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

NARCIS (Netherlands)

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

2010-01-01

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

Solo/Trio8, a membrane-associated short isoform of Trio, modulates endosome dynamics and neurite elongation.

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Sun, Ying-Jie; Nishikawa, Kaori; Yuda, Hideki; Wang, Yu-Lai; Osaka, Hitoshi; Fukazawa, Nobuna; Naito, Akira; Kudo, Yoshihisa; Wada, Keiji; Aoki, Shunsuke

2006-09-01

With DNA microarrays, we identified a gene, termed Solo, that is downregulated in the cerebellum of Purkinje cell degeneration mutant mice. Solo is a mouse homologue of rat Trio8-one of multiple Trio isoforms recently identified in rat brain. Solo/Trio8 contains N-terminal sec14-like and spectrin-like repeat domains followed by a single guanine nucleotide exchange factor 1 (GEF1) domain, but it lacks the C-terminal GEF2, immunoglobulin-like, and kinase domains that are typical of Trio. Solo/Trio8 is predominantly expressed in Purkinje neurons of the mouse brain, and expression begins following birth and increases during Purkinje neuron maturation. We identified a novel C-terminal membrane-anchoring domain in Solo/Trio8 that is required for enhanced green fluorescent protein-Solo/Trio8 localization to early endosomes (positive for both early-endosome antigen 1 [EEA1] and Rab5) in COS-7 cells and primary cultured neurons. Solo/Trio8 overexpression in COS-7 cells augmented the EEA1-positive early-endosome pool, and this effect was abolished via mutation and inactivation of the GEF domain or deletion of the C-terminal membrane-anchoring domain. Moreover, primary cultured neurons transfected with Solo/Trio8 showed increased neurite elongation that was dependent on these domains. These results suggest that Solo/Trio8 acts as an early-endosome-specific upstream activator of Rho family GTPases for neurite elongation of developing Purkinje neurons.

A novel chimeric cell-penetrating peptide with membrane-disruptive properties for efficient endosomal escape.

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Salomone, Fabrizio; Cardarelli, Francesco; Di Luca, Mariagrazia; Boccardi, Claudia; Nifosì, Riccardo; Bardi, Giuseppe; Di Bari, Lorenzo; Serresi, Michela; Beltram, Fabio

2012-11-10

Efficient endocytosis into a wide range of target cells and low toxicity make the arginine-rich Tat peptide (Tat(11): YGRKKRRQRRR, residues 47-57 of HIV-1 Tat protein) an excellent transporter for delivery purposes. Unfortunately, molecules taken up by endocytosis undergo endosomal entrapment and possible metabolic degradation. Escape from the endosome is therefore actively researched. In this context, antimicrobial peptides (AMPs) provide viable templates for the design of new membrane-disruptive motifs. In particular the Cecropin-A and Melittin hybrids (CMs) are among the smallest and most effective peptides with membrane-perturbing abilities. Here we present a novel chimeric peptide in which the Tat(11) motif is fused to the CM(18) hybrid (KWKLFKKIGAVLKVLTTG, residues 1-7 of Cecropin-A and 2-12 of Melittin). When administered to cells, CM(18)-Tat(11) combines the two desired functionalities: efficient uptake and destabilization of endocytotic-vesicle membranes. We show that this chimeric peptide effectively increases cargo-molecule cytoplasm availability and allows the subsequent intracellular localization of diverse membrane-impermeable molecules (i.e. Tat(11)-EGFP fusion protein, calcein, dextrans, and plasmidic DNA) with no detectable cytotoxicity. The present results open the way to the rational engineering of "modular" cell-penetrating peptides (CPPs) that combine (i) efficient translocation from the extracellular milieu into vesicles and (ii) efficient release of molecules from vesicles into the cytoplasm. Copyright © 2012 Elsevier B.V. All rights reserved.

Heterotrimeric G protein subunits are located on rat liver endosomes

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Van Dyke Rebecca W

2004-01-01

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

Retromer guides STxB and CD8-M6PR from early to recycling endosomes, EHD1 guides STxB from recycling endosome to Golgi

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McKenzie, Jenna E.; Raisley, Brent; Zhou, Xin; Naslavsky, Naava; Taguchi, Tomohiko; Caplan, Steve; Sheff, David

2012-01-01

Retrograde trafficking transports proteins, lipids and toxins from the plasma membrane to the Golgi and ER. To reach the Golgi, these cargos must transit the endosomal system, consisting of early endosomes, recycling endosomes, late endosomes and lysosomes. All cargos pass through early endosomes, but may take different routes to the Golgi. Retromer dependent cargos bypass the late endosomes to reach the Golgi. We compared how two very different retromer dependent cargos negotiate the endosomal sorting system. Shiga toxin B, bound to the external layer of the plasma membrane, and chimeric CD8-Mannose-6-Phosphate Receptor, which is anchored via a transmembrane domain. Both appear to pass through the recycling endosome. Ablation of the recycling endosome diverted both of these cargos to an aberrant compartment and prevented them from reaching the Golgi. Once in the recycling endosome, Shiga toxin required EHD1 to traffic to the TGN, while the CD8-Mannose-6-Phosphate Receptor was not significantly dependent on EHD1. Knockdown of retromer components left cargo in the early endosomes, suggesting that it is required for retrograde exit from this compartment. This work establishes the recycling endosome as a required step in retrograde traffic of at least these two retromer dependent cargos. Along this pathway, retromer is associated with EE to recycling endosome traffic, while EHD1 is associated with recycling endosome to TGN traffic of STxB. PMID:22540229

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

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

2016-03-18

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

Limited and selective transfer of plasma membrane glycoproteins to membrane of secondary lysosomes

International Nuclear Information System (INIS)

Haylett, T.; Thilo, L.

1986-01-01

Radioactive galactose, covalently bound to cell surface glycoconjugates on mouse macrophage cells, P388D 1 , was used as a membrane marker to study the composition, and the kinetics of exchange, of plasma membrane-derived constituents in the membrane of secondary lysosomes. Secondary lysosomes were separated from endosomes and plasma membrane by self-forming Percoll density gradients. Horseradish peroxidase, taken up by fluid-phase pinocytosis, served as a vesicle contents marker to monitor transfer of endosomal contents into secondary lysosomes. Concurrently, the fraction of plasma membrane-derived label of secondary lysosomes increased by first order kinetics from 4 PAGE, labeled molecules of M/sub r/ 160-190 kD were depleted and of the M/sub r/ 100-120 kD were enriched in lysosome membrane compared with the relative composition of label on the cell surface. No corresponding selectivity was observed for the degradation of label, with all M/sub r/ classes being affected to the same relative extent. The results indicate that endocytosis-derived transfer of plasma membrane constitutents to secondary lysosomes is a limited and selective process, and that only ∼1% of internalized membrane is recycled via a membrane pool of secondary lysosomes

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

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Jerry eCurran

2015-02-01

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

Unconventional secretion of tissue transglutaminase involves phospholipid-dependent delivery into recycling endosomes.

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Evgeny A Zemskov

2011-04-01

Full Text Available Although endosomal compartments have been suggested to play a role in unconventional protein secretion, there is scarce experimental evidence for such involvement. Here we report that recycling endosomes are essential for externalization of cytoplasmic secretory protein tissue transglutaminase (tTG. The de novo synthesized cytoplasmic tTG does not follow the classical ER/Golgi-dependent secretion pathway, but is targeted to perinuclear recycling endosomes, and is delivered inside these vesicles prior to externalization. On its route to the cell surface tTG interacts with internalized β1 integrins inside the recycling endosomes and is secreted as a complex with recycled β1 integrins. Inactivation of recycling endosomes, blocking endosome fusion with the plasma membrane, or downregulation of Rab11 GTPase that controls outbound trafficking of perinuclear recycling endosomes, all abrogate tTG secretion. The initial recruitment of cytoplasmic tTG to recycling endosomes and subsequent externalization depend on its binding to phosphoinositides on endosomal membranes. These findings begin to unravel the unconventional mechanism of tTG secretion which utilizes the long loop of endosomal recycling pathway and indicate involvement of endosomal trafficking in non-classical protein secretion.

Localization of the AP-3 adaptor complex defines a novel endosomal exit site for lysosomal membrane proteins

NARCIS (Netherlands)

Peden, A.A.; Oorschot, V.; Hesser, B.A.; Austin, C.D.; Scheller, R.H.; Klumperman, J.

2004-01-01

The adaptor protein (AP) 3 adaptor complex has been implicated in the transport of lysosomal membrane proteins, but its precise site of action has remained controversial. Here, we show by immuno-electron microscopy that AP-3 is associated with budding profiles evolving from a tubular endosomal

PIKfyve mediates the motility of late endosomes and lysosomes in neuronal dendrites.

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Tsuruta, Fuminori; Dolmetsch, Ricardo E

2015-09-25

The endosome/lysosome system in the nervous system is critically important for a variety of neuronal functions such as neurite outgrowth, retrograde transport, and synaptic plasticity. In neurons, the endosome/lysosome system is crucial for the activity-dependent internalization of membrane proteins and contributes to the regulation of lipid level on the plasma membrane. Although homeostasis of membrane dynamics plays important roles in the properties of central nervous systems, it has not been elucidated how endosome/lysosome system is regulated. Here, we report that phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) mediates the motility of late endosomes and lysosomes in neuronal dendrites. Endosomes and lysosomes are highly motile in resting neurons, however knockdown of PIKfyve led to a significant reduction in late endosomes and lysosomes motility. We also found that vesicle acidification is crucial for their motility and PIKfyve is associated with this process indirectly. These data suggest that PIKfyve mediates vesicle motility through the regulation of vesicle integrity in neurons. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Chimeric forms of furin and TGN38 are transported with the plasma membrane in the trans-Golgi network via distinct endosomal pathways.

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Mallet, W G; Maxfield, F R

1999-07-26

Furin and TGN38 are menbrane proteins that cycle between the plasma membrane and the trans-Golgi network (TGN), each maintaining a predominant distribution in the TGN. We have used chimeric proteins with an extracellular Tac domain and the cytoplasmic domain of TGN38 or furin to study the trafficking of these proteins in endosomes. Previously, we demonstrated that the postendocytic trafficking of Tac-TGN38 to the TGN is via the endocytic recycling pathway (Ghosh, R.N.,W.G. Mallet,T.T. Soe,T.E.McGraw, and F.R. Maxfield.1998.J. Cell Biol.142:923-936). Here we show that internalized Tac-furin is delivered to the TGN through late endosomes, bypassing the endocytic recycling compartment. The transport of Tac-furin from late endosomes to the TGN appears to proceed via an efficient, single-pass mechanism. Delivery of Tac-furin but not Tac-TGN38 to the TGN is blocked by nocodazole, and the two pathways are also differentially affected by wortmannin. These studies demonstrate the existence of two independentpathways for endosomal transport of proteins to the TGN from the plasma membrane.

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

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Alberto Canfrán-Duque

2016-03-01

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

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

Science.gov (United States)

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

2016-01-01

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

ER network homeostasis is critical for plant endosome streaming and endocytosis

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

2015-01-01

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

Conformational biosensors reveal GPCR signalling from endosomes

DEFF Research Database (Denmark)

Irannejad, R; Tomshine, Jin C; Tomshine, Jon R

2013-01-01

A long-held tenet of molecular pharmacology is that canonical signal transduction mediated by G-protein-coupled receptor (GPCR) coupling to heterotrimeric G proteins is confined to the plasma membrane. Evidence supporting this traditional view is based on analytical methods that provide limited...... or no subcellular resolution. It has been subsequently proposed that signalling by internalized GPCRs is restricted to G-protein-independent mechanisms such as scaffolding by arrestins, or GPCR activation elicits a discrete form of persistent G protein signalling, or that internalized GPCRs can indeed contribute...... in the early endosome membrane, and that internalized receptors contribute to the overall cellular cyclic AMP response within several minutes after agonist application. These findings provide direct support for the hypothesis that canonical GPCR signalling occurs from endosomes as well as the plasma membrane...

Augmented internalisation of ferroportin to late endosomes impairs iron uptake by enterocyte-like IEC-6 cells.

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Oates, Phillip S; Thomas, Carla

2005-08-01

Absorption of iron occurs by duodenal enterocytes, involving uptake by the divalent metal transporter-1 (DMT1) and release by ferroportin. Ferroportin responds to the hepatocyte-produced 25-amino-acid-peptide hepcidin-25 by undergoing internalisation to late endosomes that impair iron release. Ferroportin is also expressed on the apical membrane of polarised Caco-2 cells, rat intestinal cells and in IEC-6 cells (an intestinal epithelial cell line). A blocking antibody to ferroportin also impairs the uptake, but not the release, of iron. In this study IEC-6 cells were used to study the mechanism of impairment or recovery from impairment produced by the blocking antibody and the fate of DMT1 and ferroportin. Uptake of 1 muM Fe(II) was studied by adding the antibody from time 0 and after adding or removing the antibody once a steady state had been reached. Surface binding, maximum iron transport rate V(max) and transporter affinity (K(m)) were measured after impairment of iron uptake. Ferroportin and DMT1 distribution were assessed by immunofluorescence microscopy. Antibody-mediated impairment, or recovery from impairment, of Fe(II) uptake occurred within minutes. Impairment was lost when the antibody was combined with the immunizing peptide. DMT1 and ferroportin undergo internalisation to late endosomes and, in the presence of the antibody, augmented internalisation of DMT1 and ferroportin caused swelling of late endosomes. Surface binding of Fe(II) and iron transport V(max) were reduced by 50%, indicating that the antibody removed membrane-bound DMT1. The ferroportin antibody induced rapid turnover of membrane ferroportin and DMT1 and its internalisation to late endosomes, resulting in impaired Fe(II) uptake.

Positioning of AMPA Receptor-Containing Endosomes Regulates Synapse Architecture

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Marta Esteves da Silva

2015-11-01

Full Text Available Lateral diffusion in the membrane and endosomal trafficking both contribute to the addition and removal of AMPA receptors (AMPARs at postsynaptic sites. However, the spatial coordination between these mechanisms has remained unclear, because little is known about the dynamics of AMPAR-containing endosomes. In addition, how the positioning of AMPAR-containing endosomes affects synapse organization and functioning has never been directly explored. Here, we used live-cell imaging in hippocampal neuron cultures to show that intracellular AMPARs are transported in Rab11-positive recycling endosomes, which frequently enter dendritic spines and depend on the microtubule and actin cytoskeleton. By using chemically induced dimerization systems to recruit kinesin (KIF1C or myosin (MyosinV/VI motors to Rab11-positive recycling endosomes, we controlled their trafficking and found that induced removal of recycling endosomes from spines decreases surface AMPAR expression and PSD-95 clusters at synapses. Our data suggest a mechanistic link between endosome positioning and postsynaptic structure and composition.

Concerted regulation of npc2 binding to endosomal/lysosomal membranes by bis(monoacylglycero)phosphate and sphingomyelin

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Enkavi, Giray; Mikkolainen, Heikki; Güngör, Burçin

2017-01-01

remained elusive. Here, based on an extensive set of atomistic simulations and free energy calculations, we clarify the mechanism and energetics of npc2-membrane binding and characterize the roles of physiologically relevant key lipids associated with the binding process. Our results capture in atomistic......Niemann-Pick Protein C2 (npc2) is a small soluble protein critical for cholesterol transport within and from the lysosome and the late endosome. Intriguingly, npc2-mediated cholesterol transport has been shown to be modulated by lipids, yet the molecular mechanism of npc2-membrane interactions has......). This mode is associated with cholesterol uptake and release. On the other hand, the second mode (Supine) places the cholesterol binding pocket away from the membrane surface, but has overall higher membrane binding affinity. We determined that bis(monoacylglycero)phosphate (bmp) is specifically required...

Rab9-dependent retrograde transport and endosomal sorting of the endopeptidase furin

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Chia, Pei Zhi Cheryl; Gasnereau, Isabelle; Lieu, Zi Zhao; Gleeson, Paul A.

2011-01-01

The endopeptidase furin and the trans-Golgi network protein TGN38 are membrane proteins that recycle between the TGN and plasma membrane. TGN38 is transported by a retromer-dependent pathway from early endosomes to the TGN, whereas the intracellular transport of furin is poorly defined. Here we have identified the itinerary and transport requirements of furin. Using internalisation assays, we show that furin transits the early and late endosomes en route to the TGN. The GTPase Rab9 and the TGN golgin GCC185, components of the late endosome-to-TGN pathway, were required for efficient TGN retrieval of furin. By contrast, TGN38 trafficking was independent of Rab9 and GCC185. To identify the sorting signals for the early endosome-to-TGN pathway, the trafficking of furin–TGN38 chimeras was investigated. The diversion of furin from the Rab9-dependent late-endosome-to-TGN pathway to the retromer-dependent early-endosome-to-TGN pathway required both the transmembrane domain and cytoplasmic tail of TGN38. We present evidence to suggest that the length of the transmembrane domain is a contributing factor in endosomal sorting. Overall, these data show that furin uses the Rab9-dependent pathway from late endosomes and that retrograde transport directly from early endosomes is dependent on both the transmembrane domain and the cytoplasmic tail. PMID:21693586

Membrane fusion by VAMP3 and plasma membrane t-SNAREs

International Nuclear Information System (INIS)

Hu Chuan; Hardee, Deborah; Minnear, Fred

2007-01-01

Pairing of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and SNARE proteins on target membranes (t-SNAREs) mediates intracellular membrane fusion. VAMP3/cellubrevin is a v-SNARE that resides in recycling endosomes and endosome-derived transport vesicles. VAMP3 has been implicated in recycling of transferrin receptors, secretion of α-granules in platelets, and membrane trafficking during cell migration. Using a cell fusion assay, we examined membrane fusion capacity of the ternary complexes formed by VAMP3 and plasma membrane t-SNAREs syntaxin1, syntaxin4, SNAP-23 and SNAP-25. VAMP3 forms fusogenic pairing with t-SNARE complexes syntaxin1/SNAP-25, syntaxin1/SNAP-23 and syntaxin4/SNAP-25, but not with syntaxin4/SNAP-23. Deletion of the N-terminal domain of syntaxin4 enhanced membrane fusion more than two fold, indicating that the N-terminal domain negatively regulates membrane fusion. Differential membrane fusion capacities of the ternary v-/t-SNARE complexes suggest that transport vesicles containing VAMP3 have distinct membrane fusion kinetics with domains of the plasma membrane that present different t-SNARE proteins

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

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

2017-04-12

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

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

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

2015-05-18

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

On the entry of an emerging arbovirus into host cells: Mayaro virus takes the highway to the cytoplasm through fusion with early endosomes and caveolae-derived vesicles

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Carlos A.M. Carvalho

2017-04-01

Full Text Available Mayaro virus (MAYV is an emergent sylvatic alphavirus in South America, related to sporadic outbreaks of a chikungunya-like human febrile illness accompanied by severe arthralgia. Despite its high potential for urban emergence, MAYV is still an obscure virus with scarce information about its infection cycle, including the corresponding early events. Even for prototypical alphaviruses, the cell entry mechanism still has some rough edges to trim: although clathrin-mediated endocytosis is quoted as the putative route, alternative paths as distinct as direct virus genome injection through the cell plasma membrane seems to be possible. Our aim was to clarify crucial details on the entry route exploited by MAYV to gain access into the host cell. Tracking the virus since its first contact with the surface of Vero cells by fluorescence microscopy, we show that its entry occurs by a fast endocytic process and relies on fusion with acidic endosomal compartments. Moreover, blocking clathrin-mediated endocytosis or depleting cholesterol from the cell membrane leads to a strong inhibition of viral infection, as assessed by plaque assays. Following this clue, we found that early endosomes and caveolae-derived vesicles are both implicated as target membranes for MAYV fusion. Our findings unravel the very first events that culminate in a productive infection by MAYV and shed light on potential targets for a rational antiviral therapy, besides providing a better comprehension of the entry routes exploited by alphaviruses to get into the cell.

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

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Sofie Ignoul

Full Text Available BACKGROUND: The mammalian CLC protein family comprises nine members (ClC-1 to -7 and ClC-Ka, -Kb that function either as plasma membrane chloride channels or as intracellular chloride/proton antiporters, and that sustain a broad spectrum of cellular processes, such as membrane excitability, transepithelial transport, endocytosis and lysosomal degradation. In this study we focus on human ClC-6, which is structurally most related to the late endosomal/lysomal ClC-7. PRINCIPAL FINDINGS: Using a polyclonal affinity-purified antibody directed against a unique epitope in the ClC-6 COOH-terminal tail, we show that human ClC-6, when transfected in COS-1 cells, is N-glycosylated in a region that is evolutionary poorly conserved between mammalian CLC proteins and that is located between the predicted helices K and M. Three asparagine residues (N410, N422 and N432 have been defined by mutagenesis as acceptor sites for N-glycosylation, but only two of the three sites seem to be simultaneously N-glycosylated. In a differentiated human neuroblastoma cell line (SH-SY5Y, endogenous ClC-6 colocalizes with LAMP-1, a late endosomal/lysosomal marker, but not with early/recycling endosomal markers such as EEA-1 and transferrin receptor. In contrast, when transiently expressed in COS-1 or HeLa cells, human ClC-6 mainly overlaps with markers for early/recycling endosomes (transferrin receptor, EEA-1, Rab5, Rab4 and not with late endosomal/lysosomal markers (LAMP-1, Rab7. Analogously, overexpression of human ClC-6 in SH-SY5Y cells also leads to an early/recycling endosomal localization of the exogenously expressed ClC-6 protein. Finally, in transiently transfected COS-1 cells, ClC-6 copurifies with detergent-resistant membrane fractions, suggesting its partitioning in lipid rafts. Mutating a juxtamembrane string of basic amino acids (amino acids 71-75: KKGRR disturbs the association with detergent-resistant membrane fractions and also affects the segregation of ClC-6

Post-Golgi anterograde transport requires GARP-dependent endosome-to-TGN retrograde transport

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Hirata, Tetsuya; Fujita, Morihisa; Nakamura, Shota; Gotoh, Kazuyoshi; Motooka, Daisuke; Murakami, Yoshiko; Maeda, Yusuke; Kinoshita, Taroh

2015-01-01

The importance of endosome-to–trans-Golgi network (TGN) retrograde transport in the anterograde transport of proteins is unclear. In this study, genome-wide screening of the factors necessary for efficient anterograde protein transport in human haploid cells identified subunits of the Golgi-associated retrograde protein (GARP) complex, a tethering factor involved in endosome-to-TGN transport. Knockout (KO) of each of the four GARP subunits, VPS51–VPS54, in HEK293 cells caused severely defective anterograde transport of both glycosylphosphatidylinositol (GPI)-anchored and transmembrane proteins from the TGN. Overexpression of VAMP4, v-SNARE, in VPS54-KO cells partially restored not only endosome-to-TGN retrograde transport, but also anterograde transport of both GPI-anchored and transmembrane proteins. Further screening for genes whose overexpression normalized the VPS54-KO phenotype identified TMEM87A, encoding an uncharacterized Golgi-resident membrane protein. Overexpression of TMEM87A or its close homologue TMEM87B in VPS54-KO cells partially restored endosome-to-TGN retrograde transport and anterograde transport. Therefore GARP- and VAMP4-dependent endosome-to-TGN retrograde transport is required for recycling of molecules critical for efficient post-Golgi anterograde transport of cell-surface integral membrane proteins. In addition, TMEM87A and TMEM87B are involved in endosome-to-TGN retrograde transport. PMID:26157166

Arf6, Rab11 and transferrin receptor define distinct populations of recycling endosomes.

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Kobayashi, Hotaka; Fukuda, Mitsunori

2013-09-01

Recycling endosomes are key platforms for endocytic recycling that return internalized molecules back to the plasma membrane. To determine how recycling endosomes perform their functions, searching for proteins and lipids that specifically localized at recycling endosomes has often been performed by colocalization analyses between candidate molecules and conventional recycling endosome markers. However, it remains unclear whether all the conventional markers have identical localizations. Here we report finding that three well-known recycling endosome markers, i.e., Arf6, Rab11 and transferrin receptor (TfR), have different intracellular localizations in PC12 cells. The results of immunofluorescence analyses showed that the signals of endogenous Arf6, Rab11 and TfR in nerve growth factor-stimulated PC12 cells generally differed, although there was some overlapping. Our findings provide new information about recycling endosome markers, and they highlight the heterogeneity of recycling endosomes.

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

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

2003-05-12

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

Charcot-Marie-Tooth disease-linked protein SIMPLE functions with the ESCRT machinery in endosomal trafficking

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Lee, Samuel M.; Chin, Lih-Shen; Li, Lian

2012-01-01

Mutations in small integral membrane protein of lysosome/late endosome (SIMPLE) cause autosomal dominant, Charcot-Marie-Tooth disease (CMT) type 1C. The cellular function of SIMPLE is unknown and the pathogenic mechanism of SIMPLE mutations remains elusive. Here, we report that SIMPLE interacted and colocalized with endosomal sorting complex required for transport (ESCRT) components STAM1, Hrs, and TSG101 on early endosomes and functioned with the ESCRT machinery in the control of endosome-to...

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

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

2015-01-01

An emerging theme in cellular logistics is the close connection between mRNA and membrane trafficking. A prominent example is the microtubule-dependent transport of mRNAs and associated ribosomes on endosomes. This coordinated process is crucial for correct septin filamentation and efficient growth of polarised cells, such as fungal hyphae. Despite detailed knowledge on the key RNA-binding protein and the molecular motors involved, it is unclear how mRNAs are connected to membranes during transport. Here, we identify a novel factor containing a FYVE zinc finger domain for interaction with endosomal lipids and a new PAM2-like domain required for interaction with the MLLE domain of the key RNA-binding protein. Consistently, loss of this FYVE domain protein leads to specific defects in mRNA, ribosome, and septin transport without affecting general functions of endosomes or their movement. Hence, this is the first endosomal component specific for mRNP trafficking uncovering a new mechanism to couple mRNPs to endosomes. DOI: http://dx.doi.org/10.7554/eLife.06041.001 PMID:25985087

Syndapin/SDPN-1 is required for endocytic recycling and endosomal actin association in the Caenorhabditis elegans intestine

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Gleason, Adenrele M.; Nguyen, Ken C. Q.; Hall, David H.; Grant, Barth D.

2016-01-01

Syndapin/pascin-family F-BAR domain proteins bind directly to membrane lipids and are associated with actin dynamics at the plasma membrane. Previous reports also implicated mammalian syndapin 2 in endosome function during receptor recycling, but precise analysis of a putative recycling function for syndapin in mammalian systems is difficult because of its effects on the earlier step of endocytic uptake and potential redundancy among the three separate genes that encode mammalian syndapin isoforms. Here we analyze the endocytic transport function of the only Caenorhabditis elegans syndapin, SDPN-1. We find that SDPN-1 is a resident protein of the early and basolateral recycling endosomes in the C. elegans intestinal epithelium, and sdpn-1 deletion mutants display phenotypes indicating a block in basolateral recycling transport. sdpn-1 mutants accumulate abnormal endosomes positive for early endosome and recycling endosome markers that are normally separate, and such endosomes accumulate high levels of basolateral recycling cargo. Furthermore, we observed strong colocalization of endosomal SDPN-1 with the F-actin biosensor Lifeact and found that loss of SDPN-1 greatly reduced Lifeact accumulation on early endosomes. Taken together, our results provide strong evidence for an in vivo function of syndapin in endocytic recycling and suggest that syndapin promotes transport via endosomal fission. PMID:27630264

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

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

2012-01-27

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

Strategies for the Activation and Release of the Membranolytic Peptide Melittin from Liposomes Using Endosomal pH as a Trigger.

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Oude Blenke, E; Sleszynska, M; Evers, M J W; Storm, G; Martin, N I; Mastrobattista, E

2017-02-15

Endosomolytic peptides are often coupled to drug delivery systems to enhance endosomal escape, which is crucial for the delivery of macromolecular drugs that are vulnerable to degradation in the endolysosomal pathway. Melittin is a 26 amino acid peptide derived from bee venom that has a very high membranolytic activity. However, such lytic peptides also impose a significant safety risk when applied in vivo as they often have similar activity against red blood cells and other nontarget cell membranes. Our aim is to control the membrane-disrupting capacity of these peptides in time and space by physically constraining them to a nanocarrier surface in such a way that they only become activated when delivered inside acidic endosomes. To this end, a variety of chemical approaches for the coupling of lytic peptides to liposomes via functionalized PEG-lipids were explored, including maleimide-thiol chemistry, click-chemistry, and aldehyde-hydrazide chemistry. The latter enables reversible conjugation via a hydrazone bond, allowing for release of the peptide under endosomal conditions. By carefully choosing the conjugation site and by using a pH activated analog of the melittin peptide, lytic activity toward a model membrane is completely inhibited at physiological pH. At endosomal pH the activity is restored by hydrolysis of the acid-labile hydrazone bond, releasing the peptide in its most active, free form. Furthermore, using an analogue containing a nonhydrolyzable bond as a control, it was shown that the activity observed can be completely attributed to release of the peptide, validating dynamic covalent conjugation as a suitable strategy to maintain safety during circulation.

The Cdc42 guanine nucleotide exchange factor FGD6 coordinates cell polarity and endosomal membrane recycling in osteoclasts.

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Steenblock, Charlotte; Heckel, Tobias; Czupalla, Cornelia; Espírito Santo, Ana Isabel; Niehage, Christian; Sztacho, Martin; Hoflack, Bernard

2014-06-27

The initial step of bone digestion is the adhesion of osteoclasts onto bone surfaces and the assembly of podosomal belts that segregate the bone-facing ruffled membrane from other membrane domains. During bone digestion, membrane components of the ruffled border also need to be recycled after macropinocytosis of digested bone materials. How osteoclast polarity and membrane recycling are coordinated remains unknown. Here, we show that the Cdc42-guanine nucleotide exchange factor FGD6 coordinates these events through its Src-dependent interaction with different actin-based protein networks. At the plasma membrane, FGD6 couples cell adhesion and actin dynamics by regulating podosome formation through the assembly of complexes comprising the Cdc42-interactor IQGAP1, the Rho GTPase-activating protein ARHGAP10, and the integrin interactors Talin-1/2 or Filamin A. On endosomes and transcytotic vesicles, FGD6 regulates retromer-dependent membrane recycling through its interaction with the actin nucleation-promoting factor WASH. These results provide a mechanism by which a single Cdc42-exchange factor controlling different actin-based processes coordinates cell adhesion, cell polarity, and membrane recycling during bone degradation. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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

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Takahashi, Yusuke; Nada, Shigeyuki; Mori, Shunsuke; Soma-Nagae, Taeko; Oneyama, Chitose; Okada, Masato

2012-01-01

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

Overexpression of Rab22a hampers the transport between endosomes and the Golgi apparatus

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Mesa, Rosana; Magadan, Javier; Barbieri, Alejandro; Lopez, Cecilia; Stahl, Philip D.; Mayorga, Luis S.

2005-01-01

The transport and sorting of soluble and membrane-associated macromolecules arriving at endosomal compartments require a complex set of Rab proteins. Rab22a has been localized to the endocytic compartment; however, very little is known about the function of Rab22a and inconsistent results have been reported in studies performed in different cell lines. To characterize the function of Rab22a in endocytic transport, the wild-type protein (Rab22a WT), a hydrolysis-deficient mutant (Rab22a Q64L), and a mutant with reduced affinity for GTP (Rab22a S19N) were expressed in CHO cells. None of the three Rab22a constructs affected the transport of rhodamine-dextran to lysosomes, the digestion of internalized proteins, or the lysosomal localization of cathepsin D. In contrast with the mild effect of Rab22a on the endosome-lysosome route, cells expressing Rab22a WT and Rab22a Q64L presented a strong delay in the retrograde transport of cholera toxin from endosomes to the Golgi apparatus. Moreover, these cells accumulated the cation independent mannose 6-phosphate receptor in endosomes. These observations indicate that Rab22a can affect the trafficking from endosomes to the Golgi apparatus probably by promoting fusion among endosomes and impairing the proper segregation of membrane domains required for targeting to the trans-Golgi network (TGN)

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

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

2012-01-01

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

Retrogradely Transported TrkA Endosomes Signal Locally within Dendrites to Maintain Sympathetic Neuron Synapses

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Kathryn M. Lehigh

2017-04-01

Full Text Available Sympathetic neurons require NGF from their target fields for survival, axonal target innervation, dendritic growth and formation, and maintenance of synaptic inputs from preganglionic neurons. Target-derived NGF signals are propagated retrogradely, from distal axons to somata of sympathetic neurons via TrkA signaling endosomes. We report that a subset of TrkA endosomes that are transported from distal axons to cell bodies translocate into dendrites, where they are signaling competent and move bidirectionally, in close proximity to synaptic protein clusters. Using a strategy for spatially confined inhibition of TrkA kinase activity, we found that distal-axon-derived TrkA signaling endosomes are necessary within sympathetic neuron dendrites for maintenance of synapses. Thus, TrkA signaling endosomes have unique functions in different cellular compartments. Moreover, target-derived NGF mediates circuit formation and synapse maintenance through TrkA endosome signaling within dendrites to promote aggregation of postsynaptic protein complexes.

The ESCRT regulator Did2 maintains the balance between long-distance endosomal transport and endocytic trafficking.

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Carl Haag

2017-04-01

Full Text Available In highly polarised cells, like fungal hyphae, early endosomes function in both endocytosis as well as long-distance transport of various cargo including mRNA and protein complexes. However, knowledge on the crosstalk between these seemingly different trafficking processes is scarce. Here, we demonstrate that the ESCRT regulator Did2 coordinates endosomal transport in fungal hyphae of Ustilago maydis. Loss of Did2 results in defective vacuolar targeting, less processive long-distance transport and abnormal shuttling of early endosomes. Importantly, the late endosomal protein Rab7 and vacuolar protease Prc1 exhibit increased shuttling on these aberrant endosomes suggesting defects in endosomal maturation and identity. Consistently, molecular motors fail to attach efficiently explaining the disturbed processive movement. Furthermore, the endosomal mRNP linker protein Upa1 is hardly present on endosomes resulting in defects in long-distance mRNA transport. In conclusion, the ESCRT regulator Did2 coordinates precise maturation of endosomes and thus provides the correct membrane identity for efficient endosomal long-distance transport.

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

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Ewoud Bernardus Compeer

2012-03-01

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

Rapid endosomal escape of prickly nanodiamonds: implications for gene delivery

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

2015-06-01

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

Rapid endosomal escape of prickly nanodiamonds: implications for gene delivery

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

2015-01-01

The prickly nanodiamonds easily entered cells via endocytosis followed by unique intracellular translocation characteristics—quick endosomal escape followed by stable residence in cytoplasm. Endosomal membrane rupturing is identified as the major route of nanodiamonds’ escaping the vesicle confinement and to the cytoplasm. Little cytotoxicity is observed to associate with the nanodiamonds’ cytosolic release. Such features enable its application for gene delivery, which requires both effective cellular uptake and cytosolic release of the gene. Taking green fluorescent protein gene as an example, we demonstrate the successful cytosolic delivery and expression of such a gene using the prickly nanodiamonds as carrier. PMID:26123532

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

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

2015-06-30

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

Lysosomal degradation of membrane lipids.

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Kolter, Thomas; Sandhoff, Konrad

2010-05-03

The constitutive degradation of membrane components takes place in the acidic compartments of a cell, the endosomes and lysosomes. Sites of lipid degradation are intralysosomal membranes that are formed in endosomes, where the lipid composition is adjusted for degradation. Cholesterol is sorted out of the inner membranes, their content in bis(monoacylglycero)phosphate increases, and, most likely, sphingomyelin is degraded to ceramide. Together with endosomal and lysosomal lipid-binding proteins, the Niemann-Pick disease, type C2-protein, the GM2-activator, and the saposins sap-A, -B, -C, and -D, a suitable membrane lipid composition is required for degradation of complex lipids by hydrolytic enzymes. Copyright 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Disturbed vesicular trafficking of membrane proteins in prion disease.

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Uchiyama, Keiji; Miyata, Hironori; Sakaguchi, Suehiro

2013-01-01

The pathogenic mechanism of prion diseases remains unknown. We recently reported that prion infection disturbs post-Golgi trafficking of certain types of membrane proteins to the cell surface, resulting in reduced surface expression of membrane proteins and abrogating the signal from the proteins. The surface expression of the membrane proteins was reduced in the brains of mice inoculated with prions, well before abnormal symptoms became evident. Prions or pathogenic prion proteins were mainly detected in endosomal compartments, being particularly abundant in recycling endosomes. Some newly synthesized membrane proteins are delivered to the surface from the Golgi apparatus through recycling endosomes, and some endocytosed membrane proteins are delivered back to the surface through recycling endosomes. These results suggest that prions might cause neuronal dysfunctions and cell loss by disturbing post-Golgi trafficking of membrane proteins via accumulation in recycling endosomes. Interestingly, it was recently shown that delivery of a calcium channel protein to the cell surface was impaired and its function was abrogated in a mouse model of hereditary prion disease. Taken together, these results suggest that impaired delivery of membrane proteins to the cell surface is a common pathogenic event in acquired and hereditary prion diseases.

The Human ABCG1 Transporter Mobilizes Plasma Membrane and Late Endosomal Non-Sphingomyelin-Associated-Cholesterol for Efflux and Esterification

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Edward B. Neufeld

2014-12-01

Full Text Available We have previously shown that GFP-tagged human ABCG1 on the plasma membrane (PM and in late endosomes (LE mobilizes sterol on both sides of the membrane lipid bilayer, thereby increasing cellular cholesterol efflux to lipid surfaces. In the present study, we examined ABCG1-induced changes in membrane cholesterol distribution, organization, and mobility. ABCG1-GFP expression increased the amount of mobile, non-sphingomyelin(SM-associated cholesterol at the PM and LE, but not the amount of SM-associated-cholesterol or SM. ABCG1-mobilized non-SM-associated-cholesterol rapidly cycled between the PM and LE and effluxed from the PM to extracellular acceptors, or, relocated to intracellular sites of esterification. ABCG1 increased detergent-soluble pools of PM and LE cholesterol, generated detergent-resistant, non-SM-associated PM cholesterol, and increased resistance to both amphotericin B-induced (cholesterol-mediated and lysenin-induced (SM-mediated cytolysis, consistent with altered organization of both PM cholesterol and SM. ABCG1 itself resided in detergent-soluble membrane domains. We propose that PM and LE ABCG1 residing at the phase boundary between ordered (Lo and disordered (Ld membrane lipid domains alters SM and cholesterol organization thereby increasing cholesterol flux between Lo and Ld, and hence, the amount of cholesterol available for removal by acceptors on either side of the membrane bilayer for either efflux or esterification.

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

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Andreea Popa

2015-02-01

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

Inhibition of HIV-1 endocytosis allows lipid mixing at the plasma membrane, but not complete fusion

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de la Vega Michelle

2011-12-01

Full Text Available Abstract Background We recently provided evidence that HIV-1 enters HeLa-derived TZM-bl and lymphoid CEMss cells by fusing with endosomes, whereas its fusion with the plasma membrane does not proceed beyond the lipid mixing step. The mechanism of restriction of HIV-1 fusion at the cell surface and/or the factors that aid the virus entry from endosomes remain unclear. Results We examined HIV-1 fusion with a panel of target cells lines and with primary CD4+ T cells. Kinetic measurements of fusion combined with time-resolved imaging of single viruses further reinforced the notion that HIV-1 enters the cells via endocytosis and fusion with endosomes. Furthermore, we attempted to deliberately redirect virus fusion to the plasma membrane, using two experimental strategies. First, the fusion reaction was synchronized by pre-incubating the viruses with cells at reduced temperature to allow CD4 and coreceptors engagement, but not the virus uptake or fusion. Subsequent shift to a physiological temperature triggered accelerated virus uptake followed by entry from endosomes, but did not permit fusion at the cell surface. Second, blocking HIV-1 endocytosis by a small-molecule dynamin inhibitor, dynasore, resulted in transfer of viral lipids to the plasma membrane without any detectable release of the viral content into the cytosol. We also found that a higher concentration of dynasore is required to block the HIV-endosome fusion compared to virus internalization. Conclusions Our results further support the notion that HIV-1 enters disparate cell types through fusion with endosomes. The block of HIV-1 fusion with the plasma membrane at a post-lipid mixing stage shows that this membrane is not conducive to fusion pore formation and/or enlargement. The ability of dynasore to interfere with the virus-endosome fusion suggests that dynamin could be involved in two distinct steps of HIV-1 entry - endocytosis and fusion within intracellular compartments.

Modeling the signaling endosome hypothesis: Why a drive to the nucleus is better than a (random walk

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Howe Charles L

2005-10-01

Full Text Available Abstract Background Information transfer from the plasma membrane to the nucleus is a universal cell biological property. Such information is generally encoded in the form of post-translationally modified protein messengers. Textbook signaling models typically depend upon the diffusion of molecular signals from the site of initiation at the plasma membrane to the site of effector function within the nucleus. However, such models fail to consider several critical constraints placed upon diffusion by the cellular milieu, including the likelihood of signal termination by dephosphorylation. In contrast, signaling associated with retrogradely transported membrane-bounded organelles such as endosomes provides a dephosphorylation-resistant mechanism for the vectorial transmission of molecular signals. We explore the relative efficiencies of signal diffusion versus retrograde transport of signaling endosomes. Results Using large-scale Monte Carlo simulations of diffusing STAT-3 molecules coupled with probabilistic modeling of dephosphorylation kinetics we found that predicted theoretical measures of STAT-3 diffusion likely overestimate the effective range of this signal. Compared to the inherently nucleus-directed movement of retrogradely transported signaling endosomes, diffusion of STAT-3 becomes less efficient at information transfer in spatial domains greater than 200 nanometers from the plasma membrane. Conclusion Our model suggests that cells might utilize two distinct information transmission paradigms: 1 fast local signaling via diffusion over spatial domains on the order of less than 200 nanometers; 2 long-distance signaling via information packets associated with the cytoskeletal transport apparatus. Our model supports previous observations suggesting that the signaling endosome hypothesis is a subset of a more general hypothesis that the most efficient mechanism for intracellular signaling-at-a-distance involves the association of signaling

G protein-coupled receptor (GPCR) signaling via heterotrimeric G proteins from endosomes.

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Tsvetanova, Nikoleta G; Irannejad, Roshanak; von Zastrow, Mark

2015-03-13

Some G protein-coupled receptors (GPCRs), in addition to activating heterotrimeric G proteins in the plasma membrane, appear to elicit a "second wave" of G protein activation after ligand-induced internalization. We briefly summarize evidence supporting this view and then discuss what is presently known about the functional significance of GPCR-G protein activation in endosomes. Endosomal activation can shape the cellular response temporally by prolonging its overall duration, and may shape the response spatially by moving the location of intracellular second messenger production relative to effectors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Membrane's Eleven: heavy-atom derivatives of membrane-protein crystals

DEFF Research Database (Denmark)

Morth, Jens Preben; Sørensen, Thomas Lykke-Møller; Nissen, Poul

2006-01-01

A database has been assembled of heavy-atom derivatives used in the structure determination of membrane proteins. The database can serve as a guide to the design of experiments in the search for heavy-atom derivatives of new membrane-protein crystals. The database pinpoints organomercurials...

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

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Marshansky, V

2007-11-01

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

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

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Karim J Brandt

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

Plasma membrane is the site of productive HIV-1 particle assembly.

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Nolwenn Jouvenet

2006-12-01

Full Text Available Recently proposed models that have gained wide acceptance posit that HIV-1 virion morphogenesis is initiated by targeting the major structural protein (Gag to late endosomal membranes. Thereafter, late endosome-based secretory pathways are thought to deliver Gag or assembled virions to the plasma membrane (PM and extracellular milieu. We present several findings that are inconsistent with this model. Specifically, we demonstrate that HIV-1 Gag is delivered to the PM, and virions are efficiently released into the extracellular medium, when late endosome motility is abolished. Furthermore, we show that HIV-1 virions are efficiently released when assembly is rationally targeted to the PM, but not when targeted to late endosomes. Recently synthesized Gag first accumulates and assembles at the PM, but a proportion is subsequently internalized via endocytosis or phagocytosis, thus accounting for observations of endosomal localization. We conclude that HIV-1 assembly is initiated and completed at the PM, and not at endosomal membranes.

Membrane raft association is a determinant of plasma membrane localization.

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Diaz-Rohrer, Blanca B; Levental, Kandice R; Simons, Kai; Levental, Ilya

2014-06-10

The lipid raft hypothesis proposes lateral domains driven by preferential interactions between sterols, sphingolipids, and specific proteins as a central mechanism for the regulation of membrane structure and function; however, experimental limitations in defining raft composition and properties have prevented unequivocal demonstration of their functional relevance. Here, we establish a quantitative, functional relationship between raft association and subcellular protein sorting. By systematic mutation of the transmembrane and juxtamembrane domains of a model transmembrane protein, linker for activation of T-cells (LAT), we generated a panel of variants possessing a range of raft affinities. These mutations revealed palmitoylation, transmembrane domain length, and transmembrane sequence to be critical determinants of membrane raft association. Moreover, plasma membrane (PM) localization was strictly dependent on raft partitioning across the entire panel of unrelated mutants, suggesting that raft association is necessary and sufficient for PM sorting of LAT. Abrogation of raft partitioning led to mistargeting to late endosomes/lysosomes because of a failure to recycle from early endosomes. These findings identify structural determinants of raft association and validate lipid-driven domain formation as a mechanism for endosomal protein sorting.

Reorganization of the Endosomal System in Salmonella-Infected Cells: The Ultrastructure of Salmonella-Induced Tubular Compartments

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Krieger, Viktoria; Liebl, David; Zhang, Yuying; Rajashekar, Roopa; Chlanda, Petr; Giesker, Katrin; Chikkaballi, Deepak; Hensel, Michael

2014-01-01

During the intracellular life of Salmonella enterica, a unique membrane-bound compartment termed Salmonella-containing vacuole, or SCV, is formed. By means of translocated effector proteins, intracellular Salmonella also induce the formation of extensive, highly dynamic membrane tubules termed Salmonella-induced filaments or SIF. Here we report the first detailed ultrastructural analyses of the SCV and SIF by electron microscopy (EM), EM tomography and live cell correlative light and electron microscopy (CLEM). We found that a subset of SIF is composed of double membranes that enclose portions of host cell cytosol and cytoskeletal filaments within its inner lumen. Despite some morphological similarities, we found that the formation of SIF double membranes is independent from autophagy and requires the function of the effector proteins SseF and SseG. The lumen of SIF network is accessible to various types of endocytosed material and our CLEM analysis of double membrane SIF demonstrated that fluid phase markers accumulate only between the inner and outer membrane of these structures, a space continual with endosomal lumen. Our work reveals how manipulation of the endosomal membrane system by an intracellular pathogen results in a unique tubular membrane compartmentalization of the host cell, generating a shielded niche permissive for intracellular proliferation of Salmonella. PMID:25254663

Endocytosis of wheat germ agglutinin binding sites from the cell surface into a tubular endosomal network.

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Raub, T J; Koroly, M J; Roberts, R M

1990-04-01

By using fluorescence and electron microscopy, the endocytic pathway encountered by cell surface components after they had bound wheat germ agglutinin (WGA) was visualized. The majority of these components are thought to consist of sialylated glycoproteins (HMWAG) that represent a subpopulation of the total cell surface proteins but most of the externally disposed plasma membrane proteins of the cell. Examination of semi-thin sections by medium- and high-voltage electron microscopy revealed the three-dimensional organization of vesicular and tubular endosomes. Binding of either fluorescein isothiocyanate-, horseradish peroxidase-, or ferritin-conjugated WGA to cells at 4 degrees C showed that the HMWAG were distributed uniformly over the cell surface. Warming of surface-labeled cells to 37 degrees C resulted in the endocytosis of WGA into peripheral endosomes via invagination of regions of both coated and uncoated membrane. The peripheral endosome appeared as isolated complexes comprising a vesicular element (300-400 nm diam.) surrounded by and continuous with tubular cisternae (45-60 nm diam.), which did not interconnect the endosomes. After 30 min or more label also became localized in a network of anastomosing tubules (45-60 nm diam.) that were located in the centrosomal region of the cell. Endocytosed WGA-HMWAG complexes did not become associated with cisternae of the Golgi apparatus, although tubular and vesicular endosomes were noted in the vicinity of the trans-Golgi region. The accumulation of WGA-HMWAG in the endosomes within the centrosomal region was inhibited when cells were incubated at 18 degrees C. None of these compartments contained acid phosphatase activity, a result that is consistent with other data that the HMWAG do not pass through lysosomes initially. The kinetics of labeling were consistent with the interpretation that recycling of most of the WGA binding surface glycoproteins occurred rapidly from early peripheral endosomes followed by the

GGA1 regulates signal-dependent sorting of BACE1 to recycling endosomes, which moderates Aβ production

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Toh, Wei Hong; Chia, Pei Zhi Cheryl; Hossain, Mohammed Iqbal; Gleeson, Paul A.

2018-01-01

The diversion of the membrane-bound β-site amyloid precursor protein–(APP) cleaving enzyme (BACE1) from the endolysosomal pathway to recycling endosomes represents an important transport step in the regulation of amyloid beta (Aβ) production. However, the mechanisms that regulate endosome sorting of BACE1 are poorly understood. Here we assessed the transport of BACE1 from early to recycling endosomes and have identified essential roles for the sorting nexin 4 (SNX4)-mediated, signal-independent pathway and for a novel signal-mediated pathway. The signal-mediated pathway is regulated by the phosphorylation of the DXXLL-motif sequence DISLL in the cytoplasmic tail of BACE1. The phosphomimetic S498D BACE1 mutant was trafficked to recycling endosomes at a faster rate compared with wild-type BACE1 or the nonphosphorylatable S498A mutant. The rapid transit of BACE1 S498D from early endosomes was coupled with reduced levels of amyloid precursor protein processing and Aβ production, compared with the S498A mutant. We show that the adaptor, GGA1, and retromer are essential to mediate rapid trafficking of phosphorylated BACE1 to recycling endosomes. In addition, the BACE1 DISLL motif is phosphorylated and regulates endosomal trafficking, in primary neurons. Therefore, post-translational phosphorylation of DISLL enhances the exit of BACE1 from early endosomes, a pathway mediated by GGA1 and retromer, which is important in regulating Aβ production. PMID:29142073

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

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Yeates, Eniola Funmilayo Aduke; Tesco, Giuseppina

2016-07-22

The β-site amyloid precursor protein-cleaving enzyme (BACE1) is the rate-limiting enzyme in the production of amyloid-β, the toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that that depletion of the trafficking molecule Golgi-localized γ-ear-containing ARF-binding protein 3 (GGA3) results in increased BACE1 levels and activity because of impaired lysosomal degradation. We also determined that GGA3 regulation of BACE1 levels requires its ability to bind ubiquitin. Accordingly, we reported that BACE1 is ubiquitinated at lysine 501 and that lack of ubiquitination at lysine 501 produces BACE1 stabilization. Ubiquitin conjugation is a reversible process mediated by deubiquitinating enzymes. The ubiquitin-specific peptidase 8 (USP8), an endosome-associated deubiquitinating enzyme, regulates the ubiquitination, trafficking, and lysosomal degradation of several plasma membrane proteins. Here, we report that RNAi-mediated depletion of USP8 reduced levels of both ectopically expressed and endogenous BACE1 in H4 human neuroglioma cells. Moreover, USP8 depletion increased BACE1 ubiquitination, promoted BACE1 accumulation in the early endosomes and late endosomes/lysosomes, and decreased levels of BACE1 in the recycling endosomes. We also found that decreased BACE1 protein levels were accompanied by a decrease in BACE1-mediated amyloid precursor protein cleavage and amyloid-β levels. Our findings demonstrate that USP8 plays a key role in the trafficking and degradation of BACE1 by deubiquitinating lysine 501. These studies suggest that therapies able to accelerate BACE1 degradation (e.g. by increasing BACE1 ubiquitination) may represent a potential treatment for Alzheimer disease. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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

Science.gov (United States)

Yeates, Eniola Funmilayo Aduke; Tesco, Giuseppina

2016-01-01

The β-site amyloid precursor protein-cleaving enzyme (BACE1) is the rate-limiting enzyme in the production of amyloid-β, the toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that that depletion of the trafficking molecule Golgi-localized γ-ear-containing ARF-binding protein 3 (GGA3) results in increased BACE1 levels and activity because of impaired lysosomal degradation. We also determined that GGA3 regulation of BACE1 levels requires its ability to bind ubiquitin. Accordingly, we reported that BACE1 is ubiquitinated at lysine 501 and that lack of ubiquitination at lysine 501 produces BACE1 stabilization. Ubiquitin conjugation is a reversible process mediated by deubiquitinating enzymes. The ubiquitin-specific peptidase 8 (USP8), an endosome-associated deubiquitinating enzyme, regulates the ubiquitination, trafficking, and lysosomal degradation of several plasma membrane proteins. Here, we report that RNAi-mediated depletion of USP8 reduced levels of both ectopically expressed and endogenous BACE1 in H4 human neuroglioma cells. Moreover, USP8 depletion increased BACE1 ubiquitination, promoted BACE1 accumulation in the early endosomes and late endosomes/lysosomes, and decreased levels of BACE1 in the recycling endosomes. We also found that decreased BACE1 protein levels were accompanied by a decrease in BACE1-mediated amyloid precursor protein cleavage and amyloid-β levels. Our findings demonstrate that USP8 plays a key role in the trafficking and degradation of BACE1 by deubiquitinating lysine 501. These studies suggest that therapies able to accelerate BACE1 degradation (e.g. by increasing BACE1 ubiquitination) may represent a potential treatment for Alzheimer disease. PMID:27302062

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

DEFF Research Database (Denmark)

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

2012-01-01

that dimerization is achieved in part through neutralization of charged residues in the hydrophobic BAR-dimerization interface. Membrane remodelling also requires functional amphipathic helices, predicted to be present in all SNX-BARs, and the formation of high order SNX-BAR oligomers through selective 'tip...... and organizes the tubular endosomal network....

Neurotrophin signaling endosomes; biogenesis, regulation, and functions

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Yamashita, Naoya; Kuruvilla, Rejji

2016-01-01

In the nervous system, communication between neurons and their post-synaptic target cells is critical for the formation, refinement and maintenance of functional neuronal connections. Diffusible signals secreted by target tissues, exemplified by the family of neurotrophins, impinge on nerve terminals to influence diverse developmental events including neuronal survival and axonal growth. Key mechanisms of action of target-derived neurotrophins include the cell biological processes of endocytosis and retrograde trafficking of their Trk receptors from growth cones to cell bodies. In this review, we summarize the molecular mechanisms underlying this endosome-mediated signaling, focusing on the instructive role of neurotrophin signaling itself in directing its own trafficking. Recent studies have linked impaired neurotrophin trafficking to neurodevelopmental disorders, highlighting the relevance of neurotrophin endosomes in human health. PMID:27327126

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

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Hirokazu Tanaka

2013-05-01

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

Early to Late Endosome Trafficking Controls Secretion and Zymogen Activation in Rodent and Human Pancreatic Acinar Cells.

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Messenger, Scott W; Thomas, Diana Dh; Cooley, Michelle M; Jones, Elaina K; Falkowski, Michelle A; August, Benjamin K; Fernandez, Luis A; Gorelick, Fred S; Groblewski, Guy E

2015-11-01

Pancreatic acinar cells have an expanded apical endosomal system, the physiological and pathophysiological significance of which is still emerging. Phosphatidylinositol-3,5-bisphosphate (PI(3,5)P 2 ) is an essential phospholipid generated by PIKfyve, which phosphorylates phosphatidylinositol-3-phosphate (PI(3)P). PI(3,5)P 2 is necessary for maturation of early endosomes (EE) to late endosomes (LE). Inhibition of EE to LE trafficking enhances anterograde endosomal trafficking and secretion at the plasma membrane by default through a recycling endosome (RE) intermediate. We assessed the effects of modulating PIKfyve activity on apical trafficking and pancreatitis responses in pancreatic acinar cells. Inhibition of EE to LE trafficking was achieved using pharmacological inhibitors of PIKfyve, expression of dominant negative PIKfyve K1877E, or constitutively active Rab5-GTP Q79L. Anterograde endosomal trafficking was manipulated by expression of constitutively active and dominant negative Rab11a mutants. The effects of these agents on secretion, endolysosomal exocytosis of lysosome associated membrane protein (LAMP1), and trypsinogen activation in response to high-dose CCK-8, bile acids and cigarette toxin was determined. PIKfyve inhibition increased basal and stimulated secretion. Adenoviral overexpression of PIKfyve decreased secretion leading to cellular death. Expression of Rab5-GTP Q79L or Rab11a-GTP Q70L enhanced secretion. Conversely, dominant-negative Rab11a-GDP S25N reduced secretion. High-dose CCK inhibited endolysosomal exocytosis that was reversed by PIKfyve inhibition. PIKfyve inhibition blocked intracellular trypsin accumulation and cellular damage responses to high CCK-8, tobacco toxin, and bile salts in both rodent and human acini. These data demonstrate that EE-LE trafficking acutely controls acinar secretion and the intracellular activation of zymogens leading to the pathogenicity of acute pancreatitis.

Basolateral Endocytic Recycling Requires RAB-10 and AMPH-1 Mediated Recruitment of RAB-5 GAP TBC-2 to Endosomes

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Liu, Ou; Grant, Barth D.

2015-01-01

The small GTPase RAB-5/Rab5 is a master regulator of the early endosome, required for a myriad of coordinated activities, including the degradation and recycling of internalized cargo. Here we focused on the recycling function of the early endosome and the regulation of RAB-5 by GAP protein TBC-2 in the basolateral C. elegans intestine. We demonstrate that downstream basolateral recycling regulators, GTPase RAB-10/Rab10 and BAR domain protein AMPH-1/Amphiphysin, bind to TBC-2 and help to recruit it to endosomes. In the absence of RAB-10 or AMPH-1 binding to TBC-2, RAB-5 membrane association is abnormally high and recycling cargo is trapped in early endosomes. Furthermore, the loss of TBC-2 or AMPH-1 leads to abnormally high spatial overlap of RAB-5 and RAB-10. Taken together our results indicate that RAB-10 and AMPH-1 mediated down-regulation of RAB-5 is an important step in recycling, required for cargo exit from early endosomes and regulation of early endosome–recycling endosome interactions. PMID:26393361

Liposome Model Systems to Study the Endosomal Escape of Cell-Penetrating Peptides: Transport across Phospholipid Membranes Induced by a Proton Gradient

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Fatemeh Madani

2011-01-01

Full Text Available Detergent-mediated reconstitution of bacteriorhodopsin (BR into large unilamellar vesicles (LUVs was investigated, and the effects were carefully characterized for every step of the procedure. LUVs were prepared by the extrusion method, and their size and stability were examined by dynamic light scattering. BR was incorporated into the LUVs using the detergent-mediated reconstitution method and octyl glucoside (OG as detergent. The result of measuring pH outside the LUVs suggested that in the presence of light, BR pumps protons from the outside to the inside of the LUVs, creating acidic pH inside the vesicles. LUVs with 20% negatively charged headgroups were used to model endosomes with BR incorporated into the membrane. The fluorescein-labeled cell-penetrating peptide penetratin was entrapped inside these BR-containing LUVs. The light-induced proton pumping activity of BR has allowed us to observe the translocation of fluorescein-labeled penetratin across the vesicle membrane.

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

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

2016-12-01

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

Membrane localization and dynamics of geranylgeranylated Rab5 hypervariable region.

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Edler, Eileen; Schulze, Eric; Stein, Matthias

2017-08-01

The small GTPase Rab5 is a key regulator of endosomal trafficking processes and a marker for the early endosome. The C-terminal hypervariable region (HVR) of Rab5 is post-translationally modified at residues Cys 212 and Cys 213 to accommodate two geranylgeranyl anchors (C20 carbon chain length) in order to associate Rab5 with the membrane. The structural role of the HVR regarding protein-early endosome membrane recruitment is not resolved due to its high degree of flexibility and lack of crystallographic information. Here, full-atomistic and coarse-grained molecular dynamics simulations of the truncated Rab5 HVR 206-215 in three model membranes of increasing complexity (pure phospholipid bilayer, ternary membrane with cholesterol, six-component early endosome) were performed. Specific electrostatic interactions between the HVR 206-215 Arg 209 residue and the phosphate group of the inositol ring of PI(3)P were detected. This shows that PI(3)P acts as a first contact site of protein recruitment to the early endosome. The free energy change of HVR 206-215 extraction from the bilayer was largest for the physiological negatively charged membrane. 5μs coarse-grained simulations revealed an active recruitment of PI(3)P to the HVR 206-215 supporting the formation of Rab5- and PI(3)P enriched signaling platforms. Copyright © 2017 Elsevier B.V. All rights reserved.

