  12. Fuel Dependence of Benzene Pathways

    SciTech Connect

    Zhang, H; Eddings, E; Sarofim, A; Westbrook, C

    2008-07-14

    The relative importance of formation pathways for benzene, an important precursor to soot formation, was determined from the simulation of 22 premixed flames for a wide range of equivalence ratios (1.0 to 3.06), fuels (C{sub 1}-C{sub 12}), and pressures (20 to 760 torr). The maximum benzene concentrations in 15 out of these flames were well reproduced within 30% of the experimental data. Fuel structural properties were found to be critical for benzene production. Cyclohexanes and C{sub 3} and C{sub 4} fuels were found to be among the most productive in benzene formation; and long-chain normal paraffins produce the least amount of benzene. Other properties, such as equivalence ratio and combustion temperatures, were also found to be important in determining the amount of benzene produced in flames. Reaction pathways for benzene formation were examined critically in four premixed flames of structurally different fuels of acetylene, n-decane, butadiene, and cyclohexane. Reactions involving precursors, such as C{sub 3} and C{sub 4} species, were examined. Combination reactions of C{sub 3} species were identified to be the major benzene formation routes with the exception of the cyclohexane flame, in which benzene is formed exclusively from cascading fuel dehydrogenation via cyclohexene and cyclohexadiene intermediates. Acetylene addition makes a minor contribution to benzene formation, except in the butadiene flame where C{sub 4}H{sub 5} radicals are produced directly from the fuel, and in the n-decane flame where C{sub 4}H{sub 5} radicals are produced from large alkyl radical decomposition and H atom abstraction from the resulting large olefins.

  13. Human stretch reflex pathways reexamined

    PubMed Central

    Yavuz, Ş. Utku; Mrachacz-Kersting, Natalie; Sebik, Oğuz; Berna Ünver, M.; Farina, Dario

    2013-01-01

    Reflex responses of tibialis anterior motor units to stretch stimuli were investigated in human subjects. Three types of stretch stimuli were applied (tap-like, ramp-and-hold, and half-sine stretch). Stimulus-induced responses in single motor units were analyzed using the classical technique, which involved building average surface electromyogram (SEMG) and peristimulus time histograms (PSTH) from the discharge times of motor units and peristimulus frequencygrams (PSF) from the instantaneous discharge rates of single motor units. With the use of SEMG and PSTH, the tap-like stretch stimulus induced five separate reflex responses, on average. With the same single motor unit data, the PSF technique indicated that the tap stimulus induced only three reflex responses. Similar to the finding using the tap-like stretch stimuli, ramp-and-hold stimuli induced several peaks and troughs in the SEMG and PSTH. The PSF analyses displayed genuine increases in discharge rates underlying the peaks but not underlying the troughs. Half-sine stretch stimuli induced a long-lasting excitation followed by a long-lasting silent period in SEMG and PSTH. The increase in the discharge rate, however, lasted for the entire duration of the stimulus and continued during the silent period. The results are discussed in the light of the fact that the discharge rate of a motoneuron has a strong positive linear association with the effective synaptic current it receives and hence represents changes in the membrane potential more directly and accurately than the other indirect measures. This study suggests that the neuronal pathway of the human stretch reflex does not include inhibitory pathways. PMID:24225537

  14. Reaction pathways of propene pyrolysis.

    PubMed

    Qu, Yena; Su, Kehe; Wang, Xin; Liu, Yan; Zeng, Qingfeng; Cheng, Laifei; Zhang, Litong

    2010-05-01

    The gas-phase reaction pathways in preparing pyrolytic carbon with propene pyrolysis have been investigated in detail with a total number of 110 transition states and 50 intermediates. The structure of the species was determined with density functional theory at B3PW91/6-311G(d,p) level. The transition states and their linked intermediates were confirmed with frequency and the intrinsic reaction coordinates analyses. The elementary reactions were explored in the pathways of both direct and the radical attacking decompositions. The energy barriers and the reaction energies were determined with accurate model chemistry method at G3(MP2) level after an examination of the nondynamic electronic correlations. The heat capacities and entropies were obtained with statistical thermodynamics. The Gibbs free energies at 298.15 K for all the reaction steps were reported. Those at any temperature can be developed with classical thermodynamics by using the fitted (as a function of temperature) heat capacities. It was found that the most favorable paths are mainly in the radical attacking chain reactions. The chain was proposed with 26 reaction steps including two steps of the initialization of the chain to produce H and CH(3) radicals. For a typical temperature (1200 K) adopted in the experiments, the highest energy barriers were found in the production of C(3) to be 203.4 and 193.7 kJ/mol. The highest energy barriers for the production of C(2) and C were found 174.1 and 181.4 kJ/mol, respectively. These results are comparable with the most recent experimental observation of the apparent activation energy 201.9 +/- 0.6 or 137 +/- 25 kJ/mol. PMID:20082392

  15. Clathrin-Independent Pathways of Endocytosis

    PubMed Central

    Mayor, Satyajit; Parton, Robert G.; Donaldson, Julie G.

    2014-01-01

    There are many pathways of endocytosis at the cell surface that apparently operate at the same time. With the advent of new molecular genetic and imaging tools, an understanding of the different ways by which a cell may endocytose cargo is increasing by leaps and bounds. In this review we explore pathways of endocytosis that occur in the absence of clathrin. These are referred to as clathrin-independent endocytosis (CIE). Here we primarily focus on those pathways that function at the small scale in which some have distinct coats (caveolae) and others function in the absence of specific coated intermediates. We follow the trafficking itineraries of the material endocytosed by these pathways and finally discuss the functional roles that these pathways play in cell and tissue physiology. It is likely that these pathways will play key roles in the regulation of plasma membrane area and tension and also control the availability of membrane during cell migration. PMID:24890511

  16. Premetazoan origin of the Hippo signaling pathway

    PubMed Central

    Sebé-Pedrós, Arnau; Zheng, Yonggang; Ruiz-Trillo, Iñaki; Pan, Duojia

    2012-01-01

    Summary Non-aggregative multicellularity requires strict control of cell number. The Hippo signaling pathway coordinates cell proliferation and apoptosis and is a central regulator of organ size in animals. Recent studies have shown the presence of key members of the Hippo pathway in non-bilaterian animals, but failed to identify this pathway outside Metazoa. Through comparative analyses of recently sequenced holozoan genomes, we show that Hippo pathway components, such as the kinases Hippo and Warts, the co-activator Yorkie and the transcription factor Scalloped, were already present in the unicellular ancestors of animals. Remarkably, functional analysis of Hippo components of the amoeboid holozoan Capsaspora owczarzaki, performed in Drosophila, demonstrate that the growth-regulatory activity of the Hippo pathway is conserved in this unicellular lineage. Our findings show that the Hippo pathway evolved well before the origin of Metazoa and highlight the importance of Hippo signaling as a key developmental mechanism pre-dating the origin of Metazoa. PMID:22832104

  17. Stochasticity in the yeast mating pathway

    NASA Astrophysics Data System (ADS)

    Wang, Hong-Li; Fu, Zheng-Ping; Xu, Xin-Hang; Ouyang, Qi

    2009-05-01

    We report stochastic simulations of the yeast mating signal transduction pathway. The effects of intrinsic and external noise, the influence of cell-to-cell difference in the pathway capacity, and noise propagation in the pathway have been examined. The stochastic temporal behaviour of the pathway is found to be robust to the influence of inherent fluctuations, and intrinsic noise propagates in the pathway in a uniform pattern when the yeasts are treated with pheromones of different stimulus strengths and of varied fluctuations. In agreement with recent experimental findings, extrinsic noise is found to play a more prominent role than intrinsic noise in the variability of proteins. The occurrence frequency for the reactions in the pathway are also examined and a more compact network is obtained by dropping most of the reactions of least occurrence.