Desipramine induces disorder in cholesterol-rich membranes: implications for viral trafficking

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Pakkanen, Kirsi; Salonen, Emppu; Mäkelä, Anna R.; Oker-Blom, Christian; Vattulainen, Ilpo; Vuento, Matti

2009-12-01

In this study, the effect of desipramine (DMI) on phospholipid bilayers and parvoviral entry was elucidated. In atomistic molecular dynamics simulations, DMI was found to introduce disorder in cholesterol-rich phospholipid bilayers. This was manifested by a decrease in the deuterium order parameter SCD as well as an increase in the membrane area. Disordering of the membrane suggested DMI to destabilize cholesterol-rich membrane domains (rafts) in cellular conditions. To relate the raft disrupting ability of DMI with novel biological relevance, we studied the intracellular effect of DMI using canine parvovirus (CPV), a virus known to interact with endosomal membranes and sphingomyelin, as an intracellular probe. DMI was found to cause retention of the virus in intracellular vesicular structures leading to the inhibition of viral proliferation. This implies that DMI has a deleterious effect on the viral traffic. As recycling endosomes and the internal vesicles of multivesicular bodies are known to contain raft components, the effect of desipramine beyond the plasma membrane step could be caused by raft disruption leading to impaired endosomal function and possibly have direct influence on the penetration of the virus through an endosomal membrane.

Desipramine induces disorder in cholesterol-rich membranes: implications for viral trafficking

International Nuclear Information System (INIS)

Pakkanen, Kirsi; Mäkelä, Anna R; Oker-Blom, Christian; Vuento, Matti; Salonen, Emppu; Vattulainen, Ilpo

2009-01-01

In this study, the effect of desipramine (DMI) on phospholipid bilayers and parvoviral entry was elucidated. In atomistic molecular dynamics simulations, DMI was found to introduce disorder in cholesterol-rich phospholipid bilayers. This was manifested by a decrease in the deuterium order parameter S CD as well as an increase in the membrane area. Disordering of the membrane suggested DMI to destabilize cholesterol-rich membrane domains (rafts) in cellular conditions. To relate the raft disrupting ability of DMI with novel biological relevance, we studied the intracellular effect of DMI using canine parvovirus (CPV), a virus known to interact with endosomal membranes and sphingomyelin, as an intracellular probe. DMI was found to cause retention of the virus in intracellular vesicular structures leading to the inhibition of viral proliferation. This implies that DMI has a deleterious effect on the viral traffic. As recycling endosomes and the internal vesicles of multivesicular bodies are known to contain raft components, the effect of desipramine beyond the plasma membrane step could be caused by raft disruption leading to impaired endosomal function and possibly have direct influence on the penetration of the virus through an endosomal membrane

Recycling domains in plant cell morphogenesis: small GTPase effectors, plasma membrane signalling and the exocyst.

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Zárský, Viktor; Potocký, Martin

2010-04-01

The Rho/Rop small GTPase regulatory module is central for initiating exocytotically ACDs (active cortical domains) in plant cell cortex, and a growing array of Rop regulators and effectors are being discovered in plants. Structural membrane phospholipids are important constituents of cells as well as signals, and phospholipid-modifying enzymes are well known effectors of small GTPases. We have shown that PLDs (phospholipases D) and their product, PA (phosphatidic acid), belong to the regulators of the secretory pathway in plants. We have also shown that specific NOXs (NADPH oxidases) producing ROS (reactive oxygen species) are involved in cell growth as exemplified by pollen tubes and root hairs. Most plant cells exhibit several distinct plasma membrane domains (ACDs), established and maintained by endocytosis/exocytosis-driven membrane protein recycling. We proposed recently the concept of a 'recycling domain' (RD), uniting the ACD and the connected endosomal recycling compartment (endosome), as a dynamic spatiotemporal entity. We have described a putative GTPase-effector complex exocyst involved in exocytic vesicle tethering in plants. Owing to the multiplicity of its Exo70 subunits, this complex, along with many RabA GTPases (putative recycling endosome organizers), may belong to core regulators of RD organization in plants.

Sphingolipids activate membrane fusion of Semliki Forest virus in a stereospecific manner

DEFF Research Database (Denmark)

Moesby, Lise; Corver, J; Erukulla, R K

1995-01-01

The alphavirus Semliki Forest virus (SFV) enters cells through receptor-mediated endocytosis. Subsequently, triggered by the acid pH in endosomes, the viral envelope fuses with the endosomal membrane. Membrane fusion of SFV has been shown previously to be dependent on the presence of cholesterol ...

Gαs regulates Glucagon-Like Peptide 1 Receptor-mediated cyclic AMP generation at Rab5 endosomal compartment.

Science.gov (United States)

Girada, Shravan Babu; Kuna, Ramya S; Bele, Shilpak; Zhu, Zhimeng; Chakravarthi, N R; DiMarchi, Richard D; Mitra, Prasenjit

2017-10-01

Upon activation, G protein coupled receptors (GPCRs) associate with heterotrimeric G proteins at the plasma membrane to initiate second messenger signaling. Subsequently, the activated receptor experiences desensitization, internalization, and recycling back to the plasma membrane, or it undergoes lysosomal degradation. Recent reports highlight specific cases of persistent cyclic AMP generation by internalized GPCRs, although the functional significance and mechanistic details remain to be defined. Cyclic AMP generation from internalized Glucagon-Like Peptide-1 Receptor (GLP-1R) has previously been reported from our laboratory. This study aimed at deciphering the molecular mechanism by which internalized GLP-R supports sustained cyclic AMP generation upon receptor activation in pancreatic beta cells. We studied the time course of cyclic AMP generation following GLP-1R activation with particular emphasis on defining the location where cyclic AMP is generated. Detection involved a novel GLP-1 conjugate coupled with immunofluorescence using specific endosomal markers. Finally, we employed co-immunoprecipitation as well as immunofluorescence to assess the protein-protein interactions that regulate GLP-1R mediated cyclic AMP generation at endosomes. Our data reveal that prolonged association of G protein α subunit Gαs with activated GLP-1R contributed to sustained cyclic AMP generation at Rab 5 endosomal compartment. The findings provide the mechanism of endosomal cyclic AMP generation following GLP-1R activation. We identified the specific compartment that serves as an organizing center to generate endosomal cyclic AMP by internalized activated receptor complex. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

LMTK1 regulates dendritic formation by regulating movement of Rab11A-positive endosomes.

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Takano, Tetsuya; Urushibara, Tomoki; Yoshioka, Nozomu; Saito, Taro; Fukuda, Mitsunori; Tomomura, Mineko; Hisanaga, Shin-Ichi

2014-06-01

Neurons extend two types of neurites-axons and dendrites-that differ in structure and function. Although it is well understood that the cytoskeleton plays a pivotal role in neurite differentiation and extension, the mechanisms by which membrane components are supplied to growing axons or dendrites is largely unknown. We previously reported that the membrane supply to axons is regulated by lemur kinase 1 (LMTK1) through Rab11A-positive endosomes. Here we investigate the role of LMTK1 in dendrite formation. Down-regulation of LMTK1 increases dendrite growth and branching of cerebral cortical neurons in vitro and in vivo. LMTK1 knockout significantly enhances the prevalence, velocity, and run length of anterograde movement of Rab11A-positive endosomes to levels similar to those expressing constitutively active Rab11A-Q70L. Rab11A-positive endosome dynamics also increases in the cell body and growth cone of LMTK1-deficient neurons. Moreover, a nonphosphorylatable LMTK1 mutant (Ser34Ala, a Cdk5 phosphorylation site) dramatically promotes dendrite growth. Thus LMTK1 negatively controls dendritic formation by regulating Rab11A-positive endosomal trafficking in a Cdk5-dependent manner, indicating the Cdk5-LMTK1-Rab11A pathway as a regulatory mechanism of dendrite development as well as axon outgrowth. © 2014 Takano et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Two barcodes encoded by the type-1 PDZ and by phospho-Ser312 regulate retromer/WASH-mediated sorting of the ß1-adrenergic receptor from endosomes to the plasma membrane.

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Nooh, Mohammed M; Bahouth, Suleiman W

2017-01-01

Recycling of the majority of agonist-internalized GPCR is dependent on a type I-PDZ "barcode" in their C-tail. The recycling of wild-type (WT) ß 1 -AR is also dependent on its default "type-1 PDZ barcode", but trafficking of the ß 1 -AR is inhibited when PKA or its substrate serine at position 312 (Ser 312 ) are inactivated. We tested the hypothesis that phospho-Ser 312 provided a second barcode for ß 1 -AR sorting from endosomes to the plasma membrane by determining the role of retromer/WASH complexes in ß 1 -AR trafficking. Recycling of WT ß 1 -AR or WT ß 2 -AR was dependent on targeting the retromer to endosomal membranes via SNX3 and rab7a, and on complexing the retromer to the WASH pentamer via the C-tail of FAM21 (FAM21 C ). These maneuvers however, did not inhibit the recycling of a phospho-Ser 312 ß 1 -AR mimic ((S312D) ß 1 -AR). Knockdown of the trans-acting PDZ protein sorting nexin27 (SNX27) inhibited the recycling of WT ß 1 -AR and WT ß 2 -AR, but had no effect on (S312D) ß 1 -AR∆PDZ or on phosphorylation of WT ß 1 -AR by PKA at Ser 312 . However, depletion of FKBP15, a FAM21 C -binding endosomal protein, selectively inhibited WT ß 1 -AR but not ß 2 -AR recycling, suggesting divergence might exist in GPCR trafficking roadmaps. These results indicate that two barcodes are involved in sorting WT ß 1 -AR out of early endosomes. The first and antecedent "barcode" was the "type-1 PDZ", followed by a second reversible "phospho-Ser 312 " verification "barcode". This organization allows tight regulation of ß 1 -AR density to signaling intensity in conditions associated with aberrant ß 1 -AR signaling such as in hypertension and heart failure. Copyright © 2016 Elsevier Inc. All rights reserved.

Small Molecules for Early Endosome-Specific Patch Clamping.

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Chen, Cheng-Chang; Butz, Elisabeth S; Chao, Yu-Kai; Grishchuk, Yulia; Becker, Lars; Heller, Stefan; Slaugenhaupt, Susan A; Biel, Martin; Wahl-Schott, Christian; Grimm, Christian

2017-07-20

To resolve the subcellular distribution of endolysosomal ion channels, we have established a novel experimental approach to selectively patch clamp Rab5 positive early endosomes (EE) versus Rab7/LAMP1-positive late endosomes/lysosomes (LE/LY). To functionally characterize ion channels in endolysosomal membranes with the patch-clamp technique, it is important to develop techniques to selectively enlarge the respective organelles. We found here that two small molecules, wortmannin and latrunculin B, enlarge Rab5-positive EE when combined but not Rab7-, LAMP1-, or Rab11 (RE)-positive vesicles. The two compounds act rapidly, specifically, and are readily applicable in contrast to genetic approaches or previously used compounds such as vacuolin, which enlarges EE, RE, and LE/LY. We apply this approach here to measure currents mediated by TRPML channels, in particular TRPML3, which we found to be functionally active in both EE and LE/LY in overexpressing cells as well as in endogenously expressing CD11b+ lung-tissue macrophages. Copyright © 2017 Elsevier Ltd. All rights reserved.

Integrin-linked kinase and ELMO2 modulate recycling endosomes in keratinocytes.

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Ho, Ernest; Ivanova, Iordanka A; Dagnino, Lina

2016-12-01

The formation of tight cell-cell junctions is essential in the epidermis for its barrier properties. In this tissue, keratinocytes follow a differentiation program tightly associated with their movement from the innermost basal to the outer suprabasal layers, and with changes in their cell-cell adhesion profile. Intercellular adhesion in keratinocytes is mediated through cell-cell contacts, including E-cadherin-based adherens junctions. Although the mechanisms that mediate E-cadherin delivery to the plasma membrane have been widely studied in simple epithelia, this process is less well understood in the stratified epidermis. In this study, we have investigated the role of Engulfment and Cell Motility 2 (ELMO2) and integrin-linked kinase (ILK) in the positioning of E-cadherin-containing recycling endosomes during establishment of cell-cell contacts in differentiating keratinocytes. We now show that induction of keratinocyte differentiation by Ca 2+ is accompanied by localization of ELMO2 and ILK to Rab4- and Rab11a-containing recycling endosomes. The positioning of long-loop Rab11a-positive endosomes at areas adjacent to cell-cell contacts is disrupted in ELMO2- or ILK-deficient keratinocytes, and is associated with impaired localization of E-cadherin to cell borders. Our studies show a previously unrecognized role for ELMO2 and ILK in modulation of endosomal positioning, which may play key roles in epidermal sheet maintenance and permeability barrier function. Copyright © 2016 Elsevier B.V. All rights reserved.

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

OpenAIRE

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

2003-01-01

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

Pannexin2 oligomers localize in the membranes of endosomal vesicles in mammalian cells while Pannexin1 channels traffic to the plasma membrane.

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Boassa, Daniela; Nguyen, Phuong; Hu, Junru; Ellisman, Mark H; Sosinsky, Gina E

2014-01-01

Pannexin2 (Panx2) is the largest of three members of the pannexin proteins. Pannexins are topologically related to connexins and innexins, but serve different functional roles than forming gap junctions. We previously showed that pannexins form oligomeric channels but unlike connexins and innexins, they form only single membrane channels. High levels of Panx2 mRNA and protein in the Central Nervous System (CNS) have been documented. Whereas Pannexin1 (Panx1) is fairly ubiquitous and Pannexin3 (Panx3) is found in skin and connective tissue, both are fully glycosylated, traffic to the plasma membrane and have functions correlated with extracellular ATP release. Here, we describe trafficking and subcellular localizations of exogenous Panx2 and Panx1 protein expression in MDCK, HeLa, and HEK 293T cells as well as endogenous Panx1 and Panx2 patterns in the CNS. Panx2 was found in intracellular localizations, was partially N-glycosylated, and localizations were non-overlapping with Panx1. Confocal images of hippocampal sections immunolabeled for the astrocytic protein GFAP, Panx1 and Panx2 demonstrated that the two isoforms, Panx1 and Panx2, localized at different subcellular compartments in both astrocytes and neurons. Using recombinant fusions of Panx2 with appended genetic tags developed for correlated light and electron microscopy and then expressed in different cell lines, we determined that Panx2 is localized in the membrane of intracellular vesicles and not in the endoplasmic reticulum as initially indicated by calnexin colocalization experiments. Dual immunofluorescence imaging with protein markers for specific vesicle compartments showed that Panx2 vesicles are early endosomal in origin. In electron tomographic volumes, cross-sections of these vesicles displayed fine structural details and close proximity to actin filaments. Thus, pannexins expressed at different subcellular compartments likely exert distinct functional roles, particularly in the nervous system.

The homeodomain derived peptide Penetratin induces curvature of fluid membrane domains.

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Antonin Lamazière

Full Text Available BACKGROUND: Protein membrane transduction domains that are able to cross the plasma membrane are present in several transcription factors, such as the homeodomain proteins and the viral proteins such as Tat of HIV-1. Their discovery resulted in both new concepts on the cell communication during development, and the conception of cell penetrating peptide vectors for internalisation of active molecules into cells. A promising cell penetrating peptide is Penetratin, which crosses the cell membranes by a receptor and metabolic energy-independent mechanism. Recent works have claimed that Penetratin and similar peptides are internalized by endocytosis, but other endocytosis-independent mechanisms have been proposed. Endosomes or plasma membranes crossing mechanisms are not well understood. Previously, we have shown that basic peptides induce membrane invaginations suggesting a new mechanism for uptake, "physical endocytosis". METHODOLOGY/PRINCIPAL FINDINGS: Herein, we investigate the role of membrane lipid phases on Penetratin induced membrane deformations (liquid ordered such as in "raft" microdomains versus disordered fluid "non-raft" domains in membrane models. Experimental data show that zwitterionic lipid headgroups take part in the interaction with Penetratin suggesting that the external leaflet lipids of cells plasma membrane are competent for peptide interaction in the absence of net negative charges. NMR and X-ray diffraction data show that the membrane perturbations (tubulation and vesiculation are associated with an increase in membrane negative curvature. These effects on curvature were observed in the liquid disordered but not in the liquid ordered (raft-like membrane domains. CONCLUSIONS/SIGNIFICANCE: The better understanding of the internalisation mechanisms of protein transduction domains will help both the understanding of the mechanisms of cell communication and the development of potential therapeutic molecular vectors. Here we

Apolipoprotein E Regulates Amyloid Formation within Endosomes of Pigment Cells

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Guillaume van Niel

2015-10-01

Full Text Available Accumulation of toxic amyloid oligomers is a key feature in the pathogenesis of amyloid-related diseases. Formation of mature amyloid fibrils is one defense mechanism to neutralize toxic prefibrillar oligomers. This mechanism is notably influenced by apolipoprotein E variants. Cells that produce mature amyloid fibrils to serve physiological functions must exploit specific mechanisms to avoid potential accumulation of toxic species. Pigment cells have tuned their endosomes to maximize the formation of functional amyloid from the protein PMEL. Here, we show that ApoE is associated with intraluminal vesicles (ILV within endosomes and remain associated with ILVs when they are secreted as exosomes. ApoE functions in the ESCRT-independent sorting mechanism of PMEL onto ILVs and regulates the endosomal formation of PMEL amyloid fibrils in vitro and in vivo. This process secures the physiological formation of amyloid fibrils by exploiting ILVs as amyloid nucleating platforms.

Early to Late Endosome Trafficking Controls Secretion and Zymogen Activation in Rodent and Human Pancreatic Acinar CellsSummary

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Scott W. Messenger

2015-11-01

Full Text Available Background & Aims: Pancreatic acinar cells have an expanded apical endosomal system, the physiologic and pathophysiologic significance of which is still emerging. Phosphatidylinositol-3,5-bisphosphate [PI(3,5P2] is an essential phospholipid generated by phosphatidylinositol 3-phosphate 5-kinase (PIKfyve, which phosphorylates phosphatidylinositol-3-phosphate (PI3P. PI(3,5P2 is necessary for maturation of early endosomes (EE to late endosomes (LE. Inhibition of EE to LE trafficking enhances anterograde endosomal trafficking and secretion at the plasma membrane by default through a recycling endosome (RE intermediate. We assessed the effects of modulating PIKfyve activity on apical trafficking and pancreatitis responses in pancreatic acinar cells. Methods: Inhibition of EE to LE trafficking was achieved using pharmacologic inhibitors of PIKfyve, expression of dominant negative PIKfyve K1877E, or constitutively active Rab5-GTP Q79L. Anterograde endosomal trafficking was manipulated by expression of constitutively active and dominant negative Rab11a mutants. The effects of these agents on secretion, endolysosomal exocytosis of lysosome associated membrane protein (LAMP1, and trypsinogen activation in response to supramaximal cholecystokinin (CCK-8, bile acids, and cigarette toxin was determined. Results: PIKfyve inhibition increased basal and stimulated secretion. Adenoviral overexpression of PIKfyve decreased secretion leading to cellular death. Expression of Rab5-GTP Q79L or Rab11a-GTP Q70L enhanced secretion. Conversely, dominant-negative Rab11a-GDP S25N reduced secretion. High-dose CCK inhibited endolysosomal exocytosis that was reversed by PIKfyve inhibition. PIKfyve inhibition blocked intracellular trypsin accumulation and cellular damage responses to supramaximal CCK-8, tobacco toxin, and bile salts in both rodent and human acini. Conclusions: These data demonstrate that EE-LE trafficking acutely controls acinar secretion and the intracellular

Acid sphingomyelinase activity is regulated by membrane lipids and facilitates cholesterol transfer by NPC2.

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Oninla, Vincent O; Breiden, Bernadette; Babalola, Jonathan O; Sandhoff, Konrad

2014-12-01

During endocytosis, membrane components move to intraluminal vesicles of the endolysosomal compartment for digestion. At the late endosomes, cholesterol is sorted out mainly by two sterol-binding proteins, Niemann-Pick protein type C (NPC)1 and NPC2. To study the NPC2-mediated intervesicular cholesterol transfer, we developed a liposomal assay system. (Abdul-Hammed, M., B. Breiden, M. A. Adebayo, J. O. Babalola, G. Schwarzmann, and K. Sandhoff. 2010. Role of endosomal membrane lipids and NPC2 in cholesterol transfer and membrane fusion. J. Lipid Res. 51: 1747-1760.) Anionic lipids stimulate cholesterol transfer between liposomes while SM inhibits it, even in the presence of anionic bis(monoacylglycero)phosphate (BMP). Preincubation of vesicles containing SM with acid sphingomyelinase (ASM) (SM phosphodiesterase, EC 3.1.4.12) results in hydrolysis of SM to ceramide (Cer), which enhances cholesterol transfer. Besides SM, ASM also cleaves liposomal phosphatidylcholine. Anionic phospholipids derived from the plasma membrane (phosphatidylglycerol and phosphatidic acid) stimulate SM and phosphatidylcholine hydrolysis by ASM more effectively than BMP, which is generated during endocytosis. ASM-mediated hydrolysis of liposomal SM was also stimulated by incorporation of diacylglycerol (DAG), Cer, and free fatty acids into the liposomal membranes. Conversely, phosphatidylcholine hydrolysis was inhibited by incorporation of cholesterol, Cer, DAG, monoacylglycerol, and fatty acids. Our data suggest that SM degradation by ASM is required for physiological secretion of cholesterol from the late endosomal compartment, and is a key regulator of endolysosomal lipid digestion. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

Effective photo-enhancement of cellular activity of fluorophore-octaarginine antisense PNA conjugates correlates with singlet oxygen formation, endosomal escape and chromophore lipophilicity

DEFF Research Database (Denmark)

Yarani, Reza; Shiraishi, Takehiko; Nielsen, Peter E.

2018-01-01

Photochemical internalization (PCI) is a cellular drug delivery method based on the generation of light-induced reactive oxygen species (ROS) causing damage to the endosomal membrane and thereby resulting in drug release to the cytoplasm. In our study a series of antisense fluorophore octaarginin...... indicate that efficient photodynamic endosomal escape is strongly dependent on the quantum yield for photochemical singlet oxygen formation, photostability as well as the lipophilicity of the chromophore....

2,2'-Bis(monoacylglycero) PO4 (BMP), but Not 3,1'-BMP, increases membrane curvature stress to enhance α-tocopherol transfer protein binding to membranes.

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Baptist, Matilda; Panagabko, Candace; Nickels, Jonathan D; Katsaras, John; Atkinson, Jeffrey

2015-03-01

Previous work revealed that α-tocopherol transfer protein (α-TTP) co-localizes with bis(monoacylglycero)phosphate (BMP) in late endosomes. BMP is a lipid unique to late endosomes and is believed to induce membrane curvature and support the multivesicular nature of this organelle. We examined the effect of BMP on α-TTP binding to membranes using dual polarization interferometry and vesicle-binding assay. α-TTP binding to membranes is increased by the curvature-inducing lipid BMP. α-TTP binds to membranes with greater affinity when they contain the 2,2'-BMP versus 3,1'-BMP isomers.

Oilfield Produced Water Reuse and Reinjection with Membrane

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Siagian Utjok W.R.

2018-01-01

Full Text Available Produced water has become a global environmental issue due to its huge volume and toxicity that may pose detrimental effects on receiving environment. Several approaches have been proposed to provide a strategy for produced water handling such as reinjection, reuse, or discharge. With various advantages, membrane technology has been increasingly used in produced water treatment replacing the conventional technologies. However, fouling is a major drawback of membrane processes in this application which needs to be controlled. This paper gives an overview and comparison of different produced water management. Special attention is given to produced water treatment for reuse purpose. Furthermore, the use of membrane processes in produced water reuse including performance, challenges, and future outlook are discussed.

A fluorescence resonance energy transfer-based approach for investigating late endosome-lysosome retrograde fusion events.

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Kaufmann, A M; Goldman, S D B; Krise, J P

2009-03-01

Traditionally, lysosomes have been considered to be a terminal endocytic compartment. Recent studies suggest that lysosomes are quite dynamic, being able to fuse with other late endocytic compartments as well as with the plasma membrane. Here we describe a quantitative fluorescence energy transfer (FRET)-based method for assessing rates of retrograde fusion between terminal lysosomes and late endosomes in living cells. Late endosomes were specifically labeled with 800-nm latex beads that were conjugated with streptavidin and Alexa Fluor 555 (FRET donor). Terminal lysosomes were specifically labeled with 10,000-MW dextran polymers conjugated with biotin and Alexa Fluor 647 (FRET acceptor). Following late endosome-lysosome fusion, the strong binding affinity between streptavidin and biotin brought the donor and acceptor fluorophore molecules into close proximity, thereby facilitating the appearance of a FRET emission signal. Because apparent size restrictions in the endocytic pathway do not permit endocytosed latex beads from reaching terminal lysosomes in an anterograde fashion, the appearance of the FRET signal is consistent with retrograde transport of lysosomal cargo back to late endosomes. We assessed the efficiency of this transport step in fibroblasts affected by different lysosome storage disorders-Niemann-Pick type C, mucolipidosis type IV, and Sandhoff's disease, all of which have a similar lysosomal lipid accumulation phenotype. We report here, for the first time, that these disorders can be distinguished by their rate of transfer of lysosome cargos to late endosomes, and we discuss the implications of these findings for developing new therapeutic strategies.

Actin polymerization in the endosomal pathway, but not on the Coxiella-containing vacuole, is essential for pathogen growth.

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Heather E Miller

2018-04-01

Full Text Available Coxiella burnetii is an intracellular bacterium that replicates within an expansive phagolysosome-like vacuole. Fusion between the Coxiella-containing vacuole (CCV and late endosomes/multivesicular bodies requires Rab7, the HOPS tethering complex, and SNARE proteins, with actin also speculated to play a role. Here, we investigated the importance of actin in CCV fusion. Filamentous actin patches formed around the CCV membrane that were preferred sites of vesicular fusion. Accordingly, the mediators of endolysosomal fusion Rab7, VAMP7, and syntaxin 8 were concentrated in CCV actin patches. Generation of actin patches required C. burnetii type 4B secretion and host retromer function. Patches decorated with VPS29 and VPS35, components of the retromer, FAM21 and WASH, members of the WASH complex that engage the retromer, and Arp3, a component of the Arp2/3 complex that generates branched actin filaments. Depletion by siRNA of VPS35 or VPS29 reduced CCV actin patches and caused Rab7 to uniformly distribute in the CCV membrane. C. burnetii grew normally in VPS35 or VPS29 depleted cells, as well as WASH-knockout mouse embryo fibroblasts, where CCVs are devoid of actin patches. Endosome recycling to the plasma membrane and trans-Golgi of glucose transporter 1 (GLUT1 and cationic-independent mannose-6-phosphate receptor (CI-M6PR, respectively, was normal in infected cells. However, siRNA knockdown of retromer resulted in aberrant trafficking of GLUT1, but not CI-M6PR, suggesting canonical retrograde trafficking is unaffected by retromer disruption. Treatment with the specific Arp2/3 inhibitor CK-666 strongly inhibited CCV formation, an effect associated with altered endosomal trafficking of transferrin receptor. Collectively, our results show that CCV actin patches generated by retromer, WASH, and Arp2/3 are dispensable for CCV biogenesis and stability. However, Arp2/3-mediated production of actin filaments required for cargo transport within the

The translocon protein Sec61 mediates antigen transport from endosomes in the cytosol for cross-presentation to CD8(+) T cells.

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Zehner, Matthias; Marschall, Andrea L; Bos, Erik; Schloetel, Jan-Gero; Kreer, Christoph; Fehrenschild, Dagmar; Limmer, Andreas; Ossendorp, Ferry; Lang, Thorsten; Koster, Abraham J; Dübel, Stefan; Burgdorf, Sven

2015-05-19

The molecular mechanisms regulating antigen translocation into the cytosol for cross-presentation are under controversial debate, mainly because direct data is lacking. Here, we have provided direct evidence that the activity of the endoplasmic reticulum (ER) translocon protein Sec61 is essential for endosome-to-cytosol translocation. We generated a Sec61-specific intrabody, a crucial tool that trapped Sec61 in the ER and prevented its recruitment into endosomes without influencing Sec61 activity and antigen presentation in the ER. Expression of this ER intrabody inhibited antigen translocation and cross-presentation, demonstrating that endosomal Sec61 indeed mediates antigen transport across endosomal membranes. Moreover, we showed that the recruitment of Sec61 toward endosomes, and hence antigen translocation and cross-presentation, is dependent on dendritic cell activation by Toll-like receptor (TLR) ligands. These data shed light on a long-lasting question regarding antigen cross-presentation and point out a role of the ER-associated degradation machinery in compartments distinct from the ER. Copyright © 2015 Elsevier Inc. All rights reserved.

African Swine Fever Virus Undergoes Outer Envelope Disruption, Capsid Disassembly and Inner Envelope Fusion before Core Release from Multivesicular Endosomes.

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Bruno Hernáez

2016-04-01

Full Text Available African swine fever virus (ASFV is a nucleocytoplasmic large DNA virus (NCLDV that causes a highly lethal disease in domestic pigs. As other NCLDVs, the extracellular form of ASFV possesses a multilayered structure consisting of a genome-containing nucleoid successively wrapped by a thick protein core shell, an inner lipid membrane, an icosahedral protein capsid and an outer lipid envelope. This structural complexity suggests an intricate mechanism of internalization in order to deliver the virus genome into the cytoplasm. By using flow cytometry in combination with pharmacological entry inhibitors, as well as fluorescence and electron microscopy approaches, we have dissected the entry and uncoating pathway used by ASFV to infect the macrophage, its natural host cell. We found that purified extracellular ASFV is internalized by both constitutive macropinocytosis and clathrin-mediated endocytosis. Once inside the cell, ASFV particles move from early endosomes or macropinosomes to late, multivesicular endosomes where they become uncoated. Virus uncoating requires acidic pH and involves the disruption of the outer membrane as well as of the protein capsid. As a consequence, the inner viral membrane becomes exposed and fuses with the limiting endosomal membrane to release the viral core into the cytosol. Interestingly, virus fusion is dependent on virus protein pE248R, a transmembrane polypeptide of the inner envelope that shares sequence similarity with some members of the poxviral entry/fusion complex. Collective evidence supports an entry model for ASFV that might also explain the uncoating of other multienveloped icosahedral NCLDVs.

The TIP30 protein complex, arachidonic acid and coenzyme A are required for vesicle membrane fusion.

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Chengliang Zhang

Full Text Available Efficient membrane fusion has been successfully mimicked in vitro using artificial membranes and a number of cellular proteins that are currently known to participate in membrane fusion. However, these proteins are not sufficient to promote efficient fusion between biological membranes, indicating that critical fusogenic factors remain unidentified. We have recently identified a TIP30 protein complex containing TIP30, acyl-CoA synthetase long-chain family member 4 (ACSL4 and Endophilin B1 (Endo B1 that promotes the fusion of endocytic vesicles with Rab5a vesicles, which transport endosomal acidification enzymes vacuolar (H⁺-ATPases (V-ATPases to the early endosomes in vivo. Here, we demonstrate that the TIP30 protein complex facilitates the fusion of endocytic vesicles with Rab5a vesicles in vitro. Fusion of the two vesicles also depends on arachidonic acid, coenzyme A and the synthesis of arachidonyl-CoA by ACSL4. Moreover, the TIP30 complex is able to transfer arachidonyl groups onto phosphatidic acid (PA, producing a new lipid species that is capable of inducing close contact between membranes. Together, our data suggest that the TIP30 complex facilitates biological membrane fusion through modification of PA on membranes.

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

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Chao-Yang Lai

2017-01-01

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

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

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

2014-01-01

The endosomal SNARE protein syntaxin-8 interacts with the acid-sensitive potassium channel TASK-1. The functional relevance of this interaction was studied by heterologous expression of these proteins (and mutants thereof) in Xenopus oocytes and in mammalian cell lines. Coexpression of syntaxin-8 caused a fourfold reduction in TASK-1 current, a corresponding reduction in the expression of TASK-1 at the cell surface, and a marked increase in the rate of endocytosis of the channel. TASK-1 and syntaxin-8 colocalized in the early endosomal compartment, as indicated by the endosomal markers 2xFYVE and rab5. The stimulatory effect of the SNARE protein on the endocytosis of the channel was abolished when both an endocytosis signal in TASK-1 and an endocytosis signal in syntaxin-8 were mutated. A syntaxin-8 mutant that cannot assemble with other SNARE proteins had virtually the same effect as wild-type syntaxin-8. Total internal reflection fluorescence microscopy showed formation and endocytosis of vesicles containing fluorescence-tagged clathrin, TASK-1, and/or syntaxin-8. Our results suggest that the unassembled form of syntaxin-8 and the potassium channel TASK-1 are internalized via clathrin-mediated endocytosis in a cooperative manner. This implies that syntaxin-8 regulates the endocytosis of TASK-1. Our study supports the idea that endosomal SNARE proteins can have functions unrelated to membrane fusion. PMID:24743596

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

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Hendrik Fuchs

2016-07-01

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

The endosomal recycling of FgSnc1 by FgSnx41-FgSnx4 heterodimer is essential for polarized growth and pathogenicity in Fusarium graminearum.

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Zheng, Wenhui; Lin, Yahong; Fang, Wenqin; Zhao, Xu; Lou, Yi; Wang, Guanghui; Zheng, Huawei; Liang, Qifu; Abubakar, Yakubu Saddeeq; Olsson, Stefan; Zhou, Jie; Wang, Zonghua

2018-04-20

Endosomal sorting machineries regulate the transport of their cargoes among intracellular compartments. However, the molecular nature of such intracellular trafficking processes in pathogenic fungal development and pathogenicity remains unclear. Here, we dissect the roles and molecular mechanisms of two sorting nexin proteins and their cargoes in endosomal recycling in Fusarium graminearum using high-resolution microscopy and high-throughput co-immunoprecipitation strategies. We show that the sorting nexins, FgSnx41 and FgSnx4, interact with each other and assemble into a functionally interdependent heterodimer through their respective BAR domains. Further analyses demonstrate that the dimer localizes to the early endosomal membrane and coordinates endosomal sorting. The small GTPase FgRab5 regulates the correct localization of FgSnx41-FgSnx4 and is consequently required for its trafficking function. The protein FgSnc1 is a cargo of FgSnx41-FgSnx4 and regulates the fusion of secreted vesicles with the fungal growing apex and plasma membrane. In the absence of FgSnx41 or FgSnx4, FgSnc1 is mis-sorted and degraded in the vacuole, and null deletion of either component causes defects in the fungal polarized growth and virulence. Overall, for the first time, our results reveal the mechanism of FgSnc1 endosomal recycling by FgSnx41-FgSnx4 heterodimer which is essential for polarized growth and pathogenicity in F. graminearum. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

Method of producing a carbon coated ceramic membrane and associated product

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Liu, Paul K. T.; Gallaher, George R.; Wu, Jeffrey C. S.

1993-01-01

A method of producing a carbon coated ceramic membrane including passing a selected hydrocarbon vapor through a ceramic membrane and controlling ceramic membrane exposure temperature and ceramic membrane exposure time. The method produces a carbon coated ceramic membrane of reduced pore size and modified surface properties having increased chemical, thermal and hydrothermal stability over an uncoated ceramic membrane.

Effect of diphtheria toxin T-domain on endosomal pH

Directory of Open Access Journals (Sweden)

A. J. Labyntsev

2015-08-01

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

Proteolytic processing of epidermal growth factor within endosomes

International Nuclear Information System (INIS)

Gorman, R.M.; Savage, C.R. Jr.; Poretz, R.D.; Schaudies, R.P.

1986-01-01

The authors have reported previously that EGF enters 3 biochemically distinct non-lysosomal intracellular compartments prior to detection within lysosomes. Earlier studies have demonstrated that EGF is processes by sequential removal of 1, 4 and 1 aminoacyl residues at the C-terminus. The final form, which lacks the 6 residues, accumulates in secondary lysosomes. After subcellular fractionation of fibroblasts exposed to 125 I-EGF, ligand is detected with 3 non-lysosomal endocytic compartments and is fully processed prior to entrance into secondary lysosome. Following internalization, EGF enters an early endosomal compartment (E 1 ). The composition of the ligand (60%, -1 form; 40%, native form) represents an enhancement of the -1 form relative to that on the plasma membrane following the 90 min, 0 0 binding period. The proportion of different EGF forms in E 1 remains constant through the 2 min pulse and chase periods up to 30 min. However, in the ultimate endosomal compartment, E 4 , the proportion of the -6 form increases from 25% at 15 min to greater than 75% in 30 min, with a concomitant decrease of the -1 and -5 forms. Secondary lysosomes contain an EGF composition similar to that found in E 4 at 30 min. Accordingly, it appears that EGF is processed to the -6 form following passage through E 1 and during its tenure in E 4

Anterograde trafficking of KCa3.1 in polarized epithelia is Rab1- and Rab8-dependent and recycling endosome-independent.

Directory of Open Access Journals (Sweden)

Claudia A Bertuccio

Full Text Available The intermediate conductance, Ca2+-activated K+ channel (KCa3.1 targets to the basolateral (BL membrane in polarized epithelia where it plays a key role in transepithelial ion transport. However, there are no studies defining the anterograde and retrograde trafficking of KCa3.1 in polarized epithelia. Herein, we utilize Biotin Ligase Acceptor Peptide (BLAP-tagged KCa3.1 to address these trafficking steps in polarized epithelia, using MDCK, Caco-2 and FRT cells. We demonstrate that KCa3.1 is exclusively targeted to the BL membrane in these cells when grown on filter supports. Following endocytosis, KCa3.1 degradation is prevented by inhibition of lysosomal/proteosomal pathways. Further, the ubiquitylation of KCa3.1 is increased following endocytosis from the BL membrane and PR-619, a deubiquitylase inhibitor, prevents degradation, indicating KCa3.1 is targeted for degradation by ubiquitylation. We demonstrate that KCa3.1 is targeted to the BL membrane in polarized LLC-PK1 cells which lack the μ1B subunit of the AP-1 complex, indicating BL targeting of KCa3.1 is independent of μ1B. As Rabs 1, 2, 6 and 8 play roles in ER/Golgi exit and trafficking of proteins to the BL membrane, we evaluated the role of these Rabs in the trafficking of KCa3.1. In the presence of dominant negative Rab1 or Rab8, KCa3.1 cell surface expression was significantly reduced, whereas Rabs 2 and 6 had no effect. We also co-immunoprecipitated KCa3.1 with both Rab1 and Rab8. These results suggest these Rabs are necessary for the anterograde trafficking of KCa3.1. Finally, we determined whether KCa3.1 traffics directly to the BL membrane or through recycling endosomes in MDCK cells. For these studies, we used either recycling endosome ablation or dominant negative RME-1 constructs and determined that KCa3.1 is trafficked directly to the BL membrane rather than via recycling endosomes. These results are the first to describe the anterograde and retrograde trafficking of KCa3

Shoc2 is targeted to late endosomes and required for Erk1/2 activation in EGF-stimulated cells.

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Emilia Galperin

Full Text Available Shoc2 is the putative scaffold protein that interacts with RAS and RAF, and positively regulates signaling to extracellular signal-regulated protein kinases 1 and 2 (ERK1/2. To elucidate the mechanism by which Shoc2 regulates ERK1/2 activation by the epidermal growth factor (EGF receptor (EGFR, we studied subcellular localization of Shoc2. Upon EGFR activation, endogenous Shoc2 and red fluorescent protein tagged Shoc2 were translocated from the cytosol to a subset of late endosomes containing Rab7. The endosomal recruitment of Shoc2 was blocked by overexpression of a GDP-bound H-RAS (N17S mutant and RNAi knockdown of clathrin, suggesting the requirement of RAS activity and clathrin-dependent endocytosis. RNAi depletion of Shoc2 strongly inhibited activation of ERK1/2 by low, physiological EGF concentrations, which was rescued by expression of wild-type recombinant Shoc2. In contrast, the Shoc2 (S2G mutant, that is myristoylated and found in patients with the Noonan-like syndrome, did not rescue ERK1/2 activation in Shoc2-depleted cells. Shoc2 (S2G was not located in late endosomes but was present on the plasma membrane and early endosomes. These data suggest that targeting of Shoc2 to late endosomes may facilitate EGFR-induced ERK activation under physiological conditions of cell stimulation by EGF, and therefore, may be involved in the spatiotemporal regulation of signaling through the RAS-RAF module.

Pannexin2 oligomers localize into endosomal vesicles in mammalian cells while Pannexin1 channels traffic to the plasma membrane

Directory of Open Access Journals (Sweden)

Daniela eBoassa

2015-02-01

Full Text Available Pannexin2 (Panx2 is the largest of three members of the pannexin proteins. Pannexins are topologically related to connexins and innexins, but serve different functional roles than forming gap junctions. We previously showed that pannexins form oligomeric channels but unlike connexins and innexins, they form only single membrane channels. High levels of Panx2 mRNA and protein in the Central Nervous System (CNS have been documented. Whereas Pannexin1 (Panx1 is fairly ubiquitous and Pannexin3 (Panx3 is found in skin and connective tissue, both are fully glycosylated, traffic to the plasma membrane and have functions correlated with extracellular ATP release. Here, we describe trafficking and subcellular localizations of exogenous Panx2 and Panx1 protein expression in MDCK, HeLa and HEK293T cells as well as endogenous Panx1 and Panx2 patterns in the CNS. Panx2 was found in intracellular localizations, was partially N-glycosylated, and localizations were non-overlapping with Panx1. Confocal images of hippocampal sections immunolabeled for the astrocytic protein GFAP, Panx1 and Panx2 demonstrated that the two isoforms, Panx1 and Panx2, localized at different subcellular compartments in both astrocytes and neurons. Using recombinant fusions of Panx2 with appended genetic tags developed for correlated light and electron microscopy and then expressed in different cell lines, we determined that Panx2 is localized in the membrane of intracellular vesicles and not in the endoplasmic reticulum as initially indicated by calnexin colocalization experiments. Dual immunofluorescence imaging with protein markers for specific vesicle compartments showed that Panx2 vesicles are early endosomal in origin. In electron tomographic volumes, cross-sections of these vesicles displayed fine structural details and close proximity to actin filaments. Thus, pannexins expressed at different subcellular compartments likely exert distinct functional roles, particularly in the

Successive membrane separation processes simplify concentration of lipases produced by Aspergillus niger by solid-state fermentation.

Science.gov (United States)

Reinehr, Christian Oliveira; Treichel, Helen; Tres, Marcus Vinicius; Steffens, Juliana; Brião, Vandré Barbosa; Colla, Luciane Maria

2017-06-01

In this study, we developed a simplified method for producing, separating, and concentrating lipases derived from solid-state fermentation of agro-industrial residues by filamentous fungi. First, we used Aspergillus niger to produce lipases with hydrolytic activity. We analyzed the separation and concentration of enzymes using membrane separation processes. The sequential use of microfiltration and ultrafiltration processes made it possible to obtain concentrates with enzymatic activities much higher than those in the initial extract. The permeate flux was higher than 60 L/m 2 h during microfiltration using 20- and 0.45-µm membranes and during ultrafiltration using 100- and 50-kDa membranes, where fouling was reversible during the filtration steps, thereby indicating that the fouling may be removed by cleaning processes. These results demonstrate the feasibility of lipase production using A. niger by solid-state fermentation of agro-industrial residues, followed by successive tangential filtration with membranes, which simplify the separation and concentration steps that are typically required in downstream processes.

Efficient myogenic differentiation of human adipose-derived stem cells by the transduction of engineered MyoD protein

Energy Technology Data Exchange (ETDEWEB)

Sung, Min Sun [Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806 (Korea, Republic of); Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350 (Korea, Republic of); Mun, Ji-Young [Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806 (Korea, Republic of); Kwon, Ohsuk [Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806 (Korea, Republic of); Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350 (Korea, Republic of); Kwon, Ki-Sun [Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806 (Korea, Republic of); Oh, Doo-Byoung, E-mail: dboh@kribb.re.kr [Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806 (Korea, Republic of); Biosystems and Bioengineering Program, University of Science and Technology (UST), Daejeon 305-350 (Korea, Republic of)

2013-07-19

Highlights: •MyoD was engineered to contain protein transduction domain and endosome-disruptive INF7 peptide. •The engineered MyoD-IT showed efficient nuclear targeting through an endosomal escape by INF7 peptide. •By applying MyoD-IT, human adipose-derived stem cells (hASCs) were differentiated into myogenic cells. •hASCs differentiated by applying MyoD-IT fused to myotubes through co-culturing with mouse myoblasts. •Myogenic differentiation using MyoD-IT is a safe method without the concern of altering the genome. -- Abstract: Human adipose-derived stem cells (hASCs) have great potential as cell sources for the treatment of muscle disorders. To provide a safe method for the myogenic differentiation of hASCs, we engineered the MyoD protein, a key transcription factor for myogenesis. The engineered MyoD (MyoD-IT) was designed to contain the TAT protein transduction domain for cell penetration and the membrane-disrupting INF7 peptide, which is an improved version of the HA2 peptide derived from influenza. MyoD-IT showed greatly improved nuclear targeting ability through an efficient endosomal escape induced by the pH-sensitive membrane disruption of the INF7 peptide. By applying MyoD-IT to a culture, hASCs were efficiently differentiated into long spindle-shaped myogenic cells expressing myosin heavy chains. Moreover, these cells differentiated by an application of MyoD-IT fused to myotubes with high efficiency through co-culturing with mouse C2C12 myoblasts. Because internalized proteins can be degraded in cells without altering the genome, the myogenic differentiation of hASCs using MyoD-IT would be a safe and clinically applicable method.

Efficient myogenic differentiation of human adipose-derived stem cells by the transduction of engineered MyoD protein

International Nuclear Information System (INIS)

Sung, Min Sun; Mun, Ji-Young; Kwon, Ohsuk; Kwon, Ki-Sun; Oh, Doo-Byoung

2013-01-01

Highlights: •MyoD was engineered to contain protein transduction domain and endosome-disruptive INF7 peptide. •The engineered MyoD-IT showed efficient nuclear targeting through an endosomal escape by INF7 peptide. •By applying MyoD-IT, human adipose-derived stem cells (hASCs) were differentiated into myogenic cells. •hASCs differentiated by applying MyoD-IT fused to myotubes through co-culturing with mouse myoblasts. •Myogenic differentiation using MyoD-IT is a safe method without the concern of altering the genome. -- Abstract: Human adipose-derived stem cells (hASCs) have great potential as cell sources for the treatment of muscle disorders. To provide a safe method for the myogenic differentiation of hASCs, we engineered the MyoD protein, a key transcription factor for myogenesis. The engineered MyoD (MyoD-IT) was designed to contain the TAT protein transduction domain for cell penetration and the membrane-disrupting INF7 peptide, which is an improved version of the HA2 peptide derived from influenza. MyoD-IT showed greatly improved nuclear targeting ability through an efficient endosomal escape induced by the pH-sensitive membrane disruption of the INF7 peptide. By applying MyoD-IT to a culture, hASCs were efficiently differentiated into long spindle-shaped myogenic cells expressing myosin heavy chains. Moreover, these cells differentiated by an application of MyoD-IT fused to myotubes with high efficiency through co-culturing with mouse C2C12 myoblasts. Because internalized proteins can be degraded in cells without altering the genome, the myogenic differentiation of hASCs using MyoD-IT would be a safe and clinically applicable method

Ebola Viral Glycoprotein Bound to Its Endosomal Receptor Niemann-Pick C1.

Science.gov (United States)

Wang, Han; Shi, Yi; Song, Jian; Qi, Jianxun; Lu, Guangwen; Yan, Jinghua; Gao, George F

2016-01-14

Filoviruses, including Ebola and Marburg, cause fatal hemorrhagic fever in humans and primates. Understanding how these viruses enter host cells could help to develop effective therapeutics. An endosomal protein, Niemann-Pick C1 (NPC1), has been identified as a necessary entry receptor for this process, and priming of the viral glycoprotein (GP) to a fusion-competent state is a prerequisite for NPC1 binding. Here, we have determined the crystal structure of the primed GP (GPcl) of Ebola virus bound to domain C of NPC1 (NPC1-C) at a resolution of 2.3 Å. NPC1-C utilizes two protruding loops to engage a hydrophobic cavity on head of GPcl. Upon enzymatic cleavage and NPC1-C binding, conformational change in the GPcl further affects the state of the internal fusion loop, triggering membrane fusion. Our data therefore provide structural insights into filovirus entry in the late endosome and the molecular basis for design of therapeutic inhibitors of viral entry. Copyright © 2016 Elsevier Inc. All rights reserved.

Membrane crystallization for recovery of salts from produced water

DEFF Research Database (Denmark)

Quist-Jensen, Cejna Anna; Jensen, Henriette Casper; Ali, Aamer

Membrane Crystallization (MCr) is a novel technology able to recover freshwater and high-purity salts from complex solutions and therefore, is suggested for a better exploitation of wastewater streams. Unlike other membrane processes, MCr is not limited by high concentrations and, therefore, the ......, the membrane maintained its hydrophobic nature despite that produced water contained oil residues. Conductivity and HPLC was utilized to analyze the quality of the permeate stream......., the solutions can be treated to achieve saturation level. Hereby different salts can be precipitated and directly recovered from various streams. In this study, it is shown that MCr is able to treat produced water by producing clean water and simultaneously NaCl crystals. The recovered crystals exhibited high...

The influence of saponins on cell membrane cholesterol.

Science.gov (United States)

Böttger, Stefan; Melzig, Matthias F

2013-11-15

of endosomes and lysosomes. In these experiments ECV-304 cells were either incubated with (3)H-cholesterol or with (3)H-cholesterol and 5 μM saponin. After isolation of the endosomes/lysosomes their (3)H-cholesterol content was measured. A significant influence of the saponins on the cholesterol content of endosomal/lysosomal membranes was not detected. Copyright © 2013 Elsevier Ltd. All rights reserved.

RAB-10-Dependent Membrane Transport Is Required for Dendrite Arborization

Science.gov (United States)

Zou, Wei; Yadav, Smita; DeVault, Laura; Jan, Yuh Nung; Sherwood, David R.

2015-01-01

Formation of elaborately branched dendrites is necessary for the proper input and connectivity of many sensory neurons. Previous studies have revealed that dendritic growth relies heavily on ER-to-Golgi transport, Golgi outposts and endocytic recycling. How new membrane and associated cargo is delivered from the secretory and endosomal compartments to sites of active dendritic growth, however, remains unknown. Using a candidate-based genetic screen in C. elegans, we have identified the small GTPase RAB-10 as a key regulator of membrane trafficking during dendrite morphogenesis. Loss of rab-10 severely reduced proximal dendritic arborization in the multi-dendritic PVD neuron. RAB-10 acts cell-autonomously in the PVD neuron and localizes to the Golgi and early endosomes. Loss of function mutations of the exocyst complex components exoc-8 and sec-8, which regulate tethering, docking and fusion of transport vesicles at the plasma membrane, also caused proximal dendritic arborization defects and led to the accumulation of intracellular RAB-10 vesicles. In rab-10 and exoc-8 mutants, the trans-membrane proteins DMA-1 and HPO-30, which promote PVD dendrite stabilization and branching, no longer localized strongly to the proximal dendritic membranes and instead were sequestered within intracellular vesicles. Together these results suggest a crucial role for the Rab10 GTPase and the exocyst complex in controlling membrane transport from the secretory and/or endosomal compartments that is required for dendritic growth. PMID:26394140

IL4/PGE2 induction of an enlarged early endosomal compartment in mouse macrophages is Rab5-dependent

International Nuclear Information System (INIS)

Wainszelbaum, Marisa J.; Proctor, Brandon M.; Pontow, Suzanne E.; Stahl, Philip D.; Barbieri, M. Alejandro

2006-01-01

The endosomal compartment and the plasma membrane form a complex partnership that controls signal transduction and trafficking of different molecules. The specificity and functionality of the early endocytic pathway are regulated by a growing number of Rab GTPases, particularly Rab5. In this study, we demonstrate that IL4 (a Th-2 cytokine) and prostaglandin E 2 (PGE 2 ) synergistically induce Rab5 and several Rab effector proteins, including Rin1 and EEA1, and promote the formation of an enlarged early endocytic (EEE) compartment. Endosome enlargement is linked to a substantial induction of the mannose receptor (MR), a well-characterized macrophage endocytic receptor. Both MR levels and MR-mediated endocytosis are enhanced approximately 7-fold. Fluid-phase endocytosis is also elevated in treated cells. Light microscopy and fractionation studies reveal that MR colocalizes predominantly with Rab5a and partially with Rab11, an endosomal recycling pathway marker. Using retroviral expression of Rab5a:S34N, a dominant negative mutant, and siRNA Rab5a silencing, we demonstrate that Rab5a is essential for the large endosome phenotype and for localization of MR in these structures. We speculate that the EEE is maintained by activated Rab5, and that the EEE phenotype is part of some macrophage developmental program such as cell fusion, a characteristic of IL4-stimulated cells

Hsp70 facilitates trans-membrane transport of bacterial ADP-ribosylating toxins into the cytosol of mammalian cells.

Science.gov (United States)

Ernst, Katharina; Schmid, Johannes; Beck, Matthias; Hägele, Marlen; Hohwieler, Meike; Hauff, Patricia; Ückert, Anna Katharina; Anastasia, Anna; Fauler, Michael; Jank, Thomas; Aktories, Klaus; Popoff, Michel R; Schiene-Fischer, Cordelia; Kleger, Alexander; Müller, Martin; Frick, Manfred; Barth, Holger

2017-06-02

Binary enterotoxins Clostridium (C.) botulinum C2 toxin, C. perfringens iota toxin and C. difficile toxin CDT are composed of a transport (B) and a separate non-linked enzyme (A) component. Their B-components mediate endocytic uptake into mammalian cells and subsequently transport of the A-components from acidic endosomes into the cytosol, where the latter ADP-ribosylate G-actin resulting in cell rounding and cell death causing clinical symptoms. Protein folding enzymes, including Hsp90 and peptidyl-prolyl cis/trans isomerases facilitate transport of the A-components across endosomal membranes. Here, we identified Hsp70 as a novel host cell factor specifically interacting with A-components of C2, iota and CDT toxins to facilitate their transport into the cell cytosol. Pharmacological Hsp70-inhibition specifically prevented pH-dependent trans-membrane transport of A-components into the cytosol thereby protecting living cells and stem cell-derived human miniguts from intoxication. Thus, Hsp70-inhibition might lead to development of novel therapeutic strategies to treat diseases associated with bacterial ADP-ribosylating toxins.

Transport, metabolism, and endosomal trafficking-dependent regulation of intestinal fructose absorption

Science.gov (United States)

Patel, Chirag; Douard, Veronique; Yu, Shiyan; Gao, Nan; Ferraris, Ronaldo P.

2015-01-01

Dietary fructose that is linked to metabolic abnormalities can up-regulate its own absorption, but the underlying regulatory mechanisms are not known. We hypothesized that glucose transporter (GLUT) protein, member 5 (GLUT5) is the primary fructose transporter and that fructose absorption via GLUT5, metabolism via ketohexokinase (KHK), as well as GLUT5 trafficking to the apical membrane via the Ras-related protein-in-brain 11 (Rab11)a-dependent endosomes are each required for regulation. Introducing fructose but not lysine and glucose solutions into the lumen increased by 2- to 10-fold the heterogeneous nuclear RNA, mRNA, protein, and activity levels of GLUT5 in adult wild-type mice consuming chow. Levels of GLUT5 were >100-fold that of candidate apical fructose transporters GLUTs 7, 8, and 12 whose expression, and that of GLUT 2 and the sodium-dependent glucose transporter protein 1 (SGLT1), was not regulated by luminal fructose. GLUT5-knockout (KO) mice exhibited no facilitative fructose transport and no compensatory increases in activity and expression of SGLT1 and other GLUTs. Fructose could not up-regulate GLUT5 in GLUT5-KO, KHK-KO, and intestinal epithelial cell-specific Rab11a-KO mice. The fructose-specific metabolite glyceraldehyde did not increase GLUT5 expression. GLUT5 is the primary transporter responsible for facilitative absorption of fructose, and its regulation specifically requires fructose uptake and metabolism and normal GLUT5 trafficking to the apical membrane.—Patel, C., Douard, V., Yu, S., Gao, N., Ferraris, R. P. Transport, metabolism, and endosomal trafficking-dependent regulation of intestinal fructose absorption. PMID:26071406

FAM21 directs SNX27–retromer cargoes to the plasma membrane by preventing transport to the Golgi apparatus

Science.gov (United States)

Lee, Seongju; Chang, Jaerak; Blackstone, Craig

2016-01-01

The endosomal network maintains cellular homeostasis by sorting, recycling and degrading endocytosed cargoes. Retromer organizes the endosomal sorting pathway in conjunction with various sorting nexin (SNX) proteins. The SNX27–retromer complex has recently been identified as a major endosomal hub that regulates endosome-to-plasma membrane recycling by preventing lysosomal entry of cargoes. Here, we show that SNX27 directly interacts with FAM21, which also binds retromer, within the Wiskott–Aldrich syndrome protein and SCAR homologue (WASH) complex. This interaction is required for the precise localization of SNX27 at an endosomal subdomain as well as for recycling of SNX27-retromer cargoes. Furthermore, FAM21 prevents cargo transport to the Golgi apparatus by controlling levels of phosphatidylinositol 4-phosphate, which facilitates cargo dissociation at the Golgi. Together, our results demonstrate that the SNX27–retromer–WASH complex directs cargoes to the plasma membrane by blocking their transport to lysosomes and the Golgi. PMID:26956659

Plasma membrane cholesterol level and agonist-induced internalization of δ-opioid receptors; colocalization study with intracellular membrane markers of Rab family.

Science.gov (United States)

Brejchova, Jana; Vosahlikova, Miroslava; Roubalova, Lenka; Parenti, Marco; Mauri, Mario; Chernyavskiy, Oleksandr; Svoboda, Petr

2016-08-01

Decrease of cholesterol level in plasma membrane of living HEK293 cells transiently expressing FLAG-δ-OR by β-cyclodextrin (β-CDX) resulted in a slight internalization of δ-OR. Massive internalization of δ-OR induced by specific agonist DADLE was diminished in cholesterol-depleted cells. These results suggest that agonist-induced internalization of δ-OR, which has been traditionally attributed exclusively to clathrin-mediated pathway, proceeds at least partially via membrane domains. Identification of internalized pools of FLAG-δ-OR by colocalization studies with proteins of Rab family indicated the decreased presence of receptors in early endosomes (Rab5), late endosomes and lysosomes (Rab7) and fast recycling vesicles (Rab4). Slow type of recycling (Rab11) was unchanged by cholesterol depletion. As expected, agonist-induced internalization of oxytocin receptors was totally suppressed in β-CDX-treated cells. Determination of average fluorescence lifetime of TMA-DPH, the polar derivative of hydrophobic membrane probe diphenylhexatriene, in live cells by FLIM indicated a significant alteration of the overall PM structure which may be interpreted as an increased "water-accessible space" within PM area. Data obtained by studies of HEK293 cells transiently expressing FLAG-δ-OR by "antibody feeding" method were extended by analysis of the effect of cholesterol depletion on distribution of FLAG-δ-OR in sucrose density gradients prepared from HEK293 cells stably expressing FLAG-δ-OR. Major part of FLAG-δ-OR was co-localized with plasma membrane marker Na,K-ATPase and β-CDX treatment resulted in shift of PM fragments containing both FLAG-δ-OR and Na,K-ATPase to higher density. Thus, the decrease in content of the major lipid constituent of PM resulted in increased density of resulting PM fragments.

Leghemoglobin-derived radicals. Evidence for multiple protein-derived radicals and the initiation of peribacteroid membrane damage

DEFF Research Database (Denmark)

Moreau, S; Davies, Michael Jonathan; Mathieu, C

1996-01-01

, with the consequent generation of lipid-derived radicals. The formation of such radicals may result in the depletion of membrane antioxidants and the initiation of lipid peroxidation. This transfer of damage from the heme center via the protein surface to neighboring membranes may be of considerable biological......-derived phenoxyl radical present at Tyr-133 in the soybean protein and Tyr-138 in the lupin protein. To obtain further information on these protein radicals and their potential interaction with the physiologically important peribacteroid membrane (which surrounds the microsymbiont in vivo), EPR spin trapping......); these radicals may be side chain- or alpha-carbon-derived, their exact sites have not been determined. Some of these radicals are on the protein surface and may be key intermediates in the formation of protein dimers. These radicals have been shown to be capable of reacting with peribacteroid membrane fractions...

PDGF-regulated rab4-dependent recycling of alphavbeta3 integrin from early endosomes is necessary for cell adhesion and spreading.

Science.gov (United States)

Roberts, M; Barry, S; Woods, A; van der Sluijs, P; Norman, J

2001-09-18

It has been postulated that the regulation of integrin vesicular traffic facilitates cell migration by internalizing integrins at the rear of the cell and transporting them forward within vesicles for exocytosis at the leading edge to form new contacts with the extracellular matrix. The rab family of GTPases control key targeting events in the endo/exocytic pathway; therefore, these GTPases may be involved in the regulation of cell-matrix contact assembly. The endo/exocytic cycle of alphavbeta3 and alpha5beta1 integrins was studied using mouse 3T3 fibroblast cell lines. In serum-starved cells, internalized integrins were transported through rab4-positive, early endosomes and arrived at the rab11-positive, perinuclear recycling compartment approximately 30 min after endocytosis. From the recycling compartment, integrins were recycled to the plasma membrane in a rab11-dependent fashion. Following treatment with PDGF, alphavbeta3 integrin, but not alpha5beta1, was rapidly recycled directly back to the plasma membrane from the early endosomes via a rab4-dependent mechanism without the involvement of rab11. This rapid recycling pathway directed alphavbeta3 to numerous small puncta distributed evenly across the dorsal surface of the cell, and the integrin only became localized into focal complexes at later times following PDGF addition. Interestingly, inhibition of PDGF-stimulated alphavbeta3 recycling using dominant-negative rab4 mutants compromised cell adhesion and spreading on vitronectin (a ligand for alphavbeta3), but adhesion to fibronectin (a ligand for alpha5beta1 and alphavbeta3) was unchanged. We propose that growth factor-regulated, rab4-dependent recycling of alphavbeta3 integrin from early endosomes to the plasma membrane is a critical upstream event in the assembly of cell-matrix contacts.

Cost-Effective Method for Producing Self Supported Palladium Alloy Membranes for Use in Efficient Production of Coal Derived Hydrogen

Energy Technology Data Exchange (ETDEWEB)

K. Coulter

2008-03-31

Southwest Research Institute{reg_sign} (SwRI{reg_sign}) has utilized its expertise in large-area vacuum deposition methods to conduct research into the fabrication of dense, freestanding Pd-alloy membranes that are 3-5 microns thick and over 100 in{sup 2} in area. The membranes were deposited onto flexible and rigid supports that were subsequently removed and separated using novel techniques developed over the course of the project. Using these methods, the production of novel alloy compositions centered around the Pd-Cu system were developed with the objective of producing a thermally stable, nano-crystalline grain structure with the highest flux recorded as 242 SCFH/ft{sup 2} for a 2 {micro}m thick Pd{sub 53}Cu{sub 47} at 400 C and 20 psig feed pressure which when extrapolated is over twice the 2010 Department of Energy pure H{sub 2} flux target. Several membranes were made with the same permeability, but with different thicknesses and these membranes were highly selective. Researchers at the Colorado School of Mines supported the effort with extensive testing of experimental membranes as well as design and modeling of novel alloy composite structures. IdaTech provided commercial bench testing and analysis of SwRI-manufactured membranes. The completed deliverables for the project include test data on the performance of experimental membranes fabricated by vacuum deposition and several Pd-alloy membranes that were supplied to IdaTech for testing.

Role of the AP-5 adaptor protein complex in late endosome-to-Golgi retrieval.

Directory of Open Access Journals (Sweden)

Jennifer Hirst

2018-01-01

Full Text Available The AP-5 adaptor protein complex is presumed to function in membrane traffic, but so far nothing is known about its pathway or its cargo. We have used CRISPR-Cas9 to knock out the AP-5 ζ subunit gene, AP5Z1, in HeLa cells, and then analysed the phenotype by subcellular fractionation profiling and quantitative mass spectrometry. The retromer complex had an altered steady-state distribution in the knockout cells, and several Golgi proteins, including GOLIM4 and GOLM1, were depleted from vesicle-enriched fractions. Immunolocalisation showed that loss of AP-5 led to impaired retrieval of the cation-independent mannose 6-phosphate receptor (CIMPR, GOLIM4, and GOLM1 from endosomes back to the Golgi region. Knocking down the retromer complex exacerbated this phenotype. Both the CIMPR and sortilin interacted with the AP-5-associated protein SPG15 in pull-down assays, and we propose that sortilin may act as a link between Golgi proteins and the AP-5/SPG11/SPG15 complex. Together, our findings suggest that AP-5 functions in a novel sorting step out of late endosomes, acting as a backup pathway for retromer. This provides a mechanistic explanation for why mutations in AP-5/SPG11/SPG15 cause cells to accumulate aberrant endolysosomes, and highlights the role of endosome/lysosome dysfunction in the pathology of hereditary spastic paraplegia and other neurodegenerative disorders.

Mobility of tethering factor EEA1 on endosomes is decreased upon stimulation of EGF receptor endocytosis in HeLa cells

Energy Technology Data Exchange (ETDEWEB)

Kosheverova, Vera V., E-mail: kosheverova_vera@incras.ru [Institute of Cytology of RAS, 4, Tikhoretsky Ave, St. Petersburg, 194064 (Russian Federation); Kamentseva, Rimma S., E-mail: rkamentseva@yandex.ru [Institute of Cytology of RAS, 4, Tikhoretsky Ave, St. Petersburg, 194064 (Russian Federation); St. Petersburg State University, 7-9, Universitetskaya nab, St. Petersburg, 199034 (Russian Federation); Gonchar, Ilya V., E-mail: ample@mail.ru [Institute of Cytology of RAS, 4, Tikhoretsky Ave, St. Petersburg, 194064 (Russian Federation); Kharchenko, Marianna V., E-mail: mariannakharchenko@gmail.com [Institute of Cytology of RAS, 4, Tikhoretsky Ave, St. Petersburg, 194064 (Russian Federation); Kornilova, Elena S., E-mail: lenkor@mail.cytspb.rssi.ru [Institute of Cytology of RAS, 4, Tikhoretsky Ave, St. Petersburg, 194064 (Russian Federation); St. Petersburg State University, 7-9, Universitetskaya nab, St. Petersburg, 199034 (Russian Federation); Department of Medical Physics, Peter the Great St. Petersburg Polytechnic University, 29, Polytechnicheskaya, St.Petersburg, 195251 (Russian Federation)

2016-04-22

Tethering factor EEA1, mediating homotypic fusion of early endosomes, was shown to be localized in membrane-bound state both in serum-deprived and stimulated for EGF receptor endocytosis cells. However, it is not known whether dynamics behavior of EEA1 is affected by EGF stimulation. We investigated EEA1 cytosol-to-membrane exchange rate in interphase HeLa cells by FRAP analysis. The data obtained fitted two-states binding model, with the bulk of membrane-associated EEA1 protein represented by the mobile fraction both in serum-starved and EGF-stimulated cells. Fast recovery state had similar half-times in the two cases: about 1.6 s and 2.8 s, respectively. However, the recovery half-time of slowly cycled EEA1 fraction significantly increased in EGF-stimulated comparing to serum-starved cells (from 21 to 99 s). We suppose that the retardation of EEA1 fluorescence recovery upon EGF-stimulation may be due to the increase of activated Rab5 on endosomal membranes, the growth of the number of tethering events between EEA1-positive vesicles and their clustering. - Highlights: • EEA1 mobility was compared in serum-starved and EGF-stimulated interphase HeLa cells. • FRAP analysis revealed fast and slow components of EEA1 recovery in both cases. • Stimulation of EGFR endocytosis did not affect fast EEA1 turnover. • EGF stimulation significantly increased half-time of slowly exchanged EEA1 fraction.

Mobility of tethering factor EEA1 on endosomes is decreased upon stimulation of EGF receptor endocytosis in HeLa cells

International Nuclear Information System (INIS)

Kosheverova, Vera V.; Kamentseva, Rimma S.; Gonchar, Ilya V.; Kharchenko, Marianna V.; Kornilova, Elena S.

2016-01-01

Tethering factor EEA1, mediating homotypic fusion of early endosomes, was shown to be localized in membrane-bound state both in serum-deprived and stimulated for EGF receptor endocytosis cells. However, it is not known whether dynamics behavior of EEA1 is affected by EGF stimulation. We investigated EEA1 cytosol-to-membrane exchange rate in interphase HeLa cells by FRAP analysis. The data obtained fitted two-states binding model, with the bulk of membrane-associated EEA1 protein represented by the mobile fraction both in serum-starved and EGF-stimulated cells. Fast recovery state had similar half-times in the two cases: about 1.6 s and 2.8 s, respectively. However, the recovery half-time of slowly cycled EEA1 fraction significantly increased in EGF-stimulated comparing to serum-starved cells (from 21 to 99 s). We suppose that the retardation of EEA1 fluorescence recovery upon EGF-stimulation may be due to the increase of activated Rab5 on endosomal membranes, the growth of the number of tethering events between EEA1-positive vesicles and their clustering. - Highlights: • EEA1 mobility was compared in serum-starved and EGF-stimulated interphase HeLa cells. • FRAP analysis revealed fast and slow components of EEA1 recovery in both cases. • Stimulation of EGFR endocytosis did not affect fast EEA1 turnover. • EGF stimulation significantly increased half-time of slowly exchanged EEA1 fraction.

Role of the Small GTPase Rho3 in Golgi/Endosome trafficking through functional interaction with adaptin in Fission Yeast.

Directory of Open Access Journals (Sweden)

Ayako Kita

Full Text Available BACKGROUND: We had previously identified the mutant allele of apm1(+ that encodes a homolog of the mammalian µ1A subunit of the clathrin-associated adaptor protein-1 (AP-1 complex, and we demonstrated the role of Apm1 in Golgi/endosome trafficking, secretion, and vacuole fusion in fission yeast. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we isolated rho3(+, which encodes a Rho-family small GTPase, an important regulator of exocystosis, as a multicopy-suppressor of the temperature-sensitive growth of the apm1-1 mutant cells. Overexpression of Rho3 suppressed the Cl(- sensitivity and immunosuppressant sensitivity of the apm1-1 mutant cells. Overexpression of Rho3 also suppressed the fragmentation of vacuoles, and the accumulation of v-SNARE Syb1 in Golgi/endosomes and partially suppressed the defective secretion associated with apm1-deletion cells. Notably, electron microscopic observation of the rho3-deletion cells revealed the accumulation of abnormal Golgi-like structures, vacuole fragmentation, and accumulation of secretory vesicles; these phenotypes were very similar to those of the apm1-deletion cells. Furthermore, the rho3-deletion cells and apm1-deletion cells showed very similar phenotypic characteristics, including the sensitivity to the immunosuppressant FK506, the cell wall-damaging agent micafungin, Cl(-, and valproic acid. Green fluorescent protein (GFP-Rho3 was localized at Golgi/endosomes as well as the plasma membrane and division site. Finally, Rho3 was shown to form a complex with Apm1 as well as with other subunits of the clathrin-associated AP-1 complex in a GTP- and effector domain-dependent manner. CONCLUSIONS/SIGNIFICANCE: Taken together, our findings reveal a novel role of Rho3 in the regulation of Golgi/endosome trafficking and suggest that clathrin-associated adaptor protein-1 and Rho3 co-ordinate in intracellular transport in fission yeast. To the best of our knowledge, this study provides the first evidence

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

Energy Technology Data Exchange (ETDEWEB)

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

2009-09-11

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

Precursors-Derived Ceramic Membranes for High-Temperature Separation of Hydrogen

OpenAIRE

Yuji, Iwamoto

2007-01-01

This review describes recent progress in the development of hydrogen-permselective ceramic membranes derived from organometallic precursors. Microstructure and gas transport property of microporous amorphous silica-based membranes are briefly described. Then, high-temperature hydrogen permselectivity, hydrothermal stability as well as hydrogen/steam selectivity of the amorphous silica-based membranes are discussed from a viewpoint of application to membrane reactors for conversion enhancement...

Membrane emulsification to produce perfume microcapsules

Science.gov (United States)

Pan, Xuemiao

Microencapsulation is an efficient technology to deliver perfume oils from consumer products onto the surface of fabrics. Microcapsules having uniform size/mechanical strength, may provide better release performance. Membrane emulsification in a dispersion cell followed by in-situ polymerization was used to prepare narrow size distribution melamine-formaldehyde (MF) microcapsules containing several types of oil-based fragrances or ingredients. Investigated in this study are the parameters impacting to the size and size distribution of the droplets and final MF microcapsules. A pilot plant-scale cross-flow membrane system was also used to produce MF microcapsules, demonstrating that the membrane emulsification process has potential to be scaled up for industrial applications. In this study, health and environmental friendly poly (methyl methacrylate) (PMMA) microcapsules with narrow size distribution were also prepared for the first time using the dispersion cell membrane emulsification system. Characterization methods previously used for thin-shell microcapsules were expanded to analyse microcapsules with thick shells. The intrinsic mechanical properties of thick shells were determined using a micromanipulation technique and finite element analysis (FEM). The microcapsules structure was also considered in the determination of the permeability and diffusivity of the perfume oils in good solvents..

Novel polybenzimidazole derivatives for high temperature polymer electrolyte membrane fuel cell applications

Science.gov (United States)

Xiao, Lixiang

Recent advances have made polymer electrolyte membrane fuel cells (PEMFCs) a leading alternative to internal combustion engines for both stationary and transportation applications. In particular, high temperature polymer electrolyte membranes operational above 120°C without humidification offer many advantages including fast electrode kinetics, high tolerance to fuel impurities and simple thermal and water management systems. A series of polybenzimidazole (PBI) derivatives including pyridine-based PBI (PPBI) and sulfonated PBI (SPBI) homopolymers and copolymers have been synthesized using polyphosphoric acid (PPA) as both solvent and polycondensation agent. High molecular weight PBI derivative polymers were obtained with well controlled backbone structures in terms of pyridine ring content, polymer backbone rigidity and degree of sulfonation. A novel process, termed the PPA process, has been developed to prepare phosphoric acid (PA) doped PBI membranes by direct-casting of the PPA polymerization solution without isolation or re-dissolution of the polymers. The subsequent hydrolysis of PPA to PA by moisture absorbed from the atmosphere usually induced a transition from the solution-like state to a gel-like state and produced PA doped PBI membranes with a desirable suite of physiochemical properties characterized by the PA doping levels, mechanical properties and proton conductivities. The effects of the polymer backbone structure on the polymer characteristics and membrane properties, i.e., the structure-property relationships of the PBI derivative polymers have been studied. The incorporation of additional basic nitrogen containing pyridine rings and sulfonic acid groups enhanced the polymer solubility in acid and dipolar solvents while retaining the inherently high thermal stability of the PBI heteroaromatic backbone. In particular, the degradation of the SPBI polymers with reasonable high molecular weights commenced above 450°C, notably higher than other

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

NARCIS (Netherlands)

Kant, Rik Henricus Nicolaas van der

2013-01-01

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

Aggregation of nanoparticles in endosomes and lysosomes produces surface-enhanced Raman spectroscopy

Science.gov (United States)

Lucas, Leanne J.; Chen, Xiaoke K.; Smith, Aaron J.; Korbelik, Mladen; Zeng, Haishan; Lee, Patrick W. K.; Hewitt, Kevin Cecil

2015-01-01

The purpose of this study was to explore the use of surface-enhanced Raman spectroscopy (SERS) to image the distribution of epidermal growth factor receptor (EGFR) in cells. To accomplish this task, 30-nm gold nanoparticles (AuNPs) tagged with antibodies to EGFR (1012 per mL) were incubated with cells (106 per mL) of the A431 human epidermoid carcinoma and normal human bronchial epithelial cell lines. Using the 632.8-nm excitation line of a He-Ne laser, Raman spectroscopy measurements were performed using a point mapping scheme. Normal cells show little to no enhancement. SERS signals were observed inside the cytoplasm of A431 cells with an overall enhancement of 4 to 7 orders of magnitude. Raman intensity maps of the 1450 and 1583 cm-1 peaks correlate well with the expected distribution of EGFR and AuNPs, aggregated following uptake by endosomes and lysosomes. Spectral features from tyrosine and tryptophan residues dominate the SERS signals.

Enrichment of Phosphatidylethanolamine in Viral Replication Compartments via Co-opting the Endosomal Rab5 Small GTPase by a Positive-Strand RNA Virus.

Directory of Open Access Journals (Sweden)

Kai Xu

2016-10-01

Full Text Available Positive-strand RNA viruses build extensive membranous replication compartments to support replication and protect the virus from antiviral responses by the host. These viruses require host factors and various lipids to form viral replication complexes (VRCs. The VRCs built by Tomato bushy stunt virus (TBSV are enriched with phosphatidylethanolamine (PE through a previously unknown pathway. To unravel the mechanism of PE enrichment within the TBSV replication compartment, in this paper, the authors demonstrate that TBSV co-opts the guanosine triphosphate (GTP-bound active form of the endosomal Rab5 small GTPase via direct interaction with the viral replication protein. Deletion of Rab5 orthologs in a yeast model host or expression of dominant negative mutants of plant Rab5 greatly decreases TBSV replication and prevents the redistribution of PE to the sites of viral replication. We also show that enrichment of PE in the viral replication compartment is assisted by actin filaments. Interestingly, the closely related Carnation Italian ringspot virus, which replicates on the boundary membrane of mitochondria, uses a similar strategy to the peroxisomal TBSV to hijack the Rab5-positive endosomes into the viral replication compartments. Altogether, usurping the GTP-Rab5-positive endosomes allows TBSV to build a PE-enriched viral replication compartment, which is needed to support peak-level replication. Thus, the Rab family of small GTPases includes critical host factors assisting VRC assembly and genesis of the viral replication compartment.

Spatiotemporal dynamics of membrane remodeling and fusion proteins during endocytic transport.

Science.gov (United States)

Arlt, Henning; Auffarth, Kathrin; Kurre, Rainer; Lisse, Dominik; Piehler, Jacob; Ungermann, Christian

2015-04-01

Organelles of the endolysosomal system undergo multiple fission and fusion events to combine sorting of selected proteins to the vacuole with endosomal recycling. This sorting requires a consecutive remodeling of the organelle surface in the course of endosomal maturation. Here we dissect the remodeling and fusion machinery on endosomes during the process of endocytosis. We traced selected GFP-tagged endosomal proteins relative to exogenously added fluorescently labeled α-factor on its way from the plasma membrane to the vacuole. Our data reveal that the machinery of endosomal fusion and ESCRT proteins has similar temporal localization on endosomes, whereas they precede the retromer cargo recognition complex. Neither deletion of retromer nor the fusion machinery with the vacuole affects this maturation process, although the kinetics seems to be delayed due to ESCRT deletion. Of importance, in strains lacking the active Rab7-like Ypt7 or the vacuolar SNARE fusion machinery, α-factor still proceeds to late endosomes with the same kinetics. This indicates that endosomal maturation is mainly controlled by the early endosomal fusion and remodeling machinery but not the downstream Rab Ypt7 or the SNARE machinery. Our data thus provide important further understanding of endosomal biogenesis in the context of cargo sorting. © 2015 Arlt et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Stochastic acidification, activation of hemagglutinin and escape of influenza viruses from an endosome

Science.gov (United States)

Lagache, Thibault; Sieben, Christian; Meyer, Tim; Herrmann, Andreas; Holcman, David

2017-06-01

Influenza viruses enter the cell inside an endosome. During the endosomal journey, acidification triggers a conformational change of the virus spike protein hemagglutinin (HA) that results in escape of the viral genome from the endosome into the cytoplasm. It is still unclear how the interplay between acidification and HA conformation changes affects the kinetics of the viral endosomal escape. We develop here a stochastic model to estimate the change of conformation of HAs inside the endosome nanodomain. Using a Markov process, we model the arrival of protons to HA binding sites and compute the kinetics of their accumulation. We compute the Mean First Passage Time (MFPT) of the number of HA bound sites to a threshold, which is used to estimate the HA activation rate for a given pH concentration. The present analysis reveals that HA proton binding sites possess a high chemical barrier, ensuring a stability of the spike protein at sub-acidic pH. We predict that activating more than 3 adjacent HAs is necessary to trigger endosomal fusion and this configuration prevents premature release of viruses from early endosomes

Newly synthesized benzanthrone derivatives as prospective fluorescent membrane probes

International Nuclear Information System (INIS)

Zhytniakivska, Olga; Trusova, Valeriya; Gorbenko, Galyna; Kirilova, Elena; Kalnina, Inta; Kirilov, Georgiy; Kinnunen, Paavo

2014-01-01

Fluorescence spectral properties of a series of novel benzanthrone derivatives have been explored in lipid bilayers composed of zwitterionic lipid phosphatidylcholine (PC) and its mixtures with cholesterol (Chol) and anionic phospholipid cardiolipin (CL). Analysis of partition coefficients showed that all the examined compounds possess rather high lipid-associating ability, with the amidino derivatives exhibiting stronger membrane partitioning compared with the aminobenzanthrones. To understand how benzanthrone partition properties correlate with their structure, quantitative structure property relationship (QSPR) analysis was performed involving a range of quantum chemical molecular descriptors. -- Highlights: • Benzanthrone partitioning into lipid bilayer correlates with lipophilicity of the dyes. • Partition properties of benzanthrones depend on the dye dipole moment. • Amidino derivatives exhibit higher membrane affinity than aminobenzanthrones

Treatment of offshore produced water - an effective membrane process

International Nuclear Information System (INIS)

Taylor, J.; Larson, R.; Scherer, B.

1991-01-01

The conference paper describes a new membrane technology being extremely effective in separating hydrocarbons from water streams. The membrane is composed of a completely natural cellulose and is resistant to all hydrocarbons and organic solvents, and preliminary tests have shown that it is resistant to fouling by oily molecules and calcium scaling. The membrane system being designed shows good potential for the treatment of offshore produced water with a hydrocarbon content well within present and emerging standards. 6 refs., 8 figs., 3 tabs

Huntingtin coordinates the dynein-mediated dynamic positioning of endosomes and lysosomes

Science.gov (United States)

Caviston, Juliane P.; Zajac, Allison L.; Tokito, Mariko; Holzbaur, Erika L.F.

2011-01-01

Huntingtin (Htt) is a membrane-associated scaffolding protein that interacts with microtubule motors as well as actin-associated adaptor molecules. We examined a role for Htt in the dynein-mediated intracellular trafficking of endosomes and lysosomes. In HeLa cells depleted of either Htt or dynein, early, recycling, and late endosomes (LE)/lysosomes all become dispersed. Despite altered organelle localization, kinetic assays indicate only minor defects in intracellular trafficking. Expression of full-length Htt is required to restore organelle localization in Htt-depleted cells, supporting a role for Htt as a scaffold that promotes functional interactions along its length. In dynein-depleted cells, LE/lysosomes accumulate in tight patches near the cortex, apparently enmeshed by cortactin-positive actin filaments; Latrunculin B-treatment disperses these patches. Peripheral LE/lysosomes in dynein-depleted cells no longer colocalize with microtubules. Htt may be required for this off-loading, as the loss of microtubule association is not seen in Htt-depleted cells or in cells depleted of both dynein and Htt. Inhibition of kinesin-1 relocalizes peripheral LE/lysosomes induced by Htt depletion but not by dynein depletion, consistent with their detachment from microtubules upon dynein knockdown. Together, these data support a model of Htt as a facilitator of dynein-mediated trafficking that may regulate the cytoskeletal association of dynamic organelles. PMID:21169558

Staphylococcus aureus α-toxin-dependent induction of host cell death by membrane-derived vesicles.

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Bernard Thay

Full Text Available Staphylococcus aureus causes a wide spectrum of infections in humans, ranging from superficial cutaneous infections, infections in the circum-oral region, to life-threatening bacteremia. It was recently demonstrated that Gram-positive organisms such as S. aureus liberate membrane-derived vesicles (MVs, which analogously to outer membrane vesicles (OMVs of Gram-negative bacteria can play a role in delivering virulence factors to host cells. In the present study we have shown that cholesterol-dependent fusion of S. aureus MVs with the plasma membrane represents a route for delivery of a key virulence factor, α-toxin (α-hemolysin; Hla to human cells. Most S. aureus strains produce this 33-kDa pore-forming protein, which can lyse a wide range of human cells, and induce apoptosis in T-lymphocytes. Our results revealed a tight association of biologically active α-toxin with membrane-derived vesicles isolated from S. aureus strain 8325-4. Concomitantly, α-toxin contributed to HeLa cell cytotoxicity of MVs, and was the main vesicle-associated protein responsible for erythrocyte lysis. In contrast, MVs obtained from an isogenic hla mutant were significantly attenuated with regards to both causing lysis of erythrocytes and death of HeLa cells. This is to our knowledge the first recognition of an S. aureus MV-associated factor contributing to host cell cytotoxicity.

Polyoxadiazole hollow fibers for produced water treatment by direct contact membrane distillation

KAUST Repository

Xu, Jingli

2018-01-08

Treatment of produced water in the petroleum industry has been a challenge worldwide. In this study, we evaluated the use of direct contact membrane distillation (DCMD) for this purpose, removing oil and dissolved elements and supplying clean water from waste. We synthesized fluorinated polyoxadiazole, a highly hydrophobic polymer, to fabricate hollow fiber membranes, which were optimized and tested for simulated produced water and real produced water treatment. The process performance was investigated under different operating parameters, such as feed temperature, feed flow velocity and length of the membrane module for 4 days. The results indicate that by increasing feed temperature and feed flow rate the vapor flux increases. The flux decreased with increasing the length of the module due to the decrease of the driving force along the module. The fouling behavior, which corresponds to flux decline and cleaning efficiency of the membrane, was studied. The performance of the fabricated hollow fiber membranes was demonstrated for the treatment of produced water, complying with the industrial reuse and discharge limits.

Mouse polyomavirus enters early endosomes, requires their acidic pH for productive infection, and meets transferrin cargo in rab11-positive endosomes

Czech Academy of Sciences Publication Activity Database

Liebl, D.; Difato, F.; Horníková, L.; Mannová, P.; Štokrová, Jitka; Forstová, J.

2006-01-01

Roč. 80, č. 9 (2006), s. 4610-4622 ISSN 0022-538X R&D Projects: GA ČR(CZ) GA204/03/0593; GA MŠk(CZ) LC545 Institutional research plan: CEZ:AV0Z50520514 Keywords : Polyomavirus internalization and trafficking * Early endosomes * Dependence of infection on endosomal pH Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.341, year: 2006

Desipramine induces disorder in cholesterol-rich membranes

DEFF Research Database (Denmark)

Pakkanen, Kirsi; Salonen, Emppu; Mäkelä, Anna R

2009-01-01

canine parvovirus (CPV), a virus known to interact with endosomal membranes and sphingomyelin, as an intracellular probe. DMI was found to cause retention of the virus in intracellular vesicular structures leading to the inhibition of viral proliferation. This implies that DMI has a deleterious effect...

Decoupling Internalization, Acidification and Phagosmal-Endosomal/Iysosomal Phagocytosis of Internalin A coated Beads in epithelial cells

Energy Technology Data Exchange (ETDEWEB)

Blanchette, C D; Woo, Y; Thomas, C; Shen, N; Sulchek, T A; Hiddessen, A L

2008-12-22

Phagocytosis has been extensively examined in 'professional' phagocytic cells using pH sensitive dyes. However, in many of the previous studies, a separation between the end of internalization, beginning of acidification and completion of phagosomal-endosomal/lysosomal fusion was not clearly established, and in several cases, it was treated as a one-step process. In addition, very little work has been done to systematically examine phagosomal maturation in 'non-professional' phagocytic cells, such as epithelial cells. Therefore, in this study, we developed a simple and novel method to decouple and accurately measure particle internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in Madin-Darby Canine Kidney (MDCK) and Caco-2 epithelial cells. Our method was developed using a pathogen mimetic system consisting of polystyrene beads coated with Internalin A (InlA), a membrane surface protein from Listeria monocytogenes known to trigger receptor-mediated internalization. We achieved independent measurements of the rates of internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in epithelial cells by combining the InlA-coated beads (InlA-beads) with antibody quenching, pH sensitive dyes and endosomal/lysosomal dyes, as follows: the rate of InlA bead internalization was measured via antibody quenching of a pH independent dye (Alexa488) conjugated to InlA-beads, the rate at which phagosomes containing internalized InlA beads became acidified was measured using a pH dependent dye (FITC) conjugated to the beads and the rate of phagosomal-endosomal/lysosomal fusion was measured using a combination of unlabeled InlA-beads and an endosomal/lysosomal dye. By performing these independent measurements under identical experimental conditions, we were able to decouple the three processes and establish time scales for each. In a separate set of experiments, we also exploited the phagosomal acidification

Extracellular anti-angiogenic proteins augment an endosomal protein trafficking pathway to reach mitochondria and execute apoptosis in HUVECs.

Science.gov (United States)

Chen, Mo; Qiu, Tao; Wu, Jiajie; Yang, Yang; Wright, Graham D; Wu, Min; Ge, Ruowen

2018-03-09

Classic endocytosis destinations include the recycling endosome returning to the plasma membrane or the late endosome (LE) merging with lysosomes for cargo degradation. However, the anti-angiogenic proteins angiostatin and isthmin, are endocytosed and trafficked to mitochondria (Mito) to execute apoptosis of endothelial cells. How these extracellular proteins reach mitochondria remains a mystery. Through confocal and super-resolution fluorescent microscopy, we demonstrate that angiostatin and isthmin are trafficked to mitochondria through the interaction between LE and Mito. Using purified organelles, the LE-Mito interaction is confirmed through in vitro lipid-fusion assay, as well as single vesicle total internal reflection fluorescent microscopy. LE-Mito interaction enables the transfer of not only lipids but also proteins from LE to Mito. Angiostatin and isthmin augment this endosomal protein trafficking pathway and make use of it to reach mitochondria to execute apoptosis. Cell fractionation and biochemical analysis identified that the cytosolic scaffold protein Na+/H+ exchanger regulatory factor 1 (NHERF1) associated with LE and the t-SNARE protein synaptosome-associated protein 25 kDa (SNAP25) associated with Mito form an interaction complex to facilitate LE-Mito interaction. Proximity ligation assay coupled with fluorescent microscopy showed that both NHERF1 and SNAP25 are located at the contacting face between LE and Mito. RNAi knockdown of either NHERF1 or SNAP25 suppressed not only the mitochondrial trafficking of angiostatin and isthmin but also their anti-angiogenic and pro-apoptotic functions. Hence, this study reveals a previously unrealized endosomal protein trafficking pathway from LE to Mito that allows extracellular proteins to reach mitochondria and execute apoptosis.

Decoupling internalization, acidification and phagosomal-endosomal/lysosomal fusion during phagocytosis of InlA coated beads in epithelial cells.

Directory of Open Access Journals (Sweden)

Craig D Blanchette

Full Text Available BACKGROUND: Phagocytosis has been extensively examined in 'professional' phagocytic cells using pH sensitive dyes. However, in many of the previous studies, a separation between the end of internalization, beginning of acidification and completion of phagosomal-endosomal/lysosomal fusion was not clearly established. In addition, very little work has been done to systematically examine phagosomal maturation in 'non-professional' phagocytic cells. Therefore, in this study, we developed a simple method to measure and decouple particle internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in Madin-Darby Canine Kidney (MDCK and Caco-2 epithelial cells. METHODOLOGY/PRINCIPAL FINDINGS: Our method was developed using a pathogen mimetic system consisting of polystyrene beads coated with Internalin A (InlA, a membrane surface protein from Listeria monocytogenes known to trigger receptor-mediated phagocytosis. We were able to independently measure the rates of internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in epithelial cells by combining the InlA-coated beads (InlA-beads with antibody quenching, a pH sensitive dye and an endosomal/lysosomal dye. By performing these independent measurements under identical experimental conditions, we were able to decouple the three processes and establish time scales for each. In a separate set of experiments, we exploited the phagosomal acidification process to demonstrate an additional, real-time method for tracking bead binding, internalization and phagosomal acidification. CONCLUSIONS/SIGNIFICANCE: Using this method, we found that the time scales for internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion ranged from 23-32 min, 3-4 min and 74-120 min, respectively, for MDCK and Caco-2 epithelial cells. Both the static and real-time methods developed here are expected to be readily and broadly applicable, as they simply

Surface modification of cellulose acetate membrane using thermal annealing to enhance produced water treatment

Energy Technology Data Exchange (ETDEWEB)

Kusworo, T. D., E-mail: tdkusworo@che.undip.ac.id; Aryanti, N., E-mail: nita.aryanti@gmail.com; Firdaus, M. M. H.; Sukmawati, H. [Chemical Engineering, Faculty of Engineering, Diponegoro University Prof. Soedarto Street, Tembalang, Semarang, 50239, Phone/Fax : (024)7460058 (Indonesia)

2015-12-29

This study is performed primarily to investigate the effect of surface modification of cellulose acetate using thermal annealing on the enhancement of membrane performance for produced water treatment. In this study, Cellulose Acetate membranes were casted using dry/wet phase inversion technique. The effect of additive and post-treatment using thermal annealing on the membrane surface were examined for produced water treatment. Therma annealing was subjected to membrane surface at 60 and 70 °C for 5, 10 and 15 second, respectively. Membrane characterizations were done using membrane flux and rejection with produced water as a feed, Scanning Electron Microscopy (SEM) and Fourier Transform Infra Red (FTIR) analysis. Experimental results showed that asymmetric cellulose acetate membrane can be made by dry/wet phase inversion technique. The results from the Scanning Electron Microscopy (FESEM) analysis was also confirmed that polyethylene glycol as additivie in dope solution and thermal annealing was affected the morphology and membrane performance for produced water treatment, respectively. Scanning electron microscopy micrographs showed that the selective layer and the substructure of membrane became denser and more compact after the thermal annealing processes. Therefore, membrane rejection was significantly increased while the flux was slighty decreased, respectively. The best membrane performance is obtained on the composition of 18 wt % cellulose acetate, poly ethylene glycol 5 wt% with thermal annealing at 70° C for 15 second.

Surface modification of cellulose acetate membrane using thermal annealing to enhance produced water treatment

International Nuclear Information System (INIS)

Kusworo, T. D.; Aryanti, N.; Firdaus, M. M. H.; Sukmawati, H.

2015-01-01

This study is performed primarily to investigate the effect of surface modification of cellulose acetate using thermal annealing on the enhancement of membrane performance for produced water treatment. In this study, Cellulose Acetate membranes were casted using dry/wet phase inversion technique. The effect of additive and post-treatment using thermal annealing on the membrane surface were examined for produced water treatment. Therma annealing was subjected to membrane surface at 60 and 70 °C for 5, 10 and 15 second, respectively. Membrane characterizations were done using membrane flux and rejection with produced water as a feed, Scanning Electron Microscopy (SEM) and Fourier Transform Infra Red (FTIR) analysis. Experimental results showed that asymmetric cellulose acetate membrane can be made by dry/wet phase inversion technique. The results from the Scanning Electron Microscopy (FESEM) analysis was also confirmed that polyethylene glycol as additivie in dope solution and thermal annealing was affected the morphology and membrane performance for produced water treatment, respectively. Scanning electron microscopy micrographs showed that the selective layer and the substructure of membrane became denser and more compact after the thermal annealing processes. Therefore, membrane rejection was significantly increased while the flux was slighty decreased, respectively. The best membrane performance is obtained on the composition of 18 wt % cellulose acetate, poly ethylene glycol 5 wt% with thermal annealing at 70° C for 15 second

Picornavirus RNA is protected from cleavage by ribonuclease during virion uncoating and transfer across cellular and model membranes.

Science.gov (United States)

Groppelli, Elisabetta; Levy, Hazel C; Sun, Eileen; Strauss, Mike; Nicol, Clare; Gold, Sarah; Zhuang, Xiaowei; Tuthill, Tobias J; Hogle, James M; Rowlands, David J

2017-02-01

Picornaviruses are non-enveloped RNA viruses that enter cells via receptor-mediated endocytosis. Because they lack an envelope, picornaviruses face the challenge of delivering their RNA genomes across the membrane of the endocytic vesicle into the cytoplasm to initiate infection. Currently, the mechanism of genome release and translocation across membranes remains poorly understood. Within the enterovirus genus, poliovirus, rhinovirus 2, and rhinovirus 16 have been proposed to release their genomes across intact endosomal membranes through virally induced pores, whereas one study has proposed that rhinovirus 14 releases its RNA following disruption of endosomal membranes. For the more distantly related aphthovirus genus (e.g. foot-and-mouth disease viruses and equine rhinitis A virus) acidification of endosomes results in the disassembly of the virion into pentamers and in the release of the viral RNA into the lumen of the endosome, but no details have been elucidated as how the RNA crosses the vesicle membrane. However, more recent studies suggest aphthovirus RNA is released from intact particles and the dissociation to pentamers may be a late event. In this study we have investigated the RNase A sensitivity of genome translocation of poliovirus using a receptor-decorated-liposome model and the sensitivity of infection of poliovirus and equine-rhinitis A virus to co-internalized RNase A. We show that poliovirus genome translocation is insensitive to RNase A and results in little or no release into the medium in the liposome model. We also show that infectivity is not reduced by co-internalized RNase A for poliovirus and equine rhinitis A virus. Additionally, we show that all poliovirus genomes that are internalized into cells, not just those resulting in infection, are protected from RNase A. These results support a finely coordinated, directional model of viral RNA delivery that involves viral proteins and cellular membranes.

Picornavirus RNA is protected from cleavage by ribonuclease during virion uncoating and transfer across cellular and model membranes.

Directory of Open Access Journals (Sweden)

Elisabetta Groppelli

2017-02-01

Full Text Available Picornaviruses are non-enveloped RNA viruses that enter cells via receptor-mediated endocytosis. Because they lack an envelope, picornaviruses face the challenge of delivering their RNA genomes across the membrane of the endocytic vesicle into the cytoplasm to initiate infection. Currently, the mechanism of genome release and translocation across membranes remains poorly understood. Within the enterovirus genus, poliovirus, rhinovirus 2, and rhinovirus 16 have been proposed to release their genomes across intact endosomal membranes through virally induced pores, whereas one study has proposed that rhinovirus 14 releases its RNA following disruption of endosomal membranes. For the more distantly related aphthovirus genus (e.g. foot-and-mouth disease viruses and equine rhinitis A virus acidification of endosomes results in the disassembly of the virion into pentamers and in the release of the viral RNA into the lumen of the endosome, but no details have been elucidated as how the RNA crosses the vesicle membrane. However, more recent studies suggest aphthovirus RNA is released from intact particles and the dissociation to pentamers may be a late event. In this study we have investigated the RNase A sensitivity of genome translocation of poliovirus using a receptor-decorated-liposome model and the sensitivity of infection of poliovirus and equine-rhinitis A virus to co-internalized RNase A. We show that poliovirus genome translocation is insensitive to RNase A and results in little or no release into the medium in the liposome model. We also show that infectivity is not reduced by co-internalized RNase A for poliovirus and equine rhinitis A virus. Additionally, we show that all poliovirus genomes that are internalized into cells, not just those resulting in infection, are protected from RNase A. These results support a finely coordinated, directional model of viral RNA delivery that involves viral proteins and cellular membranes.

Adiponectin release and insulin receptor targeting share trans-Golgi-dependent endosomal trafficking routes

Directory of Open Access Journals (Sweden)

Maria Rödiger

2018-02-01

Full Text Available Objective: Intracellular vesicle trafficking maintains cellular structures and functions. The assembly of cargo-laden vesicles at the trans-Golgi network is initiated by the ARF family of small GTPases. Here, we demonstrate the role of the trans-Golgi localized monomeric GTPase ARFRP1 in endosomal-mediated vesicle trafficking of mature adipocytes. Methods: Control (Arfrp1flox/flox and inducible fat-specific Arfrp1 knockout (Arfrp1iAT−/− mice were metabolically characterized. In vitro experiments on mature 3T3-L1 cells and primary mouse adipocytes were conducted to validate the impact of ARFRP1 on localization of adiponectin and the insulin receptor. Finally, secretion and transferrin-based uptake and recycling assays were performed with HeLa and HeLa M-C1 cells. Results: We identified the ARFRP1-based sorting machinery to be involved in vesicle trafficking relying on the endosomal compartment for cell surface delivery. Secretion of adiponectin from fat depots was selectively reduced in Arfrp1iAT−/− mice, and Arfrp1-depleted 3T3-L1 adipocytes revealed an accumulation of adiponectin in Rab11-positive endosomes. Plasma adiponectin deficiency of Arfrp1iAT−/− mice resulted in deteriorated hepatic insulin sensitivity, increased gluconeogenesis and elevated fasting blood glucose levels. Additionally, the insulin receptor, undergoing endocytic recycling after ligand binding, was less abundant at the plasma membrane of adipocytes lacking Arfrp1. This had detrimental effects on adipose insulin signaling, followed by insufficient suppression of basal lipolytic activity and impaired adipose tissue expansion. Conclusions: Our findings suggest that adiponectin secretion and insulin receptor surface targeting utilize the same post-Golgi trafficking pathways that are essential for an appropriate systemic insulin sensitivity and glucose homeostasis. Keywords: Adiponectin, ARFRP1, Exocytosis, Insulin receptor, trans-Golgi

Glycosaminoglycan-resistant and pH-sensitive lipid-coated DNA complexes produced by detergent removal method.

Science.gov (United States)

Lehtinen, Julia; Hyvönen, Zanna; Subrizi, Astrid; Bunjes, Heike; Urtti, Arto

2008-10-21

Cationic polymers are efficient gene delivery vectors in in vitro conditions, but these carriers can fail in vivo due to interactions with extracellular polyanions, i.e. glycosaminoglycans (GAG). The aim of this study was to develop a stable gene delivery vector that is activated at the acidic endosomal pH. Cationic DNA/PEI complexes were coated by 1,2-dioleylphosphatidylethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS) (3:2 mol/mol) using two coating methods: detergent removal and mixing with liposomes prepared by ethanol injection. Only detergent removal produced lipid-coated DNA complexes that were stable against GAGs, but were membrane active at low pH towards endosome mimicking liposomes. In relation to the low cellular uptake of the coated complexes, their transfection efficacy was relatively high. PEGylation of the coated complexes increased their cellular uptake but reduced the pH-sensitivity. Detergent removal was thus a superior method for the production of stable, but acid activatable, lipid-coated DNA complexes.

Aggregation of endosomal-vacuolar compartments in the Aovps24-deleted strain in the filamentous fungus Aspergillus oryzae

International Nuclear Information System (INIS)

Tatsumi, Akinori; Shoji, Jun-ya; Kikuma, Takashi; Arioka, Manabu; Kitamoto, Katsuhiko

2007-01-01

Previously, we found that deletion of Aovps24, an ortholog of Saccharomyces cerevisiae VPS24, that encodes an ESCRT (endosomal sorting complex required for transport)-III component required for late endosomal function results in fragmented and aggregated vacuoles. Although defective late endosomal function is likely responsible for this phenotype, critical lack of our knowledge on late endosomes in filamentous fungi prevented us from further characterization. In this study, we identified late endosomes of Aspergillus oryzae, by expressing a series of fusion proteins of fluorescent proteins with orthologs of late endosomal proteins. Using these fusion proteins as markers, we observed late endosomes in the wild type strain and the Aovps24 disruptant and demonstrated that late endosomes are aberrantly aggregated in the Aovps24 disruptant. Moreover, we revealed that the aggregated late endosomes have features of vacuoles as well. As deletion of another ESCRT-III component-encoding gene, Aovps2, resulted in similar phenotypes to that in the Aovps24 disruptant, phenotypes of the Aovps24 disruptant are probably due to defective late endosomal function

Structural Basis for Endosomal Targeting by the Bro1 Domain

Science.gov (United States)

Kim, Jaewon; Sitaraman, Sujatha; Hierro, Aitor; Beach, Bridgette M.; Odorizzi, Greg; Hurley, James H.

2010-01-01

Summary Proteins delivered to the lysosome or the yeast vacuole via late endosomes are sorted by the ESCRT complexes and by associated proteins, including Alix and its yeast homolog Bro1. Alix, Bro1, and several other late endosomal proteins share a conserved 160 residue Bro1 domain whose boundaries, structure, and function have not been characterized. The crystal structure of the Bro1 domain of Bro1 reveals a folded core of 367 residues. The extended Bro1 domain is necessary and sufficient for binding to the ESCRT-III subunit Snf7 and for the recruitment of Bro1 to late endosomes. The structure resembles a boomerang with its concave face filled in and contains a triple tetratricopeptide repeat domain as a substructure. Snf7 binds to a conserved hydrophobic patch on Bro1 that is required for protein complex formation and for the protein-sorting function of Bro1. These results define a conserved mechanism whereby Bro1 domain-containing proteins are targeted to endosomes by Snf7 and its orthologs. PMID:15935782

Ceramic membrane ozonator for soluble organics removal from produced water

Science.gov (United States)

Siagian, U. W. R.; Dwipramana, A. S.; Perwira, S. B.; Khoiruddin; Wenten, I. G.

2018-01-01

In this work, the performance of ozonation for degradation of soluble organic compounds in produced water was investigated. Tubular ceramic membrane diffuser (with and without a static mixer in the lumen side) was used to facilitate contact between ozone and produced water. The ozonation was conducted at ozone flow rate of 8 L.min-1, ozone concentration of 0.4 ppm, original pH of the solution, and pressure of 1.2 bar, while the flow rates of the produced water were varied (192, 378 and 830 mL.min-1). It was found that the reduction of benzene, toluene, ethylbenzene, and xylene were 85%, 99%, 85%, and 95%, respectively. A lower liquid flow rate in a laminar state showed a better component reduction due to the longer contacting time between the liquid and the gas phase. The introduction of the static mixer in the lumen side of the membrane as a turbulence promoter provided a positive effect on the performance of the membrane diffuser. The twisted static mixer exhibited the better removal rate than the spiral static mixer.

Electrolytic process to produce sodium hypochlorite using sodium ion conductive ceramic membranes

Science.gov (United States)

Balagopal, Shekar; Malhotra, Vinod; Pendleton, Justin; Reid, Kathy Jo

2012-09-18

An electrochemical process for the production of sodium hypochlorite is disclosed. The process may potentially be used to produce sodium hypochlorite from seawater or low purity un-softened or NaCl-based salt solutions. The process utilizes a sodium ion conductive ceramic membrane, such as membranes based on NASICON-type materials, in an electrolytic cell. In the process, water is reduced at a cathode to form hydroxyl ions and hydrogen gas. Chloride ions from a sodium chloride solution are oxidized in the anolyte compartment to produce chlorine gas which reacts with water to produce hypochlorous and hydrochloric acid. Sodium ions are transported from the anolyte compartment to the catholyte compartment across the sodium ion conductive ceramic membrane. Sodium hydroxide is transported from the catholyte compartment to the anolyte compartment to produce sodium hypochlorite within the anolyte compartment.

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

Science.gov (United States)

2011-01-01

early endosome movement. In filamentous hyphae , dynein powers the minus end–directed movement of early endosomes (Steinberg and Schuster 2011...observed in time-lapse sequences (Video 1; Abenza et al., 2009). In still images, early endosomes were seen to distribute along the hyphae (Fig. 2 A...nuclear distribution along elongated hyphae and also for the microtubule minus end–directed movement of early endosomes away from the tip (Morris

L-Type Voltage-Gated Ca2+ Channels Regulate Synaptic-Activity-Triggered Recycling Endosome Fusion in Neuronal Dendrites

Directory of Open Access Journals (Sweden)

Brian G. Hiester

2017-11-01

Full Text Available The repertoire and abundance of proteins displayed on the surface of neuronal dendrites are tuned by regulated fusion of recycling endosomes (REs with the dendritic plasma membrane. While this process is critical for neuronal function and plasticity, how synaptic activity drives RE fusion remains unexplored. We demonstrate a multistep fusion mechanism that requires Ca2+ from distinct sources. NMDA receptor Ca2+ initiates RE fusion with the plasma membrane, while L-type voltage-gated Ca2+ channels (L-VGCCs regulate whether fused REs collapse into the membrane or reform without transferring their cargo to the cell surface. Accordingly, NMDA receptor activation triggered AMPA-type glutamate receptor trafficking to the dendritic surface in an L-VGCC-dependent manner. Conversely, potentiating L-VGCCs enhanced AMPA receptor surface expression only when NMDA receptors were also active. Thus L-VGCCs play a role in tuning activity-triggered surface expression of key synaptic proteins by gating the mode of RE fusion.

Extraction Socket Preservation Using Porcine-Derived Collagen Membrane Alone or Associated with Porcine-Derived Bone. Clinical Results of Randomized Controlled Study

Directory of Open Access Journals (Sweden)

Renzo Guarnieri

2017-03-01

Full Text Available Objectives: The aim of present randomized controlled clinical trial was to clinically evaluate hard tissue changes after extraction socket preservation procedures compared to natural spontaneous healing. Material and Methods: Thirty patients were enrolled in the present study and underwent single-tooth extraction in the premolar/molar areas. Ten sites were grafted with porcine-derived bone covered by collagen membrane, 10 covered by porcine-derived collagen membrane alone, and 10 underwent natural spontaneous healing. Vertical and horizontal bone changes after 3-month were evaluated at implant placement. Results: The vertical and horizontal bone changes at the extraction sockets treated with collagen membrane alone (vertical: -0.55 [SD 0.11] mm, and horizontal: -1.21 [SD 0.69] mm and collagen membrane plus porcine-derived bone (vertical: -0.37 [SD 0.7] mm, and horizontal: -0.91 [SD 0.53] mm were found significantly lower (P < 0.001, when compared to non-grafted sockets (vertical: -2.09 [SD 0.19] mm, and horizontal: -3.96 [SD 0.87] mm. In type 1 extraction sockets, in premolar sites, and in presence of vestibular bone thicknesses ≥ 1.5 mm, the use of collagen membrane alone revealed similar outcomes to those with additional graft material. Conclusions: At the re-entry surgery, extraction sockets grafted with porcine-derived bone and covered by collagen membrane, and extraction sockets covered by porcine-derived collagen membrane alone, showed significantly lower vertical and horizontal bone changes, compared to extraction sockets sites underwent natural spontaneous healing. However, a complete prevention of remodelling is not achievable, irrespective of the technique used.

Characterization of the Mammalian CORVET and HOPS Complexes and Their Modular Restructuring for Endosome Specificity

NARCIS (Netherlands)

van der Kant, Rik; Jonker, Caspar T. H.; Wijdeven, Ruud H.; Bakker, Jeroen; Janssen, Lennert; Klumperman, Judith; Neefjes, Jacques

2015-01-01

Trafficking of cargo through the endosomal system depends on endosomal fusion events mediated by SNARE proteins, Rab-GTPases, and multisubunit tethering complexes. The CORVET and HOPS tethering complexes, respectively, regulate early and late endosomal tethering and have been characterized in detail

Pd Alloy Membranes for Hydrogen Separation from Coal-Derived Syngas

National Research Council Canada - National Science Library

Alptekin, Gokhan O; DeVoss, Sarah; Amalfitano, Bob; Way, Douglas; Thoen, Paul; Lusk, Mark

2006-01-01

TDA Research Inc., in collaboration with Colorado School of Mines (CSM) is developing a sulfur and CO-tolerant membrane to produce the clean hydrogen from syngas using Pd membrane films prepared on a variety of supports (e.g...

A review of recent advances in molecular simulation of graphene-derived membranes for gas separation

Science.gov (United States)

Fatemi, Seyyed Mahmood; Abbasi, Zeynab; Rajabzadeh, Halimeh; Hashemizadeh, Seyyed Ali; Deldar, Amir Noori

2017-07-01

To obtain an ideal membrane for gas separation the following three characteristics should be considered: the membrane should be as thin as possible, be mechanically robust, and have well-defined pore sizes. These features will maximize its solvent flux, preserve it from fracture, and guarantee its selectivity. These attractive properties of graphene-derived membranes introduce them as appropriate candidates for gas separation and gas molecular-sieving processes in nanoscale dimensions. The current effort has focused on two issues, including the review of the most newly progression on drilling holes in single graphene membranes for making ultrathin membranes for gas separation, and studying functionalized nanoporous sheet and graphene-derived membranes, including doped graphene, graphene oxide, fluorographene, and reduced graphene oxide from theoretical perspectives for making functional coatings for nano ultrafiltration for gas separation. We investigated the basic mechanism of separation by membranes derived from graphene and relevant possible applications. Functionalized nanoporous membranes as novel approach are characterized by low energy cost in realizing high throughput molecular-sieving separation.

Enhanced biofouling resistance of polyethersulfone membrane surface modified with capsaicin derivative and itaconic acid

Energy Technology Data Exchange (ETDEWEB)

Wang, Jian [Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, Qingdao 266100 (China); College of Chemistry and Chemical Engineering, Ocean University of China, Qingdao 266100 (China); Gao, Xueli, E-mail: gxl_ouc@126.com [Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, Qingdao 266100 (China); College of Chemistry and Chemical Engineering, Ocean University of China, Qingdao 266100 (China); Wang, Qun; Sun, Haijing; Wang, Xiaojuan [Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, Qingdao 266100 (China); College of Chemistry and Chemical Engineering, Ocean University of China, Qingdao 266100 (China); Gao, Congjie, E-mail: gaocjie@ouc.edu.cn [Key Laboratory of Marine Chemistry Theory and Technology, Ministry of Education, Ocean University of China, Qingdao 266100 (China); College of Chemistry and Chemical Engineering, Ocean University of China, Qingdao 266100 (China)

2015-11-30

Graphical abstract: - Highlights: • PES membrane was modified with a capsaicin derivative. • UV-assisted graft polymerization was carried out on membrane surface. • The capsaicin derivative modified membrane shows better antibiofouling property. - Abstract: The culprit of biofouling is the reproduction of viable microorganisms on the membrane surface. Recently, functionalization of membrane surface with natural antibacterial agents has drawn great attention. This work presents the fabrication of antibiofouling polyethersulfone (PES) ultrafiltration (UF) membranes by UV-assisted photo grafting of capsaicin derivative (N-(4-hydroxy-3-methoxy-benzyl)-acrylamide, HMBA) and itaconic acid (IA) on the surface of PES membrane. Results of FTIR-ATR, water static contact angle (WSCA) and atomic force microscopy (AFM) analysis confirmed the successful grafting of HMBA and IA on the membrane surface. We investigated the antifouling and antibacterial properties of these membranes using BSA and Escherichia coli as the test model, respectively. During a 150-min test, the modified membranes show much lower flux decline (42.7% for PES-g-1H0I, 22.2% for PES-g-1H1I and 7.7% for PES-g-1H5I) when compared with the pristine membrane (flux declined by 77%). The modified membranes exhibit excellent antibacterial activity (nearly 100%) when UV irradiation time was 6 min. The morphological study suggested that the E. coli on the pristine membrane showed a regular and smooth surface while that on the modified membrane was disrupted, which validated the antibacterial activity of the modified membranes.

Enhanced biofouling resistance of polyethersulfone membrane surface modified with capsaicin derivative and itaconic acid

International Nuclear Information System (INIS)

Wang, Jian; Gao, Xueli; Wang, Qun; Sun, Haijing; Wang, Xiaojuan; Gao, Congjie

2015-01-01

Graphical abstract: - Highlights: • PES membrane was modified with a capsaicin derivative. • UV-assisted graft polymerization was carried out on membrane surface. • The capsaicin derivative modified membrane shows better antibiofouling property. - Abstract: The culprit of biofouling is the reproduction of viable microorganisms on the membrane surface. Recently, functionalization of membrane surface with natural antibacterial agents has drawn great attention. This work presents the fabrication of antibiofouling polyethersulfone (PES) ultrafiltration (UF) membranes by UV-assisted photo grafting of capsaicin derivative (N-(4-hydroxy-3-methoxy-benzyl)-acrylamide, HMBA) and itaconic acid (IA) on the surface of PES membrane. Results of FTIR-ATR, water static contact angle (WSCA) and atomic force microscopy (AFM) analysis confirmed the successful grafting of HMBA and IA on the membrane surface. We investigated the antifouling and antibacterial properties of these membranes using BSA and Escherichia coli as the test model, respectively. During a 150-min test, the modified membranes show much lower flux decline (42.7% for PES-g-1H0I, 22.2% for PES-g-1H1I and 7.7% for PES-g-1H5I) when compared with the pristine membrane (flux declined by 77%). The modified membranes exhibit excellent antibacterial activity (nearly 100%) when UV irradiation time was 6 min. The morphological study suggested that the E. coli on the pristine membrane showed a regular and smooth surface while that on the modified membrane was disrupted, which validated the antibacterial activity of the modified membranes.

Advanced Membrane Filtration Technology for Cost Effective Recovery of Fresh Water from Oil & Gas Produced Brine

Energy Technology Data Exchange (ETDEWEB)

David B. Burnett

2004-09-29

Produced water is a major waste generated at the oil and natural gas wells in the state of Texas. This water could be a possible source of new fresh water to meet the growing demands of the state after treatment and purification. Treatment of brine generated in oil fields or produced water with an ultrafiltration membranes were the subject of this thesis. The characterization of ultrafiltration membranes for oil and suspended solids removal of produced water, coupled with the reverse osmosis (RO) desalination of brine were studied on lab size membrane testing equipment and a field size testing unit to test whether a viable membrane system could be used to treat produced water. Oil and suspended solids were evaluated using turbidity and oil in water measurements taken periodically. The research considered the effect of pressure and flow rate on membrane performance of produced water treatment of three commercially available membranes for oily water. The study also analyzed the flux through the membrane and any effect it had on membrane performance. The research showed that an ultrafiltration membrane provided turbidity removal of over 99% and oil removal of 78% for the produced water samples. The results indicated that the ultrafiltration membranes would be asset as one of the first steps in purifying the water. Further results on selected RO membranes showed that salt rejection of greater than 97% could be achieved with satisfactory flux and at reasonable operating cost.

The subapical compartment : a traffic center in membrane polarity development

NARCIS (Netherlands)

Hoekstra, D; Tyteca, D; van IJzendoorn, SCD

2004-01-01

Spatially separated apical and basolateral plasma membrane domains that have distinct functions and molecular compositions are a characteristic feature of epithelial cell polarity. The subapical compartment (SAC), also known as the common endosome (CE), where endocytic pathways from both surfaces

Application of ceramic membranes to SAGD produced water treatment for enhanced recycle and reuse

Energy Technology Data Exchange (ETDEWEB)

Minnich, K. [Veolia Water Solutions and Technologies, Mississauga, ON (Canada)

2009-07-01

Drivers for using ceramic membranes in steam assisted gravity drainage (SAGD) include reduced investment cost; alternative treatment technologies that reduce energy and greenhouse gas emissions; and ceramic membranes can be chemically and steam cleaned. This presentation discussed the application of ceramic membranes to SAGD produced water treatment for enhanced recycle and reuse. The presentation illustrated conventional ceramic membranes as well as surface enhanced membranes and provided background information on oil separation. Other topics that were discussed included issues regarding desalter bottoms de-oiling; challenges in de-oiling oil sands produced water; CeraMem surface enhanced membranes; surface facilities and ceramic membrane opportunities; and water treatment using ceramic membranes. The presentation concluded with a discussion of the application of ceramic membranes to SAGD next steps such as a demonstration test of industrial prototype membranes for de-oiling, and pilot testing of ceramic desilication. tabs., figs.

A Role for EHD4 in the Regulation of Early Endosomal Transport

Science.gov (United States)

Sharma, Mahak; Naslavsky, Naava; Caplan, Steve

2009-01-01

All four of the C-terminal Eps15 homology domain (EHD) proteins have been implicated in the regulation of endocytic trafficking. However, the high level of amino acid sequence identity among these proteins has made it challenging to elucidate the precise function of individual EHD proteins. We demonstrate here with specific peptide antibodies that endogenous EHD4 localizes to Rab5-, early embryonic antigen 1 (EEA1)- and Arf6-containing endosomes and colocalizes with internalized transferrin in the cell periphery. Knock-down of EHD4 expression by both small interfering RNA and short hairpin RNA leads to the generation of enlarged early endosomal structures that contain Rab5 and EEA1 as well as internalized transferrin or major histocompatibility complex class I molecules. In addition, cargo destined for degradation, such as internalized low-density lipoprotein, also accumulates in the enlarged early endosomes in EHD4-depleted cells. Moreover, we have demonstrated that these enlarged early endosomes are enriched in levels of the activated GTP-bound Rab5. Finally, we show that endogenous EHD4 and EHD1 interact in cells, suggesting coordinated involvement in the regulation of receptor transport along the early endosome to endocytic recycling compartment axis. The results presented herein provide evidence that EHD4 is involved in the control of trafficking at the early endosome and regulates exit of cargo toward both the recycling compartment and the late endocytic pathway. PMID:18331452

Strategies for the Activation and Release of the Membranolytic Peptide Melittin from Liposomes Using Endosomal pH as a Trigger

NARCIS (Netherlands)

Oude Blenke, E.; Sleszynska, M.; Evers, M. J W; Storm, G.; Martin, N. I.; Mastrobattista, E.

2017-01-01

Endosomolytic peptides are often coupled to drug delivery systems to enhance endosomal escape, which is crucial for the delivery of macromolecular drugs that are vulnerable to degradation in the endolysosomal pathway. Melittin is a 26 amino acid peptide derived from bee venom that has a very high

Chitosan derivatives targeting lipid bilayers: Synthesis, biological activity and interaction with model membranes.