  18. Developmental pathways: Sonic hedgehog-Patched-GLI.

    PubMed Central

    Walterhouse, D O; Yoon, J W; Iannaccone, P M

    1999-01-01

    Developmental pathways are networks of genes that act coordinately to establish the body plan. Disruptions of genes in one pathway can have effects in related pathways and may result in serious dysmorphogenesis or cancer. Environmental exposures can be associated with poor pregnancy outcomes, including dysmorphic offspring or children with a variety of diseases. An important goal of environmental science should be reduction of these poor outcomes. This will require an understanding of the genes affected by specific exposures and the consequence of alterations in these genes or their products, which in turn will require an understanding of the pathways critical in development. The ligand Sonic hedgehog, the receptors Patched and Smoothened, and the GLI family of transcription factors represent one such pathway. This pathway illustrates several operating principles important in the consideration of developmental consequences of environmental exposures to toxins. Images Figure 1 Figure 2 PMID:10064544

  19. Pathway-Based Functional Analysis of Metagenomes

    NASA Astrophysics Data System (ADS)

    Bercovici, Sivan; Sharon, Itai; Pinter, Ron Y.; Shlomi, Tomer

    Metagenomic data enables the study of microbes and viruses through their DNA as retrieved directly from the environment in which they live. Functional analysis of metagenomes explores the abundance of gene families, pathways, and systems, rather than their taxonomy. Through such analysis researchers are able to identify those functional capabilities most important to organisms in the examined environment. Recently, a statistical framework for the functional analysis of metagenomes was described that focuses on gene families. Here we describe two pathway level computational models for functional analysis that take into account important, yet unaddressed issues such as pathway size, gene length and overlap in gene content among pathways. We test our models over carefully designed simulated data and propose novel approaches for performance evaluation. Our models significantly improve over current approach with respect to pathway ranking and the computations of relative abundance of pathways in environments.

  20. Manipulating Quantum Pathways on the Fly

    NASA Astrophysics Data System (ADS)

    Rey-de-Castro, Roberto; Leghtas, Zaki; Rabitz, Herschel

    2013-05-01

    The expectation value of a quantum system observable can be written as a sum over interfering pathway amplitudes. In this Letter, we demonstrate for the fist time adaptive manipulation of quantum pathways using the Hamiltonian encoding-observable decoding (HE-OD) technique. The principles of HE-OD are illustrated for population transfer in atomic rubidium using shaped femtosecond laser pulses. The ability to manipulate multiple pathway amplitudes is of fundamental importance in all quantum control applications.

  1. Photodegradation Pathways in Arid Ecosystems

    NASA Astrophysics Data System (ADS)

    King, J. Y.; Lin, Y.; Adair, E. C.; Brandt, L.; Carbone, M. S.

    2013-12-01

    Recent interest in improving our understanding of decomposition patterns in arid and semi-arid ecosystems and under potentially drier future conditions has led to a flurry of research related to abiotic degradation processes. Oxidation of organic matter by solar radiation (photodegradation) is one abiotic degradation process that contributes significantly to litter decomposition rates. Our meta-analysis results show that increasing solar radiation exposure corresponds to an average increase of 23% in litter mass loss rate with large variation among studies associated primarily with environmental and litter chemistry characteristics. Laboratory studies demonstrate that photodegradation results in CO2 emissions. Indirect estimates suggest that photodegradation could account for as much as 60% of ecosystem CO2 emissions from dry ecosystems, but these CO2 fluxes have not been measured in intact ecosystems. The current data suggest that photodegradation is important, not only for understanding decomposition patterns, but also for modeling organic matter turnover and ecosystem C cycling. However, the mechanisms by which photodegradation operates, along with their environmental and litter chemistry controls, are still poorly understood. Photodegradation can directly influence decomposition rates and ecosystem CO2 flux via photochemical mineralization. It can also indirectly influence biotic decomposition rates by facilitating microbial degradation through breakdown of more recalcitrant compounds into simpler substrates or by suppressing microbial activity directly. All of these pathways influence the decomposition process, but the relative importance of each is uncertain. Furthermore, a specific suite of controls regulates each of these pathways (e.g., environmental conditions such as temperature and relative humidity; physical environment such as canopy architecture and contact with soil; and litter chemistry characteristics such as lignin and cellulose content), and

  2. The pathway ontology – updates and applications

    PubMed Central

    2014-01-01

    Background The Pathway Ontology (PW) developed at the Rat Genome Database (RGD), covers all types of biological pathways, including altered and disease pathways and captures the relationships between them within the hierarchical structure of a directed acyclic graph. The ontology allows for the standardized annotation of rat, and of human and mouse genes to pathway terms. It also constitutes a vehicle for easy navigation between gene and ontology report pages, between reports and interactive pathway diagrams, between pathways directly connected within a diagram and between those that are globally related in pathway suites and suite networks. Surveys of the literature and the development of the Pathway and Disease Portals are important sources for the ongoing development of the ontology. User requests and mapping of pathways in other databases to terms in the ontology further contribute to increasing its content. Recently built automated pipelines use the mapped terms to make available the annotations generated by other groups. Results The two released pipelines – the Pathway Interaction Database (PID) Annotation Import Pipeline and the Kyoto Encyclopedia of Genes and Genomes (KEGG) Annotation Import Pipeline, make available over 7,400 and 31,000 pathway gene annotations, respectively. Building the PID pipeline lead to the addition of new terms within the signaling node, also augmented by the release of the RGD “Immune and Inflammatory Disease Portal” at that time. Building the KEGG pipeline lead to a substantial increase in the number of disease pathway terms, such as those within the ‘infectious disease pathway’ parent term category. The ‘drug pathway’ node has also seen increases in the number of terms as well as a restructuring of the node. Literature surveys, disease portal deployments and user requests have contributed and continue to contribute additional new terms across the ontology. Since first presented, the content of PW has increased by

  3. Dissecting the Cell Entry Pathway of Dengue Virus by Single-Particle Tracking in Living Cells

    PubMed Central

    Chen, Chen; van der Ende-Metselaar, Heidi; Wilschut, Jan; Zhuang, Xiaowei; Smit, Jolanda M.

    2008-01-01

    Dengue virus (DENV) is an enveloped RNA virus that causes the most common arthropod-borne infection worldwide. The mechanism by which DENV infects the host cell remains unclear. In this work, we used live-cell imaging and single-virus tracking to investigate the cell entry, endocytic trafficking, and fusion behavior of DENV. Simultaneous tracking of DENV particles and various endocytic markers revealed that DENV enters cells exclusively via clathrin-mediated endocytosis. The virus particles move along the cell surface in a diffusive manner before being captured by a pre-existing clathrin-coated pit. Upon clathrin-mediated entry, DENV particles are transported to Rab5-positive endosomes, which subsequently mature into late endosomes through acquisition of Rab7 and loss of Rab5. Fusion of the viral membrane with the endosomal membrane was primarily detected in late endosomal compartments. PMID:19096510

  4. Orexin: Pathways to obesity resistance?

    PubMed Central

    Butterick, Tammy A.; Billington, Charles J.; Kotz, Catherine M.; Nixon, Joshua P.