Science.gov (United States)

Martins, Danubia Batista; Nasário, Fábio Domingues; Silva-Gonçalves, Laiz Costa; de Oliveira Tiera, Vera Aparecida; Arcisio-Miranda, Manoel; Tiera, Marcio José; Dos Santos Cabrera, Marcia Perez

2018-02-01

The antimicrobial activity of chitosan and derivatives to human and plant pathogens represents a high-valued prospective market. Presently, two low molecular weight derivatives, endowed with hydrophobic and cationic character at different ratios were synthesized and characterized. They exhibit antimicrobial activity and increased performance in relation to the intermediate and starting compounds. However, just the derivative with higher cationic character showed cytotoxicity towards human cervical carcinoma cells. Considering cell membranes as targets, the mode of action was investigated through the interaction with model lipid vesicles mimicking bacterial, tumoral and erythrocyte membranes. Intense lytic activity and binding are demonstrated for both derivatives in anionic bilayers. The less charged compound exhibits slightly improved selectivity towards bacterial model membranes, suggesting that balancing its hydrophobic/hydrophilic character may improve efficiency. Observing the aggregation of vesicles, we hypothesize that the "charge cluster mechanism", ascribed to some antimicrobial peptides, could be applied to these chitosan derivatives. Copyright © 2017 Elsevier Ltd. All rights reserved.

Interaction of MreB-derived antimicrobial peptides with membranes.

Science.gov (United States)

Saikia, Karabi; Chaudhary, Nitin

2018-03-25

Antimicrobial peptides are critical components of defense systems in living forms. The activity is conferred largely by the selective membrane-permeabilizing ability. In our earlier work, we derived potent antimicrobial peptides from the 9-residue long, N-terminal amphipathic helix of E. coli MreB protein. The peptides display broad-spectrum activity, killing not only Gram-positive and Gram-negative bacteria but opportunistic fungus, Candida albicans as well. These results proved that membrane-binding stretches of bacterial proteins could turn out to be self-harming when applied from outside. Here, we studied the membrane-binding and membrane-perturbing potential of these peptides. Steady-state tryptophan fluorescence studies with tryptophan extended peptides, WMreB 1-9 and its N-terminal acetylated analog, Ac-WMreB 1-9 show preferential binding to negatively-charged liposomes. Both the peptides cause permeabilization of E. coli inner and outer-membranes. Tryptophan-lacking peptides, though permeabilize the outer-membrane efficiently, little permeabilization of the inner-membrane is observed. These data attest membrane-destabilization as the mechanism of rapid bacterial killing. This study is expected to motivate the research in identifying microbes' self-sequences to combat them. Copyright © 2018 Elsevier Inc. All rights reserved.

Two novel WD40 domain–containing proteins, Ere1 and Ere2, function in the retromer-mediated endosomal recycling pathway

Science.gov (United States)

Shi, Yufeng; Stefan, Christopher J.; Rue, Sarah M.; Teis, David; Emr, Scott D.

2011-01-01

Regulated secretion, nutrient uptake, and responses to extracellular signals depend on cell-surface proteins that are internalized and recycled back to the plasma membrane. However, the underlying mechanisms that govern membrane protein recycling to the cell surface are not fully known. Using a chemical-genetic screen in yeast, we show that the arginine transporter Can1 is recycled back to the cell surface via two independent pathways mediated by the sorting nexins Snx4/41/42 and the retromer complex, respectively. In addition, we identify two novel WD40-domain endosomal recycling proteins, Ere1 and Ere2, that function in the retromer pathway. Ere1 is required for Can1 recycling via the retromer-mediated pathway, but it is not required for the transport of other retromer cargoes, such as Vps10 and Ftr1. Biochemical studies reveal that Ere1 physically interacts with internalized Can1. Ere2 is present in a complex containing Ere1 on endosomes and functions as a regulator of Ere1. Taken together, our results suggest that Snx4/41/42 and the retromer comprise two independent pathways for the recycling of internalized cell-surface proteins. Moreover, a complex containing the two novel proteins Ere1 and Ere2 mediates cargo-specific recognition by the retromer pathway. PMID:21880895

Membrane Technology for Produced Water in Lea County

Energy Technology Data Exchange (ETDEWEB)

Cecilia Nelson; Ashok Ghosh

2011-06-30

Southeastern New Mexico (SENM) is rich in mineral resources, including oil and gas. Produced water is a byproduct from oil and gas recovery operations. SENM generates approximately 400 million barrels per year of produced water with total dissolved solids (TDS) as high as ~ 200,000 ppm. Typically, produced water is disposed of by transporting it to injection wells or disposal ponds, costing around $1.2 billion per year with an estimated use of 0.3 million barrels of transportation fuel. New Mexico ranks first among U.S. states in potash production. Nationally, more than 85% of all potash produced comes from the Carlsbad potash district in SENM. Potash manufacturing processes use large quantities of water, including fresh water, for solution mining. If the produced water from oilfield operations can be treated and used economically in the potash industry, it will provide a beneficial use for the produced water as well as preserve valuable water resources in an area where fresh water is scarce. The goal of this current research was to develop a prototype desalination system that economically treats produced water from oil and/or natural gas operations for the beneficial use of industries located in southeastern New Mexico. Up until now, most water cleaning technologies have been developed for treating water with much lower quantities of TDS. Seawater with TDS of around 30,000 ppm is the highest concentration that has been seriously studied by researchers. Reverse osmosis (RO) technology is widely used; however the cost remains high due to high-energy consumption. Higher water fluxes and recoveries are possible with a properly designed Forward Osmosis (FO) process as large driving forces can be induced with properly chosen membranes and draw solution. Membrane fouling and breakdown is a frequent and costly problem that drives the cost of desalination very high. The technology developed by New Mexico Tech (NMT) researchers not only protects the membrane, but has also

Purification and differentiation of human adipose-derived stem cells by membrane filtration and membrane migration methods

Science.gov (United States)

Lin, Hong Reng; Heish, Chao-Wen; Liu, Cheng-Hui; Muduli, Saradaprasan; Li, Hsing-Fen; Higuchi, Akon; Kumar, S. Suresh; Alarfaj, Abdullah A.; Munusamy, Murugan A.; Hsu, Shih-Tien; Chen, Da-Chung; Benelli, Giovanni; Murugan, Kadarkarai; Cheng, Nai-Chen; Wang, Han-Chow; Wu, Gwo-Jang

2017-01-01

Human adipose-derived stem cells (hADSCs) are easily isolated from fat tissue without ethical concerns, but differ in purity, pluripotency, differentiation ability, and stem cell marker expression, depending on the isolation method. We isolated hADSCs from a primary fat tissue solution using: (1) conventional culture, (2) a membrane filtration method, (3) a membrane migration method where the primary cell solution was permeated through membranes, adhered hADSCs were cultured, and hADSCs migrated out from the membranes. Expression of mesenchymal stem cell markers and pluripotency genes, and osteogenic differentiation were compared for hADSCs isolated by different methods using nylon mesh filter membranes with pore sizes ranging from 11 to 80 μm. hADSCs isolated by the membrane migration method had the highest MSC surface marker expression and efficient differentiation into osteoblasts. Osteogenic differentiation ability of hADSCs and MSC surface marker expression were correlated, but osteogenic differentiation ability and pluripotent gene expression were not. PMID:28071738

Evaluation of membranes for the treatment of oilfield-produced water

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Silvio Edegar Weschenfelder

2015-04-01

Full Text Available Large quantities of water are generated during oil extraction. Reinjection of this effluent is frequently the most suitable option for environmental considerations. For this purpose, the water produced must have characteristics that will prevent the plugging of the reservoir rocks, i.e., suspended solids and oil and grease content must be lower than 5 mg L-1. This work evaluated the performance of membranes for water treatment, based upon long-term experiments with real effluent, evaluating the permeate flux over time and the effluent characteristics. The results indicated that it is possible to obtain a permeate flux with suspended solids content with less than 1 mg L-1 oil and a grease concentration in the range of 1 to 3 mg L-1 using membranes with a pore size of 0,1 m. After 50 hours of permeation, the permeate fluxes obtained with the polymeric and ceramic membranes were equal to 50 and 300 L h-1 m-2, respectively. With a chemical regeneration procedure, it was possible to recover 95% of the ceramic membrane’s original permeability and 30% of the polymeric membrane’s permeability. Considering the permeate quality, the regeneration efficiency of the membrane and the fluxes achieved, it is possible to point out the use of ceramic membranes as a potential technology for treating oilfield-produced water, and it has the advantage of being a compact and robust process.

HIV-1 Envelope Glycoprotein Trafficking through the Endosomal Recycling Compartment Is Required for Particle Incorporation.

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Kirschman, Junghwa; Qi, Mingli; Ding, Lingmei; Hammonds, Jason; Dienger-Stambaugh, Krista; Wang, Jaang-Jiun; Lapierre, Lynne A; Goldenring, James R; Spearman, Paul

2018-03-01

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) encodes specific trafficking signals within its long cytoplasmic tail (CT) that regulate incorporation into HIV-1 particles. Rab11-family interacting protein 1C (FIP1C) and Rab14 are host trafficking factors required for Env particle incorporation, suggesting that Env undergoes sorting from the endosomal recycling compartment (ERC) to the site of particle assembly on the plasma membrane. We disrupted outward sorting from the ERC by expressing a C-terminal fragment of FIP1C (FIP1C 560-649 ) and examined the consequences on Env trafficking and incorporation into particles. FIP1C 560-649 reduced cell surface levels of Env and prevented its incorporation into HIV-1 particles. Remarkably, Env was trapped in an exaggerated perinuclear ERC in a CT-dependent manner. Mutation of either the Yxxϕ endocytic motif or the YW 795 motif in the CT prevented Env trapping in the ERC and restored incorporation into particles. In contrast, simian immunodeficiency virus SIVmac239 Env was not retained in the ERC, while substitution of the HIV-1 CT for the SIV CT resulted in SIV Env retention in this compartment. These results provide the first direct evidence that Env traffics through the ERC and support a model whereby HIV-1 Env is specifically targeted to the ERC prior to FIP1C- and CT-dependent outward sorting to the particle assembly site on the plasma membrane. IMPORTANCE The HIV envelope protein is an essential component of the viral particle. While many aspects of envelope protein structure and function have been established, the pathway it follows in the cell prior to reaching the site of particle assembly is not well understood. The envelope protein has a very long cytoplasmic tail that interacts with the host cell trafficking machinery. Here, we utilized a truncated form of the trafficking adaptor FIP1C protein to arrest the intracellular transport of the envelope protein, demonstrating that it becomes

Alternative movement : collaborative project has researchers looking to ceramic membranes to improve produced water treatment

Energy Technology Data Exchange (ETDEWEB)

Wells, P.

2009-10-15

Ceramic membranes have high chemical and thermal stability coupled with mechanical strength and are therefore used in a range of microfiltration, ultrafiltration and nanofiltration applications. This article described a new technology that involves the use of ceramic membranes in the treatment of produced water in thermal heavy oil recovery operations. The efficacy of advanced ceramic nano-membrane technology (CMT) is being examined in bench-scale experiments at the Southern Alberta Institute of Technology (SAIT) in collaboration with the department of chemical and petroleum engineering at the University of Calgary. In one project, next-generation ceramic membrane technology is being used as part of the overall treatment process of produced water. The project is funded through a Canadian Association of Petroleum Producers fund and the Alberta Department of Energy. It is facilitated by the Petroleum Technology Alliance Canada in an effort to find cost-effective treatment solutions for recycling produced water for the conventional oil and gas industry. The key objective is to increase the amount of produced water that can be reused rather than disposed into deep saline aquifers. The research focuses on the pre-treatment of produced water and related salt impacted water by using ceramic membranes for the removal of organic compounds for beneficial reuse downstream. Ceramic membranes consist of a multilayer system and their performance depends on the separation and permeation properties of the membrane as well as its mechanical integrity. It was concluded that the CMT findings will be beneficial to the oil and gas industry in providing practical solutions for the challenging issues associated with de-oiling and produced water treatment. 2 figs.

Biogenesis and function of T cell-derived exosomes

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Miguel Angel Alonso

2016-08-01

Full Text Available Exosomes are a particular type of extracellular vesicle, characterized by their endosomal origin as intraluminal vesicles present in large endosomes with a multivesicular structure. After these endosomes fuse with the plasma membrane, exosomes are secreted into the extracellular space. The ability of exosomes to carry and selectively deliver bioactive molecules (e.g., lipids, proteins and nucleic acids confers on them the capacity to modulate the activity of receptor cells, even if these cells are located in distant tissues or organs. Since exosomal cargo depends on cell type, a detailed understanding of the mechanisms that regulate the biochemical composition of exosomes is fundamental to a comprehensive view of exosome function. Here, we review the latest advances concerning exosome function and biogenesis in T cells, with particular focus on the mechanism of protein sorting at multivesicular endosomes. Exosomes secreted by specific T-cell subsets can modulate the activity of immune cells, including other T-cell subsets. Ceramide, tetraspanins and MAL have been revealed to be important in exosome biogenesis by T cells. These molecules, therefore, constitute potential molecular targets for artificially modulating exosome production and, hence, the immune response for therapeutic purposes.

An Inside Job: How Endosomal Na+/H+ Exchangers Link to Autism and Neurological Disease

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Kalyan C. Kondapalli

2014-06-01

Full Text Available Autism imposes a major impediment to childhood development and a huge emotional and financial burden on society. In recent years, there has been rapidly accumulating genetic evidence that links the eNHE, a subset of Na+/H+ exchangers that localize to intracellular vesicles, to a variety of neurological conditions including autism, attention deficit hyperactivity disorder, intellectual disability and epilepsy. By providing a leak pathway for protons pumped by the V-ATPase, eNHE determine luminal pH and regulate cation (Na+, K+ content in early and recycling endosomal compartments. Loss-of-function mutations in eNHE cause hyperacidification of endosomal lumen, as a result of imbalance in pump and leak pathways. Two isoforms, NHE6 and NHE9 are highly expressed in brain, including hippocampus and cortex. Here, we summarize evidence for the importance of luminal cation content and pH on processing, delivery and fate of cargo and on the surface expression and function of membrane receptors and neurotransmitter transporters, drawing upon insights from model organisms and mammalian cells. These studies lead to cellular models of eNHE activity in pre- and post-synaptic neurons and astrocytes, where they could impact synapse development and plasticity. The study of eNHE has provided new insight on the mechanism of autism and other debilitating neurological disorders and opened up new possibilities for therapeutic intervention.

The small G protein Arl5 contributes to endosome-to-Golgi traffic by aiding the recruitment of the GARP complex to the Golgi

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Cláudia Rosa-Ferreira

2015-03-01

Full Text Available The small G proteins of the Arf family play critical roles in membrane trafficking and cytoskeleton organization. However, the function of some members of the family remains poorly understood including Arl5 which is widely conserved in eukaryotes. Humans have two closely related Arl5 paralogues (Arl5a and Arl5b, and both Arl5a and Arl5b localize to the trans-Golgi with Arl5b being involved in retrograde traffic from endosomes to the Golgi apparatus. To investigate the function of Arl5, we have used Drosophila melanogaster as a model system. We find that the single Arl5 orthologue in Drosophila also localizes to the trans-Golgi, but flies lacking the Arl5 gene are viable and fertile. By using both liposome and column based affinity chromatography methods we find that Arl5 interacts with the Golgi-associated retrograde protein (GARP complex that acts in the tethering of vesicles moving from endosomes to the trans-Golgi network (TGN. In Drosophila tissues the GARP complex is partially displaced from the Golgi when Arl5 is absent, and the late endosomal compartment is enlarged. In addition, in HeLa cells GARP also becomes cytosolic upon depletion of Arl5b. These phenotypes are consistent with a role in endosome-to-Golgi traffic, but are less severe than loss of GARP itself. Thus it appears that Arl5 is one of the factors that directs the recruitment of the GARP complex to the trans-Golgi, and this function is conserved in both flies and humans.

Smart DNA vectors based on cyclodextrin polymers: compaction and endosomal release.

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Wintgens, Véronique; Leborgne, Christian; Baconnais, Sonia; Burckbuchler, Virginie; Le Cam, Eric; Scherman, Daniel; Kichler, Antoine; Amiel, Catherine

2012-02-01

Neutral β-cyclodextrin polymers (polyβCD) associated with cationic adamantyl derivatives (Ada) can be used to deliver plasmid DNA into cells. In absence of an endosomolytic agent, transfection efficiency remains low because most complexes are trapped in the endosomal compartment. We asked whether addition of an imidazole-modified Ada can increase efficiency of polyβCD/cationic Ada-based delivery system. We synthesized two adamantyl derivatives: Ada5, which has a spacer arm between the Ada moiety and a bi-cationic polar head group, and Ada6, which presents an imidazole group. Strength of association between polyβCD and Ada derivatives was evaluated by fluorimetric titration. Gel mobility shift assay, zeta potential, and dark field transmission electron microscopy experiments demonstrated the system allowed for efficient DNA compaction. In vitro transfection experiments performed on HepG2 and HEK293 cells revealed the quaternary system polyβCD/Ada5/Ada6/DNA has efficiency comparable to cationic lipid DOTAP. We successfully designed fine-tuned DNA vectors based on cyclodextrin polymers combined with two new adamantyl derivatives, leading to significant transfection associated with low toxicity.

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

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Tracy P. M. Chong

2011-03-01

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

Vps33b pathogenic mutations preferentially affect VIPAS39/SPE-39-positive endosomes.

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Tornieri, Karine; Zlatic, Stephanie A; Mullin, Ariana P; Werner, Erica; Harrison, Robert; L'hernault, Steven W; Faundez, Victor

2013-12-20

Mutations in Vps33 isoforms cause pigment dilution in mice (Vps33a, buff) and Drosophila (car) and the neurogenic arthrogryposis, renal dysfunction and cholestasis syndrome in humans (ARC1, VPS33B). The later disease is also caused by mutations in VIPAS39, (Vps33b interacting protein, apical-basolateral polarity regulator, SPE-39 homolog; ARC2), a protein that interacts with the HOmotypic fusion and Protein Sorting (HOPS) complex, a tether necessary for endosome-lysosome traffic. These syndromes offer insight into fundamental endosome traffic processes unique to metazoans. However, the molecular and cellular mechanisms underlying these mutant phenotypes remain poorly understood. Here we investigate interactions of wild-type and disease-causing mutations in VIPAS39/SPE-39 and Vps33b by yeast two hybrid, immunoprecipitation and quantitative fluorescent microscopy. We find that although few mutations prevent interaction between VIPAS39/SPE-39 and Vps33b, some mutants fragment VIPAS39/SPE-39-positive endosomes, but all mutants alter the subcellular localization of Vps33b to VIPAS39/SPE-39-positive endosomes. Our data suggest that the ARC syndrome may result through impaired VIPAS39/SPE-39 and Vps33b-dependent endosomal maturation or fusion.

Perfringolysin O Theta Toxin as a Tool to Monitor the Distribution and Inhomogeneity of Cholesterol in Cellular Membranes.

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Maekawa, Masashi; Yang, Yanbo; Fairn, Gregory D

2016-03-08

Cholesterol is an essential structural component of cellular membranes in eukaryotes. Cholesterol in the exofacial leaflet of the plasma membrane is thought to form membrane nanodomains with sphingolipids and specific proteins. Additionally, cholesterol is found in the intracellular membranes of endosomes and has crucial functions in membrane trafficking. Furthermore, cellular cholesterol homeostasis and regulation of de novo synthesis rely on transport via both vesicular and non-vesicular pathways. Thus, the ability to visualize and detect intracellular cholesterol, especially in the plasma membrane, is critical to understanding the complex biology associated with cholesterol and the nanodomains. Perfringolysin O (PFO) theta toxin is one of the toxins secreted by the anaerobic bacteria Clostridium perfringens and this toxin forms pores in the plasma membrane that causes cell lysis. It is well understood that PFO recognizes and binds to cholesterol in the exofacial leaflets of the plasma membrane, and domain 4 of PFO (D4) is sufficient for the binding of cholesterol. Recent studies have taken advantage of this high-affinity cholesterol-binding domain to create a variety of cholesterol biosensors by using a non-toxic PFO or the D4 in isolation. This review highlights the characteristics and usefulness of, and the principal findings related to, these PFO-derived cholesterol biosensors.

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

LENUS (Irish Health Repository)

Johnson, Ian R D

2015-11-10

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

Ion transport membrane reactor systems and methods for producing synthesis gas

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Repasky, John Michael

2015-05-12

Embodiments of the present invention provide cost-effective systems and methods for producing a synthesis gas product using a steam reformer system and an ion transport membrane (ITM) reactor having multiple stages, without requiring inter-stage reactant injections. Embodiments of the present invention also provide techniques for compensating for membrane performance degradation and other changes in system operating conditions that negatively affect synthesis gas production.

Tetraspanin CD63 Bridges Autophagic and Endosomal Processes To Regulate Exosomal Secretion and Intracellular Signaling of Epstein-Barr Virus LMP1

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Hurwitz, Stephanie N; Cheerathodi, Mujeeb R; Nkosi, Dingani; York, Sara B; Meckes, David G

2018-03-01

The tetraspanin protein CD63 has been recently described as a key factor in extracellular vesicle (EV) production and endosomal cargo sorting. In the context of Epstein-Barr virus (EBV) infection, CD63 is required for the efficient packaging of the major viral oncoprotein latent membrane protein 1 (LMP1) into exosomes and other EV populations and acts as a negative regulator of LMP1 intracellular signaling. Accumulating evidence has also pointed to intersections of the endosomal and autophagy pathways in maintaining cellular secretory processes and as sites for viral assembly and replication. Indeed, LMP1 can activate the mammalian target of rapamycin (mTOR) pathway to suppress host cell autophagy and facilitate cell growth and proliferation. Despite the growing recognition of cross talk between endosomes and autophagosomes and its relevance to viral infection, little is understood about the molecular mechanisms governing endosomal and autophagy convergence. Here, we demonstrate that CD63-dependent vesicle protein secretion directly opposes intracellular signaling activation downstream of LMP1, including mTOR-associated proteins. Conversely, disruption of normal autolysosomal processes increases LMP1 secretion and dampens signal transduction by the viral protein. Increases in mTOR activation following CD63 knockout are coincident with the development of serum-dependent autophagic vacuoles that are acidified in the presence of high LMP1 levels. Altogether, these findings suggest a key role of CD63 in regulating the interactions between endosomal and autophagy processes and limiting cellular signaling activity in both noninfected and virally infected cells. IMPORTANCE The close connection between extracellular vesicles and viruses is becoming rapidly and more widely appreciated. EBV, a human gamma herpesvirus that contributes to the progression of a multitude of lymphomas and carcinomas in immunocompromised or genetically susceptible populations, packages its major

Human Papillomavirus 16 Infection Induces VAP-Dependent Endosomal Tubulation.

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Siddiqa, Abida; Massimi, Paola; Pim, David; Broniarczyk, Justyna; Banks, Lawrence

2018-03-15

Human papillomavirus (HPV) infection involves complex interactions with the endocytic transport machinery, which ultimately facilitates the entry of the incoming viral genomes into the trans -Golgi network (TGN) and their subsequent nuclear entry during mitosis. The endosomal pathway is a highly dynamic intracellular transport system, which consists of vesicular compartments and tubular extensions, although it is currently unclear whether incoming viruses specifically alter the endocytic machinery. In this study, using MICAL-L1 as a marker for tubulating endosomes, we show that incoming HPV-16 virions induce a profound alteration in global levels of endocytic tubulation. In addition, we also show a critical requirement for the endoplasmic reticulum (ER)-anchored protein VAP in this process. VAP plays an essential role in actin nucleation and endosome-to-Golgi transport. Indeed, the loss of VAP results in a dramatic decrease in the level of endosomal tubulation induced by incoming HPV-16 virions. This is also accompanied by a marked reduction in virus infectivity. In VAP knockdown cells, we see that the defect in virus trafficking occurs after capsid disassembly but prior to localization at the trans -Golgi network, with the incoming virion-transduced DNA accumulating in Vps29/TGN46-positive hybrid vesicles. Taken together, these studies demonstrate that infection with HPV-16 virions induces marked alterations of endocytic transport pathways, some of which are VAP dependent and required for the endosome-to-Golgi transport of the incoming viral L2/DNA complex. IMPORTANCE Human papillomavirus infectious entry involves multiple interactions with the endocytic transport machinery. In this study, we show that incoming HPV-16 virions induce a dramatic increase in endocytic tubulation. This tubulation requires ER-associated VAP, which plays a critical role in ensuring the delivery of cargoes from the endocytic compartments to the trans -Golgi network. Indeed, the loss of

Cytosolic antibody delivery by lipid-sensitive endosomolytic peptide

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Akishiba, Misao; Takeuchi, Toshihide; Kawaguchi, Yoshimasa; Sakamoto, Kentarou; Yu, Hao-Hsin; Nakase, Ikuhiko; Takatani-Nakase, Tomoka; Madani, Fatemeh; Gräslund, Astrid; Futaki, Shiroh

2017-08-01

One of the major obstacles in intracellular targeting using antibodies is their limited release from endosomes into the cytosol. Here we report an approach to deliver proteins, which include antibodies, into cells by using endosomolytic peptides derived from the cationic and membrane-lytic spider venom peptide M-lycotoxin. The delivery peptides were developed by introducing one or two glutamic acid residues into the hydrophobic face. One peptide with the substitution of leucine by glutamic acid (L17E) was shown to enable a marked cytosolic liberation of antibodies (immunoglobulins G (IgGs)) from endosomes. The predominant membrane-perturbation mechanism of this peptide is the preferential disruption of negatively charged membranes (endosomal membranes) over neutral membranes (plasma membranes), and the endosomolytic peptide promotes the uptake by inducing macropinocytosis. The fidelity of this approach was confirmed through the intracellular delivery of a ribosome-inactivation protein (saporin), Cre recombinase and IgG delivery, which resulted in a specific labelling of the cytosolic proteins and subsequent suppression of the glucocorticoid receptor-mediated transcription. We also demonstrate the L17E-mediated cytosolic delivery of exosome-encapsulated proteins.

Transcription factor σB plays an important role in the production of extracellular membrane-derived vesicles in Listeria monocytogenes.

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Jung Hwa Lee

Full Text Available Gram-negative bacteria produce extracellular outer membrane vesicles (OMVs that interact with host cells. Unlike Gram-negative bacteria, less is known about the production and role of extracellular membrane vesicles (MVs in Gram-positive bacteria. The food-borne pathogen Listeria monocytogenes can survive under extreme environmental and energy stress conditions and the transcription factor σ(B is involved in this survival ability. Here, we first determined the production of MVs from L. monocytogenes and evaluated whether general stress transcription factor σ(B affected production of MVs in L. monocytogenes. L. monocytogenes secreted MVs during in vitro broth culture. The wild-type strain actively produced MVs approximately nine times more and also produced more intact shapes of MVs than those of the isogenic ΔsigB mutant. A proteomic analysis showed that 130 and 89 MV proteins were identified in the wild-type and ΔsigB mutant strains, respectively. Wild-type strain-derived MVs contained proteins regulated by σ(B such as transporters (OpuCA and OpuCC, stress response (Kat, metabolism (LacD, translation (InfC, and cell division protein (FtsZ. Gene Ontology (GO enrichment analysis showed that wild-type-derived MV proteins corresponded to several GO terms, including response to stress (heat, acid, and bile resistance and extracellular polysaccharide biosynthetic process, but not the ΔsigB mutant. Internalin B (InlB was almost three times more contained in MVs derived from the wild-type strain than in MVs derived from the ΔsigB mutant. Taken together, these results suggest that σ(B plays a pivotal role in the production of MVs and protein profiles contained in MVs. L. monocytogenes MVs may contribute to host infection and survival ability under various stressful conditions.

dOCRL maintains immune cell quiescence by regulating endosomal traffic.

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Steven J Del Signore

2017-10-01

Full Text Available Lowe Syndrome is a developmental disorder characterized by eye, kidney, and neurological pathologies, and is caused by mutations in the phosphatidylinositol-5-phosphatase OCRL. OCRL plays diverse roles in endocytic and endolysosomal trafficking, cytokinesis, and ciliogenesis, but it is unclear which of these cellular functions underlie specific patient symptoms. Here, we show that mutation of Drosophila OCRL causes cell-autonomous activation of hemocytes, which are macrophage-like cells of the innate immune system. Among many cell biological defects that we identified in docrl mutant hemocytes, we pinpointed the cause of innate immune cell activation to reduced Rab11-dependent recycling traffic and concomitantly increased Rab7-dependent late endosome traffic. Loss of docrl amplifies multiple immune-relevant signals, including Toll, Jun kinase, and STAT, and leads to Rab11-sensitive mis-sorting and excessive secretion of the Toll ligand Spåtzle. Thus, docrl regulation of endosomal traffic maintains hemocytes in a poised, but quiescent state, suggesting mechanisms by which endosomal misregulation of signaling may contribute to symptoms of Lowe syndrome.

Translocation mechanism of C{sub 60} and C{sub 60} derivations across a cell membrane

Energy Technology Data Exchange (ETDEWEB)

Liang, Lijun, E-mail: michael.lijunl@gmail.com [Hangzhou Dianzi University, College of Life Information Science and Instrument Engineering (China); Kang, Zhengzhong [Zhejiang University, Department of Chemistry (China); Shen, Jia-Wei, E-mail: shen.jiawei@hotmail.com [Hangzhou Normal University, School of Medicine (China)

2016-11-15

Carbon-based nanoparticles (NPs) such as fullerenes and nanotubes have been extensively studied for drug delivery in recent years. The permeation process of fullerene and its derivative molecules through membrane is essential to the utilization of fullerene-based drug delivery system, but the mechanism and the dynamics of permeation through cell membrane are still unclear. In this study, coarse-grained molecular dynamics simulations were performed to investigate the permeation process of functionalized fullerene molecules (ca. 0.72 nm) through the membrane. Our results show that single functionalized fullerene molecule in such nanoscale could permeate the lipid membrane in micro-second time scale. Pristine C{sub 60} molecules prefer to aggregate into several small clusters while C{sub 60}OH{sub 15} molecules could aggregate into one big cluster to permeate through the lipid membrane. After permeation of C{sub 60} or its derivatives into membrane, all C{sub 60} and C{sub 60}OH{sub 15} molecules disaggregated and monodispersed in the lipid membrane.

Promyelocytic leukemia bodies tether to early endosomes during mitosis.

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

2014-01-01

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

Differential endosomal sorting of a novel P2Y12 purinoreceptor mutant.

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Cunningham, Margaret R; Nisar, Shaista P; Cooke, Alexandra E; Emery, Elizabeth D; Mundell, Stuart J

2013-05-01

P2Y12 receptor internalization and recycling play an essential role in ADP-induced platelet activation. Recently, we identified a patient with a mild bleeding disorder carrying a heterozygous mutation of P2Y12 (P341A) whose P2Y12 receptor recycling was significantly compromised. Using human cell line models, we identified key proteins regulating wild-type (WT) P2Y12 recycling and investigated P2Y12 -P341A receptor traffic. Treatment with ADP resulted in delayed Rab5-dependent internalization of P341A when compared with WT P2Y12 . While WT P2Y12 rapidly recycled back to the membrane via Rab4 and Rab11 recycling pathways, limited P341A recycling was observed, which relied upon Rab11 activity. Although minimal receptor degradation was evident, P341A was localized in Rab7-positive endosomes with considerable agonist-dependent accumulation in the trans-Golgi network (TGN). Rab7 activity is known to facilitate recruitment of retromer complex proteins to endosomes to transport cargo to the TGN. Here, we identified that P341A colocalized with Vps26; depletion of which blocked limited recycling and promoted receptor degradation. This study has identified key points of divergence in the endocytic traffic of P341A versus WT-P2Y12 . Given that these pathways are retained in human platelets, this research helps define the molecular mechanisms regulating P2Y12 receptor traffic and explain the compromised receptor function in the platelets of the P2Y12 -P341A-expressing patient. © 2013 John Wiley & Sons A/S.

Enhancing endosomal escape of transduced proteins by photochemical internalisation.

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Kevin Mellert

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

Enhancing endosomal escape of transduced proteins by photochemical internalisation.

Science.gov (United States)

Mellert, Kevin; Lamla, Markus; Scheffzek, Klaus; Wittig, Rainer; Kaufmann, Dieter

2012-01-01

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

Integration of two RAB5 groups during endosomal transport in plants

Science.gov (United States)

Ebine, Kazuo; Choi, Seung-won; Ichinose, Sakura; Uemura, Tomohiro; Nakano, Akihiko

2018-01-01

RAB5 is a key regulator of endosomal functions in eukaryotic cells. Plants possess two different RAB5 groups, canonical and plant-unique types, which act via unknown counteracting mechanisms. Here, we identified an effector molecule of the plant-unique RAB5 in Arabidopsis thaliana, ARA6, which we designated PLANT-UNIQUE RAB5 EFFECTOR 2 (PUF2). Preferential colocalization with canonical RAB5 on endosomes and genetic interaction analysis indicated that PUF2 coordinates vacuolar transport with canonical RAB5, although PUF2 was identified as an effector of ARA6. Competitive binding of PUF2 with GTP-bound ARA6 and GDP-bound canonical RAB5, together interacting with the shared activating factor VPS9a, showed that ARA6 negatively regulates canonical RAB5-mediated vacuolar transport by titrating PUF2 and VPS9a. These results suggest a unique and unprecedented function for a RAB effector involving the integration of two RAB groups to orchestrate endosomal trafficking in plant cells. PMID:29749929

Membrane Disordering is not Sufficient for Membrane Permeabilization by Islet Amyloidogenic Polypeptide: Studies of IAPP(20-29) Fragments

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Brender, Jeffrey R.; Heyl, Deborah L.; Samisetti, Shyamprasad; Kotler, Samuel A.; Osborne, Joshua M.; Pesaru, Ranadheer R.; Ramamoorthy, Ayyalusamy

2013-01-01

A key factor in the development of type II diabetes is the loss of insulin-producing beta-cells. Human islet amyloid polypeptide protein (human-IAPP) is believed to play a crucial role in this process by forming small aggregates that exhibit toxicity by disrupting the cell membrane. The actual mechanism of membrane disruption is complex and appears to involve an early component before fiber formation and later component associated with fiber formation on the membrane. By comparing the peptide-lipid interactions derived from solid-state NMR experiments of two IAPP fragments that bind the membrane and cause membrane disordering to IAPP derived peptides known to cause significant early membrane permeabilization, we show here that membrane disordering is not likely to be sufficient by itself to cause the early membrane permeabilization observed by IAPP, and may play a lesser role in IAPP membrane disruption than expected. PMID:23493863

Differential subcellular membrane recruitment of Src may specify its downstream signalling

International Nuclear Information System (INIS)

Diesbach, Philippe de; Medts, Thierry; Carpentier, Sarah; D'Auria, Ludovic; Van Der Smissen, Patrick; Platek, Anna; Mettlen, Marcel; Caplanusi, Adrian; Hove, Marie-France van den; Tyteca, Donatienne; Courtoy, Pierre J.

2008-01-01

Most Src family members are diacylated and constitutively associate with membrane 'lipid rafts' that coordinate signalling. Whether the monoacylated Src, frequently hyperactive in carcinomas, also localizes at 'rafts' remains controversial. Using polarized MDCK cells expressing the thermosensitive v-Src/tsLA31 variant, we here addressed how Src tyrosine-kinase activation may impact on its (i) membrane recruitment, in particular to 'lipid rafts'; (ii) subcellular localization; and (iii) signalling. The kinetics of Src-kinase thermoactivation correlated with its recruitment from the cytosol to sedimentable membranes where Src largely resisted solubilisation by non-ionic detergents at 4 deg. C and floated into sucrose density gradients like caveolin-1 and flotillin-2, i.e. 'lipid rafts'. By immunofluorescence, activated Src showed a dual localization, at apical endosomes/macropinosomes and at the apical plasma membrane. The plasma membrane Src pool did not colocalize with caveolin-1 and flotillin-2, but extensively overlapped GM1 labelling by cholera toxin. Severe (∼ 70%) cholesterol extraction with methyl-β-cyclodextrin (MβCD) did not abolish 'rafts' floatation, but strongly decreased Src association with floating 'rafts' and abolished its localization at the apical plasma membrane. Src activation independently activated first the MAP-kinase - ERK1/2 pathway, then the PI3-kinase - Akt pathway. MAP-kinase - ERK1/2 activation was insensitive to MβCD, which suppressed Akt phosphorylation and apical endocytosis induced by Src, both depending on the PI3-kinase pathway. We therefore suggest that activated Src is recruited at two membrane compartments, allowing differential signalling, first via ERK1/2 at 'non-raft' domains on endosomes, then via PI3-kinase-Akt on a distinct set of 'rafts' at the apical plasma membrane. Whether this model is applicable to c-Src remains to be examined

Genetic reconstitution of the human Adenovirus type 2 temperature-sensitive 1 mutant defective in endosomal escape

Directory of Open Access Journals (Sweden)

Gastaldelli Michele

2009-10-01

Full Text Available Abstract Human Adenoviruses infect the upper and lower respiratory tracts, the urinary and digestive tracts, lymphoid systems and heart, and give rise to epidemic conjunctivitis. More than 51 human serotypes have been identified to-date, and classified into 6 species A-F. The species C Adenoviruses Ad2 and Ad5 (Ad2/5 cause upper and lower respiratory disease, but how viral structure relates to the selection of particular infectious uptake pathways is not known. An adenovirus mutant, Ad2-ts1 had been isolated upon chemical mutagenesis in the past, and shown to have unprocessed capsid proteins. Ad2-ts1 fails to package the viral protease L3/p23, and Ad2-ts1 virions do not efficiently escape from endosomes. It had been suggested that the C22187T point mutation leading to the substitution of the conserved proline 137 to leucine (P137L in the L3/p23 protease was at least in part responsible for this phenotype. To clarify if the C22187T mutation is necessary and sufficient for the Ad2-ts1 phenotype, we sequenced the genes encoding the structural proteins of Ad2-ts1, and confirmed that the Ad2-ts1 DNA carries the point mutation C22187T. Introduction of C22187T to the wild-type Ad2 genome in a bacterial artificial chromosome (Ad2-BAC gave Ad2-BAC46 virions with the full Ad2-ts1 phenotype. Reversion of Ad2-BAC46 gave wild-type Ad2 particles indicating that P137L is necessary and sufficient for the Ad2-ts1 phenotype. The kinetics of Ad2-ts1 uptake into cells were comparable to Ad2 suggesting similar endocytic uptake mechanisms. Surprisingly, infectious Ad2 or Ad5 but not Ad2-ts1 uptake required CALM (clathrin assembly lymphoid myeloid protein, which controls clathrin-mediated endocytosis and membrane transport between endosomes and the trans-Golgi-network. The data show that no other mutations than P137L in the viral protease are necessary to give rise to particles that are defective in capsid processing and endosomal escape. This provides a basis for

The endocytic pathways of a secretory granule membrane protein in HEK293 cells: PAM and EGF traverse a dynamic multivesicular body network together.

Science.gov (United States)

Bäck, Nils; Kanerva, Kristiina; Kurutihalli, Vishwanatha; Yanik, Andrew; Ikonen, Elina; Mains, Richard E; Eipper, Betty A

2017-08-01

Peptidylglycine α-amidating monooxygenase (PAM) is highly expressed in neurons and endocrine cells, where it catalyzes one of the final steps in the biosynthesis of bioactive peptides. PAM is also expressed in unicellular organisms such as Chlamydomonas reinhardtii, which do not store peptides in secretory granules. As for other granule membrane proteins, PAM is retrieved from the cell surface and returned to the trans-Golgi network. This pathway involves regulated entry of PAM into multivesicular body intralumenal vesicles (ILVs). The aim of this study was defining the endocytic pathways utilized by PAM in cells that do not store secretory products in granules. Using stably transfected HEK293 cells, endocytic trafficking of PAM was compared to that of the mannose 6-phosphate (MPR) and EGF (EGFR) receptors, established markers for the endosome to trans-Golgi network and degradative pathways, respectively. As in neuroendocrine cells, PAM internalized by HEK293 cells accumulated in the trans-Golgi network. Based on surface biotinylation, >70% of the PAM on the cell surface was recovered intact after a 4h chase and soluble, bifunctional PAM was produced. Endosomes containing PAM generally contained both EGFR and MPR and ultrastructural analysis confirmed that all three cargos accumulated in ILVs. PAM containing multivesicular bodies made frequent dynamic tubular contacts with younger and older multivesicular bodies. Frequent dynamic contacts were observed between lysosomes and PAM containing early endosomes and multivesicular bodies. The ancient ability of PAM to localize to ciliary membranes, which release bioactive ectosomes, may be related to its ability to accumulate in ILVs and exosomes. Copyright © 2017 Elsevier GmbH. All rights reserved.

Detection of closed influenza virus hemagglutinin fusion peptide structures in membranes by backbone {sup 13}CO-{sup 15}N rotational-echo double-resonance solid-state NMR

Energy Technology Data Exchange (ETDEWEB)

Ghosh, Ujjayini; Xie Li; Weliky, David P., E-mail: weliky@chemistry.msu.edu [Michigan State University, Department of Chemistry (United States)

2013-02-15

The influenza virus fusion peptide is the N-terminal {approx}20 residues of the HA2 subunit of the hemagglutinin protein and this peptide plays a key role in the fusion of the viral and endosomal membranes during initial infection of a cell. The fusion peptide adopts N-helix/turn/C-helix structure in both detergent and membranes with reports of both open and closed interhelical topologies. In the present study, backbone {sup 13}CO-{sup 15}N REDOR solid-state NMR was applied to the membrane-associated fusion peptide to detect the distribution of interhelical distances. The data clearly showed a large fraction of closed and semi-closed topologies and were best-fitted to a mixture of two structures that do not exchange. One of the earlier open structural models may have incorrect G13 dihedral angles derived from TALOS analysis of experimentally correct {sup 13}C shifts.

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

KAUST Repository

Alamoudi, Kholod

2017-05-19

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

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

KAUST Repository

Alamoudi, Kholod; Martins, Patricia; Croissant, Jonas G.; Patil, Sachin; Omar, Haneen; Khashab, Niveen M.

2017-01-01

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

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

Directory of Open Access Journals (Sweden)

Meritxell Reverter

2014-05-01

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

Snapin-regulated late endosomal transport is critical for efficient autophagy-lysosomal function in neurons.

Science.gov (United States)

Cai, Qian; Lu, Li; Tian, Jin-Hua; Zhu, Yi-Bing; Qiao, Haifa; Sheng, Zu-Hang

2010-10-06

Neuron maintenance and survival require late endocytic transport from distal processes to the soma where lysosomes are predominantly localized. Here, we report a role for Snapin in attaching dynein to late endosomes through its intermediate chain (DIC). snapin(-/-) neurons exhibit aberrant accumulation of immature lysosomes, clustering and impaired retrograde transport of late endosomes along processes, reduced lysosomal proteolysis due to impaired delivery of internalized proteins and hydrolase precursors from late endosomes to lysosomes, and impaired clearance of autolysosomes, combined with reduced neuron viability and neurodegeneration. The phenotypes are rescued by expressing the snapin transgene, but not the DIC-binding-defective Snapin-L99K mutant. Snapin overexpression in wild-type neurons enhances late endocytic transport and lysosomal function, whereas expressing the mutant defective in Snapin-DIC coupling shows a dominant-negative effect. Altogether, our study highlights new mechanistic insights into how Snapin-DIC coordinates retrograde transport and late endosomal-lysosomal trafficking critical for autophagy-lysosomal function, and thus neuronal homeostasis. Copyright © 2010 Elsevier Inc. All rights reserved.

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

Directory of Open Access Journals (Sweden)

Véronique Pons

2008-09-01

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

Insulin receptor membrane retention by a traceable chimeric mutant

OpenAIRE

Giudice, Jimena; Jares, Elizabeth Andrea; Coluccio Leskow, Federico

2015-01-01

Background: The insulin receptor (IR) regulates glucose homeostasis, cell growth and differentiation. It has been hypothesized that the specific signaling characteristics of IR are in part determined by ligand-receptor complexes localization. Downstream signaling could be triggered from the plasma membrane or from endosomes. Regulation of activated receptor's internalization has been proposed as the mechanism responsible for the differential isoform and ligand-specific signaling. Re...

Characterisation of transparent exopolymer particles (TEP) produced during algal bloom: A membrane treatment perspective

KAUST Repository

Villacorte, Loreen O.

2013-01-01

Algal blooms are currently a major concern of the membrane industry as it generates massive concentrations of organic matter (e.g. transparent exopolymer particles [TEP]), which can adversely affect the operation of membrane filtration systems. The goal of this study is to understand the production, composition and membrane rejection of these organic materials using different characterisation techniques. Two common species of bloom-forming freshwater and marine algae were cultivated in batch cultures for 30days and the productions of TEP and other organic matter were monitored at different growth phases. TEP production of the marine diatom, Chaetoceros affinis, produced 6-9 times more TEP than the freshwater blue-green algae, Microcystis. The organic substances produced by both algal species were dominated by biopolymeric substances such as polysaccharides (45-64%) and proteins (2-17%) while the remaining fraction comprises of low molecular weight refractory (humic-like) and/ or biogenic organic substances. MF/UF membranes mainly rejected the biopolymers but not the low molecular weight organic materials. MF membranes (0.1-0.4 lm) rejected 42-56% of biopolymers, while UF membranes (10-100 kDa) rejected 65-95% of these materials. Further analysis of rejected organic materials on the surface of the membranes revealed that polysac-charides and proteins are likely responsible for the fouling of MF/UF systems during an algal bloom situation. © 2013 Desalination Publications.

Physiochemical properties and resorption progress of porcine skin-derived collagen membranes: In vitro and in vivo analysis.

Science.gov (United States)

An, Yin-Zhe; Kim, You-Kyoung; Lim, Su-Min; Heo, Yeong-Ku; Kwon, Mi-Kyung; Cha, Jae-Kook; Lee, Jung-Seok; Jung, Ui-Won; Choi, Seong-Ho

2018-03-30

The aim of the present study was to evaluate the physiochemical properties and resorption progress of two cross-linked, porcine skin-derived collagen membranes and compare their features with those of a membrane without cross-linking (Bio-Gide ® [BG], Geistlich Biomaterials, Wolhusen, Switzerland). Three porcine skin-derived collagen membranes, dehydrothermally (DHT) cross-linked (experimental), DHT and 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (DHT/EDC) cross-linked (experimental) and BG were investigated for their morphology, enzyme resistance, and tensile strength in vitro and biodegradation in vivo. DHT and DHT/EDC membranes exhibited irregular, interconnected macro- and micropores that formed a 3D mesh, whereas BG exhibited individual collagen fibrils interlaced to form coarse collagen strands. In enzyme resistance and tensile strength tests, DHT and DHT/EDC membranes demonstrated good resistance and mechanical properties compared with BG. In vivo, all three membranes were well integrated into the surrounding connective tissue. Thus, the DHT membrane exhibited its potential as a barrier membrane for guided bone and tissue regeneration.

ESCRT-dependent degradation of ubiquitylated plasma membrane proteins in plants.

Science.gov (United States)

Isono, Erika; Kalinowska, Kamila

2017-12-01

To control the abundance of plasma membrane receptors and transporters is crucial for proper perception and response to extracellular signals from surrounding cells and the environment. Posttranslational modification of plasma membrane proteins, especially ubiquitin conjugation or ubiquitylation, is key for the determination of stability for many transmembrane proteins localized on the cell surface. The targeted degradation is ensured by a complex network of proteins among which the endosomal sorting complex required for transport (ESCRT) plays a central role. This review focuses on progresses made in recent years on the understanding of the function of the ESCRT machinery in the degradation of ubiquitylated plasma membrane proteins in plants. Copyright © 2017 Elsevier Ltd. All rights reserved.

Potential antitumor therapeutic strategies of human amniotic membrane and amniotic fluid-derived stem cells.

Science.gov (United States)

Kang, N-H; Hwang, K-A; Kim, S U; Kim, Y-B; Hyun, S-H; Jeung, E-B; Choi, K-C

2012-08-01

As stem cells are capable of self-renewal and can generate differentiated progenies for organ development, they are considered as potential source for regenerative medicine and tissue replacement after injury or disease. Along with this capacity, stem cells have the therapeutic potential for treating human diseases including cancers. According to the origins, stem cells are broadly classified into two types: embryonic stem cells (ESCs) and adult stem cells. In terms of differentiation potential, ESCs are pluripotent and adult stem cells are multipotent. Amnion, which is a membranous sac that contains the fetus and amniotic fluid and functions in protecting the developing embryo during gestation, is another stem cell source. Amnion-derived stem cells are classified as human amniotic membrane-derived epithelial stem cells, human amniotic membrane-derived mesenchymal stem cells and human amniotic fluid-derived stem cells. They are in an intermediate stage between pluripotent ESCs and lineage-restricted adult stem cells, non-tumorigenic, and contribute to low immunogenicity and anti-inflammation. Furthermore, they are easily available and do not cause any controversial issues in their recovery and applications. Not only are amnion-derived stem cells applicable in regenerative medicine, they have anticancer capacity. In non-engineered stem cells transplantation strategies, amnion-derived stem cells effectively target the tumor and suppressed the tumor growth by expressing cytotoxic cytokines. Additionally, they also have a potential as novel delivery vehicles transferring therapeutic genes to the cancer formation sites in gene-directed enzyme/prodrug combination therapy. Owing to their own advantageous properties, amnion-derived stem cells are emerging as a new candidate in anticancer therapy.

Regulation of B cell differentiation by intracellular membrane associated proteins and microRNAs: role in the antibody response

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Zheng eLou

2015-10-01

Full Text Available B cells are central to adaptive immunity and their functions in antibody responses are exquisitely regulated. As suggested by recent findings, B cell differentiation is mediated by intracellular membrane structures (including endosomes, lysosomes and autophagosomes and protein factors specifically associated with these membranes, including Rab7, Atg5 and Atg7. These factors participate in vesicle formation/trafficking, signal transduction and induction of gene expression to promote antigen presentation, CSR/SHM, and generation/maintenance of plasma cells and memory B cells. Their expression is induced in B cells activated to differentiate and further fine-tuned by immune-modulating microRNAs, which coordinates CSR/SHM, plasma cell differentiation and memory B cell differentiation. These short non-coding RNAs would individually target multiple factors associated with the same intracellular membrane compartments and collaboratively target a single factor in addition to regulate AID and Blimp-1. These, together with regulation of microRNA biogenesis and activities by endosomes and autophagosomes, show that intracellular membranes and microRNAs, two broadly relevant cell constituents, play important roles in balancing gene expression to specify B cell differentiation processes for optimal antibody responses.

Plasma-derived exosomal survivin, a plausible biomarker for early detection of prostate cancer.

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Salma Khan

Full Text Available Survivin is expressed in prostate cancer (PCa, and its downregulation sensitizes PCa cells to chemotherapeutic agents in vitro and in vivo. Small membrane-bound vesicles called exosomes, secreted from the endosomal membrane compartment, contain RNA and protein that they readily transport via exosome internalization into recipient cells. Recent progress has shown that tumor-derived exosomes play multiple roles in tumor growth and metastasis and may produce these functions via immune escape, tumor invasion and angiogenesis. Furthermore, exosome analysis may provide novel biomarkers to diagnose or monitor PCa treatment.Exosomes were purified from the plasma and serum from 39 PCa patients, 20 BPH patients, 8 prostate cancer recurrent and 16 healthy controls using ultracentrifugation and their quantities and qualities were quantified and visualized from both the plasma and the purified exosomes using ELISA and Western blotting, respectively.Survivin was significantly increased in the tumor-derived samples, compared to those from BPH and controls with virtually no difference in the quantity of Survivin detected in exosomes collected from newly diagnosed patients exhibiting low (six or high (nine Gleason scores. Exosome Survivin levels were also higher in patients that had relapsed on chemotherapy compared to controls.These studies demonstrate that Survivin exists in plasma exosomes from both normal, BPH and PCa subjects. The relative amounts of exosomal Survivin in PCa plasma was significantly higher than in those with pre-inflammatory BPH and control plasma. This differential expression of exosomal Survivin was seen with both newly diagnosed and advanced PCa subjects with high or low-grade cancers. Analysis of plasma exosomal Survivin levels may offer a convenient tool for diagnosing or monitoring PCa and may, as it is elevated in low as well as high Gleason scored samples, be used for early detection.

Membrane with Stable Nanosized Microstructure and Method for Producing same

DEFF Research Database (Denmark)

2010-01-01

The present invention provides a membrane, comprising in this order a first catalyst layer, an electronically and ionically conducting layer having a nanosized microstructure, and a second catalyst layer, characterized in that the electronically and ionically conducting layer is formed from...... an electrolyte material, a grain growth inhibitor and/or grain boundary modifier, and a method for producing same....

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

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Miranda Arnold

2016-09-01

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

Photochemical internalisation: the journey from basic scientific concept to the threshold of clinical application.

Science.gov (United States)

Adigbli, D K; MacRobert, A J

2012-08-01

Efficient delivery of therapeutic agents to subcellular targets is a major challenge in pharmacology. Physical properties including size and charge may adversely affect the cellular uptake of molecules, and consequently reduce the accessibility of intracellular targets. For example macromolecules, which do not pass freely through the phospholipid membrane, are internalised via endocytosis and subsequently retained in endosomes or lysosomes before enzymatic degradation or cell efflux. Photochemical internalisation (PCI) is a novel drug delivery technology based on light-activated release of biologically active compounds retained within endosomes/lysosomes. PCI is founded upon the principle of photodynamic therapy (PDT), which uses light to activate photosensitisers to ultimately produce reactive oxygen species (ROS) and cause local damage/cell death. In PCI, photosensitisers are selectively localised in endosomal/lysosomal membranes. PCI triggers membrane rupture facilitating release and delivery of endocytosed molecules to intracellular targets. Copyright © 2012 Elsevier Ltd. All rights reserved.

COST-EFFECTIVE METHOD FOR PRODUCING SELF SUPPORTED PALLADIUM ALLOY MEMBRANES FOR USE IN EFFICIENT PRODUCTION OF COAL DERIVED HYDROGEN

Energy Technology Data Exchange (ETDEWEB)

B. Lanning; J. Arps

2005-08-31

Efforts in this quarter were concentrated on developing vacuum processing procedures to produce thinner (<4 {micro}m-thick), defect-free films over larger areas (>100 cm{sup 2}). We continued to test three different types of rigid supporting substrates, thermally oxidized silicon (10 cm diameter), polished borosilicate glass (10 cm diameter), and soda-lime glass (>100 cm{sup 2} areas), each representing a different cost, surface roughness, and chemistry. Mechanical integrity, defect density, and release characteristics of the films, though similar for the oxidized silicon and borosilicate glass, were distinctly different for the inexpensive soda-lime (float) glass; i.e., more sensitive to surface impurities. In general, films less than 4 {micro}m-thick were shown to be very sensitive to surface condition of the supporting substrate, particularly in the case of the soda-lime glass, to the point where surface strain overrode and dominated the intrinsic bulk stresses that are produced during the growth process. Therefore, in the near term (over the next quarter), large area films (>100 cm{sup 2}) will be produced at a minimum thickness of 5 {micro}m while further development will be conducted in subsequent quarters to reduce membrane thickness in large area films. Continued hydrogen permeation experiments and characterization of 5 and 10 {micro}m-thick, Pd-Cu films, with compositions near the 60/40 (Pd/Cu phase boundary) in combination with air oxidation treatments to improve performance. Pure hydrogen permeability for an as-received, 5 {micro}m film at 400 C was determined to be 1.3 x 10{sup -4} cm{sup 3}(STP) {center_dot} cm/cm{sup 2} {center_dot} s {center_dot} cmHg{sup 0.5} at steady state. Even a membrane {approx} 10 {micro}m-thick, exhibited a steady state hydrogen flux of 32 cm{sup 3}(STP)/cm{sup 2}min after air exposure, which, when normalized for DOE's Office of Fossil Energy's specified hydrogen flux with a {Delta}P of 100 psi and a permeate

Rab11-FIP3 Regulation of Lck Endosomal Traffic Controls TCR Signal Transduction.

Science.gov (United States)

Bouchet, Jérôme; Del Río-Iñiguez, Iratxe; Vázquez-Chávez, Elena; Lasserre, Rémi; Agüera-González, Sonia; Cuche, Céline; McCaffrey, Mary W; Di Bartolo, Vincenzo; Alcover, Andrés

2017-04-01

The role of endosomes in receptor signal transduction is a long-standing question, which remains largely unanswered. The T cell Ag receptor and various components of its proximal signaling machinery are associated with distinct endosomal compartments, but how endosomal traffic affects T cell signaling remains ill-defined. In this article, we demonstrate in human T cells that the subcellular localization and function of the protein tyrosine kinase Lck depends on the Rab11 effector FIP3 (Rab11 family interacting protein-3). FIP3 overexpression or silencing and its ability to interact with Rab11 modify Lck subcellular localization and its delivery to the immunological synapse. Importantly, FIP3-dependent Lck localization controls early TCR signaling events, such as tyrosine phosphorylation of TCRζ, ZAP70, and LAT and intracellular calcium concentration, as well as IL-2 gene expression. Interestingly, FIP3 controls both steady-state and poststimulation phosphotyrosine and calcium levels. Finally, our findings indicate that FIP3 modulates TCR-CD3 cell surface expression via the regulation of steady-state Lck-mediated TCRζ phosphorylation, which in turn controls TCRζ protein levels. This may influence long-term T cell activation in response to TCR-CD3 stimulation. Therefore, our data underscore the importance of finely regulated endosomal traffic in TCR signal transduction and T cell activation leading to IL-2 production. Copyright © 2017 by The American Association of Immunologists, Inc.

The Road not Taken: Less Traveled Roads from the TGN to the Plasma Membrane.

Science.gov (United States)

Spang, Anne

2015-03-10

The trans-Golgi network functions in the distribution of cargo into different transport vesicles that are destined to endosomes, lysosomes and the plasma membrane. Over the years, it has become clear that more than one transport pathway promotes plasma membrane localization of proteins. In spite of the importance of temporal and spatial control of protein localization at the plasma membrane, the regulation of sorting into and the formation of different transport containers are still poorly understood. In this review different transport pathways, with a special emphasis on exomer-dependent transport, and concepts of regulation and sorting at the TGN are discussed.

Characterization of PEBBLEs as a Tool for Real-Time Measurement of Dictyostelium discoideum Endosomal pH

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Everett Moding

2009-01-01

Full Text Available The measurement of intracellular ion concentration change is important for understanding the cellular mechanisms for communication. Recently developed nanosensors, (Photonic Explorers for Biomedical use with Biologically Localized Embedding PEBBLEs, have a number of advantages for measuring ions in cells over established methods using microelectrodes, unbound fluorescent dyes, or NMR. PEBBLE sensors have been shown to work in principle for measuring dynamic ion changes, but few in vivo applications have been demonstrated. We modified the protocol for the fabrication of pH sensing PEBBLEs and developed a protocol for the utilization of these sensors for the monitoring of dynamic pH changes in the endosomes of slime mold Dictyostelium discoideum (D. discoideum. Oregon Green 514-CdSe Quantum Dot PEBBLEs were used to measure real-time pH inside D. discoideum endosomes during cAMP stimulation. Endosomal pH was shown to decrease during cAMP signaling, demonstrating a movement of protons into the endosomes of D. discoideum amoebae.

Viral membrane fusion

International Nuclear Information System (INIS)

Harrison, Stephen C.

2015-01-01

Membrane fusion is an essential step when enveloped viruses enter cells. Lipid bilayer fusion requires catalysis to overcome a high kinetic barrier; viral fusion proteins are the agents that fulfill this catalytic function. Despite a variety of molecular architectures, these proteins facilitate fusion by essentially the same generic mechanism. Stimulated by a signal associated with arrival at the cell to be infected (e.g., receptor or co-receptor binding, proton binding in an endosome), they undergo a series of conformational changes. A hydrophobic segment (a “fusion loop” or “fusion peptide”) engages the target-cell membrane and collapse of the bridging intermediate thus formed draws the two membranes (virus and cell) together. We know of three structural classes for viral fusion proteins. Structures for both pre- and postfusion conformations of illustrate the beginning and end points of a process that can be probed by single-virion measurements of fusion kinetics. - Highlights: • Viral fusion proteins overcome the high energy barrier to lipid bilayer merger. • Different molecular structures but the same catalytic mechanism. • Review describes properties of three known fusion-protein structural classes. • Single-virion fusion experiments elucidate mechanism

Viral membrane fusion

Energy Technology Data Exchange (ETDEWEB)

Harrison, Stephen C., E-mail: harrison@crystal.harvard.edu

2015-05-15

Membrane fusion is an essential step when enveloped viruses enter cells. Lipid bilayer fusion requires catalysis to overcome a high kinetic barrier; viral fusion proteins are the agents that fulfill this catalytic function. Despite a variety of molecular architectures, these proteins facilitate fusion by essentially the same generic mechanism. Stimulated by a signal associated with arrival at the cell to be infected (e.g., receptor or co-receptor binding, proton binding in an endosome), they undergo a series of conformational changes. A hydrophobic segment (a “fusion loop” or “fusion peptide”) engages the target-cell membrane and collapse of the bridging intermediate thus formed draws the two membranes (virus and cell) together. We know of three structural classes for viral fusion proteins. Structures for both pre- and postfusion conformations of illustrate the beginning and end points of a process that can be probed by single-virion measurements of fusion kinetics. - Highlights: • Viral fusion proteins overcome the high energy barrier to lipid bilayer merger. • Different molecular structures but the same catalytic mechanism. • Review describes properties of three known fusion-protein structural classes. • Single-virion fusion experiments elucidate mechanism.

Polyoxadiazole hollow fibers for produced water treatment by direct contact membrane distillation

KAUST Repository

Xu, Jingli; Srivatsa Bettahalli, N.M.; Chisca, Stefan; Khalid, Mohammed Khalil; Ghaffour, NorEddine; Vilagines, Ré gis; Nunes, Suzana Pereira

2018-01-01

Treatment of produced water in the petroleum industry has been a challenge worldwide. In this study, we evaluated the use of direct contact membrane distillation (DCMD) for this purpose, removing oil and dissolved elements and supplying clean water

High ethanol producing derivatives of Thermoanaerobacter ethanolicus

Science.gov (United States)

Ljungdahl, L.G.; Carriera, L.H.

1983-05-24

Derivatives of the newly discovered microorganism Thermoanaerobacter ethanolicus which under anaerobic and thermophilic conditions continuously ferment substrates such as starch, cellobiose, glucose, xylose and other sugars to produce recoverable amounts of ethanol solving the problem of fermentations yielding low concentrations of ethanol using the parent strain of the microorganism Thermoanaerobacter ethanolicus are disclosed. These new derivatives are ethanol tolerant up to 10% (v/v) ethanol during fermentation. The process includes the use of an aqueous fermentation medium, containing the substrate at a substrate concentration greater than 1% (w/v).

Herpesvirus glycoproteins undergo multiple antigenic changes before membrane fusion.

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Daniel L Glauser

Full Text Available Herpesvirus entry is a complicated process involving multiple virion glycoproteins and culminating in membrane fusion. Glycoprotein conformation changes are likely to play key roles. Studies of recombinant glycoproteins have revealed some structural features of the virion fusion machinery. However, how the virion glycoproteins change during infection remains unclear. Here using conformation-specific monoclonal antibodies we show in situ that each component of the Murid Herpesvirus-4 (MuHV-4 entry machinery--gB, gH/gL and gp150--changes in antigenicity before tegument protein release begins. Further changes then occurred upon actual membrane fusion. Thus virions revealed their final fusogenic form only in late endosomes. The substantial antigenic differences between this form and that of extracellular virions suggested that antibodies have only a limited opportunity to block virion membrane fusion.

Staphylococcus aureus ?-Toxin-Dependent Induction of Host Cell Death by Membrane-Derived Vesicles

OpenAIRE

Thay, Bernard; Wai, Sun Nyunt; Oscarsson, Jan

2013-01-01

Staphylococcus aureus causes a wide spectrum of infections in humans, ranging from superficial cutaneous infections, infections in the circum-oral region, to life-threatening bacteremia. It was recently demonstrated that Gram-positive organisms such as S. aureus liberate membrane-derived vesicles (MVs), which analogously to outer membrane vesicles (OMVs) of Gram-negative bacteria can play a role in delivering virulence factors to host cells. In the present study we have shown that cholesterol...

The N-terminal domains of Vps3 and Vps8 are critical for localization and function of the CORVET tethering complex on endosomes.

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Nadine Epp

Full Text Available Endosomal biogenesis depends on multiple fusion and fission events. For fusion, the heterohexameric CORVET complex as an effector of the endosomal Rab5/Vps21 GTPase has a central function in the initial tethering event. Here, we show that the CORVET-specific Vps3 and Vps8 subunits, which interact with Rab5/Vps21, require their N-terminal domains for localization and function. Surprisingly, CORVET may lack either one of the two N-terminal domains, but not both, to promote protein sorting via the endosome. The dually truncated complex mislocalizes to the cytosol and is impaired in endocytic protein sorting, but not in assembly. Furthermore, the endosomal localization can be rescued by overexpression of Vps21 or one of the truncated CORVET subunits, even though CORVET assembly is not impaired by loss of the N-terminal domains or in strains lacking all endosomal Rab5s and Ypt7. We thus conclude that CORVET requires only its C-terminal domains for assembly and has beyond its putative β-propeller domains additional binding sites for endosomes, which could be important to bind Vps21 and other endosome-specific factors for efficient endosome tethering.

Biogenesis of the rat hepatocyte plasma membrane in vivo: comparison of the pathways taken by apical and basolateral proteins using subcellular fractionation

International Nuclear Information System (INIS)

Bartles, J.R.; Feracci, H.M.; Stieger, B.; Hubbard, A.L.

1987-01-01

We have used pulse-chase metabolic radiolabeling with L-[ 35 S]methionine in conjunction with subcellular fractionation and specific protein immunoprecipitation techniques to compare the posttranslational transport pathways taken by endogenous domain-specific integral proteins of the rat hepatocyte plasma membrane in vivo. Our results suggest that both apical (HA 4, dipeptidylpeptidase IV, and aminopeptidase N) and basolateral (CE 9 and the asialoglycoprotein receptor [ASGP-R]) proteins reach the hepatocyte plasma membrane with similar kinetics. The mature molecular mass form of each of these proteins reaches its maximum specific radioactivity in a purified hepatocyte plasma membrane fraction after only 45 min of chase. However, at this time, the mature radiolabeled apical proteins are not associated with vesicles derived from the apical domain of the hepatocyte plasma membrane, but instead are associated with vesicles which, by several criteria, appear to be basolateral plasma membrane. These vesicles: (a) fractionate like basolateral plasma membrane in sucrose density gradients and in free-flow electrophoresis; (b) can be separated from the bulk of the likely organellar contaminants, including membranes derived from the late Golgi cisternae, transtubular network, and endosomes; (c) contain the proven basolateral constituents CE 9 and the ASGP-R, as judged by vesicle immunoadsorption using fixed Staphylococcus aureus cells and anti-ASGP-R antibodies; and (d) are oriented with their ectoplasmic surfaces facing outward, based on the results of vesicle immunoadsorption experiments using antibodies specific for the ectoplasmic domain of the ASGP-R. Only at times of chase greater than 45 min do significant amounts of the mature radiolabeled apical proteins arrive at the apical domain, and they do so at different rates

Lysosomal and endosomal heterogeneity in the liver: A comparison of the intracellular pathways of endocytosis in rat liver cells

International Nuclear Information System (INIS)

Kindberg, G.M.; Tolleshaug, H.; Gjoen, T.; Berg, T.

1991-01-01

Air-filled albumin microspheres, asialoorosomucoid and formaldehyde-treated serum albumin are selectively taken up by endocytosis in rat liver Kupffer cells, parenchymal cells and endothelial cells, respectively. Intracellular transport and degradation of endocytosed material were studied by subcellular fractionation in sucrose and Nycodenz gradients after intravenous injection of the ligand. By using ligands labeled with 125I-tyramine-cellobiose, the subcellular distribution of labeled degradation products can be studied because they are trapped at the site of formation. The results show that the kinetics of intracellular transport are different in hepatic parenchymal, endothelial and Kupffer cells. In endothelial cells, the ligand is associated with two types of endosomes during the first minutes after internalization and then is transferred rapidly to the lysosomes. In parenchymal cells, 125I-tyramine-cellobiose-asialoorosomucoid was located in a relatively slowly sedimenting vesicle during the first minute after internalization and subsequently in denser endosomes. Degradation of 125I-tyramine-cellobiose-asialoorosomucoid in parenchymal cells started later than that of 125I-tyramine-cellobiose-formaldehyde-treated serum albumin in endothelial cells. Furthermore, the ligand seemed to be transferred relatively slowly from endosomes to lysosomes, and most of the undegraded ligand was in the endosomes. The rate-limiting step of proteolysis in parenchymal cells is probably the transport from endosomes to lysosomes. In Kupffer cells, most 125I-tyramine-cellobiose-microspheres are found as undegraded material in very dense endosomes up to 3 hr after injection. After 20 hr, most of the ligand is degraded in lysosomes distributed at a lower density than the endosomes in Nycodenz and sucrose gradients

Bee venom phospholipase A2 as a membrane-binding vector for cell surface display or internalization of soluble proteins.

Science.gov (United States)

Babon, Aurélie; Wurceldorf, Thibault; Almunia, Christine; Pichard, Sylvain; Chenal, Alexandre; Buhot, Cécile; Beaumelle, Bruno; Gillet, Daniel

2016-06-15

We showed that bee venom phospholipase A2 can be used as a membrane-binding vector to anchor to the surface of cells a soluble protein fused to its C-terminus. ZZ, a two-domain derivative of staphylococcal protein A capable of binding constant regions of antibodies was fused to the C-terminus of the phospholipase or to a mutant devoid of enzymatic activity. The fusion proteins bound to the surface of cells and could themselves bind IgGs. Their fate depended on the cell type to which they bound. On the A431 carcinoma cell line the proteins remained exposed on the cell surface. In contrast, on human dendritic cells the proteins were internalized into early endosomes. Copyright © 2015 Elsevier Ltd. All rights reserved.

Rab7: roles in membrane trafficking and disease.

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Zhang, Ming; Chen, Li; Wang, Shicong; Wang, Tuanlao

2009-06-01

The endocytosis pathway controls multiple cellular and physiological events. The lysosome is the destination of newly synthesized lysosomal hydrolytic enzymes. Internalized molecules or particles are delivered to the lysosome for degradation through sequential transport along the endocytic pathway. The endocytic pathway is also emerging as a signalling platform, in addition to the well-known role of the plasma membrane for signalling. Rab7 is a late endosome-/lysosome-associated small GTPase, perhaps the only lysosomal Rab protein identified to date. Rab7 plays critical roles in the endocytic processes. Through interaction with its partners (including upstream regulators and downstream effectors), Rab7 participates in multiple regulation mechanisms in endosomal sorting, biogenesis of lysosome [or LRO (lysosome-related organelle)] and phagocytosis. These processes are closely related to substrates degradation, antigen presentation, cell signalling, cell survival and microbial pathogen infection. Consistently, mutations or dysfunctions of Rab7 result in traffic disorders, which cause various diseases, such as neuropathy, cancer and lipid metabolism disease. Rab7 also plays important roles in microbial pathogen infection and survival, as well as in participating in the life cycle of viruses. Here, we give a brief review on the central role of Rab7 in endosomal traffic and summarize the studies focusing on the participation of Rab7 in disease pathogenesis. The underlying mechanism governed by Rab7 and its partners will also be discussed.

Gαs regulates Glucagon-Like Peptide 1 Receptor-mediated cyclic AMP generation at Rab5 endosomal compartment

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Shravan Babu Girada

2017-10-01

Conclusions: The findings provide the mechanism of endosomal cyclic AMP generation following GLP-1R activation. We identified the specific compartment that serves as an organizing center to generate endosomal cyclic AMP by internalized activated receptor complex.

BACE1 protein endocytosis and trafficking are differentially regulated by ubiquitination at lysine 501 and the Di-leucine motif in the carboxyl terminus.

Science.gov (United States)

Kang, Eugene L; Biscaro, Barbara; Piazza, Fabrizio; Tesco, Giuseppina

2012-12-14

β-Site amyloid precursor protein-cleaving enzyme (BACE1) is a membrane-tethered member of the aspartyl proteases that has been identified as β-secretase. BACE1 is targeted through the secretory pathway to the plasma membrane and then is internalized to endosomes. Sorting of membrane proteins to the endosomes and lysosomes is regulated by the interaction of signals present in their carboxyl-terminal fragment with specific trafficking molecules. The BACE1 carboxyl-terminal fragment contains a di-leucine sorting signal ((495)DDISLL(500)) and a ubiquitination site at Lys-501. Here, we report that lack of ubiquitination at Lys-501 (BACE1K501R) does not affect the rate of endocytosis but produces BACE1 stabilization and accumulation of BACE1 in early and late endosomes/lysosomes as well as at the cell membrane. In contrast, the disruption of the di-leucine motif (BACE1LLAA) greatly impairs BACE1 endocytosis and produces a delayed retrograde transport of BACE1 to the trans-Golgi network (TGN) and a delayed delivery of BACE1 to the lysosomes, thus decreasing its degradation. Moreover, the combination of the lack of ubiquitination at Lys-501 and the disruption of the di-leucine motif (BACE1LLAA/KR) produces additive effects on BACE1 stabilization and defective internalization. Finally, BACE1LLAA/KR accumulates in the TGN, while its levels are decreased in EEA1-positive compartments indicating that both ubiquitination at Lys-501 and the di-leucine motif are necessary for the trafficking of BACE1 from the TGN to early endosomes. Our studies have elucidated a differential role for the di-leucine motif and ubiquitination at Lys-501 in BACE1 endocytosis, trafficking, and degradation and suggest the involvement of multiple adaptor molecules.

BACE1 Protein Endocytosis and Trafficking Are Differentially Regulated by Ubiquitination at Lysine 501 and the Di-leucine Motif in the Carboxyl Terminus*

Science.gov (United States)

Kang, Eugene L.; Biscaro, Barbara; Piazza, Fabrizio; Tesco, Giuseppina

2012-01-01

β-Site amyloid precursor protein-cleaving enzyme (BACE1) is a membrane-tethered member of the aspartyl proteases that has been identified as β-secretase. BACE1 is targeted through the secretory pathway to the plasma membrane and then is internalized to endosomes. Sorting of membrane proteins to the endosomes and lysosomes is regulated by the interaction of signals present in their carboxyl-terminal fragment with specific trafficking molecules. The BACE1 carboxyl-terminal fragment contains a di-leucine sorting signal (495DDISLL500) and a ubiquitination site at Lys-501. Here, we report that lack of ubiquitination at Lys-501 (BACE1K501R) does not affect the rate of endocytosis but produces BACE1 stabilization and accumulation of BACE1 in early and late endosomes/lysosomes as well as at the cell membrane. In contrast, the disruption of the di-leucine motif (BACE1LLAA) greatly impairs BACE1 endocytosis and produces a delayed retrograde transport of BACE1 to the trans-Golgi network (TGN) and a delayed delivery of BACE1 to the lysosomes, thus decreasing its degradation. Moreover, the combination of the lack of ubiquitination at Lys-501 and the disruption of the di-leucine motif (BACE1LLAA/KR) produces additive effects on BACE1 stabilization and defective internalization. Finally, BACE1LLAA/KR accumulates in the TGN, while its levels are decreased in EEA1-positive compartments indicating that both ubiquitination at Lys-501 and the di-leucine motif are necessary for the trafficking of BACE1 from the TGN to early endosomes. Our studies have elucidated a differential role for the di-leucine motif and ubiquitination at Lys-501 in BACE1 endocytosis, trafficking, and degradation and suggest the involvement of multiple adaptor molecules. PMID:23109336

Endosomal recognition of Lactococcus lactis G121 and its RNA by dendritic cells is key to its allergy-protective effects.

Science.gov (United States)

Stein, Karina; Brand, Stephanie; Jenckel, André; Sigmund, Anna; Chen, Zhijian James; Kirschning, Carsten J; Kauth, Marion; Heine, Holger

2017-02-01

Bacterial cowshed isolates are allergy protective in mice; however, the underlying mechanisms are largely unknown. We examined the ability of Lactococcus lactis G121 to prevent allergic inflammatory reactions. We sought to identify the ligands and pattern recognition receptors through which L lactis G121 confers allergy protection. L lactis G121-induced cytokine release and surface expression of costimulatory molecules by untreated or inhibitor-treated (bafilomycin and cytochalasin D) human monocyte-derived dendritic cells (moDCs), bone marrow-derived mouse dendritic cells (BMDCs), and moDC/naive CD4 + T-cell cocultures were analyzed by using ELISA and flow cytometry. The pathology of ovalbumin-induced acute allergic airway inflammation after adoptive transfer of BMDCs was examined by means of microscopy. L lactis G121-treated murine BMDCs and human moDCs released T H 1-polarizing cytokines and induced T H 1 T cells. Inhibiting phagocytosis and endosomal acidification in BMDCs or moDCs impaired the release of T H 1-polarizing cytokines, costimulatory molecule expression, and T-cell activation on L lactis G121 challenge. In vivo allergy protection mediated by L lactis G121 was dependent on endosomal acidification in dendritic cells (DCs). Toll-like receptor (Tlr) 13 -/- BMDCs showed a weak response to L lactis G121 and were unresponsive to its RNA. The T H 1-polarizing activity of L lactis G121-treated human DCs was blocked by TLR8-specific inhibitors, mediated by L lactis G121 RNA, and synergistically enhanced by activation of nucleotide-binding oligomerization domain-containing protein (NOD) 2. Bacterial RNA is the main driver of L lactis G121-mediated protection against experimentally induced allergy and requires both bacterial uptake by DCs and endosomal acidification. In mice L lactis G121 RNA signals through TLR13; however, the most likely intracellular receptor in human subjects is TLR8. Copyright © 2016 American Academy of Allergy, Asthma & Immunology

Bone Morphometric Evaluation around Immediately Placed Implants Covered with Porcine-Derived Pericardium Membrane: An Experimental Study in Dogs

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Ryo Jimbo

2012-01-01

Full Text Available Objective. To investigate whether porcine-derived bioresorbable pericardium membrane coverage enhances the osseointegration around implants placed in fresh extraction sockets. Study Design. Twenty-four commercially available endosseous implants were placed in the fresh extraction sockets of the mandibular first molar of mature beagles (. On one side, implants and osteotomy sites were covered with porcine-derived bioresorbable pericardium membranes, whereas on the other side, no membranes were used. After 6 weeks, samples were retrieved and were histologically processed for histomorphometric analysis. Results. The histological observation showed that bone loss and soft tissue migration in the coronal region of the implant were evident for the control group, whereas bone fill was evident up to the neck of the implant for the membrane-covered group. Bone-to-implant contact was significantly higher for the membrane-covered group compared to the control group, 75% and 45% (, respectively. Conclusion. The experimental membranes proved to regenerate bone around implants placed in fresh extraction sockets without soft tissue intrusion.

Carbon Molecular Sieve Membranes Derived from Tröger's Base-Based Microporous Polyimide for Gas Separation.

Science.gov (United States)

Wang, Zhenggong; Ren, Huiting; Zhang, Shenxiang; Zhang, Feng; Jin, Jian

2018-03-09

Carbon molecular sieve (CMS)-based membranes have attracted great attention because of their outstanding gas-separation performance. The polymer precursor is a key point for the preparation of high-performance CMS membranes. In this work, a microporous polyimide precursor containing a Tröger's base unit was used for the first time to prepare CMS membranes. By optimizing the pyrolysis procedure and the soaking temperature, three TB-CMS membranes were obtained. Gas-permeation tests revealed that the comprehensive gas-separation performance of the TB-CMS membranes was greatly enhanced relative to that of most state-of-the-art CMS membranes derived from polyimides reported so far. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Derivation of the formula for the filtration coefficient by application of Poiseuille's law in membrane transport

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Maria Jarzyńska

2011-01-01

Full Text Available On the basis of Kedem-Katchalsky equations a mathematical analysis of volume flow (Jv of a binary solution through a membrane (M is presented. Two cases of transport generators have been considered: hydrostatic (Δp as well as osmotic (Δπ pressure difference. Based on the Poiseuille's law we derive the formula for the membrane filtration coefficient (Lp which takes into account the membrane properties, kinetic viscosity and density of a solution flowing across the membrane. With use of this formula we have made model calculations of the filtration coefficient Lp and volume flow Jv for a polymer membrane in the case when the solutions on both sides of the membrane are mixed.

Matrimid® derived carbon molecular sieve hollow fiber membranes for ethylene/ethane separation

KAUST Repository

Xu, Liren; Rungta, Meha; Koros, William J.

2011-01-01

materials in realistic gas separations. The very challenging ethylene/ethane separation is the primary target of this work. Matrimid® derived CMS hollow fiber membranes have been investigated in this work. Resultant CMS fiber showed interesting separation

Characterization of membrane protein interactions in plasma membrane derived vesicles with quantitative imaging Förster resonance energy transfer.

Science.gov (United States)

Sarabipour, Sarvenaz; Del Piccolo, Nuala; Hristova, Kalina

2015-08-18

Here we describe an experimental tool, termed quantitative imaging Förster resonance energy transfer (QI-FRET), that enables the quantitative characterization of membrane protein interactions. The QI-FRET methodology allows us to acquire binding curves and calculate association constants for complex membrane proteins in the native plasma membrane environment. The method utilizes FRET detection, and thus requires that the proteins of interest are labeled with florescent proteins, either FRET donors or FRET acceptors. Since plasma membranes of cells have complex topologies precluding the acquisition of two-dimensional binding curves, the FRET measurements are performed in plasma membrane derived vesicles that bud off cells as a result of chemical or osmotic stress. The results overviewed here are acquired in vesicles produced with an osmotic vesiculation buffer developed in our laboratory, which does not utilize harsh chemicals. The concentrations of the donor-labeled and the acceptor-labeled proteins are determined, along with the FRET efficiencies, in each vesicle. The experiments utilize transient transfection, such that a wide variety of concentrations is sampled. Then, data from hundreds of vesicles are combined to yield dimerization curves. Here we discuss recent findings about the dimerization of receptor tyrosine kinases (RTKs), membrane proteins that control cell growth and differentiation via lateral dimerization in the plasma membrane. We focus on the dimerization of fibroblast growth factor receptor 3 (FGFR3), a RTK that plays a critically important role in skeletal development. We study the role of different FGFR3 domains in FGFR3 dimerization in the absence of ligand, and we show that FGFR3 extracellular domains inhibit unliganded dimerization, while contacts between the juxtamembrane domains, which connect the transmembrane domains to the kinase domains, stabilize the unliganded FGFR3 dimers. Since FGFR3 has been documented to harbor many pathogenic

Cellular vacuoles induced by Mycoplasma pneumoniae CARDS toxin originate from Rab9-associated compartments.

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Coreen Johnson

Full Text Available Recently, we identified an ADP-ribosylating and vacuolating cytotoxin in Mycoplasma pneumoniae designated Community Acquired Respiratory Distress Syndrome (CARDS toxin. In this study we show that vacuoles induced by recombinant CARDS (rCARDS toxin are acidic and derive from the endocytic pathway as determined by the uptake of neutral red and the fluid-phase marker, Lucifer yellow, respectively. Also, we demonstrate that the formation of rCARDS toxin-associated cytoplasmic vacuoles is inhibited by the vacuolar ATPase inhibitor, bafilomycin A1, and the ionophore, monensin. To examine the ontogeny of these vacuoles, we analyzed the distribution of endosomal and lysosomal membrane markers during vacuole formation and observed the enrichment of the late endosomal GTPase, Rab9, around rCARDS toxin-induced vacuoles. Immunogold-labeled Rab9 and overexpression of green fluorescent-tagged Rab9 further confirmed vacuolar association. The late endosomal- and lysosomal-associated membrane proteins, LAMP1 and LAMP2, also localized to the vacuolar membranes, while the late endosomal protein, Rab7, and early endosomal markers, Rab5 and EEA1, were excluded. HeLa cells expressing dominant-negative (DN Rab9 exhibited markedly reduced vacuole formation in the presence of rCARDS toxin, in contrast to cells expressing DN-Rab7, highlighting the importance of Rab9 function in rCARDS toxin-induced vacuolation. Our findings reveal the unique Rab9-association with rCARDS toxin-induced vacuoles and its possible relationship to the characteristic histopathology that accompanies M. pneumoniae infection.

Hierarchy of stroma-derived factors in supporting growth of stroma-dependent hemopoietic cells: membrane-bound SCF is sufficient to confer stroma competence to epithelial cells.

Science.gov (United States)

Friel, Jutta; Itoh, Katsuhiko; Bergholz, Ulla; Jücker, Manfred; Stocking, Carol; Harrison, Paul; Ostertag, Wolfram

2002-03-01

Hemopoiesis takes place in a microenvironment where hemopoietic cells are closely associated with stroma by various interactions. Stroma coregulates the proliferation and differentiation of hemopoietic cells. Stroma-hemopoietic-cell contact can be supported by locally produced membrane associated growth factors. The stroma derived growth factor, stem cell factor (SCF) is important in hemopoiesis. We examined the different biological interactions of membrane bound and soluble SCF with human hemopoietic cells expressing the SCF receptor, c-kit. To analyze the function of the SCF isoforms in inducing the proliferation of hemopoietic TF1 or Cord blood (CB) CD34+ cells we used stroma cell lines that differ in their presentation of no SCF, membrane SCF, or soluble SCF. We established a new coculture system using an epithelial cell line that excludes potential interfering effects with other known stroma encoded hemopoietic growth factors. We show that soluble SCF, in absence of membrane-bound SCF, inhibits long term clonal growth of primary or established CD34+ hemopoietic cells, whereas membrane-inserted SCF "dominantly" induces long term proliferation of these cells. We demonstrate a hierarchy of these SCF isoforms in the interaction of stroma with hemopoietic TF1 cells. Membrane-bound SCF is "dominant" over soluble SCF, whereas soluble SCF acts epistatically in interacting with hemopoietic cells compared with other stroma derived factors present in SCF deficient stroma. A hierarchy of stroma cell lines can be arranged according to their presentation of membrane SCF or soluble SCF. In our model system, membrane-bound SCF expression is sufficient to confer stroma properties to an epithelial cell line but soluble SCF does not.

An ER-Associated Pathway Defines Endosomal Architecture for Controlled Cargo Transport

NARCIS (Netherlands)

Jongsma, Marlieke L. M.; Berlin, Ilana; Wijdeven, Ruud H. M.; Janssen, Lennert; Janssen, George M. C.; Garstka, Malgorzata A.; Janssen, Hans; Mensink, Mark; van Veelen, Peter A.; Spaapen, Robbert M.; Neefjes, Jacques

2016-01-01

Through a network of progressively maturing vesicles, the endosomal system connects the cell's interior with extracellular space. Intriguingly, this network exhibits a bilateral architecture, comprised of a relatively immobile perinuclear vesicle "cloud" and a highly dynamic peripheral contingent.

Membrane curvature stress and antibacterial activity of lactoferricin derivatives.

Science.gov (United States)

Zweytick, Dagmar; Tumer, Sabine; Blondelle, Sylvie E; Lohner, Karl

2008-05-02

We have studied correlation of non-lamellar phase formation and antimicrobial activity of two cationic amphipathic peptides, termed VS1-13 and VS1-24 derived from a fragment (LF11) of human lactoferricin on Escherichia coli total lipid extracts. Compared to LF11, VS1-13 exhibits minor, but VS1-24 significantly higher antimicrobial activity. X-ray experiments demonstrated that only VS1-24 decreased the onset of cubic phase formation of dispersions of E. coli lipid extracts, significantly, down to physiological relevant temperatures. Cubic structures were identified to belong to the space groups Pn3m and Im3m. Formation of latter is enhanced in the presence of VS1-24. Additionally, the presence of this peptide caused membrane thinning in the fluid phase, which may promote cubic phase formation. VS1-24 containing a larger hydrophobic volume at the N-terminus than its less active counterpart VS1-13 seems to increase curvature stress in the bilayer and alter the behaviour of the membrane significantly enhancing disruption.

Avian Influenza Virus Infection of Immortalized Human Respiratory Epithelial Cells Depends upon a Delicate Balance between Hemagglutinin Acid Stability and Endosomal pH.

Science.gov (United States)

Daidoji, Tomo; Watanabe, Yohei; Ibrahim, Madiha S; Yasugi, Mayo; Maruyama, Hisataka; Masuda, Taisuke; Arai, Fumihito; Ohba, Tomoyuki; Honda, Ayae; Ikuta, Kazuyoshi; Nakaya, Takaaki

2015-04-24

The highly pathogenic avian influenza (AI) virus, H5N1, is a serious threat to public health worldwide. Both the currently circulating H5N1 and previously circulating AI viruses recognize avian-type receptors; however, only the H5N1 is highly infectious and virulent in humans. The mechanism(s) underlying this difference in infectivity remains unclear. The aim of this study was to clarify the mechanisms responsible for the difference in infectivity between the current and previously circulating strains. Primary human small airway epithelial cells (SAECs) were transformed with the SV40 large T-antigen to establish a series of clones (SAEC-Ts). These clones were then used to test the infectivity of AI strains. Human SAEC-Ts could be broadly categorized into two different types based on their susceptibility (high or low) to the viruses. SAEC-T clones were poorly susceptible to previously circulating AI but were completely susceptible to the currently circulating H5N1. The hemagglutinin (HA) of the current H5N1 virus showed greater membrane fusion activity at higher pH levels than that of previous AI viruses, resulting in broader cell tropism. Moreover, the endosomal pH was lower in high susceptibility SAEC-T clones than that in low susceptibility SAEC-T clones. Taken together, the results of this study suggest that the infectivity of AI viruses, including H5N1, depends upon a delicate balance between the acid sensitivity of the viral HA and the pH within the endosomes of the target cell. Thus, one of the mechanisms underlying H5N1 pathogenesis in humans relies on its ability to fuse efficiently with the endosomes in human airway epithelial cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Studies on membrane acid electrolysis for hydrogen production

Energy Technology Data Exchange (ETDEWEB)

Silva, Marco Antonio Oliveira da; Linardi, Marcelo; Saliba-Silva, Adonis Marcelo [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil). Centro de Celulas a Combustivel e Hidrogenio], Email: saliba@ipen.br

2010-07-01

Hydrogen represents great opportunity to be a substitute for fossil fuels in the future. Water as a renewable source of hydrogen is of great interest, since it is abundant and can decompose, producing only pure H{sub 2} and O{sub 2}. This decomposition of water can be accomplished by processes such as electrolysis, thermal decomposition and thermochemical cycles. The electrolysis by membrane has been proposed as a viable process for hydrogen production using thermal and electrical energy derived from nuclear energy or any renewable source like solar energy. In this work, within the context of optimization of the electrolysis process, it is intended to develop a mathematical model that can simulate and assist in parameterization of the electrolysis performed by polymer membrane electrolytic cell. The experimental process to produce hydrogen via the cell membrane, aims to optimize the amount of gas produced using renewable energy with noncarbogenic causing no harm by producing gases deleterious to the environment. (author)

Generation of a transplantable erythropoietin-producer derived from human mesenchymal stem cells.

Science.gov (United States)

Yokoo, Takashi; Fukui, Akira; Matsumoto, Kei; Ohashi, Toya; Sado, Yoshikazu; Suzuki, Hideaki; Kawamura, Tetsuya; Okabe, Masataka; Hosoya, Tatsuo; Kobayashi, Eiji

2008-06-15

Differentiation of autologous stem cells into functional transplantable tissue for organ regeneration is a promising regenerative therapeutic approach for cancer, diabetes, and many human diseases. Yet to be established, however, is differentiation into tissue capable of producing erythropoietin (EPO), which has a critical function in anemia. We report a novel EPO-producing organ-like structure (organoid) derived from human mesenchymal stem cells. Using our previously established relay culture system, a human mesenchymal stem cell-derived, human EPO-competent organoid was established in rat omentum. The organoid-derived levels of human EPO increased in response to anemia induced by rapid blood withdrawal. In addition, the presence of an organoid in rats suppressed for native (rat) EPO production enhanced recovery from anemia when compared with control animals lacking the organoid. Together these results confirmed the generation of a stem cell-derived organoid that is capable of producing EPO and sensitive to physiological regulation.

Influence of polyvinyl alcohol amount on producing in situ photo-crosslinked thioamide functionalized nanofiber membranes

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Zeytuncu Bihter

2015-01-01

Full Text Available Poly(vinyl alcohol/maleic anhydride/acryloyl thioamide monomer (PVA/MA/ATM photo-cured nanofiber membranes and pure PVA nanofiber membranes were produced by electrospinning technique. In situ UV radiation was applied during the electrospinning in order to provide polymerization during the jet flight and promote crosslinking of ATM and MA with PVA. The cross-linking was examined by Fourier-transform infrared spectroscopy (FTIR. The morphology and thermal behavior of electrospun nanofiber were characterized by scanning electron microscope (SEM and thermogravimetric analysis (TGA, respectively. The surface area of nanofiber membranes was measured by Brunauer-Emmert-Teller (BET analysis. Furthermore, water durability test was examined. Water durability test demonstrated that in situ photo-cured PVA/MA/ATM nanofiber membrane had the least average mass loss. The surface areas of PVA/MA/ATM nanofiber membranes were 160-280 m2/g. The surface area and diameter of PVA/MA/ATM nanofibers decreased as the PVA content increased. The diameter of nanofibers was obtained less than 100 nm. The results showed that the water-insoluble nanofiber membranes with better chemical and thermal resistance were obtained. These nanofiber membranes may be a promising candidate for the usage of water treatment.

Physicochemical properties and membrane biofouling of extra-cellular polysaccharide produced by a Micrococcus luteus strain.

Science.gov (United States)

Feng, Lei; Li, Xiufen; Song, Ping; Du, Guocheng; Chen, Jian

2014-07-01

The physicochemical properties of the extra-cellular polysaccharide (EPS) produced by a Micrococcus luteus strain, a dominating strain isolated from membrane biofouling layer, were determined in this study. The EPS isolated from this strain was measured to have an average molecular weight of 63,540 Da and some typical polysaccharide absorption peaks in Fourier transform infrared spectrum. Monosaccharide components of the EPS contained rhamnose, fucose, arabinose, xylose, mannose, galactose and glucose in a molar ratio of 0.2074:0.0454:0.0262:0.0446:1.7942:1.2086:0.4578. Pseudo plastic properties were also observed for the EPS through the rheological measurement. The EPS was further characterized for its behavior to cause membrane flux decline. The results showed that both flux declines for polyvinylidenefluoride (PVDF) and polypropylene membranes became more severe as EPS feed concentration increased. A higher irreversible fouling for the PVDF membrane suggested that the EPS had the larger fouling potential to this microfiltration membrane.

Apical transport of influenza A virus ribonucleoprotein requires Rab11-positive recycling endosome.

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Fumitaka Momose

Full Text Available Influenza A virus RNA genome exists as eight-segmented ribonucleoprotein complexes containing viral RNA polymerase and nucleoprotein (vRNPs. Packaging of vRNPs and virus budding take place at the apical plasma membrane (APM. However, little is known about the molecular mechanisms of apical transport of newly synthesized vRNP. Transfection of fluorescent-labeled antibody and subsequent live cell imaging revealed that punctate vRNP signals moved along microtubules rapidly but intermittently in both directions, suggestive of vesicle trafficking. Using a series of Rab family protein, we demonstrated that progeny vRNP localized to recycling endosome (RE in an active/GTP-bound Rab11-dependent manner. The vRNP interacted with Rab11 through viral RNA polymerase. The localization of vRNP to RE and subsequent accumulation to the APM were impaired by overexpression of Rab binding domains (RBD of Rab11 family interacting proteins (Rab11-FIPs. Similarly, no APM accumulation was observed by overexpression of class II Rab11-FIP mutants lacking RBD. These results suggest that the progeny vRNP makes use of Rab11-dependent RE machinery for APM trafficking.

Evidence that membrane transduction of oligoarginine does not require vesicle formation

International Nuclear Information System (INIS)

Zaro, Jennica L.; Shen Weichiang

2005-01-01

The involvement of vesicular formation processes in the membrane transduction and nuclear transport of oligoarginine is currently a subject of controversy. In this report, a novel quantitative method which allows for the selective measurement of membrane transduction excluding concurrent endocytosis was used to determine the effects of temperature, endosomal acidification, endosomolysis, and several known inhibitors of endocytic pathways on the internalization of oligoarginine. The results show that, unlike endocytosis, transduction of oligoarginine was not affected by incubation at 16 deg. C as compared to the 37 deg. C control, and was only partially inhibited at 4 deg. C incubation. Additionally, membrane transduction was not inhibited to the same extent as endocytosis following treatment with ammonium chloride, hypertonic medium, amiloride, or filipin. The endosomolytic activity of oligoarginine was investigated by examining the leakage of FITC-dextran into the cytosolic compartment, which was not higher in the presence of oligoarginine. Furthermore, ammonium chloride showed no effect on the nuclear transport of oligoarginine. The data presented in this report indicate that membrane transduction is likely to occur at the plasma membrane without the formation of membrane vesicles, and the nuclear localization involves membrane transduction, rather than endocytosis of oligoarginine

Carbon molecular sieve membranes derived from Matrimid® polyimide for nitrogen/methane separation

KAUST Repository

Ning, Xue; Koros, William J.

2014-01-01

A commercial polyimide, Matrimid® 5218, was pyrolyzed under an inert argon atmosphere to produce carbon molecular sieve (CMS) dense film membranes for nitrogen/methane separation. The resulting CMS dense film separation performance was evaluated

The Processed Amino-Terminal Fragment of Human TLR7 Acts as a Chaperone To Direct Human TLR7 into Endosomes

Science.gov (United States)

Shepherd, Dawn; Booth, Sarah; Waithe, Dominic; Reis e Sousa, Caetano

2015-01-01

TLR7 mediates innate immune responses to viral RNA in endocytic compartments. Mouse and human (h)TLR7 undergo proteolytic cleavage, resulting in the generation of a C-terminal fragment that accumulates in endosomes and associates with the signaling adaptor MyD88 upon receptor triggering by TLR7 agonists. Although mouse TLR7 is cleaved in endosomes by acidic proteases, hTLR7 processing can occur at neutral pH throughout the secretory pathway through the activity of furin-like proprotein convertases. However, the mechanisms by which cleaved hTLR7 reaches the endosomal compartment remain unclear. In this study, we demonstrate that, after hTLR7 proteolytic processing, the liberated amino (N)-terminal fragment remains bound to the C terminus through disulfide bonds and provides key trafficking information that ensures correct delivery of the complex to endosomal compartments. In the absence of the N-terminal fragment, the C-terminal fragment is redirected to the cell surface, where it is functionally inactive. Our data reveal a novel role for the N terminus of hTLR7 as a molecular chaperone that provides processed hTLR7 with the correct targeting instructions to reach the endosomal compartment, hence ensuring its biological activity and preventing inadvertent cell surface responses to self-RNA. PMID:25917086

P2X7 receptors regulate multiple types of membrane trafficking responses and non-classical secretion pathways.

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Qu, Yan; Dubyak, George R

2009-06-01

Activation of the P2X7 receptor (P2X7R) triggers a remarkably diverse array of membrane trafficking responses in leukocytes and epithelial cells. These responses result in altered profiles of cell surface lipid and protein composition that can modulate the direct interactions of P2X7R-expressing cells with other cell types in the circulation, in blood vessels, at epithelial barriers, or within sites of immune and inflammatory activation. Additionally, these responses can result in the release of bioactive proteins, lipids, and large membrane complexes into extracellular compartments for remote communication between P2X7R-expressing cells and other cells that amplify or modulate inflammation, immunity, and responses to tissue damages. This review will discuss P2X7R-mediated effects on membrane composition and trafficking in the plasma membrane (PM) and intracellular organelles, as well as actions of P2X7R in controlling various modes of non-classical secretion. It will review P2X7R regulation of: (1) phosphatidylserine distribution in the PM outer leaflet; (2) shedding of PM surface proteins; (3) release of PM-derived microvesicles or microparticles; (4) PM blebbing; (5) cell-cell fusion resulting in formation of multinucleate cells; (6) phagosome maturation and fusion with lysosomes; (7) permeability of endosomes with internalized pathogen-associated molecular patterns; (8) permeability/integrity of mitochondria; (9) exocytosis of secretory lysosomes; and (10) release of exosomes from multivesicular bodies.

Membrane-Mimic Nanoparticles for Drug and Gene Delivery

KAUST Repository

Alamoudi, Kholod

2017-12-01

Nanoscale organic particles have gained a prominent role in drug and gene delivery field. As the nature of the nanoparticle’s (NPs) surface plays a major role in their targeting efficiency, bioavailability, and cytotoxicity, membrane-mimic nanoparticles are considered highly attractive materials for in vivo and in vitro applications. Synthetic membrane vesicles (liposomes) and nanoconstructs built with native cancer cellular membrane are excellent scaffolds to improve cellular delivery. Liposomes have been extensively used due to their high loading capacity, biocompatibility and biodegradability. However, modifications with stimuli responsive materials are highly needed to improve their stability and turn them active participants in controlled delivery. Towards a nature inspired approach, reconstructed bilayers from cell membrane are a good candidate to enhance NP’s targeting ability and biocompatibility. The primary focus of this research is to develop smart responsive (lipid) membrane coated NPs with surface modifications for controlled and targeted drug and/or gene delivery for application in cancer therapy. Three approaches have been developed, namely i) liposomes as thermoresponsive nanocarriers for the delivery of genetic material; ii) magnetically photosensitive liposome hybrids and iii) biomimetic periodic mesoporous organo silica engineered for better a biocompatibility and targeting capabilities. In the first project synthetic liposomes were loaded with ammonium bicarbonate salt (ABC) and siRNA. The combination of lipids chosen and the relative ratios allowed the rapid release of the genetic material to the multi drug resistant cancer cells studied, upon external heat trigger. This design has improved the gene silencing efficiency via successful endosomal escape. In the second project, SPIO@Au nanoparticles were imbedded in the lipid bilayer to produce a photo/thermal responsive carrier that could be also used in cell imaging besides gene transfection

Human Amniotic Membrane-Derived Products in Sports Medicine: Basic Science, Early Results, and Potential Clinical Applications.

Science.gov (United States)

Riboh, Jonathan C; Saltzman, Bryan M; Yanke, Adam B; Cole, Brian J

2016-09-01

Amniotic membrane (AM)-derived products have been successfully used in ophthalmology, plastic surgery, and wound care, but little is known about their potential applications in orthopaedic sports medicine. To provide an updated review of the basic science and preclinical and clinical data supporting the use of AM-derived products and to review their current applications in sports medicine. Systematic review. A systematic search of the literature was conducted using the Medline, EMBASE, and Cochrane databases. The search term amniotic membrane was used alone and in conjunction with stem cell, orthopaedic, tissue engineering, scaffold, and sports medicine. The search identified 6870 articles, 80 of which, after screening of the titles and abstracts, were considered relevant to this study. Fifty-five articles described the anatomy, basic science, and nonorthopaedic applications of AM-derived products. Twenty-five articles described preclinical and clinical trials of AM-derived products for orthopaedic sports medicine. Because the level of evidence obtained from this search was not adequate for systematic review or meta-analysis, a current concepts review on the anatomy, physiology, and clinical uses of AM-derived products is presented. Amniotic membranes have many promising applications in sports medicine. They are a source of pluripotent cells, highly organized collagen, antifibrotic and anti-inflammatory cytokines, immunomodulators, and matrix proteins. These properties may make it beneficial when applied as tissue engineering scaffolds, improving tissue organization in healing, and treatment of the arthritic joint. The current body of evidence in sports medicine is heavily biased toward in vitro and animal studies, with little to no human clinical data. Nonetheless, 14 companies or distributors offer commercial AM products. The preparation and formulation of these products alter their biological and mechanical properties, and a thorough understanding of these

Exosomes derived from human macrophages suppress endothelial cell migration by controlling integrin trafficking.

Science.gov (United States)

Lee, Hee Doo; Kim, Yeon Hyang; Kim, Doo-Sik

2014-04-01

Integrin trafficking, including internalization, recycling, and lysosomal degradation, is crucial for the regulation of cellular functions. Exosomes, nano-sized extracellular vesicles, are believed to play important roles in intercellular communications. This study demonstrates that exosomes released from human macrophages negatively regulate endothelial cell migration through control of integrin trafficking. Macrophage-derived exosomes promote internalization of integrin β1 in primary HUVECs. The internalized integrin β1 persistently accumulates in the perinuclear region and is not recycled back to the plasma membrane. Experimental results indicate that macrophage-derived exosomes stimulate trafficking of internalized integrin β1 to lysosomal compartments with a corresponding decrease in the integrin destined for recycling endosomes, resulting in proteolytic degradation of the integrin. Moreover, ubiquitination of HUVEC integrin β1 is enhanced by the exosomes, and exosome-mediated integrin degradation is blocked by bafilomycin A, a lysosomal degradation inhibitor. Macrophage-derived exosomes were also shown to effectively suppress collagen-induced activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway and HUVEC migration, which are both dependent on integrin β1. These observations provide new insight into the functional significance of exosomes in the regulation of integrin trafficking. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Investigation of the Use of a Bio-Derived Solvent for Non-Solvent-Induced Phase Separation (NIPS Fabrication of Polysulfone Membranes

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Xiaobo Dong

2018-05-01

Full Text Available Organic solvents, such as N-methyl-2-pyrrolidone (NMP and dimethylacetamide (DMAc, have been traditionally used to fabricate polymeric membranes. These solvents may have a negative impact on the environment and human health; therefore, using renewable solvents derived from biomass is of great interest to make membrane fabrication sustainable. Methyl-5-(dimethylamino-2-methyl-5-oxopentanoate (Rhodiasolv PolarClean is a bio-derived, biodegradable, nonflammable and nonvolatile solvent. Polysulfone is a commonly used polymer to fabricate membranes due to its thermal stability, strong mechanical strength and good chemical resistance. From cloud point curves, PolarClean showed potential to be a solvent for polysulfone. Membranes prepared with PolarClean were investigated in terms of their morphology, porosity, water permeability and protein rejection, and were compared to membranes prepared with traditional solvents. The pores of polysulfone/PolarClean membranes were sponge-like, and the membranes displayed higher water flux values (176.0 ± 8.8 LMH along with slightly higher solute rejection (99.0 ± 0.51%. On the other hand, PSf/DMAc membrane pores were finger-like with lower water flux (63.1 ± 12.4 LMH and slightly lower solute rejection (96 ± 2.00% when compared to PSf/PolarClean membranes.

Flapping tail membrane in bats produces potentially important thrust during horizontal takeoffs and very slow flight.

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Rick A Adams

Full Text Available Historically, studies concerning bat flight have focused primarily on the wings. By analyzing high-speed video taken on 48 individuals of five species of vespertilionid bats, we show that the capacity to flap the tail-membrane (uropatagium in order to generate thrust and lift during takeoffs and minimal-speed flight (<1 m (s-1 was largely underestimated. Indeed, bats flapped the tail-membrane by extensive dorso-ventral fanning motions covering as much as 135 degrees of arc consistent with thrust generation by air displacement. The degree of dorsal extension of the tail-membrane, and thus the potential amount of thrust generated during platform launches, was significantly correlated with body mass (P = 0.02. Adduction of the hind limbs during upstrokes collapsed the tail-membrane thereby reducing its surface area and minimizing negative lift forces. Abduction of the hind limbs during the downstroke fully expanded the tail-membrane as it was swept ventrally. The flapping kinematics of the tail-membrane is thus consistent with expectations for an airfoil. Timing offsets between the wings and tail-membrane during downstrokes was as much as 50%, suggesting that the tail-membrane was providing thrust and perhaps lift when the wings were retracting through the upstoke phase of the wing-beat cycle. The extent to which the tail-membrane was used during takeoffs differed significantly among four vespertilionid species (P = 0.01 and aligned with predictions derived from bat ecomorphology. The extensive fanning motion of the tail membrane by vespertilionid bats has not been reported for other flying vertebrates.

Allogeneic amniotic membrane-derived mesenchymal stromal cell transplantation in a porcine model of chronic myocardial ischemia

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Kimura M

2012-01-01

Full Text Available Introduction. Amniotic membrane contains a multipotential stem cell population and is expected to possess the machinery to regulate immunological reactions. We investigated the safety and efficacy of allogeneic amniotic membrane-derived mesenchymal stromal cell (AMSC transplantation in a porcine model of chronic myocardial ischemia as a preclinical trial. Methods. Porcine AMSCs were isolated from amniotic membranes obtained by cesarean section just before delivery and were cultured to increase their numbers before transplantation. Chronic myocardial ischemia was induced by implantation of an ameroid constrictor around the left circumflex coronary artery. Four weeks after ischemia induction, nine swine were assigned to undergo either allogeneic AMSC transplantation or normal saline injection. Functional analysis was performed by echocardiography, and histological examinations were carried out by immunohistochemistry 4 weeks after AMSC transplantation. Results. Echocardiography demonstrated that left ventricular ejection fraction was significantly improved and left ventricular dilatation was well attenuated 4 weeks after AMSC transplantation. Histological assessment showed a significant reduction in percentage of fibrosis in the AMSC transplantation group. Injected allogeneic green fluorescent protein (GFP-expressing AMSCs were identified in the immunocompetent host heart without the use of any immunosuppressants 4 weeks after transplantation. Immunohistochemistry revealed that GFP colocalized with cardiac troponin T and cardiac troponin I. Conclusions. We have demonstrated that allogeneic AMSC transplantation produced histological and functional improvement in the impaired myocardium in a porcine model of chronic myocardial ischemia. The transplanted allogeneic AMSCs survived without the use of any immunosuppressants and gained cardiac phenotype through either their transdifferentiation or cell fusion.

Membrane-elasticity model of Coatless vesicle budding induced by ESCRT complexes.

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Bartosz Różycki

Full Text Available The formation of vesicles is essential for many biological processes, in particular for the trafficking of membrane proteins within cells. The Endosomal Sorting Complex Required for Transport (ESCRT directs membrane budding away from the cytosol. Unlike other vesicle formation pathways, the ESCRT-mediated budding occurs without a protein coat. Here, we propose a minimal model of ESCRT-induced vesicle budding. Our model is based on recent experimental observations from direct fluorescence microscopy imaging that show ESCRT proteins colocalized only in the neck region of membrane buds. The model, cast in the framework of membrane elasticity theory, reproduces the experimentally observed vesicle morphologies with physically meaningful parameters. In this parameter range, the minimum energy configurations of the membrane are coatless buds with ESCRTs localized in the bud neck, consistent with experiment. The minimum energy configurations agree with those seen in the fluorescence images, with respect to both bud shapes and ESCRT protein localization. On the basis of our model, we identify distinct mechanistic pathways for the ESCRT-mediated budding process. The bud size is determined by membrane material parameters, explaining the narrow yet different bud size distributions in vitro and in vivo. Our membrane elasticity model thus sheds light on the energetics and possible mechanisms of ESCRT-induced membrane budding.

Enhancement Performance of Hybrid Membrane Zeolite/PES for Produced Water Treatment With Membrane Modification Using Combination of Ulta Violet Irradiation, Composition of Zeolite and Thermal Annealing

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Djoko Kusworo Tutuk

2018-01-01

Full Text Available Produced water is a wastewater from oil production that must be treated well. Membrane is one alternative of water treatments technology based on filtration method. However, in the use of membrane, there’s no exact optimal variable that influences membrane performance.This underlying research to assess factors that influences membrane performance to obtain optimal condition. Therefore, the objectives of this study are determining the effect of variable process in membrane fabrication and several modification techniques on membrane performance. The membranes were fabricated via dry-wet phase inversion method. The process variables of this experiment are varying the Zeolite concentration by low level 1% weight and 3% weight, UV irradiation time low level 2 minutes and high level 6 minutes, thermal annealing temperature low level 160°C and high level 180°C. The experiment runs were designed using central composite design. From the research that has been perfromed, PES/Zeolit membrane has a higher permeability after being irradiated by UV light and denser pore after heating and the longer of annealing time.

Energy-producing system of the membrane potential generation in γ-irradiated Streptococcus faecalis

International Nuclear Information System (INIS)

Fomenko, B.S.

1976-01-01

γ-irradiated (20-100 krads) Str. faecalis cells exhibited increased glycolytic and ATPase activity whereas the ATP level remained unaffected by radiation. It is concluded that the radiation-induced reduction of the membrane potential in Str. faecalis, that has been earlier described, is not connected with the impairment of the energy-producing system of the potential generation

Multifaceted Roles of ALG-2 in Ca2+-Regulated Membrane Trafficking

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Masatoshi Maki

2016-08-01

Full Text Available ALG-2 (gene name: PDCD6 is a penta-EF-hand Ca2+-binding protein and interacts with a variety of proteins in a Ca2+-dependent fashion. ALG-2 recognizes different types of identified motifs in Pro-rich regions by using different hydrophobic pockets, but other unknown modes of binding are also used for non-Pro-rich proteins. Most ALG-2-interacting proteins associate directly or indirectly with the plasma membrane or organelle membranes involving the endosomal sorting complex required for transport (ESCRT system, coat protein complex II (COPII-dependent ER-to-Golgi vesicular transport, and signal transduction from membrane receptors to downstream players. Binding of ALG-2 to targets may induce conformational change of the proteins. The ALG-2 dimer may also function as a Ca2+-dependent adaptor to bridge different partners and connect the subnetwork of interacting proteins.

The PDZ protein GIPC regulates trafficking of the LPA1 receptor from APPL signaling endosomes and attenuates the cell's response to LPA.

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Tal Varsano

Full Text Available Lysophosphatidic acid (LPA mediates diverse cellular responses through the activation of at least six LPA receptors--LPA(1-6, but the interacting proteins and signaling pathways that mediate the specificity of these receptors are largely unknown. We noticed that LPA(1 contains a PDZ binding motif (SVV identical to that present in two other proteins that interact with the PDZ protein GIPC. GIPC is involved in endocytic trafficking of several receptors including TrkA, VEGFR2, lutropin and dopamine D2 receptors. Here we show that GIPC binds directly to the PDZ binding motif of LPA(1 but not that of other LPA receptors. LPA(1 colocalizes and coimmunoprecipitates with GIPC and its binding partner APPL, an activator of Akt signaling found on APPL signaling endosomes. GIPC depletion by siRNA disturbed trafficking of LPA(1 to EEA1 early endosomes and promoted LPA(1 mediated Akt signaling, cell proliferation, and cell motility. We propose that GIPC binds LPA(1 and promotes its trafficking from APPL-containing signaling endosomes to EEA1 early endosomes and thus attenuates LPA-mediated Akt signaling from APPL endosomes.

An Alphavirus E2 Membrane-Proximal Domain Promotes Envelope Protein Lateral Interactions and Virus Budding

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Emily A. Byrd

2017-11-01

Full Text Available Alphaviruses are members of a group of small enveloped RNA viruses that includes important human pathogens such as Chikungunya virus and the equine encephalitis viruses. The virus membrane is covered by a lattice composed of 80 spikes, each a trimer of heterodimers of the E2 and E1 transmembrane proteins. During virus endocytic entry, the E1 glycoprotein mediates the low-pH-dependent fusion of the virus membrane with the endosome membrane, thus initiating virus infection. While much is known about E1 structural rearrangements during membrane fusion, it is unclear how the E1/E2 dimer dissociates, a step required for the fusion reaction. A recent Alphavirus cryo-electron microscopy reconstruction revealed a previously unidentified D subdomain in the E2 ectodomain, close to the virus membrane. A loop within this region, here referred to as the D-loop, contains two highly conserved histidines, H348 and H352, which were hypothesized to play a role in dimer dissociation. We generated Semliki Forest virus mutants containing the single and double alanine substitutions H348A, H352A, and H348/352A. The three D-loop mutations caused a reduction in virus growth ranging from 1.6 to 2 log but did not significantly affect structural protein biosynthesis or transport, dimer stability, virus fusion, or specific infectivity. Instead, growth reduction was due to inhibition of a late stage of virus assembly at the plasma membrane. The virus particles that are produced show reduced thermostability compared to the wild type. We propose the E2 D-loop as a key region in establishing the E1-E2 contacts that drive glycoprotein lattice formation and promote Alphavirus budding from the plasma membrane.

Tumour-derived exosomes as a signature of pancreatic cancer - liquid biopsies as indicators of tumour progression.

Science.gov (United States)

Nuzhat, Zarin; Kinhal, Vyjayanthi; Sharma, Shayna; Rice, Gregory E; Joshi, Virendra; Salomon, Carlos

2017-03-07

Pancreatic cancer is the fourth most common cause of death due to cancer in the world. It is known to have a poor prognosis, mostly because early stages of the disease are generally asymptomatic. Progress in pancreatic cancer research has been slow, leaving several fundamental questions pertaining to diagnosis and treatment unanswered. Recent studies highlight the putative utility of tissue-specific vesicles (i.e. extracellular vesicles) in the diagnosis of disease onset and treatment monitoring in pancreatic cancer. Extracellular vesicles are membrane-limited structures derived from the cell membrane. They contain specific molecules including proteins, mRNA, microRNAs and non-coding RNAs that are secreted in the extracellular space. Extracellular vesicles can be classified according to their size and/or origin into microvesicles (~150-1000 nm) and exosomes (~40-120 nm). Microvesicles are released by budding from the plasmatic membrane, whereas exosomes are released via the endocytic pathway by fusion of multivesicular bodies with the plasmatic membrane. This endosomal origin means that exosomes contain an abundance of cell-specific biomolecules which may act as a 'fingerprint' of the cell of origin. In this review, we discuss our current knowledge in the diagnosis and treatment of pancreatic cancer, particularly the potential role of EVs in these facets of disease management. In particular, we suggest that as exosomes contain cellular protein and RNA molecules in a cell type-specific manner, they may provide extensive information about the signature of the tumour and pancreatic cancer progression.

Membrane association of the Arabidopsis ARF exchange factor GNOM involves interaction of conserved domains

DEFF Research Database (Denmark)

Anders, Nadine; Nielsen, Michael M.; Keicher, Jutta

2008-01-01

vesicle formation by activating ARF GTPases on specific membranes in animals, plants, and fungi. However, apart from the catalytic exchange activity of the SEC7 domain, the functional significance of other conserved domains is virtually unknown. Here, we show that a distinct N-terminal domain of GNOM......The GNOM protein plays a fundamental role in Arabidopsis thaliana development by regulating endosome-to-plasma membrane trafficking required for polar localization of the auxin efflux carrier PIN1. GNOM is a family member of large ARF guanine nucleotide exchange factors (ARF-GEFs), which regulate...... mediates dimerization and in addition interacts heterotypically with two other conserved domains in vivo. In contrast with N-terminal dimerization, the heterotypic interaction is essential for GNOM function, as mutations abolishing this interaction inactivate the GNOM protein and compromise its membrane...

Developmentally regulated GTP-binding protein 2 is required for stabilization of Rac1-positive membrane tubules.

Science.gov (United States)

Mani, Muralidharan; Lee, Unn Hwa; Yoon, Nal Ae; Yoon, Eun Hye; Lee, Byung Ju; Cho, Wha Ja; Park, Jeong Woo

2017-11-04

Previously we have reported that developmentally regulated GTP-binding protein 2 (DRG2) localizes on Rab5 endosomes and plays an important role in transferrin (Tfn) recycling. We here identified DRG2 as a key regulator of membrane tubule stability. At 30 min after Tfn treatment, DRG2 localized to membrane tubules which were enriched with phosphatidylinositol 4-monophosphate [PI(4)P] and did not contain Rab5. DRG2 interacted with Rac1 more strongly with GTP-bound Rac1 and tubular localization of DRG2 depended on Rac1 activity. DRG2 depletion led to destabilization of membrane tubules, while ectopic expression of DRG2 rescued the stability of the membrane tubules in DRG2-depleted cells. Our results reveal a novel mechanism for regulation of membrane tubule stability mediated by DRG2. Copyright © 2017 Elsevier Inc. All rights reserved.

Recycling endosomes in human cytotoxic T lymphocytes constitute an auxiliary intracellular trafficking pathway for newly synthesized perforin

Science.gov (United States)

Lesteberg, Kelsey E.; Orange, Jordan S.; Makedonas, George

2018-01-01

Background Although cytotoxic T lymphocytes (CTLs) store perforin within cytoplasmic secretory granules for immediate use, perforin is synthesized anew within hours of TCR stimulation. Previously, we observed new perforin protein at an immunologic synapse independent of secretory lysosomes; herein we aimed to determine how new perforin transits to the synapse if not via lytic granules. Results We analyzed antigen-specific human CTLs via imaging flow cytometry and high-resolution confocal microscopy, with attention to intracellular trafficking components and new perforin. The recycling endosome compartments identified by rab8, rab11a, rab4, and rab37 co-localized with new perforin, as well as the SNAREs vti1b and VAMP4. After ablating the function of the recycling endosome pathway, we observed a relative accumulation of new perforin in rab8 vesicles. Conclusions The recycling endosome pathway may serve as an auxiliary intracellular route for the delivery of new perforin to an immunologic synapse in order to perpetuate a cytotoxic response. PMID:28822075

ABMA, a small molecule that inhibits intracellular toxins and pathogens by interfering with late endosomal compartments.

Science.gov (United States)

Wu, Yu; Pons, Valérie; Goudet, Amélie; Panigai, Laetitia; Fischer, Annette; Herweg, Jo-Ana; Kali, Sabrina; Davey, Robert A; Laporte, Jérôme; Bouclier, Céline; Yousfi, Rahima; Aubenque, Céline; Merer, Goulven; Gobbo, Emilie; Lopez, Roman; Gillet, Cynthia; Cojean, Sandrine; Popoff, Michel R; Clayette, Pascal; Le Grand, Roger; Boulogne, Claire; Tordo, Noël; Lemichez, Emmanuel; Loiseau, Philippe M; Rudel, Thomas; Sauvaire, Didier; Cintrat, Jean-Christophe; Gillet, Daniel; Barbier, Julien

2017-11-14

Intracellular pathogenic microorganisms and toxins exploit host cell mechanisms to enter, exert their deleterious effects as well as hijack host nutrition for their development. A potential approach to treat multiple pathogen infections and that should not induce drug resistance is the use of small molecules that target host components. We identified the compound 1-adamantyl (5-bromo-2-methoxybenzyl) amine (ABMA) from a cell-based high throughput screening for its capacity to protect human cells and mice against ricin toxin without toxicity. This compound efficiently protects cells against various toxins and pathogens including viruses, intracellular bacteria and parasite. ABMA provokes Rab7-positive late endosomal compartment accumulation in mammalian cells without affecting other organelles (early endosomes, lysosomes, the Golgi apparatus, the endoplasmic reticulum or the nucleus). As the mechanism of action of ABMA is restricted to host-endosomal compartments, it reduces cell infection by pathogens that depend on this pathway to invade cells. ABMA may represent a novel class of broad-spectrum compounds with therapeutic potential against diverse severe infectious diseases.