    2016-01-01

    Obesity has increased in prevalence worldwide, attributed in part to the influences of an obesity-promoting environment and genetic factors. While obesity and overweight increasingly seem to be the norm, there remain individuals who resist obesity. We present here an overview of data supporting the idea that hypothalamic neuropeptide orexin A (OXA; hypocretin 1) may be a key component of brain mechanisms underlying obesity resistance. Prior work with models of obesity and obesity resistance in rodents has shown that increased orexin and/or orexin sensitivity is correlated with elevated spontaneous physical activity (SPA), and that orexin-induced SPA contributes to obesity resistance via increased non-exercise activity thermogenesis (NEAT). However, central hypothalamic orexin signaling mechanisms that regulate SPA remain undefined. Our ongoing studies and work of others support the hypothesis that one such mechanism may be upregulation of a hypoxia-inducible factor 1 alpha (HIF-1α)-dependent pathway, suggesting that orexin may promote obesity resistance both by increasing SPA and by influencing the metabolic state of orexin-responsive hypothalamic neurons. We discuss potential mechanisms based on both animal and in vitro pharmacological studies, in the context of elucidating potential molecular targets for obesity prevention and therapy. PMID:24005942

  5. Two pathways ensuring social harmony

    NASA Astrophysics Data System (ADS)

    Konrad, Matthias; Pamminger, Tobias; Foitzik, Susanne

    2012-08-01

    Reproductive division of labour is a characteristic trait of social insects. The dominant reproductive individual, often the queen, uses chemical communication and/or behaviour to maintain her social status. Queens of many social insects communicate their fertility status via cuticle-bound substances. As these substances usually possess a low volatility, their range in queen-worker communication is potentially limited. Here, we investigate the range and impact of behavioural and chemical queen signals on workers of the ant Temnothorax longispinosus. We compared the behaviour and ovary development of workers subjected to three different treatments: workers with direct chemical and physical contact to the queen, those solely under the influence of volatile queen substances and those entirely separated from the queen. In addition to short-ranged queen signals preventing ovary development in workers, we discovered a novel secondary pathway influencing worker behaviour. Workers with no physical contact to the queen, but exposed to volatile substances, started to develop their ovaries, but did not change their behaviour compared to workers in direct contact to the queen. In contrast, workers in queen-separated groups showed both increased ovary development and aggressive dominance interactions. We conclude that T. longispinosus queens influence worker ovary development and behaviour via two independent signals, both ensuring social harmony within the colony.

  6. Inconsistent pathways of household waste

    SciTech Connect

    Dahlen, Lisa Aberg, Helena; Lagerkvist, Anders; Berg, Per E.O.

    2009-06-15

    The aim of this study was to provide policy-makers and waste management planners with information about how recycling programs affect the quantities of specific materials recycled and disposed of. Two questions were addressed: which factors influence household waste generation and pathways? and how reliable are official waste data? Household waste flows were studied in 35 Swedish municipalities, and a wide variation in the amount of waste per capita was observed. When evaluating the effect of different waste collection policies, it was found to be important to identify site-specific factors influencing waste generation. Eleven municipal variables were investigated in an attempt to explain the variation. The amount of household waste per resident was higher in populous municipalities and when net commuting was positive. Property-close collection of dry recyclables led to increased delivery of sorted metal, plastic and paper packaging. No difference was seen in the amount of separated recyclables per capita when weight-based billing for the collection of residual waste was applied, but the amount of residual waste was lower. Sixteen sources of error in official waste statistics were identified and the results of the study emphasize the importance of reliable waste generation and composition data to underpin waste management policies.

  7. Glycosyltransferase efficiently controls phenylpropanoid pathway

    PubMed Central

    Aksamit-Stachurska, Anna; Korobczak-Sosna, Alina; Kulma, Anna; Szopa, Jan

    2008-01-01

    Background In a previous study, anthocyanin levels in potato plants were increased by manipulating genes connected with the flavonoid biosynthesis pathway. However, starch content and tuber yield were dramatically reduced in the transgenic plants, which over-expressed dihydroflavonol reductase (DFR). Results Transgenic plants over-expressing dihydroflavonol reductase (DFR) were subsequently transformed with the cDNA coding for the glycosyltransferase (UGT) of Solanum sogarandinum in order to obtain plants with a high anthocyanin content without reducing tuber yield and quality. Based on enzyme studies, the recombinant UGT is a 7-O-glycosyltransferase whose natural substrates include both anthocyanidins and flavonols such as kaempferol and quercetin. In the super-transformed plants, tuber production was much higher than in the original transgenic plants bearing only the transgene coding for DFR, and was almost the same as in the control plants. The anthocyanin level was lower than in the initial plants, but still higher than in the control plants. Unexpectedly, the super-transformed plants also produced large amounts of kaempferol, chlorogenic acid, isochlorogenic acid, sinapic acid and proanthocyanins. Conclusion In plants over-expressing both the transgene for DFR and the transgene for UGT, the synthesis of phenolic acids was diverted away from the anthocyanin branch. This represents a novel approach to manipulating phenolic acids synthesis in plants. PMID:18321380

  8. Racial discrimination & health: pathways & evidence.

    PubMed

    Ahmed, Ameena T; Mohammed, Selina A; Williams, David R

    2007-10-01

    This review provides an overview of the existing empirical research of the multiple ways by which discrimination can affect health. Institutional mechanisms of discrimination such as restricting marginalized groups to live in undesirable residential areas can have deleterious health consequences by limiting socio-economic status (SES) and creating health-damaging conditions in residential environments. Discrimination can also adversely affect health through restricting access to desirable services such as medical care and creating elevated exposure to traditional stressors such as unemployment and financial strain. Central to racism is an ideology of inferiority that can adversely affect non-dominant groups because some members of marginalized populations will accept as true the dominant society's ideology of their group's inferiority. Limited empirical research indicates that internalized racism is inversely related to health. In addition, the existence of these negative stereotypes can lead dominant group members to consciously and unconsciously discriminate against the stigmatized. An overview of the growing body of research examining the ways in which psychosocial stress generated by subjective experiences of discrimination can affect health is also provided. We review the evidence from the United States and other societies that suggest that the subjective experience of discrimination can adversely affect health and health enhancing behaviours. Advancing our understanding of the relationship between discrimination and health requires improved assessment of the phenomenon of discrimination and increased attention to identifying the psychosocial and biological pathways that may link exposure to discrimination to health status. PMID:18032807

  9. Nicotinic receptors in addiction pathways.

    PubMed

    Leslie, Frances M; Mojica, Celina Y; Reynaga, Daisy D

    2013-04-01

    Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that consist of pentameric combinations of α and β subunits. These receptors are widely distributed throughout the brain and are highly expressed in addiction circuitry. The role of nAChRs in regulating neuronal activity and motivated behavior is complex and varies both in and among brain regions. The rich diversity of central nAChRs has hampered the characterization of their structure and function with use of classic pharmacological techniques. However, recent molecular approaches using null mutant mice with specific regional lentiviral re-expression, in combination with neuroanatomical and electrophysiological techniques, have allowed the elucidation of the influence of different nAChR types on neuronal circuit activity and behavior. This review will address the influence of nAChRs on limbic dopamine circuitry and the medial habenula-interpeduncular nucleus complex, which are critical mediators of reinforced behavior. Characterization of the mechanisms underlying regulation of addiction pathways by endogenous cholinergic transmission and by nicotine may lead to the identification of new therapeutic targets for treating tobacco dependence and other addictions. PMID:23247824

  10. Greater Capital Region Career Pathways Initiative

    ERIC Educational Resources Information Center

    Workforce Strategy Center, 2005

    2005-01-01

    This case study documents the activities of the Greater Capital Region Career Pathways Initiative (CPI), a partnership to develop regional, demand driven career pathways for the biotechnology sector in and around Albany, NY. This document is written for three primary audiences. First, it provides a report to the New York State Department of Labor…

  11. Implementing Guided Pathways: Tips and Tools

    ERIC Educational Resources Information Center

    Bailey, Thomas; Jaggars, Shanna Smith; Jenkins, Davis

    2015-01-01

    A growing number of community colleges and four-year universities are seeking to improve student outcomes by redesigning academic programs and student support services following the guided pathways approach. These institutions are mapping out highly structured, educationally coherent program pathways for students to follow by starting with the end…

  12. Quicker cancer care: reshaping patient pathways.

    PubMed

    Towler, Lucy

    2009-07-01

    A new pathway has been devised for patients with ovarian cancer who attend a day-care unit for chemotherapy. This pathway, which is provided by nurses and doctors, has reduced patients' waiting time for treatment. Its implementation shows, therefore, that good clinical leadership can effect positive change. PMID:19639906

  13. Diversifying Carotenoid Biosynthetic Pathways by Directed Evolution

    PubMed Central

    Umeno, Daisuke; Tobias, Alexander V.; Arnold, Frances H.

    2005-01-01

    Microorganisms and plants synthesize a diverse array of natural products, many of which have proven indispensable to human health and well-being. Although many thousands of these have been characterized, the space of possible natural products—those that could be made biosynthetically—remains largely unexplored. For decades, this space has largely been the domain of chemists, who have synthesized scores of natural product analogs and have found many with improved or novel functions. New natural products have also been made in recombinant organisms, via engineered biosynthetic pathways. Recently, methods inspired by natural evolution have begun to be applied to the search for new natural products. These methods force pathways to evolve in convenient laboratory organisms, where the products of new pathways can be identified and characterized in high-throughput screening programs. Carotenoid biosynthetic pathways have served as a convenient experimental system with which to demonstrate these ideas. Researchers have mixed, matched, and mutated carotenoid biosynthetic enzymes and screened libraries of these “evolved” pathways for the emergence of new carotenoid products. This has led to dozens of new pathway products not previously known to be made by the assembled enzymes. These new products include whole families of carotenoids built from backbones not found in nature. This review details the strategies and specific methods that have been employed to generate new carotenoid biosynthetic pathways in the laboratory. The potential application of laboratory evolution to other biosynthetic pathways is also discussed. PMID:15755953

  14. Fuel Pathway Integration Technical Team Roadmap

    SciTech Connect

    2013-06-01

    The Fuel Pathway Integration Technical Team (FPITT) supports the U.S. DRIVE Partnership (the Partnership) in the identification and evaluation of implementation scenarios for fuel cell technology pathways, including hydrogen and fuel cell electric vehicles in the transportation sector, both during a transition period and in the long term.

  15. Women's Work Pathways Across the Life Course.

    PubMed

    Damaske, Sarah; Frech, Adrianne

    2016-04-01

    Despite numerous changes in women's employment in the latter half of the twentieth century, women's employment continues to be uneven and stalled. Drawing from data on women's weekly work hours in the National Longitudinal Survey of Youth (NLSY79), we identify significant inequality in women's labor force experiences across adulthood. We find two pathways of stable full-time work for women, three pathways of part-time employment, and a pathway of unpaid labor. A majority of women follow one of the two full-time work pathways, while fewer than 10% follow a pathway of unpaid labor. Our findings provide evidence of the lasting influence of work-family conflict and early socioeconomic advantages and disadvantages on women's work pathways. Indeed, race, poverty, educational attainment, and early family characteristics significantly shaped women's work careers. Work-family opportunities and constraints also were related to women's work hours, as were a woman's gendered beliefs and expectations. We conclude that women's employment pathways are a product of both their resources and changing social environment as well as individual agency. Significantly, we point to social stratification, gender ideologies, and work-family constraints, all working in concert, as key explanations for how women are "tracked" onto work pathways from an early age. PMID:27001314

  16. Improving College System Pathways: Project Highlights Reports

    ERIC Educational Resources Information Center

    Colleges Ontario, 2008

    2008-01-01

    In 2006, Ontario's colleges received funding from the Ministry of Training, Colleges and Universities for the Improving College System Pathways Project. The project goals were to significantly increase educational pathways within and between colleges by developing a clearer understanding of student mobility within the system; to identify the scope…

  17. Modeling biochemical pathways in the gene ontology

    PubMed Central

    Hill, David P.; D’Eustachio, Peter; Berardini, Tanya Z.; Mungall, Christopher J.; Renedo, Nikolai; Blake, Judith A.

    2016-01-01

    The concept of a biological pathway, an ordered sequence of molecular transformations, is used to collect and represent molecular knowledge for a broad span of organismal biology. Representations of biomedical pathways typically are rich but idiosyncratic presentations of organized knowledge about individual pathways. Meanwhile, biomedical ontologies and associated annotation files are powerful tools that organize molecular information in a logically rigorous form to support computational analysis. The Gene Ontology (GO), representing Molecular Functions, Biological Processes and Cellular Components, incorporates many aspects of biological pathways within its ontological representations. Here we present a methodology for extending and refining the classes in the GO for more comprehensive, consistent and integrated representation of pathways, leveraging knowledge embedded in current pathway representations such as those in the Reactome Knowledgebase and MetaCyc. With carbohydrate metabolic pathways as a use case, we discuss how our representation supports the integration of variant pathway classes into a unified ontological structure that can be used for data comparison and analysis. PMID:27589964

  18. Pathways to Success for Michigan's Opportunity Youth

    ERIC Educational Resources Information Center

    American Youth Policy Forum, 2015

    2015-01-01

    Each young person must navigate his/her own pathway into and through postsecondary education and the workforce to long-term success personalized to his/her own unique needs and desires. The pathway to long-term success is often articulated as a straight road through K-12 education into postsecondary education (either academic or technical…

  19. Optic pathway degeneration in Japanese black cattle

    PubMed Central

    CHIBA, Shiori; FUNATO, Shingo; HORIUCHI, Noriyuki; MATSUMOTO, Kotaro; INOKUMA, Hisashi; FURUOKA, Hidefumi; KOBAYASHI, Yoshiyasu

    2014-01-01

    Degeneration of the optic pathway has been reported in various animal species including cattle. We experienced a case of bilateral optic tract degeneration characterized by severe gliosis in a Japanese black cattle without any obvious visual defects. To evaluate the significance, pathological nature and pathogenesis of the lesions, we examined the optic pathway in 60 cattle (41 Japanese black, 13 Holstein and 6 crossbreed) with or without ocular abnormalities. None of these animals had optic canal stenosis. Degenerative changes with severe gliosis in the optic pathway, which includes the optic nerve, optic chiasm and optic tract, were only observed in 8 Japanese black cattle with or without ocular abnormalities. Furthermore, strong immunoreactivity of glial fibrillary acidic protein was observed in the retinal stratum opticum and ganglion cell layer in all 5 cattle in which the optic pathway lesions could be examined. As etiological research, we also examined whether the concentrations of vitamin A and vitamin B12 or bovine viral diarrhea virus (BVDV) infection was associated with optic pathway degeneration. However, our results suggested that the observed optic pathway degeneration was probably not caused by these factors. These facts indicate the presence of optic pathway degeneration characterized by severe gliosis that has never been reported in cattle without bilateral compressive lesions in the optic pathway or bilateral severe retinal atrophy. PMID:25421501

  20. The Career Pathways How-To Guide

    ERIC Educational Resources Information Center

    Jenkins, Davis; Spence, Christopher

    2006-01-01

    Career pathways is a series of connected education and training programs and support services that enable individuals to secure employment within a specific industry or occupational sector, and to advance over time to successively higher levels of education and employment in that sector. Career pathways are distinct from most educational efforts…