Tumor imaging and targeting potential of an Hsp70-derived 14-mer peptide.

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Mathias Gehrmann

Full Text Available We have previously used a unique mouse monoclonal antibody cmHsp70.1 to demonstrate the selective presence of a membrane-bound form of Hsp70 (memHsp70 on a variety of leukemia cells and on single cell suspensions derived from solid tumors of different entities, but not on non-transformed cells or cells from corresponding 'healthy' tissue. This antibody can be used to image tumors in vivo and target them for antibody-dependent cellular cytotoxicity. Tumor-specific expression of memHsp70 therefore has the potential to be exploited for theranostic purposes. Given the advantages of peptides as imaging and targeting agents, this study assessed whether a 14-mer tumor penetrating peptide (TPP; TKDNNLLGRFELSG, the sequence of which is derived from the oligomerization domain of Hsp70 which is expressed on the cell surface of tumor cells, can also be used for targeting membrane Hsp70 positive (memHsp70+ tumor cells, in vitro.The specificity of carboxy-fluorescein (CF- labeled TPP (TPP to Hsp70 was proven in an Hsp70 knockout mammary tumor cell system. TPP specifically binds to different memHsp70+ mouse and human tumor cell lines and is rapidly taken up via endosomes. Two to four-fold higher levels of CF-labeled TPP were detected in MCF7 (82% memHsp70+ and MDA-MB-231 (75% memHsp70+ cells compared to T47D cells (29% memHsp70+ that exhibit a lower Hsp70 membrane positivity. After 90 min incubation, TPP co-localized with mitochondrial membranes in memHsp70+ tumors. Although there was no evidence that any given vesicle population was specifically localized, fluorophore-labeled cmHsp70.1 antibody and TPP preferentially accumulated in the proximity of the adherent surface of cultured cells. These findings suggest a potential association between membrane Hsp70 expression and cytoskeletal elements that are involved in adherence, the establishment of intercellular synapses and/or membrane reorganization.This study demonstrates the specific binding and rapid

Balance between Apical Membrane Growth and Luminal Matrix Resistance Determines Epithelial Tubule Shape

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Bo Dong

2014-05-01

Full Text Available The morphological stability of biological tubes is crucial for the efficient circulation of fluids and gases. Failure of this stability causes irregularly shaped tubes found in multiple pathological conditions. Here, we report that Drosophila mutants of the ESCRT III component Shrub/Vps32 exhibit a strikingly elongated sinusoidal tube phenotype. This is caused by excessive apical membrane synthesis accompanied by the ectopic accumulation and overactivation of Crumbs in swollen endosomes. Furthermore, we demonstrate that the apical extracellular matrix (aECM of the tracheal tube is a viscoelastic material coupled with the apical membrane. We present a simple mechanical model in which aECM elasticity, apical membrane growth, and their interaction are three vital parameters determining the stability of biological tubes. Our findings demonstrate a mechanical role for the extracellular matrix and suggest that the interaction of the apical membrane and an elastic aECM determines the final morphology of biological tubes independent of cell shape.

Balance between apical membrane growth and luminal matrix resistance determines epithelial tubule shape.

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Dong, Bo; Hannezo, Edouard; Hayashi, Shigeo

2014-05-22

The morphological stability of biological tubes is crucial for the efficient circulation of fluids and gases. Failure of this stability causes irregularly shaped tubes found in multiple pathological conditions. Here, we report that Drosophila mutants of the ESCRT III component Shrub/Vps32 exhibit a strikingly elongated sinusoidal tube phenotype. This is caused by excessive apical membrane synthesis accompanied by the ectopic accumulation and overactivation of Crumbs in swollen endosomes. Furthermore, we demonstrate that the apical extracellular matrix (aECM) of the tracheal tube is a viscoelastic material coupled with the apical membrane. We present a simple mechanical model in which aECM elasticity, apical membrane growth, and their interaction are three vital parameters determining the stability of biological tubes. Our findings demonstrate a mechanical role for the extracellular matrix and suggest that the interaction of the apical membrane and an elastic aECM determines the final morphology of biological tubes independent of cell shape. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Amyloid precursor protein and endosomal-lysosomal dysfunction in Alzheimer's disease: inseparable partners in a multifactorial disease.

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Nixon, Ralph A

2017-07-01

Abnormalities of the endosomal-lysosomal network (ELN) are a signature feature of Alzheimer's disease (AD). These include the earliest known cytopathology that is specific to AD and that affects endosomes and induces the progressive failure of lysosomes, each of which are directly linked by distinct mechanisms to neurodegeneration. The origins of ELN dysfunction and β-amyloidogenesis closely overlap, which reflects their common genetic basis, the established early involvement of endosomes and lysosomes in amyloid precursor protein (APP) processing and clearance, and the pathologic effect of certain APP metabolites on ELN functions. Genes that promote β-amyloidogenesis in AD (APP, PSEN1/2, and APOE4) have primary effects on ELN function. The importance of primary ELN dysfunction to pathogenesis is underscored by the mutations in more than 35 ELN-related genes that, thus far, are known to cause familial neurodegenerative diseases even though different pathogenic proteins may be involved. In this article, I discuss growing evidence that implicates AD gene-driven ELN disruptions as not only the antecedent pathobiology that underlies β-amyloidogenesis but also as the essential partner with APP and its metabolites that drive the development of AD, including tauopathy, synaptic dysfunction, and neurodegeneration. The striking amelioration of diverse deficits in animal AD models by remediating ELN dysfunction further supports a need to integrate APP and ELN relationships, including the role of amyloid-β, into a broader conceptual framework of how AD arises, progresses, and may be effectively therapeutically targeted.-Nixon, R. A. Amyloid precursor protein and endosomal-lysosomal dysfunction in Alzheimer's disease: inseparable partners in a multifactorial disease. © FASEB.

Altered neurological function in mice immunized with early endosome antigen 1

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Fritzler Marvin J

2004-01-01

Full Text Available Abstract Background Autoantibodies directed against the 160 kDa endosome protein early endosome antigen 1 (EEA1 are seen in patients with neurological diseases. To determine if antibodies to EEA1 have a neuropathological effect, mice from three major histocompatability haplotype backgrounds (H2q, H2b and H2d were immunized with EEA1 (amino acids 82–1411 that was previously shown to contain the target EEA1 epitopes. The mice were then subjected to five neuro-behavioural tests: grid walking, forelimb strength, open field, reaching and rotarod. Results The immunized SWR/J mice with sustained anti-EEA1 antibodies had significantly reduced forelimb strength than the control non-immune mice of the same strain, and BALB/CJ immune mice demonstrated significantly more forelimb errors on the grid walk test than the control group. Conclusions Antibodies to recombinant EEA1 in mice may mediate neurological deficits that are consistent with clinical features of some humans that spontaneously develop anti-EEA1 autoantibodies.

Membrane Localization is Critical for Activation of the PICK1 BAR Domain

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Madsen, Kenneth L.; Eriksen, Jacob; Milan-Lobo, Laura; Han, Daniel S.; Niv, Masha Y.; Ammendrup-Johnsen, Ina; Henriksen, Ulla; Bhatia, Vikram K.; Stamou, Dimitrios; Sitte, Harald H.; McMahon, Harvey T.; Weinstein, Harel; Gether, Ulrik

2013-01-01

The PSD-95/Discs-large/ZO-1 homology (PDZ) domain protein, protein interacting with C kinase 1 (PICK1) contains a C-terminal Bin/amphiphysin/Rvs (BAR) domain mediating recognition of curved membranes; however, the molecular mechanisms controlling the activity of this domain are poorly understood. In agreement with negative regulation of the BAR domain by the N-terminal PDZ domain, PICK1 distributed evenly in the cytoplasm, whereas truncation of the PDZ domain caused BAR domain-dependent redistribution to clusters colocalizing with markers of recycling endosomal compartments. A similar clustering was observed both upon truncation of a short putative α-helical segment in the linker between the PDZ and the BAR domains and upon coexpression of PICK1 with a transmembrane PDZ ligand, including the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR2 subunit, the GluR2 C-terminus transferred to the single transmembrane protein Tac or the dopamine transporter C-terminus transferred to Tac. In contrast, transfer of the GluR2 C-terminus to cyan fluorescent protein, a cytosolic protein, did not elicit BAR domain-dependent clustering. Instead, localizing PICK1 to the membrane by introducing an N-terminal myristoylation site produced BAR domain-dependent, but ligand-independent, PICK1 clustering. The data support that in the absence of PDZ ligand, the PICK1 BAR domain is inhibited through a PDZ domain-dependent and linker-dependent mechanism. Moreover, they suggest that unmasking of the BAR domain’s membrane-binding capacity is not a consequence of ligand binding to the PDZ domain per se but results from, and coincides with, recruitment of PICK1 to a membrane compartment. PMID:18466293

Incorporation of membrane-bound, mammalian-derived immunomodulatory proteins into influenza whole virus vaccines boosts immunogenicity and protection against lethal challenge

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Roberts Paul C

2009-04-01

Full Text Available Abstract Background Influenza epidemics continue to cause morbidity and mortality within the human population despite widespread vaccination efforts. This, along with the ominous threat of an avian influenza pandemic (H5N1, demonstrates the need for a much improved, more sophisticated influenza vaccine. We have developed an in vitro model system for producing a membrane-bound Cytokine-bearing Influenza Vaccine (CYT-IVAC. Numerous cytokines are involved in directing both innate and adaptive immunity and it is our goal to utilize the properties of individual cytokines and other immunomodulatory proteins to create a more immunogenic vaccine. Results We have evaluated the immunogenicity of inactivated cytokine-bearing influenza vaccines using a mouse model of lethal influenza virus challenge. CYT-IVACs were produced by stably transfecting MDCK cell lines with mouse-derived cytokines (GM-CSF, IL-2 and IL-4 fused to the membrane-anchoring domain of the viral hemagglutinin. Influenza virus replication in these cell lines resulted in the uptake of the bioactive membrane-bound cytokines during virus budding and release. In vivo efficacy studies revealed that a single low dose of IL-2 or IL-4-bearing CYT-IVAC is superior at providing protection against lethal influenza challenge in a mouse model and provides a more balanced Th1/Th2 humoral immune response, similar to live virus infections. Conclusion We have validated the protective efficacy of CYT-IVACs in a mammalian model of influenza virus infection. This technology has broad applications in current influenza virus vaccine development and may prove particularly useful in boosting immune responses in the elderly, where current vaccines are minimally effective.

Marine-derived collagen biomaterials from echinoderm connective tissues

KAUST Repository

Ferrario, Cinzia; Leggio, Livio; Leone, Roberta; Di Benedetto, Cristiano; Guidetti, Luca; Coccè , Valentina; Ascagni, Miriam; Bonasoro, Francesco; La Porta, Caterina A.M.; Candia Carnevali, M. Daniela; Sugni, Michela

2016-01-01

The use of marine collagens is a hot topic in the field of tissue engineering. Echinoderms possess unique connective tissues (Mutable Collagenous Tissues, MCTs) which can represent an innovative source of collagen to develop collagen barrier-membranes for Guided Tissue Regeneration (GTR). In the present work we used MCTs from different echinoderm models (sea urchin, starfish and sea cucumber) to produce echinoderm-derived collagen membranes (EDCMs). Commercial membranes for GTR or soluble/reassembled (fibrillar) bovine collagen substrates were used as controls. The three EDCMs were similar among each other in terms of structure and mechanical performances and were much thinner and mechanically more resistant than the commercial membranes. Number of fibroblasts seeded on sea-urchin membranes were comparable to the bovine collagen substrates. Cell morphology on all EDCMs was similar to that of structurally comparable (reassembled) bovine collagen substrates. Overall, echinoderms, and sea urchins particularly, are alternative collagen sources to produce efficient GTR membranes. Sea urchins display a further advantage in terms of eco-sustainability by recycling tissues from food wastes.

Marine-derived collagen biomaterials from echinoderm connective tissues

KAUST Repository

Ferrario, Cinzia

2016-03-31

The use of marine collagens is a hot topic in the field of tissue engineering. Echinoderms possess unique connective tissues (Mutable Collagenous Tissues, MCTs) which can represent an innovative source of collagen to develop collagen barrier-membranes for Guided Tissue Regeneration (GTR). In the present work we used MCTs from different echinoderm models (sea urchin, starfish and sea cucumber) to produce echinoderm-derived collagen membranes (EDCMs). Commercial membranes for GTR or soluble/reassembled (fibrillar) bovine collagen substrates were used as controls. The three EDCMs were similar among each other in terms of structure and mechanical performances and were much thinner and mechanically more resistant than the commercial membranes. Number of fibroblasts seeded on sea-urchin membranes were comparable to the bovine collagen substrates. Cell morphology on all EDCMs was similar to that of structurally comparable (reassembled) bovine collagen substrates. Overall, echinoderms, and sea urchins particularly, are alternative collagen sources to produce efficient GTR membranes. Sea urchins display a further advantage in terms of eco-sustainability by recycling tissues from food wastes.

The NKG2D ligand ULBP2 is specifically regulated through an invariant chain-dependent endosomal pathway

DEFF Research Database (Denmark)

Uhlenbrock, Franziska Katharina; Hagemann-Jensen, Michael Henrik; Kehlet, Stephanie

2014-01-01

by affecting endosomal/lysosomal integrity and protein kinase C activity. The invariant chain was further essential for endosomal transport of ULBP2. This novel pathway was identified through screening experiments by which methylselenic acid was found to possess notable NKG2D ligand regulatory properties....... The protein kinase C inhibitor methylselenic acid induced MICA/B surface expression but dominantly blocked ULBP2 surface transport. Remarkably, by targeting this novel pathway we could specifically block the production of soluble ULBP2 from different, primary melanomas. Our findings strongly suggest...

Human lactoferricin derived di-peptides deploying loop structures induce apoptosis specifically in cancer cells through targeting membranous phosphatidylserine.

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Riedl, Sabrina; Leber, Regina; Rinner, Beate; Schaider, Helmut; Lohner, Karl; Zweytick, Dagmar

2015-11-01

Host defense-derived peptides have emerged as a novel strategy for the development of alternative anticancer therapies. In this study we report on characteristic features of human lactoferricin (hLFcin) derivatives which facilitate specific killing of cancer cells of melanoma, glioblastoma and rhabdomyosarcoma compared with non-specific derivatives and the synthetic peptide RW-AH. Changes in amino acid sequence of hLFcin providing 9-11 amino acids stretched derivatives LF11-316, -318 and -322 only yielded low antitumor activity. However, the addition of the repeat (di-peptide) and the retro-repeat (di-retro-peptide) sequences highly improved cancer cell toxicity up to 100% at 20 μM peptide concentration. Compared to the complete parent sequence hLFcin the derivatives showed toxicity on the melanoma cell line A375 increased by 10-fold and on the glioblastoma cell line U-87mg by 2-3-fold. Reduced killing velocity, apoptotic blebbing, activation of caspase 3/7 and formation of apoptotic DNA fragments proved that the active and cancer selective peptides, e.g. R-DIM-P-LF11-322, trigger apoptosis, whereas highly active, though non-selective peptides, such as DIM-LF11-318 and RW-AH seem to kill rapidly via necrosis inducing membrane lyses. Structural studies revealed specific toxicity on cancer cells by peptide derivatives with loop structures, whereas non-specific peptides comprised α-helical structures without loop. Model studies with the cancer membrane mimic phosphatidylserine (PS) gave strong evidence that PS only exposed by cancer cells is an important target for specific hLFcin derivatives. Other negatively charged membrane exposed molecules as sialic acid, heparan and chondroitin sulfate were shown to have minor impact on peptide activity. Copyright © 2015. Published by Elsevier B.V.

Asymmetric membranes for destabilization of oil droplets in produced water from alkaline-surfactant-polymer (ASP) flooding

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Ramlee, Azierah; Chiam, Chel-Ken; Sarbatly, Rosalam

2018-05-01

This work presents a study of destabilization of oil droplets in the produced water from alkaline-surfactant-polymer (ASP) flooding by using four types of laboratory-fabricated polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were fabricated via immersion precipitation method with ethanol (0 - 30 %, v/v) as the coagulant. The membranes with the effective area of 17.35 cm2 were tested with synthesized ASP solution as the feed in cross-flow microfiltration process. The ASP feed solution initially contained the oil droplets with radius ranged from 40 to 100 nm and the mean radius was 61 nm. Results have shown that the concentration of the ethanol in the coagulation bath affects the formation of the membrane structure and the corresponding porosity, while no significance influence on the membrane thickness. Coalescence of the oil droplets was occurred when the ASP solution permeated through the asymmetric PVDF membranes. Through the coalescence process, the oil droplets were destabilized where the radius of the oil droplets in the permeates increased to 1.5-4 µm with the corresponding mean radius ranged from 2.4 to 2.7 µm.

Endocytosis regulates membrane localization and function of the fusogen EFF-1.

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Smurova, Ksenia; Podbilewicz, Benjamin

2017-07-03

Cell fusion is essential for sexual reproduction and formation of muscles, bones, and placenta. Two families of cell fusion proteins (Syncytins and FFs) have been identified in eukaryotes. Syncytins have been shown to form the giant syncytial trophoblasts in the placenta. The FFs are essential to fuse cells in the skin, reproductive, excretory, digestive and nervous systems in nematodes. EFF-1 (Epithelial Fusion Failure 1), a member of the FF family, is a type I membrane glycoprotein that is essential for most cell fusions in C. elegans. The crystal structure of EFF-1 ectodomain reveals striking structural similarity to class II fusion glycoproteins from enveloped viruses (e.g. dengue and rubella) that mediate virus to cell fusion. We found EFF-1 to be present on the plasma membrane and in RAB-5-positive early endosomes, with EFF-1 recycling between these 2 cell compartments. Only when EFF-1 proteins transiently arrive to the surfaces of 2 adjacent cells do they dynamically interact in trans and mediate membrane fusion. EFF-1 is continuously internalized by receptor-mediated endocytosis via the activity of 2 small GTPases: RAB-5 and Dynamin. Here we propose a model that explains how EFF-1 endocytosis together with interactions in trans can control cell-cell fusion. Kontani et al. showed that vacuolar ATPase (vATPase) mutations result in EFF-1-dependent hyperfusion. 1 We propose that vATPase is required for normal degradation of EFF-1. Failure to degrade EFF-1 results in delayed hyperfusion and mislocalization to organelles that appear to be recycling endosomes. EFF-1 is also required to fuse neurons as part of the repair mechanism following injury and to prune dendrites. We speculate that EFF-1 may regulate neuronal tree like structures via endocytosis. Thus, endocytosis of cell-cell fusion proteins functions to prevent merging of cells and to sculpt organs and neurons.

A Two-Component Regulatory System Impacts Extracellular Membrane-Derived Vesicle Production in Group A Streptococcus

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Ulrike Resch

2016-11-01

Full Text Available Export of macromolecules via extracellular membrane-derived vesicles (MVs plays an important role in the biology of Gram-negative bacteria. Gram-positive bacteria have also recently been reported to produce MVs; however, the composition and mechanisms governing vesiculogenesis in Gram-positive bacteria remain undefined. Here, we describe MV production in the Gram-positive human pathogen group A streptococcus (GAS, the etiological agent of necrotizing fasciitis and streptococcal toxic shock syndrome. M1 serotype GAS isolates in culture exhibit MV structures both on the cell wall surface and in the near vicinity of bacterial cells. A comprehensive analysis of MV proteins identified both virulence-associated protein substrates of the general secretory pathway in addition to “anchorless surface proteins.” Characteristic differences in the contents, distributions, and fatty acid compositions of specific lipids between MVs and GAS cell membrane were also observed. Furthermore, deep RNA sequencing of vesicular RNAs revealed that GAS MVs contained differentially abundant RNA species relative to bacterial cellular RNA. MV production by GAS strains varied in a manner dependent on an intact two-component system, CovRS, with MV production negatively regulated by the system. Modulation of MV production through CovRS was found to be independent of both GAS cysteine protease SpeB and capsule biosynthesis. Our data provide an explanation for GAS secretion of macromolecules, including RNAs, lipids, and proteins, and illustrate a regulatory mechanism coordinating this secretory response.

Kinesin Khc-73/KIF13B modulates retrograde BMP signaling by influencing endosomal dynamics at the Drosophila neuromuscular junction.

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Liao, Edward H; Gray, Lindsay; Tsurudome, Kazuya; El-Mounzer, Wassim; Elazzouzi, Fatima; Baim, Christopher; Farzin, Sarah; Calderon, Mario R; Kauwe, Grant; Haghighi, A Pejmun

2018-01-01

Retrograde signaling is essential for neuronal growth, function and survival; however, we know little about how signaling endosomes might be directed from synaptic terminals onto retrograde axonal pathways. We have identified Khc-73, a plus-end directed microtubule motor protein, as a regulator of sorting of endosomes in Drosophila larval motor neurons. The number of synaptic boutons and the amount of neurotransmitter release at the Khc-73 mutant larval neuromuscular junction (NMJ) are normal, but we find a significant decrease in the number of presynaptic release sites. This defect in Khc-73 mutant larvae can be genetically enhanced by a partial genetic loss of Bone Morphogenic Protein (BMP) signaling or suppressed by activation of BMP signaling in motoneurons. Consistently, activation of BMP signaling that normally enhances the accumulation of phosphorylated form of BMP transcription factor Mad in the nuclei, can be suppressed by genetic removal of Khc-73. Using a number of assays including live imaging in larval motor neurons, we show that loss of Khc-73 curbs the ability of retrograde-bound endosomes to leave the synaptic area and join the retrograde axonal pathway. Our findings identify Khc-73 as a regulator of endosomal traffic at the synapse and modulator of retrograde BMP signaling in motoneurons.

Molecular determinants of the interaction between Doa1 and Hse1 involved in endosomal sorting.

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Han, Seungsu; Shin, Donghyuk; Choi, Hoon; Lee, Sangho

2014-03-28

Yeast Doa1/Ufd3 is an adaptor protein for Cdc48 (p97 in mammal), an AAA type ATPase associated with endoplasmic reticulum-associated protein degradation pathway and endosomal sorting into multivesicular bodies. Doa1 functions in the endosomal sorting by its association with Hse1, a component of endosomal sorting complex required for transport (ESCRT) system. The association of Doa1 with Hse1 was previously reported to be mediated between PFU domain of Doa1 and SH3 of Hse1. However, it remains unclear which residues are specifically involved in the interaction. Here we report that Doa1/PFU interacts with Hse1/SH3 with a moderate affinity of 5 μM. Asn-438 of Doa1/PFU and Trp-254 of Hse1/SH3 are found to be critical in the interaction while Phe-434, implicated in ubiquitin binding via a hydrophobic interaction, is not. Small-angle X-ray scattering measurements combined with molecular docking and biochemical analysis yield the solution structure of the Doa1/PFU:Hse1/SH3 complex. Taken together, our results suggest that hydrogen bonding is a major determinant in the interaction of Doa1/PFU with Hse1/SH3. Copyright © 2014 Elsevier Inc. All rights reserved.

The basic route of the nuclear translocation porcine growth hormone (GH)-growth hormone receptor (GHR) complex (pGH/GHR) in porcine hepatocytes.

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Hainan, Lan; Huilin, Liu; Khan, Mahamad; Xin, Zheng; YuJiang, Yang; Hui, Zhang; Naiquan, Yao

2018-06-08

Traditional views suggest that growth hormone and the growth hormone receptor (GH/GHR complex) exert their functions only on the plasma membrane. This paradigm, however, has been challenged by recent new findings that the GH/GHR complex could translocate into cell nuclei where they could still exhibit important physiological functions. We also reported the nuclear localization of porcine GH/GHR and their potential functions in porcine hepatocytes. However, the basic path of pGH/GHR's nuclear translocation remains unclear. Combining previous research results and our current findings, we proposed two basic routes of pGH/GHR's nuclear transportation as follows: 1) after pGH binding to GHR, pGH/GHR enters into the cytoplasm though clathrin- or caveolin-mediated endocytosis, then the pGH/GHR complex enters into early endosomes (Rab5-positive), and the endosome carries the GH/GHR complex to the endoplasmic reticulum (ER). After endosome docking on the ER, the endosome starts fission, and the pGH/GHR complex enters into the ER lumen. Then the pGH/GHR complex transports into the cytoplasm, possibly by the ERAD pathway. Subsequently, the pGH/GHR complex interacts with IMPα/β, which, in turn, mediates GH/GHR nuclear localization; 2) pGH binds with the GHR on the cell membrane and, subsequently, pGH/GHR internalizes into the cell and enters into the endosome (this endosome may belong to a class of endosomes called envelope-associated endosomes (NAE)). Then, the endosome carries the pGH/GHR to the nuclear membrane. After docking on the nuclear membrane, the pGH/GHR complex fuses with the nuclear membrane and then enters into the cell nucleus. Copyright © 2018 Elsevier Inc. All rights reserved.

Evaluation of Time-Temperature Integrators (TTIs) with Microorganism-Entrapped Microbeads Produced Using Homogenization and SPG Membrane Emulsification Techniques.

Science.gov (United States)

Rahman, A T M Mijanur; Lee, Seung Ju; Jung, Seung Won

2015-12-28

A comparative study was conducted to evaluate precision and accuracy in controlling the temperature dependence of encapsulated microbial time-temperature integrators (TTIs) developed using two different emulsification techniques. Weissela cibaria CIFP 009 cells, immobilized within 2% Na-alginate gel microbeads using homogenization (5,000, 7,000, and 10,000 rpm) and Shirasu porous glass (SPG) membrane technologies (10 μm), were applied to microbial TTIs. The prepared micobeads were characterized with respect to their size, size distribution, shape and morphology, entrapment efficiency, and bead production yield. Additionally, fermentation process parameters including growth rate were investigated. The TTI responses (changes in pH and titratable acidity (TA)) were evaluated as a function of temperature (20°C, 25°C, and 30°C). In comparison with conventional methods, SPG membrane technology was able not only to produce highly uniform, small-sized beads with the narrowest size distribution, but also the bead production yield was found to be nearly 3.0 to 4.5 times higher. However, among the TTIs produced using the homogenization technique, poor linearity (R(2)) in terms of TA was observed for the 5,000 and 7,000 rpm treatments. Consequently, microbeads produced by the SPG membrane and by homogenization at 10,000 rpm were selected for adjusting the temperature dependence. The Ea values of TTIs containing 0.5, 1.0, and 1.5 g microbeads, prepared by SPG membrane and conventional methods, were estimated to be 86.0, 83.5, and 76.6 kJ/mol, and 85.5, 73.5, and 62.2 kJ/mol, respectively. Therefore, microbial TTIs developed using SPG membrane technology are much more efficient in controlling temperature dependence.

A Class of Rigid Linker-bearing Glucosides for Membrane Protein Structural Study.

Science.gov (United States)

Sadaf, Aiman; Mortensen, Jonas S; Capaldi, Stefano; Tikhonova, Elena; Hariharan, Parameswaran; de Castro Ribeiro, Orquidea; Loland, Claus J; Guan, Lan; Byrne, Bernadette; Chae, Pil Seok

2016-03-01

Membrane proteins are amphipathic bio-macromolecules incompatible with the polar environments of aqueous media. Conventional detergents encapsulate the hydrophobic surfaces of membrane proteins allowing them to exist in aqueous solution. Membrane proteins stabilized by detergent micelles are used for structural and functional analysis. Despite the availability of a large number of detergents, only a few agents are sufficiently effective at maintaining the integrity of membrane proteins to allow successful crystallization. In the present study, we describe a novel class of synthetic amphiphiles with a branched tail group and a triglucoside head group. These head and tail groups were connected via an amide or ether linkage by using a tris(hydroxylmethyl)aminomethane (TRIS) or neopentyl glycol (NPG) linker to produce TRIS-derived triglucosides (TDTs) and NPG-derived triglucosides (NDTs), respectively. Members of this class conferred enhanced stability on target membrane proteins compared to conventional detergents. Because of straightforward synthesis of the novel agents and their favourable effects on a range of membrane proteins, these agents should be of wide applicability to membrane protein science.

Generation of stable lipid raft microdomains in the enterocyte brush border by selective endocytic removal of non-raft membrane

DEFF Research Database (Denmark)

Danielsen, E Michael; Hansen, Gert H

2013-01-01

The small intestinal brush border has an unusually high proportion of glycolipids which promote the formation of lipid raft microdomains, stabilized by various cross-linking lectins. This unique membrane organization acts to provide physical and chemical stability to the membrane that faces...... functions to enrich the contents of lipid raft components in the brush border. The lipophilic fluorescent marker FM, taken up into early endosomes in the terminal web region (TWEEs), was absent from detergent resistant membranes (DRMs), implying an association with non-raft membrane. Furthermore, neither...... major lipid raft-associated brush border enzymes nor glycolipids were detected by immunofluorescence microscopy in subapical punctae resembling TWEEs. Finally, two model raft lipids, BODIPY-lactosylceramide and BODIPY-GM1, were not endocytosed except when cholera toxin subunit B (CTB) was present...

Membrane Trafficking Modulation during Entamoeba Encystation.

Science.gov (United States)

Herman, Emily; Siegesmund, Maria A; Bottery, Michael J; van Aerle, Ronny; Shather, Maulood Mohammed; Caler, Elisabet; Dacks, Joel B; van der Giezen, Mark

2017-10-09

Entamoeba histolytica is an intestinal parasite that infects 50-100 million people and causes up to 55,000 deaths annually. The transmissive form of E. histolytica is the cyst, with a single infected individual passing up to 45 million cysts per day, making cyst production an attractive target for infection control. Lectins and chitin are secreted to form the cyst wall, although little is known about the underlying membrane trafficking processes supporting encystation. As E. histolytica does not readily form cysts in vitro, we assessed membrane trafficking gene expression during encystation in the closely related model Entamoeba invadens. Genes involved in secretion are up-regulated during cyst formation, as are some trans-Golgi network-to-endosome trafficking genes. Furthermore, endocytic and general trafficking genes are up-regulated in the mature cyst, potentially preserved as mRNA in preparation for excystation. Two divergent dynamin-related proteins found in Entamoeba are predominantly expressed during cyst formation. Phylogenetic analyses indicate that they are paralogous to, but quite distinct from, classical dynamins found in human, suggesting that they may be potential drug targets to block encystation. The membrane-trafficking machinery is clearly regulated during encystation, providing an additional facet to understanding this crucial parasitic process.

Nanothin Coculture Membranes with Tunable Pore Architecture and Thermoresponsive Functionality for Transfer-Printable Stem Cell-Derived Cardiac Sheets.

Science.gov (United States)

Ryu, Seungmi; Yoo, Jin; Jang, Yeongseon; Han, Jin; Yu, Seung Jung; Park, Jooyeon; Jung, Seon Yeop; Ahn, Kyung Hyun; Im, Sung Gap; Char, Kookheon; Kim, Byung-Soo

2015-10-27

Coculturing stem cells with the desired cell type is an effective method to promote the differentiation of stem cells. The features of the membrane used for coculturing are crucial to achieving the best outcome. Not only should the membrane act as a physical barrier that prevents the mixing of the cocultured cell populations, but it should also allow effective interactions between the cells. Unfortunately, conventional membranes used for coculture do not sufficiently meet these requirements. In addition, cell harvesting using proteolytic enzymes following coculture impairs cell viability and the extracellular matrix (ECM) produced by the cultured cells. To overcome these limitations, we developed nanothin and highly porous (NTHP) membranes, which are ∼20-fold thinner and ∼25-fold more porous than the conventional coculture membranes. The tunable pore size of NTHP membranes at the nanoscale level was found crucial for the formation of direct gap junctions-mediated contacts between the cocultured cells. Differentiation of the cocultured stem cells was dramatically enhanced with the pore size-customized NTHP membrane system compared to conventional coculture methods. This was likely due to effective physical contacts between the cocultured cells and the fast diffusion of bioactive molecules across the membrane. Also, the thermoresponsive functionality of the NTHP membranes enabled the efficient generation of homogeneous, ECM-preserved, highly viable, and transfer-printable sheets of cardiomyogenically differentiated cells. The coculture platform developed in this study would be effective for producing various types of therapeutic multilayered cell sheets that can be differentiated from stem cells.

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

Science.gov (United States)

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

2015-12-15

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

A FTIR study water in membrane of nitrocellulose prepared by phase inversion

International Nuclear Information System (INIS)

Benosmane, N.; Boutemeur, B.; Hamdi, M.

2004-01-01

Full text.Cellulose derivates were the first biopolymers used to produce synthesis membranes for technical applications, in this study the state of water in asymmetric membrane of nitrocellulose, prepared by the phase inversion process, was investigated using infrared spectroscopy (FTIR), after membrane preparation by the wet inversion process in acetone, the spectre FTIR of wet asymmetric membrane of nitrocellulose after immersion in water (after one week) is compared to the spectre of dried asymmetric membrane of nitrocellulose, the difference in spectre of dried and wet membrane indicate a weakly hydrogen-bonded to the polymer hydroxyl groups between water and hydroxyl groups in surface of membrane, the results demonstrate the amount of water species present in the surface of asymmetric membrane and heterogeneous of surface

Evaluation of a dental pulp-derived cell sheet cultured on amniotic membrane substrate.

Science.gov (United States)

Honjo, Ken-ichi; Yamamoto, Toshiro; Adachi, Tetsuya; Amemiya, Takeshi; Mazda, Osam; Kanamura, Narisato; Kita, Masakazu

2015-01-01

Mesenchymal stem cells (MSC) are transplanted for periodontal tissue regeneration, and the periodontal ligament (PDL) is regenerated using a cultured cell sheet. This cultured cell sheet is prepared using PDL-derived cells, growth factors, and amniotic membrane (AM). Dental pulp (DP)-derived cells can be easily obtained from extracted wisdom teeth, proliferate rapidly, and are less susceptible to bacterial infection than PDL-derived cells. Thus, to prepare a novel cell sheet, DP-derived cells were cultured on AM as a culture substrate for immunohistochemical examination. Wisdom teeth extracted from three adults were cut along the cement-enamel border. DP tissue was collected, minced, and primarily cultured. After three or four passage cultures, DP-derived cells were cultured on AM, followed by hematoxylin-eosin (H-E) and immunofluorescence staining. DP-derived cells cultured on AM formed a layered structure. Cells positive for vimentin, Ki-67, ZO-1, desmoplakin, CD29, 44, 105 or 146, STRO-1, collagen IV or VII or laminin 5 or α5 chain were localized. DP-derived cells proliferated on AM, while retaining the properties of DP, which allowed the cultured cell sheet to be prepared. In addition, the cultured cell sheet contained MSC, which suggests its potential application in periodontal tissue regeneration.

The ArfGAP2/3 Glo3 and ergosterol collaborate in transport of a subset of cargoes

Directory of Open Access Journals (Sweden)

Alejandro F. Estrada

2015-07-01

Full Text Available Proteins reach the plasma membrane through the secretory pathway in which the trans Golgi network (TGN acts as a sorting station. Transport from the TGN to the plasma membrane is maintained by a number of different pathways that act either directly or via the endosomal system. Here we show that a subset of cargoes depends on the ArfGAP2/3 Glo3 and ergosterol to maintain their proper localization at the plasma membrane. While interfering with neither ArfGAP2/3 activity nor ergosterol biosynthesis individually significantly altered plasma membrane localization of the tryptophan transporter Tat2, the general amino acid permease Gap1 and the v-SNARE Snc1, in a Δglo3 Δerg3 strain those proteins accumulated in internal endosomal structures. Export from the TGN to the plasma membrane and recycling from early endosomes appeared unaffected as the chitin synthase Chs3 that travels along these routes was localized normally. Our data indicate that a subset of proteins can reach the plasma membrane efficiently but after endocytosis becomes trapped in endosomal structures. Our study supports a role for ArfGAP2/3 in recycling from endosomes and in transport to the vacuole/lysosome.

PALLADIUM/COPPER ALLOY COMPOSITE MEMBRANES FOR HIGH TEMPERATURE HYDROGEN SEPARATION FROM COAL-DERIVED GAS STREAMS; F

International Nuclear Information System (INIS)

J. Douglas Way; Robert L. McCormick

2001-01-01

Recent advances have shown that Pd-Cu composite membranes are not susceptible to the mechanical, embrittlement, and poisoning problems that have prevented widespread industrial use of Pd for high temperature H(sub 2) separation. These membranes consist of a thin ((approx)10(micro)m) film of metal deposited on the inner surface of a porous metal or ceramic tube. Based on preliminary results, thin Pd(sub 60)Cu(sub 40) films are expected to exhibit hydrogen flux up to ten times larger than commercial polymer membranes for H(sub 2) separation, and resist poisoning by H(sub 2)S and other sulfur compounds typical of coal gas. Similar Pd-membranes have been operated at temperatures as high as 750 C. The overall objective of the proposed project is to demonstrate the feasibility of using sequential electroless plating to fabricate Pd(sub 60)Cu(sub 40) alloy membranes on porous supports for H(sub 2) separation. These following advantages of these membranes for processing of coal-derived gas will be demonstrated: High H(sub 2) flux; Sulfur tolerant, even at very high total sulfur levels (1000 ppm); Operation at temperatures well above 500 C; and Resistance to embrittlement and degradation by thermal cycling. The proposed research plan is designed to providing a fundamental understanding of: Factors important in membrane fabrication; Optimization of membrane structure and composition; Effect of temperature, pressure, and gas composition on H(sub 2) flux and membrane selectivity; and How this membrane technology can be integrated in coal gasification-fuel cell systems

Low oxygen biomass-derived pyrolysis oils and methods for producing the same

Science.gov (United States)

Marinangeli, Richard; Brandvold, Timothy A; Kocal, Joseph A

2013-08-27

Low oxygen biomass-derived pyrolysis oils and methods for producing them from carbonaceous biomass feedstock are provided. The carbonaceous biomass feedstock is pyrolyzed in the presence of a catalyst comprising base metal-based catalysts, noble metal-based catalysts, treated zeolitic catalysts, or combinations thereof to produce pyrolysis gases. During pyrolysis, the catalyst catalyzes a deoxygenation reaction whereby at least a portion of the oxygenated hydrocarbons in the pyrolysis gases are converted into hydrocarbons. The oxygen is removed as carbon oxides and water. A condensable portion (the vapors) of the pyrolysis gases is condensed to low oxygen biomass-derived pyrolysis oil.

Kinetic imaging of NPC1L1 and sterol trafficking between plasma membrane and recycling endosomes in hepatoma cells

DEFF Research Database (Denmark)

Hartwig Petersen, Nicole; Færgeman, Nils J; Yu, Liqing

2008-01-01

fluorescent protein (NPC1L1-EGFP) and cholesterol analogues in hepatoma cells. At steady state about 42% of NPC1L1 resided in the transferrin (Tf) positive, sterol enriched endocytic recycling compartment (ERC), while time-lapse microscopy demonstrated NPC1L1 traffic between plasma membrane and ERC...... the ERC to the plasma membrane. NPC1L1-EGFP facilitated transport of fluorescent sterols from the plasma membrane to the ERC. Insulin induced translocation of vesicles containing NPC1L1 and fluorescent sterol from the ERC to the cell membrane. Upon polarization of hepatoma cells NPC1L1 resided almost...... exclusively in the canalicular membrane, where the protein is highly mobile. Our study demonstrates dynamic trafficking of NPC1L1 between cell surface and intracellular compartments and suggests that this transport is involved in NPC1L1 mediated cellular sterol uptake....

Light-controlled endosomal escape of the novel CD133-targeting immunotoxin AC133-saporin by photochemical internalization - A minimally invasive cancer stem cell-targeting strategy.

Science.gov (United States)

Bostad, Monica; Olsen, Cathrine Elisabeth; Peng, Qian; Berg, Kristian; Høgset, Anders; Selbo, Pål Kristian

2015-05-28

The cancer stem cell (CSC) marker CD133 is an attractive target to improve antitumor therapy. We have used photochemical internalization (PCI) for the endosomal escape of the novel CD133-targeting immunotoxin AC133-saporin (PCIAC133-saporin). PCI employs an endocytic vesicle-localizing photosensitizer, which generates reactive oxygen species upon light-activation causing a rupture of the vesicle membranes and endosomal escape of entrapped drugs. Here we show that AC133-saporin co-localizes with the PCI-photosensitizer TPCS2a, which upon light exposure induces cytosolic release of AC133-saporin. PCI of picomolar levels of AC133-saporin in colorectal adenocarcinoma WiDr cells blocked cell proliferation and induced 100% inhibition of cell viability and colony forming ability at the highest light doses, whereas no cytotoxicity was obtained in the absence of light. Efficient PCI-based CD133-targeting was in addition demonstrated in the stem-cell-like, triple negative breast cancer cell line MDA-MB-231 and in the aggressive malignant melanoma cell line FEMX-1, whereas no enhanced targeting was obtained in the CD133-negative breast cancer cell line MCF-7. PCIAC133-saporin induced mainly necrosis and a minimal apoptotic response based on assessing cleavage of caspase-3 and PARP, and the TUNEL assay. PCIAC133-saporin resulted in S phase arrest and reduced LC3-II conversion compared to control treatments. Notably, co-treatment with Bafilomycin A1 and PCIAC133-saporin blocked LC3-II conversion, indicating a termination of the autophagic flux in WiDr cells. For the first time, we demonstrate laser-controlled targeting of CD133 in vivo. After only one systemic injection of AC133-saporin and TPCS2a, a strong anti-tumor response was observed after PCIAC133-saporin. The present PCI-based endosomal escape technology represents a minimally invasive strategy for spatio-temporal, light-controlled targeting of CD133+ cells in localized primary tumors or metastasis. Copyright © 2015

INTERACTION OF ALDEHYDES DERIVED FROM LIPID PEROXIDATION AND MEMBRANE PROTEINS.

Directory of Open Access Journals (Sweden)

Stefania ePizzimenti

2013-09-01

Full Text Available A great variety of compounds are formed during lipid peroxidation of polyunsaturated fatty acids of membrane phospholipids. Among them, bioactive aldehydes, such as 4-hydroxyalkenals, malondialdehyde (MDA and acrolein, have received particular attention since they have been considered as toxic messengers that can propagate and amplify oxidative injury. In the 4-hydroxyalkenal class, 4-hydroxy-2-nonenal (HNE is the most intensively studied aldehyde, in relation not only to its toxic function, but also to its physiological role. Indeed, HNE can be found at low concentrations in human tissues and plasma and participates in the control of biological processes, such as signal transduction, cell proliferation and differentiation. Moreover, at low doses, HNE exerts an anti-cancer effect, by inhibiting cell proliferation, angiogenesis, cell adhesion and by inducing differentiation and/or apoptosis in various tumor cell lines. It is very likely that a substantial fraction of the effects observed in cellular responses, induced by HNE and related aldehydes, be mediated by their interaction with proteins, resulting in the formation of covalent adducts or in the modulation of their expression and/or activity. In this review we focus on membrane proteins affected by lipid peroxidation-derived aldehydes, under physiological and pathological conditions.

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

Science.gov (United States)

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

2016-01-01

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

Wnt signaling requires retromer-dependent recycling of MIG-14/Wntless in Wnt-producing cells.

NARCIS (Netherlands)

Yang, P.T.; Lorenowicz, M.J.; Silhankova, M.; Coudreuse, D.Y.M.; Betist, M.C.; Korswagen, H.C.

2008-01-01

Wnt proteins are secreted signaling molecules that play a central role in development and adult tissue homeostasis. We have previously shown that Wnt signaling requires retromer function in Wnt-producing cells. The retromer is a multiprotein complex that mediates endosome-to-Golgi transport of

Single histidine residue in head-group region is sufficient to impart remarkable gene transfection properties to cationic lipids: evidence for histidine-mediated membrane fusion at acidic pH.

Science.gov (United States)

Kumar, V V; Pichon, C; Refregiers, M; Guerin, B; Midoux, P; Chaudhuri, A

2003-08-01

Presence of endosome-disrupting multiple histidine functionalities in the molecular architecture of cationic polymers, such as polylysine, has previously been demonstrated to significantly enhance their in vitro gene delivery efficiencies. Towards harnessing improved transfection property through covalent grafting of endosome-disrupting single histidine functionality in the molecular structure of cationic lipids, herein, we report on the design, the synthesis and the transfection efficiency of two novel nonglycerol-based histidylated cationic amphiphiles. We found that L-histidine-(N,N-di-n-hexadecylamine)ethylamide (lipid 1) and L-histidine-(N,N-di-n-hexadecylamine,-N-methyl)ethylamide (lipid 2) in combination with cholesterol gave efficient transfections into various cell lines. The transfection efficiency of Chol/lipid 1 lipoplexes into HepG2 cells was two order of magnitude higher than that of FuGENE(TM)6 and DC-Chol lipoplexes, whereas it was similar into A549, 293T7 and HeLa cells. A better efficiency was obtained with Chol/lipid 2 lipoplexes when using the cytosolic luciferase expression vector (pT7Luc) under the control of the bacterial T7 promoter. Membrane fusion activity measurements using fluorescence resonance energy transfer (FRET) technique showed that the histidine head-groups of Chol/lipid 1 liposomes mediated membrane fusion in the pH range 5-7. In addition, the transgene expression results using the T7Luc expression vector convincingly support the endosome-disrupting role of the presently described mono-histidylated cationic transfection lipids and the release of DNA into the cytosol. We conclude that covalent grafting of a single histidine amino acid residue to suitable twin-chain hydrophobic compounds is able to impart remarkable transfection properties on the resulting mono-histidylated cationic amphiphile, presumably via the endosome-disrupting characteristics of the histidine functionalities.

Derivative expansion of one-loop effective energy of stiff membranes with tension

Science.gov (United States)

Borelli, M. E. S.; Kleinert, H.; Schakel, Adriaan M. J.

1999-03-01

With help of a derivative expansion, the one-loop corrections to the energy functional of a nearly flat, stiff membrane with tension due to thermal fluctuations are calculated in the Monge parametrization. Contrary to previous studies, an arbitrary tilt of the surface is allowed to exhibit the nontrivial relations between the different, highly nonlinear terms accompanying the ultraviolet divergences. These terms are shown to have precisely the same form as those in the original energy functional, as necessary for renormalizability. Also infrared divergences arise. These, however, are shown to cancel in a nontrivial way.

Human endothelial progenitor cells internalize high-density lipoprotein.

Directory of Open Access Journals (Sweden)

Kaemisa Srisen

Full Text Available Endothelial progenitor cells (EPCs originate either directly from hematopoietic stem cells or from a subpopulation of monocytes. Controversial views about intracellular lipid traffic prompted us to analyze the uptake of human high density lipoprotein (HDL, and HDL-cholesterol in human monocytic EPCs. Fluorescence and electron microscopy were used to investigate distribution and intracellular trafficking of HDL and its associated cholesterol using fluorescent surrogates (bodipy-cholesterol and bodipy-cholesteryl oleate, cytochemical labels and fluorochromes including horseradish peroxidase and Alexa Fluor® 568. Uptake and intracellular transport of HDL were demonstrated after internalization periods from 0.5 to 4 hours. In case of HDL-Alexa Fluor® 568, bodipy-cholesterol and bodipy-cholesteryl oleate, a photooxidation method was carried out. HDL-specific reaction products were present in invaginations of the plasma membrane at each time of treatment within endocytic vesicles, in multivesicular bodies and at longer periods of uptake, also in lysosomes. Some HDL-positive endosomes were arranged in form of "strings of pearl"- like structures. HDL-positive multivesicular bodies exhibited intensive staining of limiting and vesicular membranes. Multivesicular bodies of HDL-Alexa Fluor® 568-treated EPCs showed multilamellar intra-vacuolar membranes. At all periods of treatment, labeled endocytic vesicles and organelles were apparent close to the cell surface and in perinuclear areas around the Golgi apparatus. No HDL-related particles could be demonstrated close to its cisterns. Electron tomographic reconstructions showed an accumulation of HDL-containing endosomes close to the trans-Golgi-network. HDL-derived bodipy-cholesterol was localized in endosomal vesicles, multivesicular bodies, lysosomes and in many of the stacked Golgi cisternae and the trans-Golgi-network Internalized HDL-derived bodipy-cholesteryl oleate was channeled into the lysosomal

An EPR spin-probe and spin-trap study of the free radicals produced by plant plasma membranes

Directory of Open Access Journals (Sweden)

GORAN BACIC

2005-02-01

Full Text Available Plant plasma membranes are known to produce superoxide radicals, while the production of hydroxyl radical is thought to occur only in the cell wall. In this work it was demonstrated using combined spin-trap and spin-probe EPR spectroscopic techniques, that plant plasma membranes do produce superoxide and hydroxyl radicals but by kinetically different mechanisms. The results show that superoxide and hydroxyl radicals can be detected by DMPO spin-trap and that the mechanisms and location of their production can be differentiated using the reduction of spin-probes Tempone and 7-DS. It was shown that the mechanism of production of oxygen reactive species is NADH dependent and diphenylene iodonium inhibited. The kinetics of the reduction of Tempone, combined with scavengers or the absence of NADH indicates that hydroxyl radicals are produced by a mechanism independent of that of superoxide production. It was shown that a combination of the spin-probe and spin-trap technique can be used in free radical studies of biological systems, with a number of advantages inherent to them.

Biomimetic membranes and methods of making biomimetic membranes

Science.gov (United States)

Rempe, Susan; Brinker, Jeffrey C.; Rogers, David Michael; Jiang, Ying-Bing; Yang, Shaorong

2016-11-08

The present disclosure is directed to biomimetic membranes and methods of manufacturing such membranes that include structural features that mimic the structures of cellular membrane channels and produce membrane designs capable of high selectivity and high permeability or adsorptivity. The membrane structure, material and chemistry can be selected to perform liquid separations, gas separation and capture, ion transport and adsorption for a variety of applications.

OXYGEN TRANSPORT CERAMIC MEMBRANES

International Nuclear Information System (INIS)

Dr. Sukumar Bandopadhyay; Dr. Nagendra Nagabhushana

2001-01-01

Conversion of natural gas to liquid fuels and chemicals is a major goal for the Nation as it enters the 21st Century. Technically robust and economically viable processes are needed to capture the value of the vast reserves of natural gas on Alaska's North Slope, and wean the Nation from dependence on foreign petroleum sources. Technologies that are emerging to fulfill this need are all based syngas as an intermediate. Syngas (a mixture of hydrogen and carbon monoxide) is a fundamental building block from which chemicals and fuels can be derived. Lower cost syngas translates directly into more cost-competitive fuels and chemicals. The currently practiced commercial technology for making syngas is either steam methane reforming (SMR) or a two-step process involving cryogenic oxygen separation followed by natural gas partial oxidation (POX). These high-energy, capital-intensive processes do not always produce syngas at a cost that makes its derivatives competitive with current petroleum-based fuels and chemicals. This project has the following 6 main tasks: Task 1--Design, fabricate and evaluate ceramic to metal seals based on graded ceramic powder/metal braze joints. Task 2--Evaluate the effect of defect configuration on ceramic membrane conductivity and long term chemical and structural stability. Task 3--Determine materials mechanical properties under conditions of high temperatures and reactive atmospheres. Task 4--Evaluate phase stability and thermal expansion of candidate perovskite membranes and develop techniques to support these materials on porous metal structures. Task 5--Assess the microstructure of membrane materials to evaluate the effects of vacancy-impurity association, defect clusters, and vacancy-dopant association on the membrane performance and stability. Task 6--Measure kinetics of oxygen uptake and transport in ceramic membrane materials under commercially relevant conditions using isotope labeling techniques

Endocytosis of GPI-linked membrane folate receptor-alpha.

Science.gov (United States)

Rijnboutt, S; Jansen, G; Posthuma, G; Hynes, J B; Schornagel, J H; Strous, G J

1996-01-01

GPI-linked membrane folate receptors (MFRs) have been implicated in the receptor-mediated uptake of reduced folate cofactors and folate-based chemotherapeutic drugs. We have studied the biosynthetic transport to and internalization of MFR isoform alpha in KB-cells. MFR-alpha was synthesized as a 32-kD protein and converted in a maturely glycosylated 36-38-kD protein 1 h after synthesis. 32-kD MFR-alpha was completely soluble in Triton X-100 at 0 degree C. In contrast, only 33% of the 36-38-kD species could be solubilized at these conditions whereas complete solubilization was obtained in Triton X-100 at 37 degrees C or in the presence of saponin at 0 degree C. Similar solubilization characteristics were found when MFR-alpha at the plasma membrane was labeled with a crosslinkable 125I-labeled photoaffinity-analog of folic acid as a ligand. Triton X-100-insoluble membrane domains containing MFR-alpha could be separated from soluble MFR-alpha on sucrose flotation gradients. Only Triton X-100 soluble MFR-alpha was internalized from the plasma membrane. The reduced-folate-carrier, an integral membrane protein capable of translocating (anti-)folates across membranes, was completely excluded from the Triton X-100-resistant membrane domains. Internalized MFR-alpha recycled slowly to the cell surface during which it remained soluble in Triton X-100 at 0 degree C. Using immunoelectron microscopy, we found MFR-alpha along the entire endocytic pathway: in clathrin-coated buds and vesicles, and in small and large endosomal vacuoles. In conclusion, our data indicate that a large fraction, if not all, of internalizing MFR-alpha bypasses caveolae.

Functional links between mucolipin-1 and Ca2+-dependent membrane trafficking in mucolipidosis IV

International Nuclear Information System (INIS)

LaPlante, Janice M.; Ye, C.P.; Quinn, Stephen J.; Goldin, Ehud; Brown, Edward M.; Slaugenhaupt, Susan A.; Vassilev, Peter M.

2004-01-01

Most of the membrane trafficking phenomena including those involving the interactions between endosomes and lysosomes are regulated by changes in intracellular Ca 2+ (Ca i ). These processes are disturbed in some types of mucolipidoses and other lysosomal storage disorders, such as mucolipidosis IV (MLIV), a neurological disorder that usually presents during the first year of life with blindness, cognitive impairment, and psychomotor delays. It is caused by mutations in MCOLN1, the gene encoding mucolipin-1 (MLN1), which we have recently established to represent a Ca 2+ -permeable cation channel that is transiently modulated by changes in Ca i . The cells of MLIV patients contain enlarged lysosomes that are likely associated with abnormal sorting and trafficking of these and related organelles. We studied fibroblasts from MLIV patients and found disturbed Ca 2+ signaling and large acidic organelles such as late endosomes and lysosomes (LEL) with altered cellular localization in these cells. The fusion between LEL vesicles in these cells was defective. This is a Ca 2+ -dependent process related to signaling pathways involved in regulation of Ca 2+ homeostasis and trafficking. The MLN1 channels could play a key role in Ca 2+ release from LEL vesicles, which triggers the fusion and trafficking of these organelles. The characterization of this MLN1-mediated Ca 2+ -dependent process should provide new insights into the pathophysiological mechanisms that lead to the development of MLIV and other mucolipidoses associated with similar disturbances in membrane trafficking

Mouse endometrial stromal cells produce basement-membrane components

DEFF Research Database (Denmark)

Wewer, U M; Damjanov, A; Weiss, J

1986-01-01

During mouse pregnancy, uterine stromal cells transform into morphologically distinct decidual cells under the influence of the implanting embryo and a proper hormonal environment. Mechanical stimulation of hormonally primed uterine stromal cells leads to the same morphologic alterations. The dec......During mouse pregnancy, uterine stromal cells transform into morphologically distinct decidual cells under the influence of the implanting embryo and a proper hormonal environment. Mechanical stimulation of hormonally primed uterine stromal cells leads to the same morphologic alterations....... Mouse decidual cells isolated from 6- to 7-day pregnant uteri explanted in vitro continue to synthesize basement-membrane-like extracellular matrix. Using immunohistochemistry and metabolic labeling followed by immunoprecipitation, SDS-PAGE, and fluorography, it was shown that the decidual cells...... to undergo pseudodecidualization. We thus showed that stromal cells from pregnant and nonpregnant mouse uteri synthesize significant amounts of basement-membrane components in vitro, and hence could serve as a good model for the study of normal basement-membrane components....

Increased expression of endosomal members of toll-like receptor family abrogates wound healing in patients with type 2 diabetes mellitus.

Science.gov (United States)

Singh, Kanhaiya; Agrawal, Neeraj K; Gupta, Sanjeev K; Mohan, Gyanendra; Chaturvedi, Sunanda; Singh, Kiran

2016-10-01

The inflammatory phase of wound healing cascade is an important determinant of the fate of the wound. Acute inflammation is necessary to initiate proper wound healing, while chronic inflammation abrogates wound healing. Different endosomal members of toll-like receptor (TLR) family initiate inflammatory signalling via a range of different inflammatory mediators such as interferons, internal tissue damaged-associated molecular patterns (DAMPs) and hyperactive effector T cells. Sustained signalling of TLR9 and TLR7 contributes to chronic inflammation by activating the plasmacytoid dendritic cells. Diabetic wounds are also characterised by sustained inflammatory phase. The objective of this study was to analyse the differential expression of endosomal TLRs in human diabetic wounds compared with control wounds. We analysed the differential expression of TLR7 and TLR9 both at transcriptional and translational levels in wounds of 84 patients with type 2 diabetes mellitus (T2DM) and 6 control subjects without diabetes using quantitative real-time polymerase chain reaction (RT-PCR), western blot and immunohistochemistry. TLR7 and TLR9 were significantly up-regulated in wounds of the patients with T2DM compared with the controls and were dependent on the infection status of the diabetic wounds, and wounds with microbial infection exhibited lower expression levels of endosomal TLRs. Altered endosomal TLR expression in T2DM subjects might be associated with wound healing impairment. © 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd.

A Rab11A/myosin Vb/Rab11-FIP2 complex frames two late recycling steps of langerin from the ERC to the plasma membrane.

Science.gov (United States)

Gidon, Alexandre; Bardin, Sabine; Cinquin, Bertrand; Boulanger, Jerome; Waharte, François; Heliot, Laurent; de la Salle, Henri; Hanau, Daniel; Kervrann, Charles; Goud, Bruno; Salamero, Jean

2012-06-01

A large body of knowledge relating to the constitution of Rab GTPase/Rab effector complexes and their impact on both membrane domain organization and overall membrane trafficking has been built up in recent years. However in the context of the live cell there are still many questions that remain to be answered, such as where and when these complexes assemble and where they perform their primary function(s). We describe here the dynamic processes that take place in the final steps of the Rab11A dependent recycling pathway, in the context of the membrane platform constituted by Myosin Vb, Rab11A, and Rab11-FIP2. We first confirm that a series of previously reported observations obtained during the study of a number of trafficking cargoes also apply to langerin. Langerin is a cargo molecule that traffics through Rab11A-positive membrane domains of the endosomal recycling pathway. In order to explore the relative dynamics of this set of partners, we make extensive use of a combinatory approach of Live-FRET, fast FRAP video, fast confocal and TIRF microscopy modalities. Our data show that the Myosin Vb/Rab11A/Rab11-FIP2 platform is spatially involved in the regulation of langerin trafficking at two distinct sites within live cells, first at the sorting site in the endosomal recycling compartment (ERC) where transport vesicles are formed, and subsequently, in a strict time-defined order, at the very late stage of docking/tethering and fusion of these langerin recycling vesicles to the plasma membrane. © 2012 John Wiley & Sons A/S.

Continuous esterification to produce biodiesel by SPES/PES/NWF composite catalytic membrane in flow-through membrane reactor: experimental and kinetic studies.

Science.gov (United States)

Shi, Wenying; He, Benqiao; Cao, Yuping; Li, Jianxin; Yan, Feng; Cui, Zhenyu; Zou, Zhiqun; Guo, Shiwei; Qian, Xiaomin

2013-02-01

A novel composite catalytic membrane (CCM) was prepared from sulfonated polyethersulfone (SPES) and polyethersulfone (PES) blend supported by non-woven fabrics, as a heterogeneous catalyst to produce biodiesel from continuous esterification of oleic acid with methanol in a flow-through mode. A kinetic model of esterification was established based on a plug-flow assumption. The effects of the CCM structure (thickness, area, porosity, etc.), reaction temperature and the external and internal mass transfer resistances on esterification were investigated. The results showed that the CCM structure had a significant effect on the acid conversion. The external mass transfer resistance could be neglected when the flow rate was over 1.2 ml min(-1). The internal mass transfer resistance impacted on the conversion when membrane thickness was over 1.779 mm. An oleic acid conversion kept over 98.0% for 500 h of continuous running. The conversions obtained from the model are in good agreement with the experimental data. Copyright © 2012 Elsevier Ltd. All rights reserved.

Diverse Bacterial PKS Sequences Derived From Okadaic Acid-Producing Dinoflagellates

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Kathleen S. Rein

2008-05-01

Full Text Available Okadaic acid (OA and the related dinophysistoxins are isolated from dinoflagellates of the genus Prorocentrum and Dinophysis. Bacteria of the Roseobacter group have been associated with okadaic acid producing dinoflagellates and have been previously implicated in OA production. Analysis of 16S rRNA libraries reveals that Roseobacter are the most abundant bacteria associated with OA producing dinoflagellates of the genus Prorocentrum and are not found in association with non-toxic dinoflagellates. While some polyketide synthase (PKS genes form a highly supported Prorocentrum clade, most appear to be bacterial, but unrelated to Roseobacter or Alpha-Proteobacterial PKSs or those derived from other Alveolates Karenia brevis or Crytosporidium parvum.

Diphtheria toxin translocation across cellular membranes is regulated by sphingolipids

International Nuclear Information System (INIS)

Spilsberg, Bjorn; Hanada, Kentaro; Sandvig, Kirsten

2005-01-01

Diphtheria toxin is translocated across cellular membranes when receptor-bound toxin is exposed to low pH. To study the role of sphingolipids for toxin translocation, both a mutant cell line lacking the first enzyme in de novo sphingolipid synthesis, serine palmitoyltransferase, and a specific inhibitor of the same enzyme, myriocin, were used. The serine palmitoyltransferase-deficient cell line (LY-B) was found to be 10-15 times more sensitive to diphtheria toxin than the genetically complemented cell line (LY-B/cLCB1) and the wild-type cell line (CHO-K1), both when toxin translocation directly across the plasma membrane was induced by exposing cells with surface-bound toxin to low pH, and when the toxin followed its normal route via acidified endosomes into the cytosol. Toxin binding was similar in these three cell lines. Furthermore, inhibition of serine palmitoyltransferase activity by addition of myriocin sensitized the two control cell lines (LY-B/cLCB1 and CHO-K1) to diphtheria toxin, whereas, as expected, no effect was observed in cells lacking serine palmitoyltransferase (LY-B). In conclusion, diphtheria toxin translocation is facilitated by depletion of membrane sphingolipids

Constraints on the sources of branched tetraether membrane lipids in distal marine sediments

NARCIS (Netherlands)

Weijers, J.W.H.; Schefuß, E.; Kim, J.-H.; Sinninghe Damsté, J.; Schouten, S.

2014-01-01

Branched glycerol dialkyl glycerol tetraethers (brGDGTs) are membrane lipids produced by soil bacteria and occur in near coastal marine sediments as a result of soil organic matter input. Their abundance relative to marine-derived crenarchaeol, quantified in the BIT index, generally decreases

The attenuated inflammation of MPL is due to the lack of CD14-dependent tight dimerization of the TLR4/MD2 complex at the plasma membrane.

Science.gov (United States)

Tanimura, Natsuko; Saitoh, Shin-Ichiroh; Ohto, Umeharu; Akashi-Takamura, Sachiko; Fujimoto, Yukari; Fukase, Koichi; Shimizu, Toshiyuki; Miyake, Kensuke

2014-06-01

TLR4/MD-2 senses lipid A, activating the MyD88-signaling pathway on the plasma membrane and the TRIF-signaling pathway after CD14-mediated TLR4/MD-2 internalization into endosomes. Monophosphoryl lipid A (MPL), a detoxified derivative of lipid A, is weaker than lipid A in activating the MyD88-dependent pathway. Little is known, however, about mechanisms underlying the attenuated activation of MyD88-dependent pathways. We here show that MPL was impaired in induction of CD14-dependent TLR4/MD-2 dimerization compared with lipid A. Impaired TLR4/MD-2 dimerization decreased CD14-mediated TNFα production. In contrast, MPL was comparable to lipid A in CD14-independent MyD88-dependent TNFα production and TRIF-dependent responses including cell surface CD86 up-regulation and IFNβ induction. Although CD86 up-regulation is dependent on TRIF signaling, it was induced by TLR4/MD-2 at the plasma membrane. These results revealed that the attenuated MPL responses were due to CD14-initiated responses at the plasma membrane, but not just to responses initiated by MyD88, that is, MPL was specifically unable to induce CD14-dependent TLR4/MD-2 dimerization that selectively enhances MyD88-mediated responses at the plasma membrane. © The Japanese Society for Immunology. 2013. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Intracellular Transport of Vaccinia Virus in HeLa Cells Requires WASH-VPEF/FAM21-Retromer Complexes and Recycling Molecules Rab11 and Rab22

Science.gov (United States)

Hsiao, Jye-Chian; Chu, Li-Wei; Lo, Yung-Tsun; Lee, Sue-Ping; Chen, Tzu-Jung; Huang, Cheng-Yen

2015-01-01

ABSTRACT Vaccinia virus, the prototype of the Orthopoxvirus genus in the family Poxviridae, infects a wide range of cell lines and animals. Vaccinia mature virus particles of the WR strain reportedly enter HeLa cells through fluid-phase endocytosis. However, the intracellular trafficking process of the vaccinia mature virus between cellular uptake and membrane fusion remains unknown. We used live imaging of single virus particles with a combination of various cellular vesicle markers, to track fluorescent vaccinia mature virus particle movement in cells. Furthermore, we performed functional interference assays to perturb distinct vesicle trafficking processes in order to delineate the specific route undertaken by vaccinia mature virus prior to membrane fusion and virus core uncoating in cells. Our results showed that vaccinia virus traffics to early endosomes, where recycling endosome markers Rab11 and Rab22 are recruited to participate in subsequent virus trafficking prior to virus core uncoating in the cytoplasm. Furthermore, we identified WASH-VPEF/FAM21-retromer complexes that mediate endosome fission and sorting of virus-containing vesicles prior to virus core uncoating in the cytoplasm. IMPORTANCE Vaccinia mature virions of the WR strain enter HeLa cells through fluid phase endocytosis. We previously demonstrated that virus-containing vesicles are internalized into phosphatidylinositol 3-phosphate positive macropinosomes, which are then fused with Rab5-positive early endosomes. However, the subsequent process of sorting the virion-containing vesicles prior to membrane fusion remains unclear. We dissected the intracellular trafficking pathway of vaccinia mature virions in cells up to virus core uncoating in cytoplasm. We show that vaccinia mature virions first travel to early endosomes. Subsequent trafficking events require the important endosome-tethered protein VPEF/FAM21, which recruits WASH and retromer protein complexes to the endosome. There, the complex

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

Science.gov (United States)

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

2016-01-01

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

Matrimid® derived carbon molecular sieve hollow fiber membranes for ethylene/ethane separation

KAUST Repository

Xu, Liren

2011-09-01

Carbon molecular sieve (CMS) membranes have shown promising separation performance compared to conventional polymeric membranes. Translating the very attractive separation properties from dense films to hollow fibers is important for applying CMS materials in realistic gas separations. The very challenging ethylene/ethane separation is the primary target of this work. Matrimid® derived CMS hollow fiber membranes have been investigated in this work. Resultant CMS fiber showed interesting separation performance for several gas pairs, especially high selectivity for C2H4/C2H6. Our comparative study between dense film and hollow fiber revealed very similar selectivity for both configurations; however, a significant difference exists in the effective separation layer thickness between precursor fibers and their resultant CMS fibers. SEM results showed that the deviation was essentially due to the collapse of the porous substructure of the precursor fiber. Polymer chain flexibility (relatively low glass transition temperature (Tg) for Matrimid® relative to actual CMS formation) appears to be the fundamental cause of substructure collapse. This collapse phenomenon must be addressed in all cases involving intense heat-treatment near or above Tg. We also found that the defect-free property of the precursor fiber was not a simple predictor of CMS fiber performance. Even some precursor fibers with Knudsen diffusion selectivity could be transformed into highly selective CMS fibers for the Matrimid® precursor. To overcome the permeance loss problem caused by substructure collapse, several engineering approaches were considered. Mixed gas permeation results under realistic conditions demonstrate the excellent performance of CMS hollow fiber membrane for the challenging ethylene/ethane separation. © 2011 Elsevier B.V.

High yield cell-free production of integral membrane proteins without refolding or detergents.

Science.gov (United States)

Wuu, Jessica J; Swartz, James R

2008-05-01

Integral membrane proteins act as critical cellular components and are important drug targets. However, difficulties in producing membrane proteins have hampered investigations of structure and function. In vivo production systems are often limited by cell toxicity, and previous in vitro approaches have required unnatural folding pathways using detergents or lipid solutions. To overcome these limitations, we present an improved cell-free expression system which produces high yields of integral membrane proteins without the use of detergents or refolding steps. Our cell-free reaction activates an Escherichia coli-derived cell extract for transcription and translation. Purified E. coli inner membrane vesicles supply membrane-bound components and the lipid environment required for insertion and folding. Using this system, we demonstrated successful synthesis of two complex integral membrane transporters, the tetracycline pump (TetA) and mannitol permease (MtlA), in yields of 570+/-50 microg/mL and 130+/-30 microg/mL of vesicle-associated protein, respectively. These yields are up to 400 times typical in vivo concentrations. Insertion and folding of these proteins are verified by sucrose flotation, protease digestion, and activity assays. Whereas TetA incorporates efficiently into vesicle membranes with over two-thirds of the synthesized protein being inserted, MtlA yields appear to be limited by insufficient concentrations of a membrane-associated chaperone.

pH-Triggered Conformational Switching along the Membrane Insertion Pathway of the Diphtheria Toxin T-Domain

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Alexey S. Ladokhin

2013-08-01

Full Text Available The translocation (T-domain plays a key role in the action of diphtheria toxin and is responsible for transferring the catalytic domain across the endosomal membrane into the cytosol in response to acidification. Deciphering the molecular mechanism of pH-dependent refolding and membrane insertion of the T-domain, which is considered to be a paradigm for cell entry of other bacterial toxins, reveals general physicochemical principles underlying membrane protein assembly and signaling on membrane interfaces. Structure-function studies along the T-domain insertion pathway have been affected by the presence of multiple conformations at the same time, which hinders the application of high-resolution structural techniques. Here, we review recent progress in structural, functional and thermodynamic studies of the T-domain archived using a combination of site-selective fluorescence labeling with an array of spectroscopic techniques and computer simulations. We also discuss the principles of conformational switching along the insertion pathway revealed by studies of a series of T-domain mutants with substitutions of histidine residues.

Endocytosis of Cytotoxic Granules Is Essential for Multiple Killing of Target Cells by T Lymphocytes.

Science.gov (United States)

Chang, Hsin-Fang; Bzeih, Hawraa; Schirra, Claudia; Chitirala, Praneeth; Halimani, Mahantappa; Cordat, Emmanuelle; Krause, Elmar; Rettig, Jens; Pattu, Varsha

2016-09-15

CTLs are serial killers that kill multiple target cells via exocytosis of cytotoxic granules (CGs). CG exocytosis is tightly regulated and has been investigated in great detail; however, whether CG proteins are endocytosed following exocytosis and contribute to serial killing remains unknown. By using primary CTLs derived from a knock-in mouse of the CG membrane protein Synaptobrevin2, we show that CGs are endocytosed in a clathrin- and dynamin-dependent manner. Following acidification, endocytosed CGs are recycled through early and late, but not recycling endosomes. CGs are refilled with granzyme B at the late endosome stage and polarize to subsequent synapses formed between the CTL and new target cells. Importantly, inhibiting CG endocytosis in CTLs results in a significant reduction of their cytotoxic activity. Thus, our data demonstrate that continuous endocytosis of CG membrane proteins is a prerequisite for efficient serial killing of CTLs and identify key events in this process. Copyright © 2016 by The American Association of Immunologists, Inc.

Barcoding of GPCR trafficking and signaling through the various trafficking roadmaps by compartmentalized signaling networks.

Science.gov (United States)

Bahouth, Suleiman W; Nooh, Mohammed M

2017-08-01

Proper signaling by G protein coupled receptors (GPCR) is dependent on the specific repertoire of transducing, enzymatic and regulatory kinases and phosphatases that shape its signaling output. Activation and signaling of the GPCR through its cognate G protein is impacted by G protein-coupled receptor kinase (GRK)-imprinted "barcodes" that recruit β-arrestins to regulate subsequent desensitization, biased signaling and endocytosis of the GPCR. The outcome of agonist-internalized GPCR in endosomes is also regulated by sequence motifs or "barcodes" within the GPCR that mediate its recycling to the plasma membrane or retention and eventual degradation as well as its subsequent signaling in endosomes. Given the vast number of diverse sequences in GPCR, several trafficking mechanisms for endosomal GPCR have been described. The majority of recycling GPCR, are sorted out of endosomes in a "sequence-dependent pathway" anchored around a type-1 PDZ-binding module found in their C-tails. For a subset of these GPCR, a second "barcode" imprinted onto specific GPCR serine/threonine residues by compartmentalized kinase networks was required for their efficient recycling through the "sequence-dependent pathway". Mutating the serine/threonine residues involved, produced dramatic effects on GPCR trafficking, indicating that they played a major role in setting the trafficking itinerary of these GPCR. While endosomal SNX27, retromer/WASH complexes and actin were required for efficient sorting and budding of all these GPCR, additional proteins were required for GPCR sorting via the second "barcode". Here we will review recent developments in GPCR trafficking in general and the human β 1 -adrenergic receptor in particular across the various trafficking roadmaps. In addition, we will discuss the role of GPCR trafficking in regulating endosomal GPCR signaling, which promote biochemical and physiological effects that are distinct from those generated by the GPCR signal transduction

Neuron membrane trafficking and protein kinases involved in autism and ADHD.

Science.gov (United States)

Kitagishi, Yasuko; Minami, Akari; Nakanishi, Atsuko; Ogura, Yasunori; Matsuda, Satoru

2015-01-30

A brain-enriched multi-domain scaffolding protein, neurobeachin has been identified as a candidate gene for autism patients. Mutations in the synaptic adhesion protein cell adhesion molecule 1 (CADM1) are also associated with autism spectrum disorder, a neurodevelopmental disorder of uncertain molecular origin. Potential roles of neurobeachin and CADM1 have been suggested to a function of vesicle transport in endosomal trafficking. It seems that protein kinase B (AKT) and cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) have key roles in the neuron membrane trafficking involved in the pathogenesis of autism. Attention deficit hyperactivity disorder (ADHD) is documented to dopaminergic insufficiencies, which is attributed to synaptic dysfunction of dopamine transporter (DAT). AKT is also essential for the DAT cell-surface redistribution. In the present paper, we summarize and discuss the importance of several protein kinases that regulate the membrane trafficking involved in autism and ADHD, suggesting new targets for therapeutic intervention.

Neuron Membrane Trafficking and Protein Kinases Involved in Autism and ADHD

Directory of Open Access Journals (Sweden)

Yasuko Kitagishi

2015-01-01

Full Text Available A brain-enriched multi-domain scaffolding protein, neurobeachin has been identified as a candidate gene for autism patients. Mutations in the synaptic adhesion protein cell adhesion molecule 1 (CADM1 are also associated with autism spectrum disorder, a neurodevelopmental disorder of uncertain molecular origin. Potential roles of neurobeachin and CADM1 have been suggested to a function of vesicle transport in endosomal trafficking. It seems that protein kinase B (AKT and cyclic adenosine monophosphate (cAMP-dependent protein kinase A (PKA have key roles in the neuron membrane trafficking involved in the pathogenesis of autism. Attention deficit hyperactivity disorder (ADHD is documented to dopaminergic insufficiencies, which is attributed to synaptic dysfunction of dopamine transporter (DAT. AKT is also essential for the DAT cell-surface redistribution. In the present paper, we summarize and discuss the importance of several protein kinases that regulate the membrane trafficking involved in autism and ADHD, suggesting new targets for therapeutic intervention.

A-RAF kinase functions in ARF6 regulated endocytic membrane traffic.

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Elena Nekhoroshkova

Full Text Available 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.

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

Energy Technology Data Exchange (ETDEWEB)

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

2012-09-17

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

Macromolecule absorption and cortisol secretion in newborn calves derived from in vitro produced embryos

DEFF Research Database (Denmark)

Jacobsen, H; Sangild, P T; Schmidt, M

2002-01-01

Earlier reports indicate that calves derived from in vitro produced (IVP) embryos are more susceptible to neonatal disease than calves produced after artificial insemination (AI) or natural mating. The aims of the present study were to investigate whether calves born after IVP embryos show...

A Novel Interaction Between the TLR7 and a Colchicine Derivative Revealed Through a Computational and Experimental Study

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Francesco Gentile

2018-02-01

Full Text Available The Toll-Like Receptor 7 (TLR7 is an endosomal membrane receptor involved in the innate immune system response. Its best-known small molecule activators are imidazoquinoline derivatives such as imiquimod (R-837 and resiquimod (R-848. Recently, an interaction between R-837 and the colchicine binding site of tubulin was reported. To investigate the possibility of an interaction between structural analogues of colchicine and the TLR7, a recent computational model for the dimeric form of the TLR7 receptor was used to determine a possible interaction with a colchicine derivative called CR42-24, active as a tubulin polymerization inhibitor. The estimated values of the binding energy of this molecule with respect to the TLR7 receptor were comparable to the energies of known binders as reported in a previous study. The binding to the TLR7 was further assessed by introducing genetic transformations in the TLR7 gene in cancer cell lines and exposing them to the compound. A negative shift of the IC50 value in terms of cell growth was observed in cell lines carrying the mutated TLR7 gene. The reported study suggests a possible interaction between TLR7 and a colchicine derivative, which can be explored for rational design of new drugs acting on this receptor by using a colchicine scaffold for additional modifications.

Pleiotropic Actions of Helicobacter pylori Vacuolating Cytotoxin, VacA

OpenAIRE

Isomoto, Hajime; Moss, Joel; Hirayama, Toshiya

2010-01-01

Helicobacter pylori produces a vacuolating cytotoxin, VacA, and most virulent H. pylori strains secrete VacA. VacA binds to two types of receptor-like protein tyrosine phosphatase (RPTP), RPTPα and RPTPβ, on the surface of host cells. VacA bound to RPTPβ, relocates and concentrates in lipid rafts in the plasma membrane. VacA causes vacuolization, membrane anion-selective channel and pore formation, and disruption of endosomal and lysosomal activity in host cells. Secreted VacA is processed in...

Olefins-selective asymmetric carbon molecular sieve hollow fiber membranes for hybrid membrane-distillation processes for olefin/paraffin separations

KAUST Repository

Xu, Liren

2012-12-01

In this paper, the development of asymmetric carbon molecular sieve (CMS) hollow fiber membranes and advanced processes for olefin/paraffin separations based on the CMS membranes are reported. Membrane-based olefin/paraffin separations have been pursued extensively over the past decades. CMS membranes are promising to exceed the performance upper bound of polymer materials and have demonstrated excellent stability for gas separations. Previously, a substructure collapse phenomenon was found in Matrimid ® precursor derived CMS fiber. To overcome the permeance loss due to the increased separation layer thickness, 6FDA-DAM and 6FDA/BPDA-DAM precursors were selected as potential new precursors for carbon membrane formation. Defect-free asymmetric 6FDA-DAM and 6FDA/BPDA-DAM hollow fibers were successfully fabricated from a dry-jet/wet-quench spinning process. Polymer rigidity, glass-rubber transition and asymmetric morphology were correlated. CMS hollow fiber membranes produced from 6FDA-polymer precursors showed significant improvement in permeance for ethylene/ethane and propylene/propane separations. Further studies revealed that the CMS membranes are olefins-selective, which means the membranes are able to effectively separate olefins (ethylene and propylene) from paraffins (ethane and propane). This unique feature of CMS materials enables advanced hybrid membrane-distillation process designs. By using the olefins-selective membranes, these new processes may provide advantages over previously proposed retrofitting concepts. Further applications of the membranes are explored for hydrocarbons processes. Significant energy savings and even reduced footprint may be achieved in olefins production units. © 2012 Elsevier B.V.

Endogenous RGS14 is a cytoplasmic-nuclear shuttling protein that localizes to juxtanuclear membranes and chromatin-rich regions of the nucleus

Science.gov (United States)

Hepler, John R.

2017-01-01

Regulator of G protein signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates G protein and H-Ras/MAPkinase signaling pathways to regulate synaptic plasticity important for hippocampal learning and memory. However, to date, little is known about the subcellular distribution and roles of endogenous RGS14 in a neuronal cell line. Most of what is known about RGS14 cellular behavior is based on studies of tagged, recombinant RGS14 ectopically overexpressed in unnatural host cells. Here, we report for the first time a comprehensive assessment of the subcellular distribution and dynamic localization of endogenous RGS14 in rat B35 neuroblastoma cells. Using confocal imaging and 3D-structured illumination microscopy, we find that endogenous RGS14 localizes to subcellular compartments not previously recognized in studies of recombinant RGS14. RGS14 localization was observed most notably at juxtanuclear membranes encircling the nucleus, at nuclear pore complexes (NPC) on both sides of the nuclear envelope and within intranuclear membrane channels, and within both chromatin-poor and chromatin-rich regions of the nucleus in a cell cycle-dependent manner. In addition, a subset of nuclear RGS14 localized adjacent to active RNA polymerase II. Endogenous RGS14 was absent from the plasma membrane in resting cells; however, the protein could be trafficked to the plasma membrane from juxtanuclear membranes in endosomes derived from ER/Golgi, following constitutive activation of endogenous RGS14 G protein binding partners using AlF4¯. Finally, our findings show that endogenous RGS14 behaves as a cytoplasmic-nuclear shuttling protein confirming what has been shown previously for recombinant RGS14. Taken together, the findings highlight possible cellular roles for RGS14 not previously recognized that are distinct from the regulation of conventional GPCR-G protein signaling, in particular undefined roles for RGS14 in the nucleus. PMID:28934222

The Serotonin Transporter Undergoes Constitutive Internalization and Is Primarily Sorted to Late Endosomes and Lysosomal Degradation*

Science.gov (United States)

Rahbek-Clemmensen, Troels; Bay, Tina; Eriksen, Jacob; Gether, Ulrik; Jørgensen, Trine Nygaard

2014-01-01

The serotonin transporter (SERT) plays a critical role in regulating serotonin signaling by mediating reuptake of serotonin from the extracellular space. The molecular and cellular mechanisms controlling SERT levels in the membrane remain poorly understood. To study trafficking of the surface resident SERT, two functional epitope-tagged variants were generated. Fusion of a FLAG-tagged one-transmembrane segment protein Tac to the SERT N terminus generated a transporter with an extracellular epitope suited for trafficking studies (TacSERT). Likewise, a construct with an extracellular antibody epitope was generated by introducing an HA (hemagglutinin) tag in the extracellular loop 2 of SERT (HA-SERT). By using TacSERT and HA-SERT in antibody-based internalization assays, we show that SERT undergoes constitutive internalization in a dynamin-dependent manner. Confocal images of constitutively internalized SERT demonstrated that SERT primarily co-localized with the late endosomal/lysosomal marker Rab7, whereas little co-localization was observed with the Rab11, a marker of the “long loop” recycling pathway. This sorting pattern was distinct from that of a prototypical recycling membrane protein, the β2-adrenergic receptor. Furthermore, internalized SERT co-localized with the lysosomal marker LysoTracker and not with transferrin. The sorting pattern was further confirmed by visualizing internalization of SERT using the fluorescent cocaine analog JHC1-64 and by reversible and pulse-chase biotinylation assays showing evidence for lysosomal degradation of the internalized transporter. Finally, we found that SERT internalized in response to stimulation with 12-myristate 13-acetate co-localized primarily with Rab7- and LysoTracker-positive compartments. We conclude that SERT is constitutively internalized and that the internalized transporter is sorted mainly to degradation. PMID:24973209

Intrinsic Membrane Hyperexcitability of Amyotrophic Lateral Sclerosis Patient-Derived Motor Neurons

Directory of Open Access Journals (Sweden)

Brian J. Wainger

2014-04-01

Full Text Available Amyotrophic lateral sclerosis (ALS is a fatal neurodegenerative disease of the motor nervous system. We show using multielectrode array and patch-clamp recordings that hyperexcitability detected by clinical neurophysiological studies of ALS patients is recapitulated in induced pluripotent stem cell-derived motor neurons from ALS patients harboring superoxide dismutase 1 (SOD1, C9orf72, and fused-in-sarcoma mutations. Motor neurons produced from a genetically corrected but otherwise isogenic SOD1+/+ stem cell line do not display the hyperexcitability phenotype. SOD1A4V/+ ALS patient-derived motor neurons have reduced delayed-rectifier potassium current amplitudes relative to control-derived motor neurons, a deficit that may underlie their hyperexcitability. The Kv7 channel activator retigabine both blocks the hyperexcitability and improves motor neuron survival in vitro when tested in SOD1 mutant ALS cases. Therefore, electrophysiological characterization of human stem cell-derived neurons can reveal disease-related mechanisms and identify therapeutic candidates.

Process for producing curved surface of membrane rings for large containers, particulary for prestressed concrete pressure vessels of nuclear reactors

International Nuclear Information System (INIS)

Kumpf, H.

1977-01-01

Membrane rings for large pressure vessels, particularly for prestressed-concrete pressure vessels, often have curved surfaces. The invention describes a process of producing these at site, which is particularly advantageous as the forming and installation of the vessel component coincide. According to the invention, the originally flat membrane ring is set in a predetermined position, is then pressed in sections by a forming tool (with a preformed support ring as opposite tool), and shaped. After this, the shaped parts are welded to the ring-shaped wall parts of the large vessel. The manufacture of single and double membrane rings arrangements is described. (HP) [de

Inward cholesterol gradient of the membrane system in P. falciparum-infected erythrocytes involves a dilution effect from parasite-produced lipids

Directory of Open Access Journals (Sweden)

Fuyuki Tokumasu

2014-05-01

Full Text Available Plasmodium falciparum (Pf infection remodels the human erythrocyte with new membrane systems, including a modified host erythrocyte membrane (EM, a parasitophorous vacuole membrane (PVM, a tubulovesicular network (TVN, and Maurer's clefts (MC. Here we report on the relative cholesterol contents of these membranes in parasitized normal (HbAA and hemoglobin S-containing (HbAS, HbAS erythrocytes. Results from fluorescence lifetime imaging microscopy (FLIM experiments with a cholesterol-sensitive fluorophore show that membrane cholesterol levels in parasitized erythrocytes (pRBC decrease inwardly from the EM, to the MC/TVN, to the PVM, and finally to the parasite membrane (PM. Cholesterol depletion of pRBC by methyl-β-cyclodextrin treatment caused a collapse of this gradient. Lipid and cholesterol exchange data suggest that the cholesterol gradient involves a dilution effect from non-sterol lipids produced by the parasite. FLIM signals from the PVM or PM showed little or no difference between parasitized HbAA vs HbS-containing erythrocytes that differed in lipid content, suggesting that malaria parasites may regulate the cholesterol contents of the PVM and PM independently of levels in the host cell membrane. Cholesterol levels may affect raft structures and the membrane trafficking and sorting functions that support Pf survival in HbAA, HbAS and HbSS erythrocytes.

Galacturonomannan and Golgi-derived membrane linked to growth and shaping of biogenic calcite

Science.gov (United States)

Marsh, M. E.; Ridall, A. L.; Azadi, P.; Duke, P. J.

2002-01-01

The coccolithophores are valuable models for the design and synthesis of composite materials, because the cellular machinery controlling the nucleation, growth, and patterning of their calcitic scales (coccoliths) can be examined genetically. The coccoliths are formed within the Golgi complex and are the major CaCO(3) component in limestone sediments-particularly those of the Cretaceous period. In this study, we describe mutants lacking a sulfated galacturonomannan and show that this polysaccharide in conjunction with the Golgi-derived membrane is directly linked to the growth and shaping of coccolith calcite but not to the initial orientated nucleation of the mineral phase.

Bone Healing in Extraction Sockets Covered With Collagen Membrane Alone or Associated With Porcine-Derived Bone Graft: a Comparative Histological and Histomorphometric Analysis.

Science.gov (United States)

Guarnieri, Renzo; Testarelli, Luca; Stefanelli, Luigi; De Angelis, Francesca; Mencio, Francesca; Pompa, Giorgio; Di Carlo, Stefano

2017-01-01

The present paper reports data of a randomized study aimed to analyse and compare the histologic and histomorphometric aspects of bone healing in extraction sites covered with collagen membrane alone or associated with porcine-derived bone graft. Thirty patients, with single extraction sockets without severe bone wall defects in the premolar/molar region, were included. Ten extraction sockets were grafted with porcine-derived bone and covered with collagen membrane (group 1), 10 sites were covered with collagen membrane alone (group 2), and 10 sites healed spontaneously (group 3). After 4 months of healing, 26 (8 in group 1, 9 in group 2, and 9 in group 3) bone core specimens were harvested for histologic evaluation, then dental implants were placed. Sites in the group 1 and in the group 2 showed similar histologic and histomorphometric results without significantly differences in the percentage of vital bone (57.43% [SD 4.8] vs. 60.01% [SD 3.2]), and non-mineralized connective tissue 22.99% (SD 5.3) vs. 18.53% (SD 6.2). In group 1 a 16.57% (SD 3.8) of residual material was found. Results showed that the use of collagen membrane alone or associated to porcine-derived bone improves the healing bone process compared to that of extraction sites spontaneously healed. Moreover, histomorphometric data related to bone quality, indicated that extraction sites without severe walls defects and with a vestibular bone thickness > 1.5 mm, treated with a low resorbtion rate collagen membrane alone, do not need more than 4 months for dental implant insertion.

Automated sampling and data processing derived from biomimetic membranes

DEFF Research Database (Denmark)

Perry, Mark; Vissing, Thomas; Boesen, P.

2009-01-01

data processing software to analyze and organize the large amounts of data generated. In this work, we developed an automated instrumental voltage clamp solution based on a custom-designed software controller application (the WaveManager), which enables automated on-line voltage clamp data acquisition...... applicable to long-time series experiments. We designed another software program for off-line data processing. The automation of the on-line voltage clamp data acquisition and off-line processing was furthermore integrated with a searchable database (DiscoverySheet (TM)) for efficient data management......Recent advances in biomimetic membrane systems have resulted in an increase in membrane lifetimes from hours to days and months. Long-lived membrane systems demand the development of both new automated monitoring equipment capable of measuring electrophysiological membrane characteristics and new...

The late endosomal HOPS complex anchors active G-protein signaling essential for pathogenesis in magnaporthe oryzae.

Directory of Open Access Journals (Sweden)

Ravikrishna Ramanujam

Full Text Available In Magnaporthe oryzae, the causal ascomycete of the devastating rice blast disease, the conidial germ tube tip must sense and respond to a wide array of requisite cues from the host in order to switch from polarized to isotropic growth, ultimately forming the dome-shaped infection cell known as the appressorium. Although the role for G-protein mediated Cyclic AMP signaling in appressorium formation was first identified almost two decades ago, little is known about the spatio-temporal dynamics of the cascade and how the signal is transmitted through the intracellular network during cell growth and morphogenesis. In this study, we demonstrate that the late endosomal compartments, comprising of a PI3P-rich (Phosphatidylinositol 3-phosphate highly dynamic tubulo-vesicular network, scaffold active MagA/GαS, Rgs1 (a GAP for MagA, Adenylate cyclase and Pth11 (a non-canonical GPCR in the likely absence of AKAP-like anchors during early pathogenic development in M. oryzae. Loss of HOPS component Vps39 and consequently the late endosomal function caused a disruption of adenylate cyclase localization, cAMP signaling and appressorium formation. Remarkably, exogenous cAMP rescued the appressorium formation defects associated with VPS39 deletion in M. oryzae. We propose that sequestration of key G-protein signaling components on dynamic late endosomes and/or endolysosomes, provides an effective molecular means to compartmentalize and control the spatio-temporal activation and rapid downregulation (likely via vacuolar degradation of cAMP signaling amidst changing cellular geometry during pathogenic development in M. oryzae.

Relativistic derivation of the ponderomotive force produced by two intense laser fields

International Nuclear Information System (INIS)

Stroscio, M.A.

1985-01-01

The ponderomotive force plays a fundamental role in the absorption of laser light on self-consistent plasma density profiles, in multiple-photon ionization, and in intense field electrodynamics. The relativistic corrections to the ponderomotive force of a transversely polarized electromagnetic wave lead to an approximately 20-percent reduction in the single particle ponderomotive force produced by a 10-γm 10 16 -W/cm 2 laser field. Recent experimental investigations are based on using two intense laser fields to produce desired lasermatter interactions. This paper presents the first derivation of the nonlinear relativistic ponderomotive force produced by two intense laser fields. The results demonstrate that relativistic ponderomotive forces are not additive

The functional interplay of Rab11, FIP3 and Rho proteins on the endosomal recycling pathway controls cell shape and symmetry.

Science.gov (United States)

Bouchet, Jérôme; McCaffrey, Mary W; Graziani, Andrea; Alcover, Andrés

2018-07-04

Several families of small GTPases regulate a variety of fundamental cellular processes, encompassing growth factor signal transduction, vesicular trafficking and control of the cytoskeleton. Frequently, their action is hierarchical and complementary, but much of the detail of their functional interactions remains to be clarified. It is well established that Rab family members regulate a variety of intracellular vesicle trafficking pathways. Moreover, Rho family GTPases are pivotal for the control of the actin and microtubule cytoskeleton. However, the interplay between these 2 types of GTPases has been rarely reported. We discuss here our recent findings showing that Rab11, a key regulator of endosomal recycling, and Rac1, a central actin cytoskeleton regulator involved in lamellipodium formation and cell migration, interplay on endosomes through the Rab11 effector FIP3. In the context of the rapidly reactive T lymphocytes, Rab11-Rac1 endosomal functional interplay is important to control cell shape changes and cell symmetry during lymphocyte spreading and immunological synapse formation and ultimately modulate T cell activation.

Highly efficient transition metal and nitrogen co-doped carbide-derived carbon electrocatalysts for anion exchange membrane fuel cells

Science.gov (United States)

Ratso, Sander; Kruusenberg, Ivar; Käärik, Maike; Kook, Mati; Puust, Laurits; Saar, Rando; Leis, Jaan; Tammeveski, Kaido

2018-01-01

The search for an efficient electrocatalyst for oxygen reduction reaction (ORR) to replace platinum in fuel cell cathode materials is one of the hottest topics in electrocatalysis. Among the many non-noble metal catalysts, metal/nitrogen/carbon composites made by pyrolysis of cheap materials are the most promising with control over the porosity and final structure of the catalyst a crucial point. In this work we show a method of producing a highly active ORR catalyst in alkaline media with a controllable porous structure using titanium carbide derived carbon as a base structure and dicyandiamide along with FeCl3 or CoCl2 as the dopants. The resulting transition metal-nitrogen co-doped carbide derived carbon (M/N/CDC) catalyst is highly efficient for ORR electrocatalysis with the activity in 0.1 M KOH approaching that of commercial 46.1 wt.% Pt/C. The catalyst materials are also investigated by scanning electron microscopy, Raman spectroscopy and X-ray photoelectron spectroscopy to characterise the changes in morphology and composition causing the raise in electrochemical activity. MEA performance of M/N/CDC cathode materials in H2/O2 alkaline membrane fuel cell is tested with the highest power density reached being 80 mW cm-2 compared to 90 mW cm-2 for Pt/C.

Recovery of real dye bath wastewater using integrated membrane process: considering water recovery, membrane fouling and reuse potential of membranes.

Science.gov (United States)

Balcik-Canbolat, Cigdem; Sengezer, Cisel; Sakar, Hacer; Karagunduz, Ahmet; Keskinler, Bulent

2017-11-01

It has been recognized by the whole world that textile industry which produce large amounts of wastewater with strong color and toxic organic compounds is a major problematical industry requiring effective treatment solutions. In this study, reverse osmosis (RO) membranes were tested on biologically treated real dye bath wastewater with and without pretreatment by nanofiltration (NF) membrane to recovery. Also membrane fouling and reuse potential of membranes were investigated by multiple filtrations. Obtained results showed that only NF is not suitable to produce enough quality to reuse the wastewater in a textile industry as process water while RO provide successfully enough permeate quality. The results recommend that integrated NF/RO membrane process is able to reduce membrane fouling and allow long-term operation for real dye bath wastewater.

The Us2 gene product of herpes simplex virus 2 is a membrane-associated ubiquitin-interacting protein.

Science.gov (United States)

Kang, Ming-Hsi; Roy, Bibhuti B; Finnen, Renée L; Le Sage, Valerie; Johnston, Susan M; Zhang, Hui; Banfield, Bruce W

2013-09-01

The Us2 gene encodes a tegument protein that is conserved in most members of the Alphaherpesvirinae. Previous studies on the pseudorabies virus (PRV) Us2 ortholog indicated that it is prenylated, associates with membranes, and spatially regulates the enzymatic activity of the MAP (mitogen-activated protein) kinase ERK (extracellular signal-related kinase) through direct binding and sequestration of ERK at the cytoplasmic face of the plasma membrane. Here we present an analysis of the herpes simplex virus 2 (HSV-2) Us2 ortholog and demonstrate that, like PRV Us2, HSV-2 Us2 is a virion component and that, unlike PRV Us2, it does not interact with ERK in yeast two-hybrid assays. HSV-2 Us2 lacks prenylation signals and other canonical membrane-targeting motifs yet is tightly associated with detergent-insoluble membranes and localizes predominantly to recycling endosomes. Experiments to identify cellular proteins that facilitate HSV-2 Us2 membrane association were inconclusive; however, these studies led to the identification of HSV-2 Us2 as a ubiquitin-interacting protein, providing new insight into the functions of HSV-2 Us2.

PTP1B targets the endosomal sorting machinery

DEFF Research Database (Denmark)

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

2010-01-01

Dephosphorylation and endocytic down-regulation are distinct processes that together control the signaling output of a variety of receptor tyrosine kinases (RTKs). PTP1B can directly dephosphorylate several RTKs, but it can also promote activation of downstream pathways through largely unknown...... mechanisms. These positive signaling functions likely contribute to the tumor-promoting effect of PTP1B in mouse cancer models. Here, we have identified STAM2, an endosomal protein involved in sorting activated RTKs for lysosomal degradation, as a substrate of PTP1B. PTP1B interacts with STAM2 at defined...... phosphotyrosine sites, and knockdown of PTP1B expression augments STAM2 phosphorylation. Intriguingly, manipulating the expression and phosphorylation state of STAM2 did not have a general effect on epidermal growth factor (EGF)-induced EGF receptor trafficking, degradation, or signaling. Instead, phosphorylated...

Viral RNA Degradation and Diffusion Act as a Bottleneck for the Influenza A Virus Infection Efficiency.

Directory of Open Access Journals (Sweden)

Max Schelker

2016-10-01

Full Text Available After endocytic uptake, influenza viruses transit early endosomal compartments and eventually reach late endosomes. There, the viral glycoprotein hemagglutinin (HA triggers fusion between endosomal and viral membrane, a critical step that leads to release of the viral segmented genome destined to reach the cell nucleus. Endosomal maturation is a complex process involving acidification of the endosomal lumen as well as endosome motility along microtubules. While the pH drop is clearly critical for the conformational change and membrane fusion activity of HA, the effect of intracellular transport dynamics on the progress of infection remains largely unclear. In this study, we developed a comprehensive mathematical model accounting for the first steps of influenza virus infection. We calibrated our model with experimental data and challenged its predictions using recombinant viruses with altered pH sensitivity of HA. We identified the time point of virus-endosome fusion and thereby the diffusion distance of the released viral genome to the nucleus as a critical bottleneck for efficient virus infection. Further, we concluded and supported experimentally that the viral RNA is subjected to cytosolic degradation strongly limiting the probability of a successful genome import into the nucleus.

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

NARCIS (Netherlands)

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

2016-01-01

The low-density lipoprotein receptor (LDLR) plays a pivotal role in clearing atherogenic circulating low-density lipoprotein (LDL) cholesterol. Here we show that the COMMD/CCDC22/CCDC93 (CCC) and the Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) complexes are both crucial for endosomal

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

NARCIS (Netherlands)

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

The low-density lipoprotein receptor (LDLR) plays a pivotal role in clearing atherogenic circulating low-density lipoprotein (LDL) cholesterol. Here we show that the COMMD/CCDC22/CCDC93 (CCC) and the Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) complexes are both crucial for endosomal

Kinesin-3 and dynein cooperate in long-range retrograde endosome motility along a nonuniform microtubule array

NARCIS (Netherlands)

Schuster, M.; Kilaru, S.; Fink, G.; Collemare, J.A.R.; Roger, Y.; Steinberg, G.

2011-01-01

The polarity of microtubules (MTs) determines the motors for intracellular motility, with kinesins moving to plus ends and dynein to minus ends. In elongated cells of Ustilago maydis, dynein is thought to move early endosomes (EEs) toward the septum (retrograde), whereas kinesin-3 transports them to

Spatial resolution of cAMP signaling by soluble adenylyl cyclase

Science.gov (United States)

Caldieri, Giusi

2016-01-01

G protein–coupled receptor signaling starts at the plasma membrane and continues at endosomal stations. In this issue, Inda et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201512075) show that different forms of adenylyl cyclase are activated at the plasma membrane versus endosomes, providing a rationale for the spatial encoding of cAMP signaling. PMID:27402955

Bacillus subtilis MreB orthologs self-organize into filamentous structures underneath the cell membrane in a heterologous cell system.

Directory of Open Access Journals (Sweden)

Felix Dempwolff

Full Text Available Actin-like bacterial cytoskeletal element MreB has been shown to be essential for the maintenance of rod cell shape in many bacteria. MreB forms rapidly remodelling helical filaments underneath the cell membrane in Bacillus subtilis and in other bacterial cells, and co-localizes with its two paralogs, Mbl and MreBH. We show that MreB localizes as dynamic bundles of filaments underneath the cell membrane in Drosophila S2 Schneider cells, which become highly stable when the ATPase motif in MreB is modified. In agreement with ATP-dependent filament formation, the depletion of ATP in the cells lead to rapid dissociation of MreB filaments. Extended induction of MreB resulted in the formation of membrane protrusions, showing that like actin, MreB can exert force against the cell membrane. Mbl also formed membrane associated filaments, while MreBH formed filaments within the cytosol. When co-expressed, MreB, Mbl and MreBH built up mixed filaments underneath the cell membrane. Membrane protein RodZ localized to endosomes in S2 cells, but localized to the cell membrane when co-expressed with Mbl, showing that bacterial MreB/Mbl structures can recruit a protein to the cell membrane. Thus, MreB paralogs form a self-organizing and dynamic filamentous scaffold underneath the membrane that is able to recruit other proteins to the cell surface.

Bacillus subtilis MreB orthologs self-organize into filamentous structures underneath the cell membrane in a heterologous cell system.

Science.gov (United States)

Dempwolff, Felix; Reimold, Christian; Reth, Michael; Graumann, Peter L

2011-01-01

Actin-like bacterial cytoskeletal element MreB has been shown to be essential for the maintenance of rod cell shape in many bacteria. MreB forms rapidly remodelling helical filaments underneath the cell membrane in Bacillus subtilis and in other bacterial cells, and co-localizes with its two paralogs, Mbl and MreBH. We show that MreB localizes as dynamic bundles of filaments underneath the cell membrane in Drosophila S2 Schneider cells, which become highly stable when the ATPase motif in MreB is modified. In agreement with ATP-dependent filament formation, the depletion of ATP in the cells lead to rapid dissociation of MreB filaments. Extended induction of MreB resulted in the formation of membrane protrusions, showing that like actin, MreB can exert force against the cell membrane. Mbl also formed membrane associated filaments, while MreBH formed filaments within the cytosol. When co-expressed, MreB, Mbl and MreBH built up mixed filaments underneath the cell membrane. Membrane protein RodZ localized to endosomes in S2 cells, but localized to the cell membrane when co-expressed with Mbl, showing that bacterial MreB/Mbl structures can recruit a protein to the cell membrane. Thus, MreB paralogs form a self-organizing and dynamic filamentous scaffold underneath the membrane that is able to recruit other proteins to the cell surface.

Studies of water electrolysis in polymeric membrane cells; Estudos de eletrolise aquosa em celulas de membrana polimerica

Energy Technology Data Exchange (ETDEWEB)

Oliveira-Silva, M.A.; Linardi, M.; Saliba-Silva, A.M. [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil). Centro de Celulas a Combustivel e Hidrogenio

2010-07-01

Hydrogen represents great opportunity to be a substitute for fossil fuels in the future. Water as a renewable source of hydrogen is of great interest, since it is abundant and can decompose, producing only pure H{sub 2} and O{sub 2}. This decomposition of water can be accomplished by processes such as electrolysis, thermal decomposition and thermochemical cycles. The membrane electrolysis has been proposed as a viable process for hydrogen production using thermal and electrical energy derived from nuclear energy or any renewable source like solar energy. In this work, within the context of optimization of the electrolysis process, it is intended to develop a mathematical model that can simulate and assist in parameterization of the electrolysis performed by polymer membrane electrolytic cell. The experimental process to produce hydrogen via the cell membrane, aims to optimize the amount of gas produced using renewable energy with non-carbogenic causing no harm by producing gases deleterious to the environment. (author)

Thermal analysis and its application in evaluation of fluorinated polyimide membranes for gas separation

KAUST Repository

Qiu, Wulin

2011-08-01

Seven polyimides based on (4,4′-hexafluoroisopropylidene) diphthalic anhydride, 6FDA, with different chemical structures were synthesized in a single pot two-step procedure by first producing a high molecular weight polyamic acid (PAA), followed by reaction with acetic anhydride to produce polyimide (PI). The resulting polymers were characterized using thermal analysis techniques including TGA, derivative weight analysis, TGA-MS, and DSC. The decarboxylation-induced thermal cross-linking, ester cross-linking through a diol, and ion-exchange reactions of selected polyimide membranes were investigated. Cross-linking of polymer membranes was confirmed by solubility tests and CO 2 permeability measurements. The thermal analysis provides simple and timesaving opportunities to characterize the polymer properties, the ability to optimize polymer cross-linking conditions, and to monitor polymer functionalization to develop high performance polymeric membranes for gas separations. © 2011 Elsevier Ltd. All rights reserved.

Hydrogen evolution in enzymatic photoelectrochemical cell using modified seawater electrolytes produced by membrane desalination process

Energy Technology Data Exchange (ETDEWEB)

Joo, Hyunku; Yoon, Jaekyung [Hydrogen Energy Research Center, New and Renewable Energy Research Division, Korea Institute of Energy Research, 71-2 Jang-dong, Yuseong-gu, Daejeon 305-343 (Korea); Bae, Sanghyun [Department of Environmental Engineering, Yonsei University, 234 Maeji-ri, Hungub-myun, Wonju, Gangwon-do 220-710 (Korea); Kim, Chunghwan; Kim, Suhan [Korea Institute of Water and Environment, K-Water, 462-1 Jeonmin-dong, Yuseong-gu, Daejeon 305-730 (Korea)

2009-09-15

In the near future, potential water shortages are expected to occur all over the world and this problem will have a significant influence on the availability of water for water-splitting processes, such as photocatalysis and electrolysis, as well as for drinking water. For this reason, it has been suggested that seawater could be used as an alternative for the various water industries including hydrogen production. Seawater contains a large amount of dissolved ion components, thus allowing it to be used as an electrolyte in photoelectrochemical (PEC) systems for producing hydrogen. Especially, the concentrate (retentate) stream shows higher salinity than the seawater fed to the membrane desalination process, because purified water (fresh water) is produced as the permeate stream and the waste brine is more concentrated than the original seawater. In this study, we investigated the hydrogen evolution rate in a photoelectrochemical system, including the preparation and characterization of an anodized tubular TiO{sub 2} electrode (ATTE) as both the photoanode and the cathode with the assistance of an immobilized hydrogenase enzyme and an external bias (solar cell), and the use of various qualities of seawater produced by membrane desalination processes as the electrolyte. The results showed that the rate of hydrogen evolution obtained using the nanofiltration (NF) retentate in the PEC system is ca. 105 {mu}mol/cm{sup 2} h, showing that this is an effective seawater electrolyte for hydrogen production, the optimum amount of enzyme immobilized on the cathode is ca. 3.66 units per geometrical unit area (1 cm x 1 cm), and the optimum external external bias supplied by the solar cell is 2.0 V. (author)

The dengue virus type 2 envelope protein fusion peptide is essential for membrane fusion

International Nuclear Information System (INIS)

Huang, Claire Y.-H.; Butrapet, Siritorn; Moss, Kelly J.; Childers, Thomas; Erb, Steven M.; Calvert, Amanda E.; Silengo, Shawn J.; Kinney, Richard M.; Blair, Carol D.; Roehrig, John T.

2010-01-01

The flaviviral envelope (E) protein directs virus-mediated membrane fusion. To investigate membrane fusion as a requirement for virus growth, we introduced 27 unique mutations into the fusion peptide of an infectious cDNA clone of dengue 2 virus and recovered seven stable mutant viruses. The fusion efficiency of the mutants was impaired, demonstrating for the first time the requirement for specific FP AAs in optimal fusion. Mutant viruses exhibited different growth kinetics and/or genetic stabilities in different cell types and adult mosquitoes. Virus particles could be recovered following RNA transfection of cells with four lethal mutants; however, recovered viruses could not re-infect cells. These viruses could enter cells, but internalized virus appeared to be retained in endosomal compartments of infected cells, thus suggesting a fusion blockade. Mutations of the FP also resulted in reduced virus reactivity with flavivirus group-reactive antibodies, confirming earlier reports using virus-like particles.

Carbon molecular sieve membranes derived from Matrimid® polyimide for nitrogen/methane separation

KAUST Repository

Ning, Xue

2014-01-01

A commercial polyimide, Matrimid® 5218, was pyrolyzed under an inert argon atmosphere to produce carbon molecular sieve (CMS) dense film membranes for nitrogen/methane separation. The resulting CMS dense film separation performance was evaluated using both pure and mixed N2/CH4 permeation tests. The effects of final pyrolysis temperature on N 2/CH4 separation are reported. The separation performance of all CMS dense films significantly exceeds the polymer precursor dense film. The CMS dense film pyrolyzed at 800 C shows very attractive separation performance that surpasses the polymer membrane upper bound line, with N 2 permeability of 6.8 Barrers and N2/CH4 permselectivity of 7.7 from pure gas permeation, and N2 permeability of 5.2 Barrers and N2/CH4 permselectivity of 6.0 from mixed gas permeation. The temperature dependences of permeabilities, sorption coefficients, and diffusion coefficients of the membrane were studied, and the activation energy for permeation and diffusion, as well as the apparent heats of sorption are reported. The high permselectivity of this dense film is shown to arise from a significant entropic contribution in the diffusion selectivity. The study shows that the rigid \\'slit-shaped\\' CMS pore structure can enable a strong molecular sieving effect to effectively distinguish the size and shape difference between N2 and CH4. © 2013 Elsevier Ltd. All rights reserved.

Complex N-Glycans Influence the Spatial Arrangement of Voltage Gated Potassium Channels in Membranes of Neuronal-Derived Cells.

Directory of Open Access Journals (Sweden)

M Kristen Hall

Full Text Available The intrinsic electrical properties of a neuron depend on expression of voltage gated potassium (Kv channel isoforms, as well as their distribution and density in the plasma membrane. Recently, we showed that N-glycosylation site occupancy of Kv3.1b modulated its placement in the cell body and neurites of a neuronal-derived cell line, B35 neuroblastoma cells. To extrapolate this mechanism to other N-glycosylated Kv channels, we evaluated the impact of N-glycosylation occupancy of Kv3.1a and Kv1.1 channels. Western blots revealed that wild type Kv3.1a and Kv1.1 α-subunits had complex and oligomannose N-glycans, respectively, and that abolishment of the N-glycosylation site(s generated Kv proteins without N-glycans. Total internal reflection fluorescence microscopy images revealed that N-glycans of Kv3.1a contributed to its placement in the cell membrane while N-glycans had no effect on the distribution of Kv1.1. Based on particle analysis of EGFP-Kv proteins in the adhered membrane, glycosylated forms of Kv3.1a, Kv1.1, and Kv3.1b had differences in the number, size or density of Kv protein clusters in the cell membrane of neurites and cell body of B35 cells. Differences were also observed between the unglycosylated forms of the Kv proteins. Cell dissociation assays revealed that cell-cell adhesion was increased by the presence of complex N-glycans of Kv3.1a, like Kv3.1b, whereas cell adhesion was similar in the oligomannose and unglycosylated Kv1.1 subunit containing B35 cells. Our findings provide direct evidence that N-glycans of Kv3.1 splice variants contribute to the placement of these glycoproteins in the plasma membrane of neuronal-derived cells while those of Kv1.1 were absent. Further when the cell membrane distribution of the Kv channel was modified by N-glycans then the cell-cell adhesion properties were altered. Our study demonstrates that N-glycosylation of Kv3.1a, like Kv3.1b, provides a mechanism for the distribution of these

METHOD FOR PRODUCING ISOTOPIC METHANES AND PARTIALLY HALOGENATED DERIVATIVES THEROF

Science.gov (United States)

Frazer, J.W.

1959-08-18

A method is given for producing isotopic methanes and/ or partially halogenated derivatives. Lithium hydride, deuteride, or tritide is reacted with a halogenated methane or with a halogenated methane in combination with free halogen. The process is conveniently carried out by passing a halogenated methane preferably at low pressures or in an admixture with an inert gas through a fixed bed of finely divided lithium hydride heated initially to temperatures of 100 to 200 deg C depending upon the halogenated methane used.

Finite element method (FEM) model of the mechanical stress on phospholipid membranes from shock waves produced in nanosecond electric pulses (nsEP)

Science.gov (United States)

Barnes, Ronald; Roth, Caleb C.; Shadaram, Mehdi; Beier, Hope; Ibey, Bennett L.

2015-03-01

The underlying mechanism(s) responsible for nanoporation of phospholipid membranes by nanosecond pulsed electric fields (nsEP) remains unknown. The passage of a high electric field through a conductive medium creates two primary contributing factors that may induce poration: the electric field interaction at the membrane and the shockwave produced from electrostriction of a polar submersion medium exposed to an electric field. Previous work has focused on the electric field interaction at the cell membrane, through such models as the transport lattice method. Our objective is to model the shock wave cell membrane interaction induced from the density perturbation formed at the rising edge of a high voltage pulse in a polar liquid resulting in a shock wave propagating away from the electrode toward the cell membrane. Utilizing previous data from cell membrane mechanical parameters, and nsEP generated shockwave parameters, an acoustic shock wave model based on the Helmholtz equation for sound pressure was developed and coupled to a cell membrane model with finite-element modeling in COMSOL. The acoustic structure interaction model was developed to illustrate the harmonic membrane displacements and stresses resulting from shockwave and membrane interaction based on Hooke's law. Poration is predicted by utilizing membrane mechanical breakdown parameters including cortical stress limits and hydrostatic pressure gradients.

Cellular entry of ebola virus involves uptake by a macropinocytosis-like mechanism and subsequent trafficking through early and late endosomes.

Directory of Open Access Journals (Sweden)

Mohammad F Saeed

2010-09-01

Full Text Available Zaire ebolavirus (ZEBOV, a highly pathogenic zoonotic virus, poses serious public health, ecological and potential bioterrorism threats. Currently no specific therapy or vaccine is available. Virus entry is an attractive target for therapeutic intervention. However, current knowledge of the ZEBOV entry mechanism is limited. While it is known that ZEBOV enters cells through endocytosis, which of the cellular endocytic mechanisms used remains unclear. Previous studies have produced differing outcomes, indicating potential involvement of multiple routes but many of these studies were performed using noninfectious surrogate systems such as pseudotyped retroviral particles, which may not accurately recapitulate the entry characteristics of the morphologically distinct wild type virus. Here we used replication-competent infectious ZEBOV as well as morphologically similar virus-like particles in specific infection and entry assays to demonstrate that in HEK293T and Vero cells internalization of ZEBOV is independent of clathrin, caveolae, and dynamin. Instead the uptake mechanism has features of macropinocytosis. The binding of virus to cells appears to directly stimulate fluid phase uptake as well as localized actin polymerization. Inhibition of key regulators of macropinocytosis including Pak1 and CtBP/BARS as well as treatment with the drug EIPA, which affects macropinosome formation, resulted in significant reduction in ZEBOV entry and infection. It is also shown that following internalization, the virus enters the endolysosomal pathway and is trafficked through early and late endosomes, but the exact site of membrane fusion and nucleocapsid penetration in the cytoplasm remains unclear. This study identifies the route for ZEBOV entry and identifies the key cellular factors required for the uptake of this filamentous virus. The findings greatly expand our understanding of the ZEBOV entry mechanism that can be applied to development of new

Automated sampling and data processing derived from biomimetic membranes

International Nuclear Information System (INIS)

Perry, M; Vissing, T; Hansen, J S; Nielsen, C H; Boesen, T P; Emneus, J

2009-01-01

Recent advances in biomimetic membrane systems have resulted in an increase in membrane lifetimes from hours to days and months. Long-lived membrane systems demand the development of both new automated monitoring equipment capable of measuring electrophysiological membrane characteristics and new data processing software to analyze and organize the large amounts of data generated. In this work, we developed an automated instrumental voltage clamp solution based on a custom-designed software controller application (the WaveManager), which enables automated on-line voltage clamp data acquisition applicable to long-time series experiments. We designed another software program for off-line data processing. The automation of the on-line voltage clamp data acquisition and off-line processing was furthermore integrated with a searchable database (DiscoverySheet(TM)) for efficient data management. The combined solution provides a cost efficient and fast way to acquire, process and administrate large amounts of voltage clamp data that may be too laborious and time consuming to handle manually. (communication)

Automated sampling and data processing derived from biomimetic membranes

Energy Technology Data Exchange (ETDEWEB)

Perry, M; Vissing, T; Hansen, J S; Nielsen, C H [Aquaporin A/S, Diplomvej 377, DK-2800 Kgs. Lyngby (Denmark); Boesen, T P [Xefion ApS, Kildegaardsvej 8C, DK-2900 Hellerup (Denmark); Emneus, J, E-mail: Claus.Nielsen@fysik.dtu.d [DTU Nanotech, Technical University of Denmark, DK-2800 Kgs. Lyngby (Denmark)

2009-12-15

Recent advances in biomimetic membrane systems have resulted in an increase in membrane lifetimes from hours to days and months. Long-lived membrane systems demand the development of both new automated monitoring equipment capable of measuring electrophysiological membrane characteristics and new data processing software to analyze and organize the large amounts of data generated. In this work, we developed an automated instrumental voltage clamp solution based on a custom-designed software controller application (the WaveManager), which enables automated on-line voltage clamp data acquisition applicable to long-time series experiments. We designed another software program for off-line data processing. The automation of the on-line voltage clamp data acquisition and off-line processing was furthermore integrated with a searchable database (DiscoverySheet(TM)) for efficient data management. The combined solution provides a cost efficient and fast way to acquire, process and administrate large amounts of voltage clamp data that may be too laborious and time consuming to handle manually. (communication)

Deashing of coal liquids with ceramic membrane microfiltration and diafiltration

Energy Technology Data Exchange (ETDEWEB)

Bishop, B.; Goldsmith, R. [CeraMem Corp., Waltham, MA (United States)

1995-12-31

Removal of mineral matter from liquid hydrocarbons derived from the direct liquefaction of coal is required for product acceptability. Current methods include critical solvent deashing (Rose{sup {reg_sign}} process from Kerr-McGee) and filtration (U.S. Filter leaf filter as used by British Coal). These methods produce ash reject streams containing up to 15% of the liquid hydrocarbon product. Consequently, CeraMem proposed the use of low cost, ceramic crossflow membranes for the filtration of coal liquids bottoms to remove mineral matter and subsequent diafiltration (analogous to cake washing in dead-ended filtration) for the removal of coal liquid from the solids stream. The use of these ceramic crossflow membranes overcomes the limitations of traditional polymeric crossflow membranes by having the ability to operate at elevated temperature and to withstand prolonged exposure to hydrocarbon and solvent media. In addition, CeraMem`s membrane filters are significantly less expensive than competitive ceramic membranes due to their unique construction. With these ceramic membrane filters, it may be possible to reduce the product losses associated with traditional deashing processes at an economically attractive cost. The performance of these ceramic membrane microfilters is discussed.

Imaging endosomes and autophagosomes in whole mammalian cells using correlative cryo-fluorescence and cryo-soft X-ray microscopy (cryo-CLXM)

International Nuclear Information System (INIS)

Duke, Elizabeth M.H.; Razi, Minoo; Weston, Anne; Guttmann, Peter; Werner, Stephan; Henzler, Katja; Schneider, Gerd; Tooze, Sharon A.; Collinson, Lucy M.