  1. Modeling biochemical pathways in the gene ontology.

    PubMed

    Hill, David P; D'Eustachio, Peter; Berardini, Tanya Z; Mungall, Christopher J; Renedo, Nikolai; Blake, Judith A

    2016-01-01

    The concept of a biological pathway, an ordered sequence of molecular transformations, is used to collect and represent molecular knowledge for a broad span of organismal biology. Representations of biomedical pathways typically are rich but idiosyncratic presentations of organized knowledge about individual pathways. Meanwhile, biomedical ontologies and associated annotation files are powerful tools that organize molecular information in a logically rigorous form to support computational analysis. The Gene Ontology (GO), representing Molecular Functions, Biological Processes and Cellular Components, incorporates many aspects of biological pathways within its ontological representations. Here we present a methodology for extending and refining the classes in the GO for more comprehensive, consistent and integrated representation of pathways, leveraging knowledge embedded in current pathway representations such as those in the Reactome Knowledgebase and MetaCyc. With carbohydrate metabolic pathways as a use case, we discuss how our representation supports the integration of variant pathway classes into a unified ontological structure that can be used for data comparison and analysis. PMID:27589964

  2. The metabolic pathway collection: an update.

    PubMed

    Selkov, E; Galimova, M; Goryanin, I; Gretchkin, Y; Ivanova, N; Komarov, Y; Maltsev, N; Mikhailova, N; Nenashev, V; Overbeek, R; Panyushkina, E; Pronevitch, L; Selkov, E

    1997-01-01

    The Metabolic Pathway Collection from EMP is an extraction of data from the larger Enzymes and Metabolic Pathways database (EMP). This extraction has been made publicly available in the hope that others will find it useful for a variety of purposes. The original release in October 1995 contained 1814 distinct pathways. The current collection contains 2180. Metabolic reconstructions for the first completely sequenced organisms-Haemophilus influenzae,Mycoplasma genitalium,Saccharomyces cerevisiaeandMethanococcus janaschii-are all included in the current release. All of the pathways in the collections are available as ASCII files in the form generated by the main curator, Evgeni Selkov. In addition, we are offering a more structured encoding of a subset of the collection; our initial release of this subcollection includes all of the pathways inMycoplasma genitalium, and we ultimately intend to offer the entire collection in this form as well. PMID:9016500

  3. Proton Pathways in Green Fluorescence Protein

    PubMed Central

    Agmon, Noam

    2005-01-01

    Proton pathways in green fluorescent protein (GFP) are more extended than previously reported. In the x-ray data of wild-type GFP, a two-step exit pathway exists from the active site to the protein surface, controlled by a threonine switch. A proton entry pathway begins at a glutamate-lysine cluster around Glu-5, and extends all the way to the buried Glu-222 near the active site. This structural evidence suggests that GFP may function as a portable light-driven proton-pump, with proton emitted in the excited state through the switchable exit pathway, and replenished from Glu-222 and the Glu-5 entry pathway in the ground state. PMID:15681647

  4. Pathway Analysis Incorporating Protein-Protein Interaction Networks Identified Candidate Pathways for the Seven Common Diseases.

    PubMed

    Lin, Peng-Lin; Yu, Ya-Wen; Chung, Ren-Hua

    2016-01-01

    Pathway analysis has become popular as a secondary analysis strategy for genome-wide association studies (GWAS). Most of the current pathway analysis methods aggregate signals from the main effects of single nucleotide polymorphisms (SNPs) in genes within a pathway without considering the effects of gene-gene interactions. However, gene-gene interactions can also have critical effects on complex diseases. Protein-protein interaction (PPI) networks have been used to define gene pairs for the gene-gene interaction tests. Incorporating the PPI information to define gene pairs for interaction tests within pathways can increase the power for pathway-based association tests. We propose a pathway association test, which aggregates the interaction signals in PPI networks within a pathway, for GWAS with case-control samples. Gene size is properly considered in the test so that genes do not contribute more to the test statistic simply due to their size. Simulation studies were performed to verify that the method is a valid test and can have more power than other pathway association tests in the presence of gene-gene interactions within a pathway under different scenarios. We applied the test to the Wellcome Trust Case Control Consortium GWAS datasets for seven common diseases. The most significant pathway is the chaperones modulate interferon signaling pathway for Crohn's disease (p-value = 0.0003). The pathway modulates interferon gamma, which induces the JAK/STAT pathway that is involved in Crohn's disease. Several other pathways that have functional implications for the seven diseases were also identified. The proposed test based on gene-gene interaction signals in PPI networks can be used as a complementary tool to the current existing pathway analysis methods focusing on main effects of genes. An efficient software implementing the method is freely available at http://puppi.sourceforge.net. PMID:27622767

  5. Pathway-PDT: a flexible pathway analysis tool for nuclear families

    PubMed Central

    2013-01-01

    Background Pathway analysis based on Genome-Wide Association Studies (GWAS) data has become popular as a secondary analysis strategy. Although many pathway analysis tools have been developed for case–control studies, there is no tool that can use all information from raw genotypes in general nuclear families. We developed Pathway-PDT, which uses the framework of Pedigree Disequilibrium Test (PDT) for general family data, to perform pathway analysis based on raw genotypes in family-based GWAS. Results Simulation results showed that Pathway-PDT is more powerful than the p-value based method, ALIGATOR. Pathway-PDT also can be more powerful than the PLINK set-based test when analyzing general nuclear families with multiple siblings or missing parents. Additionally, Pathway-PDT has a flexible and convenient user interface, which allows users to modify their analysis parameters as well as to apply various types of gene and pathway definitions. Conclusions The Pathway-PDT method is implemented in C++ with POSIX threads and is computationally feasible for pathway analysis with large scale family GWAS datasets. The Windows binary along with Makefile and source codes for the Linux are available at https://sourceforge.net/projects/pathway-pdt/. PMID:24006871

  6. Hippo pathway regulation of gastrointestinal tissues.

    PubMed

    Yu, Fa-Xing; Meng, Zhipeng; Plouffe, Steven W; Guan, Kun-Liang

    2015-01-01

    The Hippo pathway plays a crucial role in regulating tissue homeostasis and organ size, and its deregulation is frequently observed in human cancer. Yap is the major effector of and is inhibited by the Hippo pathway. In mouse model studies, inducible Yap expression in multiple tissues results in organ overgrowth. In the liver, knockout of upstream Hippo pathway components or transgenic expression of Yap leads to liver enlargement and hepatocellular carcinoma. In the small intestine or colon, deletion of upstream Hippo pathway components also results in expansion of intestinal progenitor cells and eventual development of adenomas. Genetic deletion of Yap in the intestine does not change the intestinal structure, but Yap is essential for intestinal repair upon certain types of tissue injury. The function of the Hippo pathway has also been studied in other gastrointestinal tissues, including the pancreas and stomach. Here we provide a brief overview of the Hippo pathway and discuss the physiological and pathological functions of this tumor suppressor pathway in gastrointestinal tissues. PMID:25293527

  7. Dysregulation of the mevalonate pathway promotes transformation

    PubMed Central

    Clendening, James W.; Pandyra, Aleks; Boutros, Paul C.; Ghamrasni, Samah El; Khosravi, Fereshteh; Trentin, Grace A.; Martirosyan, Anna; Hakem, Anne; Hakem, Razqallah; Jurisica, Igor; Penn, Linda Z.

    2010-01-01

    The importance of cancer metabolism has been appreciated for many years, but the intricacies of how metabolic pathways interconnect with oncogenic signaling are not fully understood. With a clear understanding of how metabolism contributes to tumorigenesis, we will be better able to integrate the targeting of these fundamental biochemical pathways into patient care. The mevalonate (MVA) pathway, paced by its rate-limiting enzyme, hydroxymethylglutaryl coenzyme A reductase (HMGCR), is required for the generation of several fundamental end-products including cholesterol and isoprenoids. Despite years of extensive research from the perspective of cardiovascular disease, the contribution of a dysregulated MVA pathway to human cancer remains largely unexplored. We address this issue directly by showing that dysregulation of the MVA pathway, achieved by ectopic expression of either full-length HMGCR or its novel splice variant, promotes transformation. Ectopic HMGCR accentuates growth of transformed and nontransformed cells under anchorage-independent conditions or as xenografts in immunocompromised mice and, importantly, cooperates with RAS to drive the transformation of primary mouse embryonic fibroblasts cells. We further explore whether the MVA pathway may play a role in the etiology of human cancers and show that high mRNA levels of HMGCR and additional MVA pathway genes correlate with poor prognosis in a meta-analysis of six microarray datasets of primary breast cancer. Taken together, our results suggest that HMGCR is a candidate metabolic oncogene and provide a molecular rationale for further exploring the statin family of HMGCR inhibitors as anticancer agents. PMID:20696928

  8. Targeting the Apoptosis Pathway in Hematologic Malignancies

    PubMed Central

    Zaman, Shadia; Wang, Rui; Gandhi, Varsha

    2014-01-01

    Apoptosis is a cell death program that is well-orchestrated for normal tissue homeostasis and for removal of damaged, old, or infected cells. It is regulated by intrinsic and extrinsic pathways. The intrinsic pathway responds to signals such as ultraviolet radiation or DNA damage and activates “executioner” caspases through a mitochondria-dependent pathway. The extrinsic pathway is activated by death signals induced, for example, by an infection that activates the immune system or receptor-mediated pathways. The extrinsic pathway signals also cascade down to executioner caspases that cleave target proteins and lead to cell death. Strict control of cellular apoptosis is important for the hematopoietic system as it has a high turnover rate. However, the apoptosis program is often deregulated in hematologic malignancies leading to the accumulation of malignant cells. Therefore, apoptosis pathways have been identified for development of anticancer therapeutics. We review here the proteins that have been targeted for anticancer drug development in hematologic malignancies. These include BCL-2 family proteins, death ligands and receptors, inhibitor of apoptosis family proteins, and caspases. Except for caspase activators, drugs that target each of these classes of proteins have advanced into clinical trials. PMID:24295132

  9. Brain evolution by brain pathway duplication

    PubMed Central

    Chakraborty, Mukta; Jarvis, Erich D.

    2015-01-01

    Understanding the mechanisms of evolution of brain pathways for complex behaviours is still in its infancy. Making further advances requires a deeper understanding of brain homologies, novelties and analogies. It also requires an understanding of how adaptive genetic modifications lead to restructuring of the brain. Recent advances in genomic and molecular biology techniques applied to brain research have provided exciting insights into how complex behaviours are shaped by selection of novel brain pathways and functions of the nervous system. Here, we review and further develop some insights to a new hypothesis on one mechanism that may contribute to nervous system evolution, in particular by brain pathway duplication. Like gene duplication, we propose that whole brain pathways can duplicate and the duplicated pathway diverge to take on new functions. We suggest that one mechanism of brain pathway duplication could be through gene duplication, although other mechanisms are possible. We focus on brain pathways for vocal learning and spoken language in song-learning birds and humans as example systems. This view presents a new framework for future research in our understanding of brain evolution and novel behavioural traits. PMID:26554045

  10. The Wnt pathway: emerging anticancer strategies.

    PubMed

    Gupta, Aman; Verma, Anukriti; Mishra, Ashutosh K; Wadhwa, Gulshan; Sharma, Sanjeev K; Jain, Chakresh K

    2013-05-01

    The canonical Wnt cascade has emerged as a critical regulator of cancer cells. Activation of the Wnt signaling pathway has also been associated with stem cell, thus raising the possibility of its role in embryogenesis and in the proliferation of malignant cancer cells. Wnt pathway has been reported to be involved in normal physiological processes in adult animals and integrally associated with cancer cell growth and maintenance, thus has been harnessed to devise strategies for anticancer therapy. The presence or absence of some members in this pathway, such as β-catenin, Axin or APC, has been found to involve in different types of tumors in human beings. Dysregulation of the canonical Wnt/β-catenin signaling pathway, mostly by inactivating mutations of the APC tumor suppressor, or oncogenic mutations of β-catenin, has been implicated in colorectal tumorigenesis. Further, elevated levels of β-catenin protein, a hallmark of activated canonical Wnt pathway, have been significantly observed in common forms of human malignancies, indicating that activation of the Wnt pathway may play an important role in tumor development and hence could be a crucial consideration for drug development. The paper discusses the potential therapeutic and diagnostic strategies directing on Wnt pathways on the basis of recent patents and their analysis. PMID:23432158

  11. Brain evolution by brain pathway duplication.

    PubMed

    Chakraborty, Mukta; Jarvis, Erich D

    2015-12-19

    Understanding the mechanisms of evolution of brain pathways for complex behaviours is still in its infancy. Making further advances requires a deeper understanding of brain homologies, novelties and analogies. It also requires an understanding of how adaptive genetic modifications lead to restructuring of the brain. Recent advances in genomic and molecular biology techniques applied to brain research have provided exciting insights into how complex behaviours are shaped by selection of novel brain pathways and functions of the nervous system. Here, we review and further develop some insights to a new hypothesis on one mechanism that may contribute to nervous system evolution, in particular by brain pathway duplication. Like gene duplication, we propose that whole brain pathways can duplicate and the duplicated pathway diverge to take on new functions. We suggest that one mechanism of brain pathway duplication could be through gene duplication, although other mechanisms are possible. We focus on brain pathways for vocal learning and spoken language in song-learning birds and humans as example systems. This view presents a new framework for future research in our understanding of brain evolution and novel behavioural traits. PMID:26554045

  12. Driving and dementia: a clinical decision pathway

    PubMed Central

    Carter, Kirsty; Monaghan, Sophie; O'Brien, John; Teodorczuk, Andrew; Mosimann, Urs; Taylor, John-Paul

    2015-01-01

    Objective This study aimed to develop a pathway to bring together current UK legislation, good clinical practice and appropriate management strategies that could be applied across a range of healthcare settings. Methods The pathway was constructed by a multidisciplinary clinical team based in a busy Memory Assessment Service. A process of successive iteration was used to develop the pathway, with input and refinement provided via survey and small group meetings with individuals from a wide range of regional clinical networks and diverse clinical backgrounds as well as discussion with mobility centres and Forum of Mobility Centres, UK. Results We present a succinct clinical pathway for patients with dementia, which provides a decision-making framework for how health professionals across a range of disciplines deal with patients with dementia who drive. Conclusions By integrating the latest guidance from diverse roles within older people's health services and key experts in the field, the resulting pathway reflects up-to-date policy and encompasses differing perspectives and good practice. It is potentially a generalisable pathway that can be easily adaptable for use internationally, by replacing UK legislation for local regulations. A limitation of this pathway is that it does not address the concern of mild cognitive impairment and how this condition relates to driving safety. © 2014 The Authors. International Journal of Geriatric Psychiatry published by John Wiley & Sons, Ltd. PMID:24865643

  13. Methylerythritol 4-phosphate (MEP) pathway metabolic regulation.

    PubMed

    Banerjee, A; Sharkey, T D

    2014-08-01

    Covering: up to February 2014. The methylerythritol 4-phosphate (MEP) pathway is the recently discovered source of isoprenoid precursors isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP) in most bacteria, some eukaryotic parasites, and the plastids of plant cells. The precursors lead to the formation of various isoprenoids having diverse roles in different biological processes. Some isoprenoids have important commercial uses. Isoprene, which is made in surprising abundance by some trees, plays a significant role in atmospheric chemistry. The genetic regulation of this pathway has been discussed but information about metabolic regulation is just now becoming available. This review covers metabolic regulation of the MEP pathway starting from the inputs of carbon, ATP, and reducing power. A number of different regulatory mechanisms involving intermediate metabolites and/or enzymes are discussed. Some recent data indicate that methylerythritol cyclodiphosphate (MEcDP), the fifth intermediate of this pathway, is a key metabolite. It has been found to play diverse roles in regulation within the pathway as well as coordinating other biological processes by acting as a stress regulator in bacteria and possibly a retrograde signal from plastids to the nucleus in plants. In this review we focus on the role of the MEP pathway in photosynthetic leaves during isoprene emission and more generally the metabolic regulation of the MEP pathway in both plants and bacteria. PMID:24921065

  14. Predicting microbial nitrogen pathways from basic principles.

    PubMed

    van de Leemput, Ingrid A; Veraart, Annelies J; Dakos, Vasilis; de Klein, Jeroen J M; Strous, Marc; Scheffer, Marten

    2011-06-01

    Nitrogen compounds are transformed by a complicated network of competing geochemical processes or microbial pathways, each performed by a different ecological guild of microorganisms. Complete experimental unravelling of this network requires a prohibitive experimental effort. Here we present a simple model that predicts relative rates of hypothetical nitrogen pathways, based only on the stoichiometry and energy yield of the performed redox reaction, assuming competition for resources between alternative pathways. Simulating competing pathways in hypothetical freshwater and marine sediment situations, we surprisingly found that much of the variation observed in nature can simply be predicted from these basic principles. Investigating discrepancies between observations and predictions led to two important biochemical factors that may create barriers for the viability of pathways: enzymatic costs for long pathways and high ammonium activation energy. We hypothesize that some discrepancies can be explained by non-equilibrium dynamics. The model predicted a pathway that has not been discovered in nature yet: the dismutation of nitrite to the level of nitrate and dinitrogen gas. PMID:21429064

  15. Metabolic pathways of ochratoxin A.

    PubMed

    Wu, Qinghua; Dohnal, Vlastimil; Huang, Lingli; Kuča, Kamil; Wang, Xu; Chen, Guyue; Yuan, Zonghui

    2011-01-01

    Ochratoxin A (OTA) as a carcinogenic of group 2B to humans is produced by various fungi strains as Aspergillus and Penicillium. It is one of the most common contaminant in foodstuff. OTA is nephrotoxic, hepatotoxic, teratogenic, and immunotoxic and is assumed to cause Balkan Endemic Nephropathy (BEN), a chronic kidney disease in humans when it is digested in combination with mycotoxin citrinin. The metabolism affects greatly the fates and the toxicity of a mycotoxins in humans, animals, and plants. The understanding of the metabolism of mycotoxins by the organism as fungi, yeast, bacteria and enzymes would be very helpful for the control of the contamination by the mycotoxins in foods and feeds, and understanding of the biotransformation of the mycotoxin in the body of humans, animals, plants, microorganisms would be beneficial to the risk assessment of food safety. In animals and humans, OTA can be metabolized in the kidney, liver and intestines. Hydrolysis, hydroxylation, lactone-opening and conjugation are the major metabolic pathways. OTalpha (OTα) formed by the cleavage of the peptidic bond in OTA is a major metabolite not only in animals and humans, but also in microorganisms and enzyme systems. It is considered as a nontoxic product. However, the lactone-opened product (OP-OTA), found in rodents, is higher toxic than its parent, OTA.. (4R)-4-OH-OTA is the major hydroxy product in rodents, whereas the 4S isomer is the major in pigs. 10-OH-OTA is currently found only in rabbits. Furthermore, OTA can lose the chlorine on C-5 to produce ochratoxin B (OTB), and OTB is further to 4-OH-OTB and ochratoxin β (OTβ). Ochratoxin quinine/hydroquinone (OTQ/OTHQ) is the metabolite of OTA in animals. In addition, the conjugates of OTA such as hexose and pentose conjugates can be found in animals. Such more polar metabolites make OTA to eliminate faster. Currently, a debate exits on the formation of OTA-DNA adducts. Plants can metabolize OTA as well. OH-OTA methyl ester

  16. Syngas Upgrading to Hydrocarbon Fuels Technology Pathway

    SciTech Connect

    Talmadge, M.; Biddy, M.; Dutta, A.; Jones, S.; Meyer, A.

    2013-03-01

    This technology pathway case investigates the upgrading of woody biomass derived synthesis gas (syngas) to hydrocarbon biofuels. While this specific discussion focuses on the conversion of syngas via a methanol intermediate to hydrocarbon blendstocks, there are a number of alternative conversion routes for production of hydrocarbons through a wide array of intermediates from syngas. Future work will also consider the variations to this pathway to determine the most economically viable and lowest risk conversion route. Technical barriers and key research needs have been identified that should be pursued for the syngas-to-hydrocarbon pathway to be competitive with petroleum-derived gasoline-, diesel- and jet-range hydrocarbon blendstocks.

  17. Evolution of the JAK-STAT pathway.

    PubMed

    Liongue, Clifford; Ward, Alister C

    2013-01-01

    The JAK-STAT pathway represents a finely tuned orchestra capable of rapidly facilitating an exquisite symphony of responses from a complex array of extracellular signals. This review explores the evolution of the JAK-STAT pathway: the origins of the individual domains from which it is constructed, the formation of individual components from these basic building blocks, the assembly of the components into a functional pathway, and the subsequent reiteration of this basic template to fulfill a variety of roles downstream of cytokine receptors. PMID:24058787

  18. Electron Transfer Pathways in Cholesterol Synthesis.

    PubMed

    Porter, Todd D

    2015-10-01

    Cholesterol synthesis in the endoplasmic reticulum requires electron input at multiple steps and utilizes both NADH and NADPH as the electron source. Four enzymes catalyzing five steps in the pathway require electron input: squalene monooxygenase, lanosterol demethylase, sterol 4α-methyl oxidase, and sterol C5-desaturase. The electron-donor proteins for these enzymes include cytochrome P450 reductase and the cytochrome b5 pathway. Here I review the evidence for electron donor protein requirements with these enzymes, the evidence for additional electron donor pathways, and the effect of deletion of these redox enzymes on cholesterol and lipid metabolism. PMID:26344922

  19. [Hedgehog signaling pathway and human disorders].

    PubMed

    Fujii, Katsunori; Miyashita, Toshiyuki

    2009-07-01

    The hedgehog signaling pathway plays pivotal roles in embryonic development and cancer formation. This pathway in mammals consists of multiple molecules such as Sonic Hedgehog, PTCH, SMO, and GLI. Mutations of these components result in various human malformations or tumors, i.e., holoprosencephaly, Gorlin syndrome, Greig encephalopolysyndactyly, Pallister-Hall syndrome, Rubinstein-Taybi syndrome, basal cell carcinomas, and medulloblastomas. Recently, small molecules that inhibit this signaling pathway were developed, and clinically applied to cancer therapy. Thus, understanding of these molecular relationships may facilitate the development of new therapies and treatments for diseases caused by hedgehog signaling disorders. PMID:19618878

  20. Evolution of the JAK-STAT pathway

    PubMed Central

    Liongue, Clifford; Ward, Alister C.

    2013-01-01

    The JAK-STAT pathway represents a finely tuned orchestra capable of rapidly facilitating an exquisite symphony of responses from a complex array of extracellular signals. This review explores the evolution of the JAK-STAT pathway: the origins of the individual domains from which it is constructed, the formation of individual components from these basic building blocks, the assembly of the components into a functional pathway, and the subsequent reiteration of this basic template to fulfill a variety of roles downstream of cytokine receptors. PMID:24058787

  1. Identification of disturbed pathways in heart failure based on Gibbs sampling and pathway enrichment analysis.

    PubMed

    Chen, P; Guo, L H; Guo, Y K; Qu, Z J; Gao, Y; Qiu, H

    2016-01-01

    We identified disturbed pathways in heart failure (HF) based on Gibbs sampling combined with pathway enrichment analysis. A total of 396 Markov chains (MCs) (gene count >5) were obtained. After Gibbs sampling, six differentially expressed molecular functions (DEMFs) (possibility ≥0.8) were obtained. As statistical analysis was performed on the number of individual differentially expressed genes (DEGs), we found that there were 137 DEGs with frequency of occurrence ≥2 in the DEMFs. Pathway enrichment analysis showed that these 137 DEGs were enriched in eight significant pathways under the condition of P < 0.001. The five most significant pathways were: the calcium signaling pathway (P = 9.08E-19), arrhythmogenic right ventricular cardiomyopathy (P = 5.66E-13), cardiac muscle contraction (P = 8.04E-13), hypertrophic cardiomyopathy (P = 2.55E-12), and dilated cardiomyopathy (P = 7.30E-12). In conclusion, this novel method for identifying significant pathways in HF based on Gibbs sampling combined with pathway enrichment analysis was suitable. We predict that several altered pathways (such as the calcium signaling pathway and dilated cardiomyopathy) may play important roles in HF and are potentially novel predictive and prognostic markers for HF. PMID:27173293

  2. Genetic dissection of cardiac growth control pathways

    NASA Technical Reports Server (NTRS)

    MacLellan, W. R.; Schneider, M. D.

    2000-01-01

    Cardiac muscle cells exhibit two related but distinct modes of growth that are highly regulated during development and disease. Cardiac myocytes rapidly proliferate during fetal life but exit the cell cycle irreversibly soon after birth, following which the predominant form of growth shifts from hyperplastic to hypertrophic. Much research has focused on identifying the candidate mitogens, hypertrophic agonists, and signaling pathways that mediate these processes in isolated cells. What drives the proliferative growth of embryonic myocardium in vivo and the mechanisms by which adult cardiac myocytes hypertrophy in vivo are less clear. Efforts to answer these questions have benefited from rapid progress made in techniques to manipulate the murine genome. Complementary technologies for gain- and loss-of-function now permit a mutational analysis of these growth control pathways in vivo in the intact heart. These studies have confirmed the importance of suspected pathways, have implicated unexpected pathways as well, and have led to new paradigms for the control of cardiac growth.

  3. Signalling pathways: jack of all cascades.

    PubMed

    Cahill, M A; Janknecht, R; Nordheim, A

    1996-01-01

    The transcription factors that bind the c-fos promoter element SRE are targeted by multiple, independent signalling cascades; the identities of these signalling pathways and their modes of activation are being elucidated. PMID:8805215

  4. Adverse Outcome Pathways: From Definition to Application

    EPA Science Inventory

    A challenge for both human health and ecological toxicologists is the transparent application of mechanistic (e.g., molecular, biochemical, histological) data to risk assessments. The adverse outcome pathway (AOP) is a conceptual framework designed to meet this need. Specifical...

  5. Computing folding pathways between RNA secondary structures.

    PubMed

    Dotu, Ivan; Lorenz, William A; Van Hentenryck, Pascal; Clote, Peter

    2010-03-01

    Given an RNA sequence and two designated secondary structures A, B, we describe a new algorithm that computes a nearly optimal folding pathway from A to B. The algorithm, RNAtabupath, employs a tabu semi-greedy heuristic, known to be an effective search strategy in combinatorial optimization. Folding pathways, sometimes called routes or trajectories, are computed by RNAtabupath in a fraction of the time required by the barriers program of Vienna RNA Package. We benchmark RNAtabupath with other algorithms to compute low energy folding pathways between experimentally known structures of several conformational switches. The RNApathfinder web server, source code for algorithms to compute and analyze pathways and supplementary data are available at http://bioinformatics.bc.edu/clotelab/RNApathfinder. PMID:20044352

  6. The Pentose Phosphate Pathway in Parasitic Trypanosomatids.

    PubMed

    Kovářová, Julie; Barrett, Michael P

    2016-08-01

    Parasitic trypanosomatids cause important diseases. Dissecting the biochemistry of these organisms offers a means of discovering targets against which inhibitors may be designed and developed as drugs. The pentose phosphate pathway is a key route of glucose metabolism in most organisms, providing NADPH for use as a cellular reductant and various carbohydrate intermediates used in cellular metabolism. The pathway and its enzymes have been studied in Trypanosoma brucei, Trypanosoma cruzi, and various Leishmania species. Its functions in these parasites are becoming clear. Some enzymes of the pathway are essential to the parasites and have structural features distinguishing them from their mammalian counterparts, and this has stimulated several programs of inhibitor discovery with a view to targeting the pathway with new drugs. PMID:27174163

  7. Function and Regulation in MAPK Signaling Pathways

    PubMed Central

    Chen, Raymond E.; Thorner, Jeremy

    2007-01-01

    Signaling pathways that activate different mitogen-activated protein kinases (MAPKs) elicit many of the responses that are evoked in cells by changes in certain environmental conditions and upon exposure to a variety of hormonal and other stimuli. These pathways were first elucidated in the unicellular eukaryote Saccharomyces cerevisiae (budding yeast). Studies of MAPK pathways in this organism continue to be especially informative in revealing the molecular mechanisms by which MAPK cascades operate, propagate signals, modulate cellular processes, and are controlled by regulatory factors both internal to and external to the pathways. Here we highlight recent advances and new insights about MAPK-based signaling that have been made through studies in yeast, which provide lessons directly applicable to, and that enhance our understanding of, MAPK-mediated signaling in mammalian cells. PMID:17604854

  8. Cholangiocarcinoma: Molecular Pathways and Therapeutic Opportunities

    PubMed Central

    Rizvi, Sumera; Borad, Mitesh J.; Patel, Tushar; Gores, Gregory J.

    2015-01-01

    Cholangiocarcinoma (CCA) is an aggressive biliary tract malignancy with limited treatment options and low survival rates. Currently, there are no curative medical therapies for CCA. Recent advances have enhanced our understanding of the genetic basis of this disease, and elucidated therapeutically relevant targets. Therapeutic efforts in development are directed at several key pathways due to genetic aberrations including receptor tyrosine kinase pathways, mutant IDH enzymes, the PI3K-AKT-mTOR pathway, and chromatin remodeling networks. A highly desmoplastic, hypovascular stroma is characteristic of CCAs and recent work has highlighted the importance of targeting this pathway via stromal myofibroblast depletion. Future efforts should concentrate on combination therapies with action against the cancer cell and the surrounding tumor stroma. As the mutational landscape of CCA is being illuminated, molecular profiling of patient tumors will enable identification of specific mutations and the opportunity to offer directed, personalized treatment options. PMID:25369307

  9. Virus Budding