2014-01-01

Cryo-soft X-ray tomography (cryo-SXT) is a powerful imaging technique that can extract ultrastructural information from whole, unstained mammalian cells as close to the living state as possible. Subcellular organelles including the nucleus, the Golgi apparatus and mitochondria have been identified by morphology alone, due to the similarity in contrast to transmission electron micrographs. In this study, we used cryo-SXT to image endosomes and autophagosomes, organelles that are particularly susceptible to chemical fixation artefacts during sample preparation for electron microscopy. We used two approaches to identify these compartments. For early and recycling endosomes, which are accessible to externally-loaded markers, we used an anti-transferrin receptor antibody conjugated to 10 nm gold particles. For autophagosomes, which are not accessible to externally-applied markers, we developed a correlative cryo-fluorescence and cryo-SXT workflow (cryo-CLXM) to localise GFP-LC3 and RFP-Atg9. We used a stand-alone cryo-fluorescence stage in the home laboratory to localise the cloned fluorophores, followed by cryo-soft X-ray tomography at the synchrotron to analyse cellular ultrastructure. We mapped the 3D ultrastructure of the endocytic and autophagic structures, and discovered clusters of omegasomes arising from ‘hotspots’ on the ER. Thus, immunogold markers and cryo-CLXM can be used to analyse cellular processes that are inaccessible using other imaging modalities. - Highlights: • We image whole, unstained mammalian cells using cryo-soft X-ray tomography. • Endosomes are identified using a gold marker for the transferrin receptor. • A new workflow for correlative cryo-fluorescence and cryo-SXT is used to locate early autophagosomes. • Interactions between endosomes, endoplasmic reticulum and forming autophagosomes are mapped in 3D. • Multiple omegasomes are shown to form at ‘hotspots’ on the endoplasmic reticulum

Membrane proteomics of phagosomes suggests a connection to autophagy

Energy Technology Data Exchange (ETDEWEB)

Shui, Wenqing; Sheu, Leslie; Liu, Jun; Smart, Brian; Petzold, Christopher J.; Hsieh, Tsung-yen; Pitcher, Austin; Keasling*, Jay D.; Bertozzi*, Carolyn R.

2008-11-25

Phagocytosis is the central process by which macrophage cellsinternalize and eliminate infectious microbes as well as apoptoticcells. During maturation, phagosomes containing engulfed particlesfuse with various endosomal compartments through theaction of regulatory molecules on the phagosomal membrane. Inthis study, we performed a proteomic analysis of the membranefraction from latex bead-containing (LBC) phagosomes isolatedfrom macrophages. The profile, which comprised 546 proteins,suggests diverse functions of the phagosome and potential connectionsto secretory processes, toll-like receptor signaling, andautophagy. Many identified proteins were not previously knownto reside in the phagosome. We characterized several proteins inLBC phagosomes that change in abundance on induction of autophagy,a process that has been previously implicated in the hostdefense against microbial pathogens. These observations suggestcrosstalk between autophagy and phagocytosis that may be relevantto the innate immune response of macrophages.

Genetics Home Reference: mucolipidosis type IV

Science.gov (United States)

... This protein is located in the membranes of lysosomes and endosomes, compartments within the cell that digest ... transport (trafficking) of fats (lipids) and proteins between lysosomes and endosomes. Mucolipin-1 appears to be important ...

Accumulation of dsRNA in endosomes contributes to inefficient RNA interference in the fall armyworm, Spodoptera frugiperda.

Science.gov (United States)

Yoon, June-Sun; Gurusamy, Dhandapani; Palli, Subba Reddy

2017-11-01

RNA interference (RNAi) efficiency varies among insects studied. The barriers for successful RNAi include the presence of double-stranded ribonucleases (dsRNase) in the lumen and hemolymph that could potentially digest double-stranded RNA (dsRNA) and the variability in the transport of dsRNA into and within the cells. We recently showed that the dsRNAs are transported into lepidopteran cells, but they are not processed into small interference RNAs (siRNAs) because they are trapped in acidic bodies. In the current study, we focused on the identification of acidic bodies in which dsRNAs accumulate in Sf9 cells. Time-lapse imaging studies showed that dsRNAs enter Sf9 cells and accumulate in acidic bodies within 20 min after their addition to the medium. CypHer-5E-labeled dsRNA also accumulated in the midgut and fat body dissected from Spodoptera frugiperda larvae with similar patterns observed in Sf9 cells. Pharmacological inhibitor assays showed that the dsRNAs use clathrin mediated endocytosis pathway for transport into the cells. We investigated the potential dsRNA accumulation sites employing LysoTracker and double labeling experiments using the constructs to express a fusion of green fluorescence protein with early or late endosomal marker proteins and CypHer-5E-labeled dsRNA. Interestingly, CypHer-5E-labeled dsRNA accumulated predominantly in early and late endosomes. These data suggest that entrapment of internalized dsRNA in endosomes is one of the major factors contributing to inefficient RNAi response in lepidopteran insects. Copyright © 2017 Elsevier Ltd. All rights reserved.

Recycling Endosomes and Viral Infection.

Science.gov (United States)

Vale-Costa, Sílvia; Amorim, Maria João

2016-03-08

Many viruses exploit specific arms of the endomembrane system. The unique composition of each arm prompts the development of remarkably specific interactions between viruses and sub-organelles. This review focuses on the viral-host interactions occurring on the endocytic recycling compartment (ERC), and mediated by its regulatory Ras-related in brain (Rab) GTPase Rab11. This protein regulates trafficking from the ERC and the trans-Golgi network to the plasma membrane. Such transport comprises intricate networks of proteins/lipids operating sequentially from the membrane of origin up to the cell surface. Rab11 is also emerging as a critical factor in an increasing number of infections by major animal viruses, including pathogens that provoke human disease. Understanding the interplay between the ERC and viruses is a milestone in human health. Rab11 has been associated with several steps of the viral lifecycles by unclear processes that use sophisticated diversified host machinery. For this reason, we first explore the state-of-the-art on processes regulating membrane composition and trafficking. Subsequently, this review outlines viral interactions with the ERC, highlighting current knowledge on viral-host binding partners. Finally, using examples from the few mechanistic studies available we emphasize how ERC functions are adjusted during infection to remodel cytoskeleton dynamics, innate immunity and membrane composition.

A New Strain Collection for Improved Expression of Outer Membrane Proteins

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Ina Meuskens

2017-11-01

Full Text Available Almost all integral membrane proteins found in the outer membranes of Gram-negative bacteria belong to the transmembrane β-barrel family. These proteins are not only important for nutrient uptake and homeostasis, but are also involved in such processes as adhesion, protein secretion, biofilm formation, and virulence. As surface exposed molecules, outer membrane β-barrel proteins are also potential drug and vaccine targets. High production levels of heterologously expressed proteins are desirable for biochemical and especially structural studies, but over-expression and subsequent purification of membrane proteins, including outer membrane proteins, can be challenging. Here, we present a set of deletion mutants derived from E. coli BL21(DE3 designed for the over-expression of recombinant outer membrane proteins. These strains harbor deletions of four genes encoding abundant β-barrel proteins in the outer membrane (OmpA, OmpC, OmpF, and LamB, both single and in all combinations of double, triple, and quadruple knock-outs. The sequences encoding these outer membrane proteins were deleted completely, leaving only a minimal scar sequence, thus preventing the possibility of genetic reversion. Expression tests in the quadruple mutant strain with four test proteins, including a small outer membrane β-barrel protein and variants thereof as well as two virulence-related autotransporters, showed significantly improved expression and better quality of the produced proteins over the parent strain. Differences in growth behavior and aggregation in the presence of high salt were observed, but these phenomena did not negatively influence the expression in the quadruple mutant strain when handled as we recommend. The strains produced in this study can be used for outer membrane protein production and purification, but are also uniquely useful for labeling experiments for biophysical measurements in the native membrane environment.

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

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Israel López-Reyes

2010-01-01

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

Multifunctional Cationic Lipid-Based Nanoparticles Facilitate Endosomal Escape and Reduction-Triggered Cytosolic siRNA Release

Science.gov (United States)

Gujrati, Maneesh; Malamas, Anthony; Shin, Tesia; Jin, Erlei; Sun, Lulu; Lu, Zheng-Rong

2015-01-01

Small interfering RNA (siRNA) has garnered much attention in recent years as a promising avenue for cancer gene therapy due to its ability to silence disease-related genes. Effective gene silencing is contingent upon the delivery of siRNA into the cytosol of target cells and requires the implementation of delivery systems possessing multiple functionalities to overcome delivery barriers. The present work explores the multifunctional properties and biological activity of a recently developed cationic lipid carrier, (1-aminoethyl)iminobis[N-(oleicylcysteinyl-1-amino-ethyl)propionamide]) (ECO). The physicochemical properties and biological activity of ECO/siRNA nanoparticles were assessed over a range of N/P ratios to optimize the formulation. Potent and sustained luciferase silencing in a U87 glioblastoma cell line was observed, even in the presence of serum proteins. ECO/siRNA nanoparticles exhibited pH-dependent membrane disruption at pH levels corresponding to various stages of the intracellular trafficking pathway. It was found that disulfide linkages created during nanoparticle formation enhanced the protection of siRNA from degradation and facilitated site-specific siRNA release in the cytosol by glutathione-mediated reduction. Confocal microscopy confirmed that ECO/siRNA nanoparticles readily escaped from late endosomes prior to cytosolic release of the siRNA cargo. These results demonstrate that the rationally designed multifunctionality of ECO/siRNA nanoparticles is critical for intracellular siRNA delivery and the continuing development of safe and effective delivery systems. PMID:25020033

Antimicrobial properties and membrane-active mechanism of a potential α-helical antimicrobial derived from cathelicidin PMAP-36.

Directory of Open Access Journals (Sweden)

Yinfeng Lv

Full Text Available Antimicrobial peptides (AMPs, which present in the non-specific immune system of organism, are amongst the most promising candidates for the development of novel antimicrobials. The modification of naturally occurring AMPs based on their residue composition and distribution is a simple and effective strategy for optimization of known AMPs. In this study, a series of truncated and residue-substituted derivatives of antimicrobial peptide PMAP-36 were designed and synthesized. The 24-residue truncated peptide, GI24, displayed antimicrobial activity comparable to the mother peptide PMAP-36 with MICs ranging from 1 to 4 µM, which is lower than the MICs of bee venom melittin. Although GI24 displayed high antimicrobial activity, its hemolytic activity was much lower than melittin, suggesting that GI24 have optimal cell selectivity. In addition, the crucial site of GI24 was identified through single site-mutation. An amino acid with high hydrophobicity at position 23 played an important role in guaranteeing the high antimicrobial activity of GI24. Then, lipid vesicles and whole bacteria were employed to investigate the membrane-active mechanisms. Membrane-simulating experiments showed that GI24 interacted strongly with negatively charged phospholipids and weakly with zwitterionic phospholipids, which corresponded well with the data of its biological activities. Membrane permeabilization and flow cytometry provide the evidence that GI24 killed microbial cells by permeabilizing the cell membrane and damaging membrane integrity. GI24 resulted in greater cell morphological changes and visible pores on cell membrane as determined using scanning electron microscopy (SEM and transmission electron microscope (TEM. Taken together, the peptide GI24 may provide a promising antimicrobial agent for therapeutic applications against the frequently-encountered bacteria.

In Vivo Efficacy of Measles Virus Fusion Protein-Derived Peptides Is Modulated by the Properties of Self-Assembly and Membrane Residence

Science.gov (United States)

Figueira, T. N.; Palermo, L. M.; Veiga, A. S.; Huey, D.; Alabi, C. A.; Santos, N. C.; Welsch, J. C.; Mathieu, C.; Niewiesk, S.; Moscona, A.

2016-01-01

ABSTRACT Measles virus (MV) infection is undergoing resurgence and remains one of the leading causes of death among young children worldwide despite the availability of an effective measles vaccine. MV infects its target cells by coordinated action of the MV hemagglutinin (H) and fusion (F) envelope glycoproteins; upon receptor engagement by H, the prefusion F undergoes a structural transition, extending and inserting into the target cell membrane and then refolding into a postfusion structure that fuses the viral and cell membranes. By interfering with this structural transition of F, peptides derived from the heptad repeat (HR) regions of F can inhibit MV infection at the entry stage. In previous work, we have generated potent MV fusion inhibitors by dimerizing the F-derived peptides and conjugating them to cholesterol. We have shown that prophylactic intranasal administration of our lead fusion inhibitor efficiently protects from MV infection in vivo. We show here that peptides tagged with lipophilic moieties self-assemble into nanoparticles until they reach the target cells, where they are integrated into cell membranes. The self-assembly feature enhances biodistribution and the half-life of the peptides, while integration into the target cell membrane increases fusion inhibitor potency. These factors together modulate in vivo efficacy. The results suggest a new framework for developing effective fusion inhibitory peptides. IMPORTANCE Measles virus (MV) infection causes an acute illness that may be associated with infection of the central nervous system (CNS) and severe neurological disease. No specific treatment is available. We have shown that fusion-inhibitory peptides delivered intranasally provide effective prophylaxis against MV infection. We show here that specific biophysical properties regulate the in vivo efficacy of MV F-derived peptides. PMID:27733647

Membrane distillation for milk concentration

NARCIS (Netherlands)

Moejes, S.N.; Romero Guzman, Maria; Hanemaaijer, J.H.; Barrera, K.H.; Feenstra, L.; Boxtel, van A.J.B.

2015-01-01

Membrane distillation is an emerging technology to concentrate liquid products while producing high quality water as permeate. Application for desalination has been studied extensively the past years, but membrane distillation has also potential to produce concentrated food products like

Catalytic nanoporous membranes

Science.gov (United States)

Pellin, Michael J; Hryn, John N; Elam, Jeffrey W

2013-08-27

A nanoporous catalytic membrane which displays several unique features Including pores which can go through the entire thickness of the membrane. The membrane has a higher catalytic and product selectivity than conventional catalysts. Anodic aluminum oxide (AAO) membranes serve as the catalyst substrate. This substrate is then subjected to Atomic Layer Deposition (ALD), which allows the controlled narrowing of the pores from 40 nm to 10 nm in the substrate by deposition of a preparatory material. Subsequent deposition of a catalytic layer on the inner surfaces of the pores reduces pore sizes to less than 10 nm and allows for a higher degree of reaction selectivity. The small pore sizes allow control over which molecules enter the pores, and the flow-through feature can allow for partial oxidation of reactant species as opposed to complete oxidation. A nanoporous separation membrane, produced by ALD is also provided for use in gaseous and liquid separations. The membrane has a high flow rate of material with 100% selectivity. Also provided is a method for producing a catalytic membrane having flow-through pores and discreet catalytic clusters adhering to the inside surfaces of the pores.

Treatment of Simulated Coalbed Methane Produced Water Using Direct Contact Membrane Distillation

Directory of Open Access Journals (Sweden)

Dong-Wan Cho

2016-05-01

Full Text Available Expolitation of coalbed methane (CBM involves production of a massive amount saline water that needs to be properly managed for environmental protection. In this study, direct contact membrane distillation (DCMD was utilized for treatment of CBM-produced water to remove saline components in the water. Simulated CBM waters containing varying concentrations of NaCl (1, 20, and 500 mM and NaHCO3 (1 and 25 mM were used as feed solutions under two transmembrane temperatures (Δ40 and 60 °C. In short-term distillation (~360 min, DCMD systems showed good performance with nearly 100% removal of salts for all solutes concentrations at both temperatures. The permeate flux increased with the feed temperature, but at a given temperature, it remained fairly stable throughout the whole operation. A gradual decline in permeate flux was observed at Δ60 °C at high NaHCO3 concentration (25 mM. In long-term distillation (5400 min, the presence of 25 mM NaHCO3 further decreased the flux to 25%–35% of the initial value toward the end of the operation, likely due to membrane fouling by deposition of Ca-carbonate minerals on the pore openings. Furthermore, pore wetting by the scalants occurred at the end of the experiment, and it increased the distillate conducitivity to 110 µS·cm−1. The precipitates formed on the surface were dominantly CaCO3 crystals, identified as aragonite.

New insights into membrane-active action in plasma membrane of fungal hyphae by the lipopeptide antibiotic bacillomycin L.

Science.gov (United States)

Zhang, Bao; Dong, Chunjuan; Shang, Qingmao; Han, Yuzhu; Li, Pinglan

2013-09-01

Bacillomycin L, a natural iturinic lipopeptide produced by Bacillus amyloliquefaciens, is characterized by strong antifungal activities against a variety of agronomically important filamentous fungi including Rhizoctonia solani Kühn. Prior to this study, the role of membrane permeabilization in the antimicrobial activity of bacillomycin L against plant pathogenic fungi had not been investigated. To shed light on the mechanism of this antifungal activity, the permeabilization of R. solani hyphae by bacillomycin L was investigated and compared with that by amphotericin B, a polyene antibiotic which is thought to act primarily through membrane disruption. Our results derived from electron microscopy, various fluorescent techniques and gel retardation experiments revealed that the antifungal activity of bacillomycin L may be not solely a consequence of fungal membrane permeabilization, but related to the interaction of it with intracellular targets. Our findings provide more insights into the mode of action of bacillomycin L and other iturins, which could in turn help to develop new or improved antifungal formulations or result in novel strategies to prevent fungal spoilage. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

Small interference RNA profiling reveals the essential role of human membrane trafficking genes in mediating the infectious entry of dengue virus

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Chu Justin

2010-02-01

Full Text Available Abstract Background Dengue virus (DENV is the causative agent of Dengue fever and the life-threatening Dengue Haemorrhagic fever or Dengue shock syndrome. In the absence of anti-viral agents or vaccine, there is an urgent need to develop an effective anti-viral strategy against this medically important viral pathogen. The initial interplay between DENV and the host cells may represent one of the potential anti-viral targeting sites. Currently the involvements of human membrane trafficking host genes or factors that mediate the infectious cellular entry of dengue virus are not well defined. Results In this study, we have used a targeted small interfering RNA (siRNA library to identify and profile key cellular genes involved in processes of endocytosis, cytoskeletal dynamics and endosome trafficking that are important and essential for DENV infection. The infectious entry of DENV into Huh7 cells was shown to be potently inhibited by siRNAs targeting genes associated with clathrin-mediated endocytosis. The important role of clathrin-mediated endocytosis was confirmed by the expression of well-characterized dominant-negative mutants of genes in this pathway and by using the clathrin endocytosis inhibitor chlorpromazine. Furthermore, DENV infection was shown to be sensitive to the disruption of human genes in regulating the early to late endosomal trafficking as well as the endosomal acidic pH. The importance and involvement of both actin and microtubule dynamics in mediating the infectious entry of DENV was also revealed in this study. Conclusions Together, the findings from this study have provided a detail profiling of the human membrane trafficking cellular genes and the mechanistic insight into the interplay of these host genes with DENV to initiate an infection, hence broadening our understanding on the entry pathway of this medically important viral pathogen. These data may also provide a new potential avenue for development of anti

Amniotic membrane-derived stem cells: immunomodulatory properties and potential clinical application

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Insausti CL

2014-03-01

Full Text Available Carmen L Insausti,1 Miguel Blanquer,1 Ana M García-Hernández,1 Gregorio Castellanos,2 José M Moraleda11Unidad de Trasplante Hematopoyético y Terapia Celular, 2Servicio de Cirugía, Hospital Clínico Universitario Virgen de la Arrixaca, IMIB, Campus Mare Nostrum, Universidad de Murcia, El Palmar, Murcia, SpainAbstract: Epithelial and mesenchymal cells isolated from the amniotic membrane (AM possess stem cell characteristics, differentiation potential toward lineages of different germ layers, and immunomodulatory properties. While their expansion and differentiation potential have been well studied and characterized, knowledge about their immunomodulatory properties and the mechanisms involved is still incomplete. These mechanisms have been evaluated on various target cells of the innate and the adaptive system and in animal models of different inflammatory diseases. Some results have evidenced that the immunomodulatory effect of AM-derived cells is dependent on cell-cell contact, but many of them have demonstrated that these properties are mediated through the secretion of suppressive molecules. In this review, we present an update on the described immunomodulatory properties of the derived amniotic cells and some of the proposed involved mechanisms. Furthermore, we describe some assays in animal models of different inflammatory diseases which reveal the potential use of these cells to treat such diseases.Keywords: epithelial cells, mesenchymal cells, cell therapy, immunomodulation

Heparan sulfate proteoglycans made by different basement-membrane-producing tumors have immunological and structural similarities

DEFF Research Database (Denmark)

Wewer, U M; Albrechtsen, R; Hassell, J R

1985-01-01

in the native basement membrane of surrounding normal murine tissues. Blocking and ELISA assays demonstrated that the antibodies recognized both antigens. Using techniques involving the chemical and enzymatic degradation of 35S-sulfate-labeled glycosaminoglycans, the mouse EHS tumor cells were found to produce...... proteoglycans obtained from these two sources immunoprecipitated the same precursor protein with a molecular mass of 400,000 daltons from 35S-methionine pulse-labeled cells of both tumors. Immunohistochemistry showed the heparan sulfate proteoglycan to be distributed in the extracellular matrix and also...

The hemifusion structure induced by influenza virus haemagglutinin is determined by physical properties of the target membranes.

Science.gov (United States)

Chlanda, Petr; Mekhedov, Elena; Waters, Hang; Schwartz, Cindi L; Fischer, Elizabeth R; Ryham, Rolf J; Cohen, Fredric S; Blank, Paul S; Zimmerberg, Joshua

2016-04-18

Influenza A virus haemagglutinin conformational change drives the membrane fusion of viral and endosomal membranes at low pH. Membrane fusion proceeds through an intermediate called hemifusion(1,2). For viral fusion, the hemifusion structures are not determined(3). Here, influenza virus-like particles(4) carrying wild-type haemagglutinin or haemagglutinin hemifusion mutant G1S(5) and liposome mixtures were studied at low pH by Volta phase plate cryo-electron tomography, which improves the signal-to-noise ratio close to focus. We determined two distinct hemifusion structures: a hemifusion diaphragm and a novel structure termed a 'lipidic junction'. Liposomes with lipidic junctions were ruptured with membrane edges stabilized by haemagglutinin. The rupture frequency and hemifusion diaphragm diameter were not affected by G1S mutation, but decreased when the cholesterol level in the liposomes was close to physiological concentrations. We propose that haemagglutinin induces a merger between the viral and target membranes by one of two independent pathways: a rupture-insertion pathway leading to the lipidic junction and a hemifusion-stalk pathway leading to a fusion pore. The latter is relevant under the conditions of influenza virus infection of cells. Cholesterol concentration functions as a pathway switch because of its negative spontaneous curvature in the target bilayer, as determined by continuum analysis.

The Annexin A1 Receptor FPR2 Regulates the Endosomal Export of Influenza Virus

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Fryad Rahman

2018-05-01

Full Text Available The Formyl Peptide Receptor 2 (FPR2 is a novel promising target for the treatment of influenza. During viral infection, FPR2 is activated by annexinA1, which is present in the envelope of influenza viruses; this activation promotes virus replication. Here, we investigated whether blockage of FPR2 would affect the genome trafficking of influenza virus. We found that, upon infection and cell treatment with the specific FPR2 antagonist WRW4 or the anti-FPR2 monoclonal antibody, FN-1D6-AI, influenza viruses were blocked into endosomes. This effect was independent on the strain and was observed for H1N1 and H3N2 viruses. In addition, blocking FPR2signaling in alveolar lung A549 epithelial cells with the monoclonal anti-FPR2 antibody significantly inhibited virus replication. Altogether, these results show that FPR2signaling interferes with the endosomal trafficking of influenza viruses and provides, for the first time, the proof of concept that monoclonal antibodies directed against FPR2 inhibit virus replication. Antibodies-based therapeutics have emerged as attractive reagents in infectious diseases. Thus, this study suggests that the use of anti-FPR2 antibodies against influenza hold great promise for the future.

A fluorescent glycolipid-binding peptide probe traces cholesterol dependent microdomain-derived trafficking pathways.

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Steffen Steinert

Full Text Available BACKGROUND: The uptake and intracellular trafficking of sphingolipids, which self-associate into plasma membrane microdomains, is associated with many pathological conditions, including viral and toxin infection, lipid storage disease, and neurodegenerative disease. However, the means available to label the trafficking pathways of sphingolipids in live cells are extremely limited. In order to address this problem, we have developed an exogenous, non-toxic probe consisting of a 25-amino acid sphingolipid binding domain, the SBD, derived from the amyloid peptide Abeta, and conjugated by a neutral linker with an organic fluorophore. The current work presents the characterization of the sphingolipid binding and live cell trafficking of this novel probe, the SBD peptide. SBD was the name given to a motif originally recognized by Fantini et al in a number of glycolipid-associated proteins, and was proposed to interact with sphingolipids in membrane microdomains. METHODOLOGY/PRINCIPAL FINDINGS: In accordance with Fantini's model, optimal SBD binding to membranes depends on the presence of sphingolipids and cholesterol. In synthetic membrane binding assays, SBD interacts preferentially with raft-like lipid mixtures containing sphingomyelin, cholesterol, and complex gangliosides in a pH-dependent manner, but is less glycolipid-specific than Cholera toxin B (CtxB. Using quantitative time-course colocalization in live cells, we show that the uptake and intracellular trafficking route of SBD is unlike that of either the non-raft marker Transferrin or the raft markers CtxB and Flotillin2-GFP. However, SBD traverses an endolysosomal route that partially intersects with raft-associated pathways, with a major portion being diverted at a late time point to rab11-positive recycling endosomes. Trafficking of SBD to acidified compartments is strongly disrupted by cholesterol perturbations, consistent with the regulation of sphingolipid trafficking by cholesterol

A voltage-gated calcium channel regulates lysosomal fusion with endosomes and autophagosomes and is required for neuronal homeostasis.

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Xuejun Tian

2015-03-01

Full Text Available Autophagy helps deliver sequestered intracellular cargo to lysosomes for proteolytic degradation and thereby maintains cellular homeostasis by preventing accumulation of toxic substances in cells. In a forward mosaic screen in Drosophila designed to identify genes required for neuronal function and maintenance, we identified multiple cacophony (cac mutant alleles. They exhibit an age-dependent accumulation of autophagic vacuoles (AVs in photoreceptor terminals and eventually a degeneration of the terminals and surrounding glia. cac encodes an α1 subunit of a Drosophila voltage-gated calcium channel (VGCC that is required for synaptic vesicle fusion with the plasma membrane and neurotransmitter release. Here, we show that cac mutant photoreceptor terminals accumulate AV-lysosomal fusion intermediates, suggesting that Cac is necessary for the fusion of AVs with lysosomes, a poorly defined process. Loss of another subunit of the VGCC, α2δ or straightjacket (stj, causes phenotypes very similar to those caused by the loss of cac, indicating that the VGCC is required for AV-lysosomal fusion. The role of VGCC in AV-lysosomal fusion is evolutionarily conserved, as the loss of the mouse homologues, Cacna1a and Cacna2d2, also leads to autophagic defects in mice. Moreover, we find that CACNA1A is localized to the lysosomes and that loss of lysosomal Cacna1a in cerebellar cultured neurons leads to a failure of lysosomes to fuse with endosomes and autophagosomes. Finally, we show that the lysosomal CACNA1A but not the plasma-membrane resident CACNA1A is required for lysosomal fusion. In summary, we present a model in which the VGCC plays a role in autophagy by regulating the fusion of AVs with lysosomes through its calcium channel activity and hence functions in maintaining neuronal homeostasis.

Rabies virus co-localizes with early (Rab5) and late (Rab7) endosomal proteins in neuronal and SH-SY5Y cells.

Science.gov (United States)

Ahmad, Waqas; Li, Yingying; Guo, Yidi; Wang, Xinyu; Duan, Ming; Guan, Zhenhong; Liu, Zengshan; Zhang, Maolin

2017-06-01

Rabies virus (RABV) is a highly neurotropic virus that follows clathrin-mediated endocytosis and pH-dependent pathway for trafficking and invasion into endothelial cells. Early (Rab5, EEA1) and late (Rab7, LAMP1) endosomal proteins play critical roles in endosomal sorting, maturity and targeting various molecular cargoes, but their precise functions in the early stage of RABV neuronal infection remain elusive. In this study, the relationship between enigmatic entry of RABV with these endosomal proteins into neuronal and SH-SY5Y cells was investigated. Immunofluorescence, TCID 50 titers, electron microscopy and western blotting were carried out to determine the molecular interaction of the nucleoprotein (N) of RABV with early or late endosomal proteins in these cell lines. The expression of N was also determined by down-regulating Rab5 and Rab7 in both cell lines through RNA interference. The results were indicative that N proficiently colocalized with Rab5/EEA1 and Rab7/LAMP1 in both cell lines at 24 and 48 h post-infection, while N titers significantly decreased in early infection of RABV. Down-regulation of Rab5 and Rab7 did not inhibit N expression, but it prevented productive infection via blocking the normal trafficking of RABV in a low pH environment. Ultrathin sections of cells studied by electron microscope also verified the close association of RABV with Rab5 and Rab7 in neurons. From the data it was concluded that primary entry of RABV strongly correlates with the kinetics of Rab-proteins present on early and late vesicles, which provides helpful clues to explain the early events of RABV in nerve cells.

New ways to produce porous polymeric membranes by carbon dioxide foaming

NARCIS (Netherlands)

Krause, B.; van der Vegt, N.F.A.; Wessling, Matthias

2002-01-01

As a new solvent free method for membrane formation, we have investigated the foaming of high-Tg polymers. We report two different routes for the formation of open-microcellular and open-nanoporous membrane morphologies. Porosity is introduced by expansion of carbon dioxide saturated films and

Modeling electrically active viscoelastic membranes.

Directory of Open Access Journals (Sweden)

Sitikantha Roy

Full Text Available The membrane protein prestin is native to the cochlear outer hair cell that is crucial to the ear's amplification and frequency selectivity throughout the whole acoustic frequency range. The outer hair cell exhibits interrelated dimensional changes, force generation, and electric charge transfer. Cells transfected with prestin acquire unique active properties similar to those in the native cell that have also been useful in understanding the process. Here we propose a model describing the major electromechanical features of such active membranes. The model derived from thermodynamic principles is in the form of integral relationships between the history of voltage and membrane resultants as independent variables and the charge density and strains as dependent variables. The proposed model is applied to the analysis of an active force produced by the outer hair cell in response to a harmonic electric field. Our analysis reveals the mechanism of the outer hair cell active (isometric force having an almost constant amplitude and phase up to 80 kHz. We found that the frequency-invariance of the force is a result of interplay between the electrical filtering associated with prestin and power law viscoelasticity of the surrounding membrane. Paradoxically, the membrane viscoelasticity boosts the force balancing the electrical filtering effect. We also consider various modes of electromechanical coupling in membrane with prestin associated with mechanical perturbations in the cell. We consider pressure or strains applied step-wise or at a constant rate and compute the time course of the resulting electric charge. The results obtained here are important for the analysis of electromechanical properties of membranes, cells, and biological materials as well as for a better understanding of the mechanism of hearing and the role of the protein prestin in this mechanism.

The Vici Syndrome Protein EPG5 Is a Rab7 Effector that Determines the Fusion Specificity of Autophagosomes with Late Endosomes/Lysosomes.

Science.gov (United States)

Wang, Zheng; Miao, Guangyan; Xue, Xue; Guo, Xiangyang; Yuan, Chongzhen; Wang, Zhaoyu; Zhang, Gangming; Chen, Yingyu; Feng, Du; Hu, Junjie; Zhang, Hong

2016-09-01

Mutations in the human autophagy gene EPG5 cause the multisystem disorder Vici syndrome. Here we demonstrated that EPG5 is a Rab7 effector that determines the fusion specificity of autophagosomes with late endosomes/lysosomes. EPG5 is recruited to late endosomes/lysosomes by direct interaction with Rab7 and the late endosomal/lysosomal R-SNARE VAMP7/8. EPG5 also binds to LC3/LGG-1 (mammalian and C. elegans Atg8 homolog, respectively) and to assembled STX17-SNAP29 Qabc SNARE complexes on autophagosomes. EPG5 stabilizes and facilitates the assembly of STX17-SNAP29-VAMP7/8 trans-SNARE complexes, and promotes STX17-SNAP29-VAMP7-mediated fusion of reconstituted proteoliposomes. Loss of EPG5 activity causes abnormal fusion of autophagosomes with various endocytic vesicles, in part due to elevated assembly of STX17-SNAP25-VAMP8 complexes. SNAP25 knockdown partially suppresses the autophagy defect caused by EPG5 depletion. Our study reveals that EPG5 is a Rab7 effector involved in autophagosome maturation, providing insight into the molecular mechanism underlying Vici syndrome. Copyright © 2016 Elsevier Inc. All rights reserved.

Flow and fouling in membrane filters: Effects of membrane morphology

Science.gov (United States)

Sanaei, Pejman; Cummings, Linda J.

2015-11-01

Membrane filters are widely-used in microfiltration applications. Many types of filter membranes are produced commercially, for different filtration applications, but broadly speaking the requirements are to achieve fine control of separation, with low power consumption. The answer to this problem might seem obvious: select the membrane with the largest pore size and void fraction consistent with the separation requirements. However, membrane fouling (an inevitable consequence of successful filtration) is a complicated process, which depends on many parameters other than membrane pore size and void fraction; and which itself greatly affects the filtration process and membrane functionality. In this work we formulate mathematical models that can (i) account for the membrane internal morphology (internal structure, pore size & shape, etc.); (ii) fouling of membranes with specific morphology; and (iii) make some predictions as to what type of membrane morphology might offer optimum filtration performance.

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

DEFF Research Database (Denmark)

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

2007-01-01

The endosomal-lysosomal system (ELS) has been suggested to play a role in the pathogenesis of prion diseases. The purpose of this study was to examine how experimental observations can be translated to human neuropathology and whether alterations of the ELS relate to neuropathologic changes...... correlate with regional pathology. Overloading of this system might impair the function of lysosomal enzymes and thus may mimic some features of lysosomal storage disorders. Udgivelsesdato: 2007-Jul...

Ruthenium complexes with phenylterpyridine derivatives target cell membrane and trigger death receptors-mediated apoptosis in cancer cells.

Science.gov (United States)

Deng, Zhiqin; Gao, Pan; Yu, Lianling; Ma, Bin; You, Yuanyuan; Chan, Leung; Mei, Chaoming; Chen, Tianfeng

2017-06-01

Elucidation of the communication between metal complexes and cell membrane may provide useful information for rational design of metal-based anticancer drugs. Herein we synthesized a novel class of ruthenium (Ru) complexes containing phtpy derivatives (phtpy = phenylterpyridine), analyzed their structure-activity relationship and revealed their action mechanisms. The result showed that, the increase in the planarity of hydrophobic Ru complexes significantly enhanced their lipophilicity and cellular uptake. Meanwhile, the introduction of nitro group effectively improved their anticancer efficacy. Further mechanism studies revealed that, complex (2c), firstly accumulated on cell membrane and interacted with death receptors to activate extrinsic apoptosis signaling pathway. The complex was then transported into cell cytoplasm through transferrin receptor-mediated endocytosis. Most of the intracellular 2c accumulated in cell plasma, decreasing the level of cellular ROS, inducing the activation of caspase-9 and thus intensifying the apoptosis. At the same time, the residual 2c can translocate into cell nucleus to interact with DNA, induce DNA damage, activate p53 pathway and enhance apoptosis. Comparing with cisplatin, 2c possesses prolonged circulation time in blood, comparable antitumor ability and importantly, much lower toxicity in vivo. Taken together, this study uncovers the role of membrane receptors in the anticancer actions of Ru complexes, and provides fundamental information for rational design of membrane receptor targeting anticancer drugs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Physicochemical characterization of artificial nanoerythrosomes derived from erythrocyte ghost membranes.

Science.gov (United States)

Deák, Róbert; Mihály, Judith; Szigyártó, Imola Cs; Wacha, András; Lelkes, Gábor; Bóta, Attila

2015-11-01

Colloidal stabile nanoerythrosomes with 200 nm average diameter were formed from hemoglobin-free erythrocyte ghost membrane via sonication and membrane extrusion. The incorporation of extra lipid (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC), added to the sonicated ghosts, caused significant changes in the thermotropic character of the original membranes. As a result of the increased DPPC ratio the chain melting of the hydrated DPPC system and the characteristic small angle X-ray scattering (SAXS) of the lipid bilayers appeared. Significant morphological changes were followed by transmission electron microscopy combined with freeze fracture method (FF-TEM). After the ultrasonic treatment the large entities of erythrocyte ghosts transformed into nearly spherical nanoerythrosomes with diameters between 100 and 300 nm and at the same time a great number of 10-30 nm large membrane proteins or protein clusters were dispersed in the aqueous medium. The infrared spectroscopy (FT-IR) pointed out, that the sonication did not cause changes in the secondary structures of the membrane proteins under our preparation conditions. About fivefold of extra lipid--compared to the lipid content of the original membrane--caused homogeneous dispersion of nanoerythrosomes however the shape of the vesicles was not uniform. After the addition of about tenfold of DPPC, monoform and monodisperse nanoerythrosomes became typical. The outer surfaces of these roughly spherical objects were frequently polygonal, consisting of a net of pentagons and hexagons. Copyright © 2015 Elsevier B.V. All rights reserved.

High Dehumidification Performance of Amorphous Cellulose Composite Membranes prepared from Trimethylsilyl Cellulose

KAUST Repository

Puspasari, Tiara

2018-04-11

Cellulose is widely regarded as an environmentally friendly, natural and low cost material which can significantly contribute the sustainable economic growth. In this study, cellulose composite membranes were prepared via regeneration of trimethylsilyl cellulose (TMSC), an easily synthesized cellulose derivative. The amorphous hydrophilic feature of the regenerated cellulose enabled fast permeation of water vapour. The pore-free cellulose layer thickness was adjustable by the initial TMSC concentration and acted as an efficient gas barrier. As a result, a 5,000 GPU water vapour transmission rate (WVTR) at the highest ideal selectivity of 1.1 x 106 was achieved by the membranes spin coated from a 7% (w/w) TMSC solution. The membranes maintained a 4,000 GPU WVTR with selectivity of 1.1 x 104 in the mixed-gas experiments, surpassing the performances of the previously reported composite membranes. This study provides a simple way to not only produce high performance membranes but also to advance cellulose as a low-cost and sustainable membrane material for dehumidification applications.

Plant proton pumps

DEFF Research Database (Denmark)

Gaxiola, Roberto A.; Palmgren, Michael Gjedde; Schumacher, Karin

2007-01-01

Chemiosmotic circuits of plant cells are driven by proton (H+) gradients that mediate secondary active transport of compounds across plasma and endosomal membranes. Furthermore, regulation of endosomal acidification is critical for endocytic and secretory pathways. For plants to react...

Neuronal sphingolipidoses: Membrane lipids and sphingolipid activator proteins regulate lysosomal sphingolipid catabolism.

Science.gov (United States)

Sandhoff, Konrad

2016-11-01

Glycosphingolipids and sphingolipids of cellular plasma membranes (PMs) reach luminal intra-lysosomal vesicles (LVs) for degradation mainly by pathways of endocytosis. After a sorting and maturation process (e.g. degradation of sphingomyelin (SM) and secretion of cholesterol), sphingolipids of the LVs are digested by soluble enzymes with the help of activator (lipid binding and transfer) proteins. Inherited defects of lipid-cleaving enzymes and lipid binding and transfer proteins cause manifold and fatal, often neurodegenerative diseases. The review summarizes recent findings on the regulation of sphingolipid catabolism and cholesterol secretion from the endosomal compartment by lipid modifiers, an essential stimulation by anionic membrane lipids and an inhibition of crucial steps by cholesterol and SM. Reconstitution experiments in the presence of all proteins needed, hydrolase and activator proteins, reveal an up to 10-fold increase of ganglioside catabolism just by the incorporation of anionic lipids into the ganglioside carrying membranes, whereas an additional incorporation of cholesterol inhibits GM2 catabolism substantially. It is suggested that lipid and other low molecular modifiers affect the genotype-phenotype relationship observed in patients with lysosomal diseases. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Bicaudal-D1 regulates the intracellular sorting and signalling of neurotrophin receptors.

Science.gov (United States)

Terenzio, Marco; Golding, Matthew; Russell, Matthew R G; Wicher, Krzysztof B; Rosewell, Ian; Spencer-Dene, Bradley; Ish-Horowicz, David; Schiavo, Giampietro

2014-07-17

We have identified a new function for the dynein adaptor Bicaudal D homolog 1 (BICD1) by screening a siRNA library for genes affecting the dynamics of neurotrophin receptor-containing endosomes in motor neurons (MNs). Depleting BICD1 increased the intracellular accumulation of brain-derived neurotrophic factor (BDNF)-activated TrkB and p75 neurotrophin receptor (p75(NTR)) by disrupting the endosomal sorting, reducing lysosomal degradation and increasing the co-localisation of these neurotrophin receptors with retromer-associated sorting nexin 1. The resulting re-routing of active receptors increased their recycling to the plasma membrane and altered the repertoire of signalling-competent TrkB isoforms and p75(NTR) available for ligand binding on the neuronal surface. This resulted in attenuated, but more sustained, AKT activation in response to BDNF stimulation. These data, together with our observation that Bicd1 expression is restricted to the developing nervous system when neurotrophin receptor expression peaks, indicate that BICD1 regulates neurotrophin signalling by modulating the endosomal sorting of internalised ligand-activated receptors. © 2014 The Authors.

Nanoscale domain formation of phosphatidylinositol 4-phosphate in the plasma and vacuolar membranes of living yeast cells.

Science.gov (United States)

Tomioku, Kan-Na; Shigekuni, Mikiko; Hayashi, Hiroki; Yoshida, Akane; Futagami, Taiki; Tamaki, Hisanori; Tanabe, Kenji; Fujita, Akikazu

2018-05-01

In budding yeast Saccharomyces cerevisiae, PtdIns(4)P serves as an essential signalling molecule in the Golgi complex, endosomal system, and plasma membrane, where it is involved in the control of multiple cellular functions via direct interactions with PtdIns(4)P-binding proteins. To analyse the distribution of PtdIns(4)P in yeast cells at a nanoscale level, we employed an electron microscopy technique that specifically labels PtdIns(4)P on the freeze-fracture replica of the yeast membrane. This method minimizes the possibility of artificial perturbation, because molecules in the membrane are physically immobilised in situ. We observed that PtdIns(4)P is localised on the cytoplasmic leaflet, but not the exoplasmic leaflet, of the plasma membrane, Golgi body, vacuole, and vesicular structure membranes. PtdIns(4)P labelling was not observed in the membrane of the endoplasmic reticulum, and in the outer and inner membranes of the nuclear envelope or mitochondria. PtdIns(4)P forms clusters of plasma membrane and vacuolar membrane according to point pattern analysis of immunogold labelling. There are three kinds of compartments in the cytoplasmic leaflet of the plasma membrane. In the present study, we showed that PtdIns(4)P is specifically localised in the flat undifferentiated plasma membrane compartment. In the vacuolar membrane, PtdIns(4)P was concentrated in intramembrane particle (IMP)-deficient raft-like domains, which are tightly bound to lipid droplets, but not surrounding IMP-rich non-raft domains in geometrical IMP-distributed patterns in the stationary phase. This is the first report showing microdomain formations of PtdIns(4)P in the plasma membrane and vacuolar membrane of budding yeast cells at a nanoscale level, which will illuminate the functionality of PtdIns(4)P in each membrane. Copyright © 2018 Elsevier GmbH. All rights reserved.

Metabolic Engineering of Yeast to Produce Fatty Acid-derived Biofuels: Bottlenecks and Solutions

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Jiayuan eSheng

2015-06-01

Full Text Available Fatty acid-derived biofuels can be a better solution than bioethanol to replace petroleum fuel, since they have similar energy content and combustion properties as current transportation fuels. The environmentally friendly microbial fermentation process has been used to synthesize advanced biofuels from renewable feedstock. Due to their robustness as well as the high tolerance to fermentation inhibitors and phage contamination, yeast strains such as Saccharomyces cerevisiae and Yarrowia lipolytica have attracted tremendous attention in recent studies regarding the production of fatty acid-derived biofuels, including fatty acids, fatty acid ethyl esters, fatty alcohols, and fatty alkanes. However, the native yeast strains cannot produce fatty acids and fatty acid-derived biofuels in large quantities. To this end, we have summarized recent publications in this review on metabolic engineering of yeast strains to improve the production of fatty acid-derived biofuels, identified the bottlenecks that limit the productivity of biofuels, and categorized the appropriate approaches to overcome these obstacles.

Bile acids modulate signaling by functional perturbation of plasma membrane domains.

Science.gov (United States)

Zhou, Yong; Maxwell, Kelsey N; Sezgin, Erdinc; Lu, Maryia; Liang, Hong; Hancock, John F; Dial, Elizabeth J; Lichtenberger, Lenard M; Levental, Ilya

2013-12-13

Eukaryotic cell membranes are organized into functional lipid and protein domains, the most widely studied being membrane rafts. Although rafts have been associated with numerous plasma membrane functions, the mechanisms by which these domains themselves are regulated remain undefined. Bile acids (BAs), whose primary function is the solubilization of dietary lipids for digestion and absorption, can affect cells by interacting directly with membranes. To investigate whether these interactions affected domain organization in biological membranes, we assayed the effects of BAs on biomimetic synthetic liposomes, isolated plasma membranes, and live cells. At cytotoxic concentrations, BAs dissolved synthetic and cell-derived membranes and disrupted live cell plasma membranes, implicating plasma membrane damage as the mechanism for BA cellular toxicity. At subtoxic concentrations, BAs dramatically stabilized domain separation in Giant Plasma Membrane Vesicles without affecting protein partitioning between coexisting domains. Domain stabilization was the result of BA binding to and disordering the nonraft domain, thus promoting separation by enhancing domain immiscibility. Consistent with the physical changes observed in synthetic and isolated biological membranes, BAs reorganized intact cell membranes, as evaluated by the spatial distribution of membrane-anchored Ras isoforms. Nanoclustering of K-Ras, related to nonraft membrane domains, was enhanced in intact plasma membranes, whereas the organization of H-Ras was unaffected. BA-induced changes in Ras lateral segregation potentiated EGF-induced signaling through MAPK, confirming the ability of BAs to influence cell signal transduction by altering the physical properties of the plasma membrane. These observations suggest general, membrane-mediated mechanisms by which biological amphiphiles can produce their cellular effects.

Chitosan and carboxymethyl-chitosan capping ligands: Effects on the nucleation and growth of hydroxyapatite nanoparticles for producing biocomposite membranes

Energy Technology Data Exchange (ETDEWEB)

Dumont, Vitor C.; Mansur, Alexandra A.P.; Carvalho, Sandhra M.; Medeiros Borsagli, Fernanda G.L.; Pereira, Marivalda M.; Mansur, Herman S., E-mail: hmansur@demet.ufmg.br

2016-02-01

predominant calcium phosphate phase produced during the co-precipitation aqueous process for both the chitosan and CMC biocomposites. These novel hybrid systems based on chitosan and chitosan-derivatives with nHA composites were non-cytotoxic to a human osteoblast-like model cell line (SAOS) according to MTT in vitro assays. Moreover, the CMC-nHA biocomposites revealed a striking improvement in the cell viability response compared to the CHI-nHA biocomposite, which was attributed to the much higher surface area caused by the refinement of the nanoparticles size. Thus, the results of this study demonstrate that these novel bionanocomposite membranes offer promising perspectives as biomaterials for potential repair and replacement of cartilage and bone tissues. - Highlights: • Nanohydroxyapatite particles prepared using chitosan-based ligands via aqueous route • Effects of chitosan and CMC on the nucleation and growth of hydroxyapatite particles • Biocomposites of HA nanoparticles in chitosan and O-carboxymethyl chitosan matrices • Nanocomposites were non-cytotoxic tested with SAOS cells using in vitro MTT assay • Chitosan bionanocomposites were produced for potential bone repair bioapplications.

Pituitary adenylate cyclase 1 receptor internalization and endosomal signaling mediate the pituitary adenylate cyclase activating polypeptide-induced increase in guinea pig cardiac neuron excitability.

Science.gov (United States)

Merriam, Laura A; Baran, Caitlin N; Girard, Beatrice M; Hardwick, Jean C; May, Victor; Parsons, Rodney L

2013-03-06

After G-protein-coupled receptor activation and signaling at the plasma membrane, the receptor complex is often rapidly internalized via endocytic vesicles for trafficking into various intracellular compartments and pathways. The formation of signaling endosomes is recognized as a mechanism that produces sustained intracellular signals that may be distinct from those generated at the cell surface for cellular responses including growth, differentiation, and survival. Pituitary adenylate cyclase activating polypeptide (PACAP; Adcyap1) is a potent neurotransmitter/neurotrophic peptide and mediates its diverse cellular functions in part through internalization of its cognate G-protein-coupled PAC1 receptor (PAC1R; Adcyap1r1). In the present study, we examined whether PAC1R endocytosis participates in the regulation of neuronal excitability. Although PACAP increased excitability in 90% of guinea pig cardiac neurons, pretreatment with Pitstop 2 or dynasore to inhibit clathrin and dynamin I/II, respectively, suppressed the PACAP effect. Subsequent addition of inhibitor after the PACAP-induced increase in excitability developed gradually attenuated excitability with no changes in action potential properties. Likewise, the PACAP-induced increase in excitability was markedly decreased at ambient temperature. Receptor trafficking studies with GFP-PAC1 cell lines demonstrated the efficacy of Pitstop 2, dynasore, and low temperatures at suppressing PAC1R endocytosis. In contrast, brefeldin A pretreatments to disrupt Golgi vesicle trafficking did not blunt the PACAP effect, and PACAP/PAC1R signaling still increased neuronal cAMP production even with endocytic blockade. Our results demonstrate that PACAP/PAC1R complex endocytosis is a key step for the PACAP modulation of cardiac neuron excitability.

Characterisation of the exopolysaccharide (EPS)-producing Lactobacillus paraplantarum BGCG11 and its non-EPS producing derivative strains as potential probiotics.

Science.gov (United States)

Nikolic, Milica; López, Patricia; Strahinic, Ivana; Suárez, Ana; Kojic, Milan; Fernández-García, María; Topisirovic, Ljubisa; Golic, Natasa; Ruas-Madiedo, Patricia

2012-08-17

Traditional fermented foods are the best source for the isolation of strains with specific traits to act as functional starters and to keep the biodiversity of the culture collections. Besides, these strains could be used in the formulation of foods claimed to promote health benefits, i.e. those containing probiotic microorganisms. For the rational selection of strains acting as probiotics, several in vitro tests have been proposed. In the current study, we have characterized the probiotic potential of the strain Lactobacillus paraplantarum BGCG11, isolated from a Serbian soft, white, homemade cheese, which is able to produce a "ropy" exopolysaccharide (EPS). Three novobiocin derivative strains, which have lost the ropy phenotype, were characterized as well in order to determine the putative role of the EPS in the probiotic potential. Under chemically gastrointestinal conditions, all strains were able to survive around 1-2% (10(6)-10(7)cfu/ml cultivable bacteria) only when they were included in a food matrix (1% skimmed milk). The strains were more resistant to acid conditions than to bile salts and gastric or pancreatic enzymes, which could be due to a pre-adaptation of the parental strain to acidic conditions in the cheese habitat. The ropy EPS did not improve the survival of the producing strain. On the contrary, the presence of an EPS layer surrounding the strain BGCG11 hindered its adhesion to the three epithelial intestinal cell lines tested, since the adhesion of the three non-ropy derivatives was higher than the parental one and also than that of the reference strain Lactobacillus rhamnosus GG. Aiming to propose a potential target application of these strains as probiotics, the cytokine production of peripheral blood mononuclear cells (PBMC) was analyzed. The EPS-producing L. paraplantarum BGCG11 strain showed an anti-inflammatory or immunosuppressor profile whereas the non-ropy derivative strains induced higher pro-inflammatory response. In addition, when

Adsorption of Pb(II and Cu(II by Ginkgo-Leaf-Derived Biochar Produced under Various Carbonization Temperatures and Times

Directory of Open Access Journals (Sweden)

Myoung-Eun Lee

2017-12-01

Full Text Available Ginkgo trees are common street trees in Korea, and the large amounts of leaves that fall onto the streets annually need to be cleaned and treated. Therefore, fallen gingko leaves have been used as a raw material to produce biochar for the removal of heavy metals from solutions. Gingko-leaf-derived biochar was produced under various carbonization temperatures and times. This study evaluated the physicochemical properties and adsorption characteristics of gingko-leaf-derived biochar samples produced under different carbonization conditions regarding Pb(II and Cu(II. The biochar samples that were produced at 800 °C for 90 and 120 min contained the highest oxygen- and nitrogen-substituted carbons, which might contribute to a high metal-adsorption rate. The intensity of the phosphate bond was increased with the increasing of the carbonization temperature up to 800 °C and after 90 min of carbonization. The Pb(II and Cu(II adsorption capacities were the highest when the gingko-leaf-derived biochar was produced at 800 °C, and the removal rates were 99.2% and 34.2%, respectively. The highest removal rate was achieved when the intensity of the phosphate functional group in the biochar was the highest. Therefore, the gingko-leaf-derived biochar produced at 800 °C for 90 min can be used as an effective bio-adsorbent in the removal of metals from solutions.

Effect of GAPDH-derived antimicrobial peptides on sensitive yeasts cells: membrane permeability, intracellular pH and H+-influx/-efflux rates.

Science.gov (United States)

Branco, Patrícia; Albergaria, Helena; Arneborg, Nils; Prista, Catarina

2018-05-01

Saccharomyces cerevisiae secretes antimicrobial peptides (AMPs) derived from glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which induce the death of several non-Saccharomyces yeasts. Previously, we demonstrated that the naturally secreted GAPDH-derived AMPs (i.e. saccharomycin) caused a loss of culturability and decreased the intracellular pH (pHi) of Hanseniaspora guilliermondii cells. In this study, we show that chemically synthesised analogues of saccharomycin also induce a pHi drop and loss of culturability in H. guilliermondii, although to a lesser extent than saccharomycin. To assess the underlying causes of the pHi drop, we evaluated the membrane permeability to H+ cations of H. guilliermondii cells, after being exposed to saccharomycin or its synthetic analogues. Results showed that the H+-efflux decreased by 75.6% and the H+-influx increased by 66.5% in cells exposed to saccharomycin at pH 3.5. Since H+-efflux via H+-ATPase is energy dependent, reduced glucose consumption would decrease ATP production and consequently H+-ATPase activity. However, glucose uptake rates were not affected, suggesting that the AMPs rather than affecting glucose transporters may affect directly the plasma membrane H+-ATPase or increase ATP leakage due to cell membrane disturbance. Thus, our study revealed that both saccharomycin and its synthetic analogues induced cell death of H. guilliermondii by increasing the proton influx and inhibiting the proton efflux.

Algae Derived Biofuel

Energy Technology Data Exchange (ETDEWEB)

Jahan, Kauser [Rowan Univ., Glassboro, NJ (United States)

2015-03-31

One of the most promising fuel alternatives is algae biodiesel. Algae reproduce quickly, produce oils more efficiently than crop plants, and require relatively few nutrients for growth. These nutrients can potentially be derived from inexpensive waste sources such as flue gas and wastewater, providing a mutual benefit of helping to mitigate carbon dioxide waste. Algae can also be grown on land unsuitable for agricultural purposes, eliminating competition with food sources. This project focused on cultivating select algae species under various environmental conditions to optimize oil yield. Membrane studies were also conducted to transfer carbon di-oxide more efficiently. An LCA study was also conducted to investigate the energy intensive steps in algae cultivation.

Mesenchymal stromal cells derived from cervical cancer produce high amounts of adenosine to suppress cytotoxic T lymphocyte functions

Directory of Open Access Journals (Sweden)

María de Lourdes Mora-García

2016-10-01

Full Text Available Abstract Background In recent years, immunomodulatory mechanisms of mesenchymal stem/stromal cells (MSCs from bone marrow and other “classic” sources have been described. However, the phenotypic and functional properties of tumor MSCs are poorly understood. The aim of this study was to analyze the immunosuppressive capacity of cervical cancer-derived MSCs (CeCa-MSCs on effector T lymphocytes through the purinergic pathway. Methods We determined the expression and functional activity of the membrane-associated ectonucleotidases CD39 and CD73 on CeCa-MSCs and normal cervical tissue-derived MSCs (NCx-MSCs. We also analyzed their immunosuppressive capacity to decrease proliferation, activation and effector cytotoxic T (CD8+ lymphocyte function through the generation of adenosine (Ado. Results We detected that CeCa-MSCs express higher levels of CD39 and CD73 ectonucleotidases in cell membranes compared to NCx-MSCs, and that this feature was associated with the ability to strongly suppress the proliferation, activation and effector functions of cytotoxic T-cells through the generation of large amounts of Ado from the hydrolysis of ATP, ADP and AMP nucleotides. Conclusions This study suggests that CeCa-MSCs play an important role in the suppression of the anti-tumor immune response in CeCa through the purinergic pathway.

Mesenchymal stromal cells derived from cervical cancer produce high amounts of adenosine to suppress cytotoxic T lymphocyte functions.

Science.gov (United States)

de Lourdes Mora-García, María; García-Rocha, Rosario; Morales-Ramírez, Omar; Montesinos, Juan José; Weiss-Steider, Benny; Hernández-Montes, Jorge; Ávila-Ibarra, Luis Roberto; Don-López, Christian Azucena; Velasco-Velázquez, Marco Antonio; Gutiérrez-Serrano, Vianey; Monroy-García, Alberto

2016-10-26

In recent years, immunomodulatory mechanisms of mesenchymal stem/stromal cells (MSCs) from bone marrow and other "classic" sources have been described. However, the phenotypic and functional properties of tumor MSCs are poorly understood. The aim of this study was to analyze the immunosuppressive capacity of cervical cancer-derived MSCs (CeCa-MSCs) on effector T lymphocytes through the purinergic pathway. We determined the expression and functional activity of the membrane-associated ectonucleotidases CD39 and CD73 on CeCa-MSCs and normal cervical tissue-derived MSCs (NCx-MSCs). We also analyzed their immunosuppressive capacity to decrease proliferation, activation and effector cytotoxic T (CD8+) lymphocyte function through the generation of adenosine (Ado). We detected that CeCa-MSCs express higher levels of CD39 and CD73 ectonucleotidases in cell membranes compared to NCx-MSCs, and that this feature was associated with the ability to strongly suppress the proliferation, activation and effector functions of cytotoxic T-cells through the generation of large amounts of Ado from the hydrolysis of ATP, ADP and AMP nucleotides. This study suggests that CeCa-MSCs play an important role in the suppression of the anti-tumor immune response in CeCa through the purinergic pathway.

Chitosan and carboxymethyl-chitosan capping ligands: Effects on the nucleation and growth of hydroxyapatite nanoparticles for producing biocomposite membranes.

Science.gov (United States)

Dumont, Vitor C; Mansur, Alexandra A P; Carvalho, Sandhra M; Medeiros Borsagli, Fernanda G L; Pereira, Marivalda M; Mansur, Herman S

2016-02-01

phosphate phase produced during the co-precipitation aqueous process for both the chitosan and CMC biocomposites. These novel hybrid systems based on chitosan and chitosan-derivatives with nHA composites were non-cytotoxic to a human osteoblast-like model cell line (SAOS) according to MTT in vitro assays. Moreover, the CMC-nHA biocomposites revealed a striking improvement in the cell viability response compared to the CHI-nHA biocomposite, which was attributed to the much higher surface area caused by the refinement of the nanoparticles size. Thus, the results of this study demonstrate that these novel bionanocomposite membranes offer promising perspectives as biomaterials for potential repair and replacement of cartilage and bone tissues.

Unsupported palladium alloy membranes and methods of making same

Science.gov (United States)

Way, J. Douglas; Thoen, Paul; Gade, Sabina K.

2015-06-02

The invention provides support-free palladium membranes and methods of making these membranes. Single-gas testing of the unsupported foils produced hydrogen permeabilities equivalent to thicker membranes produced by cold-rolling. Defect-free films as thin as 7.2 microns can be fabricated, with ideal H.sub.2/N.sub.2 selectivities as high as 40,000. Homogeneous membrane compositions may also be produced using these methods.

Alternative energy efficient membrane bioreactor using reciprocating submerged membrane.

Science.gov (United States)

Ho, J; Smith, S; Roh, H K

2014-01-01

A novel membrane bioreactor (MBR) pilot system, using membrane reciprocation instead of air scouring, was operated at constant high flux and daily fluctuating flux to demonstrate its application under peak and diurnal flow conditions. Low and stable transmembrane pressure was achieved at 40 l/m(2)/h (LMH) by use of repetitive membrane reciprocation. The results reveal that the inertial forces acting on the membrane fibers effectively propel foulants from the membrane surface. Reciprocation of the hollow fiber membrane is beneficial for the constant removal of solids that may build up on the membrane surface and inside the membrane bundle. The membrane reciprocation in the reciprocating MBR pilot consumed less energy than coarse air scouring used in conventional MBR systems. Specific energy consumption for the membrane reciprocation was 0.072 kWh/m(3) permeate produced at 40 LMH flux, which is 75% less than for a conventional air scouring system as reported in literature without consideration of energy consumption for biological aeration (0.29 kWh/m(3)). The daily fluctuating flux test confirmed that the membrane reciprocation is effective to handle fluctuating flux up to 50 LMH. The pilot-scale reciprocating MBR system successfully demonstrated that fouling can be controlled via 0.43 Hz membrane reciprocation with 44 mm or higher amplitude